African Journal of Biotechnology Volume 13 Number 50, 10 December, 2014 ISSN 1684-5315

ABOUT AJB

The African Journal of Biotechnology (AJB) (ISSN 1684-5315) is published weekly (one volume per year) by Academic Journals.

African Journal of Biotechnology (AJB), a new broad-based journal, is an open access journal that was founded on two key tenets: To publish the most exciting research in all areas of applied biochemistry, industrial microbiology, molecular biology, genomics and proteomics, food and agricultural technologies, and metabolic engineering. Secondly, to provide the most rapid turn-around time possible for reviewing and publishing, and to disseminate the articles freely for teaching and reference purposes. All articles published in AJB are peer- reviewed.

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George Nkem Ude, Ph.D Prof. Dr. AE Aboulata Plant Breeder & Molecular Biologist Plant Path. Res. Inst., ARC, POBox 12619, Giza, Egypt Department of Natural Sciences 30 D, El-Karama St., Alf Maskan, P.O. Box 1567, Crawford Building, Rm 003A Ain Shams, Cairo, Bowie State University Egypt 14000 Jericho Park Road Bowie, MD 20715, USA Dr. S.K Das Department of Applied Chemistry and Biotechnology, University of Fukui, Japan Editor Prof. Okoh, A. I. N. John Tonukari, Ph.D Applied and Environmental Microbiology Research Department of Biochemistry Group (AEMREG), Delta State University Department of Biochemistry and Microbiology, PMB 1 University of Fort Hare. Abraka, Nigeria P/Bag X1314 Alice 5700, South Africa

Dr. Ismail TURKOGLU Department of Biology Education, Education Faculty, Fırat University, Elazığ, Turkey

Prof T.K.Raja, PhD FRSC (UK)

Department of Biotechnology

PSG COLLEGE OF TECHNOLOGY (Autonomous)

(Affiliated to Anna University)

Coimbatore-641004, Tamilnadu,

INDIA.

Dr. George Edward Mamati Horticulture Department, Jomo Kenyatta University of Agriculture and Technology, P. O. Box 62000-00200, Nairobi, Kenya.

Dr. Gitonga Kenya Agricultural Research Institute, National Horticultural Research Center, P.O Box 220, Thika, Kenya.

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Finland Dr. G. Reza Balali Molecular Mycology and Plant Pthology Prof. Irina-Draga Caruntu Department of Biology Department of Histology University of Isfahan Gr. T. Popa University of Medicine and Pharmacy Isfahan 16, Universitatii Street, Iasi, Iran Romania Dr. Beatrice Kilel Dr. Dieudonné Nwaga P.O Box 1413 Soil Microbiology Laboratory, Manassas, VA 20108 Biotechnology Center. PO Box 812, USA Plant Biology Department, University of Yaoundé I, Yaoundé, Prof. H. Sunny Sun Cameroon Institute of Molecular Medicine National Cheng Kung University Medical College Dr. Gerardo Armando Aguado-Santacruz 1 University road Tainan 70101, Biotechnology CINVESTAV-Unidad Irapuato Taiwan Departamento Biotecnología Km 9.6 Libramiento norte Carretera Irapuato- Prof. Ima Nirwana Soelaiman León Irapuato, Department of Pharmacology Guanajuato 36500 Faculty of Medicine Mexico Universiti Kebangsaan Malaysia Jalan Raja Muda Abdul Aziz Dr. Abdolkaim H. Chehregani 50300 Kuala Lumpur, Department of Biology Malaysia Faculty of Science Bu-Ali Sina University Prof. Tunde Ogunsanwo Hamedan, Faculty of Science, Iran Olabisi Onabanjo University, Ago-Iwoye. Dr. Abir Adel Saad Nigeria Molecular oncology Department of Biotechnology Dr. Evans C. Egwim Institute of graduate Studies and Research Federal Polytechnic, Alexandria University, Bida Science Laboratory Technology Department, Egypt PMB 55, Bida, Niger State,

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Prof. Djamel Saidi Dr. Meltem Sesli Laboratoire de Physiologie de la Nutrition et de College of Tobacco Expertise, Sécurité Turkish Republic, Celal Bayar University 45210, Alimentaire Département de Biologie, Akhisar, Manisa, Faculté des Sciences, Turkey. Université d’Oran, 31000 - Algérie Algeria Dr. Idress Hamad Attitalla Omar El-Mukhtar University, Dr. Tomohide Uno Faculty of Science, Department of Biofunctional chemistry, Botany Department, Faculty of Agriculture Nada-ku, El-Beida, Libya. Kobe., Hyogo, 657-8501,

Japan Dr. Linga R. Gutha Dr. Ulises Urzúa Washington State University at Prosser, Faculty of Medicine, 24106 N Bunn Road, University of Chile Independencia 1027, Santiago, Prosser WA 99350-8694. Chile

Dr Helal Ragab Moussa Dr Takuji Ohyama Bahnay, Al-bagour, Menoufia, Faculty of Agriculture, Niigata University Egypt. Dr Mehdi Vasfi Marandi Dr VIPUL GOHEL University of Tehran DuPont Industrial Biosciences Danisco (India) Pvt Ltd Dr FÜgen DURLU-ÖZKAYA 5th Floor, Block 4B, Gazi Üniversity, Tourism Faculty, Dept. of DLF Corporate Park Gastronomy and Culinary Art DLF Phase III Gurgaon 122 002 Dr. Reza Yari Haryana (INDIA) Islamic Azad University, Boroujerd Branch

Dr. Sang-Han Lee Dr Zahra Tahmasebi Fard Department of Food Science & Biotechnology, Roudehen branche, Islamic Azad University Kyungpook National University Daegu 702-701, Dr Albert Magrí Korea. Giro Technological Centre

Dr. Bhaskar Dutta Dr Ping ZHENG DoD Biotechnology High Performance Computing Zhejiang University, Hangzhou, China Software Applications Institute (BHSAI) Dr. Kgomotso P. Sibeko U.S. Army Medical Research and Materiel University of Pretoria Command 2405 Whittier Drive Dr Greg Spear Frederick, MD 21702 Rush University Medical Center

Dr. Muhammad Akram Prof. Pilar Morata Faculty of Eastern Medicine and Surgery, University of Malaga Hamdard Al-Majeed College of Eastern Medicine, Hamdard University, Dr Jian Wu Karachi. Harbin medical university , China

Dr. M. Muruganandam Dr Hsiu-Chi Cheng Departtment of Biotechnology National Cheng Kung University and Hospital. St. Michael College of Engineering & Technology, Kalayarkoil, Prof. Pavel Kalac India. University of South Bohemia, Czech Republic

Dr. Gökhan Aydin Dr Kürsat Korkmaz Suleyman Demirel University, Ordu University, Faculty of Agriculture, Atabey Vocational School, Department of Soil Science and Plant Nutrition Isparta-Türkiye, Dr. Shuyang Yu Dr. Rajib Roychowdhury Department of Microbiology, University of Iowa Centre for Biotechnology (CBT), Address: 51 newton road, 3-730B BSB bldg. Iowa Visva Bharati, City, IA, 52246, USA West-Bengal, India. Dr. Binxing Li

Dr. Mousavi Khaneghah College of Applied Science and Technology- Applied Food Science, Tehran, Iran.

Dr. Qing Zhou Department of Biochemistry and Molecular Biology, Oregon Health and Sciences University Portland.

Dr Legesse Adane Bahiru Department of Chemistry, Jimma University, Ethiopia.

Dr James John School Of Life Sciences, Pondicherry University, Kalapet, Pondicherry

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African Journal of Biotechnology International Journal of Medicine and Medical Sciences

Table of Contents: Volume 13 Number 50, 10 December, 2014

ARTICLES

Antenna Elicitation And Behavioral Responses Of Oriental Fruit Moth, Grapholita Molesta, To Allyl Cinnamate M. Giner, M. Balcells and J. Avilla

Phytochemical, Proximate And Anti-Nutrient Compositions Of Four Leafy Vegetables Used In South Eastern Nigeria Abu Ngozi E., Ozoagudike Chinenye M. and Akaneme Florence I.

Physicochemical, Organoleptic And Nutritional Characteristics Of Four Sweet Cassava (Manihot Opi) Varieties E. Njoh Ellong, C. Billard and S. Adenet

Seed Pre-Treatment Methods For Improving Germination Of Acacia Tortilis So Hanaoka, Norio Nakawa, Norihisa Okubo, Stephen Fredrick Omondi and Jason Kariuki

Chrysodeixis Includens (Lepidoptera: Noctuidae) On Soybean Treated With Resistance Inducers Paulo Vinicius de Souza, Bruna Ribeiro Machado, Marcelo Mueller de Freitas, Fernanda Correa, André Cirilo de Sousa Almeida and Flávio Gonçalves de Jesus

Novel Carrier System For Enhancing Oral Delivery Of Metformin Adedokun, M. O., Momoh, M. A., Kenechukwu, F. C., Umeh, O. N. C., Okpalanedu, C. M. J. and Ofokansi, K. C.

Differential Responses Of Growth, Chlorophyll Content, Lipid Peroxidation And Accumulation Of Compatible Solutes To Salt Stress In Peanut (Arachis Hypogaea L.) Cultivars Meguekam Liliane Tekam, Taffouo Victor Désiré, Grigore Marius-Nicusor, Zamfirache Magdalena Maria, Youmbi Emmanuel and Amougou Akoa

Antidiabetic Activity And Acute Toxicity Evaluation Of Aqueous Leaf Extract Of Vernonia Amygdalina M. A. Momoh, M. O. Adedokun, A. T. Mora and A. A. Agboke

Vol. 13(50), pp. 4536-4540, 10 December, 2014 DOI: 10.5897/AJB2014.14143 Article Number: 4A1BB9648953 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2014 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB

Full Length Research Paper

Antenna elicitation and behavioral responses of oriental fruit moth, Grapholita molesta, to allyl cinnamate

M. Giner1,2*, M. Balcells3 and J. Avilla1,2

1Department of Crop Production and Forestry Science, University of Lleida, Rovira Roure 191, 25005 Lleida, Spain. 2Department of Crop Protection, IRTA-Centre Lleida, Rovira Roure 191, 25005 Lleida, Spain. 3Department of Chemistry, University of Lleida, Rovira Roure 191, 25005 Lleida, Spain.

Received 1 September, 2014; Accepted 28 November, 2014

Female sex pheromones have been used in pest control since the 90s; attracting males to baited traps (mass-trapping and monitoring) or avoiding (or reducing) mating in fields under mating disruption. By contrast, little is done among the use of male sex pheromones in pest control. Allyl cinnamate was evaluated as potential oriental fruit moth (Grapholita molesta, Busck) (Lepidoptera: Tortricidae) behaviour modifier, after recording positive electroantenographical responses in both male and female moths. Females over-responded in front of sources of allyl cinnamate at short distances, and both male and female showed typical pre-mating behavioural responses at mid-distances (in a wind tunnel). Males responded showing its hair-pencils and wing fanning and females started with wing fanning, curling abdomen and locating postion opposite the source of allyl cinnamate. The same effect was observed in front of trans-ethylcinnamate, the main component of G. molesta male sex pheromone. Results here indicate a putative role of male sex pheromones (or chemically related compounds as allyl cinnamate) in oriental fruit moth integrated pest control. Understanding the role of male sex pheromones and chemically related compounds could help in the development of new pest control strategies.

Key words: Electroantennography (EAG), male sex pheromone, allyl ester, trans-ethyl cinnamate, wind-tunnel, behaviour modifier.

INTRODUCTION

Grapholita molesta (Busck) (Lepidoptera: Tortricidae), the depreciating economic value of fruits (González, 2003). oriental fruit moth, is a key-pest in most stone-fruit productive Moreover, feeding wounds are easily infected by brown areas wordwide (Rotschild and Vickers, 1991), and it is rot (Garic et al., 2004; Holb, 2004), increasing fruit depre- also known to cause damages in apples at the end of the ciation and reducing its marketability. season (Kovanci et al., 2004). They firstly feed shoots, Chemical control is still being the most used technique and after in fruits, causing damage on young trees and to maintain G. molesta populations under economic

*Corresponding author. E-mail: [email protected].

Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Giner et al. 4537

thresholds, but mating disruption based on use of female behavioral assays 3 h before light off [peak of responsiveness of sex pheromones, are being greatly spread as a way to males to female sex pheromone and female gland extrusion (Baker avoid insecticide negative effects (Il‟chev et al., 2004; and Cardé, 1979)]. The mating status of females was ascertained by the presence of Devine and Furlong, 2007; Kong et al., 2014). spermatophore, which was determined after EAG recording or tunnel Female sex pheromones are used to monitor pest bioassay. Males maintained in the same case than females that populations aimed to do chemical treatments in the best were ascertained to be mated were differentially assessed than moment, to bait traps in mass-trapping and also for males that were kept in cages with no presence of females. attract-and-kill strategies (Damos et al., 2014). In all cases, females are not the target, although some authors have reported an effect on female behaviour by exposure to Chemicals their own sex pheromone (Stelinski et al., 2006; Gökçe et Allyl cinnamate and trans-ethyl cinnamate used in all bioassays al., 2007; Kuhns et al., 2012). To increase the attractive were purchased by Sigma-Aldrich (www.sigmaaldrich.com). The effect on females, some volatile compounds were success- main compound of G. molesta female sex pheromone was purchased fully added to pheromonal blends (Natale et al., 2003, by Pherobank (www.pherobank.com). All compounds had a 2004; Piñero and Dorn, 2007). minimum of 98% purity.

By contrast, few Lepidopteran male sex pheromones were studied even playing an important role in mate accep- tance (Landolt and Heath, 1989; Hillier and Vickers, Electrophysiological assays

2011). In the case of oriental fruit moth, a male sex An EAG apparatus from Syntech (www.syntech.nl) was used. pheromone is described; trans-ethyl cinnamate which is Signals after stimulus application (mV) were amplified (100×) and the main component. It is emitted by G. molesta males on filtered (DC to KHz) with an ID-2 interface (Syntech), digitized on a their hair-pencils as aphrodisiac (Birch and Hefetz, 1987), PC and analysed with the EAG2000 program. increasing mating success at short distances (Baker et Antenna was carefully cut from an insect that was previously al., 1981; Löfstedt et al., 1990). anesthetized with ice and then immobilized using a fine needle. Another cut was done at the end of the antenna using a scalpel. The study of courtship behaviour and mate acceptance Then, antenna was placed between EAG electrodes, using electrode could help to improve pest control based in the use of gel (www.parkerlabs.com/signagel.asp) to facilitate connexion synthetic pheromones. A highly stereotypic sequence of between antenna and electrodes. Each stimulus was presented by behaviours are described to occur before mating (Curkovic first applying 0.1 µg to a piece of filter paper (2 × 2 cm). The piece et al., 2006), so the interference in this sequence could of paper was then reinserted into a Pasteur pipette, which was placed so that the tip of the pipette was 5 cm from the antenna. A reduce mating and, consequently, pest populations. -1 puff of air (300 mL min ) through the pipette then carried the stimuli Allyl cinnamate, chemically related with oriental fruit to the antenna. moth male main component of pheromone (Figure 1), have At least 10 antennae per sex and mating status were used in the been described as female attractant on other two Tortricidae bioassay. Five consecutive puffs (separated by 30 s) of the allyl pests and so, an effect in oriental fruit moth was be cinnamate and control puffs with only solvent (acetone) were applied expected. to each antenna in randomized order. No fatigue was observed in any antennae used in the bioassay. This work describes for the first time allyl cinnamate as For each antenna, the response to allyl cinnamate was calculated antenna elicitator and behaviour modifier of G. molesta as the mean response to five puffs each, and was corrected by the moths, and contributes to the knowledge of oriental fruit mean of corresponding acetone puffs (EAG corrected = mean EAG moth courtship behaviour aim to be used into an inte- compound – mean EAG control). Mean corrected EAG response of grated pest management (IPM) program. Differential allyl cinnamate for each sex and state of mating were transformed effect than observed in other Tortricidae species is sug- [log (x + 1)] to normalize the data, and then compared by one-way ANOVA, followed by Tukey-Kramer HSD test (P < 0.05) using the gested due to similarities of allyl cinnamate with the main JMP 8.0.1 program (www.jmp.com). component of G. molesta male pheromone.

Behavioural assays MATERIALS AND METHODS First assay was done to assess virgin female responses to allyl Insects cinnamate at short distances. 0.1 micrograms of allyl cinnamate disolved in acetone were applied in a filter paper (2 x 2 cm) and G. molesta laboratory strain established in IRTA Research Centre then, the filter paper was introduced in a Petri dish (10 cm diameter, (Spain) in 2005 from an established colony from field-collected 5 cm height) containing female moths (n= 10, three repetitions). individuals (Dr. F. Molinari, Piacenza, Italy) was used in all bio- Female behavioural responses were recorded during 10 min, and assays. Larvae were reared on semi-artificial diet (Ivaldi-Sender insects were maintained in Petri-dishes during 24 h. Viability of 1974) under long photoperiod (16L: 8D) and 24±1°C. Pupae were insects at the end of the assay was recorded. The same, but sorted by sex and placed into plastic cages (d= 15 cm, H= 5 cm) in applying only acetone in the filter paper, was done as control (n= a separated chamber at 24±1°C under the same photoperiod to 10, three repetitions). Percent of insects showing pre-mating beha- obtained virgin adults. To obtain mated female individuals for EAG viour against allyl cinnamate or control, and mortality among recordings and wind-tunnel behaviour assay, 2-3 couples of pupae treatment and control were compared by X2 test (P < 0.05) using were kept in the same cage. the GraphPad program (www.graphpad.com). Moths were used the second or third day after emergence and in Second assay aim to assess the effect of allyl cinnamate at mid- 4538 Afr. J. Biotechnol.

distances was done in a wind tunnel (50 cm high, 200 cm long and only observed in the presence of female pheromone and 50 cm wide) situated in a room maintained at 23±2°C. Light was allyl cinnamate, and mostly started flight in the presence supplied by a fluorescent light situated on the ceiling of the room of female sex pheromone and allyl cinnamate. Contacts (200 lux) and two ventilators either on the side of the wind tunnel operated simultaneously, produced an air movement of 0.15 cm s-1 were only recorded in front of main component of female through the tunnel. At least 20 mated and 20 virgin female moths, sex pheromone (Figure 3). and 20 naïve and 20 no-naïve male moths were observed in front of each stimulus for 3 min. Each insect was assessed individually and only once. DISCUSSION Red rubber septa of 8 mm (Sigma-Aldrich, Spain) situated 150 cm from the insect starting point and using a metal stand, held at a height of 20 cm, were used to place source, baited with 10 µg of To our knowledge, this is the first report describing G. stimulus. Septa with solvent alone (10 µL acetone) were also molesta antenna elicitation by allyl cinnamate. Antenna assessed, as control. Percent of insect showing a specific elicitation by allyl cinnamate was independent of the state behaviour for each group and stimulus was compared by X2 test of mating, as previously observed in C. pomonella and L. using a contingency table (P < 0.05) with the GraphPad program. botrana (not published data). G. molesta antenna elicit- The wind tunnel was cleaned with acetone after each experi- tation was in the same range than recorded for the main mental day and used material was washed with acetone and oven- dried at 200°C overnight. components of sex pheromones giving an idea of the high affinity of allyl cinnamate to G. molesta antennae receptors. RESULTS Due to similarities in the chemical structure of allyl cinnamate and trans-ethyl cinnamate (main component of Allyl cinnamate elicited antenna of both male and female G. molesta male sex pheromone) an effect in mating G. molesta. No differences in antenna elicitation among process is suspected. Moths perceived allyl cinnamate, mated and un-mated female moths were recorded (0.788± showing “cleaning antennae” behaviour, confirming results 0.289 mV and 0.822 ± 0.379 mV, respectively) (P > 0.05). from EAG recordings, and all typical behaviours preceding Neither among naïve and no-naïve males (1.037 ± 0.308 mating were observed in presence of allyl cinnamate mV and 1.429 ± 0.289 mV, respectively) (P > 0.05). [insect movement, walking toward, hair-pencil extrusion When G. molesta female moths were introduced into and retraction, wing funning, wing vibrating, flight and Petri-dishes containing allyl cinnamate, typical behaviour quick wing movement, cleaning antenna with legs, abdo- preceding mating was triggered [insect movement, walking men movement, female touch male abdomen, end-to-end toward, hair-pencil extrusion and retraction, wing funning, position, wing directed to floor, oviposition move-ment) wing vibrating, flight and quick wing movement, cleaning (Baker, 1989)]. The energy needed to maintained these antenna with legs, abdomen movement (Baker, 1989)]. movements during long time (24 h presence of allyl Notable increase of wind fanning was observed in all cinnamate) drives to the insect death due to exhaustion. individuals (in most cases, moth remain winging on the This fact could be useful in pest control; all energy 2 Petri-dish floor moving the abdomen) (χ =196, df= 1, P < overused when allyl cinnamate is detected reduces time 0.001). The increase of moth activity conducted up to and energy that moth could actually use to find a mate, moth exhaustion; reflected by mortality observed in front copulate, find a place to ovoposit and ovoposit. If one of of allyl cinnamate (100%) compared to controls (< 10 %) these series is disrupted, a negative effect in the next 2 (χ =185, df= 1, P < 0.001) generation (population reduction) is expected. In wind-tunnel, more female moths showed the behaviour Ethyl cinnamate acts as aphrodisiac compound “clean antenna” in the presence of allyl cinnamate and onfemales and it seems that males are attracted to ethyl ethyl cinnamate than in front of control sources. No cinnamate with the aim to „sneak‟ copulations with oriented flight was recorded in front of control sources females that are actively being courted by other males and significant lower number of females showed mating (Baker and Cardé, 1979). Allyl cinnamate effect could behaviour compared to sources baited with allyl cinnamate also be related with mating success, as a difference than or ethyl cinnamate. If we compare the responses of ethyl-cinnamate - which acts at short distances (Baker et female moth that start flight, no signi-ficant differences al., 1981); it seems that could be detected and conducted were observed among control, ethyl cinnamate and the toward longer distances. Assays aim to discern the effect lowest dose of allyl cinnamate tested. By contrast, a of both components at different ratios (female sex phero- significant higher percent of females started flight in front mone and allyl cinnamate) when applied together, and in of sources baited with 50 µg of allyl cinnamate, and most field assays are necessary to ascertain this pest control of them showed an oriented flight to the source, even no proposal. If allyl cinnamate was joined to conventional contacts were recorded (Figure 2). mating disruption, an effect in female and male behaviour All males cleaned its antenna, displayed typical beha- has to be expected; not attraction would be produced (as viour that precedes mating and showed its hair-pencils in a difference than female sex pheromone), but a display of front of allyl cinnamate and the main component of sex pre-mating behaviours. The time and energy spent on (both male and female sex pheromone) in wind-tunnel, pre-mating behaviour could reduce the number of effect- while not in front of control sources. Male fluttering was tive mating and so, population in next generations. Giner et al. 4539

Figure 1. Chemical structures of allyl cinnamate and ethyl cinnamate.

Figure 2. Percent of G. molesta virgin females showing a specific behaviour in wind- tunnel assay in front of septum containing 10 µg of allyl cinnamate, 10 µg ethyl cinnamate, 100 µg of allyl cinnamate or solvent during 3 min recording. cin = Allyl cinnamate; Columns within a specific behaviour followed by the same letter are not significantly different. Χ2 test shows homogeneity, (2 x 2) P < 0.05).

Figure 3. Percent of G. molesta virgin males showing a specific behaviour in wind-tunnel assay in front of septum containing 10 µg of main component of female sex pheromone, 10 µg of allyl cinnamate, 10 µg of ethyl cinnamate, or solvent during 3 min recording. 4540 Afr. J. Biotechnol.

Additionally, allyl cinnamate is used as aroma Gökçe A, Stelinki LL, Gut LJ, Whalon ME (2007). Comparative (http://eur-lex.europa.eu/) so low toxic effects in not- behaviour and EAG responses of female obliquebanded and redblanded leafroller moth (Lepidoptera: Tortrcidae) to their sex target organisms are suspected and it could be synthesized pheromone components. Eur. J. Entomol. 104:187-194. from glycerol (Escribà et al., 2009, 2011), so its use could González RH (2003) Las polillas de la fruta en Chile (Lepidoptera: help to find an alternative to glycerol surplus Tortricidae; Pyralidae). Universidad de Chile, serie de ciencias (www.biodiesel.org). agronómicas 9. Santiago de Chile, Chile. 188pp. Hillier NK, Vickers NJ (2011). Hairpencil volatiles influence interspecific courtship and mating between two related species. J. Chem. Ecol. 37:1127-1136. Conflict of Interests Holb I (2004). Yield loss and disease development of Monilia fructigena (Aderh. & Ruhl.) in an organic apple orchard. J. Agric. Sci. 15: 6-8. Il‟chev AL, Williams DG, Milner AD (2004). Mating disruption barriers in The author(s) have not declared any conflict of interests. pome fruit for improved control of oriental fruit moth Grapholita molesta Busck (Lepidoptera: Tortricidae) in stone fruit under mating disruption. J. Appl. Entomol. 128: 126-132. ACKNOWLEDGMENTS Ivaldi-Senser C (1974). Techniques simples pour un élevage permanent de la tordouse orientale Grapholita molesta (Lepidoptera: Tortricidae) sur milieu artificial. Ann. Zool. Ecol. Anim. 6:337-343. M. G. was financed by fellowship n° BES-2008-004779. Kong WN, Li J, Fan RJ, Li SC, Ma RY (2014). Sex-pheromone- M. G., M. B. and J. A. were supported by Spanish mediated mating disruption technology for the oriental fruit moth, Ministry of Economy and Competitiveness research grant Grapholita molesta (Busck) (Lepidoptera: Tortricidae): overview and prospects. Psyche. vol. 2014, Article ID 253924, 8 pages. AGL 2010-17486. Authors are grateful to C. Gemeno Kovanci OB, Walgenbach JF, Kennedy GG (2004). Evaluation of (University of Lleida) by insect supply and wind-tunnel extended-season mating disruption of the oriental fruit moth equipment supervision. Grapholita molesta (Busck) (Lep., Tortricidae) in apples. J. Appl. Entomol. 128:664-669. Kuhns EH, Pelz-Stelinski L, Stelinski LL (2012). Reduced mating success of female tortricid moths following intense pheromone auto- REFERENCES exposure varies with sophistication of mating system. J. Chem. Ecol.

38(2):168-175. Baker TC (1989). Origin of courtship and sex pheromones of the Landolt PJ, Heath RR (1989). Attraction of female cabbage lopper oriental fruit moth and a discussion of the role of phytochemicals in moths (Lepidoptera: Noctuidae) to male-produced sex pheromone. the evolution of Lepidopteran male scents. In: Chov CH, Waller GR Ann. Entomol. Soc. Am. 82 (4):520-525. (Eds) (1989). Phytochemical Ecology: Allelochemicals, mycotoxins Löfstedt C, Vickers NJ, Roelofs WL, Baker TC (1989). Diet related and insect pheromones and allomones. Institute of Botany, Academia courtship success in the oriental fruit moth, Grapholita molesta Sinica. Monograph Series, 9. Taipei (Taiwan). (Tortricidae). OIKOS 55:402-408. Baker TC, Cardé RT (1979). Endogenous and exogenous factors Natale D, Mattiacci L, Pasqualini E, Dorn S (2004). Apple and peach affecting periodicities of female calling and male sex pheromone fruit volatiles and the apple constituent butyl hexanoate attract female response in Grapholita molesta (Busck). J. Insect Physiol. 25:943- oriental fruit moth, Cydia molesta, in the laboratory. J. Appl. Entomol. 950. 128:22-27. Baker TC, Nishida R, Roelofs WL (1981). Close-range attraction of Natale D, Mattiaci L, Hern A, Pasqualini E, Dorn S (2003). Response of female oriental fruit moth to herbal scent of male hair-pencils. female Cydia molesta (Lepidoptera: Tortricidae) to plant derived Science 214:1359-1361. volatiles. Bull. Entomol. Res. 93:335-342. Birch MC, Hefetz A (1987). Extrusible organs in male moths and their Piñero JC, Dorn S (2007). Synergism between aromatic compounds role in courtship behavior. Bull. Entomol. Soc. Am. 33:222-229. and green leaf volatiles derived from the host plant underlies female Curkovic T, Brunner JF, Landolt PJ (2006). Courtship behavior in attraction in the oriental fruit moth. Entomol. Exp. Appl. 125: 185-194. Choristoneura rosaceana and Pandemis pyrusana (Lepidoptera: Rotschild GHL, Vickers RA (1991). Biology, ecology and control of Tortricidae). Ann. Entomol. Soc. Am. 99(3):617-624. oriental fruit moth. In: Van der Geest, L.P.S., Evenhus, H.H. (Ed). Damos P, Bonsignore CP, Gardi F, Avtzis DN (2014). Phenological World Crop Pest, Tortricid Pests: their biology, natural enemies and responses and a comparative phylogenetic insight of Anarsia control. Vol. 5. Elsevier. Amsterdam, The Netherlands. pp. 389-412. lineatella and Grapholita molesta between distinct geographical Stelinski LL, Il‟ichev AL, Gut LG (2006). Antennal and behavioral regions within the Mediterranean basin. J. Appl. Entomol. 138:528- responses of virgin and mated oriental fruit moth (Lepidoptera: 538. Tortricidae) females to their sex pheromone. Ann. Entomol. Soc. Am. Devine GJ, Furlong MJ (2007). Insecticidal use: context and ecological 99(5):898-904. disturbances. Agric. Hum. Values 24:281-306.

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Vol. 13(50), pp. 4541-4546, 10 December, 2014 DOI: 10.5897/AJB2014.14220 Article Number: 2B8EF3848955 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2014 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB

Full Length Research Paper

Phytochemical, proximate and anti-nutrient compositions of four leafy vegetables used in South Eastern Nigeria

Abu Ngozi E.*, Ozoagudike Chinenye M. and Akaneme Florence I.

Department of Plant Science and Biotechnology, University of Nigeria, Nsukka, Enugu State, Nigeria.

Received 2 October, 2014; Accepted 24 November, 2014

Chemical constituents of plants are influenced by environmental factors and fluctuations just as many other polygenic traits. Four different green leafy vegetables commonly used in the diets of South Eastern Nigeria were analyzed with a view to determine the phytochemicals, proximate and anti-nutrient compositions of these ecotypes. The vegetables, of which three are spices, are scent leaf (Ocimum gratissimum L.), fluted pumpkin (Telfairia occidentalis), amaranth globe (Gongronema latifolium Benth.) and ashanti pepper (Piper guineense Schumach. and Thonn.) leaves. The preliminary phytochemical analysis indicates the presence of the phytochemicals from trace amounts to strongly present. The quantitative tests showed that O. gratissimum had significantly (P ≤ 0.05) the highest tannin content of 1074.94±0.009 mg/100 g. Significant variations were observed in all the other phytochemicals except in alkaloid and phenol content. Proximate and antinutrient compositions showed significant variation in the different vegetables. T. occidentalis had highest values in ash (13.51%), crude fibre (33.52%), protein (25.49%) and phytate (8.58 mg/100 g) contents. The results obtained in this study clearly indicate that the four leafy vegetables are readily available sources of nutrients and prove the extensive use of these vegetables in ethnomedicine; and their potential in drug formulation.

Key words: Alkaloid, ethnomedicine, proximate, spice, vegetables.

INTRODUCTION

Vegetables are the edible parts of herbaceous plants that Nigerian and provide appreciable amounts of nutritive are consumed wholly or in parts, raw or cooked as part of minerals (Ajewole, 1999). Even though the bulk of their main dish or salad; they may be aromatic, bitter or weight is water, leafy vegetables represent a veritable tasteless (Dhellot et al., 2006). They include leaves, natural pharmacy of minerals, vitamins and phyto- stems, roots, flowers, seeds, fruits, and bulbs. Leafy chemicals (George, 2003). Leafy vegetables investigated vegetables however, are source of macro and micro- in this experiment were Ocimum gratissimum, Piper nutrients that play major role in maintaining healthy living. guineense, Gongronema latifolium and Telfaria They are regular ingredients in the diet of the average occidentalis.

*Corresponding author. E-mail: [email protected] or [email protected]. Tel: +2348064664555.

Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 4542 Afr. J. Biotechnol.

T. occidentalis commonly called fluted pumpkin is a well as topical ointments (Pessoa et al., 2003; Holets et tropical vine grown in West Africa as a leafy vegetable. al., 2003). It is used as an excellent gargle for some They belong to the family Cucurbitaceae (Akoroda, 1990). throats and tonsillitis. It is also used as an expectorant The fluted gourd grows in many nations of West Africa and a cough suppressant. The plant extract is used against but is mainly cultivated in Nigeria, used primarily in soups gastrointestinal helminths of animals and man (Chitwood, and herbal medicines (Akoroda, 1990). Fluted pumpkin 2003). Phytochemical studies show that both aqueous leaves have a lot of nutritive value. This made the leaves and methanolic extracts of O. gratissimum are rich in and tender stems potentially useful as food supplements tannins, steroids, terpenoids, flavoniods and hydrogen- (Nkang et al., 2002). The leaves contain a high amount of cyanide and equally has a good antioxidant activity vitamins A and C, antioxidants, hepatoprotective and (Afolabi et al., 2007). antimicrobial properties (Oboh et al., 2006). The leaf G. latifolium (amaranth globe) belongs to the family extract is useful in the management of cholesterolemia, Asclepiadaceae and is a tropical rainforest plant primarily liver problems and impaired defense immune systems used as spice and vegetable in food (Nwosu and Malize, (Eseyin et al., 2005). The leaves are rich in iron and play 2006). It is listed among the twenty-eight medicinally a key role in the cure of anaemia; they are also noted for important vegetables of South West Nigeria (Ayodele, lactating properties and are in high demand for nursing 2008) and also as one of the aromatic plants of medicinal mothers (Okoli and Mgbeogwu, 1983). In Nigeria the importance from Nigeria (Ogunwande et al., 2007). The fresh leaves are ground and the juice used as tonic by leaves are sharp-bitter and sweet and widely used as a women that have just given birth; its high iron content leafy vegetable and as a spice for sauces, soups and assists in the replenishment of lost blood, used for treat- salads. G. latifolium is also widely used in West Africa for ment of anaemia, chronic fatigue and diabetes (Aderibigbe medicinal and nutritional purposes. Ugochukwu et al. et al., 1999; Dina et al., 2006; Alada, 2010). Longe et al. (2003) reported that aqueous and ethanolic extracts of (1983) reported that minerals such as calcium, potas- Amaranth globe had hypoglycemic, hypolipidemic and sium, magnesium, iron, sodium and phosphorus, anti- antioxidative properties while Morebise et al. (2002) oxidants and vitamins such as thiamine, riboflavin, showed that it has anti-inflammatory properties. They nicotinamide and ascorbic acid were present in fluted also have industrial uses in brewing beer. Ogundipe et al. pumpkin. Phytochemicals such as phenols were (2003) discovered that leaves of Amaranth globe were concentrated in the testa, pulp and husk and amino acids possible hop substitute for brewing beer and that water such as alanine, aspartate, glycine, leucine and many extracts of the powdered leaves gave low bittering values, others (Fasuyi and Nonyerem, 2007). but extraction of the powdered leaves with organic P. guineense (also called African pepper, Ashanti solvents significantly increased analytical bitterness to pepper, Benin pepper, false cubeb, Guinea cubeb, uziza levels comparable with hops. pepper and Guinea pepper) belonging to the family It is believed that phytochemicals may be effective in Piperaceae are semi-cultivated in countries such as combating or preventing disease due to their antioxidant Nigeria where the leaves and seeds are used as effect (Afolabi et al., 2007). The medicinal value and flavouring agent (Asawalam, et al., 2007). Studies have multiple biological functionalities of several plants are shown that apart from the use of these plants as spices defined by their phytochemical constituents (Fallah et al., and condiments, they have several other wide appli- 2005). The medical values of leafy vegetables are dictated cations in the local treatment and management of many by their phytochemical and other chemical constituents diseases. It provides oil used as aromatic in the drink (Fallah et al., 2005), However, these chemical con- industry and also medicinally (Sumathykutty et al., 1999). stituents including the phytochemicals are influenced by Okwu (2001) reported that phytochemical analyses of P. environmental factors just as many other polygenic traits. guineense showed the presence of alkaloids, flavonoids, Therefore, there is need to determine the chemical com- saponins, tannins, resins and essential oils. These phyto- positions of the eco-types grown at Enugu, South chemicals have a lot of pharmacological properties as Easthern, Nigeria. The aim of this study was to determine proved by earlier studies (Okwu, 2001). He also reported the chemical compositions, proximate, mineral and that the presence of alkaloids signified the possession of phytochemical constituents, of four leafy vegetables, out medicinal properties within the leaves. The flavonoids of which three also serve as spices, and to relate the possessed antioxidant activity, anti-inflammatory and above to their nutritional and the ethno medicinal uses by antiviral activities (Okwu, 2001). the indigenous population of South Eastern Nigeria. O. gratissimum is a herbaceous plant which belongs to the family Lamiaceae (Calixto, 2000). It is widely distri- MATERIALS AND METHODS buted in tropical and warm temperature regions (Okigbo and Ogbonnaya, 2006). O. gratissimum, also called scent Collection and identification of plant material leaf is grown in gardens and used as a tea leaf for fevers The leafy vegetables, O. gratissimum, P. guineense, G. latifolium (Okigbo and Ogbonnaya, 2006). It is germicidal and has and T. occidentalis, were collected from Enugu, in Enugu state, found wide use in toothpastes and mouth washes as South Eastern Nigeria. The specimens were properly identified and Abu et al. 4543

Table 1. Qualitative phytochemical screening of the leaves of O. gratissimum, P. guineense, G. latifolium and T. occidentalis.

Phytochemical O. gratissimum P. guineense G.latifolium T. occidentalis Alkaloid ++ ++ ++ + Glycoside ++ + + ++ Saponin + + + + Tannin +++ ++ ++ ++ Flavonoid ++ ++ ++ ++ Steroid ++ ++ + + Terpenoid ++ ++ ++ + Phenol ++ +++ +++ ++++ HNC ++ + + ++

++++ = Very strongly present; +++ = strongly present; ++ = present; + = Trace; HNC, hydrogen cyanide.

authenticated in the Department of Plant Science and Biotechnology, Similar variations were observed in the test for Tannin, University of Nigeria, Nsukka. hydrogen cyanide, terpenoid and steroid however, flavonoid was present in all the samples (Table 1). The

Preparation of plant extracts quantitative assessment of the phytochemical consti- tuents showed significant variations in the actual com- Drying process positions of the different phytochemicals in the vege- tables (Table 2). All the phytochemical components were The samples were thoroughly washed, air dried under shed for significantly different from each other at P ≤ 0.05. The seven days at room temperature, to avoid loss of active compounds. The dried leaves were ground to powder using electric grinder. The tannin content of the vegetables ranged from 716.6 powdered samples were then stored in appropriately labeled air- mg/100 g in P. guineense to 1074.94 mg/100 g in O. tight bottles for further use (Trease and Evans, 2002). gratissimum.The values for flavonoid, saponin, glycoside and steroid also ranged from 229.6 mg/100 g to 235.8 mg/100 g, 0.86 mg/g to 0.97 mg/g, 5.45 mg/g to 6.48 Extraction process mg/g and 5.51 mg/g to 9.67 mg/g, respectively (Table 2).

Aqueous extracts were used for the phytochemical analysis. The G. latifolium had the highest values in alkaloid and process was hot water method following the procedure of Handa terpenoid while the least was recorded in T. occidentalis (2008) and Handa et al. (2008). The result of the proximate composition on the leaves of O. gratissimum, P. guineense, G. latifolium and T. occidentalis showed that the moisture content ranged Phytochemical, proximate and anti-nutrient analyses from 2.58% in T. occidentalis to 9.14% in O. gratissimum

Both the proximate, antinutrients, qualitative and quantitative (Table 3). Percentage protein ranged from 17.16 to 25.49% phytochemical analyses of the leafy samples of O. gratissimum, P. among the vegetables. Variations in the percentage guineense, G. latifolium and T. occidentalis were carried out to compositions of ash content, crude fibre, fat and carbo- detect the presence of some secondary metabolites using the hydrate were also observed in the different leafy standard methods by the Association of Official Analytical vegetables. The Anti- nutrient composition showed the Chemistry (AOAC, 2002). The phytochemicals tested for were alkaloids, saponins, glycosides, tannins, steroid, terpenoid, presence of Phytate and oxalate (Table 4). The value of flavonoids, Hydrogen cyanide and Phenols. phytate in O. gratissimum (1.26 mg/100 g) differed significantly from P. guineense (1.57 mg/100 g) but there was no significant difference between the values in T. RESULTS occidentalis and G. latifolium. The values for oxalate across the vegetables ranged from 3.89 - 8.58%. The qualitative analysis showed a trace amount of alkaloid in T. occidentalis and the presence of alkaloid in the other three samples. Glycoside was present in O. DISCUSSION gratissimum and T. occidentalis but showed trace amount in P. guineense and G. latifolium. Saponin was in trace The study revealed that the extracts of these vegetables amount in all the four leaf samples. Phenol was present are rich in phytochemicals such as saponins, alkaloids, in O. gratissimum, strongly present in P. guineense and flavonoids, terpenoids, steroids, glycosides, tannins, G. latifolium but very strongly present in T. occidentalis. hydrogen cyanide and phenols. Most of these 4544 Afr. J. Biotechnol.

Table 2. Quantitative phytochemical compositions of O. gratissimum, T. occidentalis, G. latifolium and P. guineense leaves.

Tannin Flavonoid Glycoside Saponin Steroid Terpenoid Phenol HNC Alkaloid Sample (mg/100 g) (mg/100 g) (mg/g) (mg/g) (mg/g) (mg/100 g) (mg/100 g) (mg/g) (mg/100 g) O.gratissimum 1074.94a** ± 0.009* 2320.09c ± 0.008 6.39b ± 0.004 .91b ± 0.006 7.19b±0.004 11.78b ±0.006 983.88 ±0.006 .65a ± 0.005 1406.51 ± 0.006 T. occidentalis 859.96c ± 0.008 233.34b ± 0.006 6.4a ± 0.006 .89c ± 0.004 5.51d ±0.003 10.64d ±0.005 1403.23 ±0.005 .38c ± 0.002 677.51 ± 0.005 G. latifolium 906.91b ± 0.006 229.63d ± 0.004 5.84c ± 0.004 .86d ± 0.006 9.67a ±0.005 12.37a ±0.004 1291.26 ±0.005 .45b ± 0.005 1577.24 ± 0.005 P. guineense 716.63d ± 0.006 235.81a ± 0.008 5.45d ± 0.007 .97a ± 0.009 5.62c ±0.004 11.32c ±0.005 1290.33 ±0.006 .65a ± 0.006 1132.79 ± 0.006 L.S.D (P ≤ 00.05) 00.014 0.012 0.009 0.013 0.007 0.009 NS 0.008 NS

HNC, Hydrogen cyanide. * Values are mean ± SD; NS, not significant. ** Values followed by different letters are significantly different from each other.

Table 3. Proximate Composition (%) of the leaves of O. gratissimum, P. guineense, G. latifolium and T. occidentalis.

Sample Moisture Protein Ash Fat Fibre Carbohydrate O. gratissimum 9.14a ± 0.003 19.88b ± 0.004 10.58c ± 0.007 10.25c ± 0.006 12.57d ± 0.006 37.58b ± 0.018 T. occidentalis 2.58c ± 0.003 25.49a ± 0.004 13.52a ± 0.005 12.62b ± 0.004 33.52a ± 0.008 12.28d ± 0.020 G. latifolium 4.63b ± 0.005 17.16d ± 0.003 10.13d ± 0.003 10.14d ± 0.004 16.58c ± 0.005 41.36a ± 0.007 P. guineense 4.63b ± 0.004 19.36c ± 0.007 11.39b ± 0.004 14.63a ± 0.004 21.37b ± 0.004 28.62c ± 0.013 L.S.D (P ≤ 00.05) 0.007 0.009 0.009 0.008 0.011 0.029

Table 4. Anti-nutrient composition of O. gratissimum, T. occidentalis, P. guineense, and G. latifolium leaves.

Sample Oxalate (mg/100 g) Phytate (mg/100 g) O. gratissimum 1.26b ± 0.008 3.9c ± 0.005 T. occidentalis 0.94c ± 0.003 8.58a ± 0.003 G. latifolium 0.94c ± 0.006 7.8b ± 0.002 P. guineense 1.57a ± 0.003 7.8b ± 0.007 L.S.D (P ≤ 00.05) 0.009 0.01

phytochemical constituents are potent bioactive phytochemicals are said to be pharmacologically tance since tannins serve as good antioxidant compounds found in medicinal plant parts which active and their action are felt in the automatic (Okwu, 2001). are precursors for the synthesis of useful drugs. nervous system, blood vessels, respiratory system O. gratissimum had high values in all the These phytochemicals generally have a wide range and gastrointestinal tract (Louio et al, 2002). The phytochemicals tested in this study; this makes of pharmacological activities or actions (Louio et observed presence and quantity of tannins in all this vegetable and spice very important in the al., 2002). Alkaloids, the most revered of all the vegetables could be of great medicinal impor- claimed pharmacological properties and uses in Abu et al. 4545

ethnomedicine. Koleva et al. (2000) reported that the water soluble vitamins. Low amounts of phytate and oxalate medicinal values of O. gratissimum lie in the phytochemical antinutrients were detected in the vegetables. The beneficial component which produces definite physiological actions. aspects of antinutrients and other bioactives have been These components are associated with bioactivities discussed when used in low levels (Gemede and Ratta, having health impacts. From recent findings, O. gratissimum 2014) has proved to be useful in medication for people living In conclusion these vegetables contain low calories and with HIV and AIDS (Nwinyi et al., 2009). P. guineense negligible quantities of utilizable energy; hence, they are extracts are, equally, good sources of antioxidants, which ideal for obese people who can satisfy their appetite are widely believed to be important line defense against without consuming much carbohydrate. All the plants oxidative stress leading to a lot of diseases like insomnia, studied have proved to be very important because of their and diabetes. chemical constituents, this explains their wide usage by Flavonoids were abundant in the four leafy vegetables the people of South Eastern Nigeria in diets and in but had significantly the highest value in P. guineense. ethnomedicine. Even though they have anti-nutrients, their They are recognised for their anti- oxidant, anti-carcino- values are too small to be harmful, therefore, consump- genic, anti-microbial and anti- tumour properties (Del-Rio tion of these vegetables is encouraged both as food for et al., 1997). Linus (1991) reported that flavonoids which nutrients and as medicine in ethnomedecine. The authors, are naturally occurring low molecular weight polyphenolic therefore, suggest intensified research that would be compounds located in fruits and vegetables are known to directed towards harnessing the potentials of these vege- inhibit formation of plaques and streaks in arteries and as tables in drug formulation and development in addition to such hinder hypertension and other cardiovascular their daily use in diets. diseases. Del-Rio et al. (1997) noted that flavonoids are potent water-soluble antioxidants and free radical scavengers which prevent oxidative cell damage and Conflict of Interest have strong anticancer activity. 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Vol. 13(50), pp. 4547-4556, 10 December, 2014 DOI: 10.5897/AJB2013.13449 Article Number: 8AF709648960 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2014 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB

Full Length Research Paper

Physicochemical, organoleptic and nutritional characteristics of four sweet cassava (Manihot opi) varieties

E. Njoh Ellong, C. Billard and S. Adenet*

Pôle Agroalimentaire Régional de Martinique (P. A. R. M.) Habitation Petit Morne 97232 Le Lamentin, Martinique, France.

Received 5 November, 2013; Accepted 17 November, 2014

There exist a wide variety of tropical tubercles in Martinique French West Indies (F.W.I.). This study was carried out in order to investigate the development of local varieties of sweet cassava (Manihot opi). The purpose of this research was to enhance knowledge of the nutritional and agricultural characteristics of these tubers. The first step in the research project was to carry out a survey and screening of selected varieties, which was undertaken by the local department of agriculture throughout the region of Martinique. Phenotypic characterization and identification were then established. The four varieties were distinguished from each other by phenotypic, organoleptic, physicochemical and nutritional characteri- stics. Sweet cassavas in Martinique are richer in nutritional compounds and vitamin C than ordinary potatoes. Their sensory specificity, high nutritional value and suitability for industrial processing have also been highlighted. Variety KM07 seemed to offer the best compromise between size, nutritional profile and sensory characteristics and would therefore be recommended for production.

Key words: Martinique, Manihot opi, physicochemical characteristics, nutritional characteristics, sensory profile, sweet cassava, gallic acid equivalent (GAE).

INTRODUCTION

Cassava belongs to the Euphorbiaceae family and can cassava and the most cultivated because its production be divided into two categories: Arboreae and Fructicoseae. yield is higher, despite being inedible without pre-treat- Originally from South America, cassava was introduced ment. It contains toxic cyanide glycosides, which, under into Martinique by the Caribbean Indians (Jackson et al., the effect of an enzyme, are transformed into hydrogen 1991; Silvestre and Arraudeau, 1983). There are two cyanide (Nartey, 1968; Conn, 1969; McMahon et al., varieties of cassava. Manihot opi is the sweet cassava, 1995). Hydrogen cyanide (HCN) is a strong inhibitor of the roots and leaves of which are consumed fresh. many metallo-enzymes, especially cytochrome oxidase, a Manihot esculenta Crantz (Manihot utilissima) is the bitter key enzyme in cellular respiration. Cyanogenic glycosides

*Corresponding author. E-mail: [email protected]. Tel: (+596) 596 42 12 78. Fax: (+596) 596 42 12 92.

Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 4548 Afr. J. Biotechnol.

are synthesized by cassava from germination (Dunstan et as Brazil, a large-scale cassava industry has developed. al., 1996). There are high concentrations in parts of the The starch is used as a food thickener or as glue in plant that carry out photosynthetic activity and during the stationery production. The majority of cassava imported period when the plant grows the fastest (Louembe et al., to Europe is used for animal feed. Indeed, it constitutes a 1997). Not all varieties of cassava contain the same very good source of energy and the presence of fibres amount of HCN, and predictions of cyanide contents makes it very digestible. Asian countries such as exists (Sanchez et al., 2014). Sweet varieties contain 30 Thailand and Cambodia specialize in its export. to 130 ppm of HCN when raw, bitter varieties 80 to 400 In order to expand opportunities for consumption of ppm, and very bitter varieties 275 to 500 ppm of HCN cassava in the French West Indies (F.W.I.) the Trade (Nassar and Ortiz, 2006). The global detoxification Council of Guadeloupe initiated a study in 1997 in order procedures for cassava have been reviewed by Coursey to analyse the causes of the decline in production and (1973), Lancaster et al. (1982) and Padmaja (1995). The consumption of cassava. This program also defined how effects of traditional as well as modified processing tech- to boost production. With the development of new eating niques in reducing the cyanogen levels have been habits, new ways to consume cassava have been studied by several scientists (Cooke, 1982; Fukuba et al., identified, including in the form of bread (many traditional 1984; Nambisan and Sundaresan, 1985; Mahungu et al, African methods lead to making cassava starch bread) 1987; O‟Brien et al., 1992; Padmaja et al., 1994). The and cassava couscous (also based on a method of poisonous substance, present in larger quantities in the making African "attiéké") (Regez and Mulumba, 1987). peel is removed by peeling, washing or cooking, sun Other by-products were also valued, including glue made drying or fermentation. Mild method for removal of from starch and animal feed made from cassava leaves cyanogens from cassava leaves with retention of vitamins following detoxification. Investigation of ethanol and protein has been developed (Bradbury and Denton, productivity of cassava crop as a source of biofuel in 2014). The dried roots are processed into tapioca, tropical countries has been studied (Adelekan, 2013). cassava or flour. Tapioca is a fecula of cassava that is In order to promote the production and consumption of used to thicken soups, puddings, creams or custards. cassava and integrate it into the daily diet, we decided to There are many cultivars of bitter varieties, for example, study the physicochemical and nutritional properties of „Chaspagna‟, „Green cassava‟, „Pilotin‟, „Saint Vincent‟, four local varieties of cassava grown in Martinique. We etc. (Germosén-Robineau, 1999). wanted to compare the different qualities of these Cassava is now widely grown in tropical countries. It selected cultivars. This study is therefore an evaluation of grows best in wetland and sunny areas but accepts any cassava characteristics focussing on: (i) a sensorial type of soil (Jennings, 1995). It multiplies through cutting analysis of four cassava cultivars and (ii) the stems or tubers. Planting usually takes place during the physicochemical and nutritional characteristics of these rainy season. The harvest occurs 8 to 36 months later. four tubers. Cassava is a shrub growing between 1 and 3 m high with tuberous roots and white flesh rich in starch. The skin of the bitter cassava tuber is thin and adherent, whereas in MATERIALS AND METHODS the sweet variety it is thick and easily removed. Its underground part comprises feeder roots which penetrate Plant material deep into the ground (up to 1 m) and tuberous roots Varieties were provided by the local department of agriculture and located just below the soil‟s surface. These roots form a local producers. A minimum of 30 tubers were collected from each bundle of 5 to 10 plants and can reach a length of 30 to variety of cassava in order to have representative samples for each 120 cm, with a diameter of 4 to 15 cm and a weight of 1 variety. A set of physical parameters was measured on each tuber to 8 kg [it is these roots that are consumed (Cock, 1985)]. (size and weight). They were then cleaned, peeled, sliced and steamed. Physicochemical analyses were carried out on cooked Nineteen million hectares of cassava are grown slices (texture and colour) or on crushed cooked pulp (energy, worldwide, including 12 million hectares in Africa, 2 fibres, starch, polyphenols, sugars, minerals and vitamin C content). million hectares in South America and 4 million hectares in Asia. World production reached 233 million tons in 2009 (FAOSTAT, 2009). Nigeria is the largest producer Morphology and composition of sweet cassava with 36 million tons, followed by Thailand (30 million tons) Morphological and physical characterization and Indonesia (22 million tons). In 2009, Martinique produced 194 tons of cassava, covering 76 ha. Cassava The proximate analyses of cultivar size were carried out using a in Martinique is used mainly in the form of flour. The roots calliper for fruit length and width and a precision balance for of sweet varieties are eaten boiled. In addition to the use cassava weight (Shimadzu UW4200HV). Texture parameters were of tubers and leaves for food, cassava is selected for measured with a LLOYD Instruments TA plus texture analyser, using a piston 4 mm in diameter and 30 mm / min speed. The tuber starch production. The bitter cassava starch has a variety colours were determined with a Minolta CR-200 Chroma meter of uses and its properties are similar to those of potato using the three parameters (L*, a* and b*) established by the starch (Delpeuch et al., 1978). In some countries, such International Commission on Illumination: L* for lightness to Njoh Ellong et al. 4549

distinguish light colours from dark colours, a* used to classify red to cassava. 2 g of samples were dissolved in 0.5 mM ascorbic acid, 1 green colours, and b* used to classify yellow to blue colours. M NaCl. Peroxidases reacted with gaiacol 55 mM and its enzymatic activity was measured by absorption spectrometry at 470 nm every second for 20 s using a spectrophotometer (JENWAY 7305). 2 g of Physicochemical characterization of the tuber sample were dissolved in 0.18 mM citric acid, 16 mM Na2HPO4, PVPP 10%. Polyphenoloxidase reacted with catechol and its For each analysis and sample, three replicates were performed. enzymatic activity was measured by absorption spectrometry at 400 Dry matter content (DM): Flesh samples (2 g of crushed, homo- nm every second for 30 s using a spectrophotometer (JENWAY genized flesh) were collected from each cassava for DM deter- 7305). Lipoxigenase activity was determined using a spectro- mination in triplicate, using a ventilated oven at 70°C for 5 h at photometer (JENWAY 7305). reduced pressure (- 1 bar). Vitamin C: Vitamin C was measured using the K-ASCO 11/05 Ash content: Cassava ash content was calculated from a crushed Megazyme kit. This is a colorimetric method and the absorbance at sample (1 g) following heating to 525°C for 5 h as per the AOAC 578 nm was read using a spectrophotometer (JENWAY 7305). official method 923.03 (1996). Vitamin B3: Vitamin B3 was determined with the EN 15562 2009 Protein content: The protein content was determined through the method. quantification of total nitrogen using Kjeldahll‟s method. Following Cyanhydric acid: Cyanhydric acid determinations were undertaken mineralization of the sample (2 g) in 25 ml of 95% H2SO4 using a catalyst and a Turbotherm mineralizator (Gerhardt Laboratory using the HS-GC-NPD method. Systems, Königswinter, Germany), the mineralizate was distilled using soda in a Vapodest distiller (Gerhardt Laboratory Systems, Königswinter, Germany). The distillate was collected in 40 ml of Sensorial analyses boric acid 40 g.l-1 with the use of some drops of Tashiro‟s indicator. The distillate was titrated by hydrochloric acid HCl (0.1 N). Sweet cassava samples were cleaned, peeled, bleached, placed in plastic bags, frozen and warmed just before sensorial analyses. Lipid content: Lipids were extracted from the sample (2 g) after Twelve individuals were selected as panel members, all of them acid hydrolysis with 50 ml of 8 N HCl, at 80°C. The mixture was inexperienced in sensory analysis. Five 2-h training sessions were filtered and rinsed with boiling water until a neutral pH was carried out until they were able to recognize and rate the charac- obtained, after which the filters were dried. The residues were teristics of different cassava varieties according to the AFNOR placed in glass cartridges with 140 ml of petroleum benzene and 8586-1 and 8586-2 standards. Fifteen (15) attributes represented some pumice stones to extract the lipids in a Soxtherm extractor the sensory profile according to the AFNOR 13299 standard. (Gerhardt Laboratory Systems, Königswinter, Germany). After Significant differences (p<0.05) were found for the 15 sensory extraction, the cartridges were oven dried at 101°C. attributes. Homogeneity in the group was also tested by two-way analysis of variance (ANOVA) for each attribute and the training Carbohydrate content: This was obtained by finding the difference was considered complete when no panellists × samples interaction  [(dry extract – (ash + lipids + proteins)]. Carbohydrates represent was found. ANOVA were carried out with FIZZ , UNIWIN Plus v6.1  the total fibre, starch and sugar content. and StatGraphics CENTURION XV 2005 software with a confidence interval of 5%. Energy value: Energy value was determined by adding lipid, carbohydrate and protein contents. Data statistical analysis

Starch content: The starch content was measured using the K- The physical and chemical mean values of triplicate measurements TSTA 11/05 Megazyme enzymatic kit (Megazyme, Wicklow, Ireland). or analysis were statistically analysed. ANOVA, based on student The samples were crushed cooked flesh. The absorbance at 334 tests, principal component analysis (PCA) and Duncan‟s multiple nm, proportional to the amount of glucose released by the hydro- range test (DMRT) were performed using the software StatGraphics lysis of starch, was read using a spectrophotometer (JENWAY   CENTURION XV 2005 and Uniwin PLUS v6.1. 7305).

Fibre content: Fibre content was determined with the AOAC RESULTS 985.29 method.

Agronomic data Polyphenol content: Total polyphenols were determined using

Folin and Ciocalteu‟s method described in Georgé et al. (2005). The first step was a survey and screening of varieties by Only raw extracts were analysed. the local department of agriculture throughout the region Ca, K, P, Zn and Mg: Ca, K, P, Zn and Mg contents were of Martinique. Phenotypic characterization and identi- determined with the Cofrac program 60. Ca, K, P, Zn and Mg rates fication were then established. Sweet cassavas were were determined by atomic absorption spectrometry, respectively at grown on contiguous plots and in the same agro-climatic 422.7, 766.5, 430, 213.8 and 285.2 nm. conditions for a minimum of nine months. Cropping system data was collected using a delivery sheet, provided by pH: About 25 g of flesh (cooked or raw) was blended with ~ 250 ml the department of agriculture, and is summarized in deionised water (10% w/w) for 30 min using a magnetic stirrer. The pH of the blended solution was determined at ambient temperature Table 1. Cultivars „KM06’, „KM07’ and „KM08’ exhibited with a Sentix 81 (WTW) probe. phenotypic differences such as colour of the petiole. „KM06’ and „KM08’ had the same flesh colour. Enzymology: Measures were carried out on raw and cooked „KM08’ is distinguished by a pink-coloured cortex. „KMRA’ 4550 Afr. J. Biotechnol.

Table 1. Cropping system data of the four sweet cassava varieties studied.

Variety KM06 KM07 KM08 KMRA Average rainfall (mm) 1900 to 2000 mm Irrigation in extra in extra in extra no use of water Soil type Ferralitic Sandy and pumice soil Previous crop Fallow

No fungicide, insecticide and nematicide

Chemical weed control (glyphosate) around Manual weeding Treatments Weeding at will No fungicide, insecticide Satisfactory level of fertility and nematicide Phosphorus and potassium deficiency rectified through fertilizer contribution Search chlordecone negative

Dense vegetation Moderately dense vegetation Very dense vegetation Plant height: 2 m Plant height: 2.5 m Plant height: 2.5 m Flesh colour: white Flesh colour: white Flesh colour: yellow Flesh colour: white Petiole colour: purple Observations Petiole colour: green Petiole colour: green Petiole colour: red Parchment/cortex colour: Parchment/cortex Parchment/cortex colour: Parchment/cortex white colour: white White colour: pink

Table 2. Average scores out of 7 for sensorial analysis descriptors of the four sweet cassava varieties.

Sensory attribute KM06 KM07 KM08 KMRA

Colour*** 3.08 ± 1.71b 5.54 ± 1.33a 3.00 ±1.47b 3.46 ± 1.56b Colour homogeneity 5.62 ± 1.33 5.62 ± 0.87 5.38 ± 1.04 5.15 ± 0.99 Odour 4.31 ± 1.11 4.62 ± 1.12 4.77 ± 1.30 4.77 ± 1.01 Taste 4.46 ± 1.33 4.38 ± 1.45 4.38 ± 1.66 4.85 ± 1.21 Earthy taste 2.85 ± 1.57 3.00 ± 1.78 3.15 ± 1.68 3.08 ± 1.75 Saltiness 3.15 ± 1.21 2.92 ± 1.12 3.08 ± 0.86 3.15 ± 1.34

Bitterness*** 2.69 ± 1.55bc 2.08 ± 1.50c 4.15 ± 1.41a 3.00 ± 1.68b Astringency 2.85 ± 1.77 2.85 ± 1.46 3.23 ± 1.42 3.00 ± 1.68

Firm texture** 4.62 ± 1.19ab 5.15 ± 1.21a 3.62 ± 1.39c 3.85 ± 1.14bc Mealy texture 5.00 ± 1.19 4.46 ± 1.51 4.46 ± 1.05 5.38 ± 1.26 Fibrous texture 3.08 ± 1.66 3.46 ± 1.85 3.62 ± 1.39 3.08 ± 1.71 Sticky texture 4.62 ± 1.04 3.62 ± 1.50 4.08 ± 1.38 4.23 ± 1.01

Melting texture** 3.77 ± 1.36ab 2.92 ± 1.32b 4.62 ± 1.56a 3.69 ± 1.25ab

*Significant descriptors at 5%; **Significant descriptors at 1%; ***Significant descriptors at 0.1%; NS, No significant. Different letters (a-c) within the same line indicate significant differences at (p<0.05), using Duncan‟s multiple-range test.

tubers had a phenotypic appearance similar to „KM06’. The interpretation of results can highlight significant descriptors, descriptors which reveal a statistically

Sensorial analysis reliable difference between the samples. In contrast, no difference between the samples can be established for A sensorial analysis was carried out on the four insignificant descriptors. The statistical analysis of this cassavas. Thirteen (13) descriptors were selected. Table data reveals that 4 of the 13 descriptors evaluated varied 2 shows the results with the average scores out of seven significantly: colour, bitterness, firm texture and melting for these descriptors. An analysis of variance was texture (Table 2). For the other criteria evaluated, no performed to evaluate the organoleptic characteristics significant differences were found in the four varieties of and differences between the samples of cassavas cassavas. Their specific taste and smell, astringency and presented to specialized juries, and for each descriptor. mealy and sticky mouth texture were comparable. They Njoh Ellong et al. 4551

Figure 1. Plot of scoring averages for sensory analyses (out of 7) for the four sweet cassava varieties. *Significant at 5%, **significant at 1%, ***significant at 0.1%, NS: no significant.

had the following general characteristics: a consistent intermediate position. We noted, however, in terms of colour, a moderate overall intense odour, overall a weight, that the cultivar „KM08’ tended (p = 0.07) to be slightly earthy, bitter, salty, astringent taste, a firm mealy, lighter than the cultivar „KM06’. Other cassavas belonged slightly sticky, fibrous and melting texture. A profile was to the same statistical group and were placed in an inter- established for each cultivar and presented in Figure 1. mediate position. Figure 2 shows average trans-formation We noted, however, that the intense mealy mouth texture yields of the four samples of cassava. The four cultivars tended to be different (5.86%) between samples. of cassava had close average cooking yields (~ 91 ± 1%). Cassava „KM07’ differed significantly from the other four The four cultivars differed in their peel yield; „KM06’ and cultivars by its more intense colour, slightly bitter taste „KM07’ samples were close, with lower peel yields. and firm and slightly sticky mouth texture. Conversely, Conversely, „KM08’ had the highest peel yield (86%). the „KM08’ cultivar was distinguished by a more intense Finally, the overall flesh yield had the same profile as the bitterness, slightly firm and very smooth mouth feel. peel yield. This yield was average for „KM06’ and „KM07’ Cassava „KM06’ was statistically comparable to „KMRA’. cultivars, with 58%. It was highest for „KM08’. Both cultivars were placed in the middle position for these Texture analyses were performed on the four cooked four criteria. With regard to mealy mouth textures, „KM06’ slices of cassava samples. The results of the five texture and „KMRA’ cultivars tended to have the highest intensity. parameters studied are shown in Table 3. ANOVA was performed to highlight the characteristics and significant texture differences observed for each sample. This Technological characteristics analysis of the different varieties revealed that four texture parameters of the five evaluated differed significantly All samples were weighed and measured to determine between cultivars. „KM08’ cultivar was characterized by their calibre. The results are shown in Table 3. ANOVA lower hardness and cohesion and higher tensile strength was performed on the data obtained (diameter, length than other cultivars. Cassava „KMRA’ demonstrated and weight) to highlight the features and significant higher chewiness values than other cultivars. „KM07’ was differences observed for each class of samples. Analysis significantly harder. Finally, „KM06’ was in an intermediate of the results revealed that, in terms of size, varieties position for all parameters evaluated. Texture results differed statistically by 5% for their diameter (p = 0.0034). corroborate the data obtained by sensory analysis, Multiple range tests (method of Fischer minimum signi- including hardness values. Indeed, we observed the ficant differences (LSD), 95% confidence level) highlights same profile for „KM07’ and „KM08’, that is, firmer and three groups. „KM08’ had the smallest diameter and less firm, respectively. „KMRA’ the largest. „KM06’ and „KM07’ were in an The colour of a product according to the parameters 4552 Afr. J. Biotechnol.

Table 3. Physical parameters of the four sweet cassava varieties.

Diameter Hardness Elasticity Chewiness Tensile Cultivar Length (cm) Weight (g) Cohesion L a b (cm) (kgf) (mm) (kgf.mm) strength (kgf) 29.60 ± 531.67 ± KM06 5.26 ± 1.19 5.91 ± 1.39 0.06 ± 0.02 11.48 ± 2.25 5.79 ± 2.23 0.03 ± 0.02 81.96 ± 0.17 - 0.16 ± 0.02 23.41 ± 0.20 10.58 304.61

27.95 ± 376.77 ± KM07 4.57 ± 0.66 6.31 ± 1.55 0.07 ± 0.03 12.69 ± 0.55 5.56 ± 1.47 0.03 ± 0.01 78.61 ± 0.05 - 1.34 ± 0.02 46.75 ± 0.03 11.76 200.29

274.95 ± KM08 26.8 ± 5.56 4.41 ± 0.59 2.59 ± 1.00 0.15 ± 1.00 13.05 ± 0.04 4.60 ± 1.26 0.05 ± 0.01 82.1 ± 0.23 - 0.21 ± 0.01 22.35 ± 0.09 135.23

KMRA 26.65 ± 4.81 5.77 ± 0.61 467.2 ± 130.44 4.79 ± 1.03 4.79 ± 1.03 13.46 ± 4.18 11.07 ± 6.19 0.02 ± 0.01 108.04 ± 10.23 3.88 ± 1.02 32.89 ± 5.16

Figure 2. Average transformation yields of the four cassava varieties.

L*, a* and b* results in its positioning in three- 3). ANOVA was performed to bring out the that the three colour parameters varied by 5%. dimensional space. Values were measured on characteristics and statistical colour differences The parameter L* defines the brightness of cooked slices of the four cassava cultivars (Table observed for each variety. This analysis revealed product: 0 black, 100 white. There was a statistically Njoh Ellong et al. 4553

Table 4. Nutritional composition of the four sweet cassava varieties (Abbreviation used: GAE: Gallic Acid Equivalent).

Parameter KM06 KM07 KM08 KMRA Moisture (%) 55.28 54.72 65.97 59.5 Dry matter (%) 44.73 45.28 34.03 40.5 Ash (%) 1 0.93 1.04 1.2 Carbohydrates (g/100 g) 42.45 42.82 31.27 38.64 Starch (g/100 g) 8.64 18.78 7.44 20.15 Proteins (g/100 g) 1 1.34 1.57 0.56 Lipids (g/100 g) 0.29 0.2 0.16 0.13 VE (Kcal/100 g) 176.35 178.45 132.75 157.9 Total polyphenols (mg of GAE/100 g 34.8 49.95 41.8 36.75 Fibres (g/100g) 2.5 2.4 2.9 3.6 Vitamin C (mg/100 g) 17.4 29.8 14.3 18.35 Vitamin B3 (mg/100 g) 0.898 0.937 0.837 0.734 P (mg/100 g) 1.62 2.65 2.7 2.94 Ca (mg/100 g) 32.15 25.17 17.97 18.4 K (mg/100 g) 285.95 260.1 225.54 327.91 Zn (mg/100 g) 0.16 0.38 0.25 0.21 Mg (mg/100 g) 1.12 0.65 0.45 0.55 HCN (mg/kg) < 0.5 < 0.5 < 0.5 < 0.5 PPO (AU/s/100 g) 0 0 0 0 Peroxidase (Au/s/100 g) 0 0 0 0 Lipoxigenase (Au/s/100 g) 0 0 0 0 pH 5.94 6.25 6.06 5.94

significant 5% difference in average brightness amongst studied is less than 0.5 mg/kg. According to Codex the cultivars. Multiple range tests (low significant Alimentarius (CX / CF 03/09/11 December 2008), varieties differences using Fischer method (LSD), 95% confidence can be defined as sweet cassava if their cyanide hydro- level) highlighted two groups. The cultivar „KMRA’ gen content is less than 50 mg/kg. „KM06’ and „KM07’ showed the highest brightness values. „KM06’, „KM07’ cultivars have similar energy values, carbohydrates and and „KM08’ showed no difference between them. They dry matter and show the highest values. „KM08’ had the had lower brightness than „KMRA’. The parameter “a” lowest levels for these three parameters. „KMRA’ offered represents the axis from green (-60) to red (60). The the highest levels of total ash (1.2%) and starch (20.15 parameter “b” represents the axis from blue (-60) to g/100 g). „KM07’ also presented high starch content yellow (60). There was a statistically significant difference compared to „KMRA’ but the lowest level of total ash. of 5% between average a* and b* in different cultivars. Overall, all four cassava cultivars had low levels of Multiple range tests (low significant differences using vitamin B3. „KM07’ had the highest level of vitamin C Fischer method (LSD), 95% confidence level) highlighted (29.8 mg/100 g) and total polyphenols (value 49.95 mg of 2 groups for parameter a*. „KMRA’ is characterized by GAE1 /100 g), and the lowest fibre content. Levels of the highest value for a*. There was no significant vitamin C for „KM06’ and „KMRA’ were similar difference between the three other cultivars. Multiple (statistically significant). „KM08’ was also a source of range tests (low significant differences using Fischer vitamin C (14.3 mg/100 g). The highest fibre content was method (LSD), 95% confidence level) highlighted 3 found in the „KMRA’ cultivar, which is classed as rich in groups for parameter b*. „KM08’ and „KM06’ had the fibre (> 3 g/100 g). Finally, all cultivars‟ total polyphenols lowest value for b*. Conversely, „KM07’ had the highest content were higher than 34 mg of GAE/100 g. „KM06’ value for b*. „KMRA’ was in an intermediate position. had the lowest content of total polyphenols (34.8 mg of These results fit in with the sensory results because the GAE/100 g). Mineral content was highly variable „KM07’ cultivar can be distinguished by its intense colour. depending on cultivar; values were generally low. „KMRA’ was the only source of potassium with 327.91-mg/100 g.

Physicochemical analyses „KM06’ had the highest manganese and calcium contents, with 1.12 and 32.15 mg/100 g respectively. Its Nutritional analyses were performed on cooked products. zinc and phosphorus concentrations were the lowest of Table 4 summarizes the results of measured nutritional the four cultivars. „KM07’ had the highest zinc content parameters. The hydrocyanic acid content of all cultivars (0.38 mg/100 g). „KM08’ was in an intermediate position 4554 Afr. J. Biotechnol.

Table 5. Comparison between the average nutritional composition of the four cultivars of sweet cassava studied and an average sweet cassava studied globally.

Average nutritional Average data for sweet cassavas Average data for potatoes - Nutritional composition composition of the four - international nutritional international nutritional sweet cassavas studied composition tables composition tables Moisture (%) 58.87 ± 3.87 60.07 ± 2.23 78.9 ± 3.36 Ash (g/100 g) 1.04 ± 0.08 0.92 ± 0.32 0.70 ± 0.28 Energy value (kcal) 161.36 ± 16.04 150.43 ± 11.62 81.60 ± 9.05 Proteins (g/100 g) 1.12 ± 0.34 1.12 ± 0.19 2.20 ± 0.43 Lipids (g/100 g) 0.20 ± 0.05 0.27 ± 0.11 0.26 ± 0.06 Carbohydrates (g/100 g) 38.80 ± 3.84 36.15 ± 2.34 17.80 ± 1.80 Fibres (g/100 g) 2.85 ± 0.40 1.91 ± 0.55 2.05 ± 0.62 Starch (g/100 g) 13.75 ± 5.71 - 15.8 ± 1.45

Minerals

K (mg/100 g) 274.88 ± 32.06 297.60 ± 32.84 395.33 ± 108.21

Vitamins

Vitamin C (mg/100 g) 19.96 ± 4.92 28.47 ± 5.22 18.52 ± 9.51 Niacin (B3) (mg/100 g) 0.85 ± 0.07 0.67 ± 0.16 1.74 ± 0.15

for its contents of phosphorus and zinc. It had the lowest formation of polyphenol into coloured compounds. calcium, potassium and manganese contents. „KMRA’ Polyphenol oxidase is therefore a good indicator of had the highest content of potassium and phosphorus of enzymatic browning. It can be inhibited by exposure to the four cultivars, with 327.91 and 2.94 mg/100 g high temperatures for short durations (bleaching). respectively. The enzymatic activity of four cultivars was Visually, browning of the flesh was not observed during null for the three enzymes studied, polyphenoloxidase, cassava processing (during peeling, cooking and whilst peroxidase and lipoxigenase. The average pH of the four awaiting treatment). It remained the same throughout varieties is about 6.4 ± 0.12. processing. This correlates with the enzymatic activities of three enzymes, which are null. Brightness has been identified as a factor influencing results. Indeed, it relates DISCUSSION to the lighter or darker appearance of a product. The higher the value, the lighter the product and vice versa. All sweet cassavas studied in Martinique were rich in Factors a* and b* express the colour itself and therefore minerals, vitamin C and polyphenols. Vitamin C is known were not of interest here in translating enzymatic for its antioxidant properties (Padayatty et al., 2003). browning. Results were therefore obtained only from the Vitamin B3 values were also high. Vitamin B3, also called factor L*. We did not observe any change in the value of niacin, is most frequently found in meat. It plays an L* over time. This also correlates with the absence of important role in the release of energy from food, the enzyme activity and no visual observation of browning of functioning of the nervous system and also reduces cassava flesh during treatment. cholesterol (Sauve, 2008). Total polyphenols are the Table 5 compares the average nutritional composition most abundant antioxidants in our diet and foods rich in of the four varieties studied to the nutritional composition polyphenols are thought to play a role in the prevention of of sweet cassava averages based on international tables certain diseases (Scalbert et al., 2005). Therefore, based of compositions (USA, France, Germany, South Africa, on these results and the vitamin and mineral contents Denmark, Finland, Australia, Pacific and Brazil). The four observed, it can be argued that Martiniquan sweet varieties grown in Martinique were advantageously cassavas have numerous health benefits (Sarkiyari and positioned in relation to the globally identified data for Agar, 2010). their energy values, proteins, lipids, carbohydrates, fibre, The total polyphenol content and polyphenol oxidase vitamin C and niacin (vitamin B3) content. Conversely, activity lead to enzymatic browning. The higher the their potassium content is lower than the average sweet content or activity, the greater the sensitivity to enzymatic cassava worldwide. Compared to potatoes, sweet browning. Polyphenol oxidase is an enzyme with an cassavas in Martinique have greater nutritional potential. essential role in the oxidation of phenolic compounds. In Their energy value was almost twice as much as for the presence of oxygen, it is responsible for the trans- normal potatoes. Sweet cassavas also contained more Njoh Ellong et al. 4555

vitamin C and fewer lipids than potatoes. cultivation techniques but had the same phenotypic In comparison with other vegetables for the rate of total characteristics as „KM06’. There was no significant polyphenols (Brat et al., 2006), sweet cassava would be difference between the lengths of cassavas, but varieties in twelfth position, with about 40.82 mg of GAE/100 g on differed significantly with respect to their colour. „KMRA’ average, before potatoes and peas (respectively, 23.1 was the brightest and „KM07’ had a rather yellow tint. and 36.1 mg of GAE/100 g). We attempted to demon- „KM08’ was significantly narrower, had the best average strate different ways of using sweet cassavas (fresh and yield of transformation and the lowest carbohydrate processed). We selected specific criteria to conduct this content. „KM08’ was also less firm but had a cohesion analysis (size / shape, sensory profile, nutritional value, and tensile strength significantly higher than other average yield of transformation). Sweet cassavas with cultivars. „KM07’ cultivar was the hardest and had the small size and regular shape are best for selling directly highest total polyphenol content. „KMRA’ rated highest for to consumers (purchase volume, ease of cutting and chewiness. „KM06’ had the lowest total polyphenol peeling). Cultivars with uniform colour, firm and / or content. Martinique sweet cassavas are richer in nutria- melting textures and high nutritional potential also have tional compounds and vitamin C than potatoes. Sweet greater commercial potential in relation to consumers. cassavas also have a lower glycaemic index, which Conversely, sweet cassavas with large, irregular shapes, means they could be of interest for individuals affected by fibrous appearance, granular or powdery texture and / or obesity and diabetes. Fact sheets were developed for low nutritional properties are more suitable for industrial each studied cultivar combining all the data obtained, as uses (flour, mashed, flakes). Finally, when cultivars are well as recommendations in terms of value and characterized by intermediate criteria, they can be used usefulness of each cultivar. Their sensory specificity, high both as a fresh product or processed. Thus, „KM06’ is nutritional value and suitability for industrial processing most suitable for processed products because of its large have also been highlighted. In view of their potential in size, very firm, mealy and sticky texture (very unpopular terms of consumption and processing, further investi- with consumers) and nutritional characteristics - mineral gations into the qualities of these tubercles are needed. levels (high K and Ca contents), carbohydrates and dry Further studies could aid in highlighting their positive matter content. „KM07’ displays criteria that make it characteristics and thus encourage further development suitable for fresh consumption or processing: its small within professional agriculture and agribusinesses. A size, regular shape, colour and nutritional properties (high cassava research-for-development program was carried vitamin C, carbohydrates and dry matter content) make it out in the Democratic Republic of Congo and highlighted suitable for fresh consumption, whilst high dry matter impact at the farm level (Rusike et al., 2014). This work is content and texture make it suitable for processing into thus especially helpful for optimal management of crop flour or frozen products. harvesting in a region of agriculture and climatic con- Variety „KM07‟ therefore seemed to offer the best trasts. compromise between size and nutritional and sensory characteristics and it would thus be recommended for production. In contrast, „KM08’ would be better for Conflict of Interests processed products because of its slightly firm and melting texture and low nutritional potential, including The author(s) have not declared any conflict of interest. vitamin and mineral levels. Its small size and high concentration of polyphenols are the only criteria in its REFERENCES favour in terms of fresh consumption. The regular form of „KMRA’, together with its starch and high mineral content, Adelekan BA (2013). Investigation of ethanol productivity of cassava give it greater value. However, it is very fibrous and lacks crop as a sustainable source of biofuel in tropical countries. Afr. J. Biotechnol. 9(35):5643-5650. texture, which may be considered a disadvantage. Bradbury JH, Denton IC (2014). Mild method for removal of cyanogens Conducting consumer tests on these cultivars will from cassava leaves with retention of vitamins and protein. Food determine their acceptability criteria with relation to Chem. 158:417-420. consumers in Martinique. Brat P, Georgé S, Bellamy A, Du Chaffaut L, Scalbert A, Mennen L, Arnault N, Amiot MJ (2006). Daily polyphenol intake in France from fruit and vegetables. J. Nutr. 136:2368-2373. Cock JH (1985). Cassava: new potential for a neglected crop. USA. Conclusions Conn EE (1969). Cyanogenic glycosides. J. Agric. Food 17:519-526. Cooke RD (1982). Effect of cassava processing on residual cyanide. 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The occurrence of phaseolunatin in cassava (Manihot aipi and Manihot O‟Brien GM, Mbome L, Taylor AJ, Poulter NH (1992). Variations in utilissima). Proc. Roy. Doc. London. 78:152-158. cyanogens content of cassava during village processing in FAOSTAT (2009). Available on Cameroon. Food Chem. 44:131-136. . C, Dutta A, Dutta SK, Levine M (2003). Vitamin C as an Antioxidant: Fukuba H, Igarashi O, Briones CM, Mendoza ET (1984). Cyanogenic Evaluation of Its Role in Disease Prevention. J. Am. Coll. Nutr. (22) glucosides in cassava and cassava products: Determination and 1:18-35. detoxification. In: Tropical Root Crops: Post-harvest Physiology and Padmaja G (1995). Cyanide detoxification in cassava for food and feed Processing. Edition Uritani I & Reyes ED. Japan Scientific Societies uses. Crit. Rev. Food Sci. Nutr. 35(4):299-340. Press, Tokyo, Japan. pp. 225-34. Padmaja G, Matthew G, Balagopalan C (1994). Detoxification of Georgé S, Brat P, Alter P, Amiot MJ (2005). Rapid determination of cassava cyanogens during processing: An overview of research and polyphenols and vitamin C in plant-derived products. J. Agric. Food. commercial practices in Asia. In Proc II Int Scientific Meeting of the Chem. 53(5):1370-3. Cassava Biotechnology Network. Bogor, Indonesia. pp. 609-622. Germosén-Robineau L (1999). Pharmacopée végétale caribéenne, Regez PF, Mulumba M (1987). Techniques traditionnelles de la TRAMIL. Edition Gros Desormeaux. pp. 493. transformation de manioc au Zaïre. Micro bio alim processing. ISSN Jackson FLC, Jackson RT, Delumen BO, Sio FK, Dinkins L, 0759-0644. vol. 5. Muhammed AFH (1991). Cassava (manihot esculenta) in Liberia: Rusike J, Mahungu NM, Lukombo SS, Kendenga T, Bidiaka S M, Alene history, geography, traditional processing and cyanogenic glycoside A, Manyong VM (2014). Does a cassava research-for-development levels. Ecol. Food Nutr. 28:227-42. program have impact at the farm level? Evidence from the Jennings J (1995). Manihot esculenta (Euphorbiaceae). Democratic Republic of Congo. Food Policy 46:193-204. England: Lomgnian Scientific and Technical. Sánchez T, Ceballos H, Dufour D, Ortiz D, Morante N, Calle F, Davrieux Lancaster PA, Ingram JS, Lim MY, Coursey DG (1982). Traditional F (2014). Prediction of carotenoids, cyanide and dry matter contents cassava based foods: survey of processing techniques. Econ. Bot. in fresh cassava root using NIRS and Hunter color techniques. Food 36: 12-45. Chem. 151:444-451. Louembe D, Malonga M, Kobawila SC, Mavoungou O (1997). Evolution Sarkiyari S, Agar T.M. (2010). Comparative analysis on the nutritional de la teneur en composes cyanes du manioc au cours du rouissage. and anti-nutritional contents of the sweet and bitter cassava varieties. Microbiolgie, aliments, nutrition 15(1):56-60. Adv. J. Food Sci. Technol. 2(6):328-334. Mahungu NM, Yamaguchi Y, Alamazan AM, Hahn SK (1987). Sauve AA (2008). NAD+ and vitamin B3: from metabolism to therapies. Reduction of cyanide during processing of cassava into some J. Pharmacol. Exp. Ther. 324(3):883-893. traditional African foods. J. Food Agric. 1: 11-15. Scalbert A, Manach C, Morand C, Rémésy C, Jiménez L (2005). Dietary McMahon JM, Wanda LB, White L, Sayre RT (1995). Cyanogenesis in polyphenols and the prevention of diseases. Crit. Rev. Food Sci. cassava (Manihot esculenta Crantz). J. Exp. Bot. 46 (7):731-741. Nutr. 45(4):287-306. Nambisan B, Sundaresan S (1985). Effect of processing on the Silvestre P, Arraudeau M (1983). Le manioc. Edition Maisonneuve and cyanoglucoside content of cassava. J. Sci. Food Agric. 36:1197- Larose. pp. 262. 1203. Nartey F (1968). Studies on cassava Manihot utilissima Pohl. Cyanogenesis: the biosynthesis of linamarin and lotaustralin in etiolated seeding. Phytochemistry 7:1307-12. Nassar NMA, Ortiz R (2006). Cassava improvement: challenges and impacts. J. Agric. Sci. 145:163-171.

Vol. 13(50), pp. 4557-4561, 10 December, 2014 DOI: 10.5897/AJB2014.14223 Article Number: 5A4153548964 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2014 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB

Full Length Research Paper

Seed pre-treatment methods for improving germination of Acacia tortilis

So Hanaoka1*, Norio Nakawa1, Norihisa Okubo1, Stephen Fredrick Omondi2 and Jason Kariuki2

1Forest Tree Breeding Center, Forestry and Forest Products Research Institute, 3809-1 Ishi, Hitachi City, Ibaraki, 319- 1301, Japan. 2Kenya Forestry Research Institute, P.O. Box 20412 – 00200, Nairobi, Kenya.

Received 6 October, 2014; Accepted 2 December, 2014

Acacia tortilis (Forsk.) Hayne is considered as an important dryland tree species in Africa and Middle East, and establishing an effective and efficient seed-germination method is considered necessary for producing planting materials. In this study, new germination method was compared with some of the previously reported methods and evaluated in A. tortilis. In the new method, root setting was observed within 3 days and the final germination percentage was 94%; higher than 5 to 86% reported in the previous methods. New method is therefore considered to be suitable for geminating A. tortilis seeds.

Key words: Acacia tortilis, seed germination, pre-treatment of seed germination.

INTRODUCTION

Acacia tortilis (Forsk.) Hayne (subfamily Mimosoideae, resistant and grows in areas with annual rainfall ranging family Leguminosae) is one of approximately 135 African from as low as 40 mm to as much as 1200 mm, with dry acacia species. The morphology of A. tortilis varies seasons lasting 1-12 months. A. tortilis tree can be used considerably, ranging from multi-stemmed shrubs (ssp. for a variety of purposes. The pods, which are high in tortilis) to trees measuring up to 20 m in height with protein (15-20%), fall to the ground where they are eaten rounded (ssp. raddiana) or flat-topped (ssp. heteracantha by livestock and wildlife. Leaves, new shoots, and and spirocarpa) crowns. The species is widely distributed seedlings are also eaten, with leaves being available throughout the Middle East and south into eastern and throughout most of the dry season when other fodder southern Africa, in habitats ranging from semi-desert to sources are scarce. The gum is edible, and the bark can dry bushland and grassland (Allen, 2007; Hines and be used to produce strong fibre. A. tortilis starts Eckman, 1993). In Africa, A. tortilis occurs in the drier producing firewood at 8-18 years, at a rate of 50 parts of Senegal and Somalia and extends all the way kg/tree/year. Its fast growth and good coppicing ability, down to South Africa. The species is highly drought coupled with the high calorific value of its wood of about

*Corresponding author. E-mail: [email protected]. Tel: +81-294-39-7013. Fax: +81-294-39-7306.

Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 4558 Afr. J. Biotechnol.

4400-4900 kcal/kg, make it well suited for use as fire- MATERIALS AND METHODS wood and charcoal (Orwa et al., 2009; National Academy Seed materials and pre-treatments of Sciences, 1980). Given the economic value of this species, efficient and stable seedling production is Seeds of A. tortilis were collected from several mother trees in important. However, several challenges exist regarding Kibwezi, Kenya. The seeds were processed, bulked, and sent to the artificial seed germination. Forest Tree Breeding Centre (FTBC) in Japan where they were Acacia species generally have a water-impermeable stored at room temperature for two months until the following seed coat, which means that physical and/or chemical experiments were initiated: Treatment 1(Cut-Hilum: The seeds of A. tortilis are flattened and ovoid in shape, with a white hilum on the pre-germination treatments are necessary in order to over- seed coat (Figure 1a). We defined the surface with hilum as the come seed dormancy and obtain rapid and synchronous “upper side”, which we removed by nail scissors (Figure 1b); germination. For example, soaking seeds in boiling water, Treatment 2 (Cut-Side): The seed coat of the “lower side” was sulphuric acid, or mechanical scarification treatments removed by nail-scissors. Treatment 3 (Cut-Bot): Seed coat of the have been demonstrated to promote germination in “side” (that is, the seed coat between the upper and lower sides) was removed using nail scissors; Treatment 4 (Scr-Bo): The seed Acacia species (Danthu et al., 1992; Teketay, 1998; Aref, coat was first scratched using sandpaper and then placed in boiling 2000; Rodrigues et al., 2008). As with other Acacia water at 98°C. The boiling water was allowed to cool naturally to species, the seeds of A. tortilis also have a hard seed room temperature following Abari et al (2012); Treatment 5 (Bo): coat and pre-treatment is essential to overcome seed Without removing or scratching the seed coat, seeds were placed in dormancy. Loth et al. (2005) indicated that ingestion by boiling water at 98°C. The boiling water was allowed to cool herbivores or stimulation by fire may promote germination naturally to room temperature; Treatment 6 (Scr-Sul): The seed coat was scratched using sandpaper, before being immersed in of A. tortilis seeds under natural conditions. In addition, concentrated sulphuric acid solution for 20 min and washed in the study also showed that a wet environment and distilled water; Treatment 7 (Sul): Seeds were immersed in optimal temperature conditions (from 15 to 25°C) is concentrated sulphuric acid solution for 20 min and washed in suitable for germination of the species. More recently, distilled water; Treatment 8 (Scr): Seed coat was scratched using several studies have demonstrated varying levels of sandpaper; Treatment 9 (Cont): No treatment (control). success in promoting A. tortilis seed germination. For Mechanical scarification of A. tortilis seeds using sandpaper is complicated by small size of the seeds and the hardness of the example, Abari et al. (2012) reported a germination coat. We therefore only scratched the surface of the seed coat and percentage of around 80% through scarification of the did not produce deep scars on the coat. Treatments 1 to 3 are seed coat or combined use of sulphuric acid and unique to this study while treatments 4 to 8 were based on previous hydrogen peroxide. However, the germination percentage studies (Abari et al., 2012; Azazi et al., 2013). after treatment with sulphuric acid alone was low in the study. On the other hand, Azazi et al. (2013) tested a Comparison of the time to root production among treatments number of chemical treatments and found that boiling in water or treatment with sulphuric acid for more than 20 Fifty seeds were used in each of the nine treatments described min were both comparatively efficient, and the germi- above. After completing each treatment, the seeds were placed in nation percentages obtained with these treatments was water-filled Petri dishes and root setting (produced a root of > 2 mm) was observed every morning at 8:30 am for 14 days. We also approximately 75%. identified which seeds absorbed water within the first 3 days. In addition, a germination percent-tage of 52% was However, the presence of mould on some seeds at day 15, meant obtained after mechanical scarification. Although both that the experiments had to be stopped. Expected cumulative rate studies concluded that mechanical scarification, of root setting over time and their 95% confidence interval were immersion in boiling water or sulphuric acid were all calculated by the Kaplan Meier method (Measurement of the effect effective, the obtained germination percentages were of intervention over a period of time, sometimes called “time-to- event analysis”). In addition, the differences in rate of root setting relatively variable. For the sulphuric acid treatment, among the treatments were tested by Log rank test. Seeds with differences in immersion duration may have accounted roots were planted in soil containing compost and their rates of for the observed differences in germination percentages cotyledon establishment were confirmed and noted. The in both studies, with immersion for a minimum of 20 min experiments were performed in a greenhouse at a mean associated with optimal results. However, in addition to temperature of 27.9±6.9°C. being potentially dangerous and unsafe for local farmers, chemical treatments are typically difficult to achieve in Germination percentage of seeds in each treatment rural areas. Regarding treatments involving boiling water In the above experiments, observations were stopped on the 15th or mechanical scarification, it is considered likely that day. To examine germination percentages over an extended period slight differences in experimental conditions might affect of time, 100 seeds were planted in sand at a depth of 10 mm after germination percentages. If such condition-dependent each of the above treatments. Because we understood that differences do exist, then it may be difficult for local treatment 1 was more efficient than treatment 2 and 3 in above farmers to repeat experimental conditions and obtain the experiments, we did not try treatment 2 and 3 in this experiment. same the results. In this paper, we report and discuss Watering was performed once every two to three days to prevent seeds from drying. The number of seedlings was counted on the simpler, safer and more efficient method for improving 28th day and germination percentage was calculated as: (numbers germination percentages in A. tortilis seeds. of germinated seed / numbers of total seeds examined). The Hanaoka et al. 4559

Figure 1. Seeds of Acacia tortilis; a, untreated seeds; b, seeds with hilum removed. Hilum is circled.

experiments were performed in a greenhouse at a mean very quickly, affecting germination efficiency. To compre- temperature of 23.9±4.5°C. hensively test the various effects of boiling water on seed germination, conditions such as soaking time and different water temperatures should be examined (Rehman et al., RESULTS AND DISCUSSION 1999). However, complicated pretreatment conditions are not ideal for local practitioners, such as farmers. In Root setting treatment 8 (Scr), it is possible that the low rate of water absorption may have been because we only lightly The percentage of seeds that absorbed water within the scratched the seed coat. Success rate of treatment 8 first 3 days was 100, 100, 100, 82, 8, 82, 70, 8 and 10% may depend on the technique and/or device used by the for Treatments 1 to 9, respectively. The expected rates of practitioner. Given the difficulties and time consuming root setting for time in each treatment are shown in nature of using sandpaper, Abari et al. (2012) concluded Figure 2. Rate of root setting was highest in treatment 1 that this method is not recommend for promoting (Cut-Hilum), with most seeds producing roots (that is, germination in Acacia seeds. On the other hand, the produced a root of > 2 mm) within 3 days, and all seeds results of treatment 4 (Scr-Bo) indicate that combined use rooted within 5 days in our experiment. Rate of root of mechanical scarification and treatment with boiling setting was also comparatively high in treatment 2 (Cut- water may promote germination. Side) and 4 (Scr-Bo), with a cumulative rate of root In this study, we found that roots always grow from the setting peaking at approximately 9 days. In treatments 3 point where the seed coat was removed in treatment 1 (Cut-Bot), 6 (Scr-Sul) and 7 (Sul), cumulative rate of root (that is, the hilum). The seed coat is known to retain setting gradually increased, while root setting was very elasticity, even if the seed absorbs water, and our results limited in the seeds subjected to treatments 5 (Bo), 8 suggest that root setting may sometimes be prevented (Scr) and 9 (Cont). Significant differences in germination and/or take time to break through the seed coat percentages among the treatments were detected in Log- (treatment 1 (Cut-Hilum) vs. 2 (Cut-Side), 3 (Cut-Bot)). rank test (p < 0.01). Indeed, this ability to penetrate the seed coat is Abari et al. (2012) and Azazi et al. (2013), reported a considered to be the main cause of variation in rate and slightly different results; treatments 5 (Bo) and 8 (Scr) duration of germination. Consequently, by identifying the were associated with high germination percentages point of root protrusion in treatment 1, the findings of this (around 75%) in their studies. One possible reason for study will be useful in maximizing germination of A. tortilis this disparity is because the volume of boiling water used seed. All rooted seeds were planted in composted soil in treatments 4 (Scr-Bo) and 5 (Bo) in this study was and we confirmed that cotyledons opened within 3 days small as the treatments were conducted in Petri dishes. It after sowing. Therefore, the efficiency of root setting was is thus possible that the boiling water may have cooled directly linked to seedling establishment (Table 1). 4560 Afr. J. Biotechnol.

Treatment 1 Treatment 2 Treatment 3

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Figure 2. Kaplan-Meier plot for each treatment. Expected cumulative rate of root setting over time (Day) are shown in solid line, and dashed lines indicate the 95% confidence intervals.

Germination percentages 6 and 7 in our experiments showed similar germination percentages, mechanical scarification is not considered In this study, 100 seeds from each of the seven treatments to be useful in combination with sulphuric acid treatment. were sown in sand. Of these, 94, 32, 5, 86, 85, 21 and In above root setting experiments, 54 and 50% of the 6% germinated within 28 days in treatments 1 and 4 to 9, seeds in treatments 6 and 7 produced roots within 14 respectively. Significant differences in germination days, respectively. However, 86 and 85% of the seeds in percentage were observed between treatments 1 and 4, treatments 6 and 7 in this experiment germinated. These 5, 8, 9; 4 and 5; 6 and 4, 5, 8, 9; 7 and 4, 5, 8, 9; 8 and 5, results suggest that seeds that have been exposed to 9 (Ryan’s method, p < 0.01). Although the germination chemical treatment take longer to germinate, but that final percentages for treatments 4, 5 and 8 were lower than germination levels in such seeds are not necessarily those of the previous studies (Abari et al., 2012; Azazi et always low. Nevertheless, germination levels of the seeds al., 2013), this may be attributed to differences in the way in treatment 1 (Cut-Hilum) were higher than the levels of mechanical scarification was performed and how boiling seeds in treatments 6 and 7. Thus, while it may be time water was applied. On the other hand, compared to the consuming to remove seed coat (approximately five previous studies, germination percentages in treatments seconds/seed), the levels of germination that can be 6 (Scr-Sul) and 7 (Sul) were similar. Because treatments obtained using our method (that is, treatment 1; Cut-Hilum) Hanaoka et al. 4561

Table 1. The summary results of root setting experiments.

Percentage of absorbed water Percentage of root setting Treatment within 3 days (%) within 14 days (%) Treatment 1 (Cut-Hilum) 100 100 Treatment 2 (Cut-side) 100 72 Treatment 3 (Cut-Bot) 100 56 Treatment 4 (Cut-Bo) 82 70 Treatment 5 (Cut-Bo) 8 4 Treatment 6 (Cut-Sul) 82 54 Treatment 7 (Cut- Sul) 70 50 Treatment 8 (Scr) 8 6 Treatment 9 (Con) 10 2

Table 2. The results of germination in soil of Drought Tolerant Trees for Adaptation to Climate Change in Drylands of Kenya). Treatment Germination percentage (%) Treatment 1 (Cut-Hilum) 94 Treatment 4 (Scr-Bo) 32 REFERENCES

Treatment 5 (Bo) 5 Abari AK, Nasr MH, Hodjati M, Bayat D, Radmehr M (2012). Maximizing Treatment 6 (Scr-Sul) 86 seed germination in two Acacia species. J. For. Res. 23:241-244. Treatment 7 (Sul) 85 Allen DJ (2007) A traveller’s guide to the wildflowers and common trees of east Africa. Camerapix Publishers International, Singapore. ISBN: Treatment 8 (Scr) 21 1-904722-28-8 Treatment 9 (Con) 6 Aref IM (2000) Effects of pre-germination treatments and sowing depths upon germination potential of some Acacia species. Res. Bull. 95:5- 17. Azazi El, Sayed El, Sourour MM, Belal AH, Khalifa EA (2013). Improving Acacia tortilis seed germination by breaking dormancy are higher than those that can be achieved with sand- treatment. Int. J. Adv. Biol. Res. 3:103-109 paper. Our new method should therefore be considered Danthu A, Roussel J, Saar JA. (1992) Effect of different pretreatments for seed pre-treatment in A. tortilis cultivation (Table 2). on the germination of Acacia senegal seeds. Seed Sci. Technol. 20(1): 111−117. Hines DA, Eckman K (1993) Indigenous multipurpose trees of Tanzania: uses and economic benefits for people. FAO Conclusion Loth PE, Boer WF, Heitköning MA, Prins HHT (2005) Germination strategy of East African savanna tree Acacia toritlis. J. Trop. Ecol. 21:509-517. We consider that the most efficient strategy for promoting National Academy of Sciences (1980) Firewood Crops – Shrub and tree germination in A. tortilis seeds is as follows: 1) Remove species for Energy Production. NAS, Washington DC the upper seed coat, 2) soak the seed in water until root Orwa C, Mutua A, Kindt R, Jamnadass R, Anthony S (2009). protrusion, and 3) transfer the rooted seed to a pot or Agroforestree database: a tree reference and selection guide version 4.0. World Agroforestry Centre, Kenya. Downloaded from planter. Using this method, it may be possible to minimise (http://www.worldagroforestry.org/treedb/AFTPDFS/Acacia_tortilis.pdf the number of pots required and increase the probability Rehman S, Loescher RNJ, Harris PJS (2000) Dormancy breaking and of successfully producing seedlings by more than 90%. In germination of Acacia salicina Lindl seeds. Seed Sci. Technol. 27(2): addition, the amount of time required to obtain seedlings 553-558. Rodrigues DC, Kohl MC, Pedrinho DR, Arias ERA, Favero S (2008) for transplanting to pots will be approximately one week. Treatments to overcome dormancy of Acacia mangium seeds. Acta. Sci. Agron. (UEM). 30:279-283. Teketay D (1998). Germination of Acacia origena, A. pilispina and Conflict of Interests Pterolobium stellatum in response to different presowing seed treatments, temperature and light. J. Arid Environ. 38(4):551-560. The author(s) have not declared any conflict of interests.

ACKNOWLEDGEMENTS

We wish to thank Dr K. Kato and Mr S. Yamaguchi for their kind support during the experiments. This work was carried out as part of KEFRI/JICA project (Development

Vol. 13(50), pp. 4562-4567, 10 December, 2014 DOI: 10.5897/AJB2014.14169 Article Number: C12578448974 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2014 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB

Full Length Research Paper

Chrysodeixis includens (Lepidoptera: Noctuidae) on soybean treated with resistance inducers

Paulo Vinicius de Souza, Bruna Ribeiro Machado, Marcelo Mueller de Freitas, Fernanda Correa, André Cirilo de Sousa Almeida and Flávio Gonçalves de Jesus*

Instituto Federal Goiano, Rod. Prof. Geraldo Silva Nascimento, Km 2,5 CEP. 75790-000, Urutaí, Goiás, Brasil.

Received 9 September, 2014; Accepted 17 November, 2014

Plant resistance levels can be boosted to enable better pest management. The objective of this study was to evaluate resistance interaction between four soybean genotypes and three inducers resistant to integrated management of Chrysodeixis includens. The assays were performed in the entomology laboratory. The soybean genotypes were seeded and plants were treated with calcium and magnesium silicate on soil and sodium silicate and Acibenzolar-S-methyl (ASM) spray on leaves. The weight, longevity and viability of the larvae, the duration and viability of the pre-pupal stage; the weight, longevity and viability of the pupae and the duration and viability of the egg to adult stage of C. includens were observed using plants leaves. The resistant cultivars IAC 100 and IAC 17 associated with the calcium and magnesium silicate inducers and sodium silicate lengthened larval stage and induced high mortality in the C. includens adults.

Key words: Insect, pest management, plant resistance to insects.

INTRODUCTION

Anticarsia gemmatalis Hübner and Chrysodeixis (Zanuncio et al., 2010; Fagundes et al., 2013; Souza et includens Walker (Lepidoptera: Noctuidae) are major al., 2014). The alternative methods includes induction of pests in soybean in Brazil (Bueno et al., 2012; Mourão et plant resistance using silicon (Almeida et al., 2008, 2009; al., 2014). The last species has been found to occur more Lemes et al., 2011), plant resistance to insect, biological frequently and in high populations in this crop (Bueno et control or interaction between the various control al., 2012; Silva et al., 2012). These larvae feed on the methods (McPherson and Buss, 2007; Souza et al., leaves, leaving behind only the veins, which drastically 2012; Zanuncio et al., 2012; Jesus et al., 2014). reduces the leaf area, transpiration and photosynthesis of Inducers like silicon and Acibenzolar-S-methyl (ASM) soybean plants and, consequently, the yield of this crop can increase the resistance levels without in any way (Fortunato et al., 2004; Owen et al., 2013). altering the plant genome (Lemes et al., 2011; Peixoto et Chemical insecticides cause undesirable effects (Sosa- al., 2011). The silicon increases the plant resistance Gómez and Silva, 2010) which increases the reason to stimulating growth and protecting plant against biotic and find effective alternative methods for pest control abiotic’s stresses; rising silica mechanical barrier on the

*Corresponding author. E-mail: [email protected]. Tel: 55 64 3465 1900.

Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License de Souza et al. 4563

leaf tissues and trichomes as well producing phenolic with 16 treatments and 10 replications. One Petri dish (6 cm defensive compounds (Lemes et al., 2011; Cruz et al., diameter lined with moistened filter paper) constituted a replication. 2012). The ASM may activate the genes that encode The apical leaves of each soybean cultivar per treatment were placed with one newly hatched caterpillar and the biological cycle of resistance against biotic’s agents (Goussain et al., 2002; C. includens was followed. The leaves of each of the cultivars were Costa et al., 2007). daily changed. Resistance inducers have revealed promising results to The biological parameters evaluated included, a) larva stage: other insect species on different crops. Silicon application larval period and weight of the ten-day-old caterpillars; b) pupa reduce the number of nymphs on peanut plants stage: pupal period and weight of the 24-hour-old pupa and c) egg- to-adult period, longevity and the total cycle of C. includens. (Dalastra et al., 2011) and reproduction and biological parameters of Schizaphis graminum Rondani (Hemiptera: Aphididae) in sorghum and wheat (Costa et al., 2007; Statistical analyses Pereira et al., 2012). Rhopalosiphum maidis Fitch (Hemiptera: Aphididae) Values from biological parameters were submitted to variance showed a lower degree of preference for the corn plants analysis (ANOVA) as well as to the F-test. Means values were compared with the Tukey test at 5% probability employing the treated with silicon (Moraes et al., 2005) and this Sisvar software (Ferreira, 2011). treatment induced the non-preference mechanism to Aphis gossypii Glover (Hemiptera: Aphididae) in cotton plants (Alcantra et al., 2010). The calcium and RESULTS AND DISCUSSION magnesium silicate added, increased the number of trichomes in the resistant cultivars IAC 100 and IAC 17 The larval, pupal periods, weights, adults longevity and and induced non-preference by Euschistus heros development period to egg and to adult of C. includens Fabricius (Hemiptera: Pentatomidae) to these cultivars differed among the soybean cultivars tested. The (Souza et al., 2014). resistance inducers were observed to affect the pupal Thus the objective of this work was to evaluate the period, weight and development period of this insect interactions effects between resistance inducers and (Table 1). soybean cultivars on the biology of the Soybean Looper C. includens larval and pupal weights were lower Chrysodeixis includens. (98.10 and 55.70 mg, respectively) when feed to IAC 100 cultivar, and also larval period was longer (15 days). This genotype is considered highly resistant and less MATERIALS AND METHODS damaged by Spodoptera eridania Smith (Lepidoptera: Noctuidae) and the stink bug E. heros (Souza et al., The experiment was conducted in the Entomology Laboratory from 2012; Souza et al., 2014). Instituto Federal Goiano – Campus Urutaí, Goiás, Brasil with four The negative impact of the IAC 100 cultivar on C. soybean cultivars and three resistant inducers from September 2012 to July 2013. C. includens adults were collected and includens shows the indirect defense which may be due maintained in plastic cages (21.5 cm high and 14.5 cm diameter) to increase production of the secondary compounds with the BRS valiosa soybean cultivar for oviposition and the eggs induced by the herbivory performed by the lepidopteran of this insect were collected. larvae (Piubelli et al., 2003; Li et al., 2004; Piubelli et al., The C. includens ovipositions were daily removed and placed in 2005). These inducer compounds include the flavonoids, plastic cups (16.5 × 5 cm) until caterpillar eclosion occurred and mainly rutin and the genistein, which can reduce the these were reared on an artificial diet (Greene et al., 1976). The C. includens adults which emerged were placed in plastic cages (21.5 degree of feeding by the C. includens (Hoffmann-Campo cm hight and 14.5 cm to diameter) with 10% honey solution soaked et al., 2001). in a cotton wad placed in PET type soft drink cover. C. includens The development period of C. includens was longer rearing was maintained in an acclimatized room (25±2°C, 70±10% with the resistance inducer ASM (26.94 days), than with RH and 14 h photoperiod). the other treatments, Ca+Mg silicate (19.75) and Na IAC 100 and IAC 17 (resistant), BRS Conquista (moderate resistant) and BRS Jatai (susceptible) seeds were treated with silicate (20.19 days). These results in the lengthened Captan fungicide and seeded in plastics pots (5 L) on an organic duration of the life cycle of Bemisia tabaci Gennadius substrate (29) to obtain the leaves used in the experiments. (Hemiptera: Aleyrodidae) on the cucumber plants treated The treatments included spraying the plants with 0.3% ASM with the ASM (Correa et al., 2005). solution (T1) until draining; application of calcium and magnesium The larval period (14.87 days) of C. includens was silicate on the soil (T2); 1% sodium silicate (T3); and distilled water longer on the BRS Jatai (susceptible) with the resistance (T4) 15 days after plant emergence. The leaves were collected after 30 days and transported to the laboratory to study the biology of inducer ASM than that observed in the IAC 17 and BRS insect pest. Conquista (Table 2). This demonstrates the resistance induction by the ASM in plants (Goussain et al., 2002; Costa et al., 2007). Chrysodeixis includens on soybean cultivars treated with Significant interaction was observed between inducers resistant inducers and IAC 17 cultivar. The calcium and magnesium

This experiment was conducted using a completely randomized silicates lengthened the larval stage of C. includes; the design in a factorial scheme (4 cultivars × 3 inducers + untreated) phase was completed in 15.50 days and 2.6 days 4564 Afr. J. Biotechnol.

Table 1. Larval period (days), larval weight (mg), pupal period (days), pupal weight (mg), adult longevity (days) and egg-to-adult period (days) of Chrysodeixis includens (Lepidoptera: Noctuidae) in soybean cultivars treated with the resistance inducers ASM, Ca+Mg silicate, Na silicate and untreated (25°C, 70% RH and 14h photoperiod).

Cultivars (C)1 Lar. Per.2 Lar. Wei. Pup. Per. Pup. Wei. Long. eg-Ad.

BRS Jataí 13.62ab 144.00b 6.33a 126.90b 3.75ab 27.12a BRS Conquista 13.47b 166.60a 6.62a 149.20a 4.06a 27.00b IAC 17 13.84ab 154.30ab 5.96a 141.90a 3.00b 26.19a IAC 100 15.00a 98.10c 3.92b 55.70c 0.44c 6.69b F (C) 3.28* 24.41** 17.12** 157.25** 52.61** 107.45**

Inducers (I) ASM 13.37a 151.60a 6.67a 139.60a 3.12a 26.93a Ca+Mg silicate 14.50a 132.80a 6.21a 127.30a 2.50a 19.75b Na silicate 14.00a 133.40a 4.87b 99.70b 2.50a 20.19b Untreated 14.06a 145.20a 5.08b 107.10b 3.12a 20.12b F (I) 1.46ns 2.32ns 8.57** 28.72** 2.53ns 119.66** F (C x I) 2.14* 2.96** 6.62** 26.00** 5.15** 115.31** C.V. (%) 15.50 24.41 25.41 11.55 32.25 5.82

1Means followed by the same lower-case letter do not differ significantly according to Tukey’s test at 0.05 probability. **Significant at 1% probability. *Significant at 5% probability. NSNot significant. 2Lar. Per, Larval period; Lar. Wei, larval weight; Pup. Per, pupal period; Pup. Wei, pupal weight; Long, adult longevity; eg-Ad, egg-to-adult period.

Table 2. Larval period (days) and larval weight (mg) of Chrysodeixis includens (Lepidoptera: Noctuidae) in soybean cultivars treated with the resistance inducers ASM, Ca+Mg silicate, Na silicate and untreated (25ºC, 70% RH and 14 h photoperiod).

Inducers (I) Cultivars (C)1 F (C) ASM Ca + Mg Na Untreated. Larval period (days) BRS Jataí 14.87aA 13.00aB 13.12aA 13.50aAB 1.26ns BRS Conquista 12.12aB 13.50aAB 14.12aA 14.12aAB 1.51ns IAC 17 12.50bB 15.50aAB 14.50abA 12.87abB 3.36* IAC 100 14.00aAB 16.00aA 14.25aA 15.75aA 1.77ns F (I) 2.82* 3.69* 0.62ns 2.60* −

Larval weight (mg) BRS Jataí 119.80 b C 169.00 a A 147.50 ab A 139.80 ab AB 2.82* BRS Conquista 181.00 a A 152.70 a A 165.30 a A 167.60 a A 0.91ns IAC 17 172.90 a AB 134.40 a A 145.10 a A 164.80 a A 2.13ns IAC 100 132.90 a BC 75.20 b B 75.70 b B 108.80 ab B 5.35** F (I) 6.09** 11.44** 10.66** 5.11** −

1Means followed by the same lower-case letter do not differ significantly according to Tukey’s test at 0.05 probability. **Significant at 1% probability. *Significant at 5% probability. NSNot significant.

longer than that seen in the control, a fact that could be cultivars. C. includens larvae had less weight when feed explained by the higher lignin content of the soybean on the BRS Jatai (119.8 mg), showing the resistance genotype (Moraes et al., 2009) or by the lower degree of being induced in the susceptible cultivar (Table 2). On the feeding by the S. frugiperda caterpillars on corn due to other hand, the larvae weight was higher on the BRS the mechanical barrier caused by the silicon accu- Conquista (moderate resistance) (181.0 mg). This mulation (Goussain et al., 2002). difference regarding larvae weight shows that the ASM The effect of the ASM inducer ranged amongst the can induce resistance against the biotic agents, as de Souza et al. 4565

Table 3. Pupal period (days) and pupal weight (mg) of Chrysodeixis includens (Lepidoptera: Noctuidae) in soybean cultivars treated with the resistance inducers ASM, Ca+Mg silicate, Na silicate and untreated (25ºC, 70% RH and 14h photoperiod).

Inducers (I) Cultivars (C)1 F (C) ASM Ca + Mg Na Untreated Pupal period (days) BRS Jataí 6.00aA 6.50aA 6.50aAB 6.33aA 0.16ns BRS Conquista 6.67aA 6.00aA 7.00aA 6.83aA 0.55ns IAC 17 6.67abA 7.00aA 4.50bB 5.67abA 3.61* IAC 100 7.33aA 5.33aA 1.50bC 1.50bB 24.11** F (I) 0.85ns 1.44ns 17.76** 16.92** −

Pupal weight (mg) BRS Jataí 115.80aC 136.30aA 123.40aA 132.10aB 1.79ns BRS Conquista 143.80aAB 140.80aA 146.80aA 165.40aB 2.63ns IAC 17 163.20aA 145.10abA 128.60bA 130.80bB 5.44** IAC 100 135.70aBC 86.90bB 0.00cB 0.00cC 96.84** F (I) 8.26** 15.79** 96.67** 114.51** −

1Means followed by the same lower-case letter do not differ significantly according to Tukey’s test at 0.05 probability. **Significant at 1% probability. *Significant at 5% probability. NSNot significant.

reported for S. frugiperda on corn and S. graminum on using the inducer (control) showed lower values, with wheat (Goussain et al., 2002; Costa et al., 2007). 1.50 days for the pupal stage of this insect compared with The weight of the C. includens larvae was lower on the the 7.33 days with the inducer ASM, prolonging the C. IAC 100 cultivar with the use of the calcium, magnesium includens pupal stage. and sodium resistance inducers (75.2 and 75.7 mg). The The ASM inducer increased the pupal weight (163.5 lower weight of this caterpillar with the silicon inducers mg) of the C. includens on the IAC 17 to a higher degree can be explained by the Si deposition in the leaf cell than in the other treatments, more than the IAC 100 walls, forming a mechanical barrier, thus increasing the (86.90 mg) treated with the calcium and magnesium hardness of the leaf tissues and the wearing out of the silicate and the BRS Jatai (115.8mg) (Table 3). This may caterpillar mandibles, thereby reducing the degree of be related to the increased resistance of the susceptible feeding. genotype with the resistance inducer (Costa et al., 2007; The cultivars showed significant interactions without the Souza et al., 2014). use of the resistance inducers (untreated). The C. The C. includens larval mortality reached 100% with includens caterpillars were heavier (167.6 mg) on the the use of the inducer sodium silicate; however, in the BRS Conquista cultivar with the use of the IAC 100 control it prevented the pupal weight from being recorded (108.8 mg). This is similar to the resistance recorded in with the IAC 100 genotype. Such mortality revealed by the IAC 100 cultivar to insects such as C. includens and the resistant cultivar IAC 100 is explained by the stink bugs (McPherson and Buss, 2007; Souza et al., flavonoid rutin and the genistein isoflavones linked to the 2014). anti-nutritional effect and injury to cells of the digestive The pupal period of C. includens was shorter on the tract as shown for A. gemmatalis fed on a diet containing IAC 17 treated with the calcium and magnesium silicate these metabolites (Hoffmann-Campo et al., 2001; resistance inducer (7.0 days) than with the treatment with Salvador et al., 2010). sodium silicate (4.50 days) (Table 3). This may be related The increased longevity of C. includens adults on the to the length of the larval stage because each one BRS Conquista without the use of the resistance inducers depends on the earlier one to obtain, synthesize and shows a lower impact on the IAC 17 treated with the accumulate the nutritional substances (Fugi et al., 2005). sodium silicate on the adults of this insect (Table 4). The The resistance inducer, sodium silicate, increased the mortality of the insects on the IAC 100 cultivar with the pupal period of C. includens on the BRS Conquista (7.0 use of the calcium and magnesium silicate and sodium days) compared with those on the IAC 100 and IAC 17 silicate inducers and in the control disallowed the (4.5 and 1.5 days), respectively. The shorter pupal period evaluation of the longevity of this insect and thus of C. includens on this latter cultivar shows the antibiosis corroborates the beneficial effect of the interaction of the type of resistance, as observed with the cultivars with all resistant plants with the inducers to control the spread of the inducers. Sodium silicate and the treatment without E. heros on soybean (Souza et al., 2014). 4566 Afr. J. Biotechnol.

Table 4. Adult longevity (days) and egg-to-adult period (days) of Chrysodeixis includens (Lepidoptera: Noctuidae) in soybean cultivars treated with the resistance inducers ASM, Ca+Mg silicate, Na silicate and untreated (25ºC, 70% RH and 14h photoperiod).

Inducers (I) Cultivars (C)1 F (C) ASM Ca + Mg Na Test. Adult longevity (days) BRS Jataí 2.50 c BC 3.25 bc A 5.00 a A 4.25 ab AB 5.87** BRS Conquista 4.50 ab A 3.25 b A 3.50 ab A 5.00 a A 3.32* IAC 17 3.75 a AB 3.50 a A 1.50 b B 3.25 a B 5.06** IAC 100 1.75 a C 0.00 b B 0.00 b B 0.00 b C 3.72* F (I) 7.39** 13.57** 23.49** 23.59** −

Cycle egg-to-adult period (days) BRS Jataí 28.00 a A 26.00 a B 28.00 a A 26.50 a AB 2.65ns BRS Conquista 26.50 ab A 24.50 b B 28.35 a A 28.75 a A 9.25** IAC 17 26.50 ab A 28.50 a A 24.50 b B 25.25 b B 7.62** IAC 100 26.75 a A 0.00 b C 0.00 b C 0.00 b C 46.06** F (I) 1.29ns 439.06** 458.94** 454.08** −

1Means followed by the same lower-case letter do not differ significantly according to Tukey’s test at 0.05 probability. **Significant at 1% probability. *Significant at 5% probability. NSNot significant.

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Vol. 13(50), pp. 4568-4576, 10 December, 2014 DOI: 10.5897/AJB2014.13977 Article Number: 0127F1448978 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2014 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB

Full Length Research Paper

Novel carrier system for enhancing oral delivery of metformin

Adedokun, M. O.1*, Momoh, M. A.2, Kenechukwu, F. C.2, Umeh, O. N. C.3, Okpalanedu, C. M. J.2 and Ofokansi, K. C.2

1Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, University of Uyo, Uyo, Akwa Ibom State, Nigeria. 2Drug Delivery Research Unit, Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria. 3Department of Pharmaceutical Technology and Industrial Pharmacy, Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria.

Received 10 June, 2014; Accepted 2 December, 2014

This study was designed to evaluate the potential of PEGylated lipospheres as carriers for improved oral delivery of metformin hydrochloride. Lipospheres were prepared by melt-emulsification method using Phospholipon® 90H in beeswax (30%w/w) as the lipid matrix containing increasing quantities of PEG 4000 and characterized. The in vitro and in vivo release of the formulations was evaluated. Results show that the particle size and encapsulation efficiency ranged from 33.18±1.75 - 83.23±6.05 μm and 85 to 93%, respectively. Drug release showed a biphasic pattern and was found to follow the Higuchi square root model. Metformin hydrochloride -loaded lipospheres lowered basal blood glucose levels by 60% and sustained antihyperglycemia for over 20 h. This study suggests that encapsulation of metformin hydrochloride into PEGylated lipospheres could reduce its dosing frequency and the associated side effects resulting from high doses of metformin hydrochloride as seen in conventional tablet formulations.

Key words: PEGylated, lipospheres, metformin hydrochloride, anti-diabetic.

INTRODUCTION

The design of an oral controlled drug delivery system Diabetes mellitus is a chronic metabolic disorder should primarily be aimed at achieving more predictable characterized by high blood glucose concentration and increased bioavailability of drugs as there are several caused by insulin deficiency, often combined with insulin physiological difficulties, which include restraining/- resistance (Philip et al., 2009). The effective control of localizing the drug delivery system within the regions of blood glucose is the key in preventing or reversing the gastrointestinal tract and the high variable nature of diabetic complications and improving the quality of life for gastric emptying process (Prajapati et al., 2008). both type I and type II diabetic patients. Although different

*Corresponding author. E-mail: [email protected]. Tel:+234 (0) 8034396937.

Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Adedokun et al. 4569

types of oral hypoglycemic agents are commonly polyethylene glycol 4000 (Cary Roth, Karlsruhe, Germany), employed along with insulin for the treatment of diabetes Metformin hydrochloride (Farmex, Pharma Ltd, Ikeja, Lagos State, mellitus, none offers complete glycemic control (Pradeep, Nigeria), Monobasic potassium phosphate, Sodium hydroxide and Concentrated hydrochloric (BDH, Poole, England), and Distilled et al., 2010). water (Lion Water, University of Nigeria, Nsukka, Nigeria). Other Metformin HCl is an effective antidiabetic that requires reagents were of analytical grade and used without further controlled release owing to its short biological half-life purification. (1.5-4 h) (Stepensky et al., 2001; Momoh et al., 2011). It is used as monotherapy as well as an adjunct to diet in Preparation of lipid matrix the management of type II diabetes in patients whose hyperglycemia cannot be controlled by diet alone. Quite The lipid matrix consisting of 4:1 mixture of beeswax and frequently, it causes gastrointestinal problems such as Phospholipon® 90H (P90H) were prepared by fusion method nausea, stomach pain, bloating, and diarrhoea. (Attama et al., 2008). Briefly, 80 g quantity of beeswax and 20 g of Metformin hydrochloride is absorbed from the upper P90H were weighed using an electronic balance (Mettler H8, Switzerland) and melted together on a crucible at 75°C over a intestine within 6 h of administration, so repeated thermo-regulated shaking water bath (Heto, Denmark) and stirred administration is required to maintain effective plasma thoroughly to obtain a homogenous mixture. Thereafter, the lipids concentration (Momoh et al., 2013). were allowed to cool and solidify at room temperature to get a lipid Lipospheres were first reported as a particulate matrix. dispersion of solid spherical particles between 0.2-100

μm in diameter consisting of solid hydrophobic fat core Preparation of drug loaded and unloaded lipospheres such as triglycerides or fatty acids derivatives, stabilized by monolayer of phospholipids (Attama et al., 2008, The melt homogenization technique was adopted (Attama et al., 2009). The internal core contains the drug dissolved or 2008). In each case, the lipid matrix was melted at 70°C, and the dispersed in solid fat matrix. Lipospheres drug delivery aqueous phase containing PEG-4000 and polyvinyl alcohol (PVA) at the same temperature was added to the molten lipid matrix under system is an emerging carrier for both hydrophilic and gentle stirring with a magnetic stirrer (SR 1 UM 52188, Remi Equip., hydrophobic drugs. This carrier system has several India), and the mixture was further dispersed with a mixer (Ultra- advantages over other delivery systems in terms of Turrax, Germany) at 8000 rpm for 5 min to produce the hot primary physical stability, low cost of ingredients, ease of emulsion. The lipospheres suspension obtained after cooling at preparation, and scale-up, high dispensability in aqueous room temperature was then lyophilized using a freeze-dryer (Amsco medium, high entrapment efficiency, and extended GT3, Germany) in order to get water-free lipospheres. Briefly, lyophilisates of the SLMs are obtained by freezing the formulations release of entrapped drug (Attama et al., 2009). The at a pressure of 2.7 Pa and temperature of 30°C; sublimation and novelty of the work lies on the use of beeswax and drying were at 15-25°C and all these operations took 6-12 h. ® Phospholipon 90H (a homo and hetero lipid) in The above procedure was repeated using increasing amount of combination with polyvinyl alcohol to improve and PEG, (0.5, 1.0, 1.5 and 2.0 g), decreasing amount of lipid matrix; enhanced the oral delivery of metformin entrapped (4.5, 4.0, 3.5 and 3.0 g) and two concentration levels of metformin lipospheres prepared by fusion method. Recently, lipid- hydrochloride (250 and 500 mg), to obtain metformin hydrochloride- loaded lipospheres (batches A - A and B - B ). Unloaded based formulations have been used to control and 1 4 1 4 lipospheres (without drug) were similarly prepared (C1 - C4). The enhance the release of some drug molecules (Attama et formulation compositions are shown in Table 1. al., 2009; Attama and Nkemnele, 2005). Fusion method enhances the encapsulation of the drug in the core and Characterization of lipospheres improves its delivery capacity when in contact with the wall of the gastrointestinal tract (Attama et al., 2009; Particle size analysis and morphological characteristics of Philip et al., 2009). The prepared lipospheres were lipospheres evaluated for production yield, loading efficiency, morphology, particle size, pH analysis, and in vitro drug The particle size of the lipospheres was determined by com- puterized image analysis. Approximately, 3.0 mg of the lipospheres release. The anti-diabetic property of the formulations from each batch was dispersed in distilled water and smeared on a was also evaluated in alloxan-induced rat model. slide (Marinfield, Germany) using a glass rod. Each of the batches The objective of this study was to evaluate metformin on a slide was mounted and observed under a light photo-micro- hydrochloride-loaded lipospheres prepared by fusion scope (Lieca, Germany). With the aid of the software in the method for possible oral delivery of metformin, in order to photomicroscope, the projected diameters of the particles corres- ponding to the particle sizes of the lipospheres were determined achieve a controlled release and enhanced bioavai- and the average calculated. The particle morphologies were also lability. observed and photomicrographs taken. Measurement of particle size of the formulated lipospheres was repeated at intervals of 24 h, one week and four weeks after formulation. MATERIALS AND METHODS

Materials pH stability studies of the formulations

® The following materials were used: Beeswax, Phospholipon 90H With the aid of a pH meter (Digital pH Meter, Labtech), the pH (Nattermann, Germany), Polyvinylalcohol (Sigma-Aldrich, USA), values of the different batches of the lipospheres formulations 4570 Afr. J. Biotechnol.

Table 1. Formulation compositions of the lipospheres.

Batch PEG-4000 Polyvinylalcohol (PVA) Metformin HCl Lipid matrix (g)

A1 0.5 100 500 4.5

A2 1.0 100 500 4.0

A3 1.5 100 500 3.5

A4 2.0 100 500 3.0

B1 0.5 100 250 4.5

B2 1.0 100 250 4.0

B3 1.5 100 250 3.5

B4 2.0 100 250 3.0

C1 0.5 100 - 4.5

C2 1.0 100 - 4.0

C3 1.5 100 - 3.5

C4 2.0 100 - 3.0

Batches A1 - A4 and B1 - B4 contain metformin hydrochloride whereas batches C1 - C4 contain no drug.

including those of the control were measured. Measurements were nm. The amount of drug released at each time interval was also carried out at one week intervals for one month post- determined with reference to the standard Beer’s plot earlier formulation. determined for metformin hydrochloride in SIF.

Determination of encapsulation efficiency (EE %) Pharmacodynamic study

The encapsulation efficiency of each formulation was determined. A Induction of diabetes 6 ml volume each of the reconstituted lipospheres was centrifuged for 60 min at an optimized speed of 3000 rpm in order to obtain two The animal experiments in this work complied with the regulations phases (aqueous and lipid phases). A 1 ml volume of the aqueous of the Committee on Ethics on the Use of Laboratory Animals of the phase was measured out and diluted 1000-fold using distilled University of Nigeria and were in compliance with the Federation of water. The absorbance of the solutions at a wavelength of 278 nm, European Laboratory Animal Science Association and the were taken using a UV-spectrometer and the encapsulation European Community Council Directive of November 24, 1986 efficiency was calculated using the formula below: (86/609/EEC) (EEC, 1986). Rats weighing between 200 - 220 g were purchased from the Department of Biochemistry, University of Nigeria, Nsukka. The rats were all kept in standard and conditioned (1) animal cages and left for one week to acclimatize to the new laboratory environment while being fed with standard laboratory chow diet. Diabetes was induced by intravenous injection of Alloxan

dissolved in normal saline through the marginal ear vein at a dose Determination of loading capacity (LC) of 120 mg/kg (Builders et al., 2008). After 5-7 days of the Alloxan

treatment, rats with frequent urination, loss of weight, and blood LC is expressed as the ratio between the entrapped drug by the glucose levels higher than 220 mg/dL were considered diabetic and lipid and the total quantity of lipids used in the formulation and selected for the study (Builders et al., 2008). The rats were calculated as follows: monitored for persistent blood glucose elevation for 5 days (Sharma

et al., 2006). Before testing, animals were fasted overnight with free (2) access to water.

In vivo antidiabetic study In vitro drug release studies Twenty five (25) Wistar rats (either sex) of an average weight of A volume (4.0 ml) of the reconstituted lipospheres from each batch 200.0±20.0 g were used for the evaluation of the pharmacological was accurately measured and placed in the donor compartment of effects of metformin hydrochloride-loaded lipospheres after oral a Franz diffusion cell that was separated from the receptor administration. In each case, the animals were fasted for 24 h prior compartment by an artificial membrane (pore size 0.22 µm). The to oral administration of the formulations. Rats were divided into five receptor compartment was filled with simulated intestinal fluids groups of five animals each and the formulations were administered (SIF) without pancreatin (pH 7.4) and maintained at a temperature orally. Briefly, the first group received Batch A1 containing of 37±1°C by means of a thermostatically controlled water bath, metformin hydrochloride-loaded lipospheres at a dose of 7 mg/kg with agitation being provided by a magnetic stirring bar at 50 rpm. A body weight. The second group received Batch B1 also containing 2.0 ml was removed and replaced by an equal volume of the metformin hydrochloride-loaded lipospheres at a dose of 0.7 mg/kg receptor phase at predetermined time intervals. The drug contents body weight. Whereas the third group received Batch C1 (zero-drug were analyzed using a spectrophotometer at a wavelength of 283 loaded liposphere formulation), the fourth group received a Adedokun et al. 4571

Figure 1. Photomicrographs of representative batches of lipospheres containing 10% w/w PEG 4000 after 1 month. A1, B1, and C1 contain 500, 250 and 0 mg of metformin hydrochloride, respectively.

Table 2. Time-resolved pH-dependent storage study of the lipospheres (Mean±SD, n = 3).

Batch 1 week 2 weeks 3 weeks 4 weeks

A1 4.6±0.1 4.6±0.4 5.0±0.4 5.5±0.3

A2 4.6±0.2 4.6±0.3 5.0±0.3 5.3±0.3

A3 4.5±0.1 4.6±0.2 5.5±0.4 5.6±0.4

A4 4.5±0.3 4.7±0.4 5.2±0.1 5.4±0.2

B1 4.5±0.2 4.5±0.2 5.3±0.2 5.2±0.1

B2 4.2±0.1 4.3±0.3 4.7±0.4 4.9±0.0

B3 4.1±0.1 4.4±0.1 4.8±0.3 5.1±0.4

B4 4.4±0.3 4.6±0.2 5.2±0.1 5.3±0.3

C1 4.0±0.4 4.6±0.1 4.7±0.1 4.8±0.2

C2 3.5±0.2 4.6±0.3 4.6±0.2 4.7±0.2

C3 3.6±0.2 4.6±0.1 4.7±0.2 4.8±0.1

C4 3.7±0.2 4.6±0.1 4.7±0.3 4.9±0.1

Batches A1 - A4 and B1 - B4 contain 500 and 250 mg of metformin hydrochloride respectively, while batches C1 - C4 contain no drug.

commercially available metformin hydrochloride preparation of the formulated batches of the lipospheres are shown in (Mephage®) while the last group received metformin hydrochloride Figures 1 (A1, B1 and C1) and Table 2. There was a slight dispersed in distilled water (100 mg p.o). The selected formulations variation in the particle size of the lipospheres. The drug- (A1 and B1) used in the in vivo study were based on the results of our preliminary evaluation. Blood samples were taken from the tip loaded lipospheres showed higher particles size com- of the tail vein at predetermined intervals and blood glucose levels pared to drug-loaded batches. Batches in A, B and C were measured using an Accu-Check Roach, USA). Food and showed an irregular shape, sub-batches A had smooth water intake as well as urine output of the animals were measured appearance unlike the rest of the batches. However after and monitored in the course of the study (Sharma et al., 2006). a period of storage there was slight increase in the

particle size of the formulation, although the changes in Statistical data analysis the size did not altered the shape.

All experiments were performed in replicates (n= 3) using SPSS version 18 for validity of statistical analysis. Results were expressed Time-dependent pH analysis as mean ± SD and differences between means were considered significant at p< 0.05 using the analysis of variance (ANOVA). Time-dependent pH analysis was carried out to deter- mine the pH stability of the different batches of the SLMs RESULTS when stored at room temperature and at different time intervals. Table 2 shows the pH values of the various The particle size and the representatives of the morpholo- batches of the metformin hydrochloride-loaded and 4572 Afr. J. Biotechnol.

Table 3. Physicochemical properties of the lipospheres (Mean±SD, n=3).

Particles size (µm) BC EE(%) DL% YD% 24 h 1 week 4 weeks

A1 92.46 39.31 88.00 53.11±1.1 61.11±0.15 73.00±0.12

A2 90.14 30.13 79.10 49.01±0.2 57.42±0.12 69.80±1.11

A3 86.60 31.02 86.11 54.19±0.11 61.10±0.15 65.20±1.15

A4 85.50 32.47 88.20 51.87±3.01 53.01±1.15 67.22±0.00

B1 84.93 24.51 90.10 52.12±0.13 53.17±1.22 59.21±0.05

B2 83.19 24.91 79.00 55.11±3.75 60.21±1.00 63.23±1.11

B3 68.70 24.68 74.10 62.08±3.75 65.20±0.00 74.00±3.75

B4 65.22 25.15 80.01 56.01±3.75 61.11±0.15 62.23±0.30

C1 - - 89.13 54.03±5.65 57.00±1.43 63.27±2.63

C2 - - 86.21 33.18±1.75 59.20±3.75 73.21±0.15

C3 - - 88.81 43.23±0.12 63.02±0.25 81.21±5.25

C4 - - 82.03 52.22±1.85 72.11±2.15 83.23±6.05

BC = Batch code; EE = % encapsulation efficiency; D L = % drug loading and % yield value. Batches A1 - A4 and B1 - B4 contain 500 and 250 mg of metformin hydrochloride while batches C1 - C4 contain no drug (0 mg).

Figure 2. Release profile of metformin HCl from the lipospheres (Batch A) in SIF. (A1-A4= batches of the formulation contain 500 mg of metformin hydrochloride.

unloaded lipospheres. The pH varied from between 3.5 recorded for lipospheres (batches B) containing 250 mg to 4.6 after one week of preparation to between 4.7 and of the drug (Table 3). 5.6 after four weeks of preparation, while the unloaded lipospheres (C1-C4) recorded the highest variation in pH after four weeks of formulation. Results revealed that the In vitro drug release studies EE % decreased as the amount of polymer (PEG-4000) in the lipid matrix increased. Figures 2 and 3 show the in vitro release profiles of In other words, EE % decreased as the lipid base different batches of the metformin hydrochloride-loaded decreased (Table 3) but increased with increase in the lipospheres in SIF. There was an initial release of about concentration of the drug such that the maximum EE % 15-20% of drug from the metformin hydrochloride-loaded was 92.58% for lipospheres (batches A) containing 500 lipospheres within the first 0.5 to 1 h. Drug release from mg of metformin hydrochloride, as compared to 84.93% the formulations was sustained for up to 20 h with only Adedokun et al. 4573

Figure 3. Release profile of metformin HCl from the lipospheres (Batch B) in SIF. (B1-B4= batches of the formulation contain 250 mg of metformin hydrochloride.

Table 4. Release kinetics of the lipospheres.

Formulation Higuchi’s plot Korsmeyer-Peppas plot code r2 r2 (n) Mechanism of release

A1 0.947 0.896 0.605 Non-fickian

A2 0.963 0.880 0.438 Fickian

A3 0.963 0.898 0.618 Non-fickian

A4 0.956 0.865 0.603 Non-Fickian

B1 0.762 0.966 0.475 Fickian

B2 0.672 0.989 0.451 Fickian

B3 0.762 0.926 0.578 Non-fickian

B4 0.779 0.920 0.647 Non-fickian

A1 - A4 and B1 - B4 = batches of the formulation contain 500 and 250 mg, respectively of metformin hydrochloride.

50% of the metformin hydrochloride being released after chloride-loaded lipospheres lowered basal blood glucose 16 h. levels in diabetic rats by 60% and sustained hypo- glycemia for over 20 h. No reduction in blood glucose levels was observed in rats that received no treatment. Modeling of in vitro drug release Reduction in blood glucose levels was similarly observed in rats that received pure sample of metformin The release mechanism for metformin hydrochloride- hydrochloride and the commercially available metformin loaded lipospheres is shown in Table 4. The in vitro hydrochloride marketed sample (Mephage®). Although, release profile of metformin hydrochloride from the there was sustained antihyperglycemia in all cases, for lipospheres was fitted into the Higuchi equation and the about 10 h, the hypoglycemic effect was greater in rats plots showed high linearity (r2 > 0.999). that received the PEGylated microscopic lipospheres than those that received the latter (pure sample of metformin hydrochloride and Mephage®). The result here Pharmacodynamic studies further suggests that the release of metformin hydro- chloride from the lipospheres stimulated the release of Figure 4 shows the changes in blood glucose levels after insulin from the production cell or stimulated the tissue the formulations were administered to diabetic rats. At 7.0 uptake of glucose or both in a controlled manner as was mg/kg body weight, orally delivered metformin hydro- observed in the glucose lowering effect. 4574 Afr. J. Biotechnol.

Figure 4. Percentage reduction in blood glucose level in diabetes rats after oral administration of metformin hydrochloride lipospheres formulations (A1, B1 and C1 contain 500, 250 and 0 mg of metformin hydrochloride), Mkt = market sample and PD= Pure drug.

DISCUSSION the knowledge in designing a suitable formulation for the drug (Sepici, et al., 2004). This will serve as a guide for In this study, a PEGylated microscopic lipospheres the formulator on the need of adding a preservative or delivery system of metformin hydrochloride was deve- stabilizer to ensure that a stable product is achieved and loped and evaluated for enhanced oral delivery of maintained throughout the shelf-life of the product. The metformin hydrochloride. The lipospheres formed were slight increase in the pH values in all the lipospheres fairly smooth and spherical in shape, and showed some (Table 2) may not be attributable to drug degradation consistency according to the drug incorporated into the since there was also a rise in the pH of the unloaded formulation (Figure 1). There were no signs of sedimen- lipospheres. tation after the formulations stood for a period of 8 weeks. Degradation of the lipidic excipients, common to all the There was a slight increase in the particle sizes after four formulated lipospheres, to free fatty acids could occur, weeks. The increase in the particle size was directly causing a fall in the pH of the formulation rather than a proportional to the amount of drug entrapped in the rise in pH as was observed in the present study, which is formulation. This increase may be due to crystallization of consistent with earlier report (Attama and Nkemnele, the formerly molten matrices or it may be due to 2005), suggesting that it is neither the drug nor the aggregation and subsequent growth by Ostwald ripening excipients that caused the rise in pH. It would, therefore, or sintering (Attama et al., 2008; Attama et al., 2009). The be reasonable to infer that the rise in pH is due either to a increase in particle size was not significant (p>0.05). rise in the particle surface pH or the likely interaction of The pH of the different batches of lipospheres was the ions present in the medium with the components of measured at one week intervals up to one month after the formulations (Gao, et al., 2004). preparation to ascertain the variation of pH with time, The function of the formulated lipospheres is to deliver which could be a function of degradation of the drug or the active pharmaceutical ingredient (API) for further lipid component or both. In pharmaceutical formulations, absorption into the biological system (Attama et al., 2009; the initial preparation may be stable, but as time goes on, Nnamani et al., 2007). This can be expressed or degradation of either the components or the drug or both determined by the EE % and LC. Encapsulation may set in on storage through generation of unfavorable efficiency is a function of the amount of drug entrapped in pH (increase or decrease) or reactive species from the lipid base to the total weight of drug while the loading drug. capacity expresses the ratio between the entrapped drug It is therefore of paramount importance to determine and the total weight of lipid used as a carrier (Attama et the pH of maximum stability for formulations and utilize al., 2009; Nnamani et al., 2007). Adedokun et al. 4575

Results reveal that the EE % decreased as the amount of drug released can serve as a loading dose and the of polymer (PEG-4000) in the lipid matrix increased. In remaining amounts of drug released slowly over time will other words, EE % decreased as the lipid base serve as the maintenance dose. Drug release from the decreased (Table 3) but increased with increase in the formulations was sustained for up to 20 h with only 50% concentration of the drug (Table 3). This showed that the of the metformin hydrochloride being released after 16 h. polymer (PEG-4000) together with the lipid matrix This slow phase of drug release is attributable to gradual promoted drug solubilization in a concentration-depen- drug release from the core of the lipid matrices via dent manner. The lipid matrix accommodated more drugs diffusion-controlled processes (Luan, 2006). at higher drug loadings possibly due to the low crystalline Various kinetics models were used to describe the nature of the excipients. The variation in the EE % as release of metformin hydrochloride from the liposphere shown in Table 3 is a clear indication that both the lipid formulations. The criterion for selecting the most appro- and PEG contents are critical variables in the amount of priate model was chosen on the basis of goodness-of-fit drug that could be entrapped by the formulated test. The results (Table 4) revealed that diffusion is the lipospheres. likely mechanism of release of metformin hydrochloride Loading capacity, however, increased as the amount of from the lipospheres. To confirm that release of the polymer (PEG-4000) increased and as the amount of the loaded drug occurred through diffusion, the release data lipid matrix decreased. Batches A lipospheres recorded were fitted into the Korsmeyer-Peppas equation from greater loading capacity than Batches in B lipospheres which n values ranging between 0.438 and 0.647 were (Table 3). The presence of PEG-4000 in the lipospheres obtained. These values show that release of metformin batches A1-A4, B1-B4 and C1-C4 was found to be essential hydrochloride from batches A1, B1 and B2 followed in obtaining spherical particles (Figures not shown). The Fickian diffusion mechanism whereas the mechanism of yields of the lipospheres suggested that the processing release in batches A2, A3, A4, B3 and B4 was non-Fickian parameters and the fusion method employed in the (Ofokansi et al., 2007). preparation of the liposphere did not affect the yield, thus Results of the in vivo pharmacodynamic study show making the process up scalable and this could be of that percentage reduction of basal blood glucose levels of commercial interest. 47 and 49% respectively was achieved by the pure The particle size of lipospheres (C1-C4) containing no sample of metformin hydrochloride dispersed in distilled API but formulated with 10 % of PEG were slightly larger water and the commercial product (Mephage®). The order than those formulated with 30% PEG-4000. The particle of hypoglycemic activity of formulated metformin size and morphology of metformin hydrochloride - loaded hydrochloride -loaded lipospheres are batch A1 > batch lipospheres are shown in Table 3 and Figures1, B1 > PD > MK > batch C1. This shows that batches A1 respectively. and B1 possessed greater antihyperglycemic effect than Drug release is affected by the nature or design of the commercially available metformin HCl formulation and delivery system and the medium used in the release pur sample of metformin hydrochloride, an indication that study. Factors such as pH are among the most important entrapment of metformin hydrochloride into PEGylated factors affecting drug release. Other factors such as lipospheres not only improved its therapeutic effective- viscosity and gastro intestinal motility affect the in vivo ness but also extended its therapeutic action, consistent release of drugs while in vitro release is affected by with a similar reported study (Attama et al., 2009; agitation, viscosity and temperature of the medium, Nnamani et al., 2007). stirring speed of the apparatus used in the release study. Figures 2 and 3 show the in vitro release profiles of different batches of the metformin hydrochloride-loaded Conclusion lipospheres in SIF. There was an initial release of about 15-20% of drug from the metformin hydrochloride-loaded In designing a drug delivery device such as those based lipospheres within the first 0.5 to 1 h, which could be on polymers or lipids or combinations thereof, the choice attributed to drug desorption from the surface of the as well as the combination ratio of the polymers or lipids lipospheres, of drug particles that were not encapsulated or other excipients should be considered based on the in the lipid core. desired pattern, route of administration, stability, and Loosely or poorly bound drug is released rapidly while other physicochemical considerations. In this research drug entrapped within the polymer matrix (core) is work, metformin hydrochloride-loaded lipospheres were released in a more gradual fashion either through erosion successfully prepared and optimized and evaluated in a or diffusion. The initial drug release which was observed diabetic animal model. may be due to unbound drug (Defang et al., 2005; Wang, Majority of the formulated lipospheres of metformin 2006). Clinically, however, burst release may not always hydrochloride exhibited better in vivo performance than be undesirable especially in conditions such as commercially available MT formulation. In the light of the hyperglycemia where there is urgent need for lowering of importance of metformin hydrochloride in the manage- blood glucose levels. In such cases, the initial amount of ment of type II diabetes mellitus, the formulated lipospheres 4576 Afr. J. Biotechnol.

could further be exploited as an alternative dosage form Nnamani PO, Attama AA, Ibezim EC, Adikwu MU (2007). SRMS 142- for metformin hydrochloride. In view of the sustained based solid lipid microparticles: Application in oral delivery of glibenclamide to diabetic rats. Eur. J. Pharm. Biopharm. 76:68-74. hypoglycemic effect that was observed in metformin Ofokansi KC, Adikwu MU, Okore VC (2007). Preparation and evaluation hydrochloride-loaded PEGylated lipospheres, the studied of mucin-gelatin mucoadhesive microspheres for rectal delivery of formulation would be a necessary intervention in reducing ceftriaxone sodium. Drug. Dev. Ind. Pharm. 33:691-700. the dosing frequency of metformin hydrochloride with Philip AK, Srivastava M, Pathak K (2009). Buccoadhesive gels of glibenclamide: A means for achieving enhanced bioavailability. Drug concomitant reduction in the side effects associated with Deliv. 16:405-415. it. Pradeep KR, Sujatha D, Mohamed TS, Ranganayakulu D (2010). Potential antidiabetic and antioxidant activities of Morus indica and Asystasia gangetica in alloxan-induced diabetes mellitus. J. Exp. Pharmacol. 2:29-36. Conflict of Interests Prajapati ST, Patel LD, Patel DM (2008). Gastric floating matrix tablets: design and optimization using combination of polymers. Acta Pharm. The author(s) have not declared any conflict of interests. 58:221-229. Sepici A, Gurbiz I, Cerik C, Vesilida E (2004). Hypoglycemic effect of myrtic oil in normal and alloxan diabetic rabbits. J. Ethnopharmacol. 93:311-318. REFERENCES Sharma SB, Nasir A, Prablum KM, Murphy PS (2006). Anti- hyperglycemic effects of the fruit pulp of Eugenic janbolana in Attama AA, Nkemnele MO (2005). In vitro evaluation of drug release experimental diabetes mellitus. J. Ethnopharmacol. 104:367-373. from self-micro-emulsifying drug delivery systems using a novel Stepensky D, Friedman M, Srour W, Raz I, Hoffman A (2001). biodegradable homolipid from Capra hircus. Int. J. Pharm. 304: 4-10. Preclinical evaluation of pharmacokinetic-pharmacodynamic rationale Attama AA, Okafor CE, Builders PF, Okorie O (2009). Formulation and for oral CR metformin formulation. J. Control Release 71:107-115. in vitro evaluation of a PEGylated microscopic lipospheres delivery Wang Y (2006). Pluronic F127 gel effectively controls the burst release system for ceftriaxone sodium. Drug Deliv. 16:448-457. of drug from PLGA microspheres. Pharmazie 61:367-368. Attama AA, Reichl S, Müller-Goymann CC (2008). Diclofenac sodium delivery to the eye: in vitro evaluation of novel solid lipid nanoparticle formulation using human cornea construct. Int. J. Pharm. 355:307- 313. Builders PF, Kunle OO, Adikwu MU (2008). Preparation and characterization of mucinated agarose: a mucin-agarose physical crosslink. Int. J. Pharm. 356:174-180. Defang O, Shufang N, Wei L (2005). In vitro and in vivo evaluation of two extended Release preparations of combination metformin and glipizide. Drug Dev. Ind. Pharm. 31:677-685. Gao P, Guyton ME, Huang T, Bauer JM, Stefanski KJ, Lu Q (2004). Enhanced oral bioavailability of a poorly water soluble drug PNU- 91325 by super saturable formulations. Drug Dev. Ind. Pharm. 30:221-229. Luan X (2006). Key parameters affecting the initial release (burst) and encapsulation efficiency of peptide-containing poly(lactide-co- glycolide) microparticles. Int. J. Pharm. 324:168-175. Momoh MA, Adedokun MO, Adikwu MU, Kenechukwu FC, Ibezim EC, Ugwoke EE (2013). Design, characterization and evaluation of PEGylated-mucin for oral delivery of metformin hydrochloride. Afr. J. Pharm. Pharmacol. 7:347-355. Momoh MA, Adikwu MU, Ibezim EC, Attama AA (2011). Effect of metformin and Vernonia amygdalina leaf extract loaded PEGylated- mucin formulations on haematological, kidney and liver indices of healthy and diabetic rats. J. Pharm. Res. 4:3455-3459.

Vol. 13(50), pp. 4577-4585, 10 December, 2014 DOI: 10.5897/AJB2014.14248 Article Number: 082610448982 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2014 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB

Full Length Research Paper

Differential responses of growth, chlorophyll content, lipid peroxidation and accumulation of compatible solutes to salt stress in peanut (Arachis hypogaea L.) cultivars

Meguekam Liliane Tekam1, Taffouo Victor Désiré2*, Grigore Marius-Nicusor3, Zamfirache Magdalena Maria3, Youmbi Emmanuel4 and Amougou Akoa4

1Department of Biological Sciences, Higher`s Teacher Training College, University of Yaoundé I, 47 Yaoundé - Cameroon. 2Department of Botany, Faculty of Sciences, University of Douala, 24157 Douala - Cameroon. 3Department of Plant Biology, Faculty of Sciences, University of Yaoundé I, 812 Yaoundé - Cameroon. 4Faculty of Biology, University of Alexandru Ioan Cuza de Iaşi, Street, Carol I, nr., 11/ 700506, Romania.

Received 15 October, 2014; Accepted 24 November, 2014

The present study was aimed to compare differential responses of growth, chlorophyll content, lipid peroxidation and accumulation of compatible solutes in peanut (Arachis hypogaea L.) cultivars: Fleur 11 (salt-tolerant), Mbiah and PC 79-79 (moderately-tolerant) and Vanda (salt-sensitive) at the vegetative growth stage, under greenhouse conditions, in the presence of 0, 50, 100 or 200 mM NaCl. The root dry weight (RDW) and shoot dry weight (SDW) of cv. Vanda decreased significantly (p< 0.05) in salt-treated plants than those of cvs. Fleur 11, Mbiah and PC 79-79. The SDW reduction was notably noted at 100 mM NaCl in cvs. Mbiah and PC 79-79, while cv. Fleur 11 showed significantly (P<0.05) decrease in salt- treated plants only at 200 mM NaCl but had higher SDW accumulation than others. The leaf chlorophyll content increased in cvs. Fleur 11 and PC 79-79 and decreased in cv. Vanda with increasing NaCl levels. Proline (PRO) and glycine betaine (GB) contents significantly (p<0.05) increase, for all cultivars, in the stressed plants with the highest quantity in Fleur 11 and the lowest in Vanda. Malondialdehyde levels increased under salt stress in the leaves of cv. Vanda but decreased in cvsMbiah, PC 79-79 and Fleur 11 at 100 and 200 mM, respectively. Total phenolic content increased significantly (p < 0.05) at 200 mM NaCl in the leaves of cv. Vanda than others, while cv. Fleur 11 showed the lowest increase. The salt- tolerant cv. Fleur 11 exhibits a better protection mechanism against oxidative damage caused by salt stress maintaining a higher accumulation of PRO and GB than others. Higher PRO and GB accumulation in the leaves may be regarded as potential biochemical indicator for earlier selection of salt tolerant peanut and targets for improvement through transgenic approaches.

Keywords:Arachis hypogaea, glycine betaine, growth, lipid peroxidation, proline, salt stress

INTRODUCTION

Salinity stress is the most limiting factor in agricultural excessive sodium affects plant growth of many sensitive productivity in arid and semi-arid regions of the world species which comprised most of crops (Hossein and (Horie and Schroeder, 2004). In saline environment Fatemeh, 2012; Rameeh et al., 2012). The morphological, 4578 Afr. J. Biotechnol.

physiological and biochemical attributes of plants is any interaction that a plant can have with its environment altered by salinity thus limiting its growth and (Waterman and Mole, 1994). These compounds have development (Mudgal et al., 2010). Low external water been implicated to stress resistance against biotic and potential, ion toxicity and interference with the uptake of abiotic factors (Bergmann et al., 1994). Total phenolic nutrients are a range of mechanisms that inhibit plant (TP) accumulation could be a cellular adaptive growth due to salinity (Munns et al., 1995). The mechanism for scavenging oxygen free radicals during responses of plants to high soil salinity and the stress (Mohamed and Aly, 2008). The increased mechanisms of salt tolerance have been largely synthesis of PRO, TP and the antioxidant activity in dill discussed (Ruan et al., 2010; Grigore et al., 2011). seedlings exhibited a protective mechanism against the Compartmentation of ions in vacuoles and accumulation cellular structures from oxidative damage (Zahra et al., of compatible solutes in the cytoplasm are commonly 2012). proposing mechanisms of salt tolerance species (Munns, Grain legumes provide large amounts of high quality 2002). The basic mechanisms of salt tolerance in proteins which contain relatively more of the essential halophytes seem to be mostly dependent in their amino acids not supplied by cereals in which the content capacity to sequester toxic ions (Na+, Cl-) in the vacuoles of lysine and tryptophan are relatively small (Kay, 1979). and to accumulate compatible osmotica in the cytoplasm Peanuts are essential sources of fat (34 to 54%) (Le Rudulier, 2005; Grigore et al., 2011). The compatible (Nyabyenda, 2005). Legumes intervene in crop rotation solutes accumulation that are mostly seen in plants are systems and participate in biological nitrogen fixation proline (PRO) and glycine betaine (GB), but other (Delgado et al., 1994). The selection of tolerant cultivars osmolytes can be stored at high concentrations in some can be done efficiently in cultivated saline environments, species (Girija et al., 2002). An increase in salinity and thus salt tolerance mechanisms potential can be increases PRO as an adaptative change in metabolism identified within plant species which is becoming an pattern (Mudgal et al., 2010). Thylakoid and plasma increasing research priority in many countries. It is membrane integrity are protected by GB after exposure important to make a call to the ecophysiological approach to saline solutions (Rhodes et al., 1987). which can constitute an attenuation of the effect of the Peanut salt-tolerant cultivars accumulate the highest soil’s salinity on the cultivated plant performances quantity of GB while moderately-tolerant cultivar stored (Mekhaldi et al., 2008). This would lead to the search of intermediate amount and sensitive low quantity (Girija et tolerant species or varieties of plant thus imposing a al., 2002). Thus, the accumulation of osmo-protectants in mastery of the knowledge of mechanisms to their tissues of plants growing in arid or semi-arid lands, may adaptation to salinity. exhibit more tolerance to salt stress (Munns, 2002). Salt The present study was aimed to compare differential stress can lead to stomatal closure, which reduces CO2 responses of growth, physiological and biochemical availability in the leaves and inhibits carbon fixation, characteristics in peanut cultivars differing in salt exposing chloroplasts to excessive excitation energy, tolerance at the first vegetative growth stage and which in turn could increase the generation of Reactive determine biochemical indicators which could serve as Oxygen Species (ROS) and induce oxidative stress early selection criteria for tolerance of salt in peanut. (Parvaiz and Satyawati, 2008). Plants have developed a series of enzymatic and non-enzymatic detoxification MATERIALS AND METHODS systems to counteract ROS, and protect cells from oxidative damage (Sairam and Tyagi, 2004). The assess- Plant material, growth and stress conditions ment of cell membrane stability is an appropriate technique to screen plants under saline condition (Munns Experiments were performed using seeds of four peanut (Arachis et al., 2006). Salt stress increased lipid peroxidation or hypogaea L.) cultivars differing in salt tolerance; cv. Fleur 11 (salt- induced oxidative stress in plant tissues (Hernandez et tolerant), cvs. Mbiah and PC 79-79 (moderately-tolerant) and cv. Vanda (salt-sensitive). Mbiah and Vanda were provided by the al., 1993). Malondialdehyde (MDA) has been known as breeding program of the Agronomic Institute for Research and the end product of peroxidation of membrane lipids Development of Cameroon. Fleur 11 and Pc 79-79 were obtained (Sajedi et al., 2011). Increase in the level of MDA, from Senegalese Institute of Agronomic Research. Germination produced during peroxidation of membrane lipids, is often trials were conducted in 9 cm sterile Petri dishes lined with used as an indicator of oxidative damage (Azad et al., Whatman No.1 filter papers and moistened with 10 mL of distilled water. After seed surface sterilization with 70% (v/v) ethanol 2012). The salt stress was able to induce excessive solution for 15 min, followed by rinsing with distilled water, seeds generation of MDA in the root and leaf of maize seedlings were sowed in Petri dishes and placed in seed germinator at 26°C (Azad et al., 2012). Phenolic compounds are a large for 5 d. After germination, the plants were sown in pots with group of secondary metabolites, which can play a role in sterilized sand in a greenhouse (26/23°C light/dark and 51 to 61%

*Corresponding author. E-mail: [email protected].

Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Tekam et al. 4579

hygrometry), located at the Alexandru Ioan Cuza de Iaşi University, 1. [(Abs532+TBA-Abs600+TBA). (Abs532-TBA- Abs600-TBA)] = A Romania, from June to September, 2011 and March to July, 2012. 2. [(Abs440+TBA-Abs600+TBA) 0.0571] = B The pots were arranged in a complete randomized design with five 3. MDA equivalents (nmolmL-1) = (A-B/157,000)106 replicates. One plant was grown in the middle of each pot. Each Results were expressed as MDA equivalents (nmol g-1FW) and cultivar had 20 pots divided into four groups. Each group, with five represent the mean of five samples. replicates, were fertilized every two days with the nutrient solution Total phenolic (TP) content was assayed according to method of (Boldor et al., 1983) containing 49.2% Ca(CO2)2, 13.6% KH2PO4, Marigo (1973) using the Folin-Ciocalteu reagent. 1 g fresh tissues 6.0% MgSO4, 7.5% KCl and 2.5% FeCl3 added with one of the four leaf was ground at 4°C in 3 mL of 0.1 N HCl for 20 min. After NaCl concentration levels (0, 50, 100 and 200 mM) for one month. incubation the homogenate was centrifuged at 6,000 g for 40 min. Plants were harvested for physiological and biochemical analysis at The supernatant was collected, the pellet re-suspended in 3 mL of 35 days after sowing for a total of 20 plants from each treatment. 0.1 N HCl and centrifuged as previously. The two supernatant are mixed and constitute the crude extract of soluble phenol. The reaction mixture containing 15 µL of extract, 100 µL Folin-Ciocalteu Plant growth parameters determination reagents, 0.5 mL of 20% Na2CO3 was incubated at 40°C for 20 min and absorbance read at 720 nm with a BECKMAN DU 68 Plant growth (root dry weight, shoot dry weight, stems diameter spectrophotometer. A standard curve was established using -1 (SD) and total leaf area (TLA) was evaluated using twenty plants chlorogenic acid. TP content was expressed as µg g FW. from each cultivar. All tissue parts (leaves, stems, and roots) were separated, and fresh weights of these tissue parts were measured. For the determination of dry weight, these tissue parts were dried at Measurements of the chlorophyll content 65°C for 72 h. SD was measured every week on plants using a caliper. TLA was measured every week and calculated using the Chlorophyll (CHL) content in leaves was estimated after extracting formula described by Kumar et al. (2002): 20 mg of the ground material, following the procedure described by Arnon (1949). Chlorophyll of samples was extracted with 80% TLA (cm2) = L×la×0.80×N×0.662 alkaline acetone (v/v). Full extraction of chlorophyll was achieved when the sample was discoloured. The absorption of the extracts Where L = length of leaf; la = width of leaf and N = total number of was measured at 663 and 645 nm with a BECKMAN DU 68 leaves. spectrophotometer and CHL was calculated using the following formula:

Biochemical parameters determination Total chlorophyll = (20.2 x D645 + 8.02 x D663) x (50/1000) x (100/5) x 1/2 Proline (PRO) content was extracted from fresh leaves according to the method of Bates et al. (1973). Leaves samples (0.5 g) were Where, D = absorbance and expressed as mg g-1FW. homogenized in 10 mL of 3% (w/v) aqueous sulfosalicylic acid to precipitate protein. Samples were centrifuged at 18,000 g for 10 min and supernatant was used for estimation of PRO content. The Statistical analysis reaction mixture consisted of 1 mL acid ninhydrin and 1 mL of glacial acetic acid, which was boiled at 100°C for 1 h. After tubes Results obtained from all the manipulations are expressed as mean cooling in the ice, the products were extracted with 2 mL of toluene ±standard deviation and analyzed using SPSS software. Statistical by vortex mixing and the upper (toluene) phase decanted into a differences between treatment means were established using the glass basin. The absorbance was recorded at 520 nm and the PRO Fisher LSD test at p values < 0.05. Multifactorial ANOVA was used concentration was determined as µg g-1FW using a standard curve. to estimate whether cultivar, salinity level, alone or in interaction, Glycine betaine (GB) content was measured in leaves tissue had a significant influence on the measured parameters. extracts as described by Grieve and Grattan (1983). The plant tissue was finely ground, mechanically shaken with 20 mL deionised water for 24 h at 25°C. The samples were then filtered RESULTS through Miracloth and filtrates were diluted to 1:1 with 2 N H2SO4. Aliquots were kept in centrifuge tubes and cooled in ice water for 1 Growth parameters h. Cold KI-I2 reagent was added and the reactants were gently stirred with a vortex mixture. The tubes were stored at 4°C for 16 h Peanut growth was estimated by measuring root dry and then centrifuged at 10,000 g for 15 min at 0°C. The supernatant was aspirated with a fine glass tube. The periodide crystals were weight (RDW), shoot dry weight (SDW), total leaf area dissolved in 9 mL of 1, 2-dichloroethane. After 2 h, the absorbance (TLA) and stem diameter (SD) (Table 1). A significant was measured at 365 nm using GB as standard. The GB two-way interaction between the factors, salinity level and concentration was expressed as µmol g-1DW. cultivars, was observed for SDW and TLA (Table 1). The Malondialdehyde (MDA) content was determined in leaves using RDW and SDW of cv. Vanda decreased significantly (p< a modified thiobarbituric acid (TBA) assay (Hodges et al., 1999). Samples were homogenized with inert sand in 1:25 (g FW:mL) 0.05) in salt-treated plants, when compared with control 80:20 (v/v) ethanol: water, followed by centrifugation at 3,000 g for plants than those of cvs. Fleur 11, Mbiah and PC 79-79. 10 min. One millilitre of the sample was added to a test tube with 1 The SDW inhibition effect of salt was notably noted at mL of either (1) TBA solution comprised of 20% (w/v) trichloroacetic 100 mM NaCl in cvs. Mbiah and PC 79-79, while cv. acid and 0.01% butylated hydroxytoluene or (2) +TBA solution Fleur 11 showed significantly (p<0.05) decrease in salt- containing the above and 0.65% (w/v) thiobarbituric acid. Samples treated plants only at 200 mM NaCl but had higher SDW were then mixed vigorously, heated at 95°C for 25 min, cooled and centrifuged at 3,000 g for 10 min. Absorbance values were accumulation than others (Table 1). TLA of all cultivars recorded at 440, 532 and 600 nm. MDA equivalents were was negatively affected with increasing levels of salinity calculated in the following manner: (Table 1). At the highest salt concentration (200 mM 4580 Afr. J. Biotechnol.

Table 1. Peanut cultivars growth parameters at different salinity levels. Data are mean ± standard error (n =5).

Salinity level Plant dry weight (g plant-1) Total leaf area Cultivar Stem diameter (cm) (mMNaCl) Shoot Root (cm2 plant-1) 0 0.74±0.04b 0.11±0.01b 0.27±0.01a 39.83±2.33e 50 0.78±0.02a 0.14±0.02a 0.27±0.02a 36.91±2.19f Fleur 11 100 0.75±0.03b 0.14±0.02a 0.26±0.03a 29.74±2.11h 200 0.50±0.02f 0.12±0.01ab 0.25±0.02a 28.79±1.96h

0 0.65±0.02c 0.10±0.03b 0.27±0.02a 64.89±1.55a 50 0.68±0.02b 0.12±0.03ab 0.27±0.02a 58.23±2.67b Mbiah 100 0.58±0.02e 0.14±0.02a 0.26±0.03a 51.96±3.02c 200 0.43±0.01g 0.07±0.03c 0.25±0.01a 44.32±2.23d

0 0.70±0.04c 0.15±0.05a 0.27±0.01a 46.76±1.70d 50 0.67±0.05c 0.14±0.04a 0.27±0.03a 41,69±1.02e PC 79-79 100 0.43±0.03g 0.14±0.02a 0.26±0.04a 35.67±2.09f 200 0.34±0.03h 0.12±0.01ab 0.24±0.02ab 32.58±1.33g

0 0.74±0.01b 015±0.03a 0.26±0.01a 40.98±2.98e 50 0.63±0.03cd 0.06±0.01c 0.25±0.02ab 32.60±1.88g Vanda 100 0.37±0.01h 0.05±0.01c 0.24±0.02ab 26.47±1.44i 200 0.26±0.01i 0.03±0.00cd 0.23±0.01b 23.42±1.12j Cultivar (C) * * ns * Salinity level (S) *** ** * * C x S * ns ns *

Within columns, means followed by the same letter are not significantly different (p< 0.05) by Fisher LSD test; *, **, ***, Significant at p < 0.05, p < 0.01 and p < 0.001 respectively, ns not significant.

NaCl), after four weeks of salt treatment, TLA was salinity treatment (Figure 1b). GB content significantly strongly reduced in cv. Vanda compared to control plants (p<0.05) increased in presence of NaCl in all the cultivars than those of cvs. Fleur 11, Mbiah and Pc 79-79. SD of compared to control plants. This increase was more all cultivars was not affected by salinity levels except for pronounced in cv. Fleur 11 than others, while cv. Vanda Pc 79-79 and Vanda at high salinity level (200 mM) after showed the lowest increase. four weeks of salt treatment (Table 1). In general, plant NaCl salinity induced total phenolic (TP) accumulation growth was influenced by NaCl treatment except for SD in leaves in all cultivars compared to control plants and the magnitude of responses varied according to (Figure 1c) with higher values increasing salt concen- cultivars differing in salt-tolerance (Table 1). trations. TP increased significantly (p < 0.05) at higher salt level (200 mM NaCl) in cv. Vanda than others, while

Biochemical characteristics cv. Fleur 11 showed the lowest increase. A rise in malon- dialdehyde (MDA) content, an indicator of membrane Proline (PRO) content in leaf of control and NaCl damage was observed under NaCl salinity. MDA levels in stressed plants of all peanut cultivars were found at 35 the leaves increased with increasing salinity DAS and results are presented in Figure 1a. The PRO concentrations in cv. Vanda (Figure 1d). Results showed content was significantly increased in the stressed plants that MDA of the cvs. Mbiah and PC 79-79 increased at compared to control plants of all cultivars at all salinity 50 mM also, but decreased at 100 and 200 mM NaCl. levels but differences in PRO accumulation have been MDA levels in the leaves showed no change in cv. Fleur noticed during plant growth between peanut cultivars. 11 at 50 and 100 mM NaCl but decreased at 200 mM The highest increased was observed in cv. Fleur 11 while NaCl. the lowest was found in cv. Vanda; cv. Mbiah maintained higher increase of PRO content than the cv. PC 79-79. Changes in the chlorophyll content Glycine betaine (GB) content was determined in the absence (non-saline control) as well as in presence of Leaf total chlorophyll content (CHL) was substantially NaCl in leaves of all the peanut cultivars after 4 weeks of increased in cvs. Fleur 11 and PC 79-79 with increasing Tekam et al. 4581

Figure 1. (a) Proline, (b) Glycine betaine, (c) Total phenol and (d) Malondialdehyde contents in leaves of peanut cultivars in response to NaCl concentration levels. Data are mean ± standard error (n = 5). Means followed by the same letter are not significantly different (p <0.05) as determined by Fisher LSD test. Bars indicate standard error.

NaCl concentrations (Figure 2). Salt stress caused 2009; Zahra et al., 2012; Navarro et al., 2014). The gradual reduction in CHL content of cv. Vanda but reduction in growth parameters is a consequence of showed no change and decreased in cv. Mbiah at 50 and several physiological responses including modification of 100 mM NaCl, respectively, compared to control plants. ion balance, mineral nutrition, stomatal behaviour and Under stressed conditions, CHL content in cv. Vanda was photosynthetic efficiency also (Rajest et al., 1998; lower than that in other cultivars. Dadkhah, 2011). This is consistent with the reports that NaCl reduces the ability of the plant to take up water, and this leads to slow growth and then, when excessive DISCUSSION amounts of salt entering the transpiration stream will eventually injure cells in the transpiring leaves and this Plant growth may further reduce growth (Munns et al., 2006). In the present study, the growth inhibition effect of salt in growth Depletion in plant growth (RDW and SDW) under saline parameters studied was significantly noted at 50 mM stress in cv. Vanda than cvs. Fleur 11, Mbiah and PC 79- NaCl in cv. Vanda than others, while the growth of cv. 79 after 4 weeks of salt treatment (Table 1) is attributed Fleur 11 was significantly (p < 0.05) affected only at 200 to decreased water uptake followed by limited hydrolysis mM NaCl. These results demonstrate that cv. Vanda, in of food reserves from storage tissue, as well as due to common with certain other bambara groundnut cultivars impaired translocation of food reserves from storage (black seed coat and light red seed coat), is highly tissue to the developing embryo axis (Meneguzzo et al., sensitive to salt with severe effects at 50 mM NaCl 1999). Numerous studies have reported the reduction of (Levitt, 1980). Under salt stress cv. Fleur 11 was RDW and SDW stimulated by salinity (Taffouo et al., observed to have relatively higher tolerance on average 4582 Afr. J. Biotechnol.

Figure 2. Total chlorophyll content in leaves of peanut cultivars in response to NaCl concentration levels. Data are mean ± standard error (n =5). Means followed by the same letter are not significantly different (p <0.05) as determined by Fisher LSD test. Bars indicate standard error.

of all growth parameters than others. Similar obser- tolerant cv. Fleur 11 and salt-sensitive cv. Vanda during vations for plant growth were reported in Ceriops plant growth. The PRO increases with increasing salinity roxburghiana (Rajest et al., 1998) and Vigna subterranea are an adaptative change in metabolism pattern (Mudgal cv. white seed coat (Taffouo et al., 2010a) described as et al., 2010). Previously, it has been reported that Na+ salt-tolerant plant species. In the first phase of a biphasic and Cl- are efficient osmolytes for osmotic adjustment model of growth response to salinity, the vegetative and sequestered in the vacuole of a cell in salt-tolerant growth is reduced by a decrease in a soil water potential species, the osmotic balance of the cytoplasm is ensured due to water stress effect and may be regulated by by an active synthesis of the organic and soluble inhibitory signals from the roots (Munns et al., 1995). compounds (Le Rudulier, 2005; Grigore et al., 2011). This synthesis of the PRO is a mechanism of stress resistance (Greenway and Munns, 1980), because its Biochemical characteristics accumulation contributes to the acquisition of resistance by maintaining the cell turgor in many species which is The result of the present study shows that the proline responsible for the osmotic adjustment in susceptible (PRO) content was significantly (p < 0.05) increased in plants under stress (Meloni et al., 2004). The species the stressed plants compared to control plants of all growing in arid or semi-arid lands, may exhibit more cultivars at all salinity levels but differences in PRO tolerance to salt stress, because of the accumulation of accumulation have been noticed during plant growth osmo-protectants in their tissues (Munns, 2002). between peanut cultivars (Figure 1a). The highest In the present investigation, it has been noticed that the increase, which was observed at 200 mM NaCl in cv. highest accumulation of glycine betaine (GB) was found Fleur 11, is consistent with numerous studies which in salt-tolerant cv. Fleur 11 than others, while the lowest found accumulation of PRO in salt tolerant plants was observed in salt-sensitive cv. Vanda (Figure 1b). GB exposed to salt stress (Sivaramakrishnan et al., 1988; is one of many nitrogenous osmolytes accumulated Meloni et al., 2004). A positive correlation between under osmotic stress conditions in salt-tolerant plants magnitude of PRO accumulation and salt tolerance has (Girija et al., 2002). Numerous studies reported that the been suggested as an index to determine salt tolerance accumulation of GB was found to be high in salt-tolerant potentials between cultivars (Ramanjulu and Sudhakar, cultivar (Rhodes et al., 1987; Giridara Kumar et al., 2001; Giridara Kumar et al., 2003; Grigore et al., 2011). 2003), while salt-sensitive cultivar exhibited a low By contrast, it has been also reported that the salt magnitude of GB accumulation (Hitz and Hanson, 1980). sensitive cultivars accumulated significantly higher levels GB preserves thylakoid and plasma membrane integrity of PRO compared to tolerant ones (Lutts et al., 1999; of Zea mays after exposure to saline solutions (Rhodes Vaidyanathan et al., 2003). In this study, we report a et al., 1987). Thus, the accumulation of osmo-protectants significant difference in PRO accumulation between salt- in tissues of plants growing in arid or semi-arid lands, may Tekam et al. 4583

exhibit more tolerance to salt stress (Munns, 2002). Salt contrast, the CHL content was substantially increased in stress can lead to stomatal closure, which reduces CO2 Fleur 11 and PC 79-79 with increasing NaCl concen- availability in the leaves and inhibits carbon fixation, trations. Similar observations have been reported by exposing chloroplasts to excessive excitation energy, Robinson et al. (1983) and Morales et al. (1992) in salt- which in turn could increase the generation of Reactive stressed spinach and barley, respectively. Moreover, the Oxygen Species (ROS) and induce oxidative stress cultivars Fleur 11 and PC 79-79 screened for their salt (Parvaiz and Satyawati, 2008). The osmo-protectants tolerance in this study were grown under natural field that accumulate most commonly are PRO and GB, conditions of Bambey (Senegalese) and were probably although other molecules can accumulate to high exposed to different environmental constraints. The concentrations in certain species (Girija et al., 2002). interaction between these stresses could be taken as a NaCl salinity induced total phenolic content (TP) part of the adaptive mechanisms of plants to survive accumulation in leaves in all cultivars compared to control under saline conditions and high temperatures plants (Figure 1c). TP accumulation in leaves under salt (Giannakoula et al., 2012). stress could be a cellular adaptative mechanism for scavenging oxygen free radicals during stress conditions (Mohamed and Aly, 2008). Numerous studies have Conclusion reported that TP production is stimulated by NaCl (Hanen et al., 2008; Zahra et al., 2012). Antioxidants prevent lipid In general, the results of this study showed that salt oxidation and can act in different ways, including stress caused a serious decrease in plant growth by decreasing oxygen concentrations, scavenging initiating means of reduced RDW, SDW and TLA due to ionic radicals, and binding metal ions to prevent initiating toxicity and decrease osmotic potential in all peanut radical formation (Dorman et al., 2003). cultivars but the magnitude of responses varied accor- In this study, we present the evidence that salt stress is ding to cultivars. Higher osmolyte accumulation, espe- able to produce excessive quantity of malondialdehyde cially proline and glycine betaine was found in the salt (MDA) in the leaf of cv. Vanda plants than others cultivars tolerant cultivar (Fleur 11) whereas the lower in salt- (Figure 1d). Increase in the level of MDA, produced sensitive one (Vanda). The salt stress was able to during peroxidation of membrane lipids, is often used as excessively generate MDA in the leaves of Vanda plants, an indicator of oxidative damage (Azad et al., 2012). Free that is, an indicator of oxidative damage. MDA levels radical-induced peroxidation of lipid membranes is a increased under salt stress in the leaves of cv. Vanda but reflection of stress-induced damage at the cellular level decreased in cvs Mbiah, PC 79-79 and Fleur 11 at 100 (Nagest and Devaraj, 2008). As a sequel, lipid and 200 mM, respectively. These results suggest that peroxidation products such as MDA will accumulate and Fleur 11 exhibits a better protection mechanism against severe membrane damage will inevitably occur (Azad et oxidative damage caused by salt stress due to its higher al., 2012). Previously, it has been reported that there was induced activities of antioxidant enzymes and osmolyte an improvement in MDA content in leaves of salt- accumulation than Mbiah, PC 79-79 and Vanda. Fleur 11 sensitive Vigna radiata and Plantago media, but can tolerate moderate saline conditions owing to better decreased at 200 mM in salt-tolerant Plantago maritime antioxidant system. It seems that the evaluation of under salinity (Sekmen et al., 2007; Saha et al., 2010). osmolyte accumulation and antioxidant system is useful MDA level and cell membrane damage increased under for assessment of salinity tolerance of peanut cultivars. salt stress condition in salt-sensitive cv. Vanda because of elevating of ROS production (Azad et al., 2012). MDA concentration decreased at 200 mM in salt-tolerantcv. Conflict of Interests Fleur 11. These results suggested that the cv. Fleur 11 The author(s) have not declared any conflict of interests. showed a better protection mechanism against oxidative damage caused by salt stress by its higher induced activities of antioxidant enzymes than the salt-sensitive ACKNOWLEDGEMENTS cv. Vanda (Sekmen et al., 2007). Liliane Tekam Meguekam has received a half-time doctoral fellowship from AUF-Eugen Ionescu. This work Changes in the chlorophyll content was financially supported by the AUF-Eugen Ionescu 2011 and 2012 scholarship research programme. Salinity decreased the chlorophyll (CHL) content in salt- sensitive cv. Vanda (Figure 2) leaves, which is in accordance with Taffouo et al. (2010a; b), and REFERENCES Giannakoula et al. (2012). This effect of salt was attributed to a salt-induced weakening of protein-pigment- Arnon DI (1949). Copper enzymes in isolated chloroplasts. Polyphenylodase in Beta vulgaris. 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Vol. 13(50), pp. 4586-4593, 10 December, 2014 DOI: 10.5897/AJB2014.13995 Article Number: AA973C148986 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2014 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB

Full Length Research Paper

Antidiabetic activity and acute toxicity evaluation of aqueous leaf extract of Vernonia amygdalina

M. A. Momoh1, M. O. Adedokun2*, A. T. Mora3 and A. A. Agboke2

1Drug Delivery Research Unit, Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka, Nigeria. 2Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, University of Uyo, Uyo, Nigeria. 3Department of Clinical Pharmacy and Pharmacy Management, Faculty of Pharmaceutical Sciences, Kaduna State University, Kaduna, Nigeria.

Received 19 June, 2014; Accepted 28 November, 2014

The present study was aimed to explore the therapeutic dose for antidiabetic activity and toxicological evaluation of Vernonia amygdalina (Va) aqueous extract in alloxan-induced diabetic rats. Aqueous extract of leaves of Va was administered to alloxan induced diabetic rats in a dose of 150 and 300 mg/kg (orally) daily for 14 days. After this period, blood glucose level, haematological parameters and liver enzymes activities were evaluated. Also evaluated were food and water intake, urine output and body weight of the animals. The toxic effect of the aqueous leaves extract was evaluated by determining the LD50. Oral administration of the extract at graded doses of 150 and 300 mg/kg body weight showed significant decrease in the blood glucose level in diabetic rats (P<0.05). The defects in haematological and enzyme activities in the diabetic animals were restored. We concluded that at doses of 150 and 300 mg/kg, Va extract exhibited anti-hyperglycemic effect and showed statistically significant differences (p<0.05) in all the parameters evaluated. There was a significant improvement (P<0.05) in the weight of the diabetic rats, food intake and a decrease in the urine output. This study illustrates the potential usefulness of this extract and its safety on a vital organ of the body.

Key words: Alloxan, Vernonia amygdalina, diabetics, aqueous extract, rats, safety.

INTRODUCTION

Diabetes mellitus is the clinical condition of metabolic 2009). There are two types of diabetes: Type 1 diabetes, diseases characterized by hyperglycemia resulting from previously called insulin-dependent diabetes mellitus or defects in insulin secretion, insulin action, or both juvenile-onset diabetes which accounts for up to 10% of (Obimba et al., 2014; Ozougwu et al., 2013). The chronic all diagnosed diabetes which is caused by lack of insulin hyperglycemia caused by diabetes is associated with secretion by β-cells of the pancreas. Type 2 diabetes was long term damage, dysfunction and failure of various previously called non-insulin-dependent diabetes mellitus organs, especially the eyes, kidneys, nerves, heart and or adult-onset diabetes which may account for between blood vessels (International Diabetes Federation (IDF), 90 to 95% of all diagnosed cases of diabetes (Obimba et

*Corresponding author. E-mail: [email protected]. Tel. +234 (0) 8034396937.

Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Momoh et al. 4587

al., 2014; Ozougwu et al., 2013). Type 2 diabetes mellitus Nsukka, Enugu State, Nigeria and authenticated at Bioresources has become a significant health problem in both Development and Conservation Program (BDCP), Nsukka, Nigeria developed and developing countries. IDF has estimated where voucher samples were kept for reference. Healthy fresh leaves were sorted, washed to remove debris and dust particles that 285 million people around the world have diabetes. without squeezing and then air-dried for seven days (Momoh et al., This total is expected to rise to 438 million within 20 years 2013). The dried leaves were milled into a coarse powder from (World Health Organization, (WHO), 2003). The non- which 25 g was soaked with 500 ml of distilled water in a beaker pharmacological means (diet and exercise) and/or the and the mixture shaken on the laboratory bench for 24 h before pharmacological means (insulin and oral hypogly- filtering (Whatman No. 1 filter paper). The filtrate was evaporated using a rotary evaporator to obtain a solid residue (5.0 g) called the caemics) have been used in the management of diabetes aqueous extract which correspond to a percentage yield of 20%. mellitus. The obvious limitations of oral administration of This procedure was carried out in quadruplicate to obtain a total of antidiabetic agents, especially with insulin, have 20.0 g of aqueous extract. Appropriate weights of the residue were necessitated a search for alternatives among the arsenal reconstituted separately in distilled water to give the required doses of herbs available to man (Edwin et al., 2006). A large of 100, 150 and 300 mg/kg body weight used in the present study. number of herbs, spices and other plant materials have been described for the management of diabetes Phytochemical tests on the extract throughout the world (Ojiako and Nwanjo, 2006). It was in this light that the World Health Assembly adopted among Some phytochemical tests were carried out on the V. amygdalina its resolutions the support of national traditional medicine extract to determine the presence of saponins, flavonoids, program, drawing attention to herbal medicines as being alkaloids, proteins, carbohydrates, glycosides and tannins using standard methods (Sofowora, 2006). of great importance to the health of individuals and communities (Emeje et al., 2011). Plants have always been an exemplary source of drugs Preliminary evaluation of hypoglycemic activity of extract in and many of the currently available drugs have been normal healthy albino rats derived from plants. Vernonia amygdalina (family, Twenty normal Albino rats, fasted overnight, were divided into four Compositae) is a valuable shrub that is wide spread in groups of five rats each, and used in the experiment. Group A East and West African countries (Izevbigie, 2003). In served as control, and received the vehicle (distilled water only) Nigeria, it is commonly known as "bitter leaf" because the while animals of groups B, C and D received variable doses of 100, leaves and stem have a bitter taste when chewed. Its 150 and 300 mg/kg, respectively, of extract suspended in distilled leaves were used as a popular vegetable for soups water. The blood samples were collected from the lateral vein of the tail of the animals at 2, 4, 6, 8 and 12 h after administering the particularly among the ethnic groups in Nigeria. The extract and the sugar levels were measured. These were compared organic fraction extracts of the plant was shown to to the initial blood glucose level. possess cytotoxic effects on human carcinoma cells of the nasopharynx (Okolie et al., 2008). It is effective against gastrointestinal disorders (Akah and Ekekwe, Experimental design

1995) including amoebic dysentery (Moundipa et al., Albino rats (180 to 220 g) of either sex were used for the study. The 2005) possibly due to its antimicrobial and antiparasitic animals were procured from the animal house of the Faculty of activities (Gray et al., 2000; Muraina et al., 2010). Its anti- Veterinary Medicine, University of Nigeria, Nsukka, and were thrombotic and anticoagulant properties have also been allowed to acclimatize to the new environment for a period of two evaluated (Akah and Ekekwe, 1995). Oral administration weeks prior to the study. Rats housed in cages were kept in a room with controlled temperature (20 to 22°C) and a 12 h day-night cycle. of the aqueous leaf extract of the plant was found to Fresh solution of alloxan monohydrate (Sigma, USA) was prepared relieve pain and to lower body temperature (Gray et al., just prior to injection. A stock solution of it was made by dissolving 2000). V. amygdalinais are also used traditionally as an all in normal saline (0.9% w/v NaCl) at a concentration of 100 antidiabetic remedy in Nigeria by the traditional herbal mg/mL (Dhasarathan and Theriappan, 2011). After being starved practitioners (Muraina et al., 2010; Park, 2007; Atawodi, overnight, a volume equivalent to 1 mL of the stock solution was 2005). Several workers have provided some scientific administered intra-peritoneally to the animals after which the blood glucose levels were measured for days using a glucometer (ACCU- proofs in support of this practice (Abdulazeez et al., 2013; CHEK, Roche, USA). Food consumption, water intake and urine Iwuji et al., 2010) but, available scientific reports on the volume were similarly evaluated daily. The rats were considered actual dose needed for management of diabetic diabetic when the blood glucose level was raised above 200 conditions and the possible mechanism of action of this mg/100 mL of blood (Cetto et al., 2000) after 3 days post-alloxan plant were not detailed enough. Thus, we conducted this administration. All of the animal experiments adhered to the Ethical study to further investigate the effective dose for anti- Guidelines of Animal Care and Use Committee (Research Ethics Committee) of Department of Pharmaceutics, Faculty of hyperglycemic effect and to a less extent its safety on a Pharmaceutical Sciences, University of Nigeria, Nsukka, Nigeria. vital organ of the body.

Oral administration of graded dose of extract to diabetic rats MATERIALS AND METHODS

Preparation of crude extract Twenty (20) Albino rats were used for the study. They were divided into four groups of five rats per group. Based on an initial Pesticide-free fresh leaves of V. amygdalina were collected from normoglycemic study, 150 and 300 mg doses were selected for the 4588 Afr. J. Biotechnol.

Table 1. Phytochemical analysis of Vernonia amygdalina extract.

Test Present/absence Inference Alkaloids + Present Cyanogenic glycosides - Absent Cardiac glycosides ++ Present Anthracene glycosides - Absent Steroidal glycosides ++ Present Saponins + Present Tannins ++ Present Flavonoids + Present Proteins + Present Carbohydrates + Present

+ indicates presence of photochemical secondary metabolite; - indicates absence of phytochemical secondary metabolite.

antidiabetic evaluation. The first group marked A, received distilled integrity. Using a 3.2 mL automated pipette serum was dropped on water (negative control, 1.5 mL orally). The groups marked B and C the sample spot of each LFT parameter strips (ALP) Aspartate and received the extract at 150 and 300 mg dispersed in distilled water Alanine aminotransferases strip) and analysed using a Reflotron- (1.5 mL orally), respectively, while that marked D received Plus machine (Model: SN747461). glibenclamide dispersed in distilled water (2.0 mg/Kg orally). The extract was administered once daily by gastric intubation for 14 consecutive days. Statistical analysis

Statistical analysis was performed using SPSS statistical package. Acute toxicity evaluation Mean and standard errors for all data were calculated. For batch comparisons, the Student's t-test was used to determine statistically The acute toxicity studies were carried out based on Lorke's significant differences at P≤0.05. method (1983) (with slight modifications) on adult male Wastar rats of an average weight of 220 g. The animals were housed in air- conditioned quarters under a photoperiod schedule of 12 h light/12 RESULTS h dark. They were fed on standard animal pellets and had free access to water ad libitum. The rats were randomized into two groups (i and ii) of five rats each and were orally administered The results from the study showed that the aqueous leaf graded doses of the extract 3,000 and 6,000 mg/kg body weight, extract of Va extract was positive for alkaloids, saponins, respectively. The doses were 20 times the most effective dose of flavonoids, phenolics, tannins, cardiac glycosides and the aqueous extract of V. amygdalina used in the antidiabetic steroids (Table 1). evaluation. They were observed for signs of toxicity that included paw-licking, stretching, response to stimuli and mortality for the first 4 h and thereafter daily for seven days. Food consumption, water intake and urine output were also examined for 24 h. The lethal Effect of extract on fasting blood glucose level of dose was calculated as the geometric mean of doses that caused 0 normal healthy rats and 100% mortality, respectively. Results in Table 2 reveal the hypoglycemic effect of

Effect on liver enzymes and haematological parameters graded doses of aqueous extract of V. amygdalina leaves on blood glucose level (BGL) of normal rats. Rats treated Haematological parameters with 300 mg/kg of extract showed a maximum fall of 2.0% in BGL at 8 h of oral administration, whereas reduction of Haematological analysis was performed using an automatic less than 2.0% were observed with the doses of 100 and haematological analyzer (Abacus Junior, Germany). Haemoglobin 150 mg/ kg, respectively, at 12 h of administration. (Hb) count, total white blood corpuscles (WBC) and packed cell volume (PCV) were specifically determined. Statistically, the extract produced no significant decrease on the BGL of normoglycemic (non-diabetic) rats (P>0.05). Liver enzymes activity

A 3 ml volume of blood collected in a plain bottle or serum extractor Effect on the pre-and post polytriads symptoms and was used for the study. The blood was allowed to stand in an undisturbed bench for 1 h away from sunlight; this was followed by on body weight spinning for 5 min. Serum was separated from the clotted red cells, and the resulting supernatant used for the assessment of liver As shown in Table 3, there were significant differences in Momoh et al. 4589

Table 2. Effect of graded doses of aqueous extract of Vernonia amygdalina leaves on normoglycemic rats (mean ± S.D, n=3).

Blood glucose level (%) at times (h) Batch/treatment Dose mg 0 2 4 6 8 12 A (negative control) DW 100 99.8±0.2 101±0.2 103±0.2 100±0.0 102±0.3 B (extract) 100 100 99.1±0.1 98.0±0.1 97.0±0.3 99.0±0.2 98.9±0.0 C (extract) 150 100 95.4±0.0 98.7±0.4 98.0±0.4 98.0±0.1 100±0.1 D (extract) 300 100 99.1±0.2 98.3±0.2 99.4±0.3 98.0±0.2 98±0.3

Table 3. Effect on polytriad symptoms and on body weight before and after treatment with the extract in normo and hyperglycemic rats.

Before treatment After treatment with extract Parameter Normoglycemic Diabetic Normoglycemic Diabetic Urine output (ml) 6.20±1.2 97.1±1.4a* 8.10 ± 1.2 8.6 ± 0.2a* Weight (g) 180 ±0.4** 114± 0.2a* 182 ± 0.4** 176 ± 0.2a* Food intake (g) 23.0±0.6 83.0±2.3a* 24.0 ± 0.1 27.0 ± 2.3a* Water intake (ml) 21.0 ±1.3* 123 ±1.2a* 23.4 ± 0.1* 25 ± 1.2a*

a* Indicates significant difference at p < 0.05 among the diabetic treated group, and ** values were not significant (P>0.05) when compared to non-diabetic before and after treatment. All the treated diabetic rats showed significant improvement (P>0.05) compared to the nornoglycemic rats before and after. Values are mean ± SD (n = 3).

all the parameters evaluated (water and food intake, thinness of diabetic rats, the rats also maintained a quiet urine volume output, and weight gain) between the look and showed a slow response to external stimuli, that groups of diabetic and non-diabetic animals. Studies is, touch. All these observations were restored in all the have shown that food intake, urine output and decrease treated groups regardless of the concentration of the in body weight are associated to diabetes conditions due extract administered. However, the recovery rate was to impaired metabolic pathway that greatly affected the dose-dependent. physiological wellbeing of the body. In this research, aqueous extract of Va administered to the diabetes groups successfully restored the anomalies comparably In vivo experiment to the control normoglycemic rats. The food consumption of the diabetic rats increased approximately 15%, As shown in Figure 1, V. amygdalina extract orally although these animals showed less body weight (weight administered in diabetic rats allowed a significant = 114.0 g) compared to the group of non-diabetic animals decrease of glycemia compared to rats treated with water (body weight = 180.2 g). Treatment of the diabetic alone. The rats that received DW continued to have animals with Va extract led to a significant change elevated blood glucose levels within the first 4 days, (P>0.05) in their consumption of food and water as well which may be due to force feeding as this can increase as the urine output and gain in body weight, comparable the glucose level. The decrease in the blood glucose to the untreated non-diabetic rats (before administration level that was observed was due to the fasting conditions of the extract). After the administration of the extract, which caused a decrease in the blood glucose level. The there was a significant increase (P<0.05) in the body extract (150 and 300 mg) dose-dependently lowered the weight of the diabetic group comparable to the body blood glucose levels of the rats. Maximum blood glucose weight of the control group. However, there were no lowering (100 to 51%) was encountered in group C that significant change (P>0.05) in the urine output, food was treated with 300 mg of the extract on the 6th and 12th intake and gain in the body weight of the normoglycemic day, and was comparable to the blood glucose reduction group after treatment with the aqueous extract (Table 3). (100 to 54%) encountered in the group received a The results clearly indicate that the diabetic conditions standard drug (glibenclamide) at the 4th days and later which cause an increase of food and water consumption continued to show a slight increase throughout the period and urine output, which ultimately lead to weight loss of this investigation (Figure 1). Although, it was observed were generally reversed after the treatment. On physical that the standard drug showed relatively more anti- examination, the changes in non-diabetic and diabetic glycemic effect within 4 days, the extract was able to rats were apparently distinctive because, apart from the maintain a persistent steady decrease in BGL in the 4590 Afr. J. Biotechnol.

Figure 1. Blood glucose levels of alloxan-induced diabetic rats after oral administration of graded doses of the extract: (A) = distilled water (negative control, (B) = 150 mg extract, (C) = 300 mg extract and (D) = Glibenclamide 2.0 mg/kg (positive control).

Table 4. Effect of extract on haematology and liver enzymes activities (values = ±SD).

Haematological parameter Liver enzyme activity (U/L) Group Dose (mg) PCV % WBC (x 109) Hb g/dl AST ALT ALP A (DC) DW 35.3±0.21 8.4.3±0.2 13.6±0.11 132.9±0.21 201.4±0.11 239±0.31 B 100 57.0±0.14 8.9.0±0.3 16.1±0.13 101.0±0.11 104.0±0.21 219±0.14 C 150 64.2±0.11 8.5.0±0.1 17±0.12 97.1±0.31 94.0±0.13 179±0.21 D 300 65.0±0.12 8.5.0±0.2 17.2±0.11 111.0±0.14 64.0±0.11 181±0.15 E DW 69.01±0.22 7.40±0.5 20.1±0.21 62.9±0.21 41.1±0.11 142.1±0.21

DC=Diabetes control; DW= Distilled water; NC= non-diabetes control; B, C and D tests batches at different concentrations. (n = 3); ±SD = standard deviation.

glycemic state, which is one of the desired effects in the of this plant should be evaluated before proceeding to the clinical management of diabetic conditions. formulation proper and also to provide the scientific justification for the medicinal utilization of this plant. The result of the haematological evaluation on the extract Effect of the extract on haematology and liver based on the evaluated parameters (PCV, WBC and Hb) enzymes showed no significant changes (P>0.05) in the hematological and liver enzymes activities as shown in Previous studies have shown that the investigation of the Table 4. Diabetic conditions resulted in the decrease of acute toxicity is the first step in the toxicological study of the haematological parameters such PCV, Hb and WBC an unknown substance. Although, the plant in question due to glycosylation that occurred as a result of excess has been in use as a source of food in Nigeria and many sugar in the blood as seen in the diabetic control (DC) other African countries, it is very important that the safety group. Momoh et al. 4591

DISCUSSION experimental results, it could be suggested that, the extract exhibited dose dependent glucose lowering Throughout the world, diabetes is the fastest growing effects. The result of glucose lowering potentials of the metabolic disorder and is considered as a heterogeneous extract is consistent with earlier reports on the group of diseases characterized by major causes hypoglycaemic action of the extracts of V. amygdalina in affecting cardiovascular, renal, neurological and rats (Samy and Gopalakrishnakone, 2007). Nimemibo- ophthalmic systems (Chakkarwar and Manjrekar, 2005). Uadia (2003) attributed this action to tannins present in Currently available synthetic oral antihyperglycaemic the extract of V. amygdalina, whereas other researchers agents may be associated with an increased risk of presupposed a mechanism unrelated to insulin secretion unwanted effects on prolonged use (Edwin et al., 2006). from pancreatic β cell (Ebong et al., 2006). Be that as it So, there is need to investigate newer herbal medicines may, it is probable that the two mechanisms may exist; which have less side effects, easy availability and are one related to insulin production and the other targets economical (Shah et al., 2006). The age long use of peripheral carbohydrate metabolism. The former endows herbal medicines in the management of diabetic condi- it with the ability to exert hypoglycaemia in diabetic rats, tions is a common practice in many countries across the whereas the later achieves hypoglycaemia in non- globe including Nigeria. Therefore, the need to diabetic rats. Alloxan is known to mediate pancreatic β- substantiate the folkloric claim of V. amygdalina as an cells destruction via reactive oxygen species (ROS) antidiabetic agent using rat models is imperative. The generation (Szkudelsiki, 2001), depriving the animal of results show that there was statistically significant P insulin, hence causing diabetes. V. amygdalina water reduction (P<0.05) not only in the glucose level but also extract having the ability to abate this alloxan-induced in the associated polytriads symptoms. Other related diabetes must necessarily have a corrective impact on complications including weight loss were also improved the hitherto destroyed β-cells of the pancreas. It is significantly (P<0.05) in the treatment groups over time possible to suppose that the antioxidant effect reported as compared to the negative control in the present study. by Igile et al. (1994) to include luteolin, 7-O-beta Alloxan became the first diabetogenic chemical agent glucoronoside and luelin, 7-O-betaglucoside may have when scientists accidentally produced islet-cell necrosis attempted to reverse the cytotoxic effect of alloxan or at in rabbits while researching the nephrotoxicity of uric acid least to mop up the free radicals generated by alloxan derivatives. Alloxan is a specific toxin that inactivates the responsible for beta cell destruction. By so doing, the β- pancreatic β cells, provoking a state of primary deficiency cells could have started a gradual regeneration, hence of insulin without affecting other islet types (Akah and insulin production commences to start an effectual control Okafor, 1992). Hence, alloxan was selected to induce of hyperglycemia. On the other hand, the phyto- diabetes in the present study. Based on a preliminary chemicals-endowed V. amygdalina may possess some study, batches B and C were used for the in vivo study. alpha-glucosidase inhibitors as secondary plant As shown in Figure 1, the decrease of glycemia started metabolites. Such metabolites may competitively inhibit within the first few hours after oral administration of the intestinal brush border enzymes-glucosidase, as well as extract. This lag time could be due to the time required pancreatic beta-amylase with the ultimate reduction in for V. amygdalina extract to reach the site of the gastro- digestion and subsequent absorption of carbohydrates intestinal tract where the active constituent of the extract from the gut (Igile et al., 1994). Other researchers have could be absorbed. The glycemic profiles indicate that proposed a parallel mechanism for tannins in their both doses of extract and the conventional tablet explanation (Igile et al., 1994). These were further displayed similar biological activity. The hypoglycemic strengthened by a researcher (Winleman, 1998), who action of extract (150 and 300 mg) and the positive indicated a strong positive correlation between the control demonstrated rapid onset time of 2 days reaching presence of flavonoids glycosides and phytosterols in maximal activity within 12 days. No biological effect was plants and hypoglycaemic and antihyperglycemic seen in the group treated with distilled water (DW) alone, activities, respectively. It is probable that the V. the slight reduction in the blood glucose level was due to amygdalina extract may be endowed with both. In a the effect of long term glycemia. It is well known that related research by Ali et al. (1993), the author observed prolonged glycemia can cause a slight decrease in blood similar result in a different extract and the author resolved glucose level (Ojiako and Nwanjo, 2006; Abdel-Barry et that the anti-diabetes effect was due to the alkaloid al., 1997). As illustrated in Figure 1, both 150 and 300 mg present in the plant. doses administered significantly decreased blood glucose Toxicological evaluation is an extremely important part levels (P< 0.05) compared to the non-treated group. of herbal formulation and development. High dose of The real mechanism by which the extract performed its extract is usually used for the study (Momoh et al., 2013). action was not clear. The results obtained from the At the dose used for this evaluation, no toxic effect was present study clearly confirmed that the tested extract observed on treatment with excess dose 20 to 30 times possesses marked hypoglycemic activity on the alloxan- higher than the effective dose used in blood glucose level induced diabetic and non-diabetic rats. From the present reduction. The physiological behavior of the rats remained 4592 Afr. J. Biotechnol.

normal, there was no record of death and there was no Conclusion sign of reduced activity after the administration of the 6,000 mg/kg, the response to stimuli (touch) were very Our results confirm the traditional use of leaf extracts of good and sharp. The water and food intake were those V. amygdalina in treatment of diabetes mellitus. The dose not change compared to the negative control group. The of extract of V. amygdalina used in this study, was very oral LD50 of the extract was estimated to be greater than effective and safe in treatment of hyperglycemic 6,000 mg/kg body weight. Based on the result of this condition, and equally restored the imbalance in liver evaluation, we observed that this extract may be safe for enzymes markers and hematological parameters human consumption. Pharmaceutical formulations for associated with diabetes condition. Consequently, this human consumption need to undergo safety evaluation in plant extract could be used alone or in combination with animal. This is a critical part of preclinical studies and other agents in the management of diabetes. More so, it forms an integral initial dossier during drug development. could also be recommended as a food supplement. It is on these, that, clinical protocols, relevant instruction and contraindication and dosage are designed. Hematological parameters are relevant to risk Conflict of Interests evaluation as the changes in haematological system have a higher predictive value for human toxicity, when The author(s) have not declared any conflict of interests. data is translated from animal studies. The results in Table 4 indicated that there were significant changes REFERENCES (P>0.05) in the haematological parameters of the treated groups compared to the diabetic and non-diabetic control Abdel-Barry JA, Abdel-Hassan IA, Al-Hakiem MH (1997). and groups. 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