ZANCO Journal of Pure and Applied Sciences

The official scientific journal of Salahaddin University-Erbil ZJPAS (2018), 30 (5); 1-16 http://dx.doi.org/10.21271/ZJPAS.30.5.1

Antioxidant, and Antimicrobial Activities of Phenolic and Flavonoid Rich Medicinal (Fritillaria zagrica and Tulipa kurdica) Collected in Kurdistan Region of Iraq 1 2 3 Mukhlis H. Aali , Nadhum J. Ismaiel , Fadzilah A. Abdul Majid 1Department of Biology, College of Science, University of Salahaddin, Erbil, Kurdistan Region, Iraq 2Department of Biology, College of Education, University of Salahaddin, Erbil, Kurdistan Region, Iraq 3Institute of Marine Biotechnology, University Malaysia Terengganu, 21300 Kuala Terengganu, Terengganu, Malaysia.

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

Article History: Fritillaria zagrica and Tulipa kurdica had been used as traditional herbal Received: 06/05/2018 remedies since antiquity to treat human diseases in the Kurdistan region of Iraq. Accepted: 10/07/2018 This is the first report and conceive to analyze these two medicinal plants based. Published: 28/10 /2018 Methanol, 80% ethanol and aqueous extracts of two medicinal plants (Fritillaria Keywords: zagrcia and Tulipa kurdica) were evaluated for their antibacterial activity and Fritillaria zagrica, antifungal activities. We have quantified the total phenolic (TPC) and flavonoid Tulipa kurdica, (TFC) contents and their relation to antioxidants (ABTS) [2,2’-azinobis-(3- Antioxidant, ethylbenzothiazoline-6-sulfonate) and 2,2′-diphenyl-1 picryl hydrazyl Antimicrobial Activity, (DPPH)scavenging free radicals in a dose dependent method. Results showed that TPC, F. zagrica and T. kurdica extractions by using different solvents exhibited TFC. strong Antimicrobial activities against selected bacterial and fungal strains except *Corresponding Author: C. guilliermondii ATCC 6260. Aqueous bulb extract of F. zagrica and T. kurdica Mukhlis Hamad Aali showed better antibacterial activity compared to other solvent extracts. Methanol F. zagrica bulb extracts contain significantly higher phenolic content than T. kurdica. E-mail: The TPC and TFC contents were varied between the bulb extracts and the [email protected] or DPPH and ABTS scavenging activities were exhibited to be significantly [email protected] correlated with the amount of TPC and TFC.

there is always a bright yellow spot (Weathers, 1. INTRODUCTION 1911, Ruksans, 2007). F. zagrica Stapf. Kurdistan is a closed and stored pool as belongs to the Fritillaria, which is a genus of a buried treasury industrial pharmacy. There over 160 species of bulbous plants within the are a large number of medicinal plants used in monocot family , native to temperate folk medicine by the traditional herbalists to regions of the Northern Hemisphere (Liu et al., treat varieties of human ailments. Among them, 2012, Hao et al., 2015, Kiani et al., 2015). The Fritillaria zagrica and Tulipa kurdica had been species of the Fritillaria genus are distributed priced for their local traditional medicinal especially in the Mediterranean regions and resources. eastern parts of Anatolia and Thrace, the Black Sea, and Central Anatolia in Turkey, and in Fritillaria zagrica Stapf. is a species Iran, Iraq, Syria, Afghanistan, Russia, very closely allied to F. tulipifolia and F. Lebanon, Palestine, Jordan, Pakistan, , armena. Their flowers dark lurid untesselated, Japan, Greece, Bulgaria, Italy, Spain, Portugal, unchequered with a thick bloom outside, purple North Africa, and California and Oregon in the glaucous outside and at the tips of the petals USA (Tekşen and Aytaç, 2011). Fritillaria L.

2 Aali. M. et al. /ZJPAS: 2018, 30 (5): 1-16 has been commonly used in traditional Chinese terpenes and phenolic compounds that are medicine for thousands of years, contain many involved in antioxidant and free radical pharmaceutically active constituents (Hao et scavenging (Saniewski and Horbowicz, 2003, al., 2013). Sobia, 2011). Phenolic compounds are secondary plant metabolites that are found Many Fritillaria species have long been naturally in all plant materials, including plant exploited as the natural source of the widely based food products. These compounds are used traditional medicine ‘bulbus Fritillaria’ thought to be an integral part of human and (i.e., dry bulbs or a decoction of Fritillaria animal diets. They represent the most species; ‘Beimu’ in Chinese) that has long been important group of natural antioxidants (Huyut used as one of the most important antitussive, et al., 2017). Furthermore, as a result of high expectorant, and antihypertensive drugs and medicinal plants application in treating many treat tumors, hemoptysis and deficiency of diseases, now the plants are also useful as milk in traditional Chinese medicine (Li et al., novel antimicrobial agents (Khan et al., 2018). 2006, Li et al., 2013, Matsuo et al., 2013). In particular, reactive oxygen species Similarly, T. kurdica belongs to Tulipa (ROS), such as hydroxyl radicals, superoxide L. (Liliaceae), which is a genus of about 100 anions, and hydrogen peroxide, are frequently species of bulbous monocots (Bryan, 2002, generated spontaneously in the living cell Bryan, 2005, Zonneveld, 2009) naturally during metabolism and play an important role occurring from southwestern Europe and North in cell signalization (Liu et al., 2012). Africa, the Middle East to Central Asia However, excessive amount of ROS can induce (Christenhusz et al., 2013). T. kurdica closely oxidative stress, resulting in significant damage to T. humilis which has stems 4-6 inch and to cell structures and macromolecules, flowers brick red or orange red with the basal including proteins, lipids, and nucleic acids. As blotch black, late spring (Bryan, 2005). From a it is well known, ROS also contributes in medicinal and industrial viewpoint, Tulipa and development of cancer, inflammation, diabetes, Fritillaria are two of the most important genera aging, inflammation and cardiovascular disease in Liliaceae family due to their relatively rich (Marvibaigi et al., 2016, Ismail et al., 2017). pharmacological history (Li et al., 2006). In this context, the antibacterial and Likewise, the bulbs of F. zagrica and T. antioxidant properties of various medicinal kurdica have been traditionally used in clinic plants are being investigated throughout the as herbal remedies by Kurdish people who are world because of the toxicological concerns living in the mountains of Kurdistan Region- associated with the synthetic antioxidants and Iraq, due to their positive potent therapeutic preservatives (Baba and Malik, 2014). effectivities during lesion treating, wound healing, preventing and reducing the pain and However, to the best of our knowledge, removing the excessive tumor mass (lump) there is no scientific work on antimicrobial and beneath the skin of breast, armpit and underarm antioxidant activities of F. zagrica and T. with discharging abscess. kurdica medicinal plants. Therefore, the aim of this study to determine the total phenolic-, The medicinal values of the plants flavonoid- contents and to elucidate the depend on the presence of certain chemical antioxidant properties and antimicrobial substances called secondary metabolites, activities of bulb extracts of both F. zagrica involving alkaloids, tannins, flavonoids,

3 Aali. M. et al. /ZJPAS: 2018, 30 (5): 1-16 and T. kurdica medicinal plants grown in through Whatman No.1 filter papers. The Kurdistan region- Iraq. resultant bulb extracts were concentrated in a rotary evaporator in a water bath at 40 °C until the crudes solid extracts were obtained, which 2. MATERIALS AND METHODS were then freeze-dried for completing solvents removal. Finally, the obtained bulb extracts 2.1. MATERIALS were weighed, their yields were calculated, and 2.1.1 Plant Material Collection: stored at -20 °C in sealed tubes until used for further analysis. The methanol, 80 % ethanol Fritillaria zagrica and T. kurdica were and water extracts’ yields were 7.76, 2.97, 2.60 collected in April 2014 from Zine-Asterokan % for F. zagrica bulb extracts while the mountain near to Karokh mountain and Weza methanol, 80 % ethanol and water yields were village near Choman district - Erbil / Kurdistan 7.40, 4.70, 3.57 % for T. kurdica bulb extracts region from northern Iraq, respectively. The respectively. plants were classified and identified by botanists Prof. Dr. Abdul Hussain Al Khayat and Mrs. Bnar Khalid Bakr according to the 2.2 Chemicals and Microorganisms most common classification key. The voucher specimens, accession number (0007627) for F. All chemicals and reagents used in the zagrica and (0007628) for T. kurdica plants study including solvents were of analytical were deposited at the Herbarium of Department grade. Methanol and Ethanol were obtained of Biology, College of Science, Salahaddin from Score Scientific SDN BHD. 1,1- University, Kurdistan-Iraq. The plants raw Diphenyl- 2-picryl hydrazine (DPPH), 2, 2'- materials (bulbs) were washed and air-dried azino bis-(3-ethyl benzo thiazoline-6-sulphonic under shade at room temperature (20-25oC). acid) (ABTS), quercetin, catechin, Folin- After drying, the bulbs’ plants were grounded Ciocalteu’s phenol reagent, Difco™ nutrient into fine powder using a laboratory grinding broth, gallic acid, 6-hydroxy-2,5,7,8- mill and sieved with 710 um sieve, to provide tetramethylchroman- 2-carboxylic acid homogeneous powder for analyzing. Powdered (Trolox), sodium nitrite, sodium hydroxide materials were stored in bottles in a dark room (NaOH), sodium carbonate (Na2CO3), temperature and then used. aluminum chloride (AlCl3), potassium persulfate (K2S2O8), ethylenediaminetetraacetic 2.1. .2 Preparation of Freeze-Dried acid (EDTA), and ascorbic acid were Bulb Extracts purchased from Sigma Aldrich Chemicals (USA). Dimethyl sulfoxide (DMSO) was The powdered F. zagrica and T. obtained from Fisher Scientific (USA). kurdica plants bulb materials (100 g) were carried out and macerated with 1 L of The standard bacterial strains of the extracting solvents (99.9 % absolute methanol Gram negative bacteria Escherichia coli and 80% ethanol) and water in the beakers (ATCC 25922), Pseudomonas aeruginosa covered by aluminum foil and incubated in (ATCC 27853), Acinetobacter baumannii Ultrasonic bath at room temperature for two (ATCC 19606) and of the Gram positive hours. The solvents were drained out after 4 h bacteria Staphylococcus aureus (ATCC 25923) and replaced with fresh one. This procedure and Bacillus subtilis (ATCC 6633) and was repeated thrice. The extracts were then pathogenic fungal strains, Candida tropicalis separated from the sample residues by filtration (ATCC 13803), Candida albicans (ATCC

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10231), Candida guilliermondii (ATCC 6260) disc was soaked with sterilized distilled water were used and obtained from the Department (SDW) used as negative control and DMSO as of Biology, College of Science, Salahaddin a solvent control, 50-100 discs were placed in University, Erbil/ Kurdistan-Iraq. A standard small sterile air tight labeled containers and antibiotic (amikacin) was used as positive then allowed to dry for 2 hours. The sterile control. discs were placed in petri dishes. For antifungal activity, yeast suspension prepared from 24h colony by using phosphate buffer saline (PBS) 2.3 Assay of Antibacterial Activity against in compare with standard control with 6 Pathogenic Bacterial Strains concentration 41.5*10 cell/mL of yeast suspension. Then 0.1 ml of yeast suspension The antibacterial activities have been was spread over sabouraud glucose agar checked against Gram-negative E. coli (ATCC medium (SGA), then incubated at 37oC for 24- 25922), P. aeruginosa (ATCC 27853), A. 48h. Zones of inhibition were obtained by baumannii (ATCC 19606) and Gram-positive measurement of the radius from the center of S. aureus by using a modified Kirby-Bauer the disc to the edge of the x inhibition of disk diffusion method. Aliquots of bacterial growth. Measurements were made from both suspension (100 uL) were spread on Difco™ sides of the slope and their average accepted nutrient agar containing testing microorganism (Al-Refai, 2006, Kamel et al., 2014). with optical density of 0.7 at 595 nm. Filter paper disks (8 mm) saturated with 20 uL of the prepared bulb extracts F. zagrica and T. kurdica (10 mg/ mL) in different solvents were 2.5 Determination of Total Phenolic Content placed on nutrient agar seeded with tested The total phenolic content (TPC) bacteria. The plates were incubated for 24 h at quantification of F. zagrica and T. kurdica 37 ºC, and then the zones of growth inhibition plants bulb extracts were achieved by Folin- were measured (Sharma et al., 2018). All the Ciocalteu colorimetric method with slight tests were done in triplicate and mean ± S.D. modifications (Hatami et al., 2014, Marvibaigi were calculated. et al., 2016). Briefly, 100 µl of extracts (1mg/ml diluted in distilled water) were mixed 2.4 Assay of Antifungal Activity against with 100 µl of 0.2 N Folin-Ciocalteu reagents. Pathogenic Fungi After 5 min with intermittent shaking, 80 µl of

Sensitivity of Candida spp. were tested 7.5% sodium carbonate (Na2CO3) solution was against bulb extracts of F. zagrica and T. added and incubated for 2h at room kurdica (1 mg/mL) in methanol, 80% ethanol temperature. The absorbance (of the resulting and aqueous solution by using filter paper disc blue color) was measured at 765 nm against the diffusion method (agar disc diffusion method) blank. The calibration curve was prepared the filter paper disc prepared by using ordinary using the standard gallic acid solution. All TPC office two-hole puncture, paper discs with determinations were carried out three times (n approximate diameter of 6mm. were punched = 3) and the results were expressed in mg of out one by one from a sheet of filter paper, the Gallic acid equivalent (GAE/100 g of sample) disks placed in vials, sterilized by oven and (Marvibaigi et al., 2016, Ismail et al., 2017) as allowed to cool. Then sterilized discs (6 mm) shown in figure 1. were soaked in known concentration (1 mg/mL) of plant extracts; another filter paper

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2.6 Determination of Total Flavonoid (Iqbal et al., 2006, Akter et al., 2010). For each Content (TFC) determination, the stock solution (1mg/ml) was diluted to a dilution series (156 μg-1000 μg/ml) The total flavonoid content estimation with DMSO. An aliquot of each extract of F. zagrica and T. kurdica plants bulb working concentration (20 µl) was mixed with extracts were measured spectrophotometrically methanolic solution of DPPH (200 µl, 0.06 by aluminum chloride colorimetric method, mM). The mixtures were shaken vigorously which is based on the formation of a and incubated at room temperature in the dark flavonoid–aluminum complex with slightly for 30 min. A control sample was placed under modifications (Lin and Tang, 2007, Marvibaigi the same conditions. A color change from et al., 2016). Briefly, 200 µl of extract (1 violet to yellow occurred during the reaction mg/mL) was mixed with 12 µl 5% NaNO2 and time. Sample absorbance was read at 517 nm 12 µl 10% AlCl3. After 5 min incubation at using UV-VIS spectrophotometer and the ambient room temperature, 80 µl of NaOH was percentage of free radical scavenging potential added and re-incubated for 30 min. The of the different extracts against DPPH• was absorbance of samples was read at 515 nm. determined using the following equation: The calibration curve was prepared using standard catechin solution. The TFC of extracts DPPH radical scavenging activity (%) = [(Abs were expressed in mg of g catechin equivalent control- Abs sample) /Abs control] x100 (CE/ 100 g of sample) as illustrated in figure 2. The analysis was performed in triplicate and DPPH solution was used as a control mean values were reported. and ascorbic acid and quercetin were used as references standard. The percentage of DPPH scavenging versus concentration of samples was plotted. The antioxidant activity was 2.7. Antioxidant Assay expressed as median effective concentration 2.7.1. DPPH Free Radical Scavenging (EC50) where the concentration caused 50% Activity Assay reduction of DPPH. All determinations were assayed in triplicate (Brand-Williams et al., DPPH (2, 2-diphenyl-1-picrylhydrazyl 1995, Akter et al., 2010, Ismail et al., 2017). radical), is a dark-colored crystalline powder composed of stable free-radical molecules. The antioxidant activities were determined when 2.7.2. ABTS Free Radical Scavenging using DPPH as a free radical because the use of Activity Assay DPPH• provides an easy and rapid way to evaluate the antiradical activities of The free radical scavenging capacity of antioxidants (Brand-Williams et al., 1995). F. zagrica and T. kurdica plants bulb extracts Hence DPPH has major application in were also measured using TEAC (Trolox laboratory research most notably in anti- equivalent antioxidant capacity) method. The oxidant assays (Bhandari et al., 2017). The TEAC value is based on the ability of the antioxidant activity of F. zagrica and T. antioxidant to scavenge the blue-green kurdica plants bulb extracts, on the basis of the coloured ABTS•+ radical cation relative to the scavenging activity of the stable DPPH free ABTS•+ radical cation scavenging ability of the radical (DPPH•) in prepared 96 well plates, water-soluble vitamin E analogue, Trolox (Re were performed following a previously et al., 1999, Gliszczyńska-Świgło, 2006). described method with slight modifications ABTS stock solution was dissolved in water to

6 Aali. M. et al. /ZJPAS: 2018, 30 (5): 1-16 a 7 mM concentration. ABTS radical cation correlation coefficient (r) was used find the was produced by reacting ABTS stock solution relationships between the total phenolic and with 2.45 mM potassium persulfate and the flavonoid contents and various antioxidant mixture was allowed to stand in dark at room assays. The P value < 0.05 was considered temperature for 12-16 h. Prior to use, ABTS significant. radical cation was diluted in methanol until the absorbance at 0.7 ± 0.02 units. ABTS solution (300 µl) was added to 30 µl sample and 3. RESULTS AND DISCUSSION incubated for 6 min at room temperature. The 3.1. Extraction Yield absorbance was read at 734 nm. Ascorbic acid was chosen as positive control. The radical- Extracts of bulbs of both F. zagrica and scavenging activity was expressed as inhibition T. kurdica were obtained following a percentage, and calculated using the following sequential solvent extraction procedure. formula. Different organic solvents of increasing polarity including ethanol, methanol, and water ABTS Radical scavenging activity were used to determine if any of these solvents could selectively extract compounds with antioxidant and antimicrobial activities. The where Ac is the absorbance of reaction yield percentages of F. zagrica and T. kurdica without samples and As is the absorbance of bulb extracts were expressed in terms of mass tested extract. The antioxidant activity was as percentages of samples as mentioned in Table median effective concentration (EC50) where 1 and Figure 3. Extraction yield was calculated the concentration caused 50% reduction of using following formula: ABTS (Gliszczynskaswiglo, 2006, Ismail et al., Extraction yield (%) = (weight of the freeze- 2017). dried extract x 100) / (weight of the original sample).

2.8. Statistical Analysis Results showed that the methanol bulb extracts of both F. zagrica and T. kurdica Results of all analysis are presented as provide highest yields of extracts when means of triplicate ± standard deviation (SD) compared to 80 % ethanol and aqueous and/or standard error (n = 3). Median effective extracts. The higher yield of methanol extract concentrations (EC50) were statistically might be owed to the fact that methanol analyzed by GraphPad Prism 6 software. Non- possesses high vapor pressure. Although the linear regressions of log(agonist) vs. response solubility of bio-active components and the rate with variable slope (four parameters) was of mass transfer are different, the results selected for EC50 and LogEC50 estimation. validated that nature and polarity of solvent The bottom and top constrain were 0% and affect the percentage yield of the extract. This 100% respectively. Additionally, statistical was in agreement with the study by Marvibaigi, analysis was performed using GraphPad Prism et al. (2016), who reported a high yield of 6 software with two-way ANOVA and Tukey’s mistletoe (Scurrula ferruginea) extracts when multiple comparisons post hoc test and using polar solvents (Marvibaigi et al., 2016). Dunnett's multiple comparisons test. The The lowest percentage yield was exhibited for results were considered statistically significant bulb aqueous extract of F. zagrica, while the at P value < 0.05. Furthermore, Pearson highest yield was observed in the bulb

7 Aali. M. et al. /ZJPAS: 2018, 30 (5): 1-16 methanol extract of F. zagrica. In addition to Maximum zones of inhibition of F. the lowest percentage yield was recorded for zagrica aqueous extracts (15.0, 26.33, 25.33 bulb aqueous extract of T. kurdica, while the mm) were noticed against each of E. coli highest yield was revealed in the bulb methanol ATCC 25922, A. baumannii ATCC 19606 and extract of T. kurdica. Large variations were S. aureus ATCC 25923, respectively. While showed between extraction yields of methanol, maximum zones of inhibition of P. aeruginosa 80 % ethanol and aqueous extracts for both ATCC 27853 (24.0 mm) and B. subtilis ATCC medicinal plants. Also using 80% water- 6633 (27.67 mm) were found for each of F. ethanol solvent observed higher yield than zagrica methanol extract and F. zagrica using water alone to bulb extracts of both F. ethanol extracts, respectively. zagrica and T. kurdica plants. The results of other studies showed that the highest extraction Tulipa kurdica aqueous extracts were yields with aqueous solutions might be showed maximum zones of inhibition (25.0, ascribed to an increase of polarity of the 31.0, 26.67 mm) against each of E. coli ATCC solvents by adding water having a high 25922, P. aeruginosa ATCC 27853 and S. dielectric constant (Kim et al., 2004, Kallel et aureus ATCC 25923 respectively, whereas al., 2014), which corroborate results of the ethanol extracts of T. kurdica were exhibited present study. Hence, the sequence for maximum inhibition zones against A. increasing extraction yields were methanol baumannii ATCC 19606 (27.0 mm) and B. extract > 80% ethanol extract > water extract. subtilis ATCC 6633 (30.0 mm). The difference in antibacterial activity of different bulb extracts may be due to difference in solubility of bioactive compounds in various solvents and 3.2. Assay of Antimicrobial Activity against variations in cell wall structure of tested Pathogenic Organisms bacteria. The mode of action of different This is the first report on the bioactive compounds varies which represent antibacterial screening and antifungal activity the ability of these compounds to kill or inhibit of F. zagrica and T. kurdica growing from bacteria (Howell, 2007). Kurdistan Region-Iraq. Plants and their Aqueous extract of F. zagrica and T. secondary metabolites have shown great kurdica showed better antibacterial activity potential as antibacterial and antifungal source. compared to other solvent extracts. The The first step towards this goal is the in-vitro findings of the present study are in agreement antimicrobial activity. The antibacterial and with previous investigation, which concluded antifungal assay of bulb extracts of F. zagrica that water extract exhibited higher antibacterial and T. kurdica were performed against some activity than ethanol extract of leaves and bark pathogenic bacterial and fungal strains. The of Cassia alata, when tested against C. results in Table 2 and Figure 4 elucidated that albicans and S. aureus (Somchit et al., 2003, F. zagrica and T. kurdica bulb extractions by Chen et al., 2018). The broad antibacterial using different solvents had strong antibacterial activities of the Garlic (Allium sativum L.) and activities against all using bacterial strains Sida rhombifolia Linn extracts can probably be (Gram positive and Gram negative bacteria) attributed to the presence of various bio-actives according to positive standard control components such as the phenolic (Kallel et al., (amikacin). 2014) alkaloid and flavonoid compounds (Dzoyem et al., 2010). As previously mention in the present that polar solvents are frequently

8 Aali. M. et al. /ZJPAS: 2018, 30 (5): 1-16 worked for the recovery of polyphenols from a zones (18.0, 15.0, 9.0 mm) against C. tropicalis plant matrix. ATCC 13803, C. albicans ATCC 10231 and C. guilliermondii ATCC 6260, respectively. Fritillaria zagrica and T. kurdica methanol, 80% ethanol and aqueous extracts The T. kurdica bulb (methanol, 80% like many other plants showed antibacterial ethanol and aqueous) extracts showed activity. Kumaraswamy et al. (2008) revealed antifungal activities against C. tropicalis ATCC that methanol, ethanol and aqueous Betula 13803 with the maximum inhibition zone (16 utilis D extracts had significant activity against mm) of 80% ethanol extract. The T. kurdica some human pathogenic bacteria. Also Moon 80% ethanol and aqueous, extracts were et al. (2011) showed that methanol and ethanol exhibited antifungal activities against C. bulb extracts from F. unibracteata dose- albicans ATCC 10231. While The T. kurdica dependently have inhibitory effect and bulb (methanol, 80% ethanol and aqueous) increased antimicrobial activity, the strongest extracts had no antifungal effect against C. inhibition showed at the highest concentration guilliermondii ATCC 6260. of 20 mg/mL (Moon et al., 2011). Johnson and his colleagues (2011) screened five important In parallel to our finding, previous medicinal plants, and the results observed that studies were showed that T. kurdica bulb the maximum of Aloevera plant was to be extracts have potent Tuliposides and tulipalins exposed against S. aureus and E. coli, while compounds, which exhibited their antifungal Lanatacamara inactive against bacterial strains activity (Shigetomi et al., 2011). Bergman et al. (Johnson et al., 2011). However, the aqueous (1967) isolated and identified fraction of the Pongamia pinnata had more α‐ methylene‐ butyrolactone (tulipan A), a active as compared to alcoholic extract against fungitoxic substance from tulips bulb. E. coli. Accordant with Moon et al. (2011) who were concluded that methanol and ethanol bulb In other side Shigetomi et al. (2013) extracts of F. unibracteata have potent anti- demonstrated that the natural products 1- microbial activity (for example C. albicans) for tuliposide B and the lactonized aglycon (±)- the supportive treatment on respiratory tulipaline B in bulb Tulipa are potent inhibitors diseases, food poisoning, and gastroenteritis of MurA, which may partly explain the known diseases. antibacterial activity of these compounds (Shigetomi et al., 2013) and recently, a potent antibacterial activity of 6-tuliposide B has been 3.3. Total Phenolic and Flavonoid Content reported (Shigetomi et al., 2010) . Also Tuliposides in bulb Tulipa had been reported to Table 4 and 5 summarize the TPC and reveal antimicrobial activities and the TFC in methanol, 80% ethanol and water bulb formation of tulipalins played a key role in extracts of both F. zagrica and T. kurdica antimicrobial action (Shigetomi et al., 2011, medicinal plants. The TPC values varied Lim, 2014). widely, ranging from 21.83 mg GAE/100 g samples to 153.7 mg GAE/100 g samples. MF The results Table 3 and Figure 5 exhibited the highest phenolic content at 153.7 demonstrated the antifungal activities of F. mg GAE/100 g sample followed by EF (150.4 zagrica and T. kurdica bulb extractions by mg GAE/100 g sample), AF (139.4 mg using different solvents. F. zagrica methanol GAE/100 g sample), MT (32.61 mg GAE/100 extracts were observed the maximum inhibition g sample), AT (29.16 mg GAE/100 g sample)

9 Aali. M. et al. /ZJPAS: 2018, 30 (5): 1-16 and ET (21.83 mg GAE/100 g sample). In rapid way to evaluate the antiradical activities another hand, the TFC detected varied from of antioxidants. For that reason, the antioxidant 80.12 CE/100 g sample to 365.5 mg CE/100 g activities were determined using DPPH as a sample. The highest flavonoid content was in free radical (Brand-Williams et al., 1995). The MF at 365.5 mg CE/100 g sample followed by medicinal plants showing strong scavenging EF (179.2 mg CE/100 g sample), MT (130.3 capacity on DPPH•, which is possibly due to mg CE/100 g sample), AF (114.4 mg CE/100 g the hydrogen donating ability of the sample) ET (99.49 mg CE/100 g sample) and polyphenolic (phenolic and flavonoid) AT (80.12 mg CE/100 g sample). TPC and compounds in the extracts (Mazhar, 2014, TFC of F. zagrica and T. kurdica were Bhagat et al., 2011). statistically different from each other (P <0.05), The antioxidant activities of the F. these two phytochemical compounds in zagrica and T. kurdica bulb extracts were methanol, 80% ethanol and aqueous extracts of assessed by different in vitro tests; DPPH and both plants were statistically different from ABTS radical scavenging activities as each other (P <0.05) as shown in Figure 6 and presented in Figure 8. The EC50 calculated for 7. DPPH (Table 6) were (2.006 2.661 µg/ml, 2.961 µg/ml, 9.241 µg/ml, 11.88 µg/ml and Furthermore, results of the current 21.77 µg ml-1) µg/ml for MF, EF, AF, ET, MT study represented that different plant extracts and AT respectively. Meanwhile, a similar contained different levels of TPC and TFC. F. trend was mentioned (Table 6) in ABTS in zagrica contain significantly higher phenolic which the EC50 for EF, AF, MT, MF, ET, and content than T. kurdica. The total phenol and AT were (0.1535, 0.1611, 0.4435, 1.147, 1.532 flavonoid contents of the extracts were very and 2.493 µg ml-1) respectively. Scavenging of near to those most medicinal plants. Results of DPPH radicals is mechanized by the donation the current study were in agreement with the of hydrogen atom to the unpaired electron of previous studies (Marvibaigi et al., 2016), nitrogen bridge causing the purple color turn to which presented that methanolic extracts of yellowish. Meanwhile, the ABTS+ radical Scurrula ferruginea were rich in phenolic and cation undergoes the reduction process by flavonoid compounds. The presence of large hydrogen donating antioxidant and can be amounts of phenolic compounds in the spectrophotometrically measured. It is that the methanol, 80% ethanol and aqueous extracts phenolic and flavonoid content significantly may contribute to the antioxidant activities and influence the antioxidant activities (Othman et the ability to adsorb and scavenge free radicals al., 2014, Ismail et al., 2017). (Kumar et al., 2014). This original method quantified scavenging capacity by measuring a test compound at different concentrations and calculating the compound concentration 3.4. DPPH and ABTS Free Radicals required to reduce the initial DPPH Scavenging Activities and Their Correlation concentration by 50% at steady-state (EC50). to Phenolic and Flavonoid Contents A lower EC50 value is associated with a stronger DPPH and ABTS radical scavenging DPPH is a stable free radical, when capacity under the same testing conditions. antioxidant reacts with DPPH• the electron is Higher antioxidant activity is related to lower paired off and the DPPH solution is EC50 value (Liangli, 2008). Based on DPPH decolorized and DPPH• provides an easy and

10 Aali. M. et al. /ZJPAS: 2018, 30 (5): 1-16 and ABTS radical scavenging activity analysis, activity compared to the pure solvent (Turkmen all bulb extracts of F. zagrica and T. kurdica et al., 2006). represented antioxidant activity. The lowest EC50 value of DPPH was gained from Besides, the (aqueous) bulb extract methanol bulb extracts (MF) followed by illustrated low antioxidant potential (higher ethanol (EF) and aqueous (AF) bulb extracts of EC50 values of DPPH and ABTS) and also low F. zagrica while the lowest EC50 value was content of total phenols as compared to the obtained from ethanol (ET) bulb extracts other bulb extracts produced with methanol and followed by methanol (MT) and aqueous (AT) 80 % ethanol solvents. Similar results have bulb extracts of T. kurdica. The lowest EC50 been reported during the extraction of value of DPPH in methanol bulb extract may antioxidant compounds from other raw due to the fact that methanol is better solvent materials such as mango peel and seed (Dorta than the others in extracting phenolic et al., 2012), grape by-products (Lapornik et compounds from the extracts due to their al., 2005). polarity and good solubility for phenolic In the present study Table 7 represents components from different plant materials such the correlation coefficients of the possible as walnut green husk, cacao bean husk and correlation between the phenolic and flavonoid wild rice hulls (Kallel et al., 2014). Thus the contents of F. zagrica and T. kurdica bulb samples with higher total phenols content extracts and their antioxidant activities (DPPH showed the higher antioxidant properties and ABTS). It also observes the correlation (lower EC50 values). The results of present between different methods used. The TPC and study indicated that increased concentrations TFC showed a significant and positive linear resulted in enhancing the scavenging capacity correlation (p<0.05) with different antioxidant of bulb extract samples. Broad variation of activity assays (DPPH and ABTS activities). antioxidant activity may be possibly These results suggested that the antioxidant attributable to the presence of wide range of activity is more closely related to TPC than biologically active components like phenols, TFC. Our findings exhibited a strong (higher) flavonols, carotenoids and some other positive correlation between TPC and compounds (Marvibaigi et al., 2014). antioxidant activity assays (R2 = 0.8261 and Additionally, Liu et al (2012) indicated that a 0.5861 of DPPH and ABTS, respectively) and water-soluble polysaccharide (FUP-1) was also proved that the phenolic compounds were obtained from Fritillaria ussuriensis Maxim, the major contributors to the antioxidant exerts antioxidant activity not only through its capacity of the F. zagrica and T. kurdica bulb own radical-scavenging activity but also by extracts. Similarly, Turkmen et al. (2006) who boosting the enzymatic and non-enzymatic declared that the results of black mate tea antioxidant defense system of the host. showed that solvent with different polarity had From the observation, the extracted significant effect on polyphenol content and compound from the 70% ethanol and methanol antioxidant activity and a high correlation gave higher antioxidant activity compared to between polyphenol content and antioxidant the absolute solvent and Turkmen et al. (2006) activity of tea extracts was observed (Turkmen reported the same findings where they found et al., 2006). 50% and 80% of solvent mixture exhibited Moreover, Pearson correlation analysis considerably higher DPPH radicals scavenging of the results revealed a significant and positive correlation between different antioxidant

11 Aali. M. et al. /ZJPAS: 2018, 30 (5): 1-16 assays (p<0.05). The highest correlation was contributors to the antioxidant capacity of the elucidated between ABTS and DPPH (r = F. zagrica and T. kurdica bulb extracts. 0.7601), whereas ABTS that could scavenge •+ • The strong antioxidant abilities of F. zagrica ABTS was also able to scavenge DPPH . and T. kurdica along with their traditional use Furthermore, the strong correlation between in the treatment of various ailments be DPPH and ABTS methods suggests that the suggesting their power potential as natural antioxidants in the extracts react similarly with antioxidants and providing the scientific both assays. The findings of the present study rationale to obtain pure compounds and then are in agreement with previous investigation of develop new therapeutic drugs against breast Ismail et al. (2017) who reported that radical cancer with accomplishing cytotoxicity and scavenging of DPPH and ABTS were anticancer screening for further detailed positively correlated to the phenolic and analysis in the future. flavonoid content of tested herbal plants, which include Andrographis paniculata (leaves), Cinnamon zeylanicum (bark), Curcuma Acknowledgements xanthorrhiza (rhizome), Eugenia polyantha This study was supported by the (leaves) and Orthosiphon stamineus (whole University of Salahaddin Erbil/Iraq and the plant). Based on our knowledge there is no Universiti Teknologi Malaysia, in the report on TPC and TFC and antioxidant Framework of a split-side Ph.D. program. the activities of F. zagrica and T. kurdica bulb Author acknowledges with many thanks to Dr. extracts. Abbas B. Q. Salihi for the statistics analysis and the botanists Prof. Abdul Hussain Al- Khayyat (College of Education, Salahaddin 4. CONCLUSIONS University-Erbil/Iraq) and Mrs. Bnar Khalid The present study demonstrated and proven Bakr (College of Science, Salahaddin that F. zagrica and T. kurdica bulb extracts in University-Erbil/Iraq) for the plants different solvents were exhibited strong identifications and documentations. antibacterial activities against all tested Conflict of Interest (1) bacterial strains (Gram positive and Gram Table (1) Percentage yield of two medicinal bulb negative bacteria), and fungal strains except C. extracts in different solvents guilliermondii ATCC 6260. Aqueous bulb extract of F. zagrica and T. kurdica showed Yields (%) highest antibacterial activity compared to other solvent extracts. Bulb Methanol 80%Ethano Aqueous Additionally, the phenolic and flavonoid Extract l F. zagrica 7.76 ± 2.97 ± 0.15 2.60 ± content were varied between the tested bulb 0.18* 0.08 extracts and the DPPH and ABTS scavenging T. kurdica 7.40 ± 1.1* 4.70 ± 0.18 3.57 ± 0.2 activities were found to be significantly correlated with the amount of TPC and TFC. All results are means of three replicates determinations ± Moreover, methanol F. zagrica bulb extracts SD (n = 3). contain significantly higher phenolic content * Represents significant difference at p < 0.05. than T. kurdica. Also demonstrated that the phenolic compounds were the major Table (2) Antibacterial screening test [zone of inhibition (mm)] of F. zagrica and T. kurdica bulb extracts

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(10mg/mL) in different solvents against some bacterial 0.0002) and among bulb extracts of both medicinal pathogenic strains. plants (p value = 0.0153). (0): No Antifungal inhibitory activity.

Inhibition zones (mm) Bulb Table (4) Total phenolic content of two medicinal bulb Extract extracts in different solvents Gram (-ve) pathogenic bacteria TPC (mg GAE/100 g sample) Gram (+ve) pathogenic bacteria

E. coli P. A. S. B. Bulb Methanol 80%Ethano Aqueou ATCC aerugin bauman aureus subtilis Extract l s 25922 osa nii ATCC ATCC F. zagrica 153.7 ± 150.4 ± 139.4 ± ax* bx cx ATCC ATCC 25923 6633 1.415 0.65 0.50 ay 27853 19606 T. kurdica 32.61 ± 0.18 21.83 ± 29.16 ± by cy MF 12.33 ± 24.00 ± 19.00 ± 19.33 ± 24.67 ± 0.53 0.44 0.33a 0.58 1.16 a 0.33 0.33 a All results are means of three replicates determinations ± EF 12.33 ± 13.00 ± 20.67 ± 15.33 ± 27.67 ± SD (n = 3). 0.67 a 0.58 0.67 a 0.88 a 1.45 a * Represents significant difference at p < 0.05. AF 15.00 ± 19.67 ± 26.33 ± 25.33 ± 25.00 ± 0.58 a 0.88 a 0.88 0.88 0.58 a MT 20.00 ± 10.00 ± 15.00 ± 15.33 ± 25.00 ± Table (5) Total Flavonoid content of two medicinal bulb 1.16 1.16 0.58 a 0.88 a 0.58 a extracts in different solvents ET 22.00 ± 25.67 ± 27.00 21.67 ± 30.00 ± TFC (CE/100 g of sample) 1.16 0.67 ±1.00 0.88 0.58 Bulb Methanol 80% Aqueous AT 25.00 ± 31.00 ± 23.67 ± 26.67 ± 28.67 ± Extract Ethanol 0.58 1.00 0.88 1.20 0.33 F. zagrica 365.5 ± 179.2 ± 114.4 ± PC 14.67 ± 19.67 ± 18.00 ± 14.00 ± 24.67 ± 20.01 12.11 2.48 0.33 0.88 0.58 0.58 0.88 T. kurdica 130.3 ± 99.49 80.12 ± Data are means of three replicates (n = 3) ± standard 1.24 ±13.46 1.42 error. a Not statistically different from positive control (PC). All results are means of three replicates determinations ± SD (n = 3).

* Represents significant difference at p < 0.05.

Table (6) Mean of TPC, TFC and EC50 of DPPH and ABTS radical scavenging activity. Table (3) Antifungal activities [zone of inhibition (mm)] Bulb Total Total EC50 - EC50 – of F. zagrica and T. kurdica bulb extracts against some Extract phenolic flavonoid DPPH ABTS pathogenic fungal organisms (1 mg/mL). content content RSA RSA F. zagrica T. kurdica (mg (mg (µg/mL) (µg/mL) GAE/100g CE/100g Fungal Pathogenic M E A M E A sample) sample) Organism F F F T T T MF 153.7 ± 365.5 ± 2.006 1.147 C. tropicalis ATCC 18 14 15 12 16 11 1.415 20.01 13803 MT 32.61 ± 130.3 ± 11.88 0.4435 C. albicans ATCC 15 14 13 0 10 11 0.18 1.24 10231 EF 150.4 ± 179.2 ± 2.661 0.1535 C. guilliermondii 9 8 6 0 0 0 0.65 12.11 ATCC 6260 ET 21.83 ± 99.49 9.241 1.532 Data analysis by Two-way ANOVA (p<0.05). There are 0.53 ±13.46 significant differences among Candida spp. (p value = AF 139.4 ± 114.4 ± 2.961 0.1611 0.50 2.48

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AT 29.16 ± 80.12 ± 21.77 2.493 1 0 M e th a n o lic

0.44 1.42 8 0 % E th a n o lic

Each value in the table is represented as mean (n = 3). 8 A q u e o u s d

l 6

e i

Y Table (7) Pearson correlation coefficients between % 4

antioxidant activities and TPC/ TFC of F. zagrica and T. 2 kurdica bulb extracts (at significance P < 0.05). 2 0 Correlation coefficients (R ) F . za g ric a B u lb E x tra c ts T . k u rd ic a B u lb E x tra c ts Assays TPC TFC DPPH ABTS Figure 3: Percentage of F. zagrica and T. kurdica bulb TPC 1.0000 0.6528* 0.8261** 0.5861* extraction yields obtained by using different solvents. TFC 0.6528* 1.0000 0.5923* 0.1697 DPPH 0.8261** 0.5923* 1.0000 0.7601* ABTS 0.5861* 0.1697 0.7601* 1.0000

Figure 4: Antibacterial screening test of F. zagrica and T. kurdica bulb extracts in different solvents against some bacterial pathogenic strains.

Figure 1: Standard curve graph of Total phenolic content (TPC).

Figure 5: Antifungal activities of F. zagrica and T. kurdica bulb extracts against some pathogenic fungal organisms (1 mg/mL). Figure 2: Standard curve graph of Total Flavonoid Content (TFC).

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