A New Record of Glaphyrid Scarab Beetles , Eulasia Vitatta ( Fabricius , 1775) (Coleoptera, Glaphyridae) from Erbil Kurdistan Region-Iraq

ZANCO Journal of Pure and Applied Sciences

The official scientific journal of Salahaddin University-Erbil ZJPAS (2016), 28 (3); 1-4

A new record of glaphyrid scarab beetles , Eulasia vitatta ( Fabricius , 1775) (Coleoptera, Glaphyridae) From Erbil Kurdistan region-Iraq

1* 2 2 Nabeel A. MawloodP P P P Muzafar I. HamadP P and Yaseen M. AbdullahP P 1College of Agriculture , Salahaddin University-Erbil, Iraq 2 Erbil Polytechnical Univ. Technical Institute / Khabat

A R T I C L E I N F O A B S T R A C T

Article History: A new record of glayphyrid scrab beetles Eulasia vitatta(Fabricius Received: 04/10/2015 ,1775)was described in Erbil Kurdistan region-Iraq. The important taxonomic Accepted: 08/12/2015 parts , labrum , mandibles , antenna and male genitalia have been drawn .Localities, plant hosts and date of the collection have been mentioned . Published:18/06/2016 Keywords: Coleoptera ,Glaphyridae, New record ,Eulasia vitatta, Kurdistan region-Iraq

*Corresponding Author: 1* Nabeel A. MawloodP P Email:

1. Introduction abdominal terga .They taper posteriorly and are separate at the apex . These beetles are 13-18 Glaphyridae MacLeay,1819 is one of small mm in long( Triplehorn and Johnson, 2005 ) . family of Scarabaeoidea. There are some 215 They are strong fliers and are often observed species in six genera, mainly found in the Old hovering near flowers or foliage or flying over World (Hawkins ,2006).Most Glaphyridae sandy area ( Borer and Delong , 1954) genera are restricted to the Palaearctic .Glaphyridae generally feed on a large variety (Medvedev ,1960) . The family includes five of flowers. Some species of the genus Eulasia genera and approximately 190 species in the feed on Compositae (including Asteraceae). Palaearctic region, and one genus with nine Eulasia subgenus Trichopleurus species feed species in the Nearctic region (Löbl and mainly on violet spiny flower heads such as Smetana ,2006). Several species of these Onopordum and on yellow Centaurea (both beetles, which rely strongly on visual cues to Asteraceae) ( Keasar,et al., 2010) . Larvae are find flowers, are dominant pollinators of plants free-living in sandy areas (riparian and coastal with red bowl-shaped flowers in the Eastern dunes) where they feed decaying leaf litter and Mediterranean region (Shmida, 1981; Dafni et detritus that is layered in the sand ( Borer and al. , 1990). Adults of many species are brightly Delong , 1954 ;Carlson , 2002 ) . The Eulasia colored and hairy and often possess markings Truqui, 1848 is one of the and coloration resembling bees and bumble bees.The member of this groupare elongated most widespread genus of the family include and brownish and have a very hairy body , the 57 species distributed from Turkey and Iran in elytra are short exposing two or three the north and reaching Israel in the south 2 Nabeel A. et al./ ZJPAS: 2016, 28(3): 1-4

(Baraud ,1990) . Nikodym and Kral (1998) surface sparsely dark brown setoe , lateral listed18 species of Iran . Montreuil1 and Serri margins densely brown setose, anterior ( 2007 ) described two species from Iran, sparsely setose. Mandible ( Fig.1b ) high Eulasia azerbaijanica (Petrovitz), and Eulasia sclerotized , apical part with three acute chrysopyga(Faldermann). In Iraq there are not denticels ,inner denticals is the shorter , median enough taxonomic studies of the family species denticals slightly longer than the outer , upper except; Derwesh (1965) indicated five species margin of mandibles densely long dark brown and Al-Ali (1977) recorded four species . setoes , molar area densely yellow setose. Labium dark brown , Labial palps ( Fig. 1c ) densely long , dark brown setose . 1st and 2nd segments cup shaped,2nd segment 1.2 times as Materials and Methods long as 1st segment , 3rd segment cylindrical shaped 1.1 as long as 2nd segment. Maxilla ( The specimens were collected from the Fig. 1d )dark brown , cardo triangular shaped , period of April until June / 2014 from the sparsely very long dark brown setose , stipes flowers of some weeds in different localities of black triangular shaped,densely very long , Erbil Kurdistan region - Iraq. The specimens black setose ,galea yellow densely long yellow were placed in boiling water for 10-15 minutes setose , 1st-3rd segments of maxillary palps to soften their parts. Then the parts were cup shaped , sparsely dark brown setose , 2nd separated and put in 10% KOH, placed on fire segment 1.2 as long as 3rd segment,4th with shaking for about (4-5) minutes for segment elongate oval , bare 2times as long as dissolving of lipids materials of the body and 3rd segment.Antenna ( Fig. 1e ) dark brown - destroying the muscles. After that placed in black , consist of ten segments, 1st segment is distilled water for 2-3 minutes in order to the longest 4 times as long as 2nd segment , neutralize the alkali. The parts are placed in densely black , long setose , 2nd segments ethyl alcohol 25% and dissected under rounded densely, blacksetose,3rd and 4th microscope, then transferred to ethyl alcohol segments tubular shaped , 3rd segment 1,1 50%, 75% and 100% respectively for two times as long as the fourth, 8th-9th minutes for each concentration to dehydration segmentscup shaped , sub equal in length , 10th of water, then placed in xylol for two minutes, segment oval slightly longer than 10th for translucency, finely placed in Canada segment . balsam to support slides for subsequent examination under microscope. The species Thorax :Pronotum black nearly cup shaped , identified by using taxonomic key of (Baraud purple reflections ,1.4 time as long as head , ,1990). densely long pale yellow setose. Scutellum black, triangular shaped sparsely long yellow Results and Dissection setoe .Elytra black , purple reflections,surface with five bands of yellow setae, Hind wing Eulasia vitatta ( Fabricius , 1775) yellow, well-developed , M-Cu loop present with 1 apical , veins dark yellow ,upper Body: Oval , black .Length: 14.2-17.0mm . margin with row of short black setae extend into the middle, r4 and r2 absent . Legs dark Head:black , purple reflections ,densely long brown –black , procoxae oval elongate pal yellow setoe . Eyes black, kidney shaped, ,forefemur thick ,slightly shorter than tibia , half divided by a canthus . Canthus visible, outer margin with three strong spines , dorsal densely long, dark brown setose. Clypus black and ventral surface densely setose , apical with rectangular shaped ,anterior margin strait, one spur . Protarsal segment 1.8 as long as 2nd posterior margin slightly convex , anterior segment, 2nd – 4th segments nearly equal in angles oval , posterior angles acute . Labrum( length , 5th segment 1.2 times as long as Fig.1 a ) dark brown , rectangular protarsal segment ,Foreclaws moderately shaped,anterior and posterior margins straight , curved . Midlegs resemble to the forelegs 3 Nabeel A. et al./ ZJPAS: 2016, 28(3): 1-4 except ,mesocoxae conical, mesofemur slinder ,30.4.20;Kasnazan , 12.5.2014 and Sefin , mesotibia without spines ,apically with two ,14.6.2014 . long spurs . Hind legs resemble to the midlegs except ,coxae plate shaped . References

Abdomen: Consist of six black visible Al-Ali, A. S.(1977). Phytophagous and segments , densely pale yellow setose. 1st -5th entomophagous insects and mites of Iraqi abdominals sternites transverse , 1st segment Natural History Research Center, Publishing 0.8 times as long as 2nd segment , 2nd – 5th 33:142 pp. segments nearly equal in length , 6thsternite cup shaped . 1st – 5th abdominal tergites Baraud, J. (1990).Contribution a la transverse , anterior connaissance du genre Eulasia Truqi (Coleoptera: Scarabaeoidea: Glaphyridae). Revue Suisse de Zoologie . 97, 107-138.

Borror, D.J. and D.M. Delong (1954). An introduction to study of insects.Holt, Rinehart and Winston, New York. 1030.

Carlson ,D. C. (2002). Glaphyridae MacLeay (1819) In: Arnett, R.H.,

Thomas, M.C.; Skelley, P.E.; Frank. J.H Figure (1): Eulasia vitatta ( Fabricius ) .(Eds) .AmericanBeetles.Volume 2.Polyphaga: Scarabaeoidea through a. Labrum b. Mandible c. Labial palp d. Maxilla e. Antenna Curculionoidea.CRC Press, Boca Raton, 37–38. f. Male genitalia (Lateral view) g. Male genitalia (Dorsal view ) Dafni ,A ; P. Bernhardt ; A. Shmida ; Y. Ivri ; S. O. Greenbaum ; C. Toole and L. Losito ( and posterior margins straight , 5thtergite is the 1990 ). Red bowl-shaped flowers: convergence longest . surface of tergites sparsely yellow for beetle pollination in the Mediterranean setose . 1st – 5thtergites transverse , anterior region. Israel J. Bot., 39: 81–92. and posterior margins straight . 6thtergite cup shaped . Derwesh, A.I.(1965). A preliminary list of identified insects and arachnids of Iraq. Direct. Male genitalia ( Fig. 1f , g ): Male genitalia Gen. Agri. Res. Proj. Baghdad. Bull., No. 121- with well sclerotized, strongly arched basal 123. piece. Adegeause dark yellow nearly cylindrical shaped , moderately curved, length Hawkins, S. J.( 2006 ). A revision of the 2.8-3.4 mm . paramerse , brown , triangular Chilean tribe Lichniini Burmeister, 1844 1/4 times as long as adegeause . (Coleoptera: Scarabaeidae: Melolonthinae). Zootaxa,1266:1-63. Examined specimens :The specimens were collected from the flower of some weeds , Keasar, T. ; A. Harari ; G. Sabatinell ; K. Mallow, Malva rotundifolia, ; Haory cress , Keith ; A. Dafni ; O. Shavit; A. Zylbertal and Lepidium draba ; and Dwarf rocket , A.Shmida, A.( 2010 ) . Red anemone guild Diplotaxis sp. in different localities of Erbil flowers as focal places for mating and feeding governorate :Girdarasha ,20.4.2014 ; by Levant glaphyrid beetles .Biolog. J. Linn. Shaqlawa,28. 4.2014 ; Salahhaden Soc., 99, 808–817. 4 Nabeel A. et al./ ZJPAS: 2016, 28(3): 1-4

Löbl, I. and A. Smetana (2006 ). Catalogue of Palaearctic Coleoptera, Vol. 3.Scarabaeoidea – Scirtoidea – Dascilloidea – Buprestoidea – Byrrhoidea. Apollo: Stenstrup.

Medvedev , S.I. (1960) . Fauna SSSR , tome x , fase. 4. Scarabaeidae:

Euchirinae ,Dynastinae , Glaphyrinae , Trichiinae . Moscou ,p.1-398 .

Nikodym, M. and l. D. Kral (1998) . Results of the Czech biological expedition to Iran.Part 3.Coleoptera: Glaphyridae and Scarabaeidae: Sericinae. Acta Societatis Zoologicae Bohemoslovenicae,62, 343-351.

Montreuil1 , O. and S. Serri ( 2007) . Description of a new Pygopleurus species (Col.: Glaphyridae), with a list of recorded species of the genera Pygopleurus and Eulasia from Iran . J. Entomolo. Soc. Iran . 26(2): 1- 6 .

Shmida,A. ( 1981 ). Red display flowers of the Mediterranean. Israel Land and Nature ,6: 106–115.

Triplehorn , C.A. and Johnson, N.F. ( 2005 ). Borror and Delon, introduction to study of insects. 7th ed . Brooks /Cole,

Cengage Learning . Australia . 411 pp.

Acknowledgment

We sincerely thank the specialist Dr. Cosmin Manci , from Wildlife Management Consulting in Romania for their kind help in identifying of this species. We greatly indebted to the specialists in German museum of technology who confirmed the identification .We deeply express my gratitude to Prof. Dr. Mohammed S. Abdul Rassoul in Division of Entomology, Natural History Research Center

– University of Baghdad / Iraq for his kind help and continuous encouragement to this work ZANCO Journal of Pure and Applied Sciences

The official scientific journal of Salahaddin University-Erbil ZJPAS (2016), 28 (3); 5-11

Correlation of Sperm DNA Fragmentation With Some Semen Parameters Of Infertile Men In Iraqi Kurdistan Region Mowafak k. Hassan1* Mustafa S. Mustafa2* 1University of Mosul, Department of Biology ,College of Science, Mosul, Iraq. 2University of Salahadddin-Erbil Department of Environmental Sciences, College of Science, Erbil, Iraq

A R T I C L E I N F O A B S T R A C T

Article History: Background: Semen analysis is the first step in evaluation of male infertility Received: 27/07/2015 ,but it does not reflect the quality of sperm DNA therefore the evaluation of Accepted: 20/12/2015 sperm DNA integrity could add further information on the quality of spermatozoa and fertility in males. Several tests developed to assess sperm Published:18/08/2016 DNA fragmentation (SDF), the most frequently used is the Sperm Chromatin Keywords: Dispersion test (SCD). sperm DNA damage may be due to alterations in Male infertility, DNA integrity, chromatin modelling during the process of spermiogenesis, or from abortive sperm chromatin dispersion apoptosis or may be related to the action of reactive oxygen species (ROS). assay. Objective: The current study investigated the importance of assessment sperm DNA integrity in Iraqi males undergoing intracytoplasmic sperm injection (ICSI) cycles in order to predict pregnancy outcomes because sperm DNA *Corresponding Author: damage related to poor embryo development. Results: Iraqi infertile males are Mowafak k. Hassan shown to have significantly more sperm DNA fragmentation compared to Email: fertile males (%28.368± 2.149 vs %16.85 ± 1.737) in males 35 ≥ years and (%31.09± 2.468 vs %15.88± 2.502)in males 35< years. there was a significant negative correlation between SDF and sperm motility at r = -.420 accompanied with negative correlation of SDF with sperm concentration at r = - .150, No correlation was observed between SDF and ages in infertile males. Conclusion: Sperm DNA fragmentation was within the limit of fragmentation which decreased but not prevent pregnancy rates in IVF program.

1. Introduction Wetzels, 2001; Irvine et al., 2000). Clinical Infertility affect approximately 15% of a evidence showed that sperm DNA damage couples trying to conceive and male factor could be a marker of sperm quality as well as contributing in roughly half of the cease, the relationships between DNA damage and disorders of the male reproductive system have male fertility status(Zribi et al.,2011 ; Alvarez, become an important public health issue as 2003 ; Evenson et al., 2002). Sperm DNA they can cause infertility, miscarriages and resistant to many events that occur during abnormal outcomes in the offspring (Sergerie journey from the testis to time it reaches the et al.,2005; Oehninger, 2001).The quality of oocyst for fertilization(Agarwal and sperm DNA is very important in maintaining Allamaneni, 2004 ; Aitken and Sawyer, 2003). the reproductive potential of men (Agarwal and Sperm DNA damage is clearly associated with Allamaneni, 2004). A number of studies have male infertility and abnormal spermatogenesis investigated the relationship between human but small percentage of spermatozoa from sperm DNA damage and semen parameters, fertile men also possess detectable levels of such as concentration, morphology, and DNA damage (Zini et al.,2001 ; Spano et al., motility (Sills et al., 2004 ; Ramos and 2000). The degree of DNA damage correlate 6 Mowafak k et al./ ZJPAS: 2016, 28(3): 5-11 with various indices of fertility such as the al.,2003). The objective of this study was to fertilization rate , embryo cleavage rate, determine the levels of sperm DNA damage in implantation rate ,pregnancy rate and live birth Iraqi fertile and infertile males and it's rate of the offspring (Tomsu et al., 2002). The correlation with some sperm parameters. level of sperm DNA damage is related to high levels of oxidative stress found in the semen of MATERIALS AND METHODS infertile men with this condition (Saleh et The study included 40 infertile couples al.,2003). Testing DNA integrity is essential in attended the clinic of British specialized Centre fertilization and normal embryo and fetal for infertility treatment and IVF in Erbil city, development (Morris et al.,2002). Selection of Iraqi Kurdistan Region throughout period from Spermatozoa with intact DNA or with the least August 2012 to January 2013 and 16 healthy amount of DNA damage may help in assisted fertile donors proven fertility served as a conception, When spermatozoa with extensive control group . fertile and infertile males were DNA damage are used, the embryo may fail to divided into two groups according to their ages develop or implant in the uterus or it may be 35 ≥ years with mean ages 27.8 and 28.42 naturally aborted at later stage even when years for infertile and fertile males respectively sperm with minimum DNA damage are used while males at ages 35< years with mean ages fetal development can be affected of later 43.42 and 43.11 years for infertile and fertile stages, resulting in a child with congenital males respectively, all infertile males were abnormalities (Sharma et al.,2004). The routine with mean duration of infertility period at least examination of semen analysis which assess 2 ≤ years with no history of taking any sperm concentration , percentage motility and medication during the least 3 months. All morphology, does not identify subtle defects in patients had normal genital examination. sperm chromatin and sperm DNA damage (Barazani et al.,2014). Many researchers have Semen Collection: semen samples were investigated the relationship between human collected by masturbation after 3-5 days sperm DNA damage and sperm parameters, directly into a sterile dry labeled cup in private such as concentration , morphology and and quite room adjacent to semen analysis motility (Mahfouz et al.,2010). The evaluation laboratory . The specimen were placed in an of sperm DNA integrity in addition to routine incubator at 37ºc for 30 minutes to allow sperm parameters could add further liquefaction (Kvist & Björndahl, 2002) then information on the quality of spermatozoa and mixed carefully for few seconds followed by reproductive potential of males (Sheikh et microscopic examination within one hour of al.,2008 ; Perreault et al.,2003). Sperm DNA ejaculation, volume, concentration and motility fragmentation has been promoted as a was measured according to the guidelines of promising method to predict the outcome of the world health organization (WHO,2010). IVF cycl (García-Ferreyra et al.,2014 ; Muriel Sperm DNA Fragmentation (SDF): Fresh et al.,2006).sperm DNA fragmentation semen samples was collected and sperm DNA assessing to distinguish which couples are fragmentation assay performed immediately suitable for treatment by IVF (Check et once the sperm sample has obtained. SDF was al.,2005). SCD test sperm chromatin dispersion assisted by halosperm G2, a kit supplied by a simple highly reproducible and less halotech DNA company depending on sperm expensive technique, yielding results highly chromatin dispersion test(SCDT) using agarose correlated with those from other procedures microgel on a pretreated slide .the principle of like the DBD-Fish and the SCSA (Fernández et 7 Mowafak k et al./ ZJPAS: 2016, 28(3): 5-11 this method was using unfixed fresh sperm, an as shown in (Table 3). Our data referred to initial acid treatment denatures DNA in sperm presence of significant negative correlation cells, fragmented DNA following with lysis between infertile male ages with sperm motility solution to remove most of the nuclear at (r = - .702) as well as a negative correlation proteins, nucleoids with large haloes of with sperm concentration and ejaculate volume spreading DNA loops, emerging from a central at r = - .360 and r = - .349 respectively . Also core ,are produced. The nucleoids with large SDF increases with advancing age in a positive haloes of spreading DNA loops emerging from correlation at r = .052 as illustrated in (Table a central core are produced .The nucleoids 4). No significant correlation was found from sperm with fragmented DNA either do between SDF and infertile male ages (Figure 1) not show a dispersion halo or the halo is In addition, there was a significant negative minimal. correlation between SDF and sperm motility at r = -.420 (Figure 2) accompanied with negative Statistical Analysis: comparisons between correlation of SDF with sperm concentration at fertile and infertile males were tested by r = -.150 (Figure 3) , No correlation was student's t-test using SPSS11. Correlation observed between SDF and ejaculated volume between SDF and ages, sperm motility ,sperm in infertile males (Figure 4). concentration and ejaculate volume were evaluated using Pearson correlation Table 1: Some Seminal Fluid Parameters In Fertile coefficients. p≤0.05 was considered as and Infertile Males 35 ≥ Years statistically significant. parameters Mean±SE infertile Fertile RESULTS Age (years) 27.8 ±1.036 28.42±1.109 0.262 ±3.80 0.240±2.70٭ (Volume (ml The current study revealed the differences in Ejaculation some seminal fluid parameters between fertile Sperm 22.36 ± 58.85 ± 3.120 3.626٭ and infertile males according to their ages Concentration (106/ml) ,there were significant decrease in ejaculate Sperm Motility (%) 46.78 ± 58.00 ± 4.380 1.676٭ volume, sperm concentration and sperm motility of infertile males 35 ≥ years with Values are expressed as mean ± SE means reaches to 2.70±0.240 ml, 22.36 ± 3.626 (0.05> ٭sperm/ml and 46.78 ± 1.676 % to each Differences significant at (p 106 parameter respectively compared to fertile males at the same ages as shown in (Table1). Table 2: Some Seminal Fluid Parameters In Fertile Also there are significant decrease in sperm and Infertile Males 35 < Years concentration of infertile males 35< years that Parameters Mean±SE Infertile Fertile reaches 37.04± 4.224 106 sperm /ml compared with fertile males from the same ages 52.22 ± Age (years) 43.42±1.221 43.11±1.567 2.961 106 sperm /ml accompanied by Ejaculation Volume 2.32±0.194 2.73± 0.184 significant decrease in sperm motility to 37.04 (ml) ± 1.406% vs 51.33 ± 2.094% in fertile males Sperm Concentration 37.04± 4.224 52.22 ± 6 2.961 ٭ (Table 2).Results of sperm chromatin (10 /ml) Sperm Motility (%) 37.04 ± 51.33 ± 2.094 ٭ dispersion test showed that the percentage of 1.406 SDF in infertile males was significantly higher than fertile males (28.368± 2.149 vs 16.85 ± Values are expressed as mean ± SE 1.737 %) in males 35 ≥ years and (31.09± (0.05> ٭vs 15.88± 2.502%) in males 35< years Differences significant at (p 2.468% 8 Mowafak k et al./ ZJPAS: 2016, 28(3): 5-11

Normal Table (3) Percentage of SDF in fertile and Image. 1: sperm DNA fragmentation showing sperm sperm with halo width infertile males failed in IVF Programe Values are expressed as mean ± SE

Males 35 ≥ years 35 < years No. % SDF No. % SDF

Fertile 7 16.85 ± 9 15.88± 1.737 2.502

Infertile 19 28.368± 21 31.09±

2.468 ٭2.149

Values are expressed as mean ± SE

(Differences significant at ( p < 0.0 5٭

Table 4: Correlation of male age with SDF and some semen parameters Male SDF Sperm Sperm Ejaculati s (%) motility Concen on tration( Volume (%) 106 (ml) sperm/ ml) DISCUSSION Age 29.8 41.67 ± 30.07 ± 2.50 ± Sperm chromatin dispersion test (SCDT) are 0±10 8.38 19.04 .97 currently available to assess sperm DNA (Ye Infer .39 integrity which measures the susceptibility of ars) tile r= - r= - r= -.349 sperm DNA to denaturation (Fernández et r=.05 .702** .360* ⃰ al.,2003 ; Evenson et al.,1999). From the 2 p˂0.01 p˂0.05 p˂0.05 n=(4 NS results of our study we demonstrated a negative 0) correlation between sperm motility and sperm Fertil 16.3 54.25 ± 55.12 ± 3.20 ± concentration with sperm DNA fragmentation, e 1 ± 9.29 8.99 .80 these results were agreement with (Sun et 6.22 al.,1997) who demonstrated a significant n= r= -.463 r= - r= - negative correlation between semen parameters (16) r= NS .640** .634** .215 p˂0.01 p˂0.01 and DNA damage in sperm and supported by NS (Sheikh et al., 2008) which assure the presence of negative correlation between SDF and sperm Values are expressed as mean ± SE motility at r = -0.263. Fragmentation of sperm DNA may be attributed to intrinsic factors such Differences significant at (p 0.05) as oxidative stress, aging and varicocele or due > ٭ to extrinsic factors such as medication and (0.01> ٭٭Differences significant at (p environmental factors (Singh and Agarwal, 2011). Ejaculated sperm with DNA Abnormal fragmentation might result spermfrom failure of Abnormal spermatozoa to mature normally. Oxidative sperm stress and excessive production of ROS has

been shown to affect the integrity of sperm Abnormal sperm 9 Mowafak k et al./ ZJPAS: 2016, 28(3): 5-11 chromatin (Sharma et al., 2004; Aitken et reproducible and precise whereas the Comet al.,2003 ; Saleh et al., 2003) and incomplete and Tunel assays lack the consistency and mature of gametes play an important role in reliability needed in a clinical setting (Sheikh SDF due to affecting the activity of et al., 2008; Evenson and Wixon, topoisomerase II ( Sailer et al.,1995 ; 2006).Medications and radiation therapy affect Manicardi et al., 1998) as well as changes in sperm DNA integrity and male infertility genetic materials due to Apoptotic process depending on both duration and dose of (Richburg, 2000 ; VanDyke et al.,2000 ; Sinha exposure because of their cytotoxic effect on Hikim et al., 1997 ; Furuki et al.,1996 ). spermatogenic epithelium (Morris, 2002 ; Varicoceles are associated with the abnormal Sailer et al.,1995). Exposure to air pollution retention of sperm cytoplasmic droplets and may result in sperm DNA fragmentation and that these retained droplets are correlated with affecting male infertility(Specht et al., 2012 ; sperm DNA damage in infertile men (Fischer Rubes et al.,2005 ; Evenson and Wixon et al.,2003 ; Zini et al., 2000). SDF has been ,2005).Smoking is another factor affect the frequently observed among infertile couples sperm DNA integrity because smoking causes with unexplained aetiologies and with recurrent increased production of leukocyte-derived pregnancy failures and high abortion reactive oxygen species, which has adverse rates(Carrell et al.,2003 ; Host et al.,2000) . effects on mature sperm therefore SDF can be Inflammation of the epididymis and testis is considered as an independent parameter with another factor associated with SDF which prognostic value in the treatment of male result in leukocytospermia and have been infertility (Potts et al.,1999). associated with increased levels of reactive oxygen species and subsequent sperm DNA REFERENCES damage (Erenpreiss et al.,2002).Testis Agarwal, A., and SSR, Allamaneni, 2004. The effect temperature play an important role in sperm of sperm DNA damage on assisted reproduction outcomes. A review. Minerva Ginecol., 56(3): 235-245. DNA integrity (Paul et al.,2008).Sperm DNA Ahmadi, A., S.C. Ng., 1999. Fertilizing ability of damage had a significant effect on fetal DNA-damaged spermatozoa. J Exp Zool., 284(6): 696- development after implantation in ICSI (Borini 704. Aitken, R., J.D. Sawyer, 2003. The human et al., 2006) that the pregnancy rate was spermatozoon—not waving but drowning. Adv Exp Med decreased in ICSI if the sperm DNA Biol, 518: 85-98. fragmentation in infertile males is exceeded 27- Barazani, Y., A. Agarwal and S. Edmund, J.R. Sabanegh, 2014. Proteomics in the Evaluation of Male 30% (Larson-Cook et al.,2003) because of Infertility. Urology, 84(2): 255-261. impairment of blastocyst formation due to Boe-Hansen, G., B.J. Fedder, A.K. Ersboll, P. Christensen, 2006. The sperm chromatin structure assay sperm DNA fragmentation and as a result as a diagnostic tool in the human fertility clinic. Hum failed IVF cycle (Ahmadi and Ng,1999) this Reprod., 21(6): 1576-1582. results was agreement with (Virro et al.,2004) Borini, A., N. Tarozzi, D. Bizzaro, M.A. Bonu, L. Fava, C. Flamigni, G. Coticchio, 2006. Sperm DNA but disagreed with( Boe-Hansen et fragmentation: paternal effect on early post-implantation al.,2006)who stated that 27% of sperm DNA embryo development in ART. Hum Reprod., 21(11): fragmentation in infertile males did not prevent 2876-2881. Bungum, M., P. Humaidan, M. Spano, K. Jepson, L. IVF success (Boe-Hansen et al.,2006) therefore Bungum and A. Giwercman, 2004. The predictive value we can use SDF test as a diagnostic tool of of sperm chromatin structure assay (SCSA) parameters for the outcome of intrauterine insemination, IVF and male infertility which is aid in selection of ICSI. Hum Reprod, 19(6): 1401-1408. spermatozoa with less amount of fragmentation Carrell, D.,T.L. Liu, C.M. Peterson, K.P. Jones, H.H. in assisted reproductive techniques (ART) Hatasaka, B. Campbell, L. Erickson, 2003. Sperm DNA fragmentation is increased in couples with unexplained because SCDT is very simple , high recurrent pregnancy lost. Arch Androl, 49(1): 49-55. 10 Mowafak k et al./ ZJPAS: 2016, 28(3): 5-11

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The official scientific journal of Salahaddin University-Erbil ZJPAS (2016), 28 (3); 12-19

Biological Control of Tomato Damping-off Disease using Trichoderma harzianum and Bacillus subtilis HASAN HUSSEIN ALI1* and KOCHER KAREEM TAHA2* 1 College of Agriculture, University of Salahaddin- Erbil 2 General Directorate of Agriculture, University of Salahaddin- Erbil

A R T I C L E I N F O A B S T R A C T

Article History: The two biological agents Trichoderma harzianum and Bacillus subtilis Received: 27/10/2015 used for controlling of tomato damping-off disease caused by Rhizoctonia Accepted: 03/02/2016 solani (4 isolates), Acremonium sp., Fusarium solani, Fusarium oxysporum and Fusarium proliferatum. The results of laboratory tests showed inhibited the Published:16/5/2016 mycelium growth of fungal pathogens using T. harzianum which inhibited. The Keywords: inhibition of the fungal growth by bacterium B. subtilis was ranged 92.33 – 100 Biological Control, %. In the greenhouse experiment, results the bacterium B. subtilis decreased the Trichoderma harzianum, percentage of pre-emergence damping-off in a rate ranged 6.67 – 13.33 % Bacillus subtilis. compared with untreated control which ranged from 30.00 – 40.00 %. The *Corresponding Author: percentage of infection using the bio-agent T. harzianum at concentrations of 5 and 10 g/kg seed were 3.33 – 16.67 % compared with untreated control which HASAN HUSSEIN ALI ranged 30.00 – 40.00%. Regarding the post-emergence infections, the Email: percentages of infections were 0 % for all treatments compared with untreated control which ranged 0 – 6.67%. This proves that the efficiency of the two methods in controlling the percentages of infections in high degrees against all pathogenic fungi.

1. Introduction no place where the plant is free of disease, one Tomato (Lycopersicon esculentum Miller), of the main constraints to tomato production is Solanaceae is one of the most important damage caused by pathogens, including vegetable crops in all over the world, because viruses, bacteria, nematodes and fungi, which of its high nutritive value and excellent source cause severe losses in production (Barone and of vitamin A and it plays an important role in Frusciante, 2007). Seed health is an important maintaining the human health being a rich factor in the control of diseases, since an source of lycopene, ascorbic acid and phenols; infected seed is less viable, has low tomato is used in treatment of cancer germination, reduced vigor and reduced yield (Giovannucci, 1999; George et al., 2004); it is (Van Gastel et al., 1996). Various methods for economically attractive and the area under controlling such diseases have been cultivation is increasing daily. The five investigated including the use of resistant countries recorded top productions of tomato varieties, cultural practices, plant volatile were United States, China, Turkey, Italy and compounds, plant extracts and biological India. Iraq production of 913493 tons in 2005 control, one of the most important bio-control (F.A.O., 2007).Although tomato is agents is Trichoderma spp. that is the most commercially grown across the globe; there is frequently isolated soil fungi and present in 13 HASAN H. et al./ ZJPAS: 2016, 28(3): 12-19 plant root ecosystems (Harman et al., 2004). 1 = Antagonist overgrew the pathogen and The antagonistic activity of the genus covered the entire medium. Trichoderma spp. to F. solani and R. solani has been widely demonstrated in the soilless mix 2 = Antagonist overgrew at least 2/3 of the (Lewis et al., 1998). B. subtilis would make an medium surface. effective biological control agent by testing it 3 = Antagonist and pathogen each colonized against the damping-off of tomato seedlings about 1/2 of the medium surface. caused by Rhizoctonia solani (Asakaand Makoto, 1996). Isolation of pathogenic fungi 4= Pathogen colonized overgrew at least 2/3 of from tomato seeds and seedlings are the medium surface and withstood Rhizoctonia solani (4 isolates), Acremonium encroachment by antagonist. sp., Fusarium solani, Fusarium oxysporum and Fusarium proliferatum caused damping-off 5= Pathogen completely overgrew the disease (damping-off disease are usually refers antagonist and occupied the entire medium to the disintegration of stem and root tissues at surface. and below the soil line, the plant tissues become water-soaked and mushy, and the Effect of Bacillus subtilis on the growth of seedling wilts and falls over). The objective of pathogenic fungi: B. subtilis was obtained from Plant this study is control of tomato damping-off Protection Department/College of Agriculture/ disease by biological control using biocontrol University of Salahaddin. Petri dishes agents of Bacillus subtilis and Trichoderma containing PDA were inoculated with B. harzianum. subtilis culture (48 hrs. old) grown on PDA by Materials and Methods placing four drops on the outline of perpendicular lines at a distances of 1 cm from Dual culture technique the dish edge, plates were incubated at 25 °C Mycelial plugs 5mm of T. harzianum for 48 hours (Paulitz et al., 1992). The same fungus was inoculated in the right half of plates plates were also inoculated with mycelial disc contained solidified PDA (Potato Dextrose (0.5cm) of pathogenic fungi (4 days old) in the Agar) and 5mm mycelial discs for each of center, control treatments included plates pathogenic fungi R. solani (four isolates), inoculated with pathogens alone. All plates Acremonium sp., F. solani, F. oxysporum and were incubated at 25 °C for 7 days and F. proliferatum with an equal distance of 4cm calculated after full growth of pathogenic in between both plugs of the pathogen and control, the percentage of inhibition was biocontrol agent. Control treatments inoculated measured according to following formula: with each pathogenic fungus only. Plates were incubated at 25±2°C for 7 days, whereas R1- R2 cultures of Rhizoctonia solani inoculated for 3 % Inhibition = ------× 100 days. Each treatment replicated three times, pathogenic growth was recorded after contact R1 with the antagonists. R1= the growth rate of the pathogenic fungus The degree of antagonism was rated according at control treatment. to Bell, et al (1982) 5-point rating scale as follows: R2= the growth rate of the pathogenic fungus at dual culture. 14 HASAN H. et al./ ZJPAS: 2016, 28(3): 12-19

Effect of Biocont-T., Bacterial agent on examined fungi with considerable degrees disease suppression in greenhouse: according to rating scale described by (Bell et The control of disease applied in the al., 1982). The results confirmed that T. greenhouse of Grdarasha/Erbil Research harzianum had a noticeable antagonistic directorate during March, 2015, the all activity against all tested fungi particularly following experiments received the results pre Rhizoctonia solani (isolate 4), since the and post emergence damping-off after 40 days. antagonist overgrew the pathogen entirely, while R.solani (isolate 1) observed in the scale Biocont-T. a commercial formula of (Trichoderma harzianum): 2 when antagonist overgrew at least 2/3 of the medium surface (Fig. 2-A). Three concentrations (0, 5 and 10) g of Biocont-T/kg seeds were examined with 2Kg Table (1): Effect of Trichoderma harzianum on sterilized soil in pots (20 cm diameter). The the mycelial growth of pathogenic fungi according to Bell scale soil was infested with R. solani, F. solani, F. oxysporum and Acremonium sp., grown on Pathogenic Fungi Bell scale millet seeds at a rate of 0.5% before two days of sowing. Ten sterilized tomato seeds (GS-12) Rhizoctonia solani (1) 2 in 1% sodium hypochlorite (NaOcl) for 2 min., Rhizoctonia solani (2) 1 and washed twice with sterilized distilled water Rhizoctonia solani (3) 1 for 2 min., treated with (5 and 10) g of Biocont-T, then sown in infested soil for each Rhizoctonia solani (4) 3 pot and watered. The treatment replicated three Acremonium sp. 1 times. Fusarium solani 1 Effect of Bacillus subtilis bacteria: B. subtilis bacterial were screened in Fusarium oxysporum 1 greenhouse to control plant disease and B. Fusarium proliferatum 1 subtilis were grown on petri dishes contained Control (pathogenic fungi lysogeny Broth medium (LB medium) at 20 °C 5 only) for 24-48 h. A well-isolated colony 12 hours old from medium was suspended in 5ml distill water using sterilized loop. The suspension was The potential of Trichoderma spp. as adjusted to 0.5 Mcfarland (Basri and Fan, biocontrol agent phytopathogenic fungi on 2005). Hundred tomato seeds were immersed several crops is well known particularly with bacterial suspension, after 2-5 min. excess Fusarium spp. and Rhizoctonia solani liquid were removed before thickening them (Poddar et al., 2004; Rojo et al., 2007; overnight under a sterile air stream in advance Dhahir, 2013). of being planted in the greenhouse (Amein and Weber, 2002). This results was compatible with studies stated that the ability of T. harzianum in the Results and Discussion inhibition growth of R. solani and other Effect of Trichoderma harzianum on the mycelial fungi due to different mechanisms such as growth of pathogenic fungi: production of toxic metabolites or due to direct parasitism on fungal mycelium Results in Table (1) showed that T. (Barakat et al., 2007), or production of harzianum inhibited the mycelial growth of enzymes like B-1, 3 glucanases and 15 HASAN H. et al./ ZJPAS: 2016, 28(3): 12-19

chitinases which break down the fungal cell wall (Shalini and Kotasthane, 2007).

Effect of Bacillus subtilis on the mycelial growth of pathogenic fungi:

The results illustrated that the bacteria B. subtilis was completely inhibited the growth of pathogens, Rhizoctonia solani (isolate 2) and F. oxysporum inhibited with 92.33%. (Fig. 1, 2-B), The antagonistic of B. subtilis refer to production of many

antibiotics such as subtiline, bactracin, lturin and surfactin or to inducing cell wall degrading enzymes such as endochitinase, proteases and B-1, 3-glcanases which break down the fungal cell wall (Page et al.,

1982; Muis and Quimio, 2006).

LSD0.01 = 0.72

Figure (1): Inhibition effect of Bacillus subtilis on growth of Pathogenic fungi

Control T. harzianum

Figure (2): A- Inhibition of pathogenic fungi by mycelial growth of T. harzianum on PDA in vitro

16 HASAN H. et al./ ZJPAS: 2016, 28(3): 12-19

R. solani (1)

Biological control using (Biocont – T. (T. harzianum) and B. subtilis):

R. solani (2) Results submitted in Table (2) clarified that Bacillus subtilis reduced incidence pre

emergence damping-off to range 6.67-13.33% compared to ranged 30.00-40.00% in control, post emergence damping-off did not shown R. solani (3) (Fig. 3), whereas post emergence ranged 0- 6.67% in control, there is significant difference

between treatments of pre and post emergence and control. These results similar with Morsy et al.,(2009) in reducing of pre and post R. solani (4) emergence damping-off by 19.0% and 11.0%

respectively. In contrast, the disease incidence reaches to 45.0% and 20.0%, respectively with the inoculum of Fusarium solani through application of Bacillus subtilis. Regardless of Acremonium sp. defect efficiency B. subtilis may be in reducing pathogens damage. The promotion of tomato growth due to their abilities to produce phytohormones, vitamins and solublizing F. solani minerals besides, their role in direct inhibition of pathogen growth (Morsy, 2005; Zaghloul et al., 2007). Biocont – T (T. harzianum) was tested at concentrations of 5g and 10g/kg seeds, F. oxysporum pre emergence damping-off incidence ranged between 10.00-16.67% at first concentration between 3.33-13.33% at second concentration.

In contrast the disease incidence reaches to F. proliferatum ranged between 30.00% - 40.00% with the inoculum of pathogenic fungi through Control subtilis application of Trichoderma harzianum (Table B- Inhibition of 2) there is significant difference between mycelial growth treatments and control. Emerged plants of pathogenic appeared healthy with no post emergence fungi by B. subtilis on PDA damping-off incidence at both concentrations, in vitro compared to ranged between 0-6.67% in control (Fig. 3).

17 HASAN H. et al./ ZJPAS: 2016, 28(3): 12-19

showed the combination of B. subtillis and T. Control Pre Post emergence harzianum was effective in suppressing (only emergence% % fungi) damping-off pre and post emergence but it did B. T. B. T. not differ significantly from treatments with harzianu su harzianu su either of these antagonists alone. The Pathogenic bti m bti m P fungi lis lis Pr 10 os mechanism of Trichoderma and Bacillus action 10 5g/ 10g 10 5g/ e g/k 6 kg /kg 6 kg t on pathogens may be attributed to their g % cel see see cel see % see attacking and binding the pathogens by sugar l/ ds ds l/ ds ds ml ml linkage and begins to secrete extracellular 40 6.6 10. 10. 6. protease and lipase (Cal et al., 2004). Biocont- R. solani 0 0 0 .0 7 00 00 67 0 T (T.harzianum) suppressed the deleterious soil 36 13. 13. 13. 3. microbes by competing at the active sites, F.solani 0 0 0 .6 33 33 33 33 7 reduced disease development, and 33 F. 6.6 16. 13. 3. subsequently stimulated the growth and yield 0 0 0 .3 oxysporum 7 67 33 33 3 of plants (Ahmed and Upadhyay, 2009). 30 Acremoniu 10. 10. 3.3 0 0 0 .0 0 m sp. 00 00 3 0 Control (non- 0 0 0 0 0 0 0 0 inoculated fungi)

Shabir and Rubina (2010), observed that the Biocont-T. (T. harzianum) 5 g/kg seeds Antagonist of Trichoderma harzianum inhibited the mycelial growth of R. solani ranged between 85.5 and 83.0 % as compared to control, the inhibition of R. solani by Trichoderma species could probably be due to the secretion of extracellular cell degrading enzymes such as chitinase B-1, 3- glucanase, cellulose and lectin, which help mycoparasites Biocont-T. (T. harzianum) 10g/kg seeds in the colonization of their host, the inhibition of pathogen may be also been attributed to the production of secondary metabolites by antagonists such as glioviridin, viridin and gliotoxin. One of the most important characteristics necessary for acceptance and effectiveness of biocontrol agents is their Bacillus subtilis (106 cell/ml) ability to survive in the environments other than their origin and colonize plants roots during certain period of time to control plant pathogens (Nemec et al., 1996).

Hervas et al., (1998) studied the efficiency of B. subtillis and T. harzianum on pathogenic to suppress tomato diseases. Their result 18 HASAN H. et al./ ZJPAS: 2016, 28(3): 12-19

Figure (3): Effect of Biocont-T. (T. harzianum) Bell, D. K.; Walls, H. D. and Markham, C. R. and Bacillus subtilis on the suppression of (1982). The in vitro antagonism of damping-off in greenhouse: Trichoderma species against six fungal plant pathogens. Phytopathol., 72:379-382. A-control (non¬-inoculated fungi) B- Rhizoctonia solani C- Fusarium solani Cal, A.; Larena, I.; Sabuquillo, P. and D- Fusarium oxysporum E- Acremonium sp. Melgarejo, P. (2004). Biological control of tomato wilts. Recent Research Developments in Crop Science, 1: 97-115.

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The official scientific journal of Salahaddin University-Erbil ZJPAS (2016), 28 (3); 20-23

Histopathological Changes in Gills of Goldfish, Carassius auratus (Linnaeus, 1758) Infested with Icthyophthirius multifiliis Fouquet, 1876 from Erbil Aquariums Karwan Sallo Najm Al-Marjan 1* and Shamall M. A. Abdullah2* 1 M.Sc. in Parasitology, Lecturer, Department of Pharmacy. Medical Technical Institute. Hawler Polytechnique University, Kurdistan Region, Iraq. 2 Ph.D. in Parasitology, Professor, College of Agriculture, University of Salahaddin-Erbil

A R T I C L E I N F O A B S T R A C T

Article History: The present study included the identification of the histopathological Received: 03/11/2015 effects of the ciliated protozoan, Icthyophthirius multifiliis on the gills Accepted: 15/02/2016 of the goldfish Carassius auratus, collected from aquarium shop in Erbil Published:16/5/2016 City, Kurdistan Region, Iraq. Results reveal that all infested fish showed Keywords: White spot disese, color change of the gill filaments with white spots in different Histopathological changes, diameter. Histopathological observations reveals serious damages of Carassius auratus, primary lamella and secondary lamella include: increase in cells size Icthyophthirius multifiliis (hypertrophy), increase in cells number (hyperplasia), fusion of the *Corresponding Author: secondary lamella and blood congestion of the base of primary lamella. Karwan S. Al-Marjan In conclusion, the histopathological changes produced by I. multifiliis Email:[email protected] will eventually lead to reduced surface area for effective respiration and could lead to suffocation and death.

1. Introduction ciliated protozoan parasite which affect this Goldfish, Carassius auratus (Linnaeus, species of fish (Al-Tayyar et al., 2011). 1758) belong to Family Cyprinidae, is one of the most popular pets in the world, while the Icthyophthirius multufillis reach 0.6-1.2 mm in ectoparasites are the most important causative diameter and it appears as a white spot within agents affecting this species, which are causes the gills and skin, it is normally infects the economical loses for the growing industry in skin, fins and gills which is cause white spot intensive culture systems (Osman et al., 2009). disease (Ewing, 2002). There are three stages Most ectoparasites can be readily diagnosed on of the parasite during their life cycle include: direct observation and wet mount preparations an infective theront, a parasitic trophont and a while some parasitic infestation need another reproductive tomont, free-swimming theronts clinical examination to be confirmed enter into the epidermis of fish gill lamellae to (Mohammadi et al., 2012). feed on mucus and tissue then rapidly change into trophonts and cause serious damages of The most commercial ornamental fish in Iraq the underlying epithelial cells, reducing the and Kurdistan Region is C. auratus. Among surface area for respiration and could lead to the parasitic infestation, Icthyophthirius suffocation, particularly at high temperature multifiliis is the most important and abundant 21 Karwan S. et al./ ZJPAS: 2016, 28(3): 20-23

(Aladetohun et al., 2014; Ling et al., 2010). I. include histopathological examination of the multifiliis has been recorded for the first time in gills. Tissue specimens of normal gills and Iraq from Mugil dussmieri by Herzog (1969). infested gills rinsed in normal saline and fixed After that, it was reported from Lesser Zab in formalin buffer 10% for 24 hours. After river and Darbandikhan lake in Kurdistan fixation, the tissues were dehydrated in an region (Abdullah, 2002; 2005 respectively). alcohol series of ascending concentration The higher incidence percentage of infection (70%, 80%, 90% and 100%, respectively) then 100% by I. multifiliis was recorded for the first embedded in paraffin wax and sectioned at time in Iraq by Al-Tayyar et al. (2011) from 5μm. The tissue sections were stained with skin and gills of seven species of ornamental haematoxylin-eosin (H&E). (Mohammadi et fish included C. auratus. al., 2012). All slides were examined under a light compound microscope at 100X, 400 X Histopathology provides a rapid method to and 1000X magnification and the photos were detect influences of irritants and pathogens in taken with HTC-one camera model, PNO7140 different organs and it can be considered as the 5VDC-1A 801S. indicator for abnormal case of fish environment (Mohammadi et al., 2012). 3-RESULTS AND SISCUSSION

During the last years, the issue of The gills are one of the most susceptible organ histopathological study in Iraqi fishes has of the fish because of their external location received attention only from a small number of and inner most coat which are contact with the researchers include, Al-Taee and Al- water (Hadi and Alwan, 2012), therefore they Hamdani, 2008; Yasser and Naser, 2011; are liable to damage by any causative agents, Mansor et al., 2012 and Hadi and Alwan, among them ectoparasites. I. multifiliis (Fig. 2012. 1A) is the most important and abundant ciliated protozoan parasite which cause white spot The aim of this study is to identify types of diseases (Fig. 1B) of this species of fish (Al- histopathological lesions of gills associated Tayyar et al., 2011). with external parasitic protozon, I. multufillis from C. auratus. Generally, normal gill filaments or primary lamellae (PL) of the Goldfish, C. auratus gave 2-MATERIALS AND METHODS rise to rows of secondary or respiratory lamellae (SL) including the interlamellar Samples of fish belong to C. auratus were region ( Fig. 2A) and both PL and SL were collected from an aquarium shop in Erbil City, covered by the primary and secondary Kurdistan region, Iraq. Samples were epithelia, respectively, that included different transferred (as a live) to the laboratory of Erbil cell types, pavement, mucous, chloride and Medical Technical Institute as soon as possible pillar cells (Franchini et al., 1994). and were examined within 24 hours. Smears from the gill were examined for the Histopathological observation reveals the ectoparasite I. multufillis and ciliates was serious damages of primary lamella (PL) and observed under 400X without need staining secondary lamella (SL) due to attachment and (Ewing, 2002). It has been confirmed that the feeding of this parasite (trophont stage). In hosts were infested only with the I. multufillis current study, all infested fish showed color and the aquarium water samples had been free change of the gill filaments, as white color from chemical pollutants. Second step which spots in different diameter (Fig.1B). In heavy 22 Karwan S. et al./ ZJPAS: 2016, 28(3): 20-23 infestation epithelial hypertrophy and hyperplasia of different cell types were All of the above serious damages were observed in both primary and secondary due to the activity of the trophonts stage of the lamella (Fig. 2 B; C; D). Hypertrophy include parasite which contributes to osmoregulatory the enlargement of the cells of the gill lamella problems and finally death of the fish (Ling et which produced as a host response to al., 2010). infestation while hyperplasia is a case which In conclusion, the histopathological include increase in cells number (Babue et al., changes produced by I. multifiliis will 2009). Other observations included the fusion eventually lead to reduce surface area for effective respiration and could lead to of the seconzdary lamella, two or more suffocation and death. lamellae fused with each other (Fig. 2 E) and blood congestion of the base of primary 4-Achnowledgements lamella (Fig. 2 F). Our results were in Special thanks are extended to Dr. Rafl agreement with that reported by Ewing Abdulrazaq (histopathologist), Mr. Hersh Rasool (2002); Mohammadi et al. (2012). Mina and Miss. Eman Ersallan Abdullrazaq from Alzahrawy Laboratory / Erbil and Department of Medical Technical Lamoratory (M.L.T.), University of Hawler Polytechnique for the powerful support during the laboratory works.

اﻟﺘﻐﺎﯾﺮات اﻟﻨﺴﯿﺠﯿﺔ ﻓﻲ ﺧﯿﺎﺷﯿﻢ اﻟﺴﻤﻜﺔ اﻟﺬھﺒﯿﺔ، (Carassius auratus (Linnaeus,1758 اﻟﻤﺼﺎﺑﺔ ﺑﺎﻟﻄﻔﯿﻠﻲ (Icthyophthirius (Fouquet, 1876 multifiliis ﻣﻦ ﺧﺰاﻧﺎت ﻣﺪﯾﻨﺔ ارﺑﯿﻞ Figure (1): A-Trophont stage of Icthyophthirius multifiliis (400X). B- Goldfish , C. auratus, shows gill infestation with I. ﻛﺎروان ﺳﻠﻮ ﻧﺠﻢ اﻟﻤﺮﺟﺎن* و ﺷﻤﺎل ﻣﺤﻤﺪ اﻣﯿﻦ ﻋﺒﺪاﷲ** .multifiliis make white region appearances or white spots * ﻗﺴﻢ اﻟﺼﯿﺪﻟﺔ، اﻟﻤﻌﮭﺪ اﻟﻄﺒﻰ اﻟﺘﻘﻨﻰ، ﺟﺎﻣﻌﺔ ﺑﻮﻟﺘﯿﻜﻨﯿﻚ ھﻮﻟﯿﺮ.ارﺑﯿﻞ . اﻟﻌراق **ﻛﻠﯿﺔ اﻟزراﻋﺔ، ﺠﺎﻤﻌﺔ ﺼﻼح اﻟدﯿن، ارﺒﯿل. اﻟﻌراق

اﻟﺨﻼﺻﺔ اﺟﺮﯾ��ﺖ ھ��ﺬة اﻟﺪراﺳ��ﺔ ﻟﺘﺸ��ﺨﯿﺺ اﻟﺘ��ﺎﺛﯿﺮات اﻟﻨﺴ��ﯿﺠﯿﺔ ﻟﻠﮭ��ﺪﯾﺒﻲ اﻻﺑﺘ��ﺪاﺋﻲ Icthyophthirius multifiliis ﻋﻠ��ﻰ ﺧﯿﺎﺷ��ﯿﻢ اﻟﺴ��ﻤﻜﺔ اﻟﺬھﺒﯿ��ﺔ Carassius auratus: . ﺣﯿ��ﺚ ﺗ��ﻢ ﺟﻤ��ﻊ ﻧﻤ��ﺎذج ﻣ��ﻦ اﺣ��ﺪ ﻣﺤ��ﻼت ﺑﯿ��ﻊ اﺳﻤﺎك اﻟﺰﯾﻨﺔ ﻓﻲ ﻣﺪﯾﻨﺔ ارﺑﯿﻞ، اﻗﻠﯿﻢ ﻛﺮدﺳ�ﺘﺎن اﻟﻌ�ﺮاق. اظﮭ�ﺮت اﻟﻨﺘ�ﺎﺋﺞ ﺑ�ﺎن ھﻨ��ﺎك ﺗﻐﯿ��ﺮ ﻓ��ﻲ ﻟ��ﻮن اﻟﺨﯿﺎﺷ��ﯿﻢ ﻟﺠﻤﯿ��ﻊ اﻻﺳ��ﻤﺎك اﻟﻤﺼ��ﺎﺑﺔ ﻣ��ﻊ ﺑﻘ��ﻊ ﺑﯿﻀ��ﺎء ﺑﺎﺣﺠﺎم ﻣﺨﺘﻠﻔﺔ. اظﮭﺮت اﻟﺘﺎﺛﯿﺮات اﻟﻨﺴﯿﺠﯿﺔ ﺑﺎن ھﻨﺎك ﺳﻠﺴ�ﻠﺔ ﻣ�ﻦ اﻟﺘﻐﯿ�ﺮات اﻟﻨﺴﯿﺠﯿﺔ ﻟﻜ�ﻞ ﻣ�ﻦ ﺧﯿ�ﻮط اﻟﺨﯿﺎﺷ�ﯿﻢ اﻻوﻟﯿ�ﺔ واﻟﺜﺎﻧﻮﯾ�ﺔ واﻟﺘ�ﻰ ﺗﺘﻀ�ﻤﻦ: ﺗﻀ�ﺨﻢ اﻟﺨﻼﯾ�ﺎ (hypertrophy) ، زﯾ�ﺎدة ﻓ�ﻲ ﻋ��ﺪد اﻟﺨﻼﯾ�ﺎ (hyperplasia) ، اﻟﺘﺤ��ﺎم ﺑ��ﯿﻦ ﺧﯿ��ﻮط اﻟﺨﯿﺎﺷ��ﯿﻢ اﻟﺜﺎﻧﻮﯾ��ﺔ (gill fusion ) و اﺣﺘﻘ��ﺎن اﻟ��ﺪم (blood congestion) ﻓﻲ ﻗﺎﻋﺪة ﺧﯿﻮط اﻟﺨﯿﺎﺷﯿﻢ اﻻوﻟﯿﺔ. : Figure (2): A-Normal gill structure (1000X), B-Hypertrophy اﻟﻜﻠﻤ��ﺎت اﻟﺪاﻟ��ﺔ:اﻟﺘﻐ��ﺎﯾﺮات اﻟﻨﺴ��ﯿﺠﯿﺔ، ,400X), C- Hypertrophy (1000X), D-Hyperplasia (400X), E- ، Carassius auratus) Gill fusion (400X) and F- Blood congestion (100X). Icthyophthirius multifiliis

MA = macronucleus, MI = micronucleus, W= white spots, PL = primary lamellae, SL = secondary lamellae, : F ‡Ë �Ö ˇË Ü: Óà]⁄ :Ófi]“ÏÊÁçË�Ñ:Ì÷: Ófi]“ÌË: ÓËÌfi]å:ÌËÑ]”fi\�ÑˇÁö PH = primary lamellae hypertrophy, NC = normal cells, :Ô: Carassius auratus::B:Linnaeus, 1758C ECH = Epithelial cells hypertrophy, HSL = hyperplasia of the secondary lamella, SLF = secondary lamella ::BFouquet, 1876C:ÔÑˇÁ|Ìç⁄::Ìd:·ÊÁeåÊÁh fusion, BC = blood congestion of the base of the primary :2÷ Ê Ì ‚ : ÔÑ ] å: Ófi ] “] öˇÁ “: Ì ÷ ::Icthyophthirius multifiliis lamella. 23 Karwan S. et al./ ZJPAS: 2016, 28(3): 20-23

exposed to ichlorvos. Braz. Arch. Biol. Technol., : 52 (5), pp. 1291-1296. : Ewing, M.S. (2002). 3.2.8 Ichthyophthiriasis- ** * AFS Fish Health section (online). : Ì◊÷\Åe¬:Ø⁄\Å€•:Ÿ]4::ÏÊ: ·]pÖ⁄:‹ß:Á◊à:·\ÊÑ]“ Available at: w.w.w. afs- :ÔÁ”fi\Ü:FÓ”çËál:Ó”Èfl“Ìh:Î]�fi]¥Ìl:FÔÜ]âfi]⁄ÑÏÄ:ÓåÌd fhs.org/perch/resources/14069237713.2.8 Ichthyophthiriasis 2014.pdf (Accessed: —\2¬H:ÖˇÈ÷ÊÌ‚HÖˇÈ÷ÊÌ‚:Ó”Èfl“ÌiÈ ÷ˇÁl * 12 August 2015). —\2¬H:ÖˇÈ÷ÊÌ‚H:‡ËÄÄÌt˜Ìà:ÔÁ”fi\Ü:FˇŸ]“Áiç“:ÔÉˇÈ÷Á“** Franchini, A., Alessandrinii, F. and Fantin, A. (1994). Gill morphology and ATPase activity in the goldfish Carassius carassius var. auratus exposed Ìi|Ál to experimental lead intoxication. Boll. di zool., 61, pp. 29-3. :::::ÔÑ\Åzz”�÷Á“:Ófi]“ÌzzËÑÌ�ËÑ]“:ÓfiÄÖ”fi]zzçÈfliàÏÄ:Ìzz÷:ÌzzË: 8ËÖzzd: ÌzzËÏÊÌflËÉˇËÁh: ›ÌzzÒ Hadi, A.A. and Alwan, S.F. (2012). Histopathological Ichthyophthirius multifiliis ::ÓËÌfi]zzzå:ÔÌzzzh]„”ˇÈl:ÑÌzzzàÌ÷: :ÍË]hÏÑÌzzzà changes in gills, liver and kidney of fresh water :ÌizzàÌdÌ⁄:›ÌzzÒˇÁd:ÏÊ:. Carassius auratus:ÔÑˇÁzzp:Ìzz÷:Ózzà]⁄:Ófi]“ÏÁzzçËÑ fish, Tilapia zillii, exposed to aluminum. Int. J. ::::: ÔÑ]z å:Ìz ÷: ÔÑ]z ”fi\Áp: Ózà]⁄: ÓfliåˇÊÖ : Ó“ÌË]�åˇÊÖ : Ì÷: ÏÊÌË\Ö“ˇÁ“:Óà]⁄: ÔÌfiÊÁ≥ Pharm. Life Sci., 3 (11), pp. 2071-2081. Herzog, P.H. 1969. Untersuchungen über die parasiten :::::::: ÔÑ]z”fi\ÑÁö:Ìz“:kÊÌz“ÑÏÄ: ·]“Ìz⁄]ßÌÒ: Ìz÷: H—\2z¬: Ófi]izàÄÑÁ“: Ó€ˇËÑÌ‚: F: ÖˇÈ÷ÊÌ‚ der süBwasserfische des Irak. Arch. ::::::::Ìzd:Ómzà:ÔÌz�÷Ìl:ˇŸÌzöÌ÷:·]“ÏÊÁezåÁh:ÌÈzà]⁄:jzçö:Ófi]“ÏÊÁçËÑ::Ì÷:ÌËÌ‚:ºfiÏÑ Fischereiwiss., 20(2/3): 132-147. :ÍËÌfi]zzå:ÔÑ]zz”fi\ÑˇÁö:’ÌzzËÏ2ßÜ:Ìzz“:kÊÌzz“ÑÏÄ:\Ê:]‚ÏÊÑ:Ìzz‚:ÏÊ:HÜ\Ê]zzÈp:ÔÏÑ]dÌzŒ Ling, F., Wang, J., Liu, Q., Li, M., Ye, L. and Gong, X. (2010). Prevention of Ichthyophthirius ::TÌzz÷:ØiËÖzzd:Ìzz“:Ózz⁄ÏÊÊÄ:ÏÊ:ÍË]hÏÑÌzzà:Ófi]“ÏÊÁzzçËÑ:Ìzz÷ÊÊÜÏÄ:Ìzz÷:ÌzzËÌ‚:ÓfiÊÁzzd multifiliis infestation in goldfish (Carassius ::ÔÏÑ]zz⁄Ç:ÓfiÊÁdÄ]zzËÜ:F (hypertrophy) :::·]“Ìzzfi]|: ÔÏÑ]dÌzzŒ: ÓfiÊÁdÏÑÊÌzzö auratus) by potassium ferrate (VI) treatment. Vete. ( gill Ó⁄ÏÊÊÄ: Ófi]“ÏÊÁçËÑ: Ì÷ÊÊÜÏÄ: Ófi]”÷ÏÊÌ”ˇÈl: F:(hyperplasia): ·]“Ìfi]| Parasitol. 168, pp. 212-216. Mansor, N.T., Falah, A.B., Al-Jawda, J.M. and Asmar, :::Ìz÷ÊÊÜÏÄ:ÔÌz”fld:Ì÷:(blood congestion)‡ˇËÁ|: ÔÏÊÌfiÊÁdˇÁ“: ÏÊ: fusion) K.R. (2012). Histopathological Study of Some :H ÍË ] h ÏÑÌ à: Ófi ] “ÏÊ Á çË Ñ Tigris River Fish Which Infected by Parasites. F Carassius auratus,:FÍËÌfi]å:ÔÑ]”fi\�ÑˇÁö:T·]“ÌåÊÊ: Ì ◊È◊“ Iraqi J. Vet. Med., 36 (1), pp. 33-42. :Icthyophthirius multifiliis Mohammadi, F., Mousavi, M.S. and Rezaie, A. (2012). Histopathological study of parasitic infestation of skin and gill on Oscar (Astronotus ocellatus) and References discus (Symphysodon discus). Aquaculture, Abdullah, S.M.A. (2002). Ecology, taxonomy and Aquarium, Conservation and Legislation, Int. J. biology of some parasites of fishes from Lesser Biof. Soc., 5(2), pp. 88-93. Zab and Greater Zab rivers in north of Iraq. Ph. Osman, H. A. M., Maather, M., Monier, Omaima A., D. Thesis., Coll. Educa. (Ibn Al-Haitham), Univ. Abdul Ghany, T.B.I., and MONA, M. I. (2009). of Baghdad: 153. (In Arabic). Protection of Goldfish (Carassius auratus) Against Abdullah, S.M.A. (2005). Parasitic fauna of some Ichthyophthirius multifiliis by Immunization with freshwater fishes from Darbandikhan lake, North Live Theronts, Trophonts and Sonicated of Iraq. J. of Dohuk Univ., 8(1), pp. 29-35. Trophonts. J. Global Vete., 3 (4), pp. 329-334. Aladetohun, N.F., Nestor, G.S. and Emmanuel, E.B. Yasser, A.Gh. and Naser, M.D. (2011). Impact of (2014). Histopathological changes induced by pollutants on fish fromdiffrent parts of Shatt Al-Arab copepoda parasites infections on the gills of River: a histopathological study. J. Environ. monit. economically important fish mugilidae (Liza Assess falcipinnis and Mugil cephalus) from Ganvie area of Lac Nokoue, Republic of Benin. Afr. J. Environ. Sci. Technol., 8(2), pp.143-147. Al-Taee, S.K.I. and Al-Hamadani, A.H.A. (2008). Pathological study of experimental cadmium toxicity in common carp (Cyprinus carpio L.). Iraqi J. Vet. Sci., 22(2), pp. 33-42. Al-Tayyar, O.B.A., Abdul Jabaar, W., Raof, S.A. and Shather, M.M. (2011). Isolation of Ichthyophthirius multifiliis, Gyrodactylus and Copepoda: Lernaeidae Species from Six Ornamental Fish Species for the Firs Time in Iraq. Al-Anbar J. Vet. Sci., 4 (2), pp. 82-87. Babue V.S.M., Elif, C. and Erhan, U. (2009). Histopathological changes in the gill and liver tissues of freshwater fish, Cirrhinus mrigala ZANCO Journal of Pure and Applied Sciences

The official scientific journal of Salahaddin University-Erbil ZJPAS (2016), 28 (3); 24-31

Comparative Study Of Lipid Profile & Lipid Associated Fucose In Sera Of Control, Minor And Major Thalassemic Patients*

Zeyan Abdullah Ali1* , Tarik Al-Hakeem2 and Hamid Gafoory Hassan3 1College of Education, Salahaddin University, Iraq. 2College of Israa jameaa, Iraq. 3College of pharmacy, Karbala University, Iraq.

A R T I C L E I N F O A B S T R A C T

Article History: The aim of the present study is to evaluate the lipid profile and lipid associated Received: 13/12/2015 fucose in sera of minor, major thalassemic and compared that with the levels of Accepted: 18/02/2016 normal control.Cholesterol (Ch), Triglyceride (TG). High Density Lipoprotein (HDL), Low Density Lipoprotein (LDL), Very Low Density Lipoprotein Published:16/5/2016 (VLDL) and Lipid Associated Fucose (LAF) were determined in sera of 40 Keywords: control, 40 T minor and 45 T major. Lipid Profile, Lipid Associated Fucose, and Thalassemic A significant elevation in cholesterol, TG, HDL and VLDL in sera of T major Patients was found compared to the same parameters in sera of T minor and control, also a significant increase in cholesterol, TG, HDL and VLDL in sera of T *Corresponding Author: major compared to T minor, also the same lipids showed a significant increase Zeyan A. Ali in sera of T minor compared to control.On the other hand LDL in sera of T Email:[email protected] major showed a significant reduction compared to T minor and control, also a significant decrease in LDL for T minor compared to control was found, while lipid associated fucose showed a significant increase in sera of T major over that for T minor and control, also a significant high level of LAF in sera of T minor compared to control was found

1. Introduction alternatively or additionally by attached in a sub- terminal position on the epitope (7). Beta-thalassemia is considered to be the most frequent hereditary blood disorder worldwide. Actions that alter expression of receptors for Lipid abnormalities have been detected in fucoligands should enhance or reduce the different types of beta-thalassemia (1-2), and binding of fucoligands to the target cells (8). also in various hematological disorders (3). The amphipathic nature of phospholipids and Experimental sphingolipids makes them ideally suitable as the main lipid components of cell membranes. Selection of subjects and blood sampling

α-L-Fucose is a six carbon deoxy–hexose (6- Ten ml of venous blood sample was obtained deoxy-L-galactose) with a general formula of from 45 subjects of both sexes, with β- C6H12O5 , and is important component of thalassemia major, 40 with β-thalassemia glycoproyein and glycolipid produced by minor and 40 healthy individuals as control mammalian cells (4-6). group.

Fucose usually is present at the non-reducing All patients major and minor were admitted to end of the oligosaccharide, fucose may thalassemia center in Erbil and Ibn Al-Balady hospital in Baghdad for blood transfusion for 25 Zeyan A. et al./ ZJPAS: 2016, 28(3): 24-31

β-thalassemia major and routen test for patients Determination of lipid associated fucose (LAF) with β-thalassemia minor. A mixture of chloroform and methanol The blood samples which collected from all (2: 1) extracts lipid completely, were proteins subjects were transferred into plain tube for and carbohydrates will be in the polar alcoholic serum separation which was at room solvent. The protein will be precipitated by temperature for 15 minutes, centrifuged at phosphotungstic acid leaving the carbohydrate 2500 rpm for ten minutes. in the solution (15).

Determination of total cholesterol (Ch)The Statistical analysis Data presented were the cholesterol is determined according to means and standard deviations. Student Z-test Richmond method (9). was used to compare the significance of the difference in the mean values of any two Determination of triglyceride (TG) groups. (P≤0.05) was considered statistically significant. The triglyceride is determined by enzymatically hydrolyzed glycerol and fatty The overall predictive values for the results in acid according to (10-11). all studied groups were performed according to biostatistics by Daniel in 1987(16). Determination of high density lipoprotein– cholesterol (HDL-Ch) Results and discussion

Low density lipoprotein (LDL), very low Table (1) showed the results of cholesterol and density lipoprotein (VLDL) and chylomicron triglyceride levels in sera of three studied fraction are precipitated quantitatively by the groups. addition of phosphotungstic acid which contain magnesium chloride at pH 6.2, after Cholesterol levels were found to be centrifugation the supernatant contains the (4.75±0.21), (5.00±0.33) and (5.62±2.85) for cholesterol concentration in the HDL (High control, T minor and T major groups density lipoprotein). Fraction which is respectively. determined by using cholesterol kit (12). The increase in cholesterol for minor Determination of low density lipoprotein- thalassemic and major compared to control was cholesterol (LDL-Ch) noticed, still the value is within the normal range (4-6 mmole/dL). LDL-Ch can be calculated mathematically the total cholesterol, the triglycerides and the The results of triglyceride levels for control, T HDL-Ch concentration using Friedwald΄s minor and T major were (2.39±0.33), formula (13). (2.56±0.28) and (4.68±0.52) respectively.

LDL-Ch (mmole/L) = Total cholesterol – A significant increase of triglyceride in sera of TG/2.2 – HDL-Ch. minor thalassemic compared to control and a significant increase in triglyceride for major Determination of very low density lipoprotein thalassemic compared to minor thalassemic (VLDL) and control was found The present findings are in agreement with those found by other studies VLDL concentration is calculated as one – fifth as there is significant increase in plasma TG of the serum TG. level (3), which is the same as detected by (17). However other researchers did not find such VLDL mmol./L=TG/2.2 (14). differences. Ricchi et al study, reported that triglyceride were elevated in association with 26 Zeyan A. et al./ ZJPAS: 2016, 28(3): 24-31 diseases such as thalassemia, probably due to was found. This is in agreement with reported extra-hepatic lipolytic activity (18). data that it appears because of that many factors such as iron overload, liver injury, and Triglycerides lipase activities (both hepatic hormonal disturbances affects lipids pattern and extrahepatic) were significantly lower in among patients with beta-thalassemia. Other thalassemic patients. Christina speculated that explanation could be, accelerated the decreased levels of these enzymatic erythropoiesis (3). activities could play a role in determining the decrease of HDL-C observed in thalassaemic In a study (22) evaluation of several blood lipid patients (19). and lipoprotein with ß-thalassemia major showed that the majority of the patients had Data analysis revealed that only 2% of major low LDL – Ch levels and only few population thalassemic patients had total serum cholesterol had LDL – Ch levels greater than 130mg/dL > 200 mg/dl and 15% of the patients had and a considerable proportion of the patients triglyceride level > 150 mg/dl (19). had very low HDL – Ch levels.

During the past years many scientific LDL – Ch refer to a class and range of researches reported a normal lipid levels i.e lipoprotein particules varying in their size and total cholesterol and triglycerides in patients content, which carry cholesterol in the blood with ß – thalassemia, they also reported and around the body, for use by the cells. It is abnormal distribution is not more 3% of the the final stage of VLDL which is produced by total number of patients of the 192 patients the liver. LDL is formed as VLDL lipoproteins, (20). which lose triglyceride through the action of lipoprotein lipase (LPL) and become smaller Table (2) showed lipoprotein (HDL, LDL and and denser containing a higher proportion of VLDL) mean ±S.D in sera of control, minor cholesterol. Low concentration of large LDL and major thalassemic. The values of HDL in particles is the healthy pattern. Conversely, sera of control, T minor and T major were high concentrations of small LDL particles, (1.6±0.3), (1.81±0.11) and (2.73±0.28) despite the same total cholesterol content respectively. correlates with much faster growth of atheroma and progression of atherosclerosis (23). As for LDL in control, T minor and T major were (2.07±0.78), (2.03±0.11) and (0.77±0.13) Table (3) showed the results of lipid associated respectively. fucose in sera of control, minor and major thalassemic. The levels of LAF were The levels of VLDL for control, minor and (3.88±0.39), (5.03±1.91) and (8.22±1.63) for major thalassemic were (1.08±0.17), control, T minor and T major respectively. (1.16±0.11) and (2.12±0.33) respectively. A significant increase in LAF for T minor A significant increase in HDL for minor compared to control and a significant increase thalassemic compared to control and increase in LAF for T major compared to T minor and levels of major thalassemic compared to minor control was found. and control was found. This result disagrees with results of a study revealed a lower serum Increased levels of LAF in different types of HDL-Ch level in beta thalassemic patients leukemia were reported (24). compared to control (21). Serum LAF levels have been suggested to be a A significant decrease in LDL levels in sera of useful tumor marker, more satisfactory than major thalassemic compared to minor and other fucose related compounds. Some authors control was found, also a significant decrease have observed increased levels of fucose in LDL levels of T major compared to T minor containing glycoconjugates in sera of 27 Zeyan A. et al./ ZJPAS: 2016, 28(3): 24-31 malignancy patients. These investigators Table (2): HDL, LDL and VLDL in sera of three reported significantly elevated serum studied groups concentration of total fucose and its lipid associates (25- 27). HDL LDL VLDL Lipid associated fucose levels were evaluated mmo N in sera of the three major types of leukemia. Gro le/L mmol mmole/ o P P P The results of analysis revealed a significant ups e/L L . increase of LAF values in sera of all leukemic Mea Mean Mean±S patients compared with normal subjects (28). n±S. ±S.D. .D. D. On the other hand, the levels of LAF were reported to decrease in neuroblastoma and Con 4 1.60± 2.07± 1.08±0.

Yolk sac tumors after a successful treatment of trol 0 0.3 0.78 17 the malignancy (29). T 4 1.81± ≤0. 2.03± ≤0. 1.16±0. ≤0. min The diversity in the results of fucose containing 0 0.11 05 0.11 05 11 05 glycoconjugated could be due to the difficulties or inherent in studying the biological function of T carbohydrates and it is likely that many 4 2.73± ≤0. 0.77± ≤0. 2.12±0. ≤0. maj additional functions for fucosylated lipids 5 0.28 05 0.53 05 33 05 remain to be established. or Table (1): Cholesterol and triglyceride levels in ≤0. ≤0. ≤0. sera of three studied groups 05 05* 05* * TG Ch mmole/l P represent value between control, minor and Groups No. P mmole/ml P major thalassemic patients. Mean±S.D. Mean±S.D. * represent P value between minor and major Control 40 4.75±0.21 2.39±0.33 thalassemic patients T 40 5.00±0.33 ≥0.05 2.56±0.28 ≤0.05 minor Table (3): Lipid associated fucose (LAF) in sera of T major 45 5.62±0.58 ≥0.05 4.68±0.52 ≤0.05 three studied groups

≥0.05* ≤0.05* Lipid associated Group N fucose (mg/dl) P * Represent P value between minor and major s o. Mean±S.D. thalassemic patients Contr 4 3.88±0.39 ol 0

T 4 5.03±0.91 ≤0.05 minor 0

T 4 8.22±1.63 ≤0.05 major 5

≤0.05* 28 Zeyan A. et al./ ZJPAS: 2016, 28(3): 24-31

P represent value between control, minor and of α-L- Fucose injection into rabbit major thalassemic patients. periodontium, J Bagh College Dentistry, 25 (2), 119- 124. * represent P value between minor and major thalassemic patients. 7- Yaseen N. A., and Ahmed B. M., (2014). Effect of cigarette smoking on serum α-L- References: fucose and its related parameters Zanco J. Med. 1- Haghpanah S., Davania M., Samadia B., Sci., 18 (1), 596- 603. Ashrafia A., Karimi M., , (2010) Serum lipid 8- Listinsky, J. J., Siegal, Gene, P. and profiles in patients with beta-thalassemia major Listinsk, Y. and, Catherine, M., (1998) " -L- and intermedia in southern Iran JRMS, 15(3), Fucose Apotentially critical molecule in P. 150-154. pathologic processes including Neoplasia", 2- Malakar R. , Manpreet Kour, Afroze Review: Am. J. Clin. Phathol, 110, P. 425-440. Ahmed, S.N. Malviya and C.B.S. Dangi,

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6- Mohammad C. A., Al-Safi K. a., and Ahmed 13- Friedwald, W.T., Levy, R.T., and B. M., (2013) Evaluation the antioxidant effect Fredrickson, D.S., (1972) Estimation of the 29 Zeyan A. et al./ ZJPAS: 2016, 28(3): 24-31 concentration of low-density lipoprotein 20- Wilson, P.W.F., and Abbott, R.D., (1988) cholesterol in plasma, without use of the High density lipoprotein cholesterol and preparative ultracentrifuge. Clin. Chem., 18 mortality. The Framingham Heart Study. (6), P. 499-502. Arteriosclerosis, 8, P. 737-741.

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T اﻧﺨﻔﺎض ﻣﻌﻨﻮي ﻓﻲ اﻣﺼﺎل LDL و ﻣﻦ ﻧﺎﺣﯿﺔ اﺧﺮى ﻇﮭﺮت in Oral Cancer and Oral Precancerous و ﻣﺠﻤﻮﻋﺔ اﻟﺴﯿﻄﺮة. و اﯾﻀﺎ Tminor ﻋﻨﺪ ﻣﻘﺎرﻧﺘﮫ ﻣﻊ Conditions, CANCER , 113 (2), P. 336- 346. major ﻋﻨﺪ ﻣﻘﺎرﻧﺘﮫ ﻣﻊ Tminor ﻟـ LDL ﺗﺒﯿﻨﺖ اﻧﺨﻔﺎض ﻣﻌﻨﻮي ﻓﻲ 27- Miyoshi E., Moriwaki K., Terao N.,Tan C, ﻣﺠﻤﻮﻋﺔ اﻟﺴﯿﻄﺮة، ﺑﯿﻨﻤﺎ اﻟﻔﻮﻛﻮز اﻟﻤﺮﺗﺒﻂ ﻣﻊ اﻟﺪھﻮن ﻇﮭﺮت Terao M., Nakagawa T., Matsumoto H., و Tminor اﻋﻠﻰ ﻣﻦ T major اﻧﺨﻔﺎض ﻣﻌﻨﻮي ﻓﻲ اﻣﺼﺎل Shinzaki S., and Kamada Y., (2012) ﻓﻲ LAF ﻣﺠﻤﻮﻋﺔ اﻟﺴﯿﻄﺮة، و اﯾﻀﺎ زﯾﺎدة ﻣﻌﻨﻮﯾﺔ ﻟﻤﺴﺘﻮى Fucosylation Is a Promising Target for Cancer .ﻋﻨﺪ اﻟﻤﻘﺎرﻧﺘﮫ ﻣﻊ ﻣﺠﻤﻮﻋﺔ اﻟﺴﯿﻄﺮة T minor اﻣﺼﺎل Diagnosis and Therapy Biomolecules, 2, P. 34- 45.

ﺛﻮﺧﺘﮫ 28- Hassan, H.G., (2004) Lipid Associasteod alfa –L-F ucose Levelsi n Sera of Leukaemic :Á ÔÑÊ :‰î:Ófi] “:ÂÁˇ��È å::ÓfiÅ fi] �fi:‰ â÷ :‰‚:‰Ë:ÂÊ:‰flËÉËÁh:›:‰÷:jà:‰d:‰⁄ Patients Ibn-Al-Haitham J. for pure and Appl. :‰“::Ô:‰ fi ] åˇ Á | :‰fi:Ê:‰Ò:ÔÊ \ ÄÑ:ÂÜ:‰÷:ÔÑÊ :‰î:‰d:Ê \ Ö àÁ fi :ÔÜˇÁ“ÁÈ Sci, 17(3), P. 71-79. :ÂÊ::Hj àÊ Ñ Å fi :‰h:Ófi] à:‰“:ÂÊ:’Áïd:Ê:ÂÑÊ:‰ö:ÔÑˇÁ p:‰÷:‰Ë:‰‚:·]Ë]È€Èà˜]h 29- Rompannen, J., (1998) Total and lipid- ÓfiÄÖ “:‰fi\ÁˇÈl Ch, TG, HDL, LDL, VLDL :‰î:‰d:Ê \ Ö àÁ fi :ÔÜˇÁ“ÁÈ :Ê bound sialic acid in children with malignancy :‰fi:40:ÂÊ:ÂÑÊ:‰ö:Ô] È € È à˜ ] h :ÔÑˇÁ p:‰÷:Ó åˇ Á | :‰fi:45:ÔÊ \ ÄÑ:ÂÜ:‰÷:Á ÔÑÊ or infections. Anti-Cancer Res., 18, P. 2793- è] d:j àÊ Ñ Å fi :‰h:Ífi] à:‰“:40:Ÿ:‰ö:‰÷:’Áïd:Ô]È€Èà˜]h:ÔÑˇÁ p:‰÷:Ó åˇ Á | . 2798

Ô:ÂÉ ˇËÑ:‰“:kÊ:‰ “Ñ :ÂÄ Ch :FTG :FHDL Ê VLDL ÔÊ \ ÄÑ:ÂÜ:‰÷ Tmajor ÔÊ \ ÄÑ:ÂÜ::Ÿ:‰ö:‰÷:ÄÑÊ\Ñ:‰d:‰d:ÓflÈd:ÔÊ \ Ö fl Èd:Ó”ˇÈ fiÄÖ “Ä] ËÜ اﻟﺨﻼﺻﺔ Tminor ‰“:kÊ:‰ “Ñ :ÂÄ:]‚:ÂÊÑ:‰‚:F è] d:j àÊ Ñ Å fi :‰h:Ífi] à:‰“:Ê Ch :F ان اﻟﮭﺪف ﻣﻦ اﻟﺪراﺳﺔ اﻟﺤﺎﻟﯿﺔ ھﻲ ﺗﻘﺪﯾﺮ ﺻﻮرة اﻟﺪھﻮن و TG :FHDL Ê VLDL ÔÊ \ ÄÑ:ÂÜ:‰÷ Tmajor:Ófl È d:ÔÊ\ÖflÈd:Ó”ˇÈ fiÄÖ “Ä] ËÜ و Tmajor اﻟﻔﻮﻛﻮز اﻟﻤﺮﺗﺒﻂ ﻣﻊ اﻟﺪھﻮن ﻓﻲ اﻣﺼﺎل دم اﻟﻤﺮﺿﻰ i È ⁄\Ñ] l :›:‰Ò:]‚:ÂÊÑ:‰‚:ÂÊ:F è] d:j àÊ Ñ Å fi :‰h:Ífi] à:‰“:Ÿ:‰ö:‰÷:ÄÑÊ\Ñ:‰d:‰d‰: و اﺻﺤﺎء ﻛﻤﺠﻤﻮﻋﺔ اﻟﺴﯿﻄﺮة . وﺗﻢ ﺗﻌﯿﻦ اﻟﻜﻮﻟﯿﺴﺘﺮول Tminor

ÔÊ \ ÄÑ:ÂÜ:·\ÁˇÈ fi:‰÷:Ófl È d:·] ËÊ\ÖflÈd:Ó”ˇÈ fiÄÖ “Ä] ËÜ:‰fi\Ñ Tminor :‰d:‰d ، اﻟﻼﯾﺒﻮﺑﺮوﺗﯿﻨﺎت ﻋﺎﻟﯿﺔ اﻟﻜﺜﺎﻓﺔ(TG) ، اﻟﻜﻠﯿﺴﯿﺮﯾﺪات اﻟﺜﻼﺛﯿﺔ(Ch) ، ﺑﺮوﺗﯿﻨﺎت دھﻨﯿﺔ (LDL) ، ﻻﯾﺒﻮﺑﺮوﺗﯿﻨﺎت واﻃﺌﺔ اﻟﻜﺜﺎﻓﺔ(HDL) è] d:j àÊ Ñ Å fi :‰h:Ífi] à:‰“:Ÿ:‰ö:‰÷:ÄÑÊ\Ñ. و اﻟﻔﻮﻛﻮز اﻟﻤﺮﺗﺒﻂ ﻣﻊ اﻟﺪھﻮن ﻓﻲ اﻣﺼﺎل (VLDL) ﺿﺌﯿﻠﺔ اﻟﻜﺜﺎﻓﺔ

“‰:‹:“ kÊ:‰ “Ñ :ÂÄ:ÂÊ:ÂÖh:Ó“:‰Ë˜:‰÷ LDL :ÂÜ:‰÷:ÓflÈd:ÔÊ\ÖflÈd:Ó”ˇÈ fiÄÖ:“‰ و 40 ﻟﻼﺻﺤﺎء ﻛﻤﺠﻤﻮﻋﺔ Tminor ﻟـ Tmajor ،40 45 ﻟـ .اﻟﺴﯿﻄﺮة ÔÊ \ ÄÑ Tmajor ÔÊ \ ÄÑ:ÂÜ::Ÿ:‰ö:‰÷:ÄÑÊ\Ñ:‰d:‰d Tminor :‰h:Ífi] à:‰“:Ê

ÔÊ \ ÄÑ:ÂÜ:‰÷:Ófl È d:ÔÊ\ÖflÈd:Ó”ˇÈ fiÄÖ “:›:‰“::]‚:ÂÊÑ:‰‚:ÂÊ:Hè] d:j àÊ Ñ Å fi ﻓﻲ VLDL و Ch ،TG ،HDL وﺟﺪ زﯾﺎدة ﻣﻌﻨﻮﯾﺔ ﻓﻲ ﻣﺴﺘﻮﯾﺎت و Tminor ﻋﻨﺪ ﻣﻘﺎرﻧﺔ ﻧﻔﺲ اﻟﺪوال ﻓﻲ اﻣﺼﺎل Tmajor اﻣﺼﺎل Tminor :ÔÜˇÁ“ÁÈ ::›˜:‰d:F è] d:j àÊ Ñ Å fi :‰h:Ífi] à:‰“:Ÿ:‰ö:‰÷:ÄÑÊ\Ñ:‰d:‰d ، Ch , TG ﻣﺠﻤﻮﻋﺔ اﻟﺴﯿﻄﺮة، و اﯾﻀﺎ وﺟﻮد زﯾﺎدة ﻣﻌﻨﻮﯾﺔ ﻓﻲ ÔÊ \ ÄÑ:ÂÜ:‰÷:ÓflÈd:ÔÊ\ÖflÈd:Ó”ˇÈ fiÄÖ “:›:‰“:ÔÑÊ :‰î:‰d:Ê \ Ö àÁ fi Tmajor :‰d Tminor ﻋﻨﺪ اﻟﻤﻘﺎرﻧﺔ ﻣﻊ T major ﻓﻲ اﻣﺼﺎل VLDL و HDL ÔÊ \ ÄÑ:ÂÜ::Ÿ:‰ö:‰÷:ÄÑÊ\Ñ:‰d Tminor :ÂÊÑ:‰‚:F è] d:j àÊ Ñ Å fi :‰h:Ífi] à:‰“:Ê و اﯾﻀﺎ ﻧﻔﺲ ھﺬه اﻟﺪوال اﻇﮭﺮت زﯾﺎدة ﻣﻌﻨﻮﯾﺔ ﻓﻲ اﻣﺼﺎل , .ﻋﻨﺪ اﻟﻤﻘﺎرﻧﺔ ﻣﻊ ﻣﺠﻤﻮﻋﺔ اﻟﺴﯿﻄﺮة Tminor 31 Zeyan A. et al./ ZJPAS: 2016, 28(3): 24-31

:ÂÜ:·\ÁˇÈ fi:‰÷:Ófl È d:ÔÊ\ÖflÈd:Ó”ˇÈ fiÄÖ “Ä] ËÜ:ÔÑÊ :‰î:‰d:Ê \ Ö àÁ fi :ÔÜˇÁ“ÁÈ :]‚

ÔÊ \ ÄÑ Tminor è] d:j àÊ Ñ Å ﬁ :‰h:Íﬁ] à:‰“:Ÿ:‰ö:‰÷:ÄÑÊ\Ñ:‰d:‰d.

ZANCO Journal of Pure and Applied Sciences

The official scientific journal of Salahaddin University-Erbil ZJPAS (2016), 28 (3); 32-36

Characterization and quantification of some esters of fatty acids from Iris persica L. Bulbs by GC-MS analysis Hawraz Ibrahim M. Amina 1* , Faiq Hama Saeed Hussaina2*and Giovanni Vidari3* 1 Department of Chemistry, College of Science, University of Salahaddin-Erbil/Iraq. 2Department of organic chemistry, University of Pavia-Italy 3 C.I.St.R.E., University of Pavia-Italy, Via Taramelli 10, 27100 Pavia, Italy.

A R T I C L E I N F O A B S T R A C T

Article History: The present study reports the first investigation on the chemical Received: 04/01/2016 composition of the fatty acid methyl esters (FAMEs) which isolated Accepted: 18/02/2016 by middle pressure liquid chromatography (MPLC) and analyzed by gas chromatography-mass spectroscopy (GC-MS) from bulbs of Iris Published:16/5/2016 persica L. that has been collected from Kurdistan Region-Iraq, it Keywords: usually used as a Kurdish traditional medicine for the treatment of Iris persica, FAMEs, GC-MS analysis. wound inflammation and tumor. In this study, eight constituents were *Corresponding Author: identified by GC-MS from the Bulbs of I. Persica which include Hawraz Ibrahim M. Amina oleic acid methyl ester (43.54%), Palmitic acid methyl ester (31.67%) Email:[email protected] and Dodecanedioic acid dimethyl ester (12.01%) were the major constituents of the Bulbs respectively

Introduction 2006). From these techniques, gas The genus Iris (Iridaceae) comprises over 300 chromatography (GC) along with any one of a species (Sabrin R. 2012); most of them have number of detectors, offers a simple, rapid and medicinal importance and are used for the relatively inexpensive method for the treatment of some disease such as cancer, inflammation, bacterial and viral infections identification or quantification of FAMEs in lipid (Hanawa F. 1991). Moreover, a plethora of research (Kathryn E. 2012). bioactive metabolites have been isolated (Xie The relevance of fatty acids (FAs) in human diet G. 2014). and health cannot be over emphasized. The There are several approaches commonly used human brain is estimated to contain nearly 60 in which to identify fatty acids or their derivatives percent fat (Chang C. 2009). Essential fatty acids (fatty acid methyl esters, FAMEs), including gas (EFAs) found in plant origin plays vital roles in chromatography-flame ionization detector (GC- the maintenance of optimal health and brain FID), gas chromatography- mass spectrometry functions (Singh M. 2005). FAs has been reported (GC-MS), high-performance liquid to possess antioxidant activity, anti-inflammatory chromatography (HPLC), nuclear magnetic activities and anticancer activity (Olubunmi A. resonance (NMR) spectroscopy and silver ion 2015). thin-layer chromatography. These methods have Although many of the lipids and FAMEs of been used to identify the common fatty acids of various plants have been extensively investigated animal and plant origin (Glew R. 1997), with the to contain volatile components, the need still chains from 12 to 24 carbon atoms, including zero remains for unstudied plants (Nurettin Y. 2001). I. to six double bonds, as well as specialized on- persica L. is a plant belonging to the Iridaceae methylene-interrupted double bonds (Budge S. family and is widely distributed in Kurdistan 33 Hawraz I. et al./ ZJPAS: 2016, 28(3): 32-36 region-Iraq (Kassak, P. 2012). Prior to the UV1: 254 nm, UV2: 364 nm, mobile phase:, solvent initiation of this work, no study appears to have A: water, solvent B: methanol. UV detection system been carried out on FAMEs from the bulbs of I. dual wavelength with auto sampler. After persica. The objective of this study is to identify collection, the solvent of all tubes was evaporated and quantify some of FAMEs which are present using a rotary evaporator, the separation process bulbs of I. persica. afforded (15) fractions, fraction 14 was characterized to be a mixture of fatty acid methyl Experimental: esters (Lu Y. 2013). Plant material: Iris persica L. was collected in April Analysis of FAMEs: 2014 from (Korek Mountain) in the Kurdistan GC/MS Analysis: region/IRAQ. The plant was identified by two GC/MS Analyses were performed on a botanists Prof. Dr. A. H. Al-khayyat and Dr. Thermo Scientific Focus GC instrument, coupled Abdullah Sh. Sardar at the Biology Department, with a DSQ mass spectrometer, on a HP-5 fused College of Education, Salahaddin University- silica nonpolar cap. Column (30 m_0.25 mm, film Erbil/Iraq. A voucher specimen (No. 7229) was thickness 0.25 mm). Operating conditions were as deposited at Education Salahaddin University follows: injector temp., 2508; carrier gas, He; flow Herbarium (ESUH), Kurdistan. The plant raw rate, 1 mL/min; oven temp. Program, isothermal at material (Bulb) was air dried under shade place at 608 for 1 min, then 58/min to 2608, held for 5 min. room temperature. After drying, the plant part The mass scan range was 41-350 amu; (bulb) was grounded in to fine powder using a sample/solvent ratio, 1: 20; injection volume, 1 mL laboratory grinding mill, to provide homogeneous in split mode (20:1); ionization energy, 70 eV. powder for the analysis. Powdered materials were (Gloria B. 2013). stored in bottles in a dark room temperature until required. Identification FAMEs Fraction: The FAMEs of I. persica bulbs was Extraction with Methanol: identified using their MS data compared to those The dried powdered Bulbs of I. persica L. from the NIST 98, Wiley 5 MS libraries (100 g) was extracted with methanol (500 mL), using an ultrasonic bath for 20 min, then macerated Table 1: Solvent gradient in chromatographic for 3h under continuous stirring at room purification. Seg solvent Length( temperature. The procedure are repeated three mixture ment s CV) times. The mixtures were then filtered and the 1 A/B 20% B - 35% B 4 solvent removed under “vacuum” using a rotary 2 A/B 35% B - 65% B 20 evaporator to afford crude methanol extract. 3 A/B 65% B - 75% B 3 4 A/B 75% B - 100% B 3 FAMEs isolation: 5 A/B 100% B 3 Key: A = water, B = Methanol. 1 g of methanol extract of bulb of Iris persica is dissolved in mixture of methanol-water (4:1) with ultrasonic and then loaded in to the Result and Discussion: MPLC “Isolera ONE” (Biotage) with a reversed The fractionation of the methanolic phase column containing 120 g of hand packed RP- extract of I. persica bulbs is performed through 18 (25-40) µm stationary phase, by the following MPLC by the gradient elution which illustrated in condition: flow rate: 30 mL/min, maximum fraction table (1). That started from 20% to 100% of volume: 18 mL, modalities for detector: UV1+UV2, methanol. The profile of the fraction of methyl 34 Hawraz I. et al./ ZJPAS: 2016, 28(3): 32-36 esters of fatty acids (FAMEs) releaved the presence Peaks Compounds Chem. formula tR of eight peaks with different retention times. The m/z %Area actual transit time of FAMEs fraction which 1 n-Tridecanoic acid, methyl ester isolated from I. persica bulbs was around 20 C14H28O2 13.51 228 2.85 minutes. Figure (1) shows the spectrum analysis of GC in the different fractions generally peaks at 2 Dodecanedioic acid dimethyl ester 13.51 to 18.83 minuties. Fraction of FAMEs of I. C14H26O4 14.31 258 12.01 persica bulbs were identified by the relative time with respect to those known using a bank of NIST 3 Palmitoleic acid methyl ester C17H32O2 14.4 268 1.38 98, Wiley 5 data library for mass spectroscopic identification. 4 Palmetic acid, methyl ester C17H34O2 14.5 270 31.67

5 Undecanoic acid, methyl ester C12H22O2 15.36 198 1.07

6 Oleic acid, methyl ester C19H36O2 16.12 296 43.54

7 Stearic acid, methyl ester C19H38O2 16.42 298 3.6

Figure 1: GC chromatogram of fatty acid methyl esters 8 Adipic acid, bis(2-ethylhexyl) ester (FAMEs). C22H42O4 18.83 370 3.88 The results indicated that the bulbs of the Key: tR = Retention time studied plant contain high percentage of Oleic acid methyl ester (43.54%), Palmitic acid The analysis of the data in figure (2) shows methyl ester (31.67%) and Dodecanedioic acid mass spectra of majority of characterized fatty dimethyl ester (12.01%) respectively. While acid methyl esters. Dodecanedioic acid, the percentages of other FAMEs are (3.88%) dimethyl ester figure 2(A) has an abundant Adipic acid bis(2-ethylhexyl) ester, (3.6%) molecular ion (m/z=258, m/z= 227[M-31] (loss Stearic acid methyl ester, (2.85%) n- of OMe). In figure 2(B) reading the mass Tridecanoic acid methyl ester, (1.38%) spectrum of the peak at 14.5 minutes, relived Palmitoleic acid methyl ester and (1.07%) for the presence of the molecular ion at m/z = 270. Undecanoic acid methyl ester. One the other Moreover, the mass spectra of methyl esters of hand, the mass spectroscopy showed that the unbranched FAME (palmetic acid methyl bulbs of I. persica consists of five saturated ester) are characterized by fragment ions at FAMEs and three unsaturated FAMEs m/z=74 (Mclafferty rearrangement), m/z=87 (monoenes) as shown in table (2). (cuttof B) and [M-31] (loss of OMe). The results in figure 2(C) showed the peak of the molecular ion at m/z= 296, m/z= 222 [M-74], Table 2: Chemical composition, Chemical formula and reading the mass spectrum of the peak at 16.19 some properties of FAMEs of I. persica bulbs obtained from GC-MS minutes confirmed that the corresponding compound is methyl ester of oleic acid.

35 Hawraz I. et al./ ZJPAS: 2016, 28(3): 32-36

A Khayyat and Dr.Abdullah Shukur for the plant identification.

References:

Budge S. Iverson S. And Koopman H. (2006) Studying trophic ecology in marine ecosystems using fatty acids: A primer on analysis and interpretation, Marine Mammal Science, 22,

pp.759–801. B Chang C. Ke D. And Chen J. (2005) Essential fatty acids and human brain. Acta Neurol Taiwan, 18(4), pp.231-241.

Glew R. Dorothy V. Cassius L. Louis G. Garrett S. Andrzej P. and Mark M. (1997) Amino acid, fatty acid, and mineral composition of 24 indigenous plants of Burkina Faso, Journal of Food Composition and Analysis, 10, pp.205-217. C Gloria B. Mohammed I. Alessandra D. Gianluca G. Solveig T. Pietro G. Cesare D. Maria L. Faiq H. Gabriele C. and Giovanni V. (2013) Chemical Composition and Antimicrobial Activity of the Volatile Fractions from Leaves and Flowers of the Wild Iraqi Kurdish Plant Prangos peucedanifolia Fenzl, CHEMISTRY & BIODIVERSITY, 10, 274-280.

Hanawa F. Tahara S. and Mizutani J. (1991) Conclusions: Isoflavonoids produced by Iris pseudacorus leaves treated with cupric chloride, The chemical composition of the FAMEs from Phytochemistry, 30, pp.157–163. the bulb part of Iris persica L. which collected from Kurdistan/Iraq that investigated for the Kassak P. (2012) Secondary metabolites of the first time. Identification and quantification of choosen genus Iris species, Acta univ. agric. et eight FAMEs from the bulb of Iris persica by silvic. Mendel. Brun, 8, pp. 269–280. GC-MS. The major three constituents detected from the bulbs oil were Oleic acid methyl ester Kathryn E. Sarah M. Stephen J. (2012) (38.54%), Palmitic acid methyl ester (36.67%) Comparing Gas Chromatographic Techniques and Dodecanedioic acid dimethyl ester Used in Fatty Acid Profiling of Northern Fur (12.01%) respectively. Seals (Callorhinusursinus) and Steller Sea Lions (Eumetopiasjubatus) from Lovushki Acknowledgment: Island Complex, Russia, International Journal of Applied Science and Technology, 2(2), This work is a Part of Split-Site Ph.D. pp.11-21. publication and the Author acknowledges with many thanks to Prof. Abdul Hussain Al- 36 Hawraz I. et al./ ZJPAS: 2016, 28(3): 32-36

Lu Y. Wang J. Deng Z. Wu H. Deng Q. Tan H. and Cao L. (2013) Isolation and characterization of fatty acid methyl ester (FAME)-producing Streptomyces sp. S161 from sheep (Ovis aries) faeces, Lett Appl Microbiol, ,57(3), 200-5.

Nurettin Y. Zerrin K. Hasan S. and Hasan G. (2001) Characterization of Lipids and Fatty Acid Methyl Ester Contents in Leaves and Roots of Crocus vallicola. Turk J Chem, 25, pp.391-395.

Olubunmi A. Oluwaseeni A. Oluwatimilehin O. K. and Charles B. A. (2015) Direct preparation of fatty acid methyl esters and determination of in vitro antioxidant potential of lipid from fresh Sebal causarium Seed, Journal of Applied Pharmaceutical Science, 5 (3), pp. 24-28.

Sabrin R. Gamal A. and Nawal M. (2012) New Constituents from the Rhizomes of Egyptian Iris germanica L., Molecules, 17, pp.2587- 2598.

Singh M. (2005) Essential fatty acids, DHA and human Brain, Indian Jounal of Pediatric, 72, pp.239-242.

Xie G. Chen Y. Wen R. Xu J. Wu S. and Qin M. (2014) Chemical constituents from rhizomes of Iris germanica, Zhongguo Zhongyao Zazhi, 39, pp.846-850.

ZANCO Journal of Pure and Applied Sciences

The official scientific journal of Salahaddin University-Erbil ZJPAS (2016), 28 (3); 37-48

Effect of Drought Stress on Morphology, Growth and Yield of Six Bread Wheat (Triticum aestivum L.) Cultivars

Sirwa A. Qadir1 * Mohammed Q. Khursheed2 and Fahrul Zaman Huyop3

1PhD. Student, Biology Dept., Education College, University of Salahaddin – Erbil, Iraq 2Biology Dept., Education College, University of Salahaddin – Erbil, Kurdistan region, Iraq 3Faculty of Bioscience and Medical Engineering, Universiti Teknologi Malaysia UTM, Malaysia

A R T I C L E I N F O A B S T R A C T

Article History: An experiment was conducted to study the effects of three drought stress levels, mild (70%) Received: 06/08/2015 and severe (30%) with a control of 100% soil field capacity on six bread wheat cultivars(Sabeer-beg, Aras, Panda, Adena, Rabeaa and Azady) grown in Kurdistan region Accepted: 28/02/2016 and their interaction on morphological and growth parameters including; plant height, Published:16/5/2016 number of tillers, total dry weight and its partitioning, flag leaf area, relative growth rate at three growth stages tillering, stem elongation and heading. Assuming the decrease percent in Keywords: yield and its components including; spike length (cm), number of spikes plant-1, number of wheat, drought stress. grains spike-1, 1000-grain weight (g) and grain yield (g plant-1). Analyzing the variance of data shows a great reduction at sever 30% and less reduction at mild 70% field capacity as *Corresponding Author: compared with control 100% in all studied characteristics except spike length, which did not Mohammed Q. Khursheed affected due to applying drought stress levels. The cultivars also show significant variation among them in their response to drought through the morphological and growth parameters Email:[email protected] except in number of tillers at tillering stage, which finally reflected on the final yield, Azady recorded the higher yield and then Rabeaa, Adene, Aras, Panda and finally Sabeer-beg respectively under sever water stress. The combination between bread wheat cultivars and drought stress level also led to a significant variation in all studied characteristics except in plant height at stem elongation, tiller number at the three stages, flag leaf area, stem dry weight at stem elongation, number of spikes per plant and weight of 1000 grains. Finally, an overall sequence water stress tolerance was Adene> Rabeaa> Aras> Sabeer-beg> Azady>Panda.

1. Introduction moisture is identified as a major factor limiting crops including wheat production and Wheat (Triticum aestivum L.) belongs to the therefore, breeding for drought resistant wheat tribe Triticeae which is one of the largest and is an important task and objective. On the other most important tribes in the Poaceae family. It hand, effective selection criteria for identifying is grown on 200 million hectares with an drought resistant genotypes are required (Zarei average total production of 600 million metric et al., 2013). Water stress conditions can cause tons. Global average yield is around 2.7 t•ha−1 reductions in morphological and agronomical with high variability among countries and parameters, as well as disorders at regions. The highest average yields are physiological, biochemical and molecular obtained in Western Europe, with more than 8 levels (Bartels and Sunkar, 2005). t•ha−1, in contrast to less than 1 t•ha−1 in countries in Central/West Asia and North Growth and photosynthesis are two of the most Africa (Rajaram and Braun, 2009). important processes abolished, partially or completely, by water stress, and both of them In arid and semiarid regions, dry farming is are major cause of decreased crop yield often practiced for wheat production, due to (Kramer and Boyer, 1995). Plant growth is highly variable rainfall seasonally; the low soil accomplished through cell division, cell 38 Sirwa A. et al./ ZJPAS: 2016, 28(3): 37-48 enlargement and differentiation, which involve The experiment was carried out as factorial in genetic, physiological, ecological and completely randomized design with five morphological events; sensitive to drought replicates, included (18) treatments: (Taiz and Zeiger, 2006). combination of six wheat cultivar and three drought stress levels (100%, 70% and 30%) of * Part of Ph.D. thesis of the first author field capacity. The field capacity was determined by saturating the soil in the pot, The intensity of the response to water stress covering the tops with aluminum foil and depends on the stress severity and its duration, weighing daily until there was no weight loss as well as the plant developmental stage within 24, 48 and 72 hours period and on this (Anonymous, 2007); all stages of crop growth base 500ml, 350 and 150ml water added are not uniformly susceptible to water scarcity. respectively as (100%, 70% and 30%) field Moisture stress is known to reduce biomass, capacity (Kramer and Boyer, 1995). tillering ability, grains per spike and grain size Morphological and growth characteristics at any stage when it occurs. So, the overall measured at tillering, stem elongation and effect of moisture stress depends on intensity heading stages of growth respectively which and length of stress (Bukhat, 2005). included plant height, number of tillers, dry matter production and its partitioning, flag Leaf Because of increasing drought damages on area (cm2plant-1) was measured by adopting crops especially in Iraq where arid and semi- Stickler’s linear measurement method (Stickler arid climate predominates, it is essential to et al., 1961) as Leaf area=LxBx0.747 where, detect wheat cultivars that are well adapted to L= length of leaf, B = Breadth of leaf and water shortage and to determine effective and Relative growth rate (RGR) was calculated by reliable selection criteria for selecting drought formula of (Blackman, 1969), expressed as g g- tolerant cultivars. With this background, the 1day-1, RGR= logeW2–loge W1/(t2–t1) present study was undertaken to study six Where, W1 and W2 = Total dry weight per wheat cultivars for various morphological and plant (g) at time t1 and t2 respectively. Plant growth traits associated with drought tolerance yield and yield components were evaluated and identify the most tolerant one(s) against after harvesting for each treatment which water stress. included spike length (cm), number of spikes plant-1, number of grains plant-1 , 1000-grain Materials and Methods weight (g) and grains yield (g plant-1). Some drought resistance indices based on grain yield Six cultivars of bread wheat (Triticum were calculated using the relationships: Stress aestivum L.); Sabeer-beg, Aras, Panda, Adena, susceptibility index (SSI) = [1-(Ys/Yi)]/SI; Rabeaa and Azady) were grown under SI=1- Ῡs/ Ῡi (Fischer and Maurer, 1978), greenhouse conditions on 1st December 2014, Where Ys is the yield of cultivar under stress College of Science, Salahaddin University. (30% FC), Yi the yield of genotypes under Four seeds were randomly sowed per plastic non-stressed condition (100%FC), Ῡs and Ῡi pot, each containing 6Kg soil, previously are the mean yields of all genotypes under sieved and some important chemical and stressed and non-stressed conditions, physical properties of the soil are shown in respectively. Yield stability index (YSI) Table (1). Thinning was performed after the =Ys/Yi and 1- Ῡs/Ῡi is the stress intensity. 5th day of seedling emergence, leaving only Tolerance (TOL) = Yi– Ys (Hossain et al., one plant per pot. The first dose of 1990). Mean productivity (MP) = (Ys+Yi)/2 recommended fertilizer 120Kg ha-1 DAP (Hossain et al., 1990). Geometric mean (N=18% and P=46%) added prior to seed productivity (GMP) = √ (Yi) (Ys) and stress sowing and the second dose of Urea (N= 46%) tolerance index STI = (Ys) (Yi)/ (Ῡi) 2 after 60 days from sowing. (Fernandez, 1992). The results were analyzed statistically using SPSS version 16, and the mean of treatments were compared using Least 39 Sirwa A. et al./ ZJPAS: 2016, 28(3): 37-48

Significant Differences Test (L.S.D) at of reduction at 30% field capacity recorded by significant level 5%. Sabeer-beg at tillering and Aras at stem elongation, 50% reduction by Panda at 70% Table1. Some physical and chemical properties of field capacity at stem elongation, Adene the soil used in the experiment showed minimum reduction in number of Properties Values tillers with the values 0.00% at 70 % field Particle Size Sand 55 capacity and 33.33% at 30% level of drought Distribution (%) Silt 11 stress. At heading stage Aras 100% reduced its Clay 34 tillers at 70% and 30% field capacity, while Soil Texture Sandy clay Sabeer-beg 100 % decreased at 30% drought loam levels as compared with control, Rabeaa and Organic matter (%) 0.7 Azady tolerate water shortage by decreasing Electrical Conductivity (dS.m-1at 0.6 only 50% from their tiller number at 30%. The 25 °C) cultivars differ significantly; Rabeaa regarded pH 7.82 as a pioneer cultivar which tolerates both Total nitrogen (%) 0.09 drought levels and recording highest number of Available Phosphorus (ppm) 7.23 tillers 1.33 and 0.67 tillers at 70 and 30% Potassium(ppm) 80 respectively at stem elongation, the highest number of tillers recorded 1.00 and o.67 tillers by Rabeaa and Azady respectively at both Results water shortage levels at heading. The 1. Effect of drought stress, cultivars and combination of the two factors showed no their combination on morphological significant effect on number of tillers at the and growth characteristics of six bread three growth stages. wheat cultivars Tables 8,9,10 represent the total, leaves and Results of the effects of drought stress stem dry weight of the cultivars at tillering levels on the plant height of wheat cultivars stage, it is observed that both water stress were summarized in tables 2, 3 and 4 at levels significantly decrease the dry matter of tillering, stem elongation and heading stages of the wheat plant, the minimum percent decrease growth. It was observed that at tillering and at 70% in total and leaves dry weight were; stem elongation stages the water deficit levels 8.11% and 5.21% respectively recorded by did not affect significantly. At heading stage Aras and about 11.31% reduction in stem dry water deficit caused significant reduction in the weight by Azady. At 30% drought level a plant height, the lowest decrease percents were minimum reduction in total and leaves dry 1.58 and 21% recorded by Rabeaa at both weight 14.96% and 21.35% by Azady and levels of drought stress as compared with the 5.67% reduction in stem dry weight recorded control. A significant variation recorded by Rabeaa. The cultivars showed a significant between the cultivars, a highest height recorded variation among them, the highest mean of by Aras 49.33 and 49 cm at tillering and 81.33 total, leaves and stem dry weight at 70% were and 80.33cm at stem elongation at both drought 0.47, 0.26 and 0.21g, and at 30% were 0.44, stress levels 70 and 30% of field capacity 0.23 and 0.21g respectively. respectively. Sabeer-beg regarded as tallest one From tables 11,12 and 13 a significant with 88.00cm height at 70% and Aras with differences observed in dry matter and its 84.00cm at 30% at heading stage. The partitioning at stem elongation stage as affected combination of the two above mentioned by water shortage levels; lowest decrease significant factors also exhibited a significant percent of total, leaves and stem dry weight effect in decreasing this trait at tillering and were 2.53% by Rabeaa, 11.92% by Aras and - heading stage. 14.14% by Azady respectively at 70 % stress Tables 5, 6 and 7 represent the number of level. While, minimum reduction at 30% in tillers at the three growth stages, in which a total and leaves dry weight were 22.40% and significant reduce in the number of tillers 14.85% recorded by Azady, lowest stem dry recorded at the three stages with value of 100% 40 Sirwa A. et al./ ZJPAS: 2016, 28(3): 37-48 weight decrease percent was 18.07% recorded elongation to heading stage the minimum by Sabeer-beg. Variation observed among the reduction percent was 0.0126 g g-1 day-1 by cultivars, the highest mean values of total, Aras and Adene at 70% and 0.0120 g g-1 day-1 leaves and stem dry weight at 70% were; 1.69, by Rabeaa at 30% drought stress level. The 0.73 and 0.96g recorded by Azady, while at combination of the two factors exhibited a 30% were1.32 and 0.76g of total and leaves dry significant effect on both relative growth rates. weight of Azady and 0.94g of stem dry weight of Aras regarded as a highest mean values. From tables 14, 15 and 16 the minimum 2.Effect of drought stress, cultivars and their reduction percent of total, leaves and stem dry combination on yield and its components of weight at heading stage; in which 6.90% by six bread wheat cultivars. Adene, 10% by Aras and 3,86 by Panda Table 20 shows significant differences -1 observed respectively at 70% drought stress in the number of spikes plant upon drought level. At 30% minimum reduction were; stress levels, Sabeer-beg recorded zero 25.63% and 26.35% by Sabeer-beg in total and reduction at both drought stress levels, the stem dry weight, while Azady recorded a cultivars also differ significantly from each minimum decline about 14.74% in leaves dry -1 other, 2.67 and 2.00 spikes plant regarded as weight. A maximum total leaves and stem dry weight recorded at 70% were 3.50g by panda, a highest values recorded by Azady at both 0.92g by Rabeaa and 2.82g by Panda, at 30% levels of drought. 3.09 and 0.86g by Rabeaa as a highest total and From table 21 no significant variation leaves dry weight and 2.35g by Aras due to the observed in spike length due to water deficit significant variation among the cultivars. levels. The cultivars varied significantly from The reduction in flag leaf areas of the six each other, the tallest spike was 10.67cm of cultivars under the two drought stress levels Adene cultivar at 70% and 9.33cm of Aras and summarized in table 17, the minimum reduction of the flag leaf area were 1.30% by Panda at 30% stress level. Adene at 70% and 5.45% reduction by Aras Table 22 explains the significant effect recorded at 30%. The cultivars differ from each of the two drought stress levels on grain other in the flag leaf area due to applying the number plant-1. A minimum percent decrease water stress levels, the highest flag leaf area 2 was 7.41% at 70% by Sabeer-beg and 19.49% was 39.95 and 40.14 cm at 70 and 30% reduction recorded by Aras at 30%.The respectively by Aras. cultivars show a great variation, the highest Relative growth rate obtained as in tables -1 18 and 19, the mean of decrease percent of the grain number plant was 100 and 61.3 grains -1 calculated relative growth rate form tillering to plant by Azady at both drought stress levels. stem elongation stage were -2.11% and 12.04% Table 23 Summarizing the final yield respectively at 70 % and 30% field capacity. due to applying drought stress levels, a Minimum decrease percent at 70% field minimum reduction in the yield was 2.83% by capacity level -8.3% recorded by Panda and - Rabeaa at 70%, at 30% Adene scores the 18.8% by Aras at 30% of field capacity. While the mean of decrease percent from stem minimum reduction 9.36% in the yield. The elongation to heading stage were 14.59% and highest final yields were 4.08 and 3.87g at both 23.97% at 70% and 30% level of field capacity 70 and 30% scored by Azady due to the respectively. A minimum decrease was 3.43% significant difference among the cultivars. by Adene at 70% and 18.73 by Panda at 30%. From table 24 the significant difference in The cultivars show significant variation in 1000grain yield observed due to water the relative growth rate, the highest rate was shortage. The minimum reduction was 4.59 and 0.0178 g g-1 day-1 recorded by Panda at 70% -1 -1 7.05% recorded by Azady at both drought and 0.0179 g g day by Aras at 30% from stress levels respectively. The cultivars varied tillering to stem elongation, while from stem significantly among them, highest weights 41 Sirwa A. et al./ ZJPAS: 2016, 28(3): 37-48 were 59.04 and 57.52g at 70 and 30% recorded Table 3. Effect of drought stress, cultivars and their by Azady. The combination effect shows a combination on plant height (cm) at stem elongation significant role in decreasing spike length stage of six bread wheat cultivars. -1 Decrea (cm), number of grains plant , grain yield (g Drought Stress levels Decrease% se% at -1 plant ). at 70% 30% 100% 70% 30% As shown in Table 25, a greater TOL value Cultivars Mean Field Field Field Field Field Capacity Capaci Capacity Capacity Capacity was related to Panda, Azady and Sabeer-beg ty indicating that this genotype had a larger grain Sabeer- 80.33 70.67 64.00 71.67 12.03 20.33 yield reduction under stress condition and beg higher drought sensitivity; lowest TOL was Aras 82.00 81.33 80.33 81.22 0.81 2.03 found in Adene, followed by Aras and Rabeaa. Therefore, these genotypes had a lower grain Panda 53.00 50.00 49.33 50.78 5.66 6.92 yield reduction under stress condition. SSI was highest in Panda followed by Sabeer-beg and Adene 57.00 52.33 51.33 53.56 8.19 9.94 Azady and lowest in Adene. Genotypes with low SSI values (less than 1) can be considered Rabeaa 47.33 43.33 43.33 44.67 8.45 8.45 to be drought resistant. Highest MP and GMP were observed in Azady followed by Rabeaa Azady 63.67 53.00 48.67 55.11 16.75 23.56 and Adene, the least values in Sabeer-beg followed by Aras. The highest YSI and STI LSD = N.S indices were observed for Adene, Rabeaa and LSD = Mean 60.56 60.06 57.89 0.83 4.40 Aras, respectively and the least value in Panda. 6.25 Similar results were reported by Mohammadi et al. (2010 and 2011) and Zarei et al. (2013), p≤ 0.05 LSD = N.S all of whom found these parameters to be suitable for discriminating the best genotypes Table 4. Effect of drought stress, cultivars and their combination on plant height (cm) at heading stage of under drought condition. six bread wheat cultivars.

Drought Stress levels Decrease% Decrease% Table 2. Effect of drought stress, cultivars and their at 70% at 30% 100% 70% 30% combination on plant height (cm) at tillering stage of Cultivars Mean Field Field Field Field Field six bread wheat cultivars Capacity Capacity Drought Stress levels Decre Capacity Capacity Capacity Decrease ase% Sabeer- 97.00 88.00 82.33 89.11 9.28 15.12 100% 70% 30% % at at beg Cultiva Field Field Field Mea 70% 30% rs Capacit Capacit Capacit n Field Field Aras 90.67 85.00 84.00 86.56 7.35 6.25 y y y Capacity Capa city Panda 77.00 71.00 63.67 70.56 7.79 17.32 Sabeer- 40.8 44.67 41.67 36.33 6.72 18.66 beg 9 Adene 87.67 83.67 77.00 82.78 4.56 12.17 51.0 Aras 54.67 49.33 49.00 9.76 10.37 0 33.6 Rabeaa 63.33 62.33 60.67 62.11 1.58 4.21 Panda 35.00 34.67 31.33 0.95 10.48 7 33.5 Adene 36.33 33.00 31.33 9.17 13.76 Azady 77.67 75.00 67.00 73.22 3.43 13.73 6 29.0 Rabeaa 30.33 30.00 26.67 1.10 12.09 LSD = 1.10 0 35.0 LSD Azady 38.00 35.00 32.00 7.89 15.79 0 Mean 80.39 79.17 72.61 = 1.52 9.68 LSD = N.S LSD 1.51 Mean 36.94 38.67 35.94 = 4.45 7.04 p≤ 0.05 LSD = 2.68 p≤ 0.05 LSD = 5.37 3.10 42 Sirwa A. et al./ ZJPAS: 2016, 28(3): 37-48

Table 5. Effect of drought stress, cultivars Table 8. Effect of drought stress, cultivars and -1 and their combination on number of tillers their combination on total dry weight (g.plant ) plant-1 at tillering stage of six bread wheat at tillering stage of six bread wheat cultivar Drought Stress levels Decreas cultivars Decrease 100% 70% 30% e% at Drought Stress levels Decrease Decrease % at 30% Cultivar Field Field Field 70% 100% 70% 30% % at % at Mean Field s Capacit Capa Capa Field Cultiva Field Field Field Mea 70% 30% Capacity y city city Capacity rs Capacit Capacit Capacit n Field Field Sabeer- y y y Capacity Capacity 0.38 0.30 0.25 0.31 20.58 34.18 beg Sabeer- 0.33 0.33 0.00 0.22 0.00 100.00 Aras 0.43 0.40 0.23 0.35 8.11 46.33 beg Panda 0.28 0.23 0.20 0.24 17.75 28.93 Aras 0.67 0.67 0.00 0.44 0.00 100.00 Adene 0.38 0.29 0.24 0.31 22.81 35.96 Panda 0.67 0.67 0.33 0.56 0.00 50.00 Rabeaa 0.52 0.44 0.43 0.46 15.36 18.11 Adene 1.00 1.00 0.33 0.78 0.00 66.67 Azady 0.51 0.47 0.44 0.47 8.67 14.96 Rabeaa 1.00 1.00 0.67 0.89 0.00 33.33 LSD =0.014 Azady 0.67 0.67 0.67 0.67 0.00 0.00 0.3 LSD=0 LSD =0.32 LSD Mean 0.42 0.36 14.93 28.68 0 .0.019 Mean 0.72 0.72 0.33 = 0.00 53.85 p≤ 0.05 LSD = 0.033 p≤ 0.05 LSD = N.S N.S Table 9. Effect of drought stress, cultivars and their combination on leaves dry weight (g plant- 1) at tillering stage of six bread wheat cultivar Table 6. Effect of drought stress, cultivars Drought Stress level Decreas Decrease and their combination on number of tillers 100 e% at 70% 30% -1 % % at 30% plant at stem elongation stage of six bread Field Field Field 70% Cultivars Mean Field Capa Capacit wheat cultivars. Capa Field Capacit city y Drought Stress levels Decrease% Decrease% city Capacity y 100% 70% 30% at 70% at 30% Sabeer-beg 0.18 0.15 0.12 0.15 14.95 31.78 Cultivars Field Field Field Mean Field Field Aras 0.16 0.15 0.11 0.14 5.21 29.17 Capacity Capacity Capacity Capacity Capacity Panda 0.16 0.12 0.10 0.13 24.74 36.08 Sabeer- 0.33 0.33 0.00 0.22 0.00 100.00 Adene 0.25 0.17 0.14 0.19 31.58 43.42 beg Rabeaa 0.30 0.23 0.22 0.25 23.51 27.41 Aras 1.00 0.67 0.00 0.56 33.33 100.00 Azady 0.30 0.26 0.23 0.26 14.04 21.35 Panda 1.33 0.67 0.67 0.89 50.00 50.00 LSD LSD =0.011 Adene 1.00 1.00 0.67 0.89 0.00 33.33 =0.016 19.79 30.91 Rabeaa 1.67 1.33 0.67 1.22 20.00 60.00 Mean 0.22 0.18 0.16 Azady 1.33 1.00 0.67 1.00 25.00 50.00 p≤ 0.05 LSD = 0.028 LSD =0.42 LSD Mean 1.11 0.83 0.44 = 25.00 60.00 Table 10. Effect of drought stress, cultivars and p≤ 0.05 LSD = N.S 0.60 their combination on stem dry weight (g.plant-1) at tillering stage of six bread wheat cultivar Table 7. Effect of drought stress, cultivars Drought Stress levels Decrease Decrease% and their combination on number of tillers 100% 70% % at -1 30% at 30% Field Field 70% plant at heading stage of six bread wheat Cultivars Field Mean Field Capaci Capacit Field cultivars. Capacity Capacity ty y Capacity Drought Stress levels Decrease% Decrease% Sabeer- 100% 70% 30% at 70% at 30% 0.20 0.15 0.13 0.16 25.56 36.31 beg Cultivars Field Field Field Mean Field Field Aras 0.27 0.25 0.12 0.21 9.82 56.44 Capacity Capacity Capacity Capacity Capacity Panda 0.12 0.11 0.10 0.11 8.33 19.31 Sabeer- 0.33 0.33 0.00 0.22 0.00 100.00 beg Adene 0.13 0.12 0.10 0.12 5.26 21.05 Aras 0.67 0.33 0.00 0.33 50.00 100.00 Rabeaa 0.22 0.21 0.21 0.22 4.48 5.67 Panda 1.00 0.67 0.33 0.67 33.33 66.67 Azady 0.22 0.21 0.20 0.21 1.31 6.23 Adene 1.00 0.67 0.33 0.67 33.33 66.67 Rabeaa 1.33 1.00 0.67 1.00 25.00 50.00 LSD =0.0043 LSD Azady 1.33 1.00 0.67 1.00 25.00 50.00 Mean 0.19 0.18 0.14 9.28 26.08 =0.0061 LSD =0.45 LSD p≤ 0.05 LSD = 0.010 Mean 0.94 0.67 0.33 = 29.41 64.71 p≤ 0.05 LSD = N.S 0.32 43 Sirwa A. et al./ ZJPAS: 2016, 28(3): 37-48

Table 11. Effect of drought stress, cultivars and their Table 14. Effect of drought stress, cultivars and their combination on total dry weight (g.plant-1) at stem combination on total dry weight (g.plant-1) at heading elongation stage of six bread wheat cultivar stage of six bread wheat cultivar Drought Stress levels Decrease Decrease Drought Stress levels Decrease% Decrease% 100% 70% 30% % at % at 100% 70% 30% at 70% at 30% Cultiva Field Field Field Mean 70% 30% Cultivars Field Field Field Mean Field Field rs Capaci Capaci Capaci Field Field Capacity Capacity Capacity Capacity Capacity ty ty ty Capacity Capacity Sabeer- 3.19 2.47 2.37 2.68 22.38 25.63 Sabeer- beg 1.10 0.87 0.82 0.93 20.61 25.76 beg Aras 4.11 3.41 2.80 3.44 17.10 31.93 Aras 1.72 1.64 1.20 1.52 5.03 30.37 Panda 3.88 3.50 2.51 3.30 9.79 35.22 Panda 1.28 1.20 0.37 0.95 6.75 70.91 Adene 3.57 3.33 2.31 3.07 6.90 35.26 Adene 1.72 1.33 0.86 1.30 22.76 50.13 Rabeaa 4.45 3.48 3.09 3.67 21.93 30.69 Azady 4.45 3.11 3.06 3.54 30.19 31.19 Rabeaa 1.47 1.20 1.14 1.27 18.33 22.40 LSD =0.07 Azady 1.74 1.69 1.32 1.58 2.53 24.17 LSD Mean 3.94 3.22 2.69 18.44 31.75 LSD =0.096 LSD =0.10 p≤ 0.05 LSD = 0.17 Mean 1.51 1.32 0.95 =0.14 12.23 36.83 p≤ 0.05 LSD = 0.233 3 Table 15. Effect of drought stress, cultivars and their combination on leaves dry weight (g plant-1) at Table 12. Effect of drought stress, cultivars heading stage of six bread wheat cultivar and their combination on leaves dry weight Drought Stress levels Decrease% Decrease% -1 (g plant ) at stem elongation stage of six 100% 70% 30% at 70% at 30% bread wheat cultivar Cultivars Field Field Field Mean Field Field Capacity Capacity Capacity Capacity Capacity Drought Stress levels Decrease Decrease 100% 70% 30% % at % at Sabeer- 0.42 0.31 0.33 0.35 24.80 20.80 Cultiva Field Field Field 70% 30% beg Mean rs Capaci Capaci Capaci Field Field Aras 0.83 0.75 0.45 0.68 10.00 46.00 ty ty ty Capacity Capacity Panda 0.95 0.68 0.60 0.74 28.17 36.27 Adene 0.76 0.63 0.35 0.58 17.90 53.71 Sabeer- 0.31 0.20 0.17 0.22 35.87 45.65 Rabeaa 1.03 0.92 0.86 0.93 10.71 16.23 beg Aras 0.47 0.42 0.26 0.38 11.97 44.37 Azady 0.91 0.80 0.78 0.83 12.77 14.74 Panda 0.64 0.56 0.22 0.48 12.44 65.28 LSD =0.021 LSD Adene 0.72 0.60 0.33 0.55 16.28 54.42 Mean 0.82 0.68 0.56 16.67 31.12 =0.032 Rabeaa 0.70 0.56 0.56 0.61 19.14 19.14 p≤ 0.05 LSD = 0.052 Azady 0.89 0.73 0.76 0.79 18.28 14.85

LSD =0.051 LSD Table 16. Effect of drought stress, cultivars and their -1 Mean 0.62 0.51 0.38 =0.07 17.69 38.23 combination on stem dry weight (g.plant ) at heading p≤ 0.05 LSD = 0.13 1 stage of six bread wheat cultivar Drought Stress levels Decreas 100 Decrease% e% at Table 13. Effect of drought stress, cultivars 70% 30% % at 70% 30% Field Field Mea and their combination on stem dry weight (g Cultivars Field Field Field -1 Capaci Capaci n plant ) at stem elongation stage of six bread Capa Capacity Capacit ty ty wheat cultivar city y Drought Stress levels Decrea Sabeer- 2.77 2.16 2.04 2.32 22.02 26.35 Decrease se% at beg 100% 70% 30% % at 70% 30% Aras 3.28 2.66 2.35 2.76 18.90 28.35 Field Field Field Cultivars Mean Field Field Panda 2.93 2.82 1.91 2.55 3.86 34.89 Capacit Capacit Capacit Capacity Capacit Adene 2.81 2.70 1.96 2.49 3.91 30.25 y y y y Rabeaa 3.43 2.56 2.23 2.74 25.29 35.02 Sabeer- Azady 3.54 2.31 2.28 2.71 34.68 35.44 0.79 0.68 0.65 0.71 14.71 18.07 beg LSD =0.06 Aras 1.25 1.22 0.94 1.14 2.40 25.07 2 Panda 0.64 0.63 0.15 0.47 1.04 76.56 LSD . Adene 1.00 0.73 0.53 0.75 27.41 47.05 Mean 3.13 2.54 =0.0 18.91 31.92 1 Rabeaa 0.78 0.64 0.58 0.67 17.60 25.32 9 3 Azady 0.84 0.96 0.56 0.79 -14.14 34.04 p≤ 0.05 LSD = 0.15 LSD =0.093 LSD Mean 0.88 0.81 0.57 =0.13 8.39 35.85 p≤ 0.05 LSD = N.S 2 44 Sirwa A. et al./ ZJPAS: 2016, 28(3): 37-48

Table 17. Effect of drought stress, cultivars and their Table 20. Effect of drought stress, cultivars and their combination on Flag Leaf Area (cm2 plant-1) at combination on spike length (cm) of six bread wheat heading stage of six bread wheat cultivar cultivar. Drought Stress levels Decrease% Decrease% Drought Stress levels Decrease Decrease% 100% 70% 30% at 70% at 30% 100% 70% 30% % at at 70% Cultivars Field Field Field Mean Field Field Cultiv Field Field Field 30% Mean Field Capacity Capacity Capacity Capacity Capacity ars Capacit Capacit Capacit Field Capacity Sabeer- y y y Capacity 40.70 35.67 37.90 38.09 12.36 6.86 beg Sabeer 8.30 7.33 7.50 7.71 11.65 9.64 Aras 42.46 39.95 40.14 40.85 5.90 5.45 -beg Panda 38.18 34.47 31.42 34.69 9.71 17.70 Aras 7.93 8.00 9.33 8.42 -0.84 -17.65 Adene 35.67 35.30 32.92 34.66 1.30 7.95 Panda 8.60 10.33 9.33 9.42 -20.16 -8.53 Rabeaa 37.84 37.20 35.62 36.88 1.70 5.88 Adene 7.93 10.67 8.33 8.98 -34.45 -5.04 Rabea Azady 41.35 39.03 38.84 39.74 5.61 6.08 5.50 6.00 5.50 5.67 -9.09 0.00 a LSD =1.77 Azady 8.67 7.00 7.67 7.78 19.23 11.54 LSD Mean 39.20 37.12 36.14 5.29 7.80 LSD =N.S =2.5 p≤ 0.05 LSD = N.S 7.9 LSD Mean 7.82 8.22 -5.11 -1.56 4 =0.72

Table 18. Effect of drought stress, cultivars and their p≤ 0.05 LSD =1.24 combination on Relative Growth Rate g g-1 day-1 from tillering to stem elongation stage of six bread Table 21. Effect of drought stress, cultivars and their wheat cultivar. combination on number of spikes plant-1 of six bread Drought Stress levels Decrease Decrease wheat cultivar 100% 70% 30% % at 70% % at 30% Drought Stress levels Cultivar Field Field Field Decrease% Decrease% 30% Mean Field Field s Capacit Capacit Capacit 100% 70% at 70% at 30% Field Mea Capacity Capacity y y y Cultivars Field Field Field Field Capac n Sabeer- Capacity Capacity Capacity Capacity 0.0115 0.0115 0.0129 0.0120 0.04 -11.34 ity beg Sabeer- Aras 0.0150 0.0154 0.0179 0.0161 -2.38 -18.82 1.00 1.00 1.00 1.00 0.00 0.00 beg Panda 0.0165 0.0178 0.0068 0.0137 -8.31 58.93 Aras 1.33 1.00 1.00 1.11 25.00 25.00 Adene 0.0164 0.0164 0.0137 0.0155 -0.13 16.51 Panda 1.67 1.00 1.00 1.22 40.00 40.00 Rabeaa 0.0113 0.0110 0.0107 0.0110 2.64 5.08 Adene 2.00 1.33 1.33 1.56 33.33 33.33 Azady 0.0132 0.0136 0.0120 0.0129 -2.48 9.55 Rabeaa 2.67 2.33 1.67 2.22 12.50 37.50

Azady 3.00 2.67 2.00 2.56 11.11 33.33 LSD =0.00066 LSD Mean 0.0140 0.0143 0.0123 =0.0009 -2.11 12.04 LSD =0.23 LS p≤ 0.05 LSD = 0.0016 3 Mean 1.94 1.56 1.33 D 20.00 31.43 =0. p≤ 0.05 LSD =N.S Table 19. Effect of drought stress, cultivars and their 47 combination on Relative Growth Rate g g-1 day-1 Table 22. Effect of drought stress, cultivars and their from stem elongation to heading stage of six bread -1 wheat cultivar combination on number of grains plant of six bread Drought Stress levels Decrea wheat cultivar. Decreas se% Drought Stress levels Decrease% Decrease% e% at 100% 70% 30% at at 70% at 30% 100% 70% 30% 30% Cultivars Field Field Field Mean 70% Field Field Cultivars Field Field Field Mean Field Capacity Capacity Capacity Field Capacity Capacity Capacity Capacity Capacity Capacit Capaci Sabeer- y 45.00 41.67 30.33 39.00 7.41 32.59 ty beg Sabeer- Aras 39.33 34.67 31.67 35.22 11.86 19.49 0.0124 0.0101 0.0098 0.0108 18.92 21.52 beg Panda 69.67 51.00 27.33 49.33 26.79 60.77 Aras 0.0147 0.0126 0.0108 0.0127 14.25 26.65 Adene 67.67 60.00 37.33 55.00 11.33 44.83 Panda 0.0144 0.0134 0.0117 0.0132 7.33 18.73 Rabeaa 95.67 70.33 39.33 68.44 26.48 58.89 Adene 0.0131 0.0126 0.0094 0.0117 3.43 28.07 Azady 126.00 100.00 61.33 95.78 20.63 51.32 Rabeaa 0.0158 0.0133 0.0120 0.0137 15.98 23.85 LSD =3.03 LSD Azady 0.0156 0.0115 0.0117 0.0130 26.12 24.92 Mean 73.89 59.61 37.89 19.32 48.72 =4.28 LSD =0.00027 p≤ 0.05 LSD =7.42 LSD Mean 0.0143 0.0123 0.0109 14.59 23.97 =0.00039 p≤ 0.05 LSD = 0.00067

45 Sirwa A. et al./ ZJPAS: 2016, 28(3): 37-48

Table 23. Effect of drought stress, cultivars and their deleterious effect on most physiological combination on 1000 grain weight (g) of six bread processes of wheat crop, which was reflected in wheat cultivar. plant height, tillers number, leaf area, dry Drought Stress levels Decrease% Decrease% weight and the growth rate of the plant and 100% 70% 30% at 70% at 30% Cultivars Field Field Field Mean Field Field finally the accumulative effect of all exhibits Capacity Capacity Capacity Capacity Capacity on the final yield of the plant. Sabeer- 54.02 44.62 42.71 47.12 17.40 20.95 Cell growth is one of the most drought- beg sensitive physiological processes due to the Aras 61.04 53.33 52.32 55.56 12.62 14.29 Panda 54.87 48.54 46.57 49.99 11.54 15.12 reduction in turgor pressure (Taiz and Zeiger, Adene 56.93 49.84 50.05 52.27 12.44 12.07 2006). Under severe water deficiency, cell Rabeaa 62.85 55.41 48.90 55.72 11.84 22.19 elongation of higher plants can be inhibited by Azady 61.88 59.04 57.52 59.48 4.59 7.05 interruption of water flow from the xylem to LSD =2.29 the surrounding elongating cells, impaired LSD Mean 58.60 11.60 15.22 11.60 15.22 =3.24 mitosis, cell elongation and expansion p≤ 0.05 LSD =N.S (Nonami, 1998). Water provides turgidity to Table 24. Effect of drought stress and their the cell and its stress causes dehydration, which combination on grain yield (g plant-1) of six bread in fact, reduces the enlargement and expansion wheat cultivar. of cell resulting in reduction of leaf area. The Drought Stress levels Decreas Decreas reduction in leaf area certainly affects the e% at e% at 100% 70% 30% overall growth of crop because the greater the 70% 30% Cultiv Field Field Field Mea flag leaf area of a particular plant, the higher Field Field ars Capac Capac Capac n Capacit Capacit photosynthesis rate (Swati, et al. 1985). The ity ity ity y y results are similar to the findings of (Bilgi, Sabeer 2006), (Boutraa et al., 2010) and (Al-Temimi 2.39 2.00 1.75 2.05 16.30 26.88 -beg et al., 2013). Aras 2.41 2.12 2.07 2.20 12.05 14.13 One immediate response of plant to Panda 3.22 2.81 2.01 2.68 12.75 37.51 Adene 3.38 3.20 3.07 3.22 5.42 9.36 drought is a decrease in the rate of leaf Rabea 3.53 3.43 3.17 3.38 2.83 10.29 a expansion, which results in a reduced leaf area Azady 4.85 4.08 3.87 4.27 15.75 20.22 and reduction in fresh biomass and dry matter LSD =0.12 LS production in wheat (Ashraf et al., 1998). Mean 3.23 3.01 2.65 D 7.00 17.85 Drought-induced reduction in leaf area may be p≤ =0. LSD =0.30 due to suppression of leaf expansion through 0.05 17 reduction in photosynthesis, lower leaf area Table 25. Drought indices of wheat cultivars under means lower light absorption and drought stressed and non-stressed conditions. photosynthesis rate, water stress causes Cultivars Yi Ys MP TOL GMP YSI STI SSI Sabeer- stomatal closure, which reduces the 2.39 1.75 2.07 0.64 2.04 0.73 0.73 1.35 beg Aras 2.41 2.07 2.24 0.34 2.23 0.85 0.85 0.75 CO2/O2 ratio in leaves and inhibits Panda 3.22 2.01 2.61 1.21 2.54 0.62 0.62 1.90 photosynthesis (Tezara et al., 1999). This Adene 3.38 3.07 3.22 0.31 3.22 0.90 0.90 0.50 results coincide with (Abdulla and Khoshiban, Rabeaa 3.53 3.17 3.35 0.36 3.34 0.89 0.89 0.55 2007), (Khayatnezhad et al., 2010) and (Raza et al., 2012). Azady 4.85 3.87 4.36 0.98 4.33 0.79 0.79 1.05 Crop growth rates in early stages due to Discussion: the complete absence of vegetation and low 1. Effect of drought stress, cultivars and their percentage of light absorption, but with the combination on morphological and growth rapid increase in the rate of plant growth rate characteristics of six bread wheat cultivars that occurs because the level of developed Since water plays an important role in leaves and thus absorption of solar radiation plants, it is not surprising that reduced water increase (Beadle, 1987) under normal absorption and dehydration can have a irrigation, while under water deficit levels there 46 Sirwa A. et al./ ZJPAS: 2016, 28(3): 37-48 is a reduction in the RGR rate, because of the than the other genotypes evaluated in this study morphological adjustment of all cultivars under and vice versa. Furthermore, the yield drought condition including reduction in plant components performed as a simple screening height, tiller number, leaves number as well as method for evaluating the response of leaves area, which leads to minimizing the numerous bread wheat genotypes to drought solar radiation capturing and decline in the stress. Finally, an overall sequence water photosynthesis rate as well as the rate of assimilation will be declined. These results stress tolerance was Adene>Rabeaa> agreed with (Bilgi, 2006), (Akram, 2011) and Aras>Sabeer-beg> Azady>Panda. (Al-Temimi et al., 2013). References: Great variation in the morphological 1. Abd El-Moneim, A., I.N. Mohamed, A.H. Belal adjustment at different growth stages among and M.E. Atta. 2010. Screening bread wheat the cultivars have been related to genetically genotypes for drought tolerance: Germination, radical growth and mean variations among the test cultivars and different performance of yield and its components. adaptation patterns to the environment Méditerranéennes, A 95, 301-305. (Lambers and Poorter, 1992). The variation 2. Abdalla, M.M and N.H. El-Khoshiban. 2007. between cultivars and the combination effect The influence of water stress on growth, stress levels and cultivars have been reported relative water content, photosynthesis pigment, some metabolic and hormonal and well documented by several investigators content. J. Appl. Sci. Res. 3, 2062-2074. (Blum and Pnuel, 1990), (Boutraa et al., 2010), 3. Akram, M. 2011. Growth and yield (Almeselmani et al., 2011) and (Al-Temimi, components of wheat under water Stress of 2013 ). different growth stages. Bangladesh J. Agril. Res. 36(3): 455-468. 2. Effect of drought stress, cultivars and their 4. Almeselmani, M., F. Abdullah, F.Hareri, M. combination on yield and its components of six Naaesan, M. A. Ammarand O. ZuherKanbar. bread wheat cultivars 2011. Effect of drought on different physiological characters and yield Generally, following the effect of drought components in different varieties of Syrian stress on morphological, growth characteristics durum wheat. Journal of Agricultural and decline in photosynthesis, which means, Science3, 127-133. production potential and storage capacity 5. Al-Temimi, H. N., W. A. Al-Shahwany and I. reduced too. This storage, in wheat, plays a S. Alsaadawi.2013.Screening of bread wheat (Triticumaestivum L.) genotypes for drought main role in neutralizing the effect of drought tolerance under field conditions. Iraqi on grain filling (Blum, 1998) also agreed with Journal of Science, 54(3): 577-584. (Keyvan, 2010) and (Boutraa et al., 2010).The 6. Anonymous. 2007. National coordinated grain yield of any cultivar is dependent on its wheat program http://www.parc.gov.pk/ yield components as (Sheron et al., 1986) wheat.html. Pakistan Agriculture Research Council. observed that spike length and grain per spike 7. Ashraf, M.Y. 1998. Yield and yield were directly related to grain yield. Similarly, components response of wheat (Tritcium (Shams-ud-din, 1987) reported that reduction aestivum L.) genotypes grown under in spikes per plant; grains per spike and 1000- different soil water deficit conditions. Acta grain weight were directly related to reduce the Agronomica Hungarica 46, 45-51. 8. Bartels, D. and R. Sunkar.2005. “Drought and grain yield of wheat. Salt Tolerance in Plants”, Crit. Rev. Plant Significant variation existing among Sci., 24(1): 23-58. genotypes under each drought stress level 9. Beadle, C.L.1987. Plant growth analysis. In: showed the presence of much variation among J.D.O. Coomlos, S.P. long and J.M.O. Scurlock, (Eds). Techniques in Bio-Productivity and these variables in grain yield and its nd components (Abd El-Moneim et al., 2010). Photosynthesis. 2 Ed. Pp21-23. Pergamon press, Oxford, New York. From the above results, it should be mentioned 10. Bilgi, S.2006. Physiological investigations in that the genotypes which exhibited low dicoccum wheat genotypes. M.Sc. thesis. reduction in grain yield plant-1 under water University of Agricultural Sciences- dharwad. stress conditions will be considered as more 11. Blackman, V. H.1969.The Compound interest drought tolerant for one or more of these traits law and plant growth. Annals of Botany, 33: 353-360. 47 Sirwa A. et al./ ZJPAS: 2016, 28(3): 37-48

12. Blum, A. 1998. Improving wheat grain filling 25. Nonami, H.1998.Plant water relations and under stress by stem reserves mobilization. control of cell elongation at low water Euphytica. 100,77-83. potentials. J. Plant Res. 111, 373–382. 13. Blum, A. and Y. Pnuel.1990. Physiological 26. Rajaram, S. and H. J. Braun. 2009. Wheat Yield attributes associated with drought resistance Potential, In: M. P. Reynolds, J. Pietragalla and on wheat cultivars in a Mediterranean H. J. Braun, Eds., International Symposium on environment. Australian Journal of Wheat Yield Potential: Challenges to Agricultural Research 41, 799-810. International Wheat Breeding, pp. 103- 107. 14. Boutraa, T., A. Akhkha, A. A. Al-Shoaibi and 27. Raza, M.A., M.F. Sammar, I.H. Saleem, A. M. Alhejeli.2010.Effect of water stress on M.Khan, M. Jamil and M.A. growth and water use efficiency (WUE) of Khan.2012.Evaluating the drought stress some wheat cultivars (Triticum durum) tolerance efficiency of wheat (Triticum grown in Saudi Arabia. JTUSCI 3, 39-48. aestivumL.) cultivars. Russian Journal of 15. Bukhat, N. M. 2005. Studies in yield and yield Agricultural and Socio-Economic Sciences, 12 associated traits of wheat (Triticum aestivum (12): 23-29. L.) genotypes under drought conditions. M.Sc. 28. Shams-ud-din, A. K. M. 1987. Path analysis in Thesis, Department of Agronomy. Sindh bread wheat. Indian J. Agric. Sci. 1(3):237- Agriculture University, Tandojam, Pakistan. 240. 16. Fernandez, G.C.J. 1992. Effective selection 29. Sheron, Z. S., O. P. Lauthra and M. S. criteria for assessing plant stress tolerance. In: Kuhad.1986. Association of physiological and Kuo, C.G. (Ed) Proceedings of the International biochemical characters with the yield of Symposium on Adaptation of Vegetables and rainfed area. Plant Breeding Abst. 56:5427. Other Food Crops in temperature and water 30. Stickler, F. and A.W Pauli.1961.Influence of stress, Publication, Tainan, Taiwan. date of planting on yield and yield 17. Fischer, R.A. and R. Maurer.1978.Drought components of grain sorghum. Agronomy resistance in spring wheat cultivar I: Grain Journal 53, 22-23. yield responses. Aust. J. Agric. Res., 29,897- 31. Swati, M.S., H. Rehman and J. Asfar. 1985. 912. Response of Wheat (Triticum aestivum L.) 18. Hossain, A.B.S., A.G.Sears, T.S.Cox and cultivars to different levels of water stress. G.M.Paulsen.1990. Desiccation tolerance and Sarhad J. Agric. 12(1): 295-303. its relationship to assimilate partitioning in 32. Taiz, L. and F. Zeiger. 2006. Plant Physiology. winter wheat. Crop Sci., 30, 622-627. 4th edn., Sinauer Associates Inc. Publishers: 19. Keyvan, S. 2010. The effects of drought stress Massachusetts, USA. on yield, relative water content, proline, 33. Tezara, W., V.J. Mitchell, S.D. Driscoll and soluble carbohydrates and chlorophyll of D.W. Lawlor.1999. Water stress inhibits plant bread wheat cultivars. Journal of Animal & photosynthesis by decreasing coupling factor Plant Sciences.8(3): 1051- 1060. and ATP. Nature 401, 914-917. 20. Khayatnezhad, M., M. Zaefizadeh, R. Gholamin 34. Zarei, L., K. Cheghamirza and E. and S. Iamaati-e-Somarin.2010.Study of Farshadfar.2013. Evaluation of grain yield genetic diversity and path analysis for yield and some agronomic characters in durum in durum wheat genotypes under water and wheat (Triticum turgidum L.) under rainfed dry condition. World Applied Science Journal, conditions. AJCS 7(5):609-617. 9 (6): 655-666. 21. Kramer, P.J. and J.S. Boyer. 1995. Water Relations of Plants and Soils. Academic Press, @Î@&Úü@Û„b◊Ú‰ëÚô@Î@�fi˚Å@Û�Óë@ÙÒà�Ì�â@ÙâÚ�Ìâb◊ San Diego. 22. Lambers, H. and H. Poorter.1992.Inherent @îÚë@ÛflÚÁâÚi@ÎÚëÚüÎ@îÚ◊ÎÎâ@âÚçÚ€@ÊbÓ„ÎÏö�€ Ú Ë ÿ �Ó m variation in growth rate between higher plants: a search for ecological causes and @Triticum aestivum L.@Êb„@Û∏Úü@Ú€@ÂëÚô consequences. Adv. Ecol. Res., 23,187–261. 23. Mohammadi, M., R. Karimizadeh and M. Abdipour.2011.Evaluation of drought Ú¿n¿ÅÏq tolerance in bread wheat genotypes under dry land and supplemental irrigation conditions. AJCS, 5(4):487-493. @Ú€@ ÒÎÚ‰Ó�€˚ÿ�Ó€@ ˚i@ ÒÎaâÜflb¨Úˆ@ ÚÌÒÎÚ‰Ìà�ÌÏm@ ‚Úˆ 24. Mohammadi, R., M.Armion, D. Kahrizi and A. @ÖâÎaâÚi@Úi@HE@30@Î@E70I@@ �fi ˚ Å @ Û �Ó ë @ Ù Ò à �Ì â @ Î Î Ö @Û„b◊ÚÌâÚ�Ìâb◊ Amri.2010. Efficiency of screening techniques for evaluating durum wheat genotypes under @ÊbÓ„Ïö�€ÚËÿ�ÓmÎ@ Î@ &Úü@ Û‰ëÚô@ îÚë@ Î@ E100@ fi˙6„˚◊@ �fiÚüÚ€ mild drought conditions. J. Plant Prod. 4, 11- @Ûí�Ó◊@Î@�¸bˆ@Ù�¸Úü@ÙâÚiÎÎ�â@Î@w‰i@ÙÒâbflá@Î@⁄ÒÎÎ�â@ÙãâÚi@âÚçÚ€ 24. @ÛÀb„˚”@�Ûç@Ú€@ÚëÚü@ÙÒà�ÌâÎ@Ûníü@Ûí�Ó◊@Î@ÖÚ”@Î@˛Úü@ÛÿëÎÎ 48 Sirwa A. et al./ ZJPAS: 2016, 28(3): 37-48

@Ÿ„ÒÖ@ 1000@ Ûí�Ó◊@ Î@ Êb◊Ú�€Ïü@ ÙÒâbflá@ Î@ aÖÚëÚü@ ÙãaÎbÓu @Î@‚Ú◊ÚÌ@ÙÚ‹q@Úi@E30@Û�Óë@ÙÒàÌâN@N⁄ÒÎÎ�â@Û„b◊Úÿ„ÒÖ@ÛflÚÁâÚiÎ @Û„ÖäÿflÚ◊@âÚçÚ€@ÎÏiÚÁ@ÊbÌâaÖbmaÎ@ÙâÚ�Ìb◊@‚ÒÎÎÖ@ÙÚ‹q@Úi@E70 @Ùâ˚u@Ûÿ�Ó€âb◊@NaÖÚëÚü@Û„b◊ÒãaÎbÓu@ÚÀb„˚”@Ú€@Êb◊ÚmÚ�‹çÚÅ@oíü @Ú€@ Ú�u@ ÊÎaäÿçbi@ Ú◊@ Êb◊ÚmÚ�‹çÚÅ@ oíü@ âÚçÚ€@ ÎÏiâaÖbmaÎ@ &Úü @ÙÖaãbˆ@Û‰ëÚô@NaÜv‰i@Û„Öä◊@oçÎâÖ@ÛÀb„�Ï”@Ú€@Êb◊Úv‰i@ÙÒâbflá @Î@Ú»ÓiÒ�â@ämaÎÖ@E30@Û�Óë@ÙÒÖaâ@ä�Ìá@Ú€@ÎÏiÚÁ@ÛflÚÁâÚi@ÂÌämâ˙ã @NÎÏi@ ¬Úi@ 7ibç@ ìÌ6flÚ◊@ ÊbÌÎÏflÚÁ@ Ú€@ Î@ aÜ„bq@ Î@ êaâbˆ@ Î@ Ú„ÒÖÚˆ @âaÖbmaÎ@ÙÒÎÚ„ÖäÿflÚ◊@Ù˚Á@ÒÏi@Ÿ�Ó€âb◊@ÎÎÖâÚÁ@ÊaÏ�Ó„@Û„ÎÏö�€ÚË ÿ �Ó m @ÛÀb„�Ï”@Ú€@⁄ÒÎÎâ@ÙãâÚi@Ú€@Ú�u@ÊÎaäÿçbi@Ú◊@Êb◊ÚmÚ�‹çÚÅ@oíü@Ú€ @ÙâÚiÎÎ�â@Î@aÖÚëÚü@ÛÀb„�Ï”@�Ûç@âÚÁ@Ú€@Êb◊Úv‰i@ÙÒâbflá@Î@ÊÎÏià�ÌâÖ @ÙÒâbflá@Î@ÊÎÏi@à�ÌâÖ@ÛÀb„�Ï”@Ú€@ÖÚ”@ÛÿëÎÎ@Ûí�Ó◊@Î@˛bˆ@Ù˛Úü @aÜíÓÌbm˚◊@ Ú€ NŸ„ÒÖ@ âaãÚÁ@ Ûí�Ó◊@ Î@ Ÿ�Ó◊ÒÎÎ�â@ Ú€@ Êb◊Ú�€Ïü @�fi˚Å@Ú€@Îbˆ@ÛflÚ◊@˚i@ÊbÌäüâÚi@ÙÒä�ÌÏüÚi@&Úü@Û„b◊Ú‰ëÚô@ÙÜ„ÚiåÌâ NaÜ„bq<ÙÖaãbˆ<¬Úi@7ibç<êaâbˆ<Ú»ÓiÒ�â <Ú„ÒÖÚˆ@ÎÏi@ÚÌÒÏ�Óë@‚Úi

ZANCO Journal of Pure and Applied Sciences

The official scientific journal of Salahaddin University-Erbil ZJPAS (2016), 28 (3); 49-54

Theoretical Investigation of Diabetic Urine Detection by Surface Plasmon Resonance Sensor

Haidar Jalal Ismail Physics department., Education College, University of Salahadin-Erbil.

A R T I C L E I N F O A B S T R A C T

Article History: Methods of sensing glucose concentration in blood and other human fluids are Received: 18/06/2015 rely upon various physical phenomena. Nowadays, these methods have a great Accepted: 12/10/2015 concern through many researchers. Surface Plasmon Resonance (SPR), which is an important biosensing method, is an optical detection process that happens Published:16/5/2016 when a polarized light hits a prism covered by a thin metal layer. Under certain Keywords: conditions (wavelength, polarization and incidence angle) free electrons at the Surface plasmon resonance, surface of the metal absorb incident light photons and convert them into surface Diabetic Urine Glucose, metal plasmon waves. A dip in reflectivity of the light is seen under these SPR film, MATLAB. conditions. In this study, a mathematical model of Surface Plasmon Resonance *Corresponding Author: (SPR) with kretschmann configuration investigated to increase the sensitivity for urine glucose concentration in the range 10-3000 mg/dL. The numerical Haidar Jalal Ismail computation of this model rely on three-layer Fresnel equation with p- Email: polarization. Noble metals, naming Ag, Au, and Cu, that are known for their strong SPR at optical frequencies 430–770 THz, with a wide range of their thicknesses (1-71 nm) were selected as plasmonic metal in the proposed sensor to get high sensitivities. The reflected SPR spectra, response curve, and sensitivity variation with thicknesses for these metals were computed by MATLAB program. The maximum sensitivity was 163.77 o/RIU (degree/refractive index unit) for Au with thickness 41 nm. Additionally Cu, with corresponding thickness 51 nm, which has proper sensitivity, might use as a non-invasive and low cost sensor which has drawback of oxidizing and corrosion..

1. Introduction Raman scattering, and Surface Plasmon Diabetes is one of the most spread diseases in Resonance (SPR). Latters possesses larger the world, and it can be considered as a major sensitivity for low concentration of analytes cause of death due to its side effects. It`s come [Kim et al., 2013]. from irregularity in blood glucose level due to Many researchers paid attention on un-sufficient producing of insulin by pancreas designing of SPR to obtain more sensitivity. or un-responding of cells to insulin. This Interaction of incident electromagnetic wave disease expected to spread from 246 (in 2006) with the free electrons of metal surface caused to 380 (in 2025) millions [Ghosh et al., 2010]. an optical phenomena called SPR. The sensing This, perhaps, shows the important of blood layer which attached metal cause variation glucose monitoring to overcome the risking of of the refractive index that it is the base of SPR it. Glucose sensors are based on different sensing [Kumar et al., 2013]. SPR based on techniques and measure glucose level from fiber optic [Kim et al., 2014], with different various human liquids as studied in the bending angles [Kumar et al., 2013], and a literatures. The techniques include series sets of them [Kurihara and Suzuki, 2002] amperometric, polarimetric, surface enhanced were utilized to increase sensitivity for glucose 50 Smail H. et al./ ZJPAS: 2016, 28(3): 49-54 concentration, or for measuring concentration Fig.(1): a) A system of three layers with of different multi-analyte simultaneously. different dielectric constants that an Another researchers made a photonic crystal incident wave suffers from several ring resonator [Makaram et al., 2014] or transmission and reflectance in the coating Au metal layer with various material middle layer. b) Kretschmann such as ployindole [Kim et al., 2013] to obtain configuration of SPR with prism(P)- more performance for glucose measurements. metal(M)-sensing layer(S) interface. d is the metallic thickness and θ is Traces of glucose can be find in human incident angle. serums namely saliva, sweat, breath, tears, and urine. The researches mainly focused on urines Attenuated total reflection from the system due to their high availability and non-invasive (Fig.(1.a)) can be computed using Fresnel’s collections [Makaram et al., 2014]. equation as a summation of reflection in the middle layer. First, total reflected amplitude(r) In the present study, a three-layer derived as follows [Sernelius, (2001); Ploeg kretschmann configuration of SPR was and Melis, 2012]: considered. Noble metals with different thicknesses using as plasmonic metal of SPR in …(1) order to obtain maximum sensitivity for urine glucose with wide range (10-3000 mg/dL). Expanding eq.(1), and as illustrated in Fig.(1.a), one gets:

1. Theory

A simple SPR sensor (three layers) considered with Kretschmann configuration as illustrated …(2) in Figs.(1). The SPR construct simply from a thin metal layer (M) of thickness d which It can be written in different form: sandwiched between a Prism glass (P) and a …(3) dielectric sample (S). The incident angle (θ) of a beam on the metal will be detected by a where sensor which record reflectivity experimentally

…(4) Rewriting eq.(3) as an infinite series:

…(5)

Or: …(6) (a)

The wave has phase difference (2δ = 2kmzd) through traveling back and forth in the medium layer, so eq.(6) becomes:

…(7)

By rearranging the last equation:

51 Smail H. et al./ ZJPAS: 2016, 28(3): 49-54

…(8) The dielectric constants of metal layer were computed according to Drude-Lorentz model [Rakic et al., 1998] for noble metals. The following relations are useful: 3. Results and Discussion …(9) Figure 2 shows fitting curve for refractive index variations with different urine glucose By inserting equs.(8) and (9) into eq.(3), one concentrations [Sharma and Sharan, (2014)] gets: measured by Abbe refractometer that operates using Sodium D-line (589 nm). The high …(10) coefficient relation (R2=0.99) shows excellent fitting of the data, so the resulted fitting equation was used in this work, as an input where rpm and rms, according to [Kurihara and Suzuki, (2002); Mitsushio et al., 2014;], data. defined as:

…(11)

…(12)

…(13)

…(14) Fig.(2): Refractive index versus urine glucose concentration was drawn with Finally, reflectance is related to p- experimental data and their linear polarization (the surface modes of that type) regression fitting which was done and it is equal to: by the author.

In healthy human being, there is negligible urine glucose concentration (0-15 mg/dL), So, which is called Renal threshold of Glucose (RTG). Any further appearance of glucose in …(16) urine (i.e. more than 160-180 mg/dL blood glucose concentration) indicates abnormality in health that is may be due to diabetes or The last equation was used, through malfunctions of kidney or liver [Rave et al., θ changing thickness (d), incident angle ( ) and 2006; Sharma and Sharan, (2014)]. So, type of metal (medium layer), to obtain the according to literatures [Kim et al., 2014; Kim results of the present work. It is clear from et al., 2013], the proposed sensors should have above equations, that the dielectric constant of maximum sensitivity in the range 10-3000 each layer has an important role on the results mg/dL. which related to optical properties of the materials. The dielectric constant of glass Sensitivity changes for the noble metals at prism (BK7 prism glass used that has the same range of concentration with various np=1.515) and sensing layer (for bioanalyte thicknesses of plasmonic metals are shown in fluids) are equal to square of their refractive Fig.(3). Sensitivity, which is the slope of index due to diamagnetic properties of them. resonance angle on refractive index, has similar values (50-85 o/RIU) in the range of 1-15 nm. 52 Smail H. et al./ ZJPAS: 2016, 28(3): 49-54

Au and Cu have maximum sensitivity (163.77, fitting curves (their slopes) that possesses very 163.66 o/RIU) at the metal thickness 41 and 51 high correlation coefficients. nm, respectively. Sensitivities of Au and Cu decrease after reaching their maximum values 2. Conclusion by increasing their thicknesses. A linear The three levels, Kretschmann increasing of sensitivity for Ag metal occurs as configuration, p-polarized, SPR sensor are thickness increased till it maximum 161.88 o theoretically investigated as non-invasive /RIU (d=71 nm). method to detection of urine glucose concentrations using MATLAB program. The effect of using different noble metals as plasmonic metal with a wide range of their thickness 1-71 nm as a sensor was investigated to obtain maximum sensitivity for the proposed sensor for predefined range of urine glucose concentration (10-3000 mg/dL). Based on the results obtained in this study, maximum sensitivity is 163.77 o/RIU (d=41 nm) for Au metal. The whole obtained in this study show Fig.(3): Sensitivity versus thickness of linear responses of sensitivity for same range plasmonic noble metals. of concentration. It is vital to release of increasing Cu sensitivity till 51 nm thickness The SPR spectra curves for the maximum (163.66o/RIU) which can be used as a non- sensitivities of noble metals that shown in invasive, sensitive, low cost, simple sensor for Fig.(3) are drown in Figs.(4) for different urine urine glucose concentration, yet protecting it glucose concentration. Noble metals as from oxidizing and corrosion are necessary to illustrated in the literatures [Sekhon et al., achieve reliable use 2013; Tian et al., 2002] have strong SPR at optical frequencies. A better resolution for mentioned concentration (shift in resonance angle) can be seen for Au (Fig.(4-a)) with thickness 41 nm with wider FWHM (full width at half maximum). SPR of Cu metal (d=51 nm) may be proper for using as sensor for urine glucose as seen in Fig.(4-c). The minimum FWHM was seen for Ag (Fig.(4b)) as compared with others. The dip (minimum reflectance) of SPR spectra varies inversely with increasing concentration for Au and Cu palsmonic metals (Figs. (4-a) and (4-b)).

Perhaps, this can be used as a sensor after making appropriate calibration. Moreover, (a) decreasing in intensities is seen with decreasing in metal thickness as observed in SPR spectra of Mitsushio and others [Mitsushio et al., 2006]. Linear increasing in resonance angle also shown in Fig.(5) for increasing urine glucose concentration for noble metals which was drown for same SPR spectra of Figs.(4). The sensitivities for the three curves computed from 53 Smail H. et al./ ZJPAS: 2016, 28(3): 49-54

Fig.(5): Variation of resonance angles versus refractive index (response curves) and corresponding urine glucose concentrations.

Acknowledgements The author would like to express his acknowledgement to the Salahaddin University for supporting him with available tools. If anyone who needs the program please contact the author for any additional help. (b) References Ghosh P., Bhattacharjee D., Nasipuri M., & Basu K. D., (2010 ), Round- the-clock urine sugar monitoring system for diabetic patients, In IIT Kharagpur- India, Systems in Medicine and Biology (ICSMB) International Conference, 16- 18 December, IEEE. pp. 326-330. Kim H., Jang K., Lee M., (2013), Urine glucose analysis with functionalized graphene oxide as a material for amperometric sensor, 35th Annual international conference of the IEEE (c) EMBS, 3-7 july, Japan, pp. 4510-4512. Kim H., Jang K., Veerapandian M., et al., Fig.(4): Optical based SPR curves for five (2014), Reusable urine glucose sensor urine glucose concentrations for noble metals: based on functionalized graphene a. Au (d=41 nm), b. Ag (d=71 nm), c. Cu oxide conjugated Au electrode with (d=51 nm) protective layers, Biotechnology reports, Vol. 3, pp. 49-53. Urine Glucose Concentration(mg/dL Kumar L., Gupta R., et al., (2013), A new route to glucose sensing method based on surface plasmon resonance using polyindole, Plasmonics, Vol. 8, pp. 487–494. Kurihara K., Suzuki K., (2002), Theoretical understanding of an absorption-based surface plasmon resonance sensor based on Kretchmann’s theory, Anal. Chem., Vol. 74, pp. 696-701. Makaram P., Owens D., Aceros J., (2014), Trends in nanomaterial-based non- invasive diabetes sensing technology, Diagnostics, Vol. 4, pp. 27-46. Mitsushio M., Masunaga T., Higo M., (2014), Theoretical considerations on the responses of gold-deposited surface Reractive index plasmon resonance-based glass rod 54 Smail H. et al./ ZJPAS: 2016, 28(3): 49-54

sensors using a three-layer Fresnel Srivastava S. K., Verma R., Gupta B. D., equation, Plasmonics, Vol. 9, Issue 2, (2012), Surface plasmon resonance pp. 451-459. based fiber optic glucose biosensor, Mitsushio M., Miyashita K., Higo M., (2006), Proc. Of SPIE, Vol. 8531 83511Z, pp. Sensor properties and surface 1-6. characterization of the metal-deposited Tian Zh., Ren B., Wu D., (2002), Surface- SPR optical fiber sensors with Au, Ag, enhanced Raman scattering: from Cu, and Al, Sensors and Actuators A, noble to transition metals and from Vol. 125, pp. 296-304. rough surfaces to ordered Ploeg B. S., Melis K. W., (2012), Exciting nanostructures, Americal chemistry surface plasmons with a mid-IR OPO, society, Vol. 106, No. 37, pp. 9463- Bachelor assignment applied physics, 9483. university of Twente. Verma R., Gupta B. D., (2014), A novel Rakic A. D., Djurisic A. B., et al., (1998), approach for simultaneous sensing of Optical properties of metallic films for urea and glucose by SPR based optical vertical-cavity optoelectronic devices, fiber multianalyte sensor, Analyst, Vol. Applied optics, Vol. 37, No. 22, pp. 139, pp. 1449-1455. 5271-5283. Rave K., Nosk L., et al., (2006), Renal glucose excretion as a function of blood glucose concentration in subjects with type 2 diabetes- results of a hyperglycemic glucose clamp study, Nephrol Dial Transplant, Vol. 21, pp. 2166-2171. Robinson S., Bhuvaneshwari P., Saranya K., Sasiliya S., (2015), Design of photonic crystal ring resonator based biosensor for glucose concentration, IJARTET, Vol. II, special Issue XXIV, pp. 303- 305. Sharma P., and Sharan P., (2014), Photonic crystal based ring resonator sensor for detection of glucose concentration for biomedical applications, IJETAE, Vol. 4, Issue 3, pp. 702-706. Sekhon J. S., Malik H. K., Verma S. S., (2013), DDA simulations of noble metal and alloy nanocubes for tunable optical properties in biological imaging and sensing, RSC advances, Vol. 3, pp. 15427-15434. Sernelius B. E., (2001), Surface modes in physics, 1st ed., Berlin, WILEY-VCH. Srivastava S. K., Arora V., Sapra S., Gupta B. D., (2012), Localized surface plasmon resonance-based fiber optic u-shaped biosensor for the detection of blood glucose, Plasmonics, Vol. 7, pp. 261- 268.

ZANCO Journal of Pure and Applied Sciences

The official scientific journal of Salahaddin University-Erbil ZJPAS (2016), 28 (3); 55-66

Optimal Control Scheme for Flicker Mitigation in Large-Scale PMSG based Wind Energy Conversion Systems

Fadhil T. Aula Department of Electrical Engineering, College of Engineering, University of Salahaddin -Erbil

A R T I C L E I N F O A B S T R A C T

Article History: With increasing penetration of large-scale wind energy conversion systems Received: 18/06/2015 (WECS) into the grid, the power quality issues of these systems are greatly Accepted: 12/10/2015 concerned by the power system management and control. The quality of the output power of the WECS is affected by the flicker in output voltage due to Published:16/5/2016 variation of wind speed, effect of wind turbine tower and wind shear. In this Keywords: paper, an optimal control scheme is presented to mitigate the flicker in the Flicker, optimal controller, output voltage of a permanent magnet synchronous generator (PMSG) based power quality, PMSG, WECS WECS. A linear quadratic regulator (LQR) controller is designed and simulated *Corresponding Author: on 2 MW PMSG modeling using MATLAB/Simulink and MATLAB Control Toolbox. The Performances of the optimal LQR controller are assessed and discussed by means of the simulation for achieving the mitigation of the wind Fadhil T. Aula power system flicker in the output voltage and output power. Simulation results Email: [email protected] show the improvement in the quality of the output power of PMSG based WECS through using the proposed control scheme.

1. Introduction Variations of the voltage produced by Currently, wind energy conversion systems fluctuating the wind power generation make (WECS) are the most dominant source for the voltage quality issues. Fluctuation in the producing the electricity worldwide. Variable- system voltage causes a flicker depending on speed wind turbines with permanent magnet the frequency and magnitude of the fluctuation. synchronous generators (PMSG) and full-scale This produces a kind of disturbance which is back-to-back converters dramatically become called a voltage flicker or known as flicker. in use in many countries. The easy Fig. 1 shows a typical voltage fluctuation controllability, high power density, and corresponding to the flicker. The Flicker, needless to complex gear box systems are main which is produced by the voltage variation, factors of popularity of these types of causes the disturbance for the consumers as generators (Chinchilla et al., 2006). well as other generations on the grid. Flickers As the penetration of the wind power is occur during start-up and during the continuous dramatically increased into the grid, the operation of WECS. The variations in the concern of power quality of WECSs is raised generated output power due to wind speed by power system management and control. variations, the gradient of wind, and the effect Flicker is one of the biggest problems and is of the tower shadow are the main reasons for considered a serious drawback of the WECS producing the flicker (Larsson, 2002). that greatly impact the grid power quality There are many factors that affect emission (Rossetto et al., 1999). 56 Fadhil T. ZJPAS: 2016, 28(3): 55-66 of the flicker of the grid-connected of WECS representation of PMSG mathematical model. during continuous operation, such as grid Section 5 presents the LQR model of the conditions and the characteristics of the wind WECS. Section 6 discusses the simulation (Papadopoulos et al, 1998; Weihao et al, 2008). results, and Section 7 concludes this paper. Several studies have been carried out to Flicker Measurement mitigate the flicker in WECSs. The reactive power compensation using STATCOM was There are several types of voltage variations studied in (Mohod & Aware, 2010) and in such as voltage fluctuation, swells, dips, (Kasem et al, 2010), however, this technique of collapse, and sag. Some of these types are mitigation of the flicker showed its limitation directly associated with grid stability and when the impedance angle of the grid is low in protection. However, voltage variations are some networks (Scott et al, 2002). Other works considered as a power quality issue, which is focused on varying the DC-link voltage for defined by the power management in the range controlling the active power to mitigate the of ±10% of the rated system voltage (Hanzelka flicker (Hu et al, 2009), but a large capacitor or & Bien, 2005). On the other hand, the flicker is a battery in DC-link may be required in this a voltage variation with associated technique due to the fluctuation power in the physiological and pathological components DC-link. A speed controller method for flicker such as tiredness and duress due to the mitigation in PMSG is studied in (Hu et al, variation of the lamination flux during voltage 2013), the method mainly is based of filtering variations. Fig. 2 depicts a block diagram of a with the band-pass of the signal of the active flicker measurement (Meegahapola et al, power, with the improvement of this technique 2010). towards the flicker mitigation, but it has a limitation for designing the filter.

Fig. 2 Basic structure of a flicker measurement (Ackermann, 2005) A wind power system, in general, can be decomposed in multiple subsystems which are aerodynamic systems, mechanical drive train, PMSG, back-to-back converter, and the grid. A typical structure of PMSG based WECS is shown in Fig. 3 (Kim et al, 2015).

Fig. 1 Voltage Fluctuation corresponding to flicker (Lu, 2013) presented based on the mathematical model of PMSG based WECS. The model includes a linear quadratic regulator (LQR) and tuning parameters to get the optimum controller of Fig. 3 Grid-connected PMSG based WECS voltage and active power fluctuations to structure mitigate the flicker. Aerodynamic System The organization of the paper is as follows, Section 2 includes the measurement technique The wind turbine extracts power from wind of the flicker. Section 3 presents the general and then converted it into mechanical power. mathematical model of the PMSG based The amount of aerodynamic power, Pw, is WECS. Section 4 describes the state-space related to the wind speed Vw is given by 57 Fadhil T. ZJPAS: 2016, 28(3): 55-66

(Ackermann, 2005)

(7) (1) where ng is drive train ratio, np is the number of pair poles of PMSG, and wm is the mechanical speed. (2) The relation between mechanical (Tm) and where ρ is the air density, r is the blade generator (electrical) (Te) torques are given by radius, wt is the rotational speed of the turbine, λ is the tip-speed ratio, β is the pitch angle, and Cp is the power coefficient of the aerodynamic system which is given by the following (8) expression where , where Jeq is the equivalent inertia (turbine and generator inertias) referred to the generator side. (3)

PMSG Model

(4) The dynamic model of PMSG can be expressed as the following (Elbeji et al, 2014) The pitch angle β is activated only when the wind speed Vw exceeds the rated value to maintain the normal PMSG output active power. Otherwise, it stays at zero during (9) normal operation which is considered as a case in this paper. It is necessary to maintain wt at optimal rotational speed of wind turbine, wt,opt, in order to provide the maximum Pw, which is the (10) maximum power point tracking (MPPT) control. Therefore, the maximum power that related to Cp,max is given by (Qiaoming et al, 2013) (11) where Vmd, Vmq, imd, imq, Ld, and Lq are

d-q component of stator voltage, current, and (5) inductance, respectively, Rs is stator resistance, and ψm is permanent magnetic flux.

(6) The relation of generator (we) and turbine (wt) angular velocity can be expressed as

58 Fadhil T. ZJPAS: 2016, 28(3): 55-66

Fig. 4 MSC controller 2006)

(20) (12) DC-Link Model

Assuming that the power losses in the MSC, (13) GSC, filter, and transformer can be neglected, therefore, the active power transferred to the power system can be given by

(14)

(21)

(15)

(22)

(16) (23)

where Pp and PsG are PMSG and MSC active powers, respectively, PC and PG are (17) DC-link active powers, respectively, Vdc and Idc are DC-link voltage and current, respectively.

(18) Grid-Side Controller (GSC) The voltage estimation around the RL-filter (Fig. 2) can be given by (Yazdani & Iravanil, 2010) (19)

(24) where Kp1,Kp2, Kp3, Ki1, Ki2, and Ki3 are PI controllers’ gains, Vsd, Vsq, isd, and isq are d-q voltage and current at the terminal of the (25) filter. igd and igq are grid side control d-q where Vgd and Vgq are GSC d-q voltages, currents, respectively, Pp1, Vp1, and Vp2 are respectively, Rf and Lf are RL-filter resistance intermediate variables produced by PI and inductance, respectively, and ws is the RL- controllers. filter frequency. For optimal approach and to reduce non- GSC, as shown in Fig. 5, consists of PI linearity id,ref is assumed to be zero, hence, controllers that control Vdc, igd, igq, and the id,ref can be eliminated from equations (11)- reactive power, Qout, respectively. This (13). For intendance electromagnetic torque in controller can be expressed by the following (11) can be re-written as (Yazdani & Iravanil, 59 Fadhil T. ZJPAS: 2016, 28(3): 55-66 relations (Kim et al, 2015) where Kps and Kis are PI controller’s parameters, and Vp3, Vp4, Vp5, and Vp6 are PI controllers’ intermediate variables. To maintain the unity power factor, Qout,ref igd,ref is kept zero. Also, isd and isq are equal to igd V dc,ref and igq, respectively. For simplicity, Vsq is assumed to be zero. igq,ref Qout,ref State-Space Model of the PMSG Based Fig. 5 GSC controller WECS (Kim et al, 2015) The optimal control scheme requires the system to be in state-space representation, as follows

(26) x = Ax + Bu

y = Cx (35) (27) where x is the state vector (variables), is the derivative of the state vector, u is input signal, y is the output vector, A is the system matrix, B is the input matrix, and C is the (28) output matrix. The equations in previous section are nonlinear equations and need to be linearized. The linearization of these equations after re- (29) arranging can be expressed by the following state derivatives and variables; From (1) to (8), the linearization is (30)

(36)

(31) where

(32)

(33)

(34) 60 Fadhil T. ZJPAS: 2016, 28(3): 55-66

From (12),

(37) where ,

, From (13) to (18), ,

(38) where , From (24) to (34), ,

(40)

, , where ,

, ,

From (12) to (23),

(41)

where

(39) where ,

61 Fadhil T. ZJPAS: 2016, 28(3): 55-66

where , (42) , where

(48)

(43) where ,

where , , ,

where Vwo, weo, imdo, imqo, Vsdo, Vsdo,

(44)

where , ,

igdo, and igqo are initial values for linearization of system derivatives, and Xtl is the transmission inductance between PMSG , and the grid.

From (36) to (48), the system variables are:

(45) And system inputs,

where , Optimal Control Scheme

Linear quadratic regulator (LQR) is a modern technique for designing an optimal controller. LQR can be used in continuous as (46) well as in discrete systems and permits the consideration of the disturbance and noise in where , the system. The objective in this paper is to adopt LQR for the entire WECS to mitigate the flicker effect which is produced by the impact of the variation of the wind speed. This variation of the wind speed considered in this research is about a very short time occurrence which also appeared as the effect of the blade (47) shadow by the tower. The linearization of WECS is controllable 62 Fadhil T. ZJPAS: 2016, 28(3): 55-66 and observable, and has the form as in (35). The performance index for the problem of Table 1. Simulation Parameters LQR is Table 1. Simulation Parameters

Paramet Val Para Valu Para Valu (49) er ue mete e mete e r r An important part of the design the controller is choosing Q and R. There are PMSG 2 np 9 ψm 7.15 different methods available for choosing rated MW Wb values of R and Q (Javid et al, 1982; Pintea et power al, 2011). For any Q and R matrices, the solution of the LQR problem is found by r 83.5 Rs 0.00 Lf 0.00 minimizing J with respect to u (inputs), such m 867 18 H that u(t)=-Fx(t) in which (A-BF) is stable for Ω minimizing J. Fig. 6 shows the block diagram λopt 7.20 Ld 0.00 Rf 0.00 for LQR optimal control. 64 286 65 Ω F in Fig. 6 can be calculated form H , where P is a solution of the following Reccati equation (Anderson et al, ng 30 Lq 0.00 Xtl 0.23 1990) 344 84 Ω H

(50) Kp1 1 Ki1 1 Kp2 0.1

Ki2 0.01 Kp3 1 Ki3 0.2 Simulation Results and Discussion Kp4 1 Ki4 1 Kp5 1 The simulations are carried out for a 2MW,

575 V PMSG based WECS using Ki5 0.2 Kp6 1 Ki6 1 MATLAB/Control Toolbox software. The results are compared with and without LQR Kp7 1 Ki7 1.2 Jeq 8000 controller. The values of Q and R, which are kg.m 2 obtained using try and error method, are given below. Table 1 includes the values of imdo 10.2 imqo -3.3 Vsdo 780 parameters and initial values that are used in 5 A A V the simulation of the PMSG based WECS for equations (36) to (48). igdo 50 igqo -100 Vmdo 750  1 0 0 0 0 0 0 0 0 0 0 0 0  A A V    0 1 0 0 0 0 0 0 0 0 0 0 0   0 0 1 0 0 0 0 0 0 0 0 0 0  V 50 C 0.00 V 8 V   mqo dc wo  0 0 0 0.05 0 0 0 0 0 0 0 0 0  A 8 F  0 0 0 0 0.1 0 0 0 0 0 0 0 0    6  0 0 0 0 0 1 0 0 0 0 0 0 0  10 0 0    ρ 1.22 Vdco 5400 Cp.ma 0.44 Q =  0 0 0 0 0 0 1 0 0 0 0 0 0  and R = 0 104 0     5 V x 12  2   0 0 0 0 0 0 0 0.1 0 0 0 0 0   0 0 10    kg/  0 0 0 0 0 0 0 0 1 0 0 0 0  3  0 0 0 0 0 0 0 0 0 1 0 0 0  m    0 0 0 0 0 0 0 0 0 0 1 0 0   0 0 0 0 0 0 0 0 0 0 0 1 0  For the purpose of the emission of the    0 0 0 0 0 0 0 0 0 0 0 0 0.1  flicker and simulating the WECS using the 63 Fadhil T. ZJPAS: 2016, 28(3): 55-66

Output Voltage presented optimal control scheme, a wind 0.4 PI Controller speed variation has been chosen as the source LQR Controller 0.2 of producing the flicker. However, the wind speed has a variation behavior in nature, but in 0 spite of this, here another variation, flicker, is -0.2 presented as a pulse of dip value at any wind -0.4

speed, in which the wind speed is assumed to Voltage, Volt be constant for a specific short time. This pulse -0.6 has a repeated cycle with a less or a long pulse -0.8 durations depending on the wind speed that -1 rotates the turbine blades around the turbine 0 10 20 30 40 50 60 Time. sec shaft. The majority of large WECSs have three (a) Output voltage response to a single flicker blades rotate at between 30 and 60 rpm. They produce flicker at a rate of 3 Hz at 60 rpm Output Active Power

(Harding et al, 2008). For better depicting the 50 PI Controller LQR Controller effectiveness of LQR, the flicker is represented 0 by the pulses. For rotor radius 83.5 m, 30 rpm -50 rotor speed, and tower diameter around 2.4 m, -100 the duration of the pulse is about 0.0183 -150 seconds with the effective amplitude about 0.5 -200

-250 of the wind speed. Fig. 7 shows the results of Active Power, Watt the simulation for a single flicker occurrence -300 scenario between optimal controller and the -350 -400 regular PI controller of the WECS model. Fig. 0 10 20 30 40 50 60 8 shows the simulation results when the flicker Time. sec occurs at a rate of 1.5 Hz in 30 rpm turbine rotating. (b)Output active power response to a single The output active and reactive powers in Fig. flicker 7 and 8 are simulated based on equations (15) Output Reactive Power and (30), respectively in which the output 0.2 PI Controller voltage is given by LQR Controller 0.15

0.1

(51) 0.05

Reactive Power, VAR

Fig. 9 represents the variations of the DC-Link 0 voltage due to a single flicker and multiple flickers cases, respectively. 0 10 20 30 40 50 60 Time. sec

(C )Output reactive power response to a single flicker

Fig. 7 Simulation results due to a single flicker occurrence per minute

64 Fadhil T. ZJPAS: 2016, 28(3): 55-66

DC Link Voltage 250

PI Controller 200 LQR Controller Output Voltage

2 PI Controller 150 LQR Controller 0 100 -2

50 -4 Voltage, Volt

-6 0

-8

Voltage, Volt -50

-10 -100 -12 0 10 20 30 40 50 60 Time. sec -14

0 10 20 30 40 50 60 Time. sec 1. DC-Link voltage variation to a single flicker 2. From the simulation results, it is clear that (a) Output voltage response to multiple flickers the flicker has been greatly decreased as compared to the PI controller. As depicted

4 10 Output Active Power in Fig. 7, for example, in PI controller the 2 PI Controller voltage is oscillated around reference value LQR Controller 0 from 0.4V to -0.8V before damped at 50s, while it is just a very short pulse at

-2 maximum -0.2V and for only 2s in LQR case. Same results are achieved for active -4 and reactive power when using LQR, while the power jumped above -350W and -0.16 -6 VAR in PI case, respectively. Similar Active Power, Watt advantages of LQR are detected in Fig. 8 -8 with much reduction in the variability of voltage, active and reactive power in DC Link Voltage -10 1500 0 10 20 30 40 50 60 comparison to PI controllers. Furthermore, PI Controller Time. sec the PI controller provides a gap LQRbetween Controller the output1000 responses with respect to the input (b) Output active power response to multiple signals for multiple flickers occurrence, but LQR retains the output responses to follow flickers 500 the input signals with almost no steady-state errors. Moreover, the DC-link voltage, as Output Reactive Power 0 45 shownVoltage, Volt in Fig. 9, more smoothed with the PI Controller 40 LQR Controller use of LQR than PI controller. These prove -500 35 the advantages and superior of the presented

30 controller technique in term of mitigation the-1000 flicker in the PMSG based WECS. 25 0 10 20 30 40 50 60 Time. sec 20 3. 15 4. DC-Link voltage variation to multiple 10

Reactive Power, VAR flickers 5 5. Fig. 9 DC-Link voltage simulation results 0

-5 6. Conclusion 0 10 20 30 40 50 60 Time. sec 7. In this paper an optimal scheme is presented to mitigate the flicker problem in PMSG (c )Output reactive power response to based WECS. The scheme consists of LQR controller, which minimize the variation in multiple flickers Fig. voltage and the output power of WECS. The method has been applied using MATLAB 8 Simulation results due to multiple flickers and results showed the effectiveness of occurrence per minutes using the LQR for reducing the Flicker. For 65 Fadhil T. ZJPAS: 2016, 28(3): 55-66

confirming, the results have been compared Control Systems Magazine, IEEE , vol.2, with the traditional PI controller. It is clear no.3, pp.23,29 from the simulation results that LQR gives superior performance than a regular PI 18. Kasem, A.H., El-Saadany, E.F., El- controller. However, the LQR parameters R Tamaly, H.H., Wahab, M.A.A., (2010), and Q have been obtained form try and error “Power ramp rate control and flicker process, but these values could be obtained mitigation for directly grid connected wind using the estimation techniques such as turbines,” IET Renew. Power Gener., 4, genetic algorithm. Better estimations of R 261–271 and Q give much better results for optimal 19. Kim, Y.-S., Chung, I.-Y., Moon, S.-I, controller and this was left for the future (2015), ” Tuning of the PI controller works. parameters of a PMSG wind turbine to improve control performance under various 8. References wind speeds,” Energies, 8, 1406-1425 9. Ackermann, T., (2005), “Wind Power in 20. Larsson, A., (2002), " Flicker emission Power Systems,” John Wiley & Sons: of wind turbines during continuous Hoboken, NJ, USA operation," Energy Conversion, IEEE 10. Anderson, Brian D. O., Moore, John B, Transactions on , vol.17, no.1, pp.114,118 (1990), “Optimal control: Linear quadratic 21. Lu, D. Dah-Chuan, (2013),“ An Update methods,” Prentice Hall, Englewood Cliffs, on Power Quality,” InTech, Open Access, NJ available at 11. Chinchilla, M., Arnaltes, S., Burgos, http://www.intechopen.com/books/an- J.C., (2006), "Control of permanent-magnet update-on-power-quality generators applied to variable-speed wind- 22. Meegahapola, L., Fox, B., Flynn, D., energy systems connected to the grid," (2010), "Flicker mitigation strategy for Energy Conversion, IEEE Transactions on, DFIGs during variable wind conditions," vol.21, no.1, pp.130,135 Power and Energy Society General Meeting, 12. Elbeji, O., Hamed, M., Sbita, L., 2010 IEEE , vol., no., pp.1,8, 25-29 (2014), ” PMSG Wind Energy Conversion Mohod, S.W., Aware, M.V., (2010),” A System: Modeling and Control,” STATCOM-control scheme for grid International Journal of Modern Nonlinear connected wind energy system for power Theory and Application, 3, 88-97 quality improvement,” in Systems Journal, 13. Hanzelka, Z. , Bien, A., (2005), “Power IEEE , vol.4, no.3, pp.346-352 quality application guide, voltage Papadopoulos, M.P., Papathanassiou, S.A., disturbances-flicker measurement,” Copper Tentzerakis, S.T., Boulaxis, N.G., (1998), ” Development Association Investigation of the flicker emission by grid 14. Harding, G., Harding, O., Wilkins, A., connected wind turbines,” In Proceedings (2008), “ Wind turbine, flicker, and of 8th International Conference on photosensitive epilepsy: Characterizing the Harmonics Quality of Power, Vol.2, ﬂashing that may precipitate seizures and Athens, Greece, 14–18; pp. 1152–1157. optimizing guidelines to prevent them,” Pintea, A., Haoping Wang, Christov, N., Epilepsia, 49(6), 1095-1098 Borne, P., Popescu, D., Badea, A., (2011), " 15. Hu, W., Chen, Z., Wang, Y., Wang, Z., Optimal control of variable speed wind (2009), ” Flicker mitigation by active power turbines," Control & Automation (MED), control of variable-speed wind turbines with 2011 19th Mediterranean Conference on , full-scale back-to-back power converters,” vol., no., pp.838,843, 20-23 IEEE Trans. Energy Convers, 24, 640–649 Qiaoming Shi, Gang Wang, Lijun Fu, Lei Yuan, He Huang, (2013), "State-space 16. Hu, W., Zhang, Y., Chen, Z., Hu, Y., averaging model of wind turbine with (2013), ” Flicker mitigation by speed control PMSG and its virtual inertia control," of permanent magnet synchronous generator Industrial Electronics Society, IECON variable-speed wind turbines,” Energies, 6, 2013 - 39th Annual Conference of the 3807-3821 IEEE,vol.,no., pp.1880,1886, 10-13 17. Javid, S.H., Murdoch, A., Winkelman, Rossetto, L., Tenti, P., Zuccato, A., (1999), J.R., (1982), "Control design for a wind " Electromagnetic compatibility issues in turbine-generator using output feedback," industrial equipment," Industry 66 Fadhil T. ZJPAS: 2016, 28(3): 55-66

Applications Magazine, IEEE , vol.5, no.6, pp.34,46 Scott, N.C., Atkinson, D.J., Morrell, J.E., (2002), ” Use of load control to regulate voltage on distribution networks with embedded generation,” IEEE Trans. Power Syst., 17, 510–515 Weihao Hu, Zhe Chen, Yue Wang, Zhaoan Wang, (2008), ” Flicker study on variable speed wind turbines with permanent magnet synchronous generator,” In Proceedings of 13th Power Electronics and Motion Control Conference (EPE-PEMC), Poznan, Poland, 1–3 September 2008; pp. 2325–2330. Yazdani, A., Iravani, R., (2006), ” A neutral-point clamped converter system for direct-drive variable-speed wind power unit,” IEEE Trans. Energy Conversions, 21, 596–607 Yazdani, A., Iravani, R., (2010), “Voltage- Sourced Converters in Power Systems,” John Wiley & Sons, Hoboken, NJ, USA

ZANCO Journal of Pure and Applied Sciences

The official scientific journal of Salahaddin University-Erbil ZJPAS (2016), 28 (3); 67-77

Vehicular Cloud Computing: Trends and Challenges

Kayhan Zrar Ghafoor1*, Kamalrulnizam Abu Bakar2, Marwan Aziz Mohammed1 and Jaime Lloret3 1Department of Software Engineering, College of Engineering, University of Salahaddin –Erbil 2Faculty of Computer Science and Information Systems- University Technology Malaysia 3Department of Communications- University of Polytechnic - Valencia

A R T I C L E I N F O A B S T R A C T

Article History: Recently vehicular Ad hoc Networks (VANET) has attracted the attention of research Received: 23/08/2015 communities, leading car manufactures and governments due to its potential Accepted: 07/03/2016 applications and specific characteristics. Their research outcome was started with Published:16/5/2016 awareness between vehicles for collision avoidance to internet access and then expanded to vehicular multimedia communications. Moreover, vehicle’s high Keywords: computation, communication and storage resources are set a ground for vehicular VANET, mobile cloud networks to deploy these applications in the near future. Nevertheless, on- board computing, VANET cloud, traffic resources in vehicles are mostly underutilized. Vehicular Cloud Computing (VCC) is information management developed to utilize the VANET resources efficiently and hence provide subscribers *Corresponding Author: safe and infotainment services. In this article, we perform a survey of state-of-the-art Kayhan Z Ghafoor vehicular cloud computing as well as the existing techniques that utilizes cloud Email:[email protected] computing for performance improvement in VANET. We then classified the VCC based on the applications, services types and vehicular cloud organization. We present the detail for each VCC application and formation. Lastly, we discussed the open issues and research directions related to VANET cloud computing.

1. Introduction increase their efforts toward creating a Recently, the growth in the number of vehicles standardized platform for vehicular on the road has put great stress on communications (for instance, Vehicle Safety transportation systems. This abrupt growth of Communication Consortium, Network-on- vehicles has made driving unsafe and Wheels and Honda’s Advances Safety Vehicle hazardous. Thus, existing transportation Program (Olariu ,Weigle, 2009). In particular, infrastructure requires improvements in traffic the 5.9 GHz spectrum band has been allocated safety and efficiency. To accomplish this, for licensed Short Range Communication Intelligent Transportation Systems (ITS) have (DSRC) between vehicles. In addition, in the been considered to enable such diverse traffic near future, more vehicles will be embedded applications as traffic safety, cooperative traffic with devices that facilitate communication monitoring and control of traffic flow. These between vehicles, such as Wireless Access in traffic applications would become realities Vehicular Environment (WAVE) (ITS- through the emergence of VANET because it is Standards, Intelligent transportation systems, considered as a network environment of ITS. 1996). When vehicles are equipped with The increasing necessity of this network is an WAVE, they can communicate with nearby impetus for vehicle manufacturers, research communities and government agencies to 68 Kayhan Z. et al./ ZJPAS: 2016, 28(3): 67-77 cars and access points within their coverage VCC is still in its infancy stage of researching area. and need to be explored more (Arif , Olariu , Wang, Yan, Yang, Khalil , 2012). Figure 1 In addition, car manufactures have advanced shows the number of articles published in the and hence fitted out better storage, last years. computation and communication devices in the vehicles. These advances are all for the sake of In this paper, we surveyed the state-of-arts of improving traffic safety and efficiency vehicular clouds, which is considered as a (Ghafoor, Bakar, Eenennaam, Khokhar, zalez, development of traditional cloud computing 2011) (Sommer, Tonguz, Dressler, 2010) with novel features. More precisely, the aim of (Naumov, Baumann, Gross, 2006) (Ghafoor, this paper Bakar, Lloret, Khokhar, Lee, 2013). A long with this development, vehicles can also access internet services and thereby various benefits will be offered to the drivers and passengers (Hussain, Son, Eun, Kim, Oh, 2012).Thus, these advancements in vehicular technology and communication will provide wide potential applications to meet safety and comfort requirements for driver. However, the phenomenal on-board resources are remaining under-utilized by the aforementioned applications (Wang, 2007).

In an attempt to address the problem of resource under- utilizing, the authors in Figure 1: Number of publications in the last (Eltoweissy, Olariu, Younis , 2010) have years proposed the concept of vehicular cloud. VCC is defined as paradigm shift from conventional VANET to vehicular cloud in which vehicles use their on-board resources and cloud are to introduce the concepts of vehicular resources as well (Olariu , Khalil, Abuelela, clouds and highlight the prospective applications for sake of improving the 2011) (Abid , Phuong , Wang , Lee, Qaisar , efficiency of vehicular networks. We 2011). The motivation of merging mobile explained the existing techniques that cloud computing with VCC is the dynamic utilize cloud computing for performance accessibility of resources. In other words, the improvements in VANET. We then VCC follows the concept of pay as you go classified the VCC based on the model instead of buying resources and applications, services types and vehicular infrastructure. With this technology, vehicle’s cloud organization. Moreover, we explained the communication paradigm communication, computing and storage and organization of vehicular clouds. capabilities can be combined with those of Based on this though study on VCC, we other drivers or rented to the participated cars. discussed the open issues and research This concept is similar to the traditional cloud directions related to VANET cloud computing but sensing nodes in VANET are computing. To the best of our knowledge, vehicles and they travelling with high speed. this research paper is the first attempt in 69 Kayhan Z. et al./ ZJPAS: 2016, 28(3): 67-77

thoroughly reviewing state-of-arts of VCC number of solutions have already been and highlighting its challenges. proposed, with safety applications being the most prominent (Yang, Guo, Wu, 2009) , (Yang, Guo, Wu , 2008). These traffic the rest of the paper is organized safety applications are relying on Inter- as follows: Section 2 provides an overview vehicle Communications (IVC) and the of the current state of the arts of VANET. formation of VANET. In addition to the The cloud computing taxonomy and its traffic safety services, VANET offers illustration are discussed in section 3, prosperous services like internet on the followed by the discussion on vehicular wheel, high-speed tolling, information cloud computing and its architecture, where infotainment and video on demand ( Anda, we also highlight the applications of LeBrun, Ghosal, Chuah, Zhang, Vgrid vehicular clouds and the layers of the ,2005), (Karagiannis, Altintas, Ekici, vehicular clouds. Finally, section 4 Heijenk, Jarupan, Lin, Weil ,2011). Due to concludes the paper and discusses future high mobility of vehicles, however, many directions. research challenges still need to be addressed. 2: VANET The number of vehicles contending for space in existing transportation systems 3: Cloud Computing is growing rapidly. This abrupt growth of Cloud computing is a style of a new vehicles has made driving unsafe and computing paradigm that enables on- hazardous. Thus, existing transportation demand service to consumers through infrastructure requires improvements in various cloud service providers. These traffic safety and efficiency. To achieve this cloud computing services can be accessed requirement, Intelligent Transportation through internet from configured and shared Systems (ITS) have been considered to resources like computing storage and enable diverse traffic applications such as applications in a virtual manner traffic safety, cooperative traffic monitoring and control of traffic flow. Vehicular networks are considered as a network environment for ITS.

Over the past decade, the employing of vehicular communications to support traffic safety and comfort related services have earned much attention in both industry and academia. A divergent number of wireless technologies have been used such Figure 2: Mobile cloud computing as Infrared (infrared) (Kwak, Lee, 2004), communication paradigm Bluetooth (Sugiura, Dermawan, 2008) and cellular (Santa, Go´mez-Skarmeta, Some of the services are provided for free Sa´nchez-Artigas, 2008). However, IEEE while others are based on ’pay as you go’ 802.11 based solutions has witnessed to be model from hardware and system software the popular and appropriate solution. This in remote datacenters (Rodrigues, Zhou, advancement are continued as the IEEE Mendes, Lin, Lloret, 2012) (Wlodarczyk, 802.11p (Jiang, Delgrossi, Ieee, 2008), Rong, 20111) (Song, Su, 2011). The cloud (Uzcategui, Acosta-Marum, 2009) service providers manage a network amendment to the standard is attracting infrastructure of datacenters in order to increasing interest. A range of flourish combine the computing power of massive applications are required to aim the uptake number of servers. Moreover, the ’pay as of vehicular communications and massive you go’ characteristics of cloud computing 70 Kayhan Z. et al./ ZJPAS: 2016, 28(3): 67-77

enable many advantages such as non-front neighborhood cars that are involved in the investment, lower operating cost, highly cloud formation. Indeed, the drivers may scalable and easy access. Example of cloud rent their vehicle’s on-board resources to computing are Amazon S3, Google Drive the cars that are demanding specific service and Microsoft SkyDrive and they could for stipulated time period (Eltoweissy, Olariu, provide virtualized infinite amount of Younis, 2010). In such situations, vehicles resources. have the capability to cooperate for the sake of solving problems that would happen in With the existence of a pool of centralized system. For instance, a driver resources in mobile environment, a new raise the question to vehicular cloud computing field is emerged called Mobile regarding the cause of traffic jam in the next Cloud Computing (MCC) (Gupta, Gupta, road segment. In response to this query, the 2012). In this environment, every mobile vehicular cloud create, maintain and device can act as mobile client or service propagate this information to the relevant provider. It is witnessed that the popularity vehicles. Internet cloud also can be used to of mobile devices (e.g., tablet computer, i query this type of information but at the Pad, and smartphone) for internet access are expensed of inordinate amount of time for increasing day-by-day as compared to querying and searching information in the stationary personal computers. This is global cloud. Thus, vehicular cloud because significant improvements of mobile computing has many advantages as devices have shown in terms of hardware, compared to the conventional cloud operating system and transmission 2011).،computing (Gerla, 2012) (Lin, Shen capability. Recently many mobile application like real time services, instant More significantly, vehicular clouds email checking, social networking can be have various crucial applications ranging performed ubiquity through mobile device. from traffic safety, environment sensing, These mobile devices effectively form and information distribution to the mobile cloud to access internet cloud commercial advertisement and traffic ubiquity. For instance, a mobile device infotainment. For instance, vehicles will interested in downloading an application capture information like traffic condition, from nearby mobile node. This information road condition, cars in the vicinity, is stored and propagated in the mobile cloud environment condition and surveillance (Gerla, 2012). Moreover, there are many videos via equipped sensors. This group of services of MCC such as Googles gmail, vehicles in a specific road segment can Maps and Navigation systems for mobile, exchange and maintain this information Voice Search, and some applications on an rather than uploading to the internet cloud. Android platform, that offers great Among group of vehicles, participated cars applications to the end users. The general can excess the cloud data in the VC. architecture of MCC is illustrated in Figure 2 below (Gupta, Gupta, 2012). Recently, car manufacturing companies and governments of several 3.1: Vehicular Cloud Computing countries attempt to promote the cloud A massive number of resource-rich computing in the vehicular networking. For vehicles that are travelling on the highways example, in the USA, the Federal and urban areas possess internet access Communications Commissions allocated services, on-board storage, high Dedicated Short Range Communications computation and sensing capabilities. In our (DSRC) with a range of 75 MHz of the daily life, many vehicles spend a substantial spectrum (5.850 to 5.925 GHz) in support time in malls, parking garages and drive of vehicular networking (Ghafoor, Bakar, ways. With these features, vehicles can be Eenennaam, Khokhar, zalez, 2011) (K. utilized as a cloud node within Ghafoor, K. Bakar, S. Salleh, K. Lee, M. 71 Kayhan Z. et al./ ZJPAS: 2016, 28(3): 67-77

Mohamad, M. Kamat, M. Arshad, 2012) (K. Ghafoor, K. Bakar , 2010).

Moreover, roadside infrastructures such as inductive loop detectors, video cameras, acoustic tracking systems, microwave radar sensors, and access points are also helpful for VCC. As part of a project, Ford motor company combines Figure 3: Taxonomy of the literature on social networks, GPS location awareness, vehicular cloud computing and real- time vehicle data in ways that help drivers go where they want efficiently by condition, road condition or other using the cloud (2012). Ford is equipped parameters and these collected data will be with cloud features called Ford SYNC, shared with neighborhood vehicles in the which connects customers to real time vicinity. Perhaps beacon frame is used to traffic updates, information, turn-by-turn synchronize and handshake among vehicles driving directions, business, sports, weather, and hence sharing operational data. The and news through voice commands. second layer enables communication Recently, Toyota and Microsoft announced between vehicles and vehicular clouds. As a new, 12 million partnership to bring cloud vehicles are equipped with IEEE 802.11p computing to Toyota vehicles (Liu, transceivers, they can exchange information 2011).The partnership will equip Toyota either vehicle-to- vehicle (V2V) or vehicle- vehicles with the latest technology to access to-infrastructure (V2I) by using Wi- Fi, telecommunications information, streaming IEEE 802.11p, WiMAX or 3G cellular music, energy management, and GPS communications. services, while on the road. We also consider another motor company named In V2V architecture, cars General Motors. The General Motor communicate with each other in the vicinity perception and vehicular control groups are in order to enable traffic safety for the engaged in developing a vehicle to vehicle drivers and passengers as well as comfort communication system. This system relies related applications. As it was witnessed in on DSRC with a range of about one-quarter the past decade, this communication has mile (400 m) in all directions, with radios important role in reducing number of connected to traffic lights or construction crashes, collisions in vehicular scenarios. zones. The technology will provide viable VCC also can be formed among V2V solutions for essential safety information for communication scenarios, in case of drivers. Figure 3 presents a taxonomy of abnormal road condition or driver vehicular cloud computing. behaviour, a vehicle that observed this traffic abnormality will send an event- 3.1.1: Architecture of Vehicular Clouds driven emergency message to the formed The vehicular cloud computing vehicular cloud storage and hence all composed of three layers of vehicles that are registered within this VC communication. As can be seen in Figure 4, will receive the emergency message. Event- the three communication layers are on- driven emergency message contains the board layer, communication layer and cloud positional information of the location computing layer. In the on-board layer, a where abnormal situation occurs (Chen, vehicle could sense the environment Lee, Wu, Kuo, 2009) (Yang, Guo, Wu, 2008) (Yang, Guo, Wu, 2009) (Xu, Mak, Ko, Sengupta, 2004). Moreover, VANET utilizes fixed gateways such as WLAN access points, WiMAX base stations or 3G 72 Kayhan Z. et al./ ZJPAS: 2016, 28(3): 67-77

networks in order to exchange packets same vehicular cloud. In the same way, among vehicles and internet. The V2I is some vehicles have sufficient amount of complementary to V2V architecture and can storage while others need extra storage. raise driver safety and reduce traffic deaths Storage as a service can offer storage to and injuries by implementing collision the vehicles which need extra storage. avoidance and warning systems (Bordley, Cooperation as a service also is used to Cherry, Stephens, Zimmer, Petrolino, provide various services to the drivers via 2012). As V2I requires high cost of V2V communication. infrastructure installation, V2V is considered more feasible (Huang, Fallah, 3.1.2: Organization of Vehicular Cloud Sengupta, Krishnan, 2010). It is worth Computing: The vehicular cloud is mentioning that traditional vehicular formed as follow networks consists of the lower two layers: on-board and communication layers. 3.1.2.1: Static VC Moreover, the cloud computing layer In our daily life we spend a provides communication at the cloud level substantial amount of time on the street and through roadside unit or mobile gateways. parking lot. On the roads, the traffic The cloud computing layer of situation is dynamic and is varied from vehicular cloud consists of three sub-layers normal to abnormal scenarios such as traffic i.e cloud as an infrastructure, cloud as a congestion and accidents. In such abnormal platform and cloud as service. Cloud as an traffic conditions, VC is mimic to the infrastructure provides Computation and traditional cloud computing services. This storage capabilities for vehicles. The sensed similarity between VC and traditional cloud data is classified based upon the type of computing is also true when vehicles information and then stored in the storage remain static in malls or parking garages. unit of a vehicle. Then, vehicles use their Consider an information technology company that offers IT services and solutions to its customers. In such company many vehicles of staff members remain idle in the parking lot and their computational resources also remain un- utilized. The companies’ top management may request the formation of static vehicular cloud by providing rewards to those employees who want to rent the on-board resources of their vehicles. As a result, the formed static VC

Figure 4: Vehicular cloud computing will take benefit from the cooperation of on- architecture board resources of vehicles that are participating in the cloud formation. Figure computation capability to analyze the data 5 shows the static formation of vehicular that is stored in the storage unit. Cloud as cloud. As can be seen in Figure 5, vehicles a platform consists of development tools are parked in front of the company and this that are hosted in the cloud and accessed company offers incentive to the drivers who via a group of vehicles within the same will rent on-board resources of their cloud. Moreover, the cloud as a service vehicles. This kind of VC formation is offers network as service, cooperation as a called stationery service and storage as a service (Eltoweissy, Olariu, Younis, 2010). Some vehicles have internet access while they are moving. Those vehicles can share this luxury with neighbor vehicles within the 73 Kayhan Z. et al./ ZJPAS: 2016, 28(3): 67-77

4: Applications of Vehicular Cloud Computing In this section several possible implementation scenarios and the application outcome of VCC are discussed (Olariu , Khalil, Abuelela, 2011) (Abid , Phuong , Wang , Lee, Qaisar , 2011) (Gerla ,2012) (Alazawi, Altowaijri, Mehmood, Figure 5. Static Vehicular cloud formation Abdljabar ,2011): VC since all vehicles are static. 3.1.2.2: Dynamic VC Advances in vehicular networks in one hand and cloud computing in the other motivates the necessity of dynamic formation of VC. As vehicles are travelling Figure 6. Dynamic Vehicular cloud formation with high mobility and the network changes rapidly, the mobility parameter play an important role in VC formation. Mobile vehicular cloud would be formed among vehicles with similar mobility. The reason is that among similar velocity vehicles the relative velocity will be reduced and the cloud will long lasted. Thus, moving

vehicles can make a cloud of storage and Figure 7. Urban surveillance system in VC computation resources autonomously. The process of dynamic VC formation is mimic to the dynamic cluster formation in 4.1: Datacenter Formation VANET. In our daily life, people are spending Dynamic VC is formed by first hours in malls for shopping purpose in electing a vehicle that has smallest value of holidays or weekends. As illustrated in relative mobility to the nearby cars. The Figure 7, many cars are parked in the elected vehicle is known as cloud head and parking garage and their owners spend will form the VC by inviting the neighbor substantial amount of time for shopping. vehicles to join its VC. The invited During the time of shopping, these cars neighbor vehicles should response back the remain idle with underutilized request from the cloud head. If the number computation resources. These on-borad of vehicles are sufficient, the cloud head resources can shared/rented to the interested will declares the cloud formation. Thus, VC customers with permission of the cars enables a seamless integration and pooling owners. This process can be handled by the of resources of the vehicles that are mall IT management department. For registered in the VC. With this capability, instance, each vehicle has Ethernet interface the VC provides a large computer cluster and in each parking spot the mall and a huge distributed data storage facility management should provide Ethernet cable. (Olariu , Khalil, Abuelela, 2011). In contrast If a driver intends to rent the resources of its to stationary formation of VC, Figure 6 car, he need to connect the Ethernet cable to illustrates that the neighbor vehicles are vehicle’s Ethernet interface. In this case, the involved in formation of dynamic vehicular mall management should reward drivers cloud. This is called dynamic VC since all who rented the on-board resources of their vehicles within the VC are travelling on the vehicles for the sake of static VC formation. roads. Some people are travelling from a country to the other one. When they travel, 74 Kayhan Z. et al./ ZJPAS: 2016, 28(3): 67-77

they left their vehicles in the parking lot of city in Iraq. The proposed work leverage an airport. It is a great opportunity to exploit vehicular networks and MCC for the the resources of these vehicles in order to evacuation procedure in case of form a VC in a parking spot. For instance, unpredicted disaster. the airport management section can offer power source, storage or Ethernet connection to the vehicles that are interested 4.3: Traffic Management to rent those resources for a stipulated time Nowadays the on-board navigator period. The participated vehicles in a VC can estimate the traffic fluidity and patterns will reveal their arrival and departure times. from MC and forwards the optimal route to This information helps the airport the vehicles. The navigator service enables management to schedule the shared on- traffic management through the board resources. Thus, long-term parking communication between on-board and lot and sharing travel plan by participated server navigator. The on-board navigators cars will provide plentiful, stable and long- send time, position and position of term resources (Olariu , Khalil, Abuelela, destination in a fixed rate to a server 2011). navigator in internet. The server responses back the best route based on the estimated 4.2: Disaster Management load and delay on the incremental route to The reaction to emergency situations the destination. This cooperation is is very important as its consequences are considered as an interaction between VC very catastrophic. For instance, the Japan and internet cloud. More precisely, the VC earthquake, 11 September in USA and other through on-board navigator senses the disasters. The total cost of Japan tsunami is traffic congestion of road segments whereas approximately about 300 billion USD the internet cloud through server navigator (Alazawi, Altowaijri, Mehmood, Abdljabar, estimates the traffic flow on those roads. 2011). Certainly, communication technology With the help of such applications, vehicles has crucial role in responding emergency can compute local traffic of roads and situations and hence reducing human death determine the reasons of traffic jam in order toll. VCC can be used to manage disaster to address the traffic deadlock and react events by forming vehicular clouds that are accordingly (Gerla, 2012). participating in the evacuation operation. The evacuation system should provide a 4.4: Urban Surveillance safe area for the people when a disaster Vehicles in VANET are equipped situation occurs in a city. For instance, with sensors and they communicate with evacuation zones should be chosen based on each other for mobile surveillance and the low travelling time and availability of environment monitoring. Traditional urban resources surveillance systems for monitoring security attacks rely on the fixed roadside infrastructure. This method is not efficient as compared to the mobile surveillance via vehicles. With cooperation of VCC video surveillance can be done on-demand via vehicles that are travelling on the urban Figure 8. Urban surveillance system in VC roads and highways. For urban surveillance, self-organized vehicles participating in surveillance system will form a vehicular such as medicine, food and water. The cloud and they can report any suspected authors in (Alazawi, Altowaijri, Mehmood, activity such as terrorist attack to a specific Abdljabar, 2011) proposed intelligent zone. Mobile surveillance can also be used disaster management system for Ramadi in investigations after the event has 75 Kayhan Z. et al./ ZJPAS: 2016, 28(3): 67-77

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The official scientific journal of Salahaddin University-Erbil ZJPAS (2016), 28 (3); 78-85

Speaker Recognition Using Discrete Wavelet Transform and Artificial Neural Networks Amin A. Abdul Fattah1* and Layla M. Salih2 1Department of Electrical Engineering, College of Engineering, Salahaddin University-Erbil, Iraq 2Department of Electrical Engineering, College of Engineering,, University-Erbil, Iraq

A R T I C L E I N F O A B S T R A C T

Article History: In recent years biometrics has emerged as applied scientific discipline with the Received: 18/06/2015 objective of automatically capturing personal identifying characteristics that Accepted: 12/10/2015 distinguish one individual from another and using the measurements for security, surveillance, and forensic application. Speaker recognition is the Published:16/5/2016 process of automatically recognizing who is speaking based on individual Keywords: information included in the speech waves. This paper presents the speaker Speaker recognition, identification method based on Discrete Wavelet Transform (DWT) and text-dependent, Artificial Neural Networks (ANN). In this study the DWT is used to extract a feature extraction, speaker's discriminative features from the mathematical representation of the DWT, speech signal. These feature vectors are used to train a feedforward neural ANN network which is used to model the speakers and make the decision task. A database of 20 speakers (10 male and 10 female) has been used with a *Corresponding Author: vocabulary of Kurdish words. The system led to 100% identification rate for Amin A. Abdul Fattah text-dependent and 80% identification rate for text-independent. Email:[email protected] University of Kirkuk / Iraq

1. Introduction due to the anatomical differences in speech- producing organs, and partly due to Thee speech signal contains many levels of idiosyncrasies of the speaker, such as speaking information. Primarily a message is conveyed habits and emotional state (Guruprasad et al, via the spoken words. At other levels, speech 2003) conveys information about the language being spoken, the emotion, gender, and the identity of Speaker recognition encompasses the speaker. While speech recognition sets its verification and identification. Automatic goals at recognizing the spoken words in speaker verification (ASV) is the use of a speech, the aim of automatic speaker machine to verify a person's claimed identity recognition is to identify the speaker by from his voice. In automatic speaker extraction, characterization and recognition of identification (ASI), there is no a priori identity the information contained in the speech signal. claim, and the system decide who the person is, There are two main factors that make speaker what group the person is a member of, or (in recognition a compelling biometric; (1) Speech the open-set case) that the person is unknown is natural signal to produce that is not (Campbell, 1997). considered threatening by the users to provide, and (2) the telephone system provide a familiar A generic speaker recognition system is network of sensors for obtaining and delivering shown in figure 1, where the desired features the speech signal (Singh, 2003). Speaker- are first extracted from the speech signal. specific variations in speech signal are partly These features are then used as input to a 79 Smail H. et al./ ZJPAS: 2016, 28(3): 78-85 classifier, which makes the final decision al, 2007; Shafik et al, 2009; Abdalla and Ali, regarding verification or identification (Farrell 2010; Daqrouq et al, 2009; Pawar et al, 2014) et al, 1994). are concentrating on the wavelet transform for speaker feature extraction stage. Wavelets have emerged as a new and powerful tool for nonstationary signal analysis. One of the main properties of this technique is

Figure 1: Speaker recognition system its ability to analyze signal with different levels of resolution. Fast transients can be analyzed with short windows, while slowly varying Speaker recognition methods can also phenomena can be observed with longer time be divided into text-independent and text- windows (Greenberg et al, 2004). dependent methods. In a text-independent In this paper, we consider a hybrid system, speaker models capture characteristics approach for isolated word speaker of somebody's speech which show up identification, where the Discrete Wavelet irrespective of what one is saying. In a text- Transform (DWT) is used as a feature dependent system, on the other hand, the extraction component. Also, in order to further recognition of the speaker's identity is based on reduce the dimensionality of the extracted his or her speaking one or more specific feature vectors, statistics over the set of phrases, like passwords, card numbers, PIN wavelet coefficients are used. Artificial Neural codes, etc.( Reda and Aoued, 2005). The Network (ANN) is well-known as a technique current approach is aimed at text-dependent that has the ability to classify nonlinear and text-independent speaker identification. problem. An (ANN) is used for pattern recognition. Some of the recent works on speaker recognition depend on classical features 2. The Database including formant frequencies (Daqrouq et al, 2009), Mel Frequency Cepstral Coefficients A database is created for Kurdish language (MFCC) (Kaur and Sharma, 2014), MFCC and using 20 speakers (10 male, 10 female). The inverted MFCC (Singh and Rajan, 2011), LPC- database was taken from Salahaddin University Residual and MFCC (Curipe and / Engineering College (students and staff). Camacho,2013), Linear Prediction Cepstral Each speaker utters 9 Kurdish words twice, one Coefficient (LPCC) and MFCC (Kumar and for the training phase and the other for the Lahudkar, 2015), LPC and MFCC (Subhashini recognizing phase. and Pratap, 2014), LPC, LPCC, and RASTA- 3. Discrete Wavelet Transform (DWT) PLP (Visalakshi and Dhanalakshmi, 2014). Wavelet analysis is a windowing However, these methods have some technique with variable-sized regions, which disadvantages. These methods accept signal allows the use of long time intervals where we stationary within a given time frame and may want more precise low-frequency information, therefore lack the ability to analyze the and shorter regions where we want high- localized events correctly. Moreover, the LPC frequency information (Misiti et al, 2000) method accepts a particular linear (all-pole) For many signals, the low-frequency model of speech/word production which part contains the most important part. It gives strictly speaking is not the case (Avci, 2006). an identity to a signal. Consider the human Literature on various studies reveals that in voice, if, we remove the high-frequency case of the above parameters, the feature vector components, the voice sounds different, but we dimensions and computational complexity are can still tell what’s being said. In wavelet higher to a great extent. analysis, we often speak of approximations and Some researchers (Woo et al, 2001; details. The approximations are the high-scale, Zheng and Ching, 2004; Avci, 2006; Al-Ani et low- frequency components of the signal. The 80 Smail H. et al./ ZJPAS: 2016, 28(3): 78-85 details are the low-scale, high frequency are used to encode the speech signals. Figure 2 components. The DWT is defined by the shows the decomposition and the following equation: reconstruction of three levels by using DWT.

(1)

Where (t) is a time function with finite (a) Decomposition energy and fast decay called the mother wavelet. The DWT analysis can be performed using a fast, pyramidal algorithm related to multirate filterbanks. As a multirate filterbank the DWT can be viewed as a constant Q filterbank with octave spacing between the centers of the filters. Each subband contains half the samples of the neighboring higher (b) Reconstruction frequency subband. In the pyramidal algorithm Figure 2: Wavelet decomposition & the signal is analyzed at different frequency reconstruction for 3 levels bands with different resolution by decomposing the signal into a coarse 4. Artificial Neural Networks approximation and detail information. The coarse approximation is then further Artificial Neural Networks (ANNs) are decomposed using the same wavelet computational models which attempt to mimic decomposition step. This is achieved by the learning function of the brain. ANNs are successive highpass and lowpass filtering of composed of a set of neurons or processing the time domain signal and is defined by the units which are connected together by means of following equations (Krishnan and Anto, connecting weights. ANNs which are 2009): structured to learn and generalize so that the network may learn by continuous adjustment of the weights of the connections (Topping et al, 1998). (2)

Feedforward neural networks (FNN) have been widely used for (3)various tasks, such as pattern recognition, function approximation, dynamical modeling, data mining, and time series forecasting, to name just a few. The training of FNN is mainly undertaken using the backpropagation (BP) - based learning Where y [n], y [n] are the outputs of the low high algorithms (Yu et al, 2002). lowpass (h) and highpass (g) filters, respectively after subsampling by 2. Because The typical backpropagation network of the downsampling the number of resulting has an input layer, an output layer, and at least wavelet coefficients is exactly the same as the one hidden layer. There is no theoretical limit number of input points. A variety of different on the number of hidden layers but typically wavelet families have been proposed in the there is just one or two as shown in figure 3 literature (Tzanetakis et al, 2001). In this study (Alsmadi et al, 2009). the daubechies (Db2 - Db5) and Haar wavelet 81 Smail H. et al./ ZJPAS: 2016, 28(3): 78-85

Where, N = number of samples in each frame The result of the windowing is given by: Y(n) = X(n)* w(n)

5.2 Feature Extraction The energy in each frame in each level of decomposition is calculated with the application of equation (5) (Kotnik and Kacic, Figure 3: An MLP neural network. 2007). In order to further reduce the dimensionality of the extracted feature vectors, 5. Materials and Methodology: statistics over the set of the wavelet 5.1 Preprocessing: coefficients are used (Tzanetakis et al 2001). In The sound files contained in the this study, the standard deviation of the energy database are .WAV files recorded with overall the frames for each level of the microphone sampled at 11025 Hz with a decomposition levels was calculated. The resolution of 16 bits per sample. These sampled deviated energy values are collected in a vector signals can capture all frequencies up to 5 kHz, of 9 elements. Now, the features for each which cover most energy of sounds that are speaker are calculated and the first step of generated by humans (Reda and Aoued, 2005). recognition is done. This sampling rate was chosen for recording n which is twice the frequency of the original 2 S signal and follows the Nyquist rule of ∑ i i=1 sampling. P = n The speech signal must be broken up Where Si: is an element in the into small frames with time length in the range approximation coefficient set of (20-40) millisecond. In this step the n: is the number of the elements in the continuous speech signal is blocked into frames approximation coefficient set of N samples, with adjacent frames being separated by M (M < N). The human speech 5.3 The Classification Process: production is known to exhibit quasi-stationary The classification step in automatic speaker behavior over a short period of time (20-40) identification systems is in fact a feature msec. matching process between the features of a Then windowing is performed on each new speaker and the features saved in the individual frame so as to minimize the signal database. ANN is widely used for feature discontinuities at the beginning and end of each matching. Multi-layer perceptrons (MLPs) frame (Kabir and Ahsan, 2007). In this study consisting of an input layer, one or more Hamming window was used, which has the hidden layers and an output layer can be used form: for this purpose. Each neuron in the neural network is characterized by an activation function and its bias, and each connection   2πn   − =between − two neurons by a weight factor ( ) = 0,54 0,46cos  n 0, , N 1 w n   N −1 (Shafik et al, 2009). In this study feedforward  (4)  0 otherwisebackpropagation neural network is used with the training condition and the structure of the ANN is tabulated in table 1. 82 Smail H. et al./ ZJPAS: 2016, 28(3): 78-85

Figure 4: Training of the neural network using Db4 wavelet as feature extractor. Table 1: Parameters used for the ANN Straight line – Goal, falling curve – Training 6. Experimental Results Function Description Network type Feed forward backpropagation In this study, trails were made to No. of layers Three layers: Input, one hidden and output identify speakers by comparing a sample of No. of neurons in 9-Input, 22- Hidden and 20 layers Output their voices to a database included speech data Activation tansig, logsig function files of 20 speakers (10 male and 10 female). Performance 0.0001 The speech files consist of nine isolated function No. of epochs 1477 Kurdish words. Each speaker repeats each

word twice, one for training phase and the Training a neural network is other for testing phase. Five of the utterances accomplished by adjusting its weights using a training algorithm. The training algorithm were selected for training for both text- adapts the weights by attempting to minimize dependent and text-independent identification. the sum of the squared error between a desired output and the actual output of the output The selected data set includes the words neurons given by the following equation: (Bayaneet, Bash, Choneet, Ewarat, Bash, Jwana, Shereen, Haween, and Zstan), the (6) words (Bayaneet, Bash, Choneet, Ewarat, and

Jwana) are used as training set. Figure 5 shows Where Do and Yo are the desired and actual outputs of the oth output neuron. K is the the speech sample signals for the first speaker number of output neurons. Each weight in the and its wavelet channels. ANN is adjusted by adding an increment to reduce E as rapidly as possible. The adjustment is carried out over several training iterations until a satisfactorily small value of E is obtained or a given number of epochs is reached (Shafik et al, 2009) (see figure 4).

(a)

83 Smail H. et al./ ZJPAS: 2016, 28(3): 78-85

Wavelet Text - Text - Basis Dependent Independent Haar 70% 65.00% Db2 82% 76.00% Db3 84% 77% Db4 100% 80% Db5 85% 87%

(b) Figure 5: (a) Speech samples of speaker number 1, (b) A frame from the word “Choneet” and its wavelet channels

The performance of the speaker identification system may be affected by the type of the wavelet basis function. Haar and

(Db2- Db5) type of wavelet function for Figure 6: The correct recognition rates for text- feature extraction were used in this study. A dependent and text-independent using different feature vector of size 9 is obtained in the eight wavelet basis The reliability of the pattern recognition levels of the decomposition for each speech system is measured by testing the system with sample. The feature vectors of five words are hundreds of input speech samples of the speakers with varying quantities of noise. collected to form a training set. This training Noisy signals at different SNRs are used for set is given as an input to ANN classifier for recognizing the speakers. Results are shown in table 3, the graph is shown in figure 7. the training purpose. The system is then tested Table 3: Details of the system performance at by the same five words from the recognition set different levels of noise for the case of text-dependent and the remaining utterances are used for the case of Training Testing Text- Text- Dependent Independent text-independent. Results are shown in table 2, Original Noisy 27% 30% signal set signals at the graph is shown in figure 6. SNR=10dB Original Noisy 37% 43% signal set signals at Table 2: Recognition rates for text- SNR=15dB Original Noisy 53% 58% dependent and text-independent signal set signals at SNR=20dB identification. Original Noisy 84% 78% signal set signals at SNR=25dB Original Noisy 94% 85% signal set signals at SNR=30dB 84 Smail H. et al./ ZJPAS: 2016, 28(3): 78-85

Genetic algorithms, Fuzzy logic approaches etc. can also be used and a comparative study of these classifiers can be performed as an extension of this work.

References

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The official scientific journal of Salahaddin University-Erbil ZJPAS (2016), 28 (3); 86-96

Speaker Recognition Using Discrete Wavelet Transform and Artificial Neural Networks Azeen K. Mohammad 1* and Gawhar Ibraheem Khidhir2* 1 College of Engineering University of Salahaddin -Erbil 21 College of Engineering University of Salahaddin -Erbil

A R T I C L E I N F O A B S T R A C T

Article History: Pickup and delivery problems constitute an important class of vehicle routing problems Received: 18/06/2015 in which objects have to be collected and distributed. This paper discusses a heuristic approach called Repetitive Nearest Neighbour Algorithm to solve Travelling Salesman Accepted: 12/10/2015 Problem, to determine the tour the vehicle should follow so that the total distance Published:16/5/2016 travelled is minimized. The algorithm was tested in planning the best route which Keywords: connects a set number of shops; the shops were distributed within an area of 20km by 20km, also the algorithm used to locate the best seed when computing the optimum tour. Travelling Salesman Problem, The overall minimum cost computed for cases of different shop numbers, between 10 Pickup and Delivery, Vehicle and 500 shops. The coordinates for each shop on an X,Y map was generated and Routing, The nearest neighbor recorded, then a distance matrix was calculated which shows the cost of traveling algorithm between all shops in km. The computation time for the algorithm was recorded for each case. The aim of this paper is to apply a Traveling Salesman Problem (TSP) algorithm to *Corresponding Author: solve real world problems by helping traffic managers of freight transport companies to Azeen K. Mohammad reduce operational costs via shorter routes and optimized distribution maps.. In order to Email:[email protected] do so effectively, TSP implementation is to be optimized to reduce computation time. In this paper, a nearest neighbour greedy algorithm based approach for solving the TSP effectively is presented

1. Introduction should he visit them to minimize the distance travelled? TSP is applied in many different In several physical distribution problems, places such as warehousing, material handling goods must be picked at an origin and and facility planning (Byung-In Kim et al). delivered to a destination. Examples include the pickup and delivery of fast courier, of some Homaifar states that “one approach which medical supplies, the pickup and delivery of would certainly find the optimal solution of soft drinks, etc. This problem differs from any TSP is the application of exhaustive classical transportation problems because it enumeration and evaluation.” The procedure deals with precedence constraints between the consists of generating all possible tours and customers to be visited (Renaud et al). evaluating their corresponding tour length. The tour with the smallest length is selected as the The Travelling Salesman Problem (TSP) is a best, which is guaranteed to be optimal as it deceptively simple combinatorial problem. It will require the smallest amount of traveling can be stated very simply: A salesman spends time assuming similar conditions. However, by his time visiting N cities (or nodes) cyclically. using this approach, if an algorithm could In one tour he visits each city just once, and identify and evaluate one tour per nanosecond finishes up where he started. In what order (or one billion tours per second), it would 87 Azeen K. et al./ ZJPAS: 2016, 28(3): 86-96 require almost ten million years to evaluate all good solution. Typically, with optimization of the tours in a 25-city, number of possible problems, recursions are avoided by employing tours = 3.2×1023. a bound to the optimal value: if a good solution was already identiﬁed and the current bound is An algorithm that will give a solution in a worse, there is no point in further recursion shorter amount of time is a better approach. (Leo Liberti). This method for global The traveling salesman problem is hard to optimization is non-convex problems but it’s solve problem, so there is no known algorithm often slow; exponential worst case that will solve it in polynomial time. Solution performance, but (with luck) can (sometimes) optimality needs to be sacrificed in order to get work well. The basic idea is rely on two a good answer in a shorter time. (Bryant and subroutines that compute a lower and an upper Benjamin) Many algorithms have been tried for bound on the optimal value over a given region the traveling salesman problem. Some of these upper bound can be found by choosing any algorithms are described next. point in the region, or by a local optimization method and lower bound can be found from The branch and bound algorithm converts the convex relaxation, duality, Lipchitz or other asymmetric traveling salesman problem into an bounds, etc. (Prof. S. Boyd). assignment problem. Consider a number n of cities to be visited each once. The cost to find a Greedy Algorithms are a method of finding a solution to the assignment problem for n cities feasible solution to the traveling salesman is quite large and has an asymptotic complexity problem. The algorithm creates a list of all of n3. If this is a compete tour, then the edges in the graph and then orders them from algorithm has found the solution to the smallest cost to largest cost. It then chooses the asymmetric traveling salesman problem too. If edges with smallest cost first, providing they not, then the problem is divided in several sub- do not create a cycle. The greedy algorithm problems. Each of these sub-problems excludes gives feasible solutions however they are not some arcs of a sub-tour, thus excluding the always good (Bryant and Benjamin). Greedy sub-tour itself. The way the algorithm chooses algorithm is the simplest improvement which arc to delete is called branching rules. It algorithm. It starts with the departure Node 1. is very important that there are no duplicate Then the algorithm calculates all the distances sub-problems generated so that the total to other n-1 nodes. Go to the next closest node. number of the sub-problems is minimized. Take the current node as the departing node, Carpaneto and Toth have proposed a rule that and select the next nearest node from the guarantees that the sub-problems are remaining n-2 nodes. The process continues independent. They consider the included arc set until all the nodes are visited once and only and select a minimum number of arcs that do once then back to Node 1. When the algorithm not belong to that set (The Traveling Salesman is terminated, the sequence is returned as the Problem). best tour; and its associated OFV is the final solution. The advantage of this algorithm is its The Branch-and-Bound algorithm explicitly simplicity to understand and implement. explores a large search space by recursively Sometime, it may lead to a very good solution branching on disjunctions of possible values when the problem size is small. Also, because assigned to various decisions. Some of the this algorithm does not entail any exchange of recursions can be avoided if it can establish nodes, it saves considerably much that no subsequent recursive call contains a computational time (Byung-In Kim et al). The 88 Azeen K. et al./ ZJPAS: 2016, 28(3): 86-96

Greedy heuristic gradually constructs a tour by visited city to the last city in the tour until all repeatedly selecting the shortest edge and the cities are visited. adding it to the tour as long as it doesn’t create a cycle with less than N edges, or increases the This is perhaps the simplest and most degree of any node to more than 2.The same straightforward TSP heuristic. The key to this edge not allowed to be added twice (Christian algorithm is to always visit the nearest city Nilsson). (Christian Nilsson). The nearest neighbor algorithm (Rosenkrantz, Stearns, and Philip M. The Nearest Neighbor (NN) algorithm is Lewis, 1977) follows a very simple greedy similar to the greedy algorithm in its simple procedure: The algorithm starts with a tour approach. A starting city, seed, is arbitrarily containing a randomly chosen node and then chosen and then the algorithm advances to the always adds to the last node in the tour the city closest to it that does not create cycle. The nearest not yet visited node. The algorithm algorithm continues this procedure until all stops when all nodes are on the tour. An cities are in the tour. This algorithm also does extension to this algorithm is to repeat it with not always give good solutions because often each node as the starting point and then return the last edge added to the tour (that is, the edge the best tour found. This heuristic is called leading to the last city numbered n) can be Repetitive Nearest Neighbor. quite large (Bryant and Benjamin). The reason for that behavior is that one city’s, named n, For example assuming four nodes (n=1,2,3,4) distance to the other cities might be larger than which represent four shops. A salesman the distant between any other two cities; hence starting from shop (1), as a seed, wishes to visit city n is placed at the end of the tour from the each of rest n-1 shops once. The cost of beginning and the distance of the last travel traveling from on shop to another is taken as depends on the selection of city n-1 while city the distance between them. Distances or costs n is predetermined. The nearest neighbor between all shop pairs are presumed known. algorithm is easy to implement and executes The possible solutions for the tour are, as quickly, but it can sometimes miss shorter shown in Figure 1, listed in Table 1: routes which are easily noticed with human insight due to its greedy nature. It may not find a feasible tour at all, even when one exists. The nearest neighbor algorithm still has shortcoming, and the shortcoming is that it quickly yields a short tour, but usually not the optimal one (Oloruntoyin Sefiu Taiwo et al). The NN algorithm was first introduced by J. G. Skellam. The work on the NN was continued by P.J. Clark and F.C. Evans. The NN method compares the distribution of distances that occur from a data point to its nearest neighbor Figure 1: Possible routes connecting four in a given data set with a randomly distributed shops, setting shop 1 as seed data set. The first strategy that has been introduced and used for solving TSP problem was NN algorithm. NN starts with a randomly chosen city and it adds the nearest but not yet 89 Azeen K. et al./ ZJPAS: 2016, 28(3): 86-96

Table 1: Possible routes connecting four shops, 2-Related work setting shop 1 as seed The problem is to determine the tour the Solution No. Route vehicle should follow so that the total distance 1 (1234) travelled is minimized, each load at a pickup point is picked up, each delivery point receives 2 (1243) its shipment and the vehicle capacity is not violated (Anily and Bramel). Researches have    3 (1 3 2 4) been done to minimize the travel time and cost 4 (1342) in the long run, using the expected value of travel time and cost to define this evaluation. In 5 (1423) one paper submitted by (Wang,Guo,Zheng,&Yang,2015), several 6 (1432) consistent comparison methods are presented to define the relationship between uncertain In this study, each shop is selected as a seed variables, which use expected-value, α- once. Choosing another shop as a seed will optimistic value and α-pessimistic value of result in 6 additional possible solutions. For uncertain variables respectively, as the each seed, only one of the six solutions has the expected value of uncertain variable is widely minimum distance. This means that for four used in real-life problem (ZutongWanga et al). shops each taken as a seed once, 24 possible solutions arise with four seeds. The overall best Solution algorithms for the TSP are divided in solution is the rout with the minimum total the literatures into exact and heuristics. distance and the starting shop which that rout Heuristics can also be combined with exact belongs to would be the best seed. The solution methods yielding efficient hybrid difficulty for such approach is entirely schemes. Most modern algorithms able to computational, since a solution must exists. An tackle large instances of the TSP employ effective algorithm is one which computes all heuristics in some of the solution phases. For a possible routes taken all possible seeds and review of approaches to solve the TSP before returns the best rout and best seed in minimum 1992, the reader is referred to the computation time. comprehensive work of Laporte (1992). A more recent review with developments and an There are many other algorithms available used updated set of modern areas of applications is for solving TSP , But their efficiencies vary included in Bektas (2006) and Saharidis (2014) from case to case, from size to size: such as (Georgios K.D. Saharidisa et al). exact algorithms, heuristic algorithms, approximate algorithms and meta heuristics Currently the only known method guaranteed algorithms. The heuristic algorithms for TSP to optimally solve the travelling salesman can be classified as Construction Algorithms; problem of any size, is by enumerating each Improvement Algorithms; and Hybrid possible tour and searching for the tour with Algorithms. Other TSP algorithms are smallest cost. Each possible tour is a described in (G Kizilateş) (Kizilateş and permutation of 123 . . . n, where n is the Nuriyeva)(Xiao and Jiang-qing) (Kumbharana number of cities, so therefore the number of and Pandey). tours is n! When n gets large, it becomes impossible to find the cost of every tour in 90 Azeen K. et al./ ZJPAS: 2016, 28(3): 86-96 polynomial time. Such a method, which will is known as less-than-truckload. In Jian (2007), end up giving the optimal solution, is obviously Yaohua, and Jingbo tried to reduce the cost not very feasible because of the time while maintaining a good service level. This is consumption required to calculate all the tours. very similar to the problem presented here, but It can be seen that even for small instances, the without time windows. Also, between the last time consumption is extremely high if we want pickup and the first delivery there is a break- to find every possible tour. Instead use an bull-terminal where the load is organized, and a approximation algorithm, which in less time genetic algorithm is applied (Bryant and will end up giving a result that isn’t necessarily Benjamin). the shortest tour, but instead a tour that is close to the best tour (Kumbharana and Pandey). 3. Computational Algorithm This paper discusses a new implementation of The main objective of the system a heuristic to solve the TSP in MATLAB. The described in this paper consists of helping VRP (Vehicle Routing Problem) is considered traffic managers of freight transport a one-to-many problem. One of the most companies to reduce operational costs by extensively studied variants is the VRP with optimizing the loading of freight in vehicles, capacity and time window constraints optimizing routes and grouping orders (CVRPTW), in which the routes start and following certain load/unload procedures in finish at a warehouse), in which vehicles have such a way that pick-up and delivery a limited capacity and customers must be activities can be previously planned. Certain served in a specific time interval (Toth & Vigo, restrictions were taken into account which 2002). Other variant is Open VRP (OVRP) that are: studies the non-return of vehicles to the warehouse once the working day has finished (MirHassani & Abolghasemi, 2011).  The Vehicle Routing Problem is an A peculiarity of the problem is that the distance Open VRP. between the origin of the orders and the  The shops are distributed within a destination is very long; this is known as the town with an area of 20km by 20km. “long haul transportation” problem. The references to this problem found in the  Shops are connecting by straight literature are scarce. For Caramia and lines as roads. Guerriero (2009) the routes can be multimodal, the goal is to find the set of the shortest and The nearest neighbor principle for cheapest routes, and the solution must satisfy solving the traveling salesman problem was constraints such as capacity, time windows and used for finding the minimum distance of a so on. In (2003) Çalikan and Hall develop an route connecting a specified number of efficient model which reduces distance, shops, locations. The shops were distributed balances freight and tries to ensure that routes within a town with an area of 20km by 20km finish near the drivers’ homes (Juan-Antonio map, an average town area. Each shop Siciliaa et al). A further characteristic is the location is stored as X and Y coordinates on case in which the freight from different the map. As the main factor for deciding the customers must be consolidated in the same order of the shops on the route is the vehicle, and the goods from each individual distance, the map is assumed to be a road- customer cannot therefore fill the vehicle. This 91 Azeen K. et al./ ZJPAS: 2016, 28(3): 86-96

free map in which any two locations can be connected via straight path; this will simplify A random algorithm was used to generate the variables of the problem in order to focus randomly distributed x and y coordinates for n on the behavior of the algorithm for different number of shops. distributions. The following parameters are introduced for the algorithm.

After the distance matrix is developed, the location of each element in the matrix is fixed and all possible routes are evaluated by the algorithm. The Pseudo code for finding the

route with the minimum traveled distance is as follows:

1. Initialize S=1 as a seed counter. 2. Initialize counter for route cost measurement of the seed s. 3. Take i=S 4. Select the shop number i with the

coordinates as the starting point of the rout (seed). 5. In the D matrix, locate the minimum The coordinate' parameters are used to value in the raw, which belongs specify, for n shops, all the possible distances to, this represent the next shop to travel as follows: to, with the minimum traveled distance. 6. Add the cost to the counter .

7. In the D matrix, locate the j column and the j row for the next shop.

8. Remove the raw and column which belongs to. 9. Store shop number j as the next point to visit. 10. Locate the element in the D matrix, bring the raw and column which belongs to the start of the matrix making element the first element. 11. Store j as the new i 12. Repeat steps 5-8 until only one element remains in the D matrix, last shop to visit.

13. Take S=S+1. It should be noted that according to 14. Repeat Steps 2-13 until S=n (i.e. until all shops where taken as seed for a Equation (1), tour).

92 Azeen K. et al./ ZJPAS: 2016, 28(3): 86-96

 J, the colors This algorithm will provide an array of matrix and the best route, A possible minimum cost routes: are in correspondence to the order of the shops in the route, as in Figure (2b). The overall minimum traveled distance from A to J is the sum of distances between consecutive shops.

Each value represents the minimum cost of a route taking shop i as a seed.

Figure (3) plots the minimum cost solution for each shop when taken as the The overall minimum traveled distance seed. The overall minimum solution is route is the element with the minimum indicated in green, where shop number 2 is value. The corresponding best seed is the shop the seed. number i. Table (2): Shops coordinates shop Number X- Y-Coordinate Coordinate (m) The Algorithm code was developed and 1 13115 14121 rapidly tested using MATLAB Software. 2 714 637 3 16983 5538 4 18680 923 5 13575 1943 4. Results and discussion 6 15155 16469 7 14863 13897 The algorithm was tested for its ability to find 8 7845 6342 the best solution for n number of shops. The 9 13110 19004 area of the map was maintained as 20Km by 10 3424 689 20 Km for different number of shops. The algorithm was capable of locating the best seed and the minimum solution for each case study.

Table (2) lists a random generated coordinates for 10 shops to evaluate the algorithm, the same principle applies to any Figure 2a: 10 Shops distribution number of shops in the real world, having Figure 2b: Best route there coordinates being known. Distance Table (3): Distance matrix (in m) and best tour matrix was generated and the best route could be evaluated with the best seed. Figure Sh 1 2 3 4 5 6 7 8 9 1

(2a) shows the distribution of shops on the 1 0 183 941 143 121 311 176 939 488 165 2 183 0 169 179 129 214 193 913 221 271 map and Figure (2b) shows the best route 3 941 169 0 491 495 110 862 917 140 144 that connects all shops with the minimum 4 143 179 491 0 520 159 135 121 189 152 distance. Table (3) includes the distance

93 Azeen K. et al./ ZJPAS: 2016, 28(3): 86-96

5 121 129 495 520 0 146 120 722 170 102 behavior as the number of shops to visit 6 311 214 110 159 146 0 258 124 325 196 increases. This continuous increasing is 7 176 193 862 135 120 258 0 103 540 174 shown in Figure (6). 8 939 913 917 121 722 124 103 0 137 717 9 488 221 140 189 170 325 540 137 0 207 1 165 271 144 152 102 196 174 717 207 0 Be H A E F D I G C J B st

Figure 4a: 140 Shops distribution Figure 2b: Best route

Table 4: Results for optimizing the route between 140 shops Best Seed 8 Overall 1980 Computationii C t 165.88 8 → 79 → 35→ 97→ 16→ 88→ 95→ 663→ Figure 3: Minimum cost solutions 96→ 69→ 79→669→ 651→ 89→ 99 for each shop as starting point, seed → 63→ 668→ 6→ 695→ 39→ 91→ 655→ The same method could be 98→ 689→ 99→ 59→ 51→ 699→ 659 applied to find the best solution for any number of shops n within a map. Figure → 86→ 98→ 93→ 693→ 98→ 19→ 58→ (4a) and (4b) where obtained by running 89→ 87→ 9→ 669→ 59→ 688→ 76 the algorithm for 140 shops within the Ro → 667→ 89→ 5→ 686→ 666→ 8→ 31→ map. The obtained results for this case ute 669→ 65→ 99→ 685→15→ 17→ 93 are shown in Table (4). →683→ 99→ 85→ 698→665→87→ 19→ To test the time efficiency of the algorithm 97→ 78→ 7→ 81→ 688→ 11→ 657 for larger set of shops, the search for best solution was carried out repeatedly starting → 689→ 38→ 689→ 75→ 79→89→ 18→ from 10 shops until 500 shops, increasing 81→ 71→ 1→ 656→ 661→ 89→ 699 the number of shops by 10 for each new run. The best overall solution and the → 91→ 659→ 88→ 83→ 9→699→ 13→ 59→ computation time were returned each time 57→ 88→ 36→ 66→ 698→ 98→ 19 in MATLAB allowing for a plot for the → 56→39→ 699→ 99→ 68→ 88→ 61→ computation time and the minimum overall 659→ 18→ 38→ 691→ 658→ 69→ 668 cost. The results suggest that increasing the number of shops will not reduce the → 68→ 697→ 658→ 77→85→ 681→96→ effectiveness of the algorithm to find the 58→ 3→ 55→ 73→ 67→ 86→ 696 optimum solution as shown in Figure (5).

The Computation time was found to

increase while following an exponential 94 Azeen K. et al./ ZJPAS: 2016, 28(3): 86-96

developed to be repeated for each shop. This means that the computation time will increase as the numbers of shop increase. However, the percentage of increasing was found to steadily reduce and stabilize as the number of shops increases. Therefore, the nearest neighborhood algorithm can be effectively used for larger number of shops.

Figure 5: Minimum route cost for best seed (MRC) Vs. Number of shops to visit (NOS)

Figure 7: % increasing of solution cost and computation time with increasing the number of shops Figure 6: Computation time for best solution (CT) Vs. Number of shops to visit (NOS) Conclusion

Figure (7) plots the percentage of increasing This research studied the utilization for both the solution cost and the computation of computational algorithms for optimizing time as the number of shops to visit increases the route planning for pickups and deliveries by comparing, using the standared equation of for linear paths. The study was based on percentage of change between two sequenced optimizing the Nearest Neighbor algorithm to data [(difference between sequenced solve a traveling salesman problem with values/previous value)*100] the returned considerable efficiency and educed results from MATLAB each time the number computational time. The proposed algorithm of shops increases. The Solution cost increases optimized the nearest neighbor method by are lower rate with the increasing of shops to selecting each shop as a seed once, which is visit. Some solutions for larger number of titled “Repetitive Nearest Neighbor shops had less cost than previous solutions Algorithm” RNNA. After running the RNNA with fewer shops. This is related to the location for different scenarios, where different and coordinate of each shop. Having the shops number of shops where distributed within an closer to each other will reduce the solution area of a size 20km by 20km map, the cost. This means that the solution cost is a following conclusions where reached: function of both the number of shops and the 1- The RNNA is an effective algorithm location of each shop. As for the computation for finding the overall optimum route time, the computation time depends on the to follow each time. The returned repetitions of the algorithm. The pseudo code is 95 Azeen K. et al./ ZJPAS: 2016, 28(3): 86-96

route is both the shortest and the one Oloruntoyin Kehinde Sade, with the best seed to start from. “IMPLEMENTATION OF HEURISTICS FOR SOLVING TRAVELLING 2- Theoretically, having the required SALESMAN PROBLEM USING computational recourses, the RNNA NEAREST NEIGHBOUR AND NEAREST can be effectively used to solve the INSERTION APPROACHES” International Journal of Advance Research Volume 1, TSP for finding the best route for any Issue 3, March 2013. number of shops. [3] Byung-In Kim, Jae-Ik Shim and Min 3- The distribution nature of the shops, Zhang, “Comparison of TSP Algorithms” nods to visit, within a map will not louisville.edu December affect the efficiency of the algorithm; 1998. neither will it return a closed route which does not include all the shops [4] Kylie Bryant and Arthur Benjamin, to visit. The algorithm will always Advisor, “Genetic Algorithms and the return the route which includes all the Traveling Salesman Problem” math.hmc.edu December 2000. shops in a map.

4- The solution cost for the algorithm [5] “The Traveling Salesman Problem” www.csd.uoc.gr/~hy583/papers/ch11.pdf increases as the number of shops

increases; however, the increasing [6] Leo Liberti, “Branch-and-Bound for the percentage will decay as the number Travelling Salesman Problem” LIX, Ecole of shops to run the RNN algorithm Polytechnique, F-91128 for increases. This is due to the fact Palaiseau, France that when the number of shops Email:[email protected] March 15, increases, the number of connections 2011

increases allowing for a better [7] Prof. S. Boyd, “Brunch and bound minimum distance selection. method” stanford.edu/class/ee364b /lectures/bb_slides.pdf 5- In terms of computational time, the RNNA was effective in returning the overall best route [8] Christian Nilsson, with the best seed for a moderate number of “Heuristics for the Traveling shops. The computational time will increase as Salesman Problem” the number of shops increases; however, the https://web.tuke.sk/ fei- percentage of increasing will decay as the cit/butka/hop/htsp.pdf number of shops to run the RNN algorithm for [9] Zoha Ahmadi Danesh, “Modelos y increases. métodos de la distribución de mercancías” bibing.us.es/proyectos/abreproy/. +E.T.S. References Ingenieros Universidad de Sevilla

[1] Jacques Renaud, Fayez F. Boctor and [10] G Kizilateş, “The nearest neighbour Jamal Ouenniche, “A heuristic for the algorithms for TSP” pickup and delivery traveling salesman link.springer.com/chapter /10.1007%2F978-3- Problem” Computers & Operations 319-00951-3_11 Research 27 (2000) 905-916.

[11] Gözde Kizilateş and Fidan Nuriyeva, [2] Oloruntoyin Sefiu Taiwo, Ojo Josiah, “A Comparative Study between the Nearest Amao Taiwo, Salawudeen Dkhrullahi and 96 Azeen K. et al./ ZJPAS: 2016, 28(3): 86-96

Neighbour and Genetic Algorithms” Advances in Computational Science, Engineering and Information Technology Volume 225, 2013, pp 111-118.

[12] Zhang Xiao and Wang Jiang-qing, “Hybrid Ant Algorithm and Applications for Vehicle Routing Problem” International Conference on Solid State Devices and Materials Science, April 1-2, 2012, Macao Volume 25, 2012, Pages 1892–1899.

[13] Sharad N. Kumbharana and Prof. Gopal M. Pandey, “Solving Travelling Salesman Problem using Firefly Algorithm” International Journal for Research in Science & Advanced Technologies Issue-2, Volume-2, 053- 057.

[14] Shoshana Anily and Julien Bramel, “Approximation Algorithms for the Capacitated Traveling Salesman Problem with Pickups and Deliveries” Wiley & Sons, Inc.

[15] ZutongWanga, JianshengGuoa, MingfaZhengb and YingWanga, “Uncertain multi objective traveling salesman problem” European Journal of Operational Research 241 (2015) 478–489.

[16] Georgios K.D. Saharidisa, George Kolomvosb and George Liberopoulosa, “Modelling and solution approach for the environmental travelling salesman problem” Transport Research Arena, Paris 2014.

[17] Juan-Antonio Siciliaa, Beatriz Royoa, Emilio Larrodéa, and Alberto Frailea, “A decision support system for a long-distance routing problem based on the ant colony optimization metaheuristic” 111 ( 2014 ) 1035 – 1044.

ZANCO Journal of Pure and Applied Sciences

The official scientific journal of Salahaddin University-Erbil ZJPAS (2016), 28 (3); 97-103

Kinetic studies of bioethanol production from wheat straw

Sulaiman G.Muhamad1* , Lawen S. Esmail2* and Shelear H. Hasan3* 1Chemistry Dept, College of Science, University of Salahaddin -Erbil 2 Food Technology Dept, College of Agriculture, University of Salahaddin- Erbil

A R T I C L E I N F O A B S T R A C T

Article History: Different concentration of sulfuric acid at varied temperature for different Received: 18/06/2015 hydrolysis time and enzymatic saccharification were evaluated for conversion Accepted: 12/10/2015 of wheat straw cellulose and hemicellulose to monomeric sugars. The maximum yield of sugars (Glucose : 32.95, Xylose: 28.19 , Arabinose: 5.21 Published:16/5/2016 gm/100gm total Sugar 265.404 gm/l ) from wheat straw is achieved by Keywords: hydrolysis process with 15% H2SO4 at 90oC for 60 minutes. The results show Bioethanol, wheat straw, dilute the highest yield of ethanol production (91.363gm/l) obtained after 60 h of acid hydrolysis and fermentation at 35oC. The fermentation efficiency (FE%) at this condition is fermentation. equal to 68.37%.The kinetics of fermentation process was studied at *Corresponding Author: temperature ranging between 25 – 45oC in a stirred conical flask which served as a batch reactor. The graphical method was employed to predict the order of Sulaiman G. Muhamad the fermentation reaction. The results obtained suggested a first order reaction Email:[email protected] and reaction rate constant (k) equal to 0.0818 h-1.The activation energy estimated from Arrhenius equation is 12.704 kJ/mol.

1. Introduction straw and sugarcane bagasse are becoming attractive feedstocks for bioethanol production Bioethanol is a form of quasi-renewable energy since their utilisation is not competitive with that can be produced from agricultural food (Lei Wanga, et al. 2013). feedstock. It can be either used as fuels themselves or blended with petrol based diesel Bioethanol is mainly produced by the sugar and gasoline to enhance the oxygen content of fermentation process, although it can also be such fuels. Bioethanol fuel is completely produced by the chemical process of reacting composed of biological products. The ethylene with steam. Biomass wastes contain a combustion of bio ethanol results in a clean complex mixture of carbohydrate polymers emission: Heat, Steam and Carbon Dioxide from the plant cell walls known as cellulose, (Luis F et al. ,2009).Bioethanol is liquid fuels hemi cellulose and lignin (Bustamante M. et made from plant materials. They can be used in al., 2008 and Camarero S et al., 1996). There transport fuel instead of conventional fossil are tow principle methods of extracting sugars fuels.Other reasons which lead to research and from biomass. These include acid hydrolysis development its in bioethanol production of the (with dilute and concentrated acids) and second generation are: a shortage of world oil enzymatic hydrolysis (Canam, T et al., 2011 reserves, increasing fuel prices and reduction and Dias, A et al., 2010). Hydrolysis process is of the greenhouse effect (Heike Kahr et al., an important step because it determines the 2012). Bioethanol is made by fermenting the amount of glucose produced as a substrate to sugar and starch components of plant by- be fermented into ethanol (Greta Radeva et al., products - mainly sugarcane and crops like 2011). Sulfuric acid is the most used acid grain, using yeast. It is also made from among other acids that can be used such as agricultural wastes such as corn stover, wheat 98 Sulaiman G.. et al./ ZJPAS: 2016, 28(3): 97-103 hydrochloric acid , nitric acid or phosphoric hydrolysis of cellulose that is naturally acid(Tucker, M. et al.,2003). occurring wheat straw as agricultural wastes in Kurdistan and to evaluate their kinetics. The sugar formed in the hydrolysis is fermented into ethanol. The most common Materials and Methods microorganism for this purpose is Saccharomyces cerevisiae, which is the same Biomass that is used in ordinary baking yeast. The yeast contains an enzyme called invertase, which Wheat straw was taken from a local farmer acts as a catalyst and helps to convert the in Erbil city. It was dried in oven at 55oC for sucrose sugars into glucose and fructose 24 h and smashed to the sizes of 80 meshes by (Galbe, M et al., 2002 ).Fermentation is a bio- Caulatti type MFC,Germany. chemical conversion process in which the biomass is decomposed using micro- Preparation of yeast culture organisms (bacteria or enzymes). These microorganisms can typically use the 6-carbon Two hundred grams of sugar and flour were sugars, one of the most common being glucose. respectively added to 250ml of water. The Therefore, cellulosic biomass materials solution was heated to 100oC for 45minutes, containing high levels of glucose or precursors and then cooled to room temperature. 20g of to glucose are the easiest to convert to dried yeast (Saccharomyces cerevisiae) was bioethanol. Microorganisms, termed added to the mixture. The yeast culture was ethanologens, presently convert an inadequate maintained in the refrigerator at 20oC until portion of the sugars from biomass to required. bioethanol. There are a number of microorganisms that produce significant Dilute acid hydrolysis of wheat straw (greater than 1% w/v) quantities of bioethanol (Stewart G et al., 1987). In this study the Acid hydrolysis of raw materials was carried bioethanol production from wheat straw was out according to the method described by investigated and improved straw using dilute (Gupta et al., 2009) as follows: To 10 gm of H2SO4 and enzymatic saccharification. wheat wheat straw was added 50 ml of sulfuric acid straw was used because it is the most widely solution (The acid concentrations tested were grown cereal in Kurdistan. Only a small from, 5%, 10% , 15% and 20% (v/v) to portion of wheat straw is used for animal feed, determine the optimum concentration on most of the wheat straw is still left on the fields hydrolysis before fermentation.) in a 250 ml or disposed of as waste.Wheat straw contains conical flask which served as a batch reactor 35–45% cellulose, 20–30% hemicellulose and and placed in a water bath shaker set at a 8– 15% lignin and can also serve as a low cost temperature of 80, 90,100OC with an agitation attractive feedstock for production of fuel speed of 150 rpm. This operation was carried alcohol(Badal C et al 2005). out for 2 h, and at intervals of 20 min, samples were withdrawn to determine the Glucose , Researches have been done on the separation Xylose, Arabinose and total sugar of cellulose, hemicellulose and lignin concentration. The hydrolyzed wheat straw components from wheat straw and structural solution was adjusted to pH 7 with 10 M characterization of the hemicellulose fraction NaOH before enzymatic saccharification. (Ould JM, 1984 and Schmidt AS et al., 1998). Also, a few reports are available on the Enzymatic saccharification production of ethanol from wheat straw hydrolyzates (Ahring BK et al.,1996 and - The enzymatic saccharification(using Cellulase Delgenes JP et al .,1990).The objective of this and b-glucosidase) of the diluted acid study is to examine the effects of sulfuric acid pretreated wheat straw was performed by concentration and temperature on the shaking slowly (100 rpm) at 45oC after 99 Sulaiman G.. et al./ ZJPAS: 2016, 28(3): 97-103 adjusting the pH to 5.0 with NaOH and adding Total reducing sugars enzymes at each enzyme dose of 2 ml/100 g dry solid of wheat straw, unless otherwise Reducing sugars were determined by the DNS stated for 72 hours . For pectinase solids, the (dinitrosalicylic acid) method (Miller, G 1959). This enzyme dose was 1 g/100 g dry solid. Samples method is based upon the color which forms when (1 ml) were withdrawn and kept at -30C before sugars reduce 3,5-dinitrosalicylic acid (DNSA) to3- processing for analysis (Novozyme 188) . amino-5- nitrosalicylic acid.

Ethanol Fermentation Estimation of ethanol

About 100 mL of the hydrolysis solution Ethanol produced in the fermentation medium and some nutrients, namely, 3 g/L yeast was estimated by potassium dichromate oxidation extract, 5 g/L peptone, 25 g/L KH2PO4, 0.3 method (Firoz Md et al., 2012). Potassium g/L MgCl2, and 0.25 g/L CaCl2, were added dichromate (33.882 g/l), ferrous ammonium into 250 mL conceal flasks.. The resulting sulphate (135.5 g/l) and diphenylamine (0.5 g/100 slurry was inoculated with 1mL of activated ml concentrated H SO ) solutions were used as yeast. The experiments were performed in 2 4 duplicates in a constant-temperature water bath reagent for estimation of ethanol concentration. at 25o, 30o ,35o and 40o C for 12,24,36,48 The fermented sample was diluted ten times with ,60and72 h distilled water. Ten ml of the diluted sample was

titrated against K2Cr2O7 (10 ml) containing

Analytical procedures concentrated H2SO4 (5-6 ml). Then the diluted formation sample was titrated against freshly Analysis of monomeric sugar prepared ferrous ammonium sulphate solution with diphenylamine as an indicator. Appearance of Concentration of sugar determined in Quality control director- Ministry of trade and industry green colour indicated the end point of the .High-performance liquid chromatography titration. Burette reading (amount ferrous (HPLC) was used to determine the ammonium sulphate) was recorded to calculate concentrations of monomeric sugar (Xylose, the amount of ethanol present in the sample. Glucose, and Arabinose,) using Aminex HPX- 87P column (Bio-Rad, Richmond, USA) with a Result and discussion: refractive index detector. The analysis condition was at 85 ° C using Milli-Q water as Hydrolysis of Wheat straw the eluent with a flow rate of 0.6 ml/min. Effect of acid dose Peaks area of samples were indentified and quantified by comparison with retention times Generally, glucan is the major component of of analytical standards Glucose, Xylose, and Wheat straw followed by Xylan and acid-insoluble Arabinose ).The standard HPLC chromatogram lignin. Arabinan, galactan and mannan accounted of the sugar used in this work was shown in fig 1. for only a small amount of the biomass composition (Pothira et al., 2014 ).The effects of

acid dose (5% ,10%,15% and 20% H2SO4 , v/v) on hydrolysis of Wheat straw at 90o C for 60 mints have been shown in Table 1 .

Fig.1 HPLC chromatogram of standard sugars used in this work 100 Sulaiman G.. et al./ ZJPAS: 2016, 28(3): 97-103

Arabi Effect of temperature: nose Xylose The result of the sugars yield obtained from the H2SO plus (gm/ hydrolysis process with 15% H2SO4 at different 4% Glucose galactose 100g Total temperatures (70oC, 80oC, 90oC and 100oC) for 60 (v/v (gm/100gm (gm/100g m sugar(g minutes are presented in Figure 3.Experemntal %) DS) m DS) DS) m/l) results in this figure show that increasing temperature of hydrolysis process lead to 5 27.27 28.16 5.9 233.32 increasing the Glucose production. The highest 10 30.42 28.41 4.35 260.72 yield of Glucose (32.958 gm/100gm DS) was obtained from the hydrolysis at 90oC. This was 15 32.95 28.19 5.21 265.41 followed by a yield of 32.65 gm/100 gmDS at 100oC. Therefore, we select 90oC as the optimal 20 32.65 27.93 4.83 261.64 temperature for hydrolysis of wheat straws in this Table1: amount of sugar yield after hydrolysis of wheat work. o straw using different amount of H2SO4 at 90 C for 60 mints. 35 Glucose 30 Xylose plus

) galactose The result indicates that Glucose was the main 25 Arabinose component in the hydrolysis liquor. The maximum 20 100 gm DS / 15 gm yield of Glucose was 32.95 g /100g DS in the case (

S 10 of 15% acid and the maximum yield of Xylose was 5

28.41g /100g DS in the case of 10% acid for the 0 70 80 90 100 same condition. The experimental data indicated Tempreture that Glucose and total sugar yields increase with Fig. 3 Effect of temperature on sugars yield from increasing of acid amount . Xylose and Arabinose hydrolysis process of wheat straw by using 15% H2SO4 yields in the hydrolyzates gave similar results with for 60 mints. increasing of acid amount as shown in fig 2 . No detectable quantities of Furfural, Fructose and Effect of hydrolysis time: Sucrose were present in these hydrolyzates .The The effect of time on the hydrolysis process have data in this study indicates that 15% (v/v) H2SO4 been cared out for 120 mints at 90oC using gave good yields of sugars after hydrolysis and 15%H SO . The concentration of sugars were efficiency of the process was not increased 2 4 measured every 20 mints. The results showed that significantly by increasing acid amount rather than as the hydrolysis process time increased, lead to this amount. increasers the concentration of sugars up to 60

Glucose 35 mints as shown in fig. 4 . After 60 min the Xylose plus 30 galactose Arabinose concentration of sugars was not increased ) 25 significantly by increasing the time of hydrolysis 20 gm DS 100 / 15 gm (

S 10

0 5 10 15 20 % Aci de

Fig.2. Effect of H2SO4 % on sugars yield from the hydrolysis process of wheat straw at 90oC for 60 mints.

101 Sulaiman G.. et al./ ZJPAS: 2016, 28(3): 97-103

35 Fermentation Efficiency was calculated from the 30 ratio between the practical produced ethanol and 25 ) Glucose the theoretical ethanol produced (total produced 20 Xylose plus sugars × 0.511) (Wu X et al.,2006). gm DS 15 galactose 100 / Arabinose 10 gm

( The kinetics of fermentation process was studied S 5 by Monitoring total sugar consumption during 0 0 20 40 60 80 100 120 fermentation process. The graphical method Time (min) (AJANI et al.,2011)

Fig. 4 Time courses of sugars production from hydrolysis process of o was employed to predict the order of the wheat straw by using 15% H2SO4 at 90 C. fermentation. The plot of ln [S]/[So]versus The hydrolysis results in this study indicates fermentation time, (where, [S] is the that 15% (v/v) H2SO4 gave good yields of sugars concentration of total Sugar at time t, [So]is the (32.958 gm of Glucose , 28.19 gm of Xylose and concentration of total sugar before fermentation ), 5.215 gm of Arabinose per 100gm of DS) at 90oC give a straight line behavior ,which indications of for 60minutes. The results of hydrolysis process in the validity of first-order reaction kinetics for sugar this work is in conformity with the works of (Lirong fermentation as shown in fig 6. et al.,2012 and AJANI et al.,2011) Time(h) 0 0 10 20 30 40 50 60 70 80 Fermentation process: -0.2 -0.4 y = -0.0181x R2 = 0.9204 0 -0.6 S In this work the fermentation of waste straw /

ln S -0.8 have been don in deferent time period and -1 different temperature. Fig 5 shows the -1.2 concentration of ethanol production and -1.4 remaining sugar in fermentation process of hydrolyzed solution at different time. Fig.6 Plotting of ln [S]/[S0] versus time of fermentation process at 35oC. 300 S(gm/l) E(gm/l) 250 The reaction rate constant (k) was determined 200 from the slope of the straight line and equal to 150 0.0818 h-.Table 2 shows the results of temperature Conc. (gm/l)Conc. 100

50 effect on ethanol yield and FE% in fermentation

0 process. 0 10 20 30 40 50 60 70 80 Time(h) T oC E(gm/l) FE% Fig5. Concentration of ethanol and sugar during fermentation process at 35oC. 25 49.368 36.4

The results show the highest yield of ethanol 30 67.716 49.93 production (91.363gm/l) obtained after 60 h of 35 91.364 68.37 fermentation at 35oC . The fermentation efficiency (FE%) at this point is equal to 68.37. Theoretically, 40 58.443 43.09 100 grams of glucose will produce 51.4 g of bioethanol and 48.8 g of carbon dioxide 45 47.924 35.33 (AYHAN,2005). However, in practice, the Table 2 :the results of temperature effect on ethanol microorganisms use some of the Glucose for growth and the actual yield is less than 100%. yield and FE% in fermentation process 102 Sulaiman G.. et al./ ZJPAS: 2016, 28(3): 97-103

As shown in table 2. The optimal quantity of 3- The highest yield of Glucose was obtained from ethanol (FE% =68.37) is 91.364g/l which is the hydrolysis at 90oC. obtained at a temperature value of 35oC and reaction time of 60h . The temperature 4- The highest yield of ethanol production o dependence of the rate constant of the obtained after 60 h of fermentation at 35 C. fermentation process is followed in correspondence with the Arrhenius equation (Orozco et al.2007). References:

1-Ahring BK, Jensen K, Nielsen P, Bijerre AB, Schmidt AS. Pretreat- ment of wheat straw and conversion of ln K= (- Ea ⁄ RT) + ln A xylose and xylan to ethanol by thermophilic anaerobic bacteria. Bioresour Technol ,58(1996) p 107–13

1/T -3.5 2-AJANI, A O; AGARRY, S. E; AGBEDE, O. O, A 0.0031 0.00315 0.0032 0.00325 0.0033 0.00335 0.0034 -3.6 Comparative Kinetic Study of Acidic Hydrolysis of -3.7 y = -1562.7x + 1.0615 -3.8 Wastes Cellulose from Agricultural Derived Biomass, J. R2 = 0.9194 -3.9 ln K Appl. Sci. Environ. Manage. 15 ( 2011) , p 531 – 537. -4

-4.1

-4.2 3-AYHAN DEMiRBA,Bioethanol from Cellulosic -4.3 Materials: A Renewable Motor Fuel from Biomass, Energy Sources, 21( 2005) p 23

Fig.7.Application of Arrhenius equation on 4-Badal C. Saha, Loren B. Iten, Michael A. Cotta, Y. fermentation process Victor Wu, Dilute acid pretreatment, enzymatic saccharification and fermentation of wheat straw to As shown in fig.7 Arrhenius equation is used to ethanol ,Process Biochemistry 40 (2005) p 3693– determine the activation energy (E) and pre- 3700. exponential factor (А) of ethanol formation. The value of the estimated activation energy is 12.704 5-Bustamante, M., Moral, R., Paredes, C, Perez- kJ/mol, while the value of pre-exponential factor is Espinosa, A., Moreno-Caselles, J., Perez- Murcia, M.,Agrochemical characterisation of the solid by- 173.44 min -1. The results obtained indicate that products and residues from the winery and distillery the fermentation process occurs in an energetically industry. Waste Manage. 28 (2008),p 372–380. homogeneous system providing identical ,nezيnez, M.J., Martيaccessibility of the reagent to the hydrolyzing sites. 6-Camarero, S., Bockle, B., Mart This suggests that the process investigated can be A.T.,Manganese-mediated lignin degradation by considered a pseudo-homogeneous catalytic one Pleurotus pulmonarius. Appl. Environ. Microbiol. 62 described by a first order reaction equation.( Greta (1996) 1070– 1072. Radeva et al. ,2011 ) 7-Canam, T., Town, J.R., Tsang, A., McAllister, T.A., Dumonceaux, T.J., Biological pretreatment with a Conclusion: cellobiose dehydrogenase-deficient strain of From the results of this work we can conclude the Trametes versicolor enhances the biofuel potential of canola straw. Bioresour. Technol.102 (2011),p 10020– flowing: 10027. 1- The Glucose and total sugar yields increase with 8-Delgenes JP, Moletta R, Navarro JM. Acid hydrolysis of increasing of H2SO4 concentration in hydrolysis wheat straw and process considerations for ethanol process of wheat straw. fermentation by Pichia stipitis Y7124. Proc Biochem ,25(1990) p 132–5 2-The best time of Hydrolysis of Wheat straw by

H2SO4 is 60 mints. 103 Sulaiman G.. et al./ ZJPAS: 2016, 28(3): 97-103

9-Dias, A.A., Freitas, G.S., Marques, G.S.M., Sampaio, A., 19-Orozco, A.; Ahmad, M.; Rooney, D.; Walker, G. Fraga, I.S., Rodrigues, M.A.M., Evtuguin, D.V., Bezerra, Dilute,Acid Hydrolysis of Cellulose and Cellulosic Bio- R.M.F., Enzymatic saccharification of biologically pre- waste Using a Microwave Reactor System. Trans I treated wheat straw with white rot fungi. Bioresour. ChemE, Part B, Process Safety and Environmental Technol. 101(2010) p 6045–6050 Protection 85 (2007) p 446-449.

10-Firoz Md. Ahmed1, Sabita Rezwana Rahman and 20-Ould JM. Alkaline peroxide delignification of Donald James Gomes , Saccharification of Sugarcane agricultural residues to enhance enzymatic Bagasse by Enzymatic Treatment for bioethanol saccharification. Biotechnol Bioeng,24 (1984) p 46– Production, Malaysian Journal of Microbiology, 52. 8(2012), pp. 97-10 21-Pothiraj, Ramasubramanian Arumugam and 11-Galbe, M.; Zacchi, G. A review of the production of Muthukrishnan Gobinath, Sustaining ethanol ethanol from softwood Applied Biochemistry and production from lime pretreated water hyacinth Biotechnology, 59(2002) p 618-628 biomass using mono and co-cultures of isolated fungal strains with Pichia stipitis, Bioresources and 12-Greta Radeva, Ivo Valchev and Stoiko Petrin , inetic Bioprocessing (2014) 1:27 Dependences of Two-Stage Dilute Acid and Enzyme Hydrolysis of Paulownia tomentosa, J. Chem. Chem. 22-Schmidt AS, Thomsen AB. Optimization of wet Eng. 5 (2011) 985-989 oxidation pretreat-ment of wheat straw. Bioresour Technol, 64(1998) p 139–51. 13-Gupta R, Sharma KK, Kuhad RC ,Simultaneous saccharification and fermentation of Prosopis 23-Stewart GG, Russell I. Biochemistry and genetics of jutiflora, a woody substrate, for the production of carbohydrate utilization by industrial yeast strains. cellulosic ethanol by Saccharomyces cerevisiae and Pure Appl Chemm, 59(1987) p 1493–500. Pichia stipitis- NCIM 3498. Bioresour. Technol; 100 (2009) p 1214-1220 24-Tucker, M.P., Kim, K.H., Newman, M.M., Nguyen, Q.A.. Effects of temperature and moisture on dilute- 14-Heike Kahr, Alexander Jäger and Christof acid steam explosion pre-treatment of corn Stover an Lanzerstorfer, Bioethanol Production from Steam cellulase enzyme digestibility. Appl. Biochem. Explosion Pretreated Straw , Bioethanol, (2012) Biotechnol. 105(2003) p. 165-178. University of Applied Sciences Upper Austria,p 153 25-Wu X, Zhao R, Wang D, Bean S R, Seib P A, Tuinstra 15-Lei Wanga, , , Jade Littlewoodb, Richard J. Murphy. M R, .Effects of amylose, corn protein, and corn fiber Environmental sustainability of bioethanol production contents on production of ethanol from starch-rich from wheat straw in the UK, Renewable and media. Cereal Chem, 83 (2006) 5, p 569-575. Sustainable Energy Reviews , 28 ( 2013), p 715–725

16-Lirong Han, Juntao Feng, Shuangxi Zhang, Zhiqing Ma, Yonghong Wang, Xing Zhang, ALKALI PRETREATED OF WHEAT STRAW AND ITS ENZYMATIC

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The official scientific journal of Salahaddin University-Erbil ZJPAS (2016), 28 (3); 104-113

Soil and Municipal Solid Waste Leachate Characterization at Erbil Anaerobic Landfill Site

Yousif Ismael Maulood1* and Shuokr Qarani Aziz 2 1College of Engineerimg., University of Salahaddin–Erbil 2 College of Engineerimg., University of Salahaddin–Erbil

A R T I C L E I N F O A B S T R A C T

Article History: Soil and fresh leachate samples were collected from Erbil Landfill Site (ELS), Received: 18/06/2015 Iraq. ELS is anaerobic landfill opened in 2001. Field permeability test for soil Accepted: 12/10/2015 hydraulic conductivity was performed directly in the site. Meanwhile, other examinations for liquid limit, plastic limit, particle size distribution, etc., were Published:16/5/2016 conducted at the laboratory. Unprocessed leachate samples were analyzed for Keywords: pH, electrical conductivity, biochemical oxygen demand, as well as chloride, Sanitary landfill, landfill solids, sodium, potassium, and calcium contents, among others. The sorptivity, leachate, clay, anaerobic, steady state infiltration rate, and hydraulic conductivity of the soil samples stabilized were , respectively. Raw leachate *Corresponding Author: analysis identified the leachate as mature. The soil at ELS was found to be light Yousif Ismael Maulood brown clayey gravel with sand and light brown gravelly lean clay layers with Email: low permeability. In addition, the stabilized leachate from ELS requires [email protected] treatment prior to disposal to the natural environment. ELS is considered a level 2 anaerobic landfill.

1. Introduction 2014). The generation of extremely polluted Municipal solid waste (MSW) disposal leachate that can percolate into the ground systems include open dumping, sanitary and contaminate water sources and soil is landfill, composting, and incineration. principally a focal undesirable feature Sanitary landfill is the best public MSW associated with municipal sanitary landfill disposal technique because of such benefits disposal systems (Aziz et al., 2010; Bashir et as easy disposal method, low budget, and al., 2012; Pillai et al., 2014). landscape-restoring effect on holes from mineral workings. However, the production The nature of landfill leachate depends on of highly contaminated landfill leachate is a the kind of MSW being buried, site weather, chief shortcoming of this technique. Gas and landfill age, used soil for capping, moisture inert solids comprise other products of content, site hydrology, pH, stage of landfills (Aziz et al., 2010; Mojiri et al., decomposition in the landfill, and 2014). Landfill leachate is a liquid produced biodegradability ratio. In addition, leachate principally by the percolation of can be classified as young, intermediate, and precipitations (or other disposed mature. As landfill age increases, the water/wastewater) through an open landfill biodegradable fraction of organic pollutants or through the cap of a completed site. in leachate markedly decreases as an Leachates may contain large quantities of outcome of anaerobic decomposition that organic pollutants, nitrogen compounds occurs at the landfill site. Consequently, (e.g., ammonia), suspended solids, heavy mature leachate has considerably greater metals, inorganic salts, phenols, and amount of refractory organic materials than phosphorus (Renou et al., 2008; Aziz et al., young leachate (Aziz et al., 2012). 105 Yousif I. et al./ ZJPAS: 2016, 28(3): 104-113

The most common method of waste disposal of in valleys and covered daily by local in Erbil City is dumping on land. This material from ELS. At times, disposed technique of MSW treatment usually demolition materials and construction requires large area and proper drainage. wastes are used to cover the disposed MSW. Considering the lack of standards for testing At present, Erbil Province has 10 districts disposed and compressed MSW, laboratory and 34 sub-districts. As a consequence, tests on disposed MSW are performed by more than 40 open dumpsites and landfills applying standard soil tests. However, these exist in Erbil Province, Iraq. Most of the tests usually assume that traditional soil landfills only consist of dumping grounds mechanics theories of compressibility apply without any environmental protection. to disposed MSW (Dixon and Jones, 2005; Increment of population numbers, growth of Canizal et al., 2011). A study investigated investment and economy, industrialization, certain engineering characteristics such as rising number of hospitals, among others shear strength, compressibility, and effect of have caused environmental difficulties in compaction process of old and recent solid Erbil City, particularly regarding solid waste waste disposal sites in Erbil City (Hummadi, treatment. Furthermore, published works in 2012). In a study of the influence of landfill this area are scarce. The present study was leachate infiltration on the changes of conducted to fill the knowledge gap in the chemical parameters of the soil, the article existing literature. The goals of the present presented the results of geochemical work included; to examine the research of clean soil and contaminated soil characteristics of soil at ELS, to study by leachate from the landfill. Two types of certain physical and chemical characteristics soil (brown with a stony foundation and of fresh landfill leachate at ELS, and to loamy) were studied. The obtained results define the status of ELS. Prior field showed that the total acidity, mineral observation study on the aforementioned alkalinity and chloride content in the studied parameters at ELS has not been conducted. soils were reduced after the contamination with the landfill leachate. While total 2. Methodology hardness and total alkalinity increased. It has been found that the effect of infiltration of 2.1. Site characteristics the landfill leachate was the significant ELS is located near Kani-Qrzhala Sub-district reduction of mineral substances due to their (from left-side of Erbil-Mosul main road) in washout of the soil profile. It has also been Erbil City, Iraq. The geographical coordinates shown that the studied landfill leachate are 36°10'23''N and 43°35'32''E. ELS is contained substantial quantities of approximately 15 km from Erbil City center. substances favourable to the plant growth The landfill, which opened in 2001, has a total (Gałko, 2015). However, owing to the lack site area of 37 ha. A majority part of the area of recognized documents, landfills require has been used. The site receives more than further scientific observations. Thus, this 1000 tons of MSW daily. The buried MSW is study was conducted to investigate various mixed without proper separation of the geotechnical properties such as index components. Up to date, recognized documents properties, particle size distribution, field on the MSW characteristics in Erbil City are density, void ratio, porosity, hydraulic very limited. A small fraction of recyclable conductivity, quality of produced leachate, materials, such as plastic, glass, and metals, are and landfill status. being separated by scavengers. Owing to the A site visit with laboratory tests revealed lack of scientific sanitary landfill design in this that the natural soil of Erbil Landfill Site site, the raw landfill leachate is directly (ELS) is composed of clayey gravel with disposed to the natural environment and forms sand and gravelly lean clay layers. The gas that is emitted to the atmosphere without terrain of ELS is made up of hills and any gas collection system. valleys. Collected MSW is usually disposed 106 Yousif I. et al./ ZJPAS: 2016, 28(3): 104-113

2.2. Soil and leachate sampling In addition to the experiments in Section 2.3, further examinations, such as field Soil samples were collected from ELS by permeability of the soil to measure field removing the surface debris and subsurface hydraulic conductivity, were conducted soil. For the purpose of sample collection, directly at ELS. digs to a depth of approximately 50 cm and 100 cm were used. Hand auger was used for 2.4.1. Permeability making digs. Approximately 30 kg of soil sample was collected in clean containers and Permeability is commonly measured in labelled. The collected samples were terms of the rate of water flow through the transferred to the soil laboratory and soil in a given period of time. This analyzed for soil physical properties. characteristic is usually expressed as a Meanwhile, field permeability tests of the permeability rate (coefficient of soil were conducted onsite. permeability or hydraulic conductivity k) in centimeters per second. Soils are permeable Leachate samples were collected from because of the existence of interconnected ELS. Samples were obtained from the voids, through which water can flow from natural pond in which raw leachate was high-energy to low-energy points. The study accumulated. The collected samples were of the water flow through permeable soil immediately transported to the laboratory media is important in soil mechanics. In and then stored in a cold room at 4 °C prior 1856, Darcy published a simple equation for to use for experimental purposes to the discharge velocity of water through minimize biological and chemical reactions saturated soils, which may be expressed (APHA, 2005). Raw landfill leachate as , samples were collected at ELS on January 2013, October 2013, and December 2014. (Das and Sobhan, 2014) Throughout periods of samples collection, ambient temperature and temperature of the where collected samples were 16 oC to 23 oC and 18.5 oC to 26.1 oC, receptively. Commonly, v = discharge velocity, which is the quantity the weather was sunny during collection of of water flowing in unit time. However, the the samples. Three different dates were actual velocity of water (that is, the chosen to examine the formed landfill seepage velocity) through the void spaces is leachate at ELS at different times. greater than v. 2.3. Soil characterization k = hydraulic conductivity (otherwise known For the determination of physical properties as the coefficient of permeability) of the collected samples, the soil samples were transported to Soil Laboratory, Civil Engineering Department, College of Engineering, University of Salahaddin-Erbil, Iraq. The samples were analyzed for particle The quantity of flow q through a cross- size distribution, liquid limit, plastic limit, section of area A is stated as and plasticity index. In addition, other tests such as field density, field water content, q = kiA (volume/time) void ratio, and porosity were conducted, and the classification group symbol and group Darcy’s equation considered flow only through homogeneous soils. In reality, soils name were determined. are stratified or layered with different soil 2.4. Field experiments of soil types. In calculating flow through layered soils, an average or equivalent hydraulic conductivity representing the whole soil 107 Yousif I. et al./ ZJPAS: 2016, 28(3): 104-113

mass is determined from the hydraulic This test method simulates the "field- conductivity of each layer. The value of k is saturated" condition. The values stated in SI influenced by the void ratio, pore size, units are to be regarded as standard unless interconnected pore space, particle size other units are specifically provided. By distribution, homogeneity of the soil mass, tradition in U.S. practice, hydraulic properties of the pore fluid, stress level, and conductivity is reported in cm/s, although the amount of undissolved gas in the pore the common SI units for hydraulic fluid (Budhu, 2015). Most natural soils are conductivity are m/s (ASTM, 2011). anisotropic with respect to the coefficient of permeability, and the degree of anisotropy 2.5. Leachate characterization depends on the type of soil and the nature of The untreated leachate samples were its deposition. In most cases, the anisotropy characterized in terms of pH, electrical is more predominant in clayey soils conductivity (EC) (µmhos/cm), biochemical compared to granular soils. In anisotropic oxygen demand (BOD) (mg/L), turbidity soils, the directions of the maximum and (FTU), total solids (mg/L), total suspended minimum permeabilities are generally at solids (mg/L), total volatile solids (mg/L), right angles to each other, maximum total non-volatile solids (mg/l), total acidity permeability being in the horizontal (mg/L), total alkalinity (mg/L), direction. It is sometimes difficult to obtain phenolphthalein alkalinity (mg/L), total undisturbed soil specimens from the field. hardness (mg/L), bicarbonate alkalinity For large construction projects, it is (mg/L), carbonate alkalinity (mg/L), advisable to conduct permeability tests in hydroxide alkalinity (mg/L), total dissolved situ to fined field coefficients of 2– salts (mg/L), SO (mg/L), chloride (mg/L), permeability (Das, 2014). 4 calcium (mg/L), nitrate (mg/L), sodium (mg/L), and potassium (mg/L). Parameter measurements were conducted according to 2.4.2 Hydraulic conductivity the Standard Method of Water and Wastewater (APHA, 2005), Table 1 shows This test method covers field measurement the method used for each parameter. The of limiting values for vertical and horizontal obtained parameter values were compared hydraulic conductivities (also referred to with data published by previous researchers. as coefficients of permeability) of porous Experiments such as BOD5, chloride, total materials. This test method is utilized for hardness, total acidity, and total alkalinity natural deposits above the water table by were determined by titration methods ordinary borehole tests. However, its (chemical method). Meanwhile, the rest of resulting value is the apparent conductivity. the experiments such as pH, EC, turbidity, For this test method, a distinction must be and total and suspended solids were established between "saturated" (Ks) and conducted using instruments in the Sanitary "field-saturated" (Kfs) hydraulic and Environmental Laboratory. pH was conductivity. True saturated conditions measured using a pH meter type Hl seldom occur in the vadose zone except 8424/HANNA. A turbidity meter type Lp where impermeable layers result in the 2000/HANNA was used for measuring presence of perched water tables. During turbidity in the samples. EC in the samples infiltration events, a "field-saturated" was measured by Wissenschaftlich- condition develops. True saturation does not Teshnischewerkstatten (WTW-LF42). occur because of entrapped air. The Furthermore, an electrical balance, entrapped air prevents water from moving in evaporating dish, filter paper, and oven were air-filled pores that, in turn, may reduce the used to measure total and suspended solids hydraulic conductivity measured in the field in the collected water samples. by as much as a factor of two compared with conditions in which trapped air is absent. 108 Yousif I. et al./ ZJPAS: 2016, 28(3): 104-113

and to the rate of percolation (movement of water through the soil). Pore size and the number of pores are closely related to soil texture and structure and also influence soil permeability. From Table 3, the field permeability test indicates that the hydraulic conductivity (k) was . Produced landfill leachate usually changes the properties of the original soil. To examine this fact at ELS, on the permeability of contaminated soil samples at the site was determined. Results revealed that formed leachate significantly decreased the hydraulic conductivity of the soil. The decrease in hydraulic conductivity was caused by reduction of the effective porosity, which is attributable to pore clogging (Pillai, et al., 2014). Several siting factors influence the leachate evaluation. Among the more important factors are foundation soil properties, foundation soil thickness, location of receptors, and geohydrology. Foundation soils that are in a reduced state and have high pH, high organic matter, high mineral oxides, 3. Result and Discussion and high acid volatile sulfides retard 3.1. Soil characteristics contaminant mobility by increasing contaminant retention. Foundation soils with The soil of the experimental site at low permeability restrict leachate flow. These ELS was found to include light brown clayey properties are more common in fine-grained gravel with sand and light brown gravelly lean soils. Thicker foundations of fine-grained soils clay layers. The physical properties for both provide greater retention of contaminants types of the soils are given in Table 2, and (Schroeder et al., 2003). particle size distribution curves are shown in Figure 1. The results of sorptivity, infiltration rate, hydraulic conductivity, soil porosity, and water content tests for ELS soil are presented in Table 3 and Figures 2 to 3. The determined sorptivity, steady-state infiltration rate, and hydraulic conductivity values were

, respectively. Conventionally, a number of factors affect soil permeability. These factors are sometimes extremely localized, such as cracks and holes, and calculating representative values of permeability from actual measurements is difficult. The size of the soil pores is considerably important with regard to the rate of infiltration (movement of water into the soil) 109 Yousif I. et al./ ZJPAS: 2016, 28(3): 104-113

termed sorptivity and is proportional to the square root of time. In the present work, sorptivity was measured along a 0.5 meter stretch, and the data showed that sorptivity value was equal to . The value of sorptivity was obtained from the slope of the initial part of the curve shown in Figure 2, which is consistent with the literature (Minasny and McBratney, 2000). The results of steady-state infiltration rates in the field that have been determined for ELS are shown in Figure 3 and Table 3. Accurate determination of infiltration rates is essential for reliable prediction of surface runoff and saturated hydraulic conductivity of the surface layer. The major soil types in the study area were light brown clayey gravel with sand and light brown gravelly lean clay, which presented steady-state infiltration rates of The measured flow (q) was . 0.00004 m/s. The results obtained from the Meanwhile, the estimated parameters were present work can be used to estimate surface used to predict the depth of infiltrated water, runoff, saturated hydraulic conductivity of the which is calculated as the volume infiltrated surface layers, and groundwater pollution. The starting from initial depth (Ho), taking into steady-state infiltration rates shown in Figure 3 account the surface area of infiltration. A represent field infiltration behavior. The trend consequence of lower estimated Ho is a line of cumulative infiltration increases with decrease in the calculated cumulative time, which follows the pattern of the normal infiltration. The cumulative infiltration depth infiltration curve obtained from the collected calculated for the time interval from to data. An R2 value of 0.98 showed an excellent the first measured value is illustrated in relationship between the fitted line and the Figure 2. The sorptivity quantifies the effect of steady state infiltration rate curve, as seen in capillarity on liquid movement in a porous Figure 3. material such as soil. In 1957, John Philip introduced the term sorptivity and defined it as a measure of the capacity of the medium to absorb or desorb liquid by capillarity. Sorptivity is widely used in characterizing soils and porous materials used in construction. When water is applied to dry soil, most of the water is initially absorbed by the capillary potential of the soil matrix. Capillary force dominates the initial water infiltration process. Subsequently, as infiltration proceeds, gravitational force dominates the infiltration process. Sorptivity is related to infiltration driven by capillary forces alone. Cumulative absorption or desorption of water into or out of a horizontal (minimal gravity effects) column of soil with uniform properties and moisture content has been 110 Yousif I. et al./ ZJPAS: 2016, 28(3): 104-113

parameters such as BOD5 of 32 mg/L and pH of 8.05, the obtained results agree with the published works (Worrell and Vesilind, 2010; Kostova, 2006; Alvarez-Vazquez et al., 2004). Due to decomposition of buried materials in landfills, BOD5 changes with the age of landfill and decreases with the age of landfills (Aziz, 2011). Practically, biological treatment processes are inefficient for treatment of leachates with low BOD5 and low biodegradability fraction (BOD5/COD). Consequently, combined physical- chemical with biological processes were applied for treatment of stabilized leachate (Aziz et al., 2011). Researchers reported that stabilized

landfill leachate aged greater than 10 years 3.2 Leachate characterization and status of typically contains high amounts of NH3-N ELS (>400 mg/L), moderately high strength of COD (<4000 mg/L), and a low BOD5/COD ratio Besides landfill operational practices, (<0.1) (Mojiri et al. 2014; Bashir et al., 2012). leachate characterization typically varies with Compared with a typical composition of strong climatic regions. The main features that untreated wastewater reported by (Davis, influence leachate quality include: 1) 2011), alkalinity of 1200 mg/L, chloride of composition of disposed MSW; 2) design, 3200 mg/L, total suspended solids of 2000 operation, and age of the landfill; 3) mg/L, total dissolved solids of 16000 mg/L, availability of moisture and oxygen; and 4) site volatile solids of 5000 mg/L, and total non- hydrology (Aziz, 2011). As landfill age volatile (fixed) solids of 13000 mg/L for fresh increases, the biodegradable fraction of organic landfill leachate from ELS exceeded the contaminants in leachate inevitably decreases reported parameters for strong raw wastewater as an outcome of the anaerobic decomposition (Davis, 2011). Thus, the produced untreated occurring in the landfill site. Therefore, mature landfill leachate at ELS is considered highly or stabilized leachate contains considerably polluted wastewater. The results obtained in greater amounts of refractory organics than the current work are consistent with published young leachate (Bashir et al., 2012; Aziz et al., works on other landfill leachates (Mojiri et al., 2011). Depending on the age of ELS, which 2014; Aziz et al., 2011; Aziz et al. 2010). was opened in 2001, the untreated landfill Table 5 illustrates the formed landfill leachate formed at ELS is considered stabilized leachate from Kulim landfill site, Malaysia leachate. (Aziz et al., 2011). Total acidity, alkalinity, and Analysis results of the collected raw landfill hardness in the present study were 360 mg/L, leachate from ELS are given in Table 4. 1200 mg/L, and 1680 mg/L, respectively. Based on the obtained EC value of 10170 While, greater values of 2158 mg/L for total µmhos/cm, the produced landfill leachate from acidity, 15350 mg/L for total alkalinity, and ELS is considered to be in acid formation 1893 mg/L for total hardness were stated by phase. The yielded EC value is agree with the Aziz et al. (2011). On the other hand, achieved reported data by Aziz et al. (2010). Meanwhile, total solids (18000 mg/L), total dissolved solids a pH value of 8.05 revealed that the leachate (2000 mg/L), total dissolved solids (16000 was in methane formation phase, which mg/L) in this research were greater than the actually agrees with the existence of burned measured values of total solids, total suspended gas in the site. Published data by Mojiri et al., solids, and total dissolved solids for Kulim (2014); Aziz et al. (2011); Aziz et al (2010) Landfill site (Aziz et al., 2011). Chloride figure confirmed the obtained pH value .Furthermore, of 3200 mg/L in the current work was greater the leachate is considered stabilized/mature or than the chloride value (324 mg/L) published old leachate because of certain measured by Aziz et al. (2011). According to the 111 Yousif I. et al./ ZJPAS: 2016, 28(3): 104-113 concentration of calcium in the present study, ground water inflow) (m.year-1). 2 greater and lesser values were reported by Aziz A = area of site, m et al. (2011) and Mojiri et al. (2014). E = Evapotranspirative losses (evaporation plus minimal transpiration) (m.year-1). a = absorptive capacity of the waste (as received). W = volume of waste deposited 3 (m .year-1). Data on the amount of rainfall and temperature was obtained near Kani-Qrzhala site, Erbil, Iraq. Rainfall data were obtained from Directorate of Metrological in Erbil City. The above equation was applied for determining the amount of produced leachate from ELS. Produced landfill leachates from ELS during 2001 to 2012 are given in Table 6. Area of the ELS is 37 hectares. For the aforementioned equation, value of a is equal to 0.1 (Aziz et al., 2002). Generation rate of produced solid waste in Erbil city assumed to be 1kg/c.d. Based on 3.3. Quantity of produced landfill leachate the data achieved in the site visiting, generated The amount of precipitation that enters the solid waste from about 1 500 000 persons landfill through infiltration or falls directly (inside Erbil City center and surrounded area to on the disposed MSW and the amount of ELS such as Makhmoor and Khabat areas) liquid that the waste originally contained has disposed at ELS. A variation of produced a primary effect on leachate generation. landfill leachate can be noticed in the Table 6. Further, types and components of disposed The dissimilarity of the obtained data was due MSW have a noticeable effect on the to the effect of the variables in the equation, produced leachate. Information about particularly variation of precipitations. The generation rate of leachate is significant, average figure for the formed leachate at ELS 3 especially for the treatment processes of was 158.68 m /d which is a huge amount and leachate. For estimating the quantity of it needs scientific method for collection and produced leachate from the landfill areas, treatment to avoid pollution of environment the following water balance equation may (particularly water sources and soil). be used (Aziz et al., 2002).

Lo = [I− E]A− aW

Where:

Lo = free leachate retained at the site (equivalent to leachate production minus leachate leaving the site) 3 m .year-1). I = total liquid input (precipitation plus liquid waste plus any surface or 112 Yousif I. et al./ ZJPAS: 2016, 28(3): 104-113

Infiltration from a Borehole, DOI: 10.1520/C33– 08, D6391-06, pp. 1-12. Aziz, S.Q., Aziz, H.A, Yusoff, M.S., Bashir, M.J.K., and Umar, M. (2010). Leachate characterization in semi-aerobic and anaerobic sanitary landfills: A comparative study, J. Environ. Manage. 91, 2608– 2614. Aziz, H.A., Adlan, M.N., Amilin, K., Yusoff, M.S, and Ramly, N. H. (2002). Quantification of generation rate of leachate from semi-aerobic landfill: Field data, Proceedings, the 5th Asian Symposium on Academic Activities for Waste Management (AAAWM), Kuala Lumpur, 9–12 September, 2002. Aziz, S.Q., Aziz, H.A., Yusoff, M.S., and Bashir, M. J. K. (2011). Landfill leachate treatment using powdered activated carbon augmented sequencing batch reactor (SBR) process: Optimization by Response Surface Methodology. J. Hazard. Mater, 189, 404–413. Aziz, S.Q., Aziz, H.A., and Yusoff, M.S. (2012). Landfill Leachate Treatment Using Sequencing Batch Reactor Process, LAMBERT Academic Publishing, GmbH & Co. KG, Germany. Aziz, S.Q., Aziz, H.A., Bashir, M.J.K., and Mojiri, A. 4. Conclusions (2014). Wastewater Engineering: Advanced wastewater treatment systems (Eds.:, H. Abdul Soil sample analysis from ELS Aziz, A. Mojiri), International of Scientific indicated that the soil was light brown clayey Research Books, IJSR Publications, Penang, gravel with sand and light brown gravelly lean Malaysia, pp. 208-224. clay layers. Permeability test results showed Aziz, S.Q., PhD Thesis (2011). Landfill leachate treatment using powdered activated carbon that the hydraulic conductivity was equal augmented sequencing batch reactor (SBR) -5 to , which is considered as low process, University Sains Malaysia, Malaysia. Bashir, M. J. K., Aziz, H.A., Aziz, S. Q., Abu Amr, S. S. degree of permeability and presents poor (2012). An overview of electro-oxidation processes drainage. Soil was contaminated with leachate, performance in stabilized landfill leachate treatment, thus leading to groundwater pollution. The Desalin. Water Treat. 51, 2170-2184. untreated leachate produced from ELS is Budhu M. (2015). Soil Mechanics Fundamentals. Metric considered as stabilized leachate. In addition, version, First Edition, John Wiley & Sons, Ltd, PO19 8SQ, United Kingdom. mature landfill leachate from ELS requires Canizl, J., Lapena, P., J. Castro, J., A. DA COSTA and treatment prior to disposal to the natural Sagaseta C. (2011). Determination of Shear environment. Commonly, ELS can be Strength of MSW. Field Tests vs. Laboratory th categorized as an anaerobic landfill in the Tests, 4 International Workshop on Hydro- methane formation phase (Level 2). Physico-Mechanics of Landfills Santander, Spain. Das B. M. and Sobhan K. (2014). Principles of Geotechnical Engineering, Eighth Edition, Stamford, CT 06902, USA. Das B. M. (2014). Advanced Soil Mechanics, Fourth 5. References Edition, Taylor & Francis Group, CRC Press Web Alvarez-Vazquez, H., Jefferson, B., and Judd, S.J. site at http://www.crcpress.com (2004). Membrane bioreactors vs conventional Davis, M.L. (2011). Water and wastewater engineering- biological treatment of landfill leachate: A brief Design principles and practice, McGraw-Hill review, Journal of Chemical Technology and International Edition. Biotechnology, 79 (10), 1043–1049. Dixon, N., and Jones, D. R. V. (2005). Engineering APHA (2005). Standard Methods for the Examination of properties of municipal solid waste, Geotext. Water a+A2:A12nd Waste Water, 21st edition. Geomembr, 23(3), 205–233. American Public Health Association, Washington, Envirnmental Pollution Control Centre (EPCC), Osaka DC. Prefecture (2011). Maintenance and improvement ASTM Standards (2011). Standard Test Method for of landfill site, the role of landfill site [Accessed: Field Measurement of Hydraulic Conductivity 31 May 2011], Available: Limits of Porous Materials Using Two Stages of 113 Yousif I. et al./ ZJPAS: 2016, 28(3): 104-113

http://www.menlh.go.id/apec_vc/osaka/eastjava/w st_mng_en/page1.html. Environmental Quality (2009). Control of Pollution from Solid Waste Transfer Station and Landfill, Regulations under the Laws of Malaysia-Malaysia Environmental Quality Act, Malaysia Gałko G. (2015). The Influence of Infiltration of Leachate from Landfills on the Changes of Chemical Parameters of the Soil, J. of Ecological Engineering. 16 (4), 198–205 Humadi, R. A. (2012). Engineering Characteristics of Erbil Municipal Solid Waste Materials, J. Zanco / Salahaddin University–Erbil, 24 (3), 65-74. Kostoa, I. (2006). Leachate from sanitary landfills– origin, characteristics, treatment, University of Architecture, Civil Engineering and Geodesy, “Iskar’s Summer School”–Borovetz, 26-29 July 2006. Minany, B., McBratney, A. B. (2000). Estimation of sorptivity from disc-permeameter measurements. J. Geoderma. 95, 305–324. Mojiri, A., Aziz, H.A., Zaman, N.Q., Aziz, S.Q., and Zahed, M.A. (2014). Powdered ZELIAC augmented sequencing batch reactors (SBR) process for co-treatment of landfill leachate and domestic wastewater, J. Environ. Manage. 139, 1-14. Pillai, S., Peter, A. E., Sunil, B.M., and Shrihari, S. (2014). Soil Pollution near a Municipal Solid Waste Disposal Site in India. International Conference on Biological, Civil and Environmental Engineering (BCEE-2014) March 17-18, Dubai (UAE). Renou, S., Givaudan, G.J., Poulain, S., Dirassouyan, F., Moulin, P. (2008). Landfill leachate treatment: Review and Opportunity, J. Hazard. Mater. 150, 468-493. Schroeder, P. R., and Aziz, N. M. (2003). Effects of confined disposal facility and vadose zone characteristics on leachate quality,. DOER Technical Notes Collection (ERDC TN-DOER- C31), U.S. Army Engineer Research and Development Center, Vicksburg, MS. www.wes.army.mil/el/dots/doer Worrell, W.A., and Vesilind, P.A. (2010). Solid waste engineering. Second edition. Cengage Learning products are presented in Canada by Nelson Education, LTD. Printed in the United States of America.

ZANCO Journal of Pure and Applied Sciences

The official scientific journal of Salahaddin University-Erbil ZJPAS (2016), 28 (3); 134-141

Metabolic Syndrome in Overweight Kurdish Women

Sawsan Aziz Abdullah

Pharmacy Dept., Medical Technical Inistitute., Hawler Polytechnique University. Erbil/Iraq

A R T I C L E I N F O A B S T R A C T

Article History: The present study aimed to assess metabolic syndrome in overweight adult Received: 18/06/2015 female in Erbil city. The study conducted on the hundred and fifty six Kurdish Accepted: 12/10/2015 women with four age groups, the age range was (20 –40 years). Metabolic syndrome was diagnosed using Adult Treatment Panel- III (ATP-III) Published:16/5/2016 guidelines. The prevalence of metabolic syndrome was 35.8%. The mean total Keywords: cholesterol (TC), triglyceride (TG), very low density lipoprotein (VLDL), waist Metabolic syndrome, circumference (WC) and fasting blood glucose levels (FBS) were significantly Overweight, Kurdish women. higher in subjects with metabolic syndrome when compared with subjects *Corresponding Author: without metabolic syndrome, while mean high density lipoprotein HDL- Sawsan A. Abdullah cholesterol was lower in the subjects with metabolic syndrome. The other parameters like low density lipoprotein LDL-cholesterol, systolic blood Email:[email protected] pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP) were not showing any significant differences between the two groups. In conclusion the results suggest that high waist circumference was the most frequent factor among overweight Kurdish adult women with metabolic syndrome.

1. Introduction countries and is associated with increased morbidity and mortality from cardiovascular The Metabolic syndrome was initially disease (Cameron and Zimmet, 2004). The observed in 1923 by Kyln. Then the concept of metabolic syndrome is described by the syndrome X reintroduced for the clustering of clustering of several risk factors for cardiovascular risk factors like hypertension, cardiovascular disease (CVD) such as glucose intolerance, high triglycerides and low hypertension, dyslipidemia, obesity high density lipoprotein (HDL) concentration (particularly central obesity), insulin resistance (Reaven and Banting, 1988). World Health and high fasting plasma glucose (Katzmarzyk Organization (WHO), in 1998 proposed a et al., 2005). Adipose tissue is not only a definition for the syndrome and chose to call it simple body to store fat, it is an active the " metabolic syndrome" (Albert and endocrine organ and part of innate immune Zimmet, 1998). Obesity is a multifactorial, system that affects many physiological and complex disease that occurs as a result of pathological mechanisms such as glucose chronic excessive energy intake supported by homeostasis, inflammation, angiogenesis, cell genetic and environmental factors (Ford et al., proliferation and differentiation (Rasouli, 2002; Gupta et al., 2003; Sowers, 2003). 2008). The endocrine role of adipocytes Excess weight and obesity are considered as a carrying it with the activated macrophages major health problem in several developed and infiltrated in adipose tissue , altered expression undeveloped countries (Somaya and Mustafa, of adipokines associated with obesity leads to 2005). Furtheremore, excess weight is the most induction of systemic inflammation and a low common nutritional disorder in industrialized degree of dyslipidemia, which together can 135 Sawsan A. ZJPAS: 2016, 28(3): 134-141 lead to rapid and early atherosclerosis (Kosuge height in meters (Ncep, 2001; Gupta et al., et al., 2008). The prevalence of the metabolic 2003). Weight was measured while subjects syndrome is increased generously by the were minimally clothed without shoes, using epidemic increase of obesity around the world digital scale. Height was measured in standing (Park et al., 2009). position using tape meter. Subjects with BMI between 20-24.9 Kg/m2 were considered Metabolic syndrome is a serious health normal weight and those with a BMI of 25- condition, people with it have a higher risk of 29.9 Kg/m2 were classified as overweight, diseases related to fatty buildup in artery walls while those with a BMI ≥ 30 Kg/m2 were such as coronary heart diseases and heart defined as obese. Subjects with BMI greater attack, also stroke and peripheral vascular than 45 Kg/m2 were considered very obese diseases are other examples (Taheri et al., (Trevisian et al., 1998; Dalton et al., 2003). 2012). Metabolic syndrome is a condition often Waist circumference was measured at the point seen in people who are obese or overweight. halve way between the lower border of ribs and This condition can affect blood sugar , the iliac crest in a horizontal plane (Marjani et cholesterol level and blood pressure and can al., 2012). Subjects with Waist circumference significantly increase the risk of developing more than 89cm were considered as too high in heart diseases and diabetes. Metabolic women and more than 102cm in men (Ainy et syndrome is now recognized as a major al, 2007). contributing factor behind the two major causes of death heart disease and cancer (Verbeeten et Adult women considered to have metabolic al., 2012). syndrome if they had any three or more of the following according to the Adult Treatment Since few studies has been carried out on Panel III (ATP III Criteria ), abdominal the prevalence of metabolic syndrome in obesity or waist circumference > 88cm, Kurdish women, so the aim of the present hypertriglyceridaemia or serum triglyceride study was to determine the percentage of level > 150 mg /dL, HDL-cholesterol < 50 overweight female adults with metabolic mg/dL, high blood pressure or SBP > 130 syndrome and to determine the connection of mmHg and /or DBP > 85 mmHg or on excess weight with risk factors of treatment for hypertension, high fasting cardiovascular disease, such as dyslipidemia, glucose or serum glucose level > 110mg/ dL or high blood pressure and hyperglycemia. On the on treatment for diabetes (Ramachandran et al., other hand, this study aimed to evaluate the 2003). effect of age on the incidence of metabolic syndrome. Blood samples from the women were collected by venous method after fasting MATERIALS AND METHODS overnight and analyzed for glucose level using glucometer (Accu-Chek, Roch diagnostic This study was performed in Erbil city on Gmbh, Mannheim,Germany) (Kobayashi et al., 156 overweight Kurdish adult women with four 1993). Serum TC was determined by the age groups (20-24, 25-29, 30-34 and 35-40 enzymatic method (Paulis et al., 2007) using years), each group with the following commercial laboratory kit obtained from individual numbers successively (37, 41, 41, BIOLABO. SA, (France). Serum TC was 37). calculated by the following equation:

The World Health Organization has Serum TC (mg/dL) = (Optical density of formulated an index for defining obesity test/Optical density of standered) x Standered known as the body mass index (BMI), it is concentration. based on the patient's height in meters and weight in kilograms; BMI = weight in Triglyceride kit also was obtained from kilograms divided by the square of person's BIOLABO. SA, (France) (Berdanier, 1991). 136 Sawsan A. ZJPAS: 2016, 28(3): 134-141

Serum TG was calculated according to the most frequent component was abdominal following equation:- obesity (waist circumference > 89cm has a percentage of 37.8%). The prevalence of Serum TG (mg/dL) = (Optical density of test / fasting blood glucose, TG, HDL- Optical density of standered) x Standered cholesterol,TC and high blood pressure were concentration. 31.4%, 30.1%, 28.8%, 21.1% and 4.4% respectively. Table 3 shows the distribution of Serum VLDL (mg/dL) was calculated from TG metabolic syndrome among age groups in by TG/5. overweight adult women, the prevalence of metabolic syndrome was highest in the age Serum HDL was determined by using HDL group (35-40 years) overweight adult women. enzymatic kit (Chawla, 2003). obtained from BIOLABO. SA, (France). Low density This study shows that %35.8 of this lipoprotein cholesterol level was calculated population has metabolic syndrome. using the Friedwald formula (Ritter and Mant, Differences in the prevalence of metabolic 1999). syndrome can result from different interpretation of the syndrome, sociological LDL=TC-(HDL cholesterol + (TAG/5)). and environmental, genetic factors and life style (Eshtiaghi et al. 2010). Studies among Systolic BP and DBP were measured using Korean and Chinese populations showed that sphygmomanometer. Mean AP was calculated prevalence of metabolic syndrome were 13.8% according to the following equation:- and 19.3% respectively (Gu et al., 2005; Kim et al., 2007). Marjani et al., (2012) showed that MAP= DBP + 1/3 (SBP – DBP). prevalence of metabolic syndrome was 20.62% among Fars ethnic women. Results indicate The results were reported as percentages and that the prevalence of metabolic syndrome in mean ± SD. The statistical analysis was done the present study is higher than that of other with SPSS-16 version software. The results studies. The unhealthy lifestyle habits, such as were evaluated by using independent student t lack of exercise, poorly managed stress and a and chi square tests. Statistical significance high calorie-nutrient deficient diet are was considered at p<0.05. responsible for the increased rate of the syndrome, this is also mentioned by (Gupta et RESULTS AND DISCUSSION al., 2003; Cheta and Panaite, 2006). Body fat that accumulate in the stomach area described Among the total number of 156 as abdominal obesity is more of a health risk overweight women studied, 56 of them were than body fat that builds up in buttocks and have metabolic syndrome with a prevalence of thigh areas (Azizi et al., 2004). A high waist (35.8%), and a hundred of them were circumference is a sign of a condition called considered to not have metabolic syndrome metabolic syndrome (Taheri et al., 2012). The with a prevalence of (64.1%). The age range of present study on the components of metabolic the subjects was (20-40 years). As it was syndrome showed that the most frequent shown in table 1 the mean TG, TC, fasting change of components of metabolic syndrome blood glucose and waist circumference were was abdominal obesity (waist circumference > significantly higher in subjects with metabolic 89 cm with a frequency of 37.8% ) which is not syndrome, but the mean HDL-cholesterol was in agreement with the findings in Canada significantly lower in this group, while there (Meigs, 2000), USA (Ford et al., 2003) and were no significant differences in the other Turkey (Eshtiaghi et al., 2010) in which they parameters between the subjects with and observed that the most common change was without metabolic syndrome. Table 2 shows high prevalence of low HDL-cholesterol the prevalence of the components of metabolic (Sacks and Campos, 2011). syndrome among overweight adult women, the 137 Sawsan A. ZJPAS: 2016, 28(3): 134-141

A study showed that extra fat mass rather metabolic syndrome correctly, treatment of than excess body weight was highly correlated metabolic syndrome is carried out on the basis with abnormal metabolism (Despres et al., of taking into consideration all components of 2001). Many studies reported that abdominal metabolic syndrome (obesity, hypertension, obesity is positively associated with the risk of hyperlipidemia and diabetes) this is also cardiovascular occurrences (De Koning et al., mentioned by Ruderman et al., 1998. 2007; Hu et al., 2007). In the present study, it Treatment of obesity is integrated, the leading was been also shown that women were significance has the balanced meal or adequate overweight and the results showed that it is diet. The main goal of obesity treatment is the easier to obtain metabolic syndrome in patients body weight loss and it's maintenance on the with abdominal obesity. It was been noticed defined level. The initial aim is 5-15% that the probability to develop metabolic reduction of body weight during six months disorders increase with obesity level. For (Tan et al., 2004; Oh et al., 2004). example, in comparison with normal weight subjects the probability to have metabolic In conclusion, the results suggested that syndrome increases 5.2 times for overweight increasing weight especially central obesity is subjects (Grundy et al., 2004; Kahn et al., positively associated with metabolic syndrome. 2005). On the other hand, the incidence of metabolic syndrome is increased with increasing age in This study also illustrate that the prevalence adult overweight Kurdish women. Also, the of metabolic syndrome is increased with data of the present study suggested that the increasing age and it is highest in age group prevalence of metabolic syndrome will (35-45), This is due to the effects of some increase unless effective obesity prevention unhealthy habits like lack of exercise (Lemieux efforts are implemented. Therefore, physicians et al., 2011), poorly managed stress and over- should screen metabolic syndrome in nutrition in older ages (Onat et al., 1992; overweight individuals in the prevention of Henefeld and Leonardt, 1998; Kaplan, 1998; diabetes and cardiovascular diseases. Lipsy, 2003). It is important to reduce the health risks among the subjects with metabolic RECOMMENDATION syndrome by monitoring and controlling blood glucose and blood lipid profile and to loss 1. Further studies could be recommended to weight through regulating diet and physical investigate the effects of overweighting on the exercise (Torok et al., 2011). On the other incidence of metabolic syndrome. hand, despite an increased accumulation of adipose tissue, some moderately overweight 2. More detailed researches are needed to study subjects do not show the classical the exact mechanism of metabolic syndrome. complications of obesity, this is particularly true for females, for these subjects just 3. Further works are needed for improving the reducing body weight is enough to improve complications of metabolic syndrome such as their clinical condition. This must take in hyperglycemia and dyslipidemia. consideration because obesity and insulin resistance were thoroughly to be the ''Common soil'' of metabolic syndrome this is also mentioned by Nariman and Rusudan, 2009; Liu REFRENCES et al., 2009. Ainy, E., Mirmiran P., Zahedi S. and Azizi F., The prevalence of metabolic syndrome (2007). Prevalence of metabolic syndrome varies with gender, age, ethnic background and during menopausal transition Tehranian residence and is higher in urban areas than in women. TLGS; 58: 150-5. rural areas (Hanley et al., 2002; Yannakonlia et al., 2003). It is necessary to diagnose the 138 Sawsan A. ZJPAS: 2016, 28(3): 134-141

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Somaya, A.J. and Mustafa A.M., (2005). Table 1: Baseline data of overweight women Metabolic syndrome in abdominal obesity. (subjects with metabolic syndrome and Naim Health Center. subjects without metabolic syndrome).

Sowers, J.R., (2003). Obesity as a cardiovascular risk factor. American Journal of Medicine; 8(15): 37-41. Subjects Subjects Subjects Statistical with without evaluation

Tan, C.E., Ma S., Wai D., Chew S.K. and Tai metabolic metabolic syndrome syndrome (t-test) E.S., (2004). Can you apply the national cholesterol education program Adult Treatment (Mean ± (Mean ± Panel III: Definition of the metabolic syndrome Parameters SD) SD) Asians ? Diabetes Care., 27:1182-1186. BMI ( kg/ m2) 29.6 ± 27. 1 ± p < 0.065 Taheri, E., Saedisomeolia A., Djalali M. and 2.96 19.9 Madani M., (2012). The relationship between vitamin D and obesity in diabetic patients. WC (cm) 96 ± 8. 33 90.4 ± p < 0.002 Journal of Diabetes and Metabolic Disorder ; 12.2 11(16). 2

Trevisian, M., Liu J., Bahsas F.B. and Mennoti FBS (mg/ dL) 130.6 ± 96 ± p < 0.005 A., (1998). Syndrom X and mortality : Risk 49.7 27.1 factors and life expectancy. American J. 1 6 Epidemiol; 10: 958-966. TG ( mg/ dL) 250 ± 117 ± 53 p < 0.003 61.3 Torok, K., Szelenyi Z. and Molnar D., (2011). Low physical performance in obese boys with metabolic syndrome. Int. J. Obes. Metab. VLDL (mg/ dL) 40 ± 16.7 27 ± 13.3 p < 0.005 Disord; 25:966-970. TC (mg/ dL) 227 ± 47 163 ± p < 0.026 Verbeeten, K.C., Elks C.E., Daneman D. and 58.3 Ong, K.K. (2011). Association between 4 childhood obesity and subsequent Type 1 HDL-Chol. 37 ± 11 45.69 ± p < 0.001 diabetes. Diabetic Medicine; 28: 10-18. (mg/dL) 15.7 3 WHO, Technical report: Obesity preventing and managing. the global epidemic. Geneva, 3- LDL-Chol. 120 ± 107 ± N.S. 5 june, 2000. (mg/dL) 38.6 63.3

World Health Organization. Prevention and SBP ( mm Hg) 120 ± 111 ± N.S. management of the Global Epidemic of 15.3 17.2 Obesity Report of the WHO Consultation on 9 Obesity. WHO: Geneva, 1998 ( Technical Report Series, No. 894). DBP ( mm Hg) 78.3 ± 65 ± 9.44 N.S. 11.5 Yannakonlia, M., Yannakonris N. and Bluher 9 S., (2003). Body fat mass and macronutrient intake interrelation to circulating soluble MAP ( mm 89.3 ± 80.4 ± N.S. receptor. J. Clin. Endocrinol. Metab., 88:1730- Hg) 9.81 10.1 1736. BMI: Body mass index, WC: Waist circumference, FBS: Fasting blood glucose , TG: 141 Sawsan A. ZJPAS: 2016, 28(3): 134-141

Triglyceride, VLDL: Very low density lipoprotein, TC: Total cholesterol, HDL-Chol.: High density lipoprotein cholesterol, LDL-Chol.: Age groups Subjects Subjects Low density lipoprotein, SBP: Systolic blood in year with MS without MS pressure and DBP: Diastolic blood pressure, (n=56) (n=100) MAP: Mean arterial pressure.

N.S.: Non-significant.p-value less than 0.05 was considered significant. 20-24 n (%) 5 ( 8.92)

Table 2: Prevalence of the components of 32 ( 32 ) metabolic syndrome in overweight women. 25-29 n (%) 12 (21.4) 29 ( 29 )

Metabolic syndrome n. % 30-34 n (%) 19 (33.9)* 22 ( 22 )

35-40 n (%) 20 (35.7) 17 (17) Waist circumference > 89 cm 37.8 59

MS: Metabolic syndrome.

49 31.4 * p-value less than 0.05 was considered Fasting blood sugar > 110 mg/dL significant.

47 30.1 Triglyceride > 150 mg/dL

High density lipoprotein- cholesterol < 50 mg/ dL 45 28.8

Total cholesterol > 200 mg/dL 33 21.1

Systolic blood pressure > 130 mm Hg and diastolic blood pressure > 85 mm Hg 7 4.4

n.: number of samples.

%: prevalence.

Table 3: Distribution of metabolic syndrome among age groups in overweight women in Erbil city:- ZANCO Journal of Pure and Applied Sciences

The official scientific journal of Salahaddin University-Erbil ZJPAS (2016), 28 (3); 142-151

Multivariate statistical characterization of water quality analysis for Erbil wastewater channel

Jamal K.Mohammaed Amine1* and Yahya A. Shekha

Environmental Science Department, College of Science, University of Salahaddin – Erbil,

A R T I C L E I N F O A B S T R A C T

Article History: Multivariate statistical analysis, such as principal component analysis (PCA), Received: 18/06/2015 factor analysis (FA) and cluster analysis (CA), were applied for evaluate the Accepted: 12/10/2015 water quality of Erbil wastewater channel. Seventeen variables have been monitored in three selected sites on monthly basis for one year. Results Published:16/5/2016 revealed that the most important variables are those related to salinity factors, Keywords: temporal hardness and mineral salts according to PCA/FA techniques. In Water quality, factor analysis, addition to, CA results showed that dissimilarity of site 3 from other two sites, principal component analysis, and most similarity between ions related to processes of hydrogeochemical and cluster analysis, wastewater. mineralization. *Corresponding Author: Generally, water quality for irrigation purposes comes under good water type Jamal K. Mohammaed Amine (86% of total samples) to moderate type (16%) depending on EC- SAR values Email:[email protected] guidelines. Increases in ions concentrations for EC, TDS and hardness were observed from upstream toward downstream of Erbil wastewater channel.

1. Introduction storm runoff rain water, in addition to Assessment of water and wastewater is very industrial and agricultural runoff. In medial and crucial to safeguard public health and the downstream it being used for farm irrigation environment. However, monitoring water before discharged to Greater Zab River. quality parameters is important for safety According to Erbil city general directorate of assessment of the environment and human water in 2012 they produced about 61000 public health in particular (Igbinosa and Okoh, m3.day-1 and every person in city will receive 2009). Monitoring of stream water physico- more than 400 litter.day-1 for domestic chemical characteristics and heavy metals consumption. Therefore, the quantity of concentration is necessary to establish the level sewage discharge differs from time to other, as of contamination in wastewater, with a mean, it may reaches 77760 m3.day-1 during increasing urbanization and industrialization, dry season and 108000 m3.day-1 during rainfall there has been a rapid increase in industrial season (Mustafa and Sabir, 2001). effluent discharge into the stream water, leading to increase pollution load (Sekabira The application of different multivariate etal., 2010). statistical techniques, such as cluster analysis (CA), principal component analysis (PCA) and Erbil wastewater channel is important stream factor analysis (FA) helps in the interpretation that recipient of Erbil city untreated sewage, of complex data matrices to better understand 143 Jamal K et al./ ZJPAS: 2016, 28(3): 142-151 the water quality and ecological status of the evaporation at 180 ºC), total hardness, calcium studied systems, allows the identification of and magnesium (EDTA-2Na titrimetric -2 possible factors sources that influences water method), Cl- (argentometric method), SO4 systems and offers a valuable tool for reliable (turbidimetric method), HCO3 (titrimetric management of water resources, as well as method), Na+ and K+ (flame photometric rapid solution to pollution problems (Pejman method), trace metals (Cd, Pb, Cu and Zn) by etal., 2009). (Atomic absorption spectroscopy Philips Sp9) after concentrating 1L of samples (with Many studies have been conducted about addition of HNO3). While, sodicity, including Erbil wastewater monitoring and its suitability the Na% and SAR were calculated according to for different purposes (Shekha, 1994; Aziz the formulas given by (Pescod, 1992) as etal., 2001; Ali, 2002; Ganjo etal., 2006; Sofi, follow: 2007; Shekha, 2008; Shekha etal., 2010), but most of them were not included CC, PCA and Na x 100 FA for this reason the study was selected to assess levels of physico- chemical parameters Sodium percentage % Na = ------in order to determine the source of pollutants Na + K + Mg + Ca and on quality of water in stream by using multivariate statistical methods, as well as, to evaluate their suitability for irrigation purposes. Sodium Adsorption Ratio (SAR) =

Materials and methods: C. Multivariate statistical methods A. Study area: Erbil wastewater channel which extended from southwest of Erbil city with their Physico- chemical factors were compared elongation for more than 50 km passes through statistically by using one way analysis of vast farmlands, orchards and several villages. variance (ANOVA). Duncan test was used to After Gameshtapa village wastewater effluent examine multiple comparisons. Correlation discharges into Greater Zab River. Generally, was evaluated for significance using a Pearson Erbil sewer system is constructed for storm product moment correlation test procedure. water and in most cases domestic sewers are Principal component analysis/ factor analysis connected illegally with storm sewer. The (PCA) was performed to relate different width of channel ranges from 2-4m with depth studied parameters and cluster analysis (CA) to more than 1m at different locations (Shekha evaluate similarities and dissimilarities etal., 2010). between sampling sites and variables, all mathematical and statistical calculation were B. Sample collection: implemented using (SPSS version 20) program Water samples were collected regularly on and Microsoft Office Excell 2007. monthly basis from 3 sites along Erbil wastewater channel during May 2010- April 2011 (Figure 1). Standard techniques were used (APHA, 1998) to analyze the different physico- chemical parameters: pH, electrical conductivity in field by using (pH meter Philips 4014 and EC meter Philips 4025 respectively), total dissolved solids (TDS, 144 Jamal K et al./ ZJPAS: 2016, 28(3): 142-151

the soil or by runoff from dumps at the source of the stream or by effluent discharges into the stream (Essington, 2005). The presence of ions of inorganic compounds makes water to exhibit high conductivities (Rump, 1999). The electrical conductivity (EC) of water samples ranged from 486- 1032 µs. cm-1 throughout the studied period. Higher EC were observed in downstream channel (site 3) with trend to increase from site 1 to site 3. Based on irrigation purposes in most cases EC classified as C2 medium type water and some cases as C3 saline type water, which can be used to irrigate some tolerate plant for salinity water.

Table (1): Physical-chemical properties of Erbil wastewater channel, data represented as mean± S.E. with Figure (1): Map showing southwest of Erbil minimum and maximum values, governorate, and sampling sites along the Erbil during studied period. wastewater channel (Google earth). Sodium Adsorption Ratio (SAR) is a relatively Results and Discussion: proportion of sodium ions to calcium and Hydrochemistry of Erbil wastewater channel

Descriptive characteristics (minimum, maximum, mean and standard error) for the studied chemical parameters are given in table (1).

The sewage water samples exhibited from neutral to slightly alkaline pH in the range of 6.99 to 7.98. The minimum value observed at site 1 and maximum value at site 3 (Figure 2). There is a significant difference (P≤0.05) between site 1 with sites 2 and 3. Generally, the obtained pH values fall within safe limit for crop production (Ayers and Westcot, 1994). Previous studies (Shekha, 1994; Amin and Aziz, 2005; Sofi, 2007) recorded similar results in the main sewage channel of Erbil city.

Electrical conductivity is a measure of the ability of water to conduct an electrical current (Golterman etal., 1978). It is sensitive to variations of dissolved solids mostly minerals salts, degrees to which they dissociate into ions, the amount of electrical charge, ion mobility and the temperature of the solution. The high conductivities could be attributed to high minerals salt concentration magnesium. Sodium effects soil structure which comes from the dissolution of minerals in dispersion of clay particles because it is a 145 Jamal K et al./ ZJPAS: 2016, 28(3): 142-151 monovalent cation and high hydration shell, mainly Total dissolved solids (TDS) showed same on clay and fine silt fractions. As the trend of fluctuation as noticed in EC values by percentage of sodium rises in relation to other increases from upstream to downstream of cations, lead to reduced infiltration that cause a sewage channel (Figure 4). TDS mean values decrease in water supply to crop between were 345, 380 and 387 mg.l-1 respectively. irrigations and reduced soil aeration capacity The relative high ranges of EC and TDS values (Ayers and Westcot, 1994). SAR values ranged inthe sewage water may be related to nature of from (0.32- 1.84, 1.14- 2.16 and 1.14- 1.80 municipality pollutants, industrial wastes and Meq.l-1) for sites 1, 2 and 3 respectively. The land use activities in the area (Hammer, 1996). water quality characterized by low SAR and High TDS concentrations automatically lies in first class (S1) according to (Richards, influenced the quality of the received water 1954) classification, which can be considered body. Elevated TDS can be toxic to freshwater as safe type for irrigation, since the maximum animals by causing osmotic stress and affecting SAR value not exceed 3. These results are the osmoregulatory capability of the organisms lower than obtained by (Bahri, 1998). (Igbinosa and Okoh, 2009). Similar results Generally, about 84% of the total water were noticed by (Amin and Aziz, 2005, samples fall in the C2- S1 zone of the salinity Shekha, 2008), while, its lower than recorded that regarded as medium salinity type. This by (Ganjo etal., 2006) at the same polluted comes under the good water quality zone for channel. TDS values it considered as good irrigation. While, 16% of water samples fall in type and entirely safe for irrigation as classified the C3- S1 (high salinity type) that comes by (Todd, 1980). ). Erbil wastewater can be regarded as a weak type according to (Pescod, 1992). There is high correlation coefficient observed between EC and TDS in this investigation. The TDS is related to conductivity in that if they are high, the EC also be high (Duqqah, 2002; Bhardwaj etal., 2010).

Figure (3): Classification of irrigation waters under moderate water quality zone for (Richards, 1969). Circles denoted the chemical irrigation (Figure 3) (Richards, 1969). data of the water sampling sites depending on Figure (2): pH variation in Erbil wastewater mean value.

Total hardness mean values varies from 277.9, 287 and 296.3 mg CaCO3.l-1 from upstream to downstream respectively. There is no significant difference (P≤0.05) between studied sites. Water quality can be regarded as hard to very hard water (Todd, 1980).

Sodium and potassium are common felsic cation occurrences in nature, with high channel during studied period. dissolvent in water (Hem, 1985). The mean 146 Jamal K et al./ ZJPAS: 2016, 28(3): 142-151 values of Na+ and K+ from upstream to while site 3 has more Mg2+concentration than downstream were 1.73, 2.07 and 1.91 for Na others sites, with no significant differences and 0.21, 0.22 and 0.20 Meq.l-1 respectively. (P≤0.05) between studied sites. These results Highest Na+ concentration than K in water are lower than noted by (Shekha, 1994; Lak, samples may be due to domestic effluent 2007; Bahri, 1998). characteristic that contain higher concentration of Na+ (Wetzel, 1975) or to higher resistance Chloride is one of the major anions found in of K minerals to weathering in relation to Na water and sewage. Its presence in large minerals (Davis and Dewiest, 1966). There is a amounts may be due to natural processes such trend to increase concentration for both ions as the passage of water through natural salt from site 1 to site 2 then reduction toward site formations in the earth or it may be an 3. Same phenomenon was observed by indication of pollution from industrial and (Shekha, 1994) at same polluted channel, he domestic wastes. Any change in normal commented that this increase to runoff from chloride content of natural water should be surrounding area and reduction to reason for suspecting pollution from one of sedimentation of these ions. Higher values of these sources (Ntengwe, 2006). The site with Na+ and K+ was recorded by (Goklap etal., the highest chloride levels is site 1 with mean 2011; Ganjo etal., 2006). While, similar ranges value 59.7 mg.l-1 followed by site 2 and 3 with were observed by many researches (Shekha, 56.3 and 47.2 mg.l-1 respectively. (Sawyer and 1994; Lak, 2007 and Sofi, 2007) in the same McCarty, 1978) commented that human excreta increase the amount of Cl- in sewage. polluted channel. Decrease in chloride concentration was observed from upstream to downstream. There is a significant difference (P≤0.05) between site

1 and site 3. These results come in accordance with that observed by Amin and Aziz (2005). According to Pescod (1992) guidelines the water is entirely safe for irrigation.

The results of sulphate (SO4-2) showed increases in their values from site 1 (922 mg.l- 1) to site 2 (1001.6 mg.l-1) to site 3 (1309 Figure (4): Shows increase in EC µS.cm-1, TDS mg.l1 mg.l-1). The quality of stream water is affected -1 and Harness CaCO3.l concentrations by sulphates. The concentration of sulphate from upstream to downstream of exceed maximum permissible level for Erbil wastewater channel. irrigation purposes (Todd, 1980). Moreover, SO4-2 concentration was always higher than Calcium regarded as an important and dominant those of Ca+2. cation in water, magnesium comes after Calcium, and both cations are necessary element for growth Alkalinity is a measure of the buffering of plants and animals (Hem, 1985). They react with capacity of water (Kiely, 1997). Most alkalinity carbonate to produce water hardness (Rosozka, value is due to bicarbonate ion. Generally, low 2+ 2+ 1980). The Ca and Mg minimum and maximum concentration of HCO3 was recorded in Erbil concentration of wastewater was 1.44- 4.60 and wastewater channel during studied period with 0.1- 0.42 mg.l-1 respectively. Site 2 characterized by 2+ mean values of 0.089, 0.096 and 0.087 Meq.l-1 highest Ca value than other studied sites, respectively for studied sites. These values are 147 Jamal K et al./ ZJPAS: 2016, 28(3): 142-151 lower than recorded by previous studies on the Principal component analysis/ factor analysis same channel (Shekha, 1994; Amin and Aziz, was performed on the normalized dataset (17 2005; Ganjo etal., 2006). The water quality is variables) for three different sites in twelve non-restricted to use for irrigation according to months, in order to identified important water guideline of (Pescod, 1992). High sulphate quality parameters. An eigenvalue gives a values in water caused Ca2+precipitation measure of the significance of the factor: the which lead to relatively increase in Na+ factors with the highest eigenvalues are the contain in soil that adsorbs to clay surfaces and most significant. Eigenvalues of 1.0 or greater effects on nutrients balanced in plants (Kovda, are considered significant (Shrestha and 1973). Kazama, 2007) classification of factor loading in thus “strong”, “moderate” and “weak”, Sodium considered being the most important corresponding to absolute loading values of > factor in determining the suitability of water 0.75, 0.75- 0.5 and 0.5- 0.3, respectively (Liu for irrigation (Rhoades and Merill, 1975). etal., 2003). Corresponding, variables loading Maximum sodium percent (Na%) was 47.2% and explained variance with rotated factors are recorded in site 2. According to (Wilcox, 1955) presented in (Table, 2). The results showed that the wastewater classified as good type of water first six components accounted for more than for irrigation. These results come with results 80% of the total qualitative differences among that obtained by (Amin and Aziz, 2005), while, its the sites. The first component PC1 accounted lower than noted by (Ganjo et al., 2006) at the for 25.3% of the whole dataset, has strong same polluted channel. positive loading on Na, Na% and SAR, which Generally, the mean concentrations of ions in shows that the quality of water in these sites is wastewater were observed in the order: SO4-2> affected by salinity factor, that may be comes Cl> Na+> HCO3> Ca+2> Mg+2> K+. from domestic uses and soap component (Wetzel, 1975). For the second component PC2 Water quality for Erbil wastewater channel is associated with strong positive loading to characterized by dominance of the following total hardness and bicarbonate as the most heavy metals according to their content in significant parameters and moderate positive sequences: Pb> Zn> Cd> Cu with highest mean loading to Mg and Cl ions (Figures 5 and 6). values of 50.74, 32.05, 25.7 and 14.02 µg.l-1 These variables are accounts for temporary respectively. Lak (2007) found heavy metal hardness of water. While, third component PC3 dominance in the same polluted channel in the is strongly positive loading with SO4-2 and following order: Zn> Pb> Cu> Cd. From moderately positive loading with pH, EC, Pb, previous results its can be concluded that the and negatively with K+ ions, it can be related water channel is polluted by heavy metals that to mineral salts and weathering ( Shrestha etal., comes from human activities and industrial 2008). effluent (Pradhan and Shirodkar, 2009). Sites and variables similarity Data structure determination and source identification The similarity between sites were obtained through cluster analysis using Wards method PCA is a powerful pattern recognition (linkage between groups) with Euclidian technique that attempts to explain the variance distance as a similarity measure and were of a large dataset of intercorrelated variables amalgamated into dendogram plots (Figure 7). with a smaller set independent variables As it shown there are similarity associations (Principal component) (Simeonov etal., 2003). between site 1 and 2, both of them linked with 148 Jamal K et al./ ZJPAS: 2016, 28(3): 142-151 site 3. Generally, in most studied variables site Figure (5):- Principal component analysis (PCA) 2 considered as intermediate stage between scatterplot for Erbil wastewater samples on the sites 1 and 3 in both increasing value of basis of studied water variable characteristics. variables or decreasing from upstream of

Figure (6):- Principal component analysis

(PCA) scatterplot for Erbil wastewater

samples on the channel toward the downstream. basis of studied water Figure (8) showed the similarity degree between studied variables in which two clusters of three variable groups were formed First group linked TDS, characteristics . EC and total hardness The higher concentration of TDS related to EC and hardness ions is an Figure (7): indication of man- made pollution Similarity dendogram among sampling

sites along Erbil wastewater channel from cluster analysis.

Figure (8): Similarity dendogram among variables due to domestic waste (Fisher and Mullican, concentrations of Erbil wastewater - effluent from cluster analysis. 1997). Second group association between SO4 2, Na%, Cd and Pb were obtained. While, third group consists of association between three subgroups; in which association found between + + +2 K , HCO3, Mg2+with Cl, SAR, Na , pH, Ca , Cu all with Zn. These ions suggest the associated with a combination of various hydrogeochemical processes and augment more mineralized water (Bhardwaj etal., 2010).

Conclusion: 149 Jamal K et al./ ZJPAS: 2016, 28(3): 142-151

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The official scientific journal of Salahaddin University-Erbil ZJPAS (2016), 28 (3); 152-160

Hemodynamic and Renal Effects of Bosentan and Losartan in Endothelin-1 And Angiotensin II Induced Hypertensive Rats

Sarkawt Hamad Ameen Hamad 1* and Ismail Mustafa Maulood2 1Biology Department, Faculty of Science and Engineering, Soran University, Soran, Iraq; 2 Department of Electrical Engineering, College of Engineering,, University-Erbil, Iraq

A R T I C L E I N F O A B S T R A C T

Article History: Endothelin-1 (ET-1) and Angiotensin II (Ang II) have great roles in the development of hypertension. The present study was designed to investigate the short term hemodynamic and renal Received: 18/06/2015 effects of bosentan and losartan in ET-1 and Ang II infused rats. The study included two Accepted: 12/10/2015 experiments. First experiment included: Group 1 saline infusion, Group 2 ET-1 infusion, Group 3 Bosentan + ET-1, and Group 4 Losartan + ET-1. While second experiment includes: Group 1 Published:16/5/2016 Saline infusion, Group 2 Ang II infusion, Group 3 Losartan + Ang II, and Group 4 Bosentan + Ang Keywords: II. Blood pressure was monitored every ten minutes for sixty minutes after equilibration period and Endothelin-1, Angiotensin II, urine samples were taken every twenty minutes. Mean arterial pressure (MAP) significantly raised after thirty minutes in ET-1 infusion. Bosentan slightly decreased MAP, while losartan infusion Hypertension ,Bosentan , significantly reduced it. Furthermore, Ang II significantly decreased H.R at 10 minute after losartan infusion. Neither losartan nor bosentan significantly changed serum NO level as compared with ET-1 and AngII infused rats. Serum calcium concentration significantly elevated in ET-1 induced *Corresponding Author: hypertensive rats as compared with saline group. Sodium excretion rate tended to increase significantly in losartan group at twenty minutes and in bosentan group in both twenty and sixty Sarkawt H. Hamad minutes as compared with ET-1 group. Furthermore, potassium excretion rate significantly Email: increased in both time intervals. In conclusions: Intravenous infusion of ET-1 could increase MAP. Surprisingly, losartan rather than bosentan caused significantly reduction in MAP in ET-1 induces hypertension rats. In contrast to bosentan administration, which caused a slight decrease in K+ excretion rate, bolus infusion of losartan significantly elevated it as compared with Ang II infused rats.

1. Introduction central role in the pathogenesis of proteinuria and glomerulosclerosis in the kidney, which is Endothelin-1 is the most potent known mediated via activation of the ETA receptor vasoconstrictor. It is first discovered by that leads to natriuresis and diuresis action (Yanagisawa et al., 1988). Beside ET-1, other (Kohan, 2010; Kohan et al., 2011). (Verhaar et vasoconstrictors have great roles in al., 1998) demonstrated that ET-1 binds with cardiovascular diseases one of them is Ang II. ETB receptor and increases NO and It is one of the main substances in the renin- prostacyclin, for this reason, administration of angiotensin-system (RAS), produced from ET-1 intravenously has a biphasic response. In angiotensin I (Ang I) by action of angiotensin contrast, NO inhibits the ET-1 expression also converting enzyme (ACE). ET-1 has multiple NO antagonizes ET-1 by lowering the physiological roles in body organs through concentration of ET-1 than required elicit widely distribution of ET-1 receptors over vasodilation (Khimji and Rockey, 2010). tissues (Stow et al., 2011). The most common ET-1 function is a potent vasoconstrictor in Because most of the physiological most vascular beds (Potts et al., 2013). actions of Ang II occur via AT1 receptor, so that blocking AT1 is considered more In addition to growth promoting and significant than AT2 receptors like ACEIs, vasoconstriction properties of ET-1, it has a angiotensin II receptor blockers decrease blood 153 Sarkawt H. et al./ ZJPAS: 2016, 28(3): 152-160 pressure but not affect heart rate (Sookoian et Immediately after insertion of cannula the al., 2005). Although, Ang II stimulates adrenal normal saline was infused. cortex aldosterone, which causes elevated Na+ and water retention, then, it leads to volume Procedure for cannulation of carotid artery load (Knights et al., 2006), and the result is elevation of SBP. There is few research about The common carotid arteries and two vagus the relation of ET-1 and Ang II actions, nerves run together on either side (right and therefore, this work was designed to study how left) of trachea. Carotid artery had been raised, bosentan affect Ang II and how losartan is back head of forceps placed under it, the related with ET-1 actions in induced cephalic end of artery was tied with a ligature hypertensive rats. So, the aim of the present to prevent blood flow toward the brain and study is to investigate the short term Bulldog (Larca, Germany) was clamped hemodynamic and renal effects of bosentan and cardiac end of the carotid artery. A small cut losartan in ET-1 and Ang II infused rats. was made near cephalic end by fine scissor (Eye max, Germany) through this fine rupture a Materials and Methods polythene tube (ID 0.58 mm, OD 0.96 mm. England) filled with heparinized normal saline Animals (0.9% sodium chloride) and 10 IU/ml was inserted gently, pulled toward cardiac end till Forty-eight male rats weights between reach Bulldog, a thread loosely tied around 300 – 400 grams were used, overnight fasted them and then clamp slowly released. (8-12 hrs.) and animals were brought to the laboratory at the day of surgery. Six rats were Recording invasive blood pressure and heart kept for each plastic cage. The employed rate experimental animals were met the criteria of ethic rules of the supervising committee of Direct blood pressure was recorded through College of Science. The animals were cannulated right carotid artery. For this anesthetized by intraperitonial infusion of purpose, the cannulated artery had been combination Ketamine hydrochloride 80 attached to three-way stopcock connected to mg/Kg (Trittau, Germany) and Xylasin the pressure transducer (Memscap SP 844, 12mg/Kg (Interchem, Holland). The depth of Norway) and a syringe (Ayset 10ml) filled with anesthesia was monitored by loosing reflex. heparinized saline. Software was designed (Lab The anesthesia was remained for 1.5 – 2 hours Chart 6, Blood pressure module, ADInstrument and a supplement dose was used if necessary. Australia) were analyzed those signals into Rat’s body temperature controlled between 35- mean arterial pressure and heart rate. 37 oC Experimental design Cannualation of femoral vein for intravenous infusion Two experiments were carried out. In experiment I, hypertensive rats were developed Small incision (2-3 cm) was made on the right by ET-1 infusion. The experimental rats were thigh using concave sterile scissors, outer layer divided into two groups. In the first group, of skin was removed and the matrix of collagen animals were infused 15 ml/Kg/h and the BP fibers interlaced with elastic fibers of the was monitored each 10 minutes until sixty dermis and cleaned carefully, then a 27 G ½ minutes. The rats from the second group were needle filled with heparinized normal saline 10 subdivided into three subgroups, each subgroup IU/ml was inserted into the vessel which is includes six rats. The first subgroup served as a attached to the polythene tube (ID 0.58 mm, positive control (Animals were infused by ET- OD 0.96 mm. England) that was connected to 1, 520 ng/min/Kg). The second subgroup were infusion pump (Advance series 1200 infusion injected with bolus infusion of bosentan 10 system, USA) through syringe 10 ml size. mg/0.5 ml/Kg while, the last subgroup was 154 Sarkawt H. et al./ ZJPAS: 2016, 28(3): 152-160 infused bolus infusion of losartan 10 Determination of serum NO mg/0.5ml/Kg. The same design was done in experiment II, but the model of hypertension This determination relied on Griess reagent was developed by Ang II infusion (320 system which is based on the chemical ng/min/Kg). reaction, using sulfanilamide and N-1- napthylethylenediamine dihydrochloride Determination of serum Ca2+ (NED) under acidic (phosphoric acid) conditions. Calcium in all specimens was determined spectrophotometrically using BioLabo kits, Determination of Packed cell volume (PCV) France. Three quarters of the heparinized capillary Determination of electrolytes excretion rates tubes were filled with whole blood and closed by commercial clay then centrifuged by micro- Excretion rates were found by following hematocrit centrifuge (Hawksley, England) for equations: 5 minutes at 1000g .The PCV was read using microhaematocrit reader. Sodium excretion rate (mEq/hr/Kg) = Urine flow (ml/hr/Kg b.w) * Na+ concentration in Statistical analysis Urine mEq/L All data were expressed as means ± standard Potassium excretion rate (mEq/hr/Kg) = Urine error (SE) and statistical analysis was carried flow (ml/hr/Kg b.w) * K+ concentration in out using available statistical software Urine mEq/L (statistical package for social science (SPSS) version 16). Data analysis was made using one way analysis of variance (ANOVA). The comparisons among groups were done using Duncan post hoc test. P values <0.05 were considered as significant.

Results

Statistical analysis revealed that, MAP was significantly increased (P<0.05) in ET-1 infused rats after thirty, fifty and sixty minutes compared to the saline infusion (Figure 1, A). On the other hand, it was raised significantly when Ang II infused but immediately after only 10 minutes of infusion. Bolus infusion of bosentan caused a slightly decrease in MAP, whereas losartan infusion significantly decreased MAP comparing to ET-1 infused rats (Figure1, A). Furthermore, (Figure 2, A) shows that MAP tended to reduce significantly (P<0.01) at thirty minute time interval when losartan injected in Ang II infused rats, while bosentan infusion did not reduce MAP significantly. Neither bosentan nor losartan significantly changed H.R in ET-1 infused rats as compared with saline group (Figure 1,B),while Ang II infusion at 20 minutes 155 Sarkawt H. et al./ ZJPAS: 2016, 28(3): 152-160 significantly decreased it when compared with ng/min/Kg) was infused for one hour. Data are control group. However, both ET-1 and Ang II expressed mean ± standard error (mean ± receptor antagonists did not alter H.R SEM). #P<0.05 vs saline group and *P<0.05 vs significantly (Figure 2, B ). Ang II groups .One way ANOVA and post- Figure 1: Effects of bosentan and losartan hoc Duncan were used. on mean arterial blood pressure (MAP), A) and heart rate H.R ,B) in ET-1 induced Table 1 and 2, shows that serum NO level in hypertensive rats. ET-1 and Ang II induced hypertensive rats slightly decreased but not significantly Saline (15ml/kg/h) was infused, Endothelin-1 (P>0.05) as compared with saline group. (ET-1) (520 ng/min/Kg) was infused for one Neither losartan nor bosentan significantly hour, bolus infusion losartan (10 mg/Kg) increased serum NO level as compared with immediately Endothelin-1 (520 ng/min/Kg was ET-1 or Ang II infused rats. PCV value was infused for one hour, and bolus infusion markedly increased (P<0.05) in ET-1 group, bosentan (10 mg/Kg) then Endothelin-1 (520 whereas in bosentan and losartan groups it ng/min/Kg) was infused for one hour. Data are was slightly decreased as compared with ET-1 expressed mean ± standard error (mean ± infused rats (Table 1). Serum calcium SEM). #P<0.05 vs saline group and *P<0.05 vs concentration significantly (P<0.01) elevated in Endothelin-1 group .One way ANOVA and ET-1 induced hypertensive rats as compared post-hoc Duncan were used with saline group (Table 1).

Table 1: Effects of bosentan and losartan on serum NO, PCV percentage, and serum Ca2+ in ET-1 induced hypertensive rats Paramet NO PCV * Ca2+ * * ers

Group µmole/L (%) (mg/dL)

Saline 15.50 ± 45.40 ± 8.791 ±

2.069a 1.400a 0.248a

ET-1 12.57 ± 50.83 ± 9.647 ± a b b 1.638 1.137 0.081 ET-1 + 14.25 ± 49.66 ± 9.897 ± Bosentan 1.701a 0.557b 0.233b

ET-1 + 14.41 ± 47.00 ± 9.804 ± Losartan 0.757a 2.073ab 0.245b

Figure2: Effects of bosentan and losartan on NO, nitric oxide; Ca2+, Calcium; PCV, Packed cell mean arterial blood pressure (MAP) ,A) and volume; Data expressed as (Mean ± SE) and ANOVA heart rate (H.R), B) in Ang II induced was used for analysis post-hock Duncan. * P<0.05 vs saline. ** P<0.01 vs saline. hypertensive rats.

Saline (15ml/h/kg) was infused, Angiotensin II (Ang II) (320 ng/min/kg) was infused for one Table 2: Effects of bosentan and losartan on hour, bolus infusion losartan (10 mg/Kg) serum NO, PCV, and serum Ca2+ immediately Ang II (320 ng/min/Kg was concentration in Ang II induced infused for one hour, and bolus infusion hypertension rats bosentan (10 mg/Kg) then Ang II (320 156 Sarkawt H. et al./ ZJPAS: 2016, 28(3): 152-160

Parame NO PCV Ca2+ ters

Group µmol/L (%) (mg/dL) Saline 15.50 ± 45.40 ± 8.791 ±

2.069a 1.400a 0.248a Ang II 13.58 ± 49.16 ± 8.771 ± 1.491a 1.973a 0.189a

Ang II 13.25 ± 46.50 ± 8.839 ± + 0.834a 1.586a 0.259a Losarta n

a Ang II 12.78± 49.40 ± 0.245 + 0.596 a 1.469a Bosenta n Figure 4: Effects of bosentan and losartan on

sodium (A)and potassium(B) excretion rates in Ang II induced hypertensive rats. NO, nitric oxide; Ca2+, Calcium; PCV, Packed cell volume; Data expressed as (Mean ± SE) Saline, rats were infused (15ml/h/Kg); Ang II, and ANOVA was used for analysis post-hock Angiotensin II infustion (320 ng/min/Kg; Ang Duncan. * P<0.05 vs saline. ** P<0.01 vs II + Bosentan, bolus bosentan (10 mg/Kg) was saline. infused then Ang II infusion (320 ng/min/Kg); Ang II + Losartan, bolus Losartan was infused Figure 3 A, shows that sodium excretion rate then Angiotensin II infusion (320 ng/min/Kg). tended to increase significantly (P<0.05) in Data expressed as (Mean ± SE) and ANOVA losartan group at twenty minutes and in was used for analysis post-hock Duncan. * bosentan group in both twenty and sixty P<0.05. minutes as compared with ET-1 group. Furthermore, potassium excretion rate Discussion significantly (P<0.05) increased at twenty minutes in losartan group and at sixty minutes in bosentan group as compared with ET-1 infused rats (Figure 3, B). On the other hand, Ang II infusion caused a significant increase in Hypertension was induced by infusion of ET-1 K+ excretion rate at sixty minutes as compared or Ang II in two different sub experiments to with saline group (Figure 4, B). In contrast to investigate the therapeutic impacts effects of bosentan administration which caused a slight bosentan and losartan on hemodynamic and decrease in K+ excretion rate, bolus infusion of renal parameters. There is now evidence that, losartan significantly (P<0.05) elevated it as ET-1 enhances free radical production (Viel et compared with Ang II infused rats. al., 2008)and such oxidative stress increases blood pressure (Rodrigo et al., 2007). Another possible mechanism for elevating blood

pressure is that ET-1 infusion ought to inhibit

of natriuresis as reduced Na+ and K+ excretion rate (Figure 3, A and B). However, the action

ET-1 on hypertension is still controversial, but our experiment by infusion ET-1 explained

some hemodynamic effects. For instance, 157 Sarkawt H. et al./ ZJPAS: 2016, 28(3): 152-160 infusion of ET-1 for sixty minutes caused aldosterone, which causes elevated Na+ and rising MBP. For explaining that, several water retention, then, it cause to volume load mechanisms may contributes to ET-1 through (Knights et al., 2006) and the result is elevation ETA blood vessels constriction which caused of BP. Statistical analysis revealed that an increases in peripheral vascular resistance significant elevated MAP was observed at the and then increased BP . ET-1 induced beginning then it reduced at the end of the vasoconstriction after binding to ETA receptors experiment. The possible mechanism for this occurs via G protein-dependent signal finding may be due to the pressure-natriuresis transduction pathway of phospholipase C through ET-1 A receptor (Kittikulsuth et al., activation second messengers Inositol 2011) and pressure-diuresis mechanism (Kohan triphosphate (IP3 ) and diacylglycerol (DAG) et al., 2011) showed that even small increases and the IP3 releases Ca2+ from endoplasmic in arterial pressure often causes marked reticulum (Xuan and Glass, 1996). increase in urinary excretion of sodium and water. Interestingly, losartan rather than Thus, ET-1 has direct inotropic and bosentan reduces BP even though ET-1 had chronotropic effects on heart (Sakai et al., been infused for increasing BP. In Ang II 1999), thereby increases blood pressure. It was induced hypertension, bosentan also had no cleared that, ET-1 increases neurohormonal any role in reducing BP. Serum NO acts activate reflex and increase erythropoietin, then against ET-1 effects through dilating blood BP pressure tended to increase (Kohan et al., vessels and decreases the elevated BP (Khimji 2011). Because ET-1 acts in different and Rockey, 2010). The present results showed pathways, it requires enough time to elevate that intravenous infusion of ET-1could blood pressure, especially MAP ( Figure 1, A). decrease slightly serum NO (Table 1). Also, Bolus pre infusion of bosentan and losartan both bosentan and losartan increased serum NO could decrease BP, but only losartan reached level. The possible reason of this reduction of the level of significance (P<0.05). The only serum NO level may be through NOS possible mechanism to illustrate that, ET-1 has inhibition. However, ET-1 receptor elevate NO strong positive feedback on Ang II (Yoshida et production through its ETB (Stow et al., 2011), al., 1992). Therefore, we supposed that the but the reduction in our results may be due to short-term effect of rising blood pressure by the action of ETA receptor (Verhaar et al., ET-1, might enhancing Ang II activity. 1998). The physiological elevation of NO Therefore, blocking Ang II receptors would levels by losartan bolus infusion may also be diminish the actions of ET-1. Another possible due to the reduction of ET-1 and Ang II actions mechanism of weak hypotensive effect of as they have roles for reducing NO level. bosentan might to block both ETA and ETB which could not increase serum NO level For the first time, we showed that (Table 1). intravenous infusion of ET-1 for one hour, could increase PCV significantly (P<0.05), In experiment II, intravenous infusion of and at the same time bolus pre infusion, either Ang II continuously for one hour caused bosentan or losartan could slightly decrease increases MAP from ten minutes after infusion PCV. The possible explained mechanism is until the end of the experiment, but the rising in that, ET-1 decreases pressure natriuresis by BP was not equal in all time intervals. Ang II constricting nephrons and increases Bowman’s activation AT1 receptor causes constriction in hydrostatic pressure and then decreases GFR blood vessels, and increase peripheral (Elmarakby et al., 2003). The second possible resistance that leads to elevation of BP . mechanism of PCV reduction after ET-1 Therefore, blocking AT1 by pre infusion of infusion by inhibition Na+-K+ ATPase through losartan could decrease MAP. Also, Ang II by ETB receptor, thereby more water enters the direct positive inotropic and chronotropic RBC lead to elevation PCV (Prasanna et al., effects on heart (Modgil et al., 2012) . 2001). However, Ang II stimulates adrenal cortex 158 Sarkawt H. et al./ ZJPAS: 2016, 28(3): 152-160

However, previous studies explained excretion rate may be due to decreasing GFR that ET-1 decrease serum Ca2+ through and reducing urine excretion rates (Quadri, parathyroid hormone (PTH) inhibition (Chang 2013). This is clearly observed in the current et al., 2000), but the rational explanation for result (Figure 4, B). The present result is also increasing serum Ca2+ by short term ET-1 consistent with the finding of (Cho et al., infusion is that ET-1 directly causes the influx 2012), who showed that, Ang II increases of Ca2+ into the cell through L-type calcium sodium-2 chloride- potassium co transporter channel via IP3 mediation (Bkaily et al., 2011). mechanism, so that potassium reabsorption In contrast to previous studies of natriuretic increased and then urine potassium excretion effects of ET-1 (Bugaj et al., 2008), the present decreased. result showed that intravenous systemic infusion of ET-1 caused antinatriuretic effects, In conclusion, intravenous infusion of because it decreased Na+ excretion rate in ET- Ang II immediately (after 5 minutes) elevated 1 compared to saline group. (Nakano and MAP, produced upstroke, while MAP tended Pollock, 2009) demonstrated that ET-1 has to elevate after twenty minutes of ET-1 natriuretic effect in female rats but not in male infusion. ET-1 worked in several pathways to rats, and the present study conducted on male, perform hypertensive action, it mostly decrease therefore the decrease of urine concentration serum NO level. Interestingly, losartan rather and excretion might be due to sex difference. than bosentan reduces BP even ET-1 had been infused for increasing BP. In contrast to In the present result bosentan could previous studies of natriuretic effects of ET-1, increase Na+ excretion rate, because of the present result showed that intravenous removing of ET-1 effects on renal blood systemic infusion of ET-1 caused vessels. However, the exact relation between antinatriuretic effects, because it could ET-1 and K+ homeostasis is not well decrease Na+ excretion rate in ET-1 compared explained, but the possible mechanism of to saline group. In contrast to bosentan reduction in K+ excretion rate by ET-1 administration, which caused a slight decrease infusion is stimulation of 20 in K+ excretion rate, bolus infusion of losartan hydroxyeicosateraenoic acid (20-HETE) significantly elevated it as compared with Ang formation, which in turn can inhibit Na+-K+ II infused rats. ATPase activity (Escalante et al., 2002) and consequently K+ excretion may be reduced. Ang II has mutual signaling pathways with NO, Ang II via activation of AT2 lead to NO Acknowledgements production in the endothelial cells (Yan et al., 2003).While, the present result demonstrated We appreciate Savino Company, branch of that continuous infusion of Ang II decreases (Cipla-India) company as they supplied serum NO slightly. Ang II through AT1 Bosentan as gift for our research. receptor constricted blood vessels then decrease the serum NO concentration and lead References to BP elevation and then endothelial cell function diminished . Bkaily, G, L Avedanian, J Al-Khoury, C Provost, M Nader, P D'Orleans-Juste, and D At the first 20 minutes of Ang II infusion, Na+ Jacques( 2011). Nuclear membrane receptors excretion rate tended to increase. The pressure for ET-1 in cardiovascular function. Am J natriuresis mechanism may be the possible Physiol Regul Integr Comp Physiol, 300:17. reason behind the present finding. The present finding of Ang II inducing natriuresis is similar Bugaj, V, O Pochynyuk, E Mironova, A to ET-1 effects. Because both them increases Vandewalle, JL Medina, and JD Stockand, BP and consequently elevated Na+ excretion. (2008). Regulation of the epithelial Na+ The depressant effects of Ang II on potassium 159 Sarkawt H. et al./ ZJPAS: 2016, 28(3): 152-160 channel by endothelin-1 in rat collecting duct. Modgil, A, Q Zhang, A Pingili, N Singh, F Am J Physiol Renal Physiol, 295:30. Yao, J Ge, L Guo, C Xuan, ST O'Rourke, and C Sun, (2012). Angiotensin-(1-7) attenuates the Chang, JM, SJ Hwang, JC Tsai, and YH Lai, chronotropic response to angiotensin II via (2000). In vivo effect of endothelin-1 on stimulation of PTEN in the spontaneously plasma calcium and parathyroid hormone hypertensive rat neurons. Am J Physiol Heart concentrations. J Endocrinol, 165:179-184. Circ Physiol, 302:23.

Cho, HM, DY Lee, HY Kim, HA Lee, YM Nakano, D, and DM Pollock, (2009). Seok, and IK Kim, (2012). Upregulation of the Contribution of endothelin A receptors in Na(+)-K(+)-2Cl(-) cotransporter 1 via histone endothelin 1-dependent natriuresis in female modification in the aortas of angiotensin II- rats. Hypertension, 53:324-330. induced hypertensive rats. Hypertens Res, 35:819-824. Potts, LB, PD Bradley, W Xu, L Kuo, and TW Hein, (2013). Role of endothelium in Elmarakby, AA, P Morsing, and DM Pollock, vasomotor responses to endothelin system and (2003). Enalapril attenuates endothelin-1- protein kinase C activation in porcine retinal induced hypertension via increased kinin arterioles. Invest Ophthalmol Vis Sci, 54:7587- survival. Am J Physiol Heart Circ Physiol, 7594. 284:6. Prasanna, G, A Dibas, C Hulet, and T Yorio, Escalante, BA, JC McGiff, and AO Oyekan, (2001). Inhibition of Na(+)/K(+)-atpase by (2002). Role of cytochrome P-450 arachidonate endothelin-1 in human nonpigmented ciliary metabolites in endothelin signaling in rat epithelial cells. J Pharmacol Exp Ther, proximal tubule. Am J Physiol Renal Physiol, 296:966-971. 282:F144-150. Quadri, S, (2013). Augmentation of Heme Khimji, AK, and DC Rockey, (2010). Oxygenase Promotes Acute Angiotensin II Endothelin--biology and disease. Cell Signal, Induced Hypertension. Clinical and 22:1615-1625. Experimental Medical Sciences, Vol. 1:21 - 43.

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ZANCO Journal of Pure and Applied Sciences

The official scientific journal of Salahaddin University-Erbil ZJPAS (2016), 28 (3); 161-174

Cytotoxicity and Cytogenetic Effects of Local Purslane (Portulaca oleracea) Leaf Crude Extracts on Normal and Cancer Cell Lines In vitro Zakaria Ahmed Shekha 1* and Hazha Jamal Hidayat 2 1 College of Education / Science Departments, Biology Department 2Research Center, University Salahaddin, Hawler, Kurdistan Region, Iraq.

A R T I C L E I N F O A B S T R A C T

Article History: The present study was carried out to evaluate the cytotoxic effects of aqueous Received: 18/06/2015 and ethanol crude extracts of the leaf of Portulaca oleracea L. against two Accepted: 12/10/2015 malignant cell lines which were Murine mammary adenocarcinoma (AMN3), human Rhabdomyosarcoma (RD) and one normal cell line which was kidney Published:16/5/2016 epithelium of African green monkey (Vero). Also the study included the Keywords: cytogenetic effects of aqueous and ethanol crude extracts of P. oleracea leaf on Portulaca oleracea, cytotoxicity, the mitotic index of Vero, AMN3 and RD cell lines In vitro. After plant sample Mitotic index, Vero cell, RD treatment were observed and have reported through several assays such as cell and AMN3 cell trypan blue exlusion assay for cell viability, cytotoxicity assay by crystal violet *Corresponding Author: and mitotic index assay. The aqueous crude extract was found to be more effective antiproliferate agent than the ethanol crude extract. Both extracts Zakaria A Shekha exhibited time-dependent cytotoxic effects against AMN3 and RD cancer cell Email: lines. In which AMN3 was more sensitive to the extracts than RD. All concentrations of aqueous extract were affected AMN3 cell line at 72hrs. of exposure, but for ethanol extract the last dose (0.01µg/ ml) was not effective. While the Vero normal cell line showed resistance toward all concentrations of both extracts except the first dose (10000µg/ ml), at all time of exposure (24, 48 and 72)hrs.

1. Introduction human cancers may be attributable to Cancer is a genetic disease. It is characterized by environmental, lifestyle factors such as tobacco, unrestrained growth and reproduction of cells, loss alcohol, and dietary habits (Sulaiman, 2000, of contact inhibition, and eventually metastasis. All Murthy and Mathew 2004) . of these changes represent underlying mutation or in appropriate expression of genes involved in the Portulaca oleracea L. (Purslane) has been used as a control of the cell cycle and related processes kind of food and medicinal plant for thousands of (Ness and Knight 2004). The bombardment of years in China. P. oleracea is very important Halabja city in Iraqi Kurdistan region in 1988 with because of its special medical function and all its chemical weapons, caused more than 5000 therapeutic values are attributed to the presence martyrs and 10000 injury victim survivors are still of many biologically active compounds which suffering from a variety of health complains, which include; flavonoids (Apigenin, kaempferol, include cancers, and other health complains like quercetin, luteolin, myricetin, genistein, and respiratory, ophthalmological, dermatological, genistin), Alkaloids, Coumarins, anthraquinone reproductive, and immunological complains (Hama glycoside, cardiac glycoside, and high content of ω- et al., 2009). It is widely held that (80- 90 %), of 3 fatty acids (Sharma et al,. 2011). 162 Zakaria A. et al./ ZJPAS: 2016, 28(3): 161-174

2- Material and methods appeared to be colorless. The solution was collected in a clean bottle, and then dried in rotary 2.1- Plant collections evaporator at 40 °C. This final crude extract stored The vegetative part of Portulaca oleraceae was at -20 °C until using in the experiment. collected from Khabat/ Hawler (Erbil) city- Kurdistan region/ Iraq in August 2010. The plant was washed then the leaves were separated and 2.4- Ethanol extracts preparation left to dry at room temperature in the shade for nearly three weeks. Dried leaves were ground to The ethanol extract of Portulaca oleraceae leaf fine powder. Using blender then passed through was prepared as the same way as aqueous extract, except of using 80 % ethyl alcohol instead fine pored mesh sieve to provide homogeneous of DDW (Karimi et al., 2009). From 100 grams of powder. plant powder 30 grams of aqueous crude extract 2.2 - plant classification were obtained, and 18 grams of ethanol crude extract obtained from other 100 grams of plant The plant was classified by botanist in the biology powder. department of college of education, scientific departments 2.5- Cell lines

Kingdom: Plantae (Plants( Vero is the normal cell line was initiated from kidney of a normal adult African green monkey Sub kingdom: Tracheobionta (Vascular Plants( (Cercopitecus aethiops) on 27th March 1962, by Division: Spermatophyta (Seed Plant) Yasummura and Kawakita at the Chiba University, Japan. The AMN3 cancer cell line was derived from Sub division: Angiospermae spontaneous mammary adenocarcinoma from female Balb/ C mice, and RD human cancer cell line Class: Dicotyledoneae was derived from a biopsy specimen obtained from Order: Caryophyllales pelvic rhabdomyosarcoma. All the cell lines were supplied by Tissue Culture Unit/ Iraqi Center for Family: Portulacaceae Cancer and Medical Genetic Research (ICCMGR, Baghdad/ Iraq) then maintained on MEM, and Genus: Portulaca RPMI media with 10 % of fetal bovine serum Species: P. olaracea L. (Townsend and Guest, culture, 1% penicillin- Streptomycin, cells were 1966). incubated at 37 ˚C in a humidified atmosphere of %5 CO2 in air.

2.6- Viable cell counting 2.3- Aqueous extract preparations Viable cell counting, accomplished by using trypan Fifty grams of macerated dried leaves were placed blue exclusion. Dead cells unlike viable cells take in 500ml volumetric flask then 400 ml of deionized up trypan blue within a few seconds making them distilled water (DDW), added(Karimi et al., 2009). easily distinguishable under the microscope from The volumetric flask was incubated in Ultrasonic viable cells which were metabolitically alive had a bath for two hours (Claver et al., 2000). Then the cell membrane that was active selective in the suspension was filtered through a clean gauze and materials that pass in to the cytoplasm (Freshney, then through a Bückner funnel several times. The 1994). residues re extracted for 48 and then 72 hours. The filtrations were performed daily, until the solution 2.7- Cell growths assay 163 Zakaria A. et al./ ZJPAS: 2016, 28(3): 161-174

The dry crud extracts were dissolved in 3- Results and Discussion maintenance media were used to obtain serial dilutions of (10.000 μg/ ml, 1000 μg/ ml, 100 μg/ 3.1- Cell growth assay ml, 10 μg/ ml, 1 μg/ ml, 0.1 μg/ ml, 0.01 μg/ ml) The effect of different concentrations of aqueous and 0 μg/ml as a control to study the effects of and ethanol crude extracts of P. oleracea on (Vero, aqueous and ethanolic crude extracts on different AMN3 and RD) cell lines in vitro cell lines. Cell monolayer were trypsinized washed with PBS buffer, Cells cultured (50000 cells/ ml for Figure (1): shows the mean of optical densities of AMN3, 65000 cells/ ml for RD, and 50000 cells/ ml aqueous crude extract on the cell lines (Vero, for Vero) in the microtitration plate were exposed AMN3 and RD) at 24hrs exposure revealing the to a range of plant extract concentrations during reduction of the viability of cell culture. RD cell the log phase of growth and the effect was lines was more sensitive to the plant extract in the determined of optical density of solution was first three concentrations (10000 µg/ ml, 1000 µg/ measured at 492nm using a microplate reader ml, and 100 µg/ ml) While AMN3 more than RD, in (ELISA)(Freshney, 1994). the fourth concentration also, if they compared to their controls (0 µg/ ml). But in the four last 2.8- Mitotic index concentrations, (10, 1, 0.1 and 0.01) µg/ ml, the RD The number of cells which arrested in the cancer cells not changed. But the Vero cells were Metaphase were counted and also the total slightly sensitive to the first concentration (10000 µg/ ml). The figure (2) shows the effect of ethanol number of cells (1000 cells were counted) (Subber crude extract, nearly had the same effect, but and Juma, 1999, Lima et al., 2005), then the AMN3 cells were more sensitive than RD, they mitotic index was calculated as follow: inhibited by most of the concentrations, while RD Number of cells in mitotic phase was resistant in the last three concentrations (1, 0.1 and 0.01) µg/ ml). But Vero cells were sensitive Mitotic index (%) = ———————— x 100 in the first concentration (10000 µg/ ml) only, if Total number of cells compared to the control.

2.9- Statistical analysis FIgure (3) Shows the effect of aqueous crude extract of P. oleracea at 48hrs of exposure showing The results were analyzed with a prepared no important changes in Vero normal cell line program of SPSS, version 18 (Susan et al., 1997). except in the first concentration (10000µg/ ml), if Factorial experiment with a completely compared to the control. While AMN3 cancer cell randomized design (CRD) were used for the reason line was more sensitive to all concentrations of different factors; such as two types of crude except the final (0.01 µg/ ml). But RD cancer cells extracts; (aqueous and ethanol), three types of cell were resistant to the last three concentrations (1 lines; Vero (normal), AMN3 and RD (cancer), three µg/ ml, 0.1 µg/ ml, and 0.01 µg/ ml). Also in the periods; (24, 48 and 72) hrs. the eight figure (4): by the use of ethanol extract at the concentrations of the extracts; (0 µg/ ml as 48hrs of exposure. The Vero cells were inhibited control, 10000 µg/ ml, 1000 µg/ ml, 100 µg/ ml, 10 it's growth with the first (10000 µg/ ml) and fifth (1 µg/ ml, 1 µg/ ml, 0.1 µg/ ml and 0.01 µg/ ml) and µg/ ml) concentrations. But the two cancer cells five replications for each concentrations. were showed their sensitivities especially AMN3 in the first four (10000, 1000, 100, 10) µg/ ml, and

sixth (0.1 µg/ ml) concentrations, but RD cancer cells like aqueous extract were inhibited their growths in the first four (10000, 1000, 100, 10) µg/ ml concentrations. But in the last three 164 Zakaria A. et al./ ZJPAS: 2016, 28(3): 161-174 concentrations (1, 0.1, 0.01) µg/ ml of the RD cells factors such as tobacco, alcohol, and dietary habits have no important changes observed. But at the 72 (Sulaiman, 2000, Murthy and Mathew 2004) . hrs of exposure, in the use of aqueous extract the growth of AMN3 cancer cell line was inhibited with Portulaca oleracea L. (Purslane) has been used as a kind of food and medicinal plant for thousands of all concentrations. Also the RD cells were inhibited by most of the concentrations except the last two years in China. P. oleracea is very important (0.1, 0.01) µg/ ml concentrations, while the Vero because of its special medical function and all its therapeutic values are attributed to the presence normal cell line was inhibited slightly by the first two (10000, 1000) µg/ ml, concentrations, as of many biologically active compounds which showing in the figure (5). But in the figure (6) by include; flavonoids (Apigenin, kaempferol, quercetin, luteolin, myricetin, genistein, and the use of ethanol extracts the Vero cells were sensitive to the first concentration (10000 µg/ ml) genistin), Alkaloids, Coumarins, anthraquinone only. But the growth of AMN3 cancer cell line was glycoside, cardiac glycoside, and high content of ω- inhibited by all concentrations just the last (0.01 3 fatty acids (Sharma et al,. 2011). µg/ ml) one. While the RD cancer cell line was inhibited it's growth with the first three (10000, 1000, 100) µg/ ml, and fifth (1 µg/ ml) 2- Material and methods concentrations, if they compared to their controls. 2.1- Plant collections It is revealed that in all six figures; the two The vegetative part of Portulaca oleraceae was cancer cell lines AMN3 and RD were more collected from Khabat/ Hawler (Erbil) city- inhibited their growths with aqueous extract than Kurdistan region/ Iraq in August 2010. The plant ethanol extract, and slightly changes were was washed then the leaves were separated and recorded to Vero normal cell line especially at the left to dry at room temperature in the shade for first higher concentration. And the reduction of nearly three weeks. Dried leaves were ground to cell viability was increased by increasing fine powder. Using blender then passed through concentrations, which were dose dependent fine pored mesh sieve to provide homogeneous manner. And the reduction of viability of cell powder. culture also increased by increasing time of exposure.Cancer is a genetic disease. It is 2.2 - plant classification characterized by unrestrained growth and reproduction of cells, loss of contact inhibition, and The plant was classified by botanist in the biology eventually metastasis. All of these changes department of college of education, scientific represent underlying mutation or in appropriate departments expression of genes involved in the control of the Kingdom: Plantae (Plants( cell cycle and related processes (Ness and Knight 2004). The bombardment of Halabja city in Iraqi Sub kingdom: Tracheobionta (Vascular Plants( Kurdistan region in 1988 with chemical weapons, caused more than 5000 martyrs and 10000 injury Division: Spermatophyta (Seed Plant) victim survivors are still suffering from a variety of Sub division: Angiospermae health complains, which include cancers, and other health complains like respiratory, Class: Dicotyledoneae ophthalmological, dermatological, reproductive, and immunological complains (Hama et al., 2009). Order: Caryophyllales It is widely held that (80- 90 %), of human cancers Family: Portulacaceae may be attributable to environmental, lifestyle 165 Zakaria A. et al./ ZJPAS: 2016, 28(3): 161-174

Genus: Portulaca culture, 1% penicillin- Streptomycin, cells were incubated at 37 ˚C in a humidified atmosphere of Species: P. olaracea L. (Townsend and Guest, %5 CO2 in air. 1966). 2.6- Viable cell counting

Viable cell counting, accomplished by using trypan 2.3- Aqueous extract preparations blue exclusion. Dead cells unlike viable cells take Fifty grams of macerated dried leaves were placed up trypan blue within a few seconds making them in 500ml volumetric flask then 400 ml of deionized easily distinguishable under the microscope from distilled water (DDW), added(Karimi et al., 2009). viable cells which were metabolitically alive had a The volumetric flask was incubated in Ultrasonic cell membrane that was active selective in the bath for two hours (Claver et al., 2000). Then the materials that pass in to the cytoplasm (Freshney, suspension was filtered through a clean gauze and 1994). then through a Bückner funnel several times. The 2.7- Cell growths assay residues re extracted for 48 and then 72 hours. The filtrations were performed daily, until the solution The dry crud extracts were dissolved in appeared to be colorless. The solution was maintenance media were used to obtain serial collected in a clean bottle, and then dried in rotary dilutions of (10.000 μg/ ml, 1000 μg/ ml, 100 μg/ evaporator at 40 °C. This final crude extract stored ml, 10 μg/ ml, 1 μg/ ml, 0.1 μg/ ml, 0.01 μg/ ml) at -20 °C until using in the experiment. and 0 μg/ml as a control to study the effects of aqueous and ethanolic crude extracts on different 2.4- Ethanol extracts preparation cell lines. Cell monolayer were trypsinized washed The ethanol extract of Portulaca oleraceae leaf with PBS buffer, Cells cultured (50000 cells/ ml for was prepared as the same way as aqueous AMN3, 65000 cells/ ml for RD, and 50000 cells/ ml extract, except of using 80 % ethyl alcohol instead for Vero) in the microtitration plate were exposed of DDW (Karimi et al., 2009). From 100 grams of to a range of plant extract concentrations during plant powder 30 grams of aqueous crude extract the log phase of growth and the effect was were obtained, and 18 grams of ethanol crude determined of optical density of solution was extract obtained from other 100 grams of plant measured at 492nm using a microplate reader powder. (ELISA)(Freshney, 1994).

2.5- Cell lines 2.8- Mitotic index

Vero is the normal cell line was initiated from The number of cells which arrested in the kidney of a normal adult African green monkey Metaphase were counted and also the total (Cercopitecus aethiops) on 27th March 1962, by number of cells (1000 cells were counted) (Subber Yasummura and Kawakita at the Chiba University, and Juma, 1999, Lima et al., 2005), then the Japan. The AMN3 cancer cell line was derived from mitotic index was calculated as follow: spontaneous mammary adenocarcinoma from Number of cells in mitotic phase------x 100 female Balb/ C mice, and RD human cancer cell line was derived from a biopsy specimen obtained from Total number of cells pelvic rhabdomyosarcoma. All the cell lines were supplied by Tissue Culture Unit/ Iraqi Center for Cancer and Medical Genetic Research (ICCMGR, Baghdad/ Iraq) then maintained on MEM, and RPMI media with 10 % of fetal bovine serum 166 Zakaria A. et al./ ZJPAS: 2016, 28(3): 161-174

2.9- Statistical analysis compared to the control. While AMN3 cancer cell line was more sensitive to all concentrations The results were analyzed with a prepared except the final (0.01 µg/ ml). But RD cancer cells program of SPSS, version 18 (Susan et al., 1997). were resistant to the last three concentrations (1 Factorial experiment with a completely µg/ ml, 0.1 µg/ ml, and 0.01 µg/ ml). Also in the randomized design (CRD) were used for the reason figure (4): by the use of ethanol extract at the of different factors; such as two types of crude 48hrs of exposure. The Vero cells were inhibited extracts; (aqueous and ethanol), three types of cell it's growth with the first (10000 µg/ ml) and fifth (1 lines; Vero (normal), AMN3 and RD (cancer), three µg/ ml) concentrations. But the two cancer cells periods; (24, 48 and 72) hrs. the eight were showed their sensitivities especially AMN3 in concentrations of the extracts; (0 µg/ ml as the first four (10000, 1000, 100, 10) µg/ ml, and control, 10000 µg/ ml, 1000 µg/ ml, 100 µg/ ml, 10 sixth (0.1 µg/ ml) concentrations, but RD cancer µg/ ml, 1 µg/ ml, 0.1 µg/ ml and 0.01 µg/ ml) and cells like aqueous extract were inhibited their five replications for each concentrations. growths in the first four (10000, 1000, 100, 10) µg/ 3- Results and Discussion ml concentrations. But in the last three concentrations (1, 0.1, 0.01) µg/ ml of the RD cells 3.1- Cell growth assay have no important changes observed. But at the 72 hrs of exposure, in the use of aqueous extract the The effect of different concentrations of aqueous growth of AMN3 cancer cell line was inhibited with and ethanol crude extracts of P. oleracea on (Vero, all concentrations. Also the RD cells were inhibited AMN3 and RD) cell lines in vitro by most of the concentrations except the last two Figure (1): shows the mean of optical densities of (0.1, 0.01) µg/ ml concentrations, while the Vero aqueous crude extract on the cell lines (Vero, normal cell line was inhibited slightly by the first AMN3 and RD) at 24hrs exposure revealing the two (10000, 1000) µg/ ml, concentrations, as reduction of the viability of cell culture. RD cell showing in the figure (5). But in the figure (6) by lines was more sensitive to the plant extract in the the use of ethanol extracts the Vero cells were first three concentrations (10000 µg/ ml, 1000 µg/ sensitive to the first concentration (10000 µg/ ml) ml, and 100 µg/ ml) While AMN3 more than RD, in only. But the growth of AMN3 cancer cell line was the fourth concentration also, if they compared to inhibited by all concentrations just the last (0.01 their controls (0 µg/ ml). But in the four last µg/ ml) one. While the RD cancer cell line was concentrations, (10, 1, 0.1 and 0.01) µg/ ml, the RD inhibited it's growth with the first three (10000, cancer cells not changed. But the Vero cells were 1000, 100) µg/ ml, and fifth (1 µg/ ml) slightly sensitive to the first concentration (10000 concentrations, if they compared to their controls. µg/ ml). The figure (2) shows the effect of ethanol It is revealed that in all six figures; the two crude extract, nearly had the same effect, but cancer cell lines AMN3 and RD were more AMN3 cells were more sensitive than RD, they inhibited their growths with aqueous extract than inhibited by most of the concentrations, while RD ethanol extract, and slightly changes were was resistant in the last three concentrations (1, recorded to Vero normal cell line especially at the 0.1 and 0.01) µg/ ml). But Vero cells were sensitive first higher concentration. And the reduction of cell viability was increased by increasing in the first concentration (10000 µg/ ml) only, if concentrations, which were dose dependent compared to the control. manner. And the reduction of viability of cell culture also increased by increasing time of FIgure (3) Shows the effect of aqueous crude exposure. extract of P. oleracea at 48hrs of exposure showing no important changes in Vero normal cell line except in the first concentration (10000µg/ ml), if 167 Zakaria A. et al./ ZJPAS: 2016, 28(3): 161-174

Fig. (4): Mean of optical densities of (Vero, AMN3 and RD) Fig. (1): Mean of optical densities of (Vero, AMN3 and RD) cell lines after exposure to different concentrations of cell lines after exposure to different concentrations of ethanol crude extract of P. oleracea for 48hrs of exposure. aqueous crude extract of P. oleracea for 24hrs of exposure.

Fig. (5): Mean of optical densities of (Vero, AMN3 and RD) cell lines after exposure to different concentrations of aqueous crude extract of P. oleracea for 72hrs of exposure Fig. (2): Mean of optical densities of (Vero, AMN3 and RD) cell lines after exposure to different concentrations of ethanol crude extract of P. oleracea for 24hrs of exposure.

Fig. (6): Mean of optical densities of (Vero, AMN3 and RD) cell lines after exposure to different concentrations of ethanol crude extract of P. oleracea for 72hrs of exposure. Fig. (3): Mean of optical densities of (Vero, AMN3 and RD) cell lines after exposure to different concentrations of Table (1): Mean ± S.E of the optical densities for Vero, aqueous crude extract of P. oleracea for 48hrs of AMN3 and RD cell lines respectively after treatment by exposure. different concentrations of aqueous and ethanol crude extracts in a cell growth assay in vitro:- 168 Zakaria A. et al./ ZJPAS: 2016, 28(3): 161-174

normal cell

Cell lines line. The Aqueous Extract Ethanol Extract inhibition Conc. µg/ ml Vero AMN3 RD Vero AMN3 RD C0 0.3328±0.0168 0.3008±0.0043 0.3644±0.0012 0.3286±0.0147 0.3008±0.0043 0.3600±0.0053 activity of C1 0.1798±0.0042∗∗ 0.0736±0.0031∗∗ 0.0838±0.0104∗∗ 0.2298±0.0147∗∗ 0.0686±0.0048∗∗ 0.0910±0.0073 both ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ extracts C2 0.3029±0.0042 0.0626±0.0014 0.1388±0.0150 0.3322±0.0337 0.1106±0.0061 0.1902±0.0090 ∗∗ (aqueous C3 0.3116±0.0085∗∗ 0.1166±0.0092∗∗ 0.2322±0.0270∗∗ 0.3060±0.0329∗∗ 0.1516±0.0184∗∗ 0.2308±0.0228 ∗∗ and C4 0.3470±0.0339 0.2170±0.0043∗∗ 0.3446±0.0101 0.3250±0.0399 0.1918±0.0122∗∗ 0.2700±0.0168 24 hrs. ∗∗ ethaolic) 0.3252±0.0214 0.2798±0.0146∗∗ 0.3494±0.0040 0.3384±0.0231 0.1718±0.0156∗∗ 0.3454±0.0151 C5 against ∗∗ ∗∗ C6 0.3540±0.0236 0.2828±0.0094 0.3458±0.0142 0.3462±0.0275 0.2796±0.0110 0.3398±0.0082 AMN3 0.3506±0.0169 0.2876±0.0092 0.3662±0.0099 0.3454±0.0281 0.2800±0.0209∗∗ 0.3554±0.0188

C7 C0 0.2036±0.0048 0.2912±0.0044 0.3270±0.0044 0.2036±0.0048 0.2912±0.0044 0.3270±0.0044 C1 0.1316±0.0070∗∗ 0.0670±0.0082∗∗ 0.0688±0.0021∗∗ 0.1386±0.0079∗∗ 0.0718±0.0043∗∗ 0.1108±0.0229 ∗∗ C2 0.1694±0.0065∗∗ 0.0736±0.0103∗∗ 0.0880±0.0198∗∗ 0.2188±0.0081 0.0804±0.0104∗∗ 0.1382±0.0094 ∗∗ C3 0.2050±0.0099 0.1158±0.0198∗∗ 0.1556±0.0293∗∗ 0.2114±0.0075 0.1110±0.0098∗∗ 0.1710±0.0205 ∗∗ 0.2048±0.0133 0.1846±0.0116∗∗ 0.2142±0.0105∗∗ 0.2146±0.0084 0.1438±0.0011∗∗ 0.2378±0.0046 48hrs. C4 ∗∗ C5 0.2122±0.0231 0.1972±0.0043∗∗ 0.3064±0.0115 0.1686±0.0074∗∗ 0.2854±0.0147 0.2902±0.0058 ∗∗ C6 0.1934±0.0080 0.1982±0.0021∗∗ 0.3144±0.0217 0.2066±0.0062 0.1954±0.0050∗∗ 0.3096±0.0027 0.1854±0.0092 0.2780±0.0042 0.3068±0.0035 0.2098±0.0069 0.2784±0.0120 0.3069±0.0085 C7 C0 0.1374±0.0039 0.2436±0.0035 0.3256±0.0076 0.1454±0.0039 0.2436±0.0035 0.3246±0.0080 C1 0.0618±0.0059∗∗ 0.0666±0.0012∗∗ 0.0632±0.0090∗∗ 0.0702±0.0032∗∗ 0.0742±0.0031∗∗ 0.0666±0.0209 ∗∗ C2 0.1008±0.0098∗∗ 0.0832±0.0052∗∗ 0.0906±0.0088∗∗ 0.1520±0.0049 0.0900±0.0042∗∗ 0.1170±0.0157 ∗∗

C3 0.1478±0.0040 0.0938±0.0068∗∗ 0.1186±0.0311∗∗ 0.1568±0.0069 0.1330±0.0155∗∗ 0.1462±0.0230 ∗∗ 0.1596±0.0037 0.1256±0.0028∗∗ 0.1372±0.0173∗∗ 0.1498±0.0055 0.1686±0.0131∗∗ 0.2942±0.0198 72hrs. C4 ∗∗ C5 0.1520±0.0017 0.1668±0.0024∗∗ 0.1628±0.0280∗∗ 0.1438±0.0041 0.1628±0.0118∗∗ 0.1608±0.0113 ∗∗ C6 0.1542±0.0023 0.1590±0.0049∗∗ 0.3084±0.0195 0.1440±0.0074 0.1646±0.0111∗∗ 0.3094±0.0292 0.1566±0.0036 0.1404±0.0244∗∗ 0.3130±0.0324 0.1642±0.0024 0.2286±0.0073 0.2856±0.0268

C7 ∗∗ LSD 0.021 0.0148 0.021 0.021 0.014 0.021

cancer cells were more than against RD cancer C0= 0 µg/ ml (Control), C1= 10.000 µg/ ml, C2=1000 µg/ ml, cells. It means AMN3 was more sensitive to C3=100 µg/ ml, C4=10 µg/ ml, C5= 1 µg/ ml, C6= 0.1 µg/ ml, aqueous and ethanolic crude extracts than RD, C7= 0.01 µg/ ml. this can be attributed to the sensitivity of AMN3 to the extracts more than RD, and/ or Results of the present study as in table (1) may be due to sensitivity of death receptors of showed that most concentrations were effective AMN3 plasma membrane to the active against two cancer cell lines (AMN3 and RD) compounds (especially flavonoids) in both especially at 72hrs of aqueous and ethanolic extracts of P. oleracea. and RD receptors may extracts. While the concentration (10.000 µg/ be less sensitive to the compounds. But Vero ml) was affect the Vero (normal cell line), but (normal) cell line was resistant toward all the other concentrations were safe to the concentrations of both extracts except the first 169 Zakaria A. et al./ ZJPAS: 2016, 28(3): 161-174 concentration (10000 µg/ ml). So the Vero The significant activity of aqueous and (normal) cell line was used for comparison ethanolic crude extracts of P. oleracea are with cancer cell lines. thought to be a result of the induction of cell death by apoptosis as our result indicate. Also the results showed that the growth Flavonoids would induce apoptosis in cancer inhibition of the aqueous and ethanolic crude cells as a desired strategy of controlling extracts of P. oleracea which exhibited time- cancers (Talib and Mohasneh, 2010). Our dependent inhibitory effects on AMN3 and RD results would support the previous studies cancer cell lines. The viability reaches it's especially DNA fragmentation, nuclear lowest, with extended time of exposure for 48 condensation, and cell shrinkage which were and 72hrs. Also concentration of active clearly observed, cytoplasmic disruption, compounds of both extracts were needed to cytoplasmic vaculation, pyknosis and reduce the cell viability, the higher karyorrhexis. Flavonoids may exert regulatory concentrations were strongly affected the activities in cells through actions at different AMN3 and RD cancer cell lines. Besides the signal transduction pathways such as; cyclin aqueous extract of P. oleracea at 72hrs. of dependent kinase (cdk), caspases, Bcl2 and exposure had the highest inhibitory effect Bax family members, epidermal growth factor/ against cancer cell lines than ethanolic extract epidermal growth factor receptor, at any time of exposure. The higher doses at phosphatidylinositol-3-kinase/ Akt, mitogen longer time of exposure had stronger inhibitory activated protein kinase (MAPK), and nuclear effect against cancer cell lines than lower doses factor kappa B (NFkB), which may affect at shorter time of exposure. Also the aqueous cellular function by modulating genes or extract of P. oleracea in higher doses were phosphorylating proteins (Das et al,. 2010). stronger affected the cancer cells than ethanolic Flavonoids also contribute to cancer prevention extract and lower doses. All these findings may by other mechanisms such as radical due to more solubility of active compounds in scavenging, detoxification of mutagenic water more than ethanol, and functioning of xenobiotics and inhibition of topoisomerase these compounds when they present at higher cyclin-dependent kinases, protein kinases concentrations, and at longer time of exposure. including phosphotidylinositol 3-kinase. 3.2- Cytological study Flavonoids recently showed that inhibited fatty acid synthase (FAS) activity and lipogenesis, The phenotypic characteristics of (Vero, this effect is strongly associated with growth AMN3 and RD) cell lines after exposed to arrest and cell death. Blockage of FAS by the aqueous and ethanolic crude extracts of P. chemical inhibitors will inhibits proliferation oleraceae were evaluated by using inverted and cytotoxic for various tumors cell lines in microscope. The results showed the different vitro and in vivo. Expression of FAS is cytological changes such as chromatin markedly increased in various human cancers, condensation, pyknosis, karyorrhexis, including; prostate, breast, ovary, cytoplasmic vaculation, cell shrinkage and endometrium, colon and lung (Brusselman et cytoplasmic disruption and these changes were al., 2005). depended on the different concentrations of the plant extracts and the periods of exposure (Fig.7-8-9). 170 Zakaria A. et al./ ZJPAS: 2016, 28(3): 161-174

i ii iii

Fig. 7 Fig.

(11): IC50 values for the cytotoxicity effects of P. oleracea i ii ethanol crude extract on (Vero, AMN3 and RD) cell lines for 72 hours of expos

Fig. 8 i ii

Fig. (12): Mean of mitotic index of (Vero AMN3 and Fig. 9 RD) cell lines after exposure to different concentrations of Figure 7. (i, ii, iii) The untreated Vero (normal) cells, AMN3 and RD cancer cell aqueous crude extract of P. oleracea for 24hrs lines tend to form a confluent monolayer respectively. The shape of AMN3 cancer cells was oval, while RD cancer cells appeared elongate to spindle in shape. Figure 8. (i):AMN3 cells treated by aqueous crude extract of P. oleracea 10000 µg/ ml for 48hrs Shows (a): Masses of the dead cells, (b): Disrupted apoptotic cells. (ii): AMN3 cells treated by aqueous extract of P. oleracea 10000 µg/ ml for 72hrs Shows (a): Completely dead cells (b): Cell shrinkage (400X Crystal violet). Figure 9. (i): AMN3 cells treated by Ethanolic extract of P. oleracea10000 µg/ ml for 72hrs. Shows (a): pyknosis, (b): swelling and condensation of nuclear chromatin, (c): Disruption of plasma membrane, cytoplasm and karyorrhexis, (d): completely disruption of monolayer, (e): multinucleated giant cells, (ii): RD cells treated by ethanolic extract of P. oleracea1000 µg/ ml for 72hrs Shows (a): pyknosis, (b): Cell swelling (400X Crystal violet).

Fig. (13): Mean of mitotic index of (Vero AMN3 and RD) cell lines after exposure to different concentrations of ethanol crude extract of P. oleracea for 24hrs

Fig. (10): IC50 values for the cytotoxicity effects of P. oleracea aqueous crude extract on (Vero, AMN3 and RD) cell lines for 72 hours of exposure

171 Zakaria A. et al./ ZJPAS: 2016, 28(3): 161-174

1 2

Fig. (14): Mean of mitotic index of (Vero, AMN3 and RD) cell lines after exposure to different concentrations of aqueous crude extract of P. oleracea for 48hrs Fig. (18): A: Vero cells during (1): metaphase and (2): prophas counted for mitotic index (400 X). B: Chromosomes of Vero cells 8 (1000 X) untreated to plant extracts

6 VE Aqueous crude extract of P. oleracea causes 4 RO 50% cell death in the two (AMN3, RD) cancer 2 cell lines tested. The best IC50 values obtained

Mean MI 0 were (27.33, 34.15 and 38.50) µg/ml for the µg/ml) three concentrations (10000, 1000 and 100) ١٠٠٠ Concentrations١٠٠٠٠ ٠ µg/ml), respectively causing 50% of the AMN3

Fig.(15): Mean of mitotic index of (Vero,AMN3 and cancer cell line to die. But for RD cancer cell RD) cell lines after exposur to different line the IC50 values were (21.25, 27.82 and concentrations oethano crude extract of P. 39.43 µg/ml) for the three concentrations oleracea for 48hrs. (10000, 1000 and 0.1) µg/ml, respectively caused RD cancer cell to die, as in figure 10. 8 6 In the figure 11, Ethanol crude extract of P. VER 4 O oleracea causes 50% cell death in the two 2Mean MI (AMN3, RD) cancer cell lines tested. The best

0 IC50 values obtained were (30.45 and 36.94) µg/ml for the two concentrations (10000 and ١٠٠٠ ١٠٠٠٠ ٠ Concentrations (µg/ml) 1000) µg/ml), respectively causing 50% of the Fig.(16): Mean of mitotic index of (Vero AMN3 and RD) cell lines after exposure to different concentrations of AMN3 cancer cell line to die. The IC50 values aqueous crude extract of P. oleracea for 72hrs for RD cancer cell line were (20.51 and 35.67)

µg/ml for the two concentrations (10000 and 8 1000 µg/ml), respectively causing 50% RD cell

6 death. But the IC50 values for the Vero normal VER 4 O cell line were (46.78 and 46.90) µg/ml for

Mean MI aqueous and ethanol crude extracts respectively 2 causing 50% cell death except in the highest 0 (10000) µg/ml concentration. (µg/ml) ١٠٠٠ Concentrations١٠٠٠٠ ٠

3.3- The effect of two higher concentrations of aqueous and ethanol crude extracts of P. Fig.(17): Mean of mitotic index of (Vero, AMN3 and RD) cell lines after exposure oleracea on the mitotic index of Vero, AMN to different concentrations of ethanol crude

172 Zakaria A. et al./ ZJPAS: 2016, 28(3): 161-174

3 and RD cell lines: solubility of active compounds in water than ethanol. The Vero cell line was more resistant The figures (12 and 13), show the mean of than AMN3 and RD cancer cell lines, because mitotic index of (Vero, AMN3 and RD) cell the cell division (mitosis) in AMN3 and RD lines. In which the cells were exposed to two cancer cells were highly decrease by both higher concentrations (10.000 and 1000) µg/ concentrations (10000 and 1000) µg/ ml. of ml of aqueous and ethanol crude extracts of P. aqueous and ethanolic crude extracts, and for oleracea for 24hrs. In which the mitosis (cell the three periods of exposure (24, 48 and 72) division) of the two cancer cell lines (AMN3 hrs especially at 72hrs. This was due to the and RD) were significantly (P<0.01) decreased effect of active constituents found in P. in aqueous extract more than ethanol extract. oleracea functioning as antiproliferative and But the Vero (normal cell line) was slightly apoptotic and cytotoxic agents, such as decreased and not affected at1000 µg/ ml. if flavonoids, alkaloids, anthraquinones, compared to their controls. In case of 48hrs of coumarins..etc. It is demonstrated that dietary exposure the cell division of two cancer cell flavonoids inhibit the proliferation of various lines AMN3 and RD were significantly cancer cells and tumor growth in animal (P<0.01) decreased more than 24hrs. if models, and protect against cancer induction in compared to their controls, as shown in figures several human tissues (Szliszka et al., 2008). (14 and 15). But in the 72hrs of exposure of Several flavonoids regulate the genes that are aqueous and ethanol crude extracts, the mitotic critical for the control of proliferation, cell index was high significantly (P<0.01) affected cycle and apoptosis pathway in cancer cells. and decreased in both AMN3 and RD cancer Recent reports demonstrated that apigenin and cell lines also Vero was slightly decreased as genistein caused a G2/ M arrest of the cell compare with controls, figures (16 and 17). cycle in several human and murine cell lines. Quercetin has been shown to inhibit cell cycle 3.4- Cytogenetic effect of aqueous and progression at the G1-S phase in human gastric ethanolic crude extract of P. oleracea cancer cell line HGC-27 (Lepley et al., 1996). on Vero, AMN3 and RD cell lines in The molecular mechanism of cell cycle vitro: arrest by apigenin was conducted by inhibiting cdc2 The study of mitotic index was used to of cyclin B1 and phosphorylation of p34 determine the rate of cell division. In this study protein in two mouse skin cancer keratinocyte the first two higher concentrations (10000 µg/ cell lines (C50 and 308) and HL-60 human ml and 1000 µg/ ml) of aqueous and ethanolic mylogenous leukemia cells. Since cyclin B- cdc2 crude extracts were tested on the three cell p34 complexes regulate G2/ M progression. cdc2 lines Vero (normal) cell line, and two cancer Inactivation of cyclin B-p34 is required for cell lines (AMN3 and RD) for three periods of exit from mitosis and results from degradation exposure (24, 48 and 72) hrs. In which the rate of cyclin B (Lepley et al., 1996). Also apigenin of cell division of Vero cell line was slightly arrests the cell cycle at the G2/ M phase decreased when exposed to the high dose of accompanied by decrease of cyclin B1 in plant extract 10000 µg/ ml for 72 hrs and 48 human colorectal cancer cells (Townsend and hrs of exposure. But at 24hrs there were no any Guest 1966). And induce p21 level resulting in important changes in cell division. In which the cell cycle arrest and apoptosis in human aqueous extract was more effective than prostate cancer LNCap cells (Shukla and Gupta ethanolic extract, this is may be due to the high 2010). 173 Zakaria A. et al./ ZJPAS: 2016, 28(3): 161-174

Luteolin decrease DNA synthesis in cell cycle arrest properties against the two human colon cancer cell line HF29 in a (AMN3 and RD) cancer cell lines, and did concentration dependent manner. Luteolin not have any important effects toward the inhibited cyclin-dependent kinase (cdk) activity (Vero) normal cell line. resulting in G1 arrest. And also promote G2/ M phase arrest in the cell cycle. This was References happened in variety of cancers like; human thyroid cancer cell line, HL-60 human myloid 1. Brusselmans, K.; Vrolix, R.; Verhoeven, G. leukemia, Miapaca-2 pancreatic tumor cells, and Swinnen, J. V. (2005). Induction of cancer cell apoptosis by flavonoids Is associated with HepG2 human hepatocellular carcinoma, their ability to inhibit fatty acid synthase luteolin induced a G1 cell cycle arrest in OCM- activity. The Journal of Biological Chemistry. 1 human melanoma cells, and G2/ M phase 280 (7): 5636-5645. 2. Claver, I. P.; Zhang, H.; Li, Q.; Kexue, Z. and arrest in SW480 colon cancer (Lima et al., Zhou, H. (2010). Optimization of Ultrasonic 2005). extraction of poly saccharides from chines Maltedsorghum using response surface methodology. Pakistan Journal of Nutrition. 9(4): 336-342. 3.5- Conclusions 3. Das, A.; Banik, N. L. and Ray, S. K. (2010). Flavonoids activated caspases for apoptosis in From the results of the present study, the human glioblastoma T98G and U87MG cells following conclusions could be considered: but not in human normal astrocytes. Cancer. 116: 164-176. 4. Freshney, R (1994) Culture of animal cells.(3rd 1- Most concentrations were effective against ed).wiley-Liss. USA. two cancer cell lines especially at 72 hrs. of 5. Hama, S. A.; Al-Jaff, B. M. and Mahmud, B. M. (2009). Common health complains among exposure. chemical bombardment survivors in Halabja. Journal of Duhok University. 12 (1): 312-316. 2- The AMN3 cancer cell line was the most 6. Karimi, G.; Khoei, A.; omidi, A.; Kalantari, sensitive to the inhibitory effects of aqueous M.; Babaei, J.; Taghiabadi, E. and Razari, B. M. (2009). Protective effect of aqueous and and ethanolic crude extracts of P. oleracea. ethanolic extracts of Portulaca oleracea against While the RD cancer cell line showed less cisplatin induced nephrotoxicity. Iranian Journal of Basic Medical Sciences. 13 (2): 31- response toward both extracts, especially at 35. low concentrations. 7. Lima, E. M.; Rissino, J. D.; Guimarães, A. C.; Overal, D. J.; Khayat, A. S.; Souza, P. C.; 3- Aqueous extract was more effective Christofolini, D. M.; Smith, M. A. and Burbano, R. R. (2005). Drifter technique: a new antiproliferative agent against two malignant method to obtain metaphases in Hep-2 cell line cell lines as compared to ethanolic extract. cultures. Biology and Technology. 48 (4): 537- 540. 4- The Vero normal cell line was the most 8. Lim, D. Y.; Jeong, Y.; Tyner, A. L. and Park, J. H. (2007). Induction of cell cycle arrest and resistant cell line toward both extracts, apoptosis in HT-29 human colon cancer cells except at higher concentrations, for all by the dietary compound luteolin. Animal periods of exposure. Journal Physiology of Gastrointestinal and Liver Physiology. 292: 66-75. 9. Lepley, D. M.; Li, B.; Birt, D.F. and Pelling, J. 5- Both aqueous and ethanolic crude extracts of C. (1996). The chemopreventive flavonoid Portulaca oleracea had the potent inhibitory 10. Murthy, N. S. and Mathew, A. (2004). Cancer effects on mitotic index in both cancer cell epidemiology, prevention and control. Current Science. 86 (4): 518-527. lines and at all periods of exposure. 11. Ness, B. D. and Knight, J. A. (2004). Encyclopedia of genetics. Salem Press, Inc. 6- Active constituents of P. oleracea had the California. USA. cytotoxic, apoptotic, antiproliferative and 174 Zakaria A. et al./ ZJPAS: 2016, 28(3): 161-174

12. Szliszka, E.; Czuba, Z. P.; Jernas, K. and Krol, W. (2008). Dietary flavonoids sensitize HeLa cells to Tumor necrosis factor-Related Apoptosis-Inducing Ligands (TRAIL). International Journal of Molecular Sciences. 9: 56-64. 13. Sulaiman, K. M. (2000). The genetic and histological effects of Cigarette smoke on Albino laboratory mice Mus musculus. M.Sc. thesis. College of Education/ University of Salahaddin/ Erbil- Iraq. 14. Sharma, M. M.; Singh, A.; Verma, R. N.; Ali, D. Z. and Batra, A. (2011). Influence of PGRS for the in vitro plant regeneration and flowering in Portulaca oleracea (L.): A medicinal and ornamental plant. International Journal of Botany. 7(1): 103-107. 15. Shubber, E. K. and Juma, A. S. (1999). Cytogenetic effects of plant extract of Urtica dioca on mouse somatic cells. The Nucleus. 42 (3): 182- 187. 16. Susan, G. B.; Voelki, K. E.; Anderson, T. W. and Finn, J. (1997). SPSS. Guide to the new statistical analysis of data. Springer. New York. 17. Shukla, S. and Gupta, S. (2006). Molecular targets for apigenin-induced cell cycle arrest and apoptosis in prostate cancer cell Xenograft. Molecular of Cancer Therapy. 5 (4): 843-852. 18. Shukla, S. and Gupta, S. (2010). Apigenin: A promising molecule for cancer prevention. Pharmaceutical Research. 27 (6): 962-978. 19. Talib, W. H. and Mahasneh, A. M. (2010). Antiproliferative activity of plant extracts used against cancer in traditional medicine. Scientia Pharmaceutica. 78: 33-45. 20. Townsend, C. C. and Guest, E. (1966). Flora of Iraq. Ministry of Agriculture. Baghdad/ Iraq. 2: 8-11. 21. Zhong, Y.; Krisanapun, C.; Lee, S. H.; Nualsanit, T.; Sams, C.; Peungvicha, P. and Baek, S. J. (2010). Molecular targets of apigenin in colorectal cancer cells: Involvement of p21, NAG-1 and p53. European Journal of Cancer. 46 (18): 3365-3374.

ZANCO Journal of Pure and Applied Sciences

The official scientific journal of Salahaddin University-Erbil ZJPAS (2016), 28 (3); 175-185

Synthesis and preliminary evaluation of the antibacterial activity of some new compounds from nucleophilic acyl substitution and ring opening reactions of 5(4H)-oxazolones

Aras Najmaddin Hamad Pharmaceutical Chemistry Department, College of Pharmacy, Hawler Medical University, Erbil- Iraq.

A R T I C L E I N F O A B S T R A C T

Article History: A new group of 1, 3-imidazolones incorporating a sulfonamide moiety and N- Received: 18/06/2015 substituted dehydroamino acid derivatives were efficaciously synthesized Accepted: 12/10/2015 from the nucleophilic acyl substitution and ring opening reaction of 4- arylidene-2-phenyl-5(4H)-oxazolones (1) using an appropriate synthetic Published:16/5/2016 strategy. All synthesized compounds were screened for their in vitro Keywords: antibacterial activities against two identifiable strains using cup plate method. Imidazolinones, Sulfonamides, Thereby, it was found that the benzocaine and ester derivatives of N-substituted N-substituted dehydroamino dehydroamino acids (3 and 6-9) revealed promising antibacterial activity acids, Esters, Amides, against both strains, while none of the synthesized 1, 3-imidazolones (2) Antibacterials exhibited likely result against Escherichia coli and Staphylococcus aureus. Supplementary to these, the constitutions of the newly synthesized 1, 3- *Corresponding Author: imidazolones and N-substituted dehydroamino acid derivatives have been Aras N. Hamad confirmed on the basis of their IR, 1H- and 13C-NMR spectral data Email: dr.aras_naj@ pha.hmu.edu.krd

1. Introduction and oxygen as hetero atoms and they play a The treatment of infectious diseases vital role in the field of medicinal chemistry still remains an important and challenging [Niraj et al 2015] , and have long been used in problem because of a combination of factors a wide variety of reactions as precursors in the including emerging infectious diseases and the manufacturing of various biologically active increasing number of multi-drug resistant molecules with antibacterial [Mohammad et al microbial pathogens [Chawla et al 2010, 2013 ], antifungal [Ezabadi et al 2008 ], Panneer et al 2011 and Alaa and Ibrahim antiviral [Zhuo et al 2010 ], anticancer, anti- 2013]. In spite of a large number of antibiotics inflammatory [Sondhi et al 2000], and chemotherapeutics available for medical anticonvulsant [Siddiqui et al 2010 and use, at the same time the emergence of old and Akgulet al 2007] and carbonic anhydrase new antibiotic resistance created in the last inhibitor properties [Smaine et al 2008, Di et al decades revealed a substantial medical need for 2010 and Surpuran et al 1998]; as well as new classes of antimicrobial agents [Ravi et al being key intermediates for the synthesis of 2014 and Natalia et al 2014], and is very α,β-Dehydroamino acids [Ahmad et al 2008]. important to explore additional sources for The α, β-Dehydroamino acids are naturally substances with potential antimicrobial occurring non-coded amino acids [Dawid activity, which could possibly have different Siodłak 2015] found in peptides of fungal modes of activity or affect different sites in the and microbial origin [Aydin et al 1985] and bacterial and fungal cells [Kumudini et from marine organisms [Hamann and Scheuer al2011]. 1993and Goetz et al 1997], in which they Oxazolones are five-membered contribute with a catalytic role in the active heterocyclic compounds containing nitrogen sites of some enzymes. On the other hand 176 Aras N. /ZJPAS: 2016, 28(3): 175-185 they have a long history of being used as was heated under reflux condition for 8-10 h important precursors for the manufacture of (The reaction progress was monitored by TLC unnatural D-α-amino acids and non- using 1:1 mixture of benzene-ethyl acetate). proteinaceous α-amino acids using asymmetric The resulting solution was cooled and then hydrogenation technology. Such amino acids poured onto ice-aqueous HCl. The solid that are increasingly important intermediates in the was obtained filtered off and washed with cold pharmaceutical, agrichemical, and water several times, air dried and crystallized flavor/fragrance industries [William A. N. from absolute ethanol. 1994] and N-substituted dehydroaminoacid 4-[4-(4-Methyl-benzylidene)-5-oxo-2-phenyl- derivatives are also suitable candidates as 4,5-dihydro-imidazol-1-yl]-N-thiazol-2-yl- pharmacologically active compounds and used benzenesulfonamide (2b): for the treatment and prevention of hepatitis C A white solid (67% ); 122-124 mp virus (HCV) infection [ hua et al 2005]and in °C; IR (KBr, cm−1): 1659.4 (CO), 1137.1 addition to that, they posesss free radical and 1344.1 (sym and asym, SO2), 3262.8 scavenging activity [Sibel 1999]. Looking to (NH); 1H-NMR(300 MHz, DMSO): 2.376(s, the diversified biological activities an attempt 3H, CH3), 6.871(s, H, Benzylic), 7.286-7.509 was made on the way to synthesis a series of (m, 11 H, Ar-H), 7.884 (d, Ar-H), 8.275 (d, 2H, new 1, 3-imidazolones incorporating a Ar-H), 12.88 (s, 1H, SO2NH); Broad band 13 sulfonamide moiety and N-substituted C-NMR(300 MHz, DMSO): 21.325 (CH3); dehydroamino acid derivatives in order to 108.455, 114.744 (C4,5-Thiazole); 124.543, attaining better therapeutic agents. 126.780, 128.081, 128.396, 128.469 ,128.886, 129.559, 131.305, 131.474, , 141.002, 141.860, Experimental (Materials and Methods): 159.662 (C, Ar-H); 132.435 (C-Benzylic); Melting point were determined by open 137.449 (C4- Imidazolone); 161.015 (C2- capillary tube method on a Gallen Kamp Imidazolone); 168.974 (C2, Thiazole); 169.196 13 electrothermal apparatus and are uncorrected. (CO); DEPT C- NMR: 21.325 (CH3); IR spectra were recorded on a Thermo- 108.455, 115.938 (C4,5-Thiazole); 132.230 Mattson-300 Spectrophotometer, as KBr disc (C- Benzylic); 125.009, 126.662, 127.879, (Chemistry Department, College of Education 128.097, 128.622, 128.796, 130.166, 132.230 and College of Science, Salahadden (CH, Ar-H). University/Erbil). 1H- and 13C-NMR spectra 4-[4-(4-Methoxy-benzylidene)-5-oxo-2- were measured using a Bruker ultra shield 300 phenyl-4,5-dihydro-imidazol-1-yl]-N-thiazol- MHz with internal TMS (Central Lab., AL-al- 2-yl-benzenesulfonamide (2c): Bayt University/ Jordon); chemical shifts are A golden solid (68% ); 155-156 mp given in ppm. °C; IR (KBr, cm−1): 1658.8 (CO), 1139.8 1. Synthesis of 4-substetuted and 1340.6 (sym and asym SO2), 3261.9 benzylidene-2-phenyl-5(4H)- (NH); 1H-NMR (300 MHz, DMSO): 3.844 (s, oxazolones (1): 3H, OCH3). 6.868 (s, H, Benzylic), 7.052- 4-substetuted benzylidene -2-phenyl- 7.503 (m, 11H, Ar-H), 7.889 (d, 2H, Ar-H), 5(4H)-oxazolones (1) were prepared previously 8.368 (d, 2H, Ar-H), 12.80 (s, 1H, SO2NH); as shown by [Aras et al under press] via the Broad band 13C-NMR(300 MHz, DMSO): reaction of the aryl aldehyde and N-benzoyl 55.403 (OCH3); 108.441, 114.555 (C4,5- glycine in acetic anhydride at 100 °C. Thiazole); 124.557, 124.543, 126.775, 2. General Procedure for synthesis of 4- 128.041, 128.440, 128.530, 128.806, 131.325, (substituted benzylidene-5-oxo-2- 134.501, 136.038, 136.144, 141.808, 158.762 phenyl-4,5-dihydro-imidazol-1-yl)-N- (C, Ar-H); 131.325 (C-Benzylic); 137.525 (C4- thiazol-2-yl-benzenesulfonamide (2) Imidazolone); 161.443 (C2-Imidazolone); [Patel et al 2010]: 168.979 ( C2-Thiazole); 169.131 (CO); DEPT 13 A mixture of 4-substetuted benzylidene C- NMR: 55.403 (OCH3); 108.458, 114.558 -2-phenyl-5(4H)-oxazolones (1) (1 mmol) and (C4,5-Thiazole); 131.335 (C-Benzylic); sulfathiazole (1 mmol) in dry pyridine (35 mL) 177 Aras N. /ZJPAS: 2016, 28(3): 175-185

124.557, 126.780, 128.048, 128.445, 128.534, 4.28 (q, 2H, CH3CH2), 1.75(s, 3H, CH3ArH), 128.811, 134.507 (CH, Ar-H). 6.01-8.15 (m, 13H, Ar-H and 1H, Benzylic, 4-[4-(4-Dimethylamino-benzylidene)-5-oxo- 1H, NHCO-ArH ), 10.17 (s, 1H, NHCO-ArH); 2-phenyl-4,5-dihydro-imidazol-1-yl]-N- 13C-NMR (300 MHz, DMSO): 14.076, 20.400, thiazol-2-yl-benzenesulfonamide (2f): 60.892, 113.558, 120.199, 121.309, 121.475, A firebrick solid (60%); 111-113 mp 127.690, 127.983, 128.537, 128.948, 131.117, °C; IR (KBr, cm−1): 1659.2 (CO), 1141.2 and 131.824, 132.566, 133.426, 133.880, 151.975, 1 1338.5 (sym and asym SO2),3252.6 (NH), H- 164.915, 166.566, 167.765. NMR(300 MHz, DMSO): 3.054 (s, 6H, Ethyl-4-[2-Benzoylamino-3-(p-chloro- N(CH3)2), 6.864 (s, H, Benzylic), 7.200 -7.667 phenyl)-acryloylamino] benzoate (3e): (m, 11H, Ar-H); 7.866 (d, 2H, Ar-H), 8.245(d, A white solid (89%); 117 mp °C; IR (KBr, −1 2H, Ar-H), 12.844 (s, 1H, SO2NH); Broad cm ): 1647,22 and 1726.67(NHCO and band 13C-NMR(300 MHz, DMSO): 23.928 COO), 3251.15 and 3415.35 (NH); 1H-NMR (N(CH3)2); 108.425, 111.820 (C4,5-Thiazole); (300 MHz, DMSO): 1.26(t, 3H, CH3CH2), 124.540, 126.717, 127.220, 128.402, 128.893, 4.20 (q, 2H, CH3CH2), 5.88-7.985 (m, 13H, 129.205, 132.890, 133.422, 134.753, 137.866, Ar-H and 1H, Benzylic, 1H, NHCO-ArH ), 144.179, 152.318 (C, Ar-H); 130.839, (C- 10.14 (s, 1H, NHCO-ArH); 13C-NMR (300 Benzylic); 137.866 (C4-Imidazolone); 156.091 MHz, DMSO): 14.341, 60.894, 112.592, (C2- Imidazolone); 159.663 ( C2-Thiazole); 126.892, 127.685, 128.005, 128.466, 128.637, 167.299 (CO); DEPT 13C- NMR: 23.928 128.924, 130.761, 130.983, 131.151, 131.400, (N(CH3)2); 108.440, 111.812 (C4,5-Thiazole); 131.830, 132.576, 133.429, 133.709, 164.922, 130.846 (C-Benzylic); 124.536, 126.722, 166.247, 174.319. 127.219, 127.969, 128.579, 129.214, , 132.891, 4. General Procedure for synthesis of 134.756 (CH, Ar-H). N-[1-(2-Hydroxy-ethylcarbamoyl)-2- 3. Ethyl-4-(2-Benzoylamino-3- substituted aryl-ethenyl]benzamide Substituted phenyl-acryloylamino) (4) [Bingjie et al 2014]: benzoate (3) [ Osman et al A mixture of 4-substetuted benzylidene 2013]: -2-phenyl-5(4H)-oxazolones (1) (1 mmol) and A mixture of 4-substetuted benzylidene -2- ethanolamine (2 mmol) in 15ml chloroform phenyl-5(4H)-oxazolones (1) (1 mmol) and was stirred over night at 0 to 5 oC, and then the benzocain (1 mmol) in absolute ethanol (20 resulting reaction mixture was washed with 15 mL) was refluxed for 6 h. The reaction mixture mL HCl solution (10%) , followed by 20 mL was left to cool, and then poured over ice; the sodium bicarbonate (10%). The separated solid that separated out was filtered off, dried organic layer was concentrated and the and recrystallized from ethanol-ether with ratio resulting residue was purified in a mixture of (4:1). ethylacetate and petroleum ether (50:50) to Ethyl-4-(2-Benzoylamino-3-phenyl- give enamides as white solids in up to 90 % acryloylamino) benzoate (3a): yield. A white solid (82% ); 120 mp °C; IR N-[1-(2-Hydroxy-ethylcarbamoyl)-2-(p-tolyl) (KBr, cm−1): 1647.26 and 1718.27 (NHCO ethenyl]-benzamide (4b): and COO), 3235.52, 3411.79, (NH); 1H-NMR A white solid (95%); mp 123 °C; IR −1 (300 MHz, DMSO): 1.29 (t, 3H, CH3CH2), (KBr, cm ): 1651.41, 1718.36 (NHCO), 1 4.33 (q, 2H, CH3CH2), 5.97-8.09 (m, 14H, 3253.87, 3411.53 (OH and NH); H-NMR (300 Ar-H and 1H, Benzylic, 1H, NHCO-ArH ), MHz, DMSO): 2.53(s, 3H, CH3), 3.269- 10.15 (s, 1H, NHCO-ArH) 3.431(t, 2H, CH2-NH), 3.451-3.622 (t, 2H, Ethyl-4-[(2-Benzoylamino-3-(p-tolyl)- CH2-OH), 3.7 (s, 1H, OH), 7.13-8.0 (m, 9H, acryloylamino] benzoate (3b): Ar-H and 1H, Benzylic), 8.15(s, 1H,CO NH- 13 A white solid (80%); 131 mp °C; IR CH2), 9.9 (s, 1H, NHCO-ArH); C-NMR (300 (KBr, cm−1): 1649.74 and 1725.81 (NHCO and MHz, DMSO): 20.816, 42.025, 59.731, COO), 3226.29 and 3412.92 (NH); 1H-NMR 122.251, 127.832, 128.269, 129.038, 129.240, (300 MHz, DMSO): 1.34(t, 3H, CH3CH2), 178 Aras N. /ZJPAS: 2016, 28(3): 175-185

131.439, 131.616, 133.713, 134.234, 138.251, A white solid (90%); 128 mp °C; IR 165.150, 165.749. (KBr, cm−1): 1645.56 and 1684.73 (CONH, N-[1-(2-Hydroxy-ethylcarbamoyl)-2-(p- CON) , 3306.45 (NH); 1H-NMR (300 MHz, methoxyphenyl) ethenyl]-benzamide (4c): DMSO): 1.5-1.6( m, 6H, piperidine), 3.35- A white solid (94%); mp 126 °C; IR 3.45( m, 4H, piperidine), 6.33 (s, 1H, (KBr, cm−1): 1646,36, 1715.87 (NHCO), Benzylic), 7.2-7.9 ( m, 9H, Ar-H), 10.3(s, 1H, 3232.74, 3417.30, (OH and NH); 1H-NMR NH-CO). (300 MHz, DMSO): 3.25-3.45(t, 2H, CH2- N-[2-(4-Chloro-phenyl)-1-(piperidine-1- NH), 3.41-3.5 (t, 2H, CH2OH), 3.8 (s, 1H, carbonyl)-ethenyl]-benzamide (5e): OH), 7.5-9.1(m, 9H, Ar-H and 1H, Benzylic, A White solid (91%); 149 mp °C; IR −1 1H, CO NH-CH2), 10.85 (b, 1H, NHCO-ArH); (KBr, cm ): 1637.91 and 1652.95 (CONH, 13C-NMR (300 MHz, DMSO): 42.907, 55.951, CON), 3267.82(NH); 1H-NMR (300 MHz, 60.586, 114.753, 127.509, 128.578, 128.677, DMSO): 1.55-1.6( m, 6H, piperidine), 3.35- 129.076, 130.111, 131.779, 132.384, 134.596, 3.45( m, 4H, piperidine), 6.35 (s, 1H, 160.309, 165.643, 166.135. Benzylic), 6.95 7.05 (d,2H, Ar-H), 8.1-8.2(d, N-[1-(2-Hydroxy-ethylcarbamoyl)-2-(o- 2H, Ar-H), 7.5-7.6 ( m, 5H, Ar-H), 10.25(s, Chlorophenyl) ethenyl]-benzamide (4d): 1H, NH-CO); 13C-NMR (300 MHz, DMSO): A white solid (96%); mp130 °C; IR (KBr, 24.20, 25.22, 55.259, 113.964, 122.919, cm−1): 1645.87, 1713.06 (NHCO), 3274.09, 126.838, 127.739, 128.196, 128.385, 130.560, 3411.02 (OH and NH); 1H-NMR (300 MHz, 130.993, 131.748, 133.186, 165.391, 167.268. DMSO): 3.269-3.431(t, 2H, CH2-NH), 3.451- 6. Synthesis of Alkyl -2-Benzoylamino- 3.622 (t, 2H, CH2OH), 3.7 (s, 1H, OH), 7.15- 3-(substituted phenyl) acrylate (6,7,8 7.9(m, 10H, Ar-H and 1H, Benzylic), 8.2 (s, and 9) [Bingjie et al 2014]: 1H, CO NH-CH2), 9.8 (s, 1H, NHCO-ArH); A suspend solution of 4-substetuted 13C-NMR (300 MHz, DMSO): 42.050, 59.599, benzylidene -2-phenyl-5(4H)-oxazolones (1) (1 124.471, 127.395, 128.214, 128.466, 129.603, mmol) and sodium carbonate ( 2 mmol) in 10 129.841, 130.011, 131.681, 131.983, 132.825, mL of the corresponding alcohol was warmed 133.153, 133.524, 164.643, 165.891. on a water bath at 50 to 60 oC for 5-10 min 5. General Procedure for synthesis of and then the reaction mixture stirred overnight N-[2-substutedaryl-1-(piperidine-1- at room temperature, and then the alcohol carbonyl)- ethenyl]-benzamide (5) solvent was removed by evaporation, and the [Bingjie et al 2014]: resulting residue was washed with water three A mixture of 4-substetuted times, dried and purified in ethyl acetate to benzylidene-2-phenyl-5(4H)-oxazolones (1) (1 give white esters in up to 90% yield. mmol) and piperidine (2 mmol) in 15 mL Methyl-2-Benzoylamino-3-(phenyl) acrylate acetonitrile was stirred over night at 0 to 5 oC. (6a): Then the mixture was poured onto ice-aqueous A white solid ( 96% ); 135 mp °C; IR HCl (to neutralize the remaining of piperidine ) (KBr, cm−1); 1636.63 and 1715.94 (NHCO and and the solid that separated was filtered off and COO), 3414.49 (NH); 1H-NMR (300 MHz, recrystallized from methanol. DMSO): 3.85 (s, 3H, CH3-O), 7.25-7.88 (m, N-[2-Phenyl-1-(piperidine-1-carbonyl)- 10H, Ar-H and 1H, Benzylic), 10.26 (s, 1H, ethenyl]-benzamide (5a): NHCO-ArH); 13C-NMR (300 MHz, DMSO): A white solid (89%); mp 139 °C; IR 52.307, 127.176, 127.698, 128.505, 128.676, (KBr, cm−1): 1642.01and 1657.09 (CONH, 131.442, 131.569, 131.975, 132.381, 133.100, CON), 3263.56 (NH); 1H-NMR (300 MHz, 133.931, 165.150, 166.285. DMSO): 1.45-1.55( m, 6H, piperidine), 3.2- Methyl-2-Benzoylamino-3-(4-methylphenyl) 3.35( m, 4H, piperidine), 6.25 (s, 1H, acrylate (6b): Benzylic), 7.3-7.9 ( m, 10H, Ar-H), 10.2(s, 1H, A white solid (92%); 120 mp °C; IR NH-CO). (KBr, cm−1): 1682.27 and 1719.05 (NHCO and N-[2-(2-Chloro-phenyl)-1-(piperidine-1- COO), 3414.14(NH); 1H-NMR (300 MHz, carbonyl)-ethenyl]-benzamide (5d): DMSO): 2.29(s, 3H, CH3-Ar-H), 3.72 (s, 3H, 179 Aras N. /ZJPAS: 2016, 28(3): 175-185

CH3-O), 7.21-7.92 (m, 9H, Ar-H and 1H, NMR (300 MHz, DMSO):14.024, 61.126, Benzylic), 10.04 (s, 1H, NHCO-ArH); 13C- 127.240, 127.618, 127.650, 128.018, 128.447, NMR (300 MHz, DMSO): 20.915, 52.171, 129.281, 129.574, 129.832, 130.635, 131.764, 125.641, 127.635, 128.486, 129.225, 129.869, 131.928, 133.126, 133.375, 164.572, 166.312. 130.403, 131.861, 133.298, 133.510, 139.475, Ethyl-2-Benzoylamino-3-(4- 165.540, 166.029. chlorophenyl)acrylate (7e): A white solid (93%); 109 mp °C; IR Methyl-2-Benzoylamino-3-(2-chlorophenyl) (KBr, cm−1): 1637.94 and 1718.37 (NHCO and acrylate (6d): COO), 3413.17 (NH); 1H NMR (300 MHz, A white solid ( 1%); 119 mp °C; IR DMSO): 1.15-1.3(t, 3H, CH3CH2), 4.15-4.26 −1 (KBr, cm ): 1652.27 and 1717.83 (NHCO and (q, 2H, CH3CH2), 7.35-8.05 (m, 9H, Ar-H and COO), 3406.68(NH); 1H NMR (300 MHz, 1H, Benzylic), 10.12 (s, 1H, NHCO-ArH); 13C DMSO): 3.78 (s, 3H, CH3-O), 7.15-7.98 (m, NMR (300 MHz, DMSO):14.081, 60.894, 9H, Ar-H and 1H, Benzylic), 10.20 (s, 1H, 127.685, 128.005, 128.466, 128.924, 130.761, NHCO-ArH); 13C-NMR (300 MHz, DMSO): 131.151, 131.400, 131.830, 132.576, 133.709, 52.297, 127.683, 128.029, 128.459, 128.894, 164.922, 166.247. 129.387, 129.803, 130.708, 131.420, 132.001, Isopropyl-2-Benzoylamino-3-(phenyl) 132.454, 132.961, 133.406, 165.100, 166.240. acrylate (8a): Ethyl-2-Benzoylamino-3-(phenyl) acrylate A white solid (88%); 137 mp °C; IR (7a): (KBr, cm−1): 1649.74 and 1725.81 (NHCO and A white solid (90%); 126 mp °C; IR COO), 3412.92 (NH); 1H-NMR (300 MHz, −1 (KBr, cm ): 1635.77 and 1712.82 (NHCO and DMSO): 1.21-1.28(d, 6H, (CH3)2CH), 4.85- 1 COO), 3409.31 (NH); H-NMR (300 MHz, 5.02 (m, 1H, CH(CH3)2), 7.25-7.89 (m, 9H, DMSO): 1.24(t, 3H, CH3CH2), 4.18 (q, 2H, Ar-H and 1H, Benzylic), 10.25 (s, 1H, NHCO- 13 CH3CH2), 7.37-8.15 (m, 10H, Ar-H and 1H, ArH); C-NMR (300 MHz, DMSO): 23.178, Benzylic), 10.17 (s, 1H, NHCO-ArH); 13C- 127.876, 128.274, 128.486,128.827, 129.001, NMR (300 MHz, DMSO):14.102, 60.574, 129.594, 129.869, 130.201, 130.622, 131.861, 126.918, 127.137, 127.831, 128.389, 128.816, 133.510, 139.475, 165.540, 166.029. 131.799, 131.938, 133.529, 136.189, 138.741, Isopropyl-2-Benzoylamino-3-(2- 164.426, 166.009. chlorophenyl) acrylate (8d): Ethyl-2-Benzoylamino-3-(p-tolyl) acrylate A white solid (85%); 132 mp °C; IR (7b): (KBr, cm−1): 1646.36 and 1715.87 (NHCO and A white solid ( 90% ); 139 mp °C; IR COO), 3417.30 (NH); 1H-NMR (300 MHz, −1 (KBr, cm ): 1644.82 and 1722.31 (NHCO and DMSO): 1.15-1.3(d, 6H, (CH3)2CH), 4.12- 1 COO), 3414.49 (NH); H-NMR (300 MHz, 4.26 (m, H, CH(CH3)2), 7.13-7.95 (m, 9H, Ar- DMSO): 1.15-1.35- (t, 3H, CH3CH2), 2.45(s, H and 1H, Benzylic), 10.21 (s, 1H, NHCO- 13 3H, CH3-ArH), 4.11-4.35 (q, 2H, CH3CH2), ArH); C-NMR (300 MHz, DMSO): 21.511, 7.15-8.15 (m, 9H, Ar-H and 1H, Benzylic), 68.600, 126.464, 127.204, 127.670, 128.399, 10.01 (s, 1H, NHCO-ArH); 13C-NMR (300 129.531, 129.875, 130.403, 130.824, 131.762, MHz, DMSO): 14.103, 20.910, 60.759, 131.939, 133.244, 133.499, 164.289, 166.425. 126.015, 127.597, 128.471, 129.213, 129.836, Isopropyl-2-Benzoylamino-3-(4- 130.679, 131.793, 133.124, 133.451, 139.372, chlorophenyl) acrylate (8e): 164.991, 166.111. A white solid (89%); mp128 °C; IR Ethyl-2-Benzoylamino-3-(2- (KBr, cm−1): 1642.27 and 1718.36 (NHCO and chlorophenyl)acrylate (7d): COO), 3411.53(NH); 1H-NMR (300 MHz, A white solid (95 %); 127 mp °C; IR DMSO): 1.21-1.28(d, 6H, (CH3)2CH), 4.85- −1 (KBr, cm ): 1638.67 and 1718.45 (NHCO and 5.02 (m, 1H, CH(CH3)2), 7.25-7.89 (m, 9H, COO), 3411.42 (NH); 1H-NMR (300 MHz, Ar-H and 1H, Benzylic), 10.25 (s, 1H, NHCO- 13 DMSO): 1.25 (t, 3H, CH3CH2), 4.25 (q, 2H, ArH); C-NMR (300 MHz, DMSO): 21.561, CH3CH2), 7.15-7.79 (m, 9H, Ar-H and 1H, 68.379, 127.645, 128.434, 128.606, 129.742, Benzylic), 10.16 (s, 1H, NHCO-ArH); 13C- 180 Aras N. /ZJPAS: 2016, 28(3): 175-185

130.122, 131.347, 131.717, 132.439 , 132.704, CH3CH2CH2CH2), 4.15(t, 3H, 133.594, 164.484, 166.362. CH3CH2CH2CH2), 7.55-7.92 (m, 9H, Ar-H Butyl -2-Benzoylamino-3-(phenyl) acrylate and 1H, Benzylic), 10.20 (s, 1H, NHCO-ArH); (9a): 13C NMR (300 MHz, DMSO): (300 MHz, A white solid (88%); 141 mp °C; IR DMSO-H): 13.461, 18.523, 30.137, 64.574, (KBr, cm−1): 1645.82 and 1713.06 (NHCO 127.589, 127.640, 128.470, 128.660, 131.247, and COO), 3411.02 (NH); 1H-NMR (300 MHz, 131.444, 131.895, 132.418, 133.264, 133.872, DMSO): 0.89(t, 3H, CH3CH2CH2CH2), 164.891, 166.303. 1.55(sextet, 2H, CH3CH2CH2CH2), Antibacterial activity: 1.68(quentet, 3H, CH3CH2CH2CH2), 4.61(t, Cup plate method using Mueller-Hinton 2H, CH3CH2CH2CH2), 7.28-7.85 (m, 10H, agar medium [Ramesh and Ranirukmini 2010 Ar-H and 1H, Benzylic), 10.22 (s, 1H, NHCO- and Bibi et al 2005] was employed to study the ArH); 13C-NMR (300 MHz, DMSO): 13.476, preliminary antibacterial activity of the 18.528, 30.153, 64.482, 126.957, 127.598, synthesized compounds (2-9) against E. coli 128.468, 128.622, 129.461, 129.824, 131.837, and S. aureus. 0.05g of each test compound 132.842, 133.446,133.566, 165.023, 166.285. was dissolved in 10 mL of 20 % of tween 80, Butyl -2-Benzoylamino-3-(4-methylphenyl) and then half of the solution is taken and acrylate (9b): completed with another 5 mL of 20 % tween A white solid (88%); 122 mp °C; IR 80, to prepare two different concentrations with (KBr, cm−1): 1649.82 and 1718.06 (NHCO 250 and 500 μg . The cups were made in Petri and COO), 3409.98 (NH); 1H NMR (300 plates using sterile cork borer (6mm) and 0.1 MHz, DMSO): 0.87(t, 3H, CH3CH2CH2CH2), mL of each tested compound was added 1.43 (sextet, 2H, CH3CH2CH2CH2), 1.73 separately into each well; and then bacterial (quentet, 3H, CH3CH2CH2CH2), 4.23(t, 3H, plates were incubated at 37 ̊ C 24 hrs. CH3CH2CH2CH2), 2.26 ( s, 3H, Amikacin, cefalotin, tetracycline, CH3ArH),7.65-7.79 (m, 9H, Ar-H and 1H, chloramphenicol, nalidixic acid and Benzylic), 10.21 (s, 1H, NHCO-ArH); 13C- vancomycin have been used as standard NMR (300 MHz, DMSO):13.482, 18.530, reference drugs (30 mg of each drug) and 20% 20.916, 30.166, 64.409, 126.025, 127.569, of tween 80 as a control which did not reveal 128.467, 129.224, 129.862, 130.643, 131.797, any inhibition. The Zone of inhibition 133.157, 133.443, 139.405, 165.096, 166.256. fashioned by each compound was measured in Butyl -2-Benzoylamino-3-(2-chlorophenyl) mm. acrylate (9d): A white solid (90%); 129 mp °C; 1H- Results and discussion: NMR (300 MHz, DMSO): 0.71(t, 3H, The impartial of this study was to CH3CH2CH2CH2), 1.38 (sextet, 2H, synthesize, characterize and evaluate the CH3CH2CH2CH2), 1.75 (quentet, 3H, antibacterial activity of some new 1, 3- CH3CH2 CH2CH2), 4.33(t, 3H, imidazolones incorporating a sulfonamide CH3CH2CH2CH2), 7.37-7.75 (m, 9H, Ar-H moiety and dehydroamino acid derivatives. The and 1H, Benzylic), 10.16 (s, 1H, NHCO-ArH); required 4-arylidene-2-phenyl-5(4H)- 13C-NMR (300 MHz, DMSO): 13.440, 18.522, oxazolones (1) for this study were readily 30.064, 64.724, 127.254, 127.666, 128.425, prepared through Erlenmeyer-Polchl reaction 129.262, 129.574, 129.845, 130.668, 130.971, as outlined by [Christopher et al 2013] in 131.687, 131.915, 133.117, 133.389, 164.631, Scheme (1). This study commenced by using 166.391. sulfathiazole as the model substrate to convert Butyl -2-Benzoylamino-3-(4-chlorophenyl) the oxazolone ring in 4-arylidene-2-phenyl- acrylate (9e): 5(4H)-oxazolones (1) into new 1, 3- A white solid ( 91% ); 113mp °C; 1H imidazolones (2). Since the ring NMR (300 MHz, DMSO): 0.85(t, 3H, transformation of oxazolones into 1, 3- CH3CH2CH2CH2), 1.33(sextet, 2H, imidazolones has been successful, especially in CH3CH2CH2CH2), 1.65(quentet, 3H, pyridine [Desai et al 2009], it has been 181 Aras N. /ZJPAS: 2016, 28(3): 175-185 obvious to follow the same strategy in the opening reaction with nucleophiles, the reaction of sulfathiazole with 4-arylidene-2- reaction of 4-arylidene-2-phenyl-5(4H)- phenyl-5(4H)-oxazolones (1) to the oxazolones (1) with more stronger corresponding 4-(substituted benzylidene-5- nucleophiles, ethanolamine and pipyridine was oxo-2-phenyl-4, 5-dihydro-imidazol-1-yl)-N- carried out at 0 to 5 oC and the reaction thiazol-2-yl-benzenesulfonamide (2). afforded new amide derivatives of The structures of the obtained 4- dehydroamino acids, N-[1-(2-Hydroxy- (substituted benzylidene-5-oxo-2-phenyl-4, 5- ethylcarbamoyl)-2-substituted aryl-ethenyl] dihydro-imidazol-1-yl)-N-thiazol-2-yl- benzamide (4) and N-[2-substituted aryl-1- benzenesulfonamide (2) were elucidated by (piperidine-1-carbonyl)- ethenyl]-benzamide various spectroscopic techniques. For (5), respectively in excellent yield as example, the infrared spectral data of represented in Scheme (2). The infrared compound (2c) revealed the most important spectra of the formed amide derivatives of features of the sulfonamide moiety at 1139.8 dehydroamino acids (4 and 5) confirm the ring and 1340.6 cm-1 for the symmetric and opening reaction of the oxazolone ring in (1) asymmetric vibrations of the sulfonyl group with the nucleophiles by showing the broad and at 3261.9 cm-1 for the N-H stretching of the absorption band at about 3410 cm-1 due to NH sulfonamide moiety, and 1658.8 cm-1 for the of the amide group. This ring opening reaction carbonyl group of the imidazolone ring. The was in settlement with the H1and 13C-NMR 1H- and 13C-NMR spectral data of compound data. The 1H-NMR spectrum of N-(Z)-[1-(2- (2c) Figure (1 and 2) have supported the Hydroxy-ethylcarbamoyl)-2-(p-tolyl) ethenyl]- infrared finding by displaying signals at benzamide (4b) Figure (4) has revealed several 169.131 ppm due to carbonyl carbon atom of features of note, for example the spectrum the imidazolone ring and 12.80 ppm due to displayed a characteristic signal at 10.2 ppm N-H group of the sulfonamide moiety. Further due to the aromatic NH of the amid group and verification for the formation of compound (2c) also the broad peak at 3.7 ppm and two was attained from the singlet signal at 3.884 neighbor triplets at 3.25 and 3.45 ppm which ppm due to the methoxy,s hydrogen atoms and attributed to the hydroxyl and the ethylene the signal at 55.403 ppm due to the methoxy,s moieties. The spectrum has also showed a carbon atom, and also the depicted signals in singlet signal at 2.53 ppm due to the methyl DEPT 13C-NMR spectral data of compound group at position 4 in the N-phenyl benzamide (2c) Figure (2) are in agreement with the moiety. On the other hand, the 13C-NMR number of methine’s and methyl’s carbon spectrum which depicted in Figure (5) atoms of the structure. confirmed the 1H-NMR outcome by showing Since opening of the heterocyclic ring signals at 20.816, 42.025, 59.731 ppm for the in the 4-arylidene-2-phenyl-5(4H)-oxazolones methyl and ethylene carbon atoms. (1) with various nucleophiles has received a In a similar fashion, the ring opening large amount of attention as a key step in the reaction of the oxazolone in (1) was synthesis of a wide variety of beneficial successfully extended with various alcohols in compounds and to further expand in the presence of sodium carbonate at room understanding of ring opening reaction, the temperature to synthesize a series of esters study was extended on the synthesis of a series derivatives of dehydroamino acids (6-9) in of new α,β-dehydroamino acid derivatives excellent yield, Scheme (2). Herein, it is using various alcohols and nitrogen believed that the ring opening reaction of nucleophiles. The reaction of 4-arylidene-2- oxazolone in (1) with alcohols in the presence phenyl-5(4H)-oxazolones (1) with benzocaine of sodium carbonate may proceed via the in ethanol under reflux condition resulted in the following mechanistic pathway. formation of ethyl-(Z)-4-(2-Benzoylamino-3- Substituted phenyl-acryloylamino) benzoate (3) in very good yields as in Scheme (2). Supplementary to this understanding in ring 182 Aras N. /ZJPAS: 2016, 28(3): 175-185

Na Na Na R (A) Sulfathiazole, Pyridine, Reflux; (B) H Na OH Ph Ph Benzocaine, Ethanol, Reflux; (C) C Ph O H o C C Na N Ph N Ph Ethanolamine, Chloroform, 0-5 C; (D) H o N O Pipyridine, Acetonitrile, 0-5 C; (E) The C R O R O O O O O corresponding alcohol, Sodium Carbonate, Ph H RT. The structures of the esters of dehydroamino acids (6-9) were confirmed by The antibacterial profile of all the various spectroscopic techniques. For synthesized compounds (2-9), Table (1), example, the infrared spectrum of (9a) support revealed that most of the prepared compounds the ring opening reaction by desplysing a broad peak at 3411.02cm-1 due to the NH of possessed moderate to potent and significant the amide group, in addition; the spectrum has antibacterial activity towards gram–positive also revealed two strong absorption bands at Staphylococcus aureus and gram negative -1 1645.82 and 1713.06 cm assigned to the Escherichia coli and were almost competitive stretching vibrations of the carbonyl group in with the standard drugs, Table (2). The highest the amide and ester linkages. The 1H-NMR activity was observed for ester derivatives of spectrum of (9a) which presented five characteristic signals at 0.89, 1.55, 1.68 and N-phenyl dehydroamino acids (6-9), followed 4.61 ppm due to protons of the butyl group and by benzocaine derivatives of N-phenyl at 10.22 due to the (NH) of the amide linkage, dehydroamino acids (3) and then ethanol amine supporting that the ring opening reaction had and pipyridine derivatives of N-phenyl taken place with butanol in the presence of dehydroamino acids (4 and 5) while the 1, 3- sodium bicarbonate at room temperature. imidazolones (2) showed no remarkable Additional evidence for this ring opening activities against both microorganisms. A reaction was provided by the 13C-NMR spectrum Figure (7), which shows four signals comparison of antibacterial activities of the in the aliphatic region at 13.476, 18.528, prepared compounds with their structures 30.153, 64.482 of the butyl,s carbon atoms and discovered that the carbon carbon double bonds at 166.285 for the carbon atome of the amide that incorporating a strong electron linkage. O withdrawing group, as in ester and benzocaine O H S H N C O derivatives of N-phenyl dehydroamino acids R C C N S N N O A (1) N (2) C (6-9 and 3), caused significant activity against b=4-CH3, c=4-OCH3, a= H, b=4-CH3, c=4-OCH3, O f=4-N(CH3)2 d= 2-Cl, e=4-Cl, f=4-N(CH3)2 R microorganisms especially Staphylococcus R aureus, and this is an interesting point in the H N E R O O (6) H study and it needs further exploration to find O O a= H, b=4-CH3, N d= 2-Cl O B R O out the in vivo therapeutic application of the N O (3) H a= H, b=4-CH3 H N e=4-Cl prepared compounds. E R O (7) O O a= H, b=4-CH , 3 C H d= 2-Cl, e=4-Cl N

R HO O (4) N O Table 1. Diameter of inhibited zones in H b=4-CH3, c=4-OCH3, H N d= 2-Cl E millimeters as a measure of antibacterial R O (8) O O a= H, activity of the synthesized compounds (2-9): d= 2-Cl, e=4-Cl, H N D R O N O H N (5) E a= H, d= 2-Cl, e=4-Cl O O O (9) a= H, b=4-CH3, d= 2-Cl, e=4-Cl Scheme 1: The reaction conditions and reagents in the synthesis of compounds (2 and 9). 183 Aras N. /ZJPAS: 2016, 28(3): 175-185

E. coli S. E. coli S. imidazolone system with sulfonamide moiety Co aureus Co aureus as coupling components and also mp 25 50 25 500 mp 25 50 25 500 dehydroamino acid derivatives could be .s 0 0 0 µg/ .s 0 0 0 µg/ beneficial for derivatization to develop more µg µg/ µg mL µg µg/ µg/ mL effective antibacterial agents. /m mL /m /m mL mL L L L 2b - 4 5 8 6b 9 11 9 22 2c - - - 7 6d 7 13 5 21 2f - - - 5 7a 4 7 10 20 3a - 5 16 20 7b 8 11 15 20 3b - - 11 22 7d 5 15 17 20 3e 4 4 18 24 7e 8 17 24 24 4b - 6 4 10 8a 14 17 24 28 4c - - 6 8 8d 9 16 26 29 4d - - 8 8 8e 10 15 25 25 Figure 1: 1 H-NMR spectrum of compound 5a - 4 7 10 9a 8 12 19 20 (2c) 5d - - 7 12 9b 6 10 18 19 5e - - 9 12 9d 10 12 17 22 6a 6 9 10 19 9e 5 8 18 25

Table 2: Diameter of inhibited zones in millimeters as a measure of antibacterial activity of the medium and some standard drug molecules: Figure 2: Broad band and dept. 13C-NMR spectrum of compound (2c)

Drugs E. coli S. aureus 20% tween 80 - - Amikacin 19–26 20–26 Cefalotin 15–21 29–37 Tetracycline 18–25 24–30

Chloramphenicol 21–27 19–26 Figure 3: 1H and 13C-NMR spectrum of Nalidixic acid 22–28 - compound (3b) Vancomycin - 17–21

Conclusions A group of 1, 3-imidazolones combining with a sulfonamide moiety and N- substituted dehydroaminoacid derivatives have been synthesized with a view to get a good antibacterial activity. The results of the antibacterial study indicate that the synthesized Figure 4: 1H-NMR spectrum of compound system reveal good antibacterial against gram– (4b) positive Staphylococcus aureus microorganism and compounds (3 and 6-9) equipotent with the standard drugs except nalidixic acid. Moreover, presence of electron with drawing groups in the system raises the activity. Hence, there is adequate scope for further study in developing such compounds as a good lead activity and in addition to that compounds having 1, 3-

184 Aras N. /ZJPAS: 2016, 28(3): 175-185

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The official scientific journal of Salahaddin University-Erbil ZJPAS (2016), 28 (3); 186-190

Description, Distribution and Checklist of European Earwig Forficula auricularia L. (Dermaptera: Forficulidae) in Erbil Governorate, Kurdistan region, Iraq

Nawzad B. Kadir 1College of Agriculture, University of Salahaddin-Erbil

A R T I C L E I N F O A B S T R A C T

Article History: The purpose of this study was to checklist, distribution and Received: 31/01/2016 description of Earwig, Forficula auricularia L., 1758 in Erbil Kurdistan Accepted: 12/04/2016 region-Iraq. The important taxonomic parts; Mandibles, Maxilla, Antenna, Published:16/5/2016 Keywords: Male and Female cerci have been drawn. Localities, plant hosts and date of Dermaptera, Distribution, the collection have been mentioned. Description, Forficula auricularia

L., Kurdistan region-Iraq. *Corresponding Author: Nawzad B. Kadir

Email:bnawzad1332TU @yahoo.com U32T

1. Introduction the expanded wings of the earwigs resemble somewhat the shape of the human ear (Imms, Earwigs make up the insect order Dermaptera 1964). Linnaeous (1758) is the first who and are found throughout the America, Africa, recorded the species .Recently been proposed Eurasia, Australia and New Zealand. With as a potential biological control agent for about 2,000 species (John, 2010). Zhang, aphids and psyllids in pear orchards (Lenfant et (2011) in 12 families, they are one of the al., 1994).The general characteristics which smaller insect orders. The common name, distinguish the earwing are, the body elongate, earwig, seems to have originated scientific dorso-ventrally flattened. Head Prognathous. order name, "skin wings." Some groups are Antennae are filiform and segmented. Biting- tiny parasites on mammals and lack the typical type mouthparts. Ocelli absent. Compound pincers. Earwigs rarely use their flying ability. eyes in most species, reduced or absent in some Earwigs are mostly nocturnal and often hide in taxa,a pair of forceps pincers on their abdomen, small, moist crevices during the day, and are and membranous hind wings folded underneath active at night, feeding on a wide variety of short forewings (Borer and Delong, 1954. insects and plants. Damage to foliage, flowers, Imms, 1964; Gillot, 1995).In Iraq there are not and various crops is commonly blamed on any taxonomic study of the family except; earwigs, especially the common earwig Derwesh (1965) indicated five species, and Al- Forficula auricularia (Richard et al., 2010).The Ali (1977) recorded two species. species occasionally enter the ears of sleeping humans and even penetrate the brain; that the Materials and Methods forceps resemble the pincers used to puncture human ears for the insertion of earrings; or that 187 Nawzad B./ ZJPAS: 2016, 28(3): 186-190

The study is based on, is represented by 30 Results and Discussion specimens which collected in some localities of Erbil province (Kurdistan region-Iraq)by Hand picking, pitfall traps and sweeping net the flowers of some weeds and plants (Richard et 1. Check list of Dermaptera in Erbil Kurdistan al., 2010. Yang et al., 1994) from the period of region-Iraq March till July2015.Also some samples were checked from the following museum: a. Family:Forficulidae (common earwig)

CASU: College of Agriculture – Salahaddin 1. Forficulaauricularia L. University 2. F. lucasi Dohrn CESU: College of Education – Salahaddin University b. Family:Labiduridae

CSSU: College of Science– Salahaddin 1. Labidura riparia Pallas University Key of the families IMEDR: Insect Museum. Erbil Directorate of Research 1. Second tarsal segment extending beneath base of third segment, may be dilated and broader than third segment; antennae with 12- 16segments The specimens were placed in boiling water for 10-15 minutes to soften their parts. Then the Forficulidae parts were separated and put in 10% KOH, placed on fire with shaking for about (4-5) - Second tarsal segment not dilated and not minutes for dissolving of lipids materials of the extending beneath base of third segment; body and destroying the muscles. After that antennae with 10-31 segments samples were placed in distilled water for 2-3 minutes in order to neutralize the alkali. The Labiduridae parts are placed in ethyl alcohol 25% and dissected under microscope, then transferred to Description ethyl alcohol 50%, 75% and 100% respectively for two minutes for each concentration to Forficula auriculariaL., 1758, Syst. Nat. (10) 2: dehydration of water, then placed in xylol for 423. two minutes, for translucency, finely placed in D.P.X solution to support slides for Body: Flattened, dark brown .Length12.4-17.4 subsequent examination under dissecting mm. microscope. The important parts drawing by a digital camera (Ucmas series microscope Head: Nearly taprozoid, dark brown, bare. camera) was used to photographing the Frons dark brown, smooth, bare. Compound important parts. The measured proportions of eyes, dark brown, oval shaped. Length is 0.6- body parts are given in points of an eyepiece 0.8 mm. Labrum (Fig. 1a) transverse surface linear micrometer in a binocular microscope sparsely setose, anterior margin densely setose. (Lane and Grosskey, 1993). The species Mandible (Fig. 1b) triangular shaped, dark identified by using taxonomic key of brown - black, apical part with two denticals, (Hoffman, 1987). ventral margin moderately concave at the middle, with two small teeth.Maxillae (Fig. 1c) dark yellow,lacinia and galea hook like shaped, maxillary palp have four segments, sparsely 188 Nawzad B./ ZJPAS: 2016, 28(3): 186-190 yellow setose,1st segment is the shortest,2nd - being represented in both sexes by a pair of 4th segments nearly tubular, 3ed segment 1.1 plates at the base of the cerci. the segments 2-7 times as long as the 2nd segment and 1.3 times are taprozoid shaped, 1st sternite 1.3 times as as long as fourth segment. Labium (Fig. 1d) long as 2nd, 3rd sternite 1,1 times as long as yellow, mentum rectangular, prementam oval 2nd, 4th and 5th sternites nearly same length, shaped, bilubed; labial palps with three 6th sternite1.2 times as long as 7th pygidium segments; 1st segments cup shaped, 2nd-3rd elongated, triangular, with truncate apex. segments oval elongate, 2nd segment 2 times as long as 1stsegment, which nearly same Terminal abdominal segments and Male length with 3rd segment. Antennae (Fig. 1e) genitalia filiform type, yellow consist 13 segments, 1st segment is the longest, 2.5 times as long as The 8th segment is the longest, 2nd segment which is the shortest and same rectangular. Tergite 8 cup shaped, posterior length with 3rd segment, 5th segment 1.2times margin moderately convex. 8th sternite 2.2 as long 4th segment, 6th – 10th segments times as long as the 7th. Anal cercus in the nearly same length, 11th segment 1.1 as long as male (Fig. 1f) brown forceps like, Length 3.2- 12th segment, 13th segment nearly same length 3.6 mm, basal part broad nearly rectangular, with 12th segment. 0.8-1.2 mm long, ventral margin smooth, one– third of apical part slender strongly curved,2.2 Thorax -2.6 mm long. Male genitalia (Fig. 1h) yellow, Phallobase nearly rectangular, paramers Pronutum transverse, dark brown, parallel- cylindrical shaped, moderately curved, apical sided, lateral sides yellow, anterior margin part oval, the space 0.1mm. phalloapodem strait, posterior margin moderately convex. parallel, needle lick. Anterior angles nearly L – shaped, posterior angles rounded. Prosternum dark brown–black, smooth and bare. Prosternal process nearly rectangular, smooth. Fore wing dark yellow, Female very short 1.5 mm as long as pronotam, the posterior margin is straight. Legs yellow, fore The female resembles to male except the femur cylindrical shaped sparsely yellow following characteristic: setose; foretibia tubular shaped 1.1mm as long as the femur, sparsely yellow setose. The tarsus The 7th sternum completely conceals the 8th of formula is 3-3-3; Protarsus brown, 1st segment the cerci (Fig.1g), nearly straight. Length 1.8 – is the longest densely yellow setose; the 2.4 mm. ventral margin with five small 2ndsegment pad shaped (lobed) extending tentacles at 1/6 of apical part. distally below the third tarsal segment; 3ed segment tubular shaped and very short, 4th segments tubular shaped 0.8mm as long as the 1st segment, claws hook shaped. Middle leg Distribution and hind legs nearly resembles to the forelegs except the femur and tibia slightly longer. The specimens were collected by the following methods during the period 1 March. 2015, till 1 Abdomen July. 2015.

Oval elongated, dark brown, consist of eight 1. Hand picking from homes, apartments and visible segments the widest area is at the agriculture fields from the center and around of middle. The 1st sternum is always wanting, Erbil governorate. while sterna 2 to 9 in the male and 2 to 7 in the female are clearly visible. The 9th sternum in 2. Pitfall traps in Girdarasha field. the male largely overlies the 10th, the latter 189 Nawzad B./ ZJPAS: 2016, 28(3): 186-190

3. Sweeping net of the flowers of some weeds DERWESH, A. I. (1965). A preliminary list of and plants in (Shaqlawa, Girdarasha, Ankawa, identified insects and arachnids of Iraq, Direct. Koshtapa and Kasnazan), especially mustard, Gen. Agr. Res. Proj. Baghdad. Bull., No. 121- Brassica rapa, Haory cress, Lepidium draba; 123, 128pp. Dwarf rocket, Diplotaxis sp.; Mallow,Malva rotundifoli GILLOT, C. (1995). Entomology. 3rd ed. Springer. The Netherlands. 831pp.

JOHN, L. C. (2010). European earwig Forficula auricularia Linnaeus (Insecta: Dermaptera: Forficulidae). Entomology and Nematology Department, 3: 1-5. IMMS, A. D. (1964). A general text book of Entomology, London Methienand Co. Ltd.

LANE, R. P. and CROSSKEY, R. W. (1993). Medical insects and Arachnids. The Natural History Museum. Chapman and Hall, London. 723pp.

LENFANT, C.; YOUSSOUFI, L; CHEN, A; FAIVREDT, X; and SAUPHANOR, S. (1994). Potentialitespredatrices de Forfiala auricularia sur le Psylle du Poirier Cacopsyllapyri,Bntomologia Experiïnentaliset Applicata.73: 51-60.

LINNAEUS, C. (1758). Systema Naturae (10) 2: 423. No. 1.Holmiae (Stockholm). References RICHARD S. Z.; STRENGE, D; LANDOL AL-ALI, A. S. (1977). Phytophagous and Tand, P. J., LOONEY, C. (2010). European entomophagous insects and mites of Iraq, earwig, Forficula auricularia L. (Dermaptera: Natural History Research Center, Publication Forficulidae),the Hanover Reach National No. 33:142 pp. Monument, Washington State 70(4), 441-445.

BORROR, D.J. and DELONG, D. M. (1954). YANG J. T.; CHAO J. T. and LIU W. Y. An introduction to study of insects, Holt, (1994): Collecting crickets (Orthoptera: Rinehart and Winston, New York. 1030pp. Gryllidae) using peanut butter bait traps. J.

Orthoptera Research 3: 87-89. HOFFMAN, K. M. (1987). Earwigs

(Dermaptera) of South Carolina, with a key to ZHANG, Z. Q. (2011). "Phylum Arthropoda the eastern North American species and a von Siebold, 1848 In: Zhang, Z. - Q. (Ed.) checklist of the North American fauna,Proc. Animal biodiversity: An outline of higher-level Entomol. Soc. Wash, 89: 1-14. classification and survey of taxonomic

richness"Zootaxa, 3148: 99–103. 190 Nawzad B./ ZJPAS: 2016, 28(3): 186-190

Forficula أﻧﺘﺸﺎر ووﺻﻒ أﺑﺮة اﻟﻌﺠﻮز اﻷورﺑﻲ أﻗﻠﯿﻢ أرﺑﯿﻞ، ﻣﺤﺎﻓﻈﺔ ﻓﻲ .auricularia L ﻛﻮردﺳﺘﺎن- اﻟﻌﺮاق

ﻧﻮزاد ﺑﺎوﻛﺮ ﻗﺎدر

ﻗﺴﻢ وﻗﺎﯾﺔ اﻟﻨﺒﺎت – ﻛﻠﯿﺔ اﻟﺰراﻋﺔ – ﺟﺎﻣﻌﺔ ﺻﻼح اﻟﺪﯾﻦ / أرﺑﯿﻞ

[email protected]

اﻟﺨﻼﺻﺔ

ھﺪف ھﺬا اﻟﺒﺤﺚ ﻟﺪراﺳﺔ اﻧﺘﺸﺎر ووﺻﻒ أﺑﺮة اﻟﻌﺠﻮز اﻷورﺑﻲ ,.Forficula auricularia L (1758) ﻓﻲ ﻣﺤﺎﻓﻈﺔ أرﺑﯿﻞ، أﻗﻠﯿﻢ ﻛﻮردﺳﺘﺎن- اﻟﻌﺮاق. ﺗﻢ رﺳﻢ اﻻﺟﺰاء اﻟﻤﮭﻤﺔ ﻣﺜﻞ اﻟﻔﻜﻮك اﻟﻌﻠﯿﺎ، اﻟﻤﻠﻤﺲ اﻟﻔﻜﻲ، ﻗﺮوﻧﺎ اﻻﺳﺘﺸﻌﺎر، اﻟﻘﺮون اﻟﺸﺮﺟﯿﺔ ﻟﻠﺬﻛﺮ واﻷﻧﺜﻰ، واﻟﺴﻮءة اﻟﺬﻛﺮﯾﺔ. ﺗﻤﺖ اﻻﺷﺎرة اﻟﻰ ﻣﻨﺎﻃﻖ ﺟﻤﻊ اﻟﺤﺸﺮة وﺗﺎرﯾﺨﮭﺎ وﻋﻮاﺋﻠﮭﺎ اﻟﻨﺒﺎﺗﯿﺔ.

اﻟﻜﻠﻤﺎت اﻟﻤﻔﺘﺎﺣﯿﺔ: أﻧﺘﺸﺎر، وﺻﻒ أﺑﺮة اﻟﻌﺠﻮز اﻷورﺑﻲ .Forficula auricularia L، أرﺑﯿﻞ، إﻗﻠﯿﻢ ﻛﻮردﺳﺘﺎن –اﻟﻌﺮاق. Acknowledgment

I sincerely thank the specialist Dr. Nabeel A. Mawlood from College of Agriculture, Salahaddin University for kind help in identifying of this species. I deeply express my gratitude to Prof. Dr. Mohammed S. Abdul Rassoul in Division of Entomology, Natural History Research Center – University of Baghdad / Iraq for his kind help and continuous encourag'ement to this work.