Terminalia catappa In Vitro Azrul, L. M. Effendy, A. W. M.
2011 2nd International Conference on Biotechnology and Food Science IPCBEE vol.7 (2011) © (2011) IACSIT Press, Singapore Determination of Anthelmintic Potential in Terminalia catappa by Modified Selected In Vitro Bioassay Azrul, L. M. Effendy, A. W. M. Department of Agrotechnology, Faculty of Agrotechnology and Food Science; Institute of Marine Biotechnology Universiti Malaysia Terengganu Kuala Terengganu, Terengganu, Malaysia [email protected] Institute of Marine Biotechnology; Department of Biological Sciences, Faculty of Science and Technology, Universiti Malaysia Terengganu Kuala Terengganu, Terengganu, Malaysia [email protected] Adzemi, M.A. Imelda, L. V. Department of Agrotechnology Faculty of Agrotechnology and Food Science Universiti Malaysia Terengganu Kuala Terengganu, Terengganu, Malaysia [email protected] Haematology and Parasitology Unit Veterinary Research Institute 59, Jalan Sultan Azlan Shah Ipoh, Perak, Malaysia [email protected] Nurulaini, R. Haematology and Parasitology Unit Veterinary Research Institute 59, Jalan Sultan Azlan Shah Ipoh, Perak, Malaysia [email protected] of diseases become a main factor that reduces number of goats’ production. There are a lot of problems and diseases in goats . According to Department of Veterinary Services of Malaysia, parasitic worm or helminths infection is one of major cause for reduced productivity in livestock industry. To overcome this problem, the conventional method is by using the chemical known as anthelmintic or dewormer that based on drugs . However, according to , these manufactured anthelmintics had some serious disadvantages, such as non availability in some developing countries including Malaysia, high cost, and risk of misuse leading to drug resistance, environmental pollution and food residues. According to this phenomenon especially the resistance, naturally produced plant anthelmintics offer an alternative that can overcome some of these problems. The studies about the alternative anthelmintic are becoming urgent because of the rapid escalation of anthelmintic resistance worldwide [3,4]. Besides that, the use of plant as anthelmintic is both sustainable and environmentally acceptable [2,5]. Traditional knowledge about the anthelmintic plants was widely explored nowadays. Thus, in this study, potential plant that claimed to have anthelmintic potential based on ethnobotanical knowledge was used as an experimental plant namely Terminalia catappa or locally known as Ketapang . T. catappa or Abstract—In this study, anthelmintic potential from Terminalia catappa leaves were determined using selected in vitro bioassay that was modified based on other established assays. Larvae was distributed at a concentration of 50 L3 (n=±50) per well in a 96 multiwells plate, incubated with diluted crude extract of T. catappa at a ratio of 1:1 at 20°C for 3 hours and 5 hours. Control was conducted using PBS as positive control and distilled water as negative control. After incubation, larvae motility were observed and counted using inverted microscope. All the non-motile L3 were identified in order to ensure survivality and motility of the larvae. Results showed that after 3 hours, reduction percentage for T. colubriformis, C. curticei and H. contortus was 70%, 63% and 73% respectively while at 5 hours incubation, reduction percentage for each species was at 77%, 67% and 80% respectively. Reduction percentage is calculated by comparing the number of L3 before and after incubation period. Control showed no reduction in terms of motility with standard deviation at 5-10%. From the results, it could be suggested that T. catappa leaves are a potential alternative to be used as anthelmintic. Keywords-Terminalia catappa, larvae motility, reduction percentage, anthelmintic I. INTRODUCTION Goats are one of major important animals in livestock industry in worldwide . Unfortunatley, nowadays, a range 165 locally knownn as Ketapangg tree in Malayysia is a plant widely distributed onn tropical and subtropical s beeaches . Thiis plant has a vast nattural distributiion in near marine-coastal m area of the Indian Occean, through tropical Asia,, and into the Pacific Ocean. It exttends from thhe Seychelless through Inddia, the Andamans annd adjacent isslands, and thhroughout Southeast Asia (Myanm mar, Thailand,, the Malay Peninsula, P Viietnam, Philippines, and Indonesiia) to Papuaa New Guineea and northern Austtralia as far soouth as the Troopic of Capriccorn . The leaves off this plant havve been used as a a folk remeedies in India and Phhilippine to trreat dermatitiss, helminthiassis and hepatitits . From previouus study, we know that thiis plant bioactiviities such as antiooxidant, had severaal hepatoprotecttive, anthelminntic and antiiinflammatory.. There are also few phytochemiccal research sttudies of thiss plant, olatile and tannnin . focused on vo Previous studies s had ddescribed a rannge of in vitrro tests that can be coonducted to evvaluate the anthhelmintic efficcacy of any potentiaal plants. Inn this studyy, the preliiminary modification was done to the larval mootility assay . The a larval miigration principles of the egg hatchh assay  and minalia test  were referred to study the effficacy of Term a three taargeted catappa leavees as natural aanthelmintic against species of gasstrointestinal nematode n obtained from naaturally infected Boer goats. The objeective of thiis study, is to determinne the anthlemintic potential off this plant against threee most matode in rum minants important speecies of gastroointestinal nem which are Haemonchuus contortus,, Trichostroongylus colubriformis and Cooperiaa curticei. Moodification waas done to the selectedd anthelminticc bioassays that establishedd before to give more choices c of in vitro v anthelmiintic bioassayss in the future. II. prrepared for eaach species aas it have to incubated forr two different incubaation period, w which is 3 hrss and 5 hrs. At A the w taking outt and ennd of the incuubation periodd, the well was beeing gently shaaked to make sure the worm ms is not in dorman m mode. Observaation of mottile and surv vived larvae was coonducted usingg inverted miccroscope. III. RESULTS p US-letter paaper size. If you are using A4-sized paper, p please close thiss template andd download thhe file for A4 paper CPS_A4_form mat”. foormat called “C A. Maintainingg the Integrityy of the Specifi fications The templatte is used to fformat your paper p and stylle the texxt. All marginns, column wiidths, line spaaces, and text fonts arre prescribed; please do nnot alter them m. You may note peeculiarities. Foor example, thhe head margin n in this templlate Fiigure 1. The perrcentage of larvaee differentiation based b on speciess from the whoole samples MATER RIALS AND ME ETHODS m T. catappaa leaves weree freshly colleected at the marinecoastal area of Kuala T Terengganu beach, b eastco oast of M The sampling proocess was con nducted Peninsular Malaysia. according to the t correct aggronomy practtices. Drying process p and followed by cutting prrocess were doone according g to the FAO Protocool  and prroceed to the next level unntil the crude powderr of T. catapp ppa was prepaared. Crude powder p was used in the anthelminntic bioassay with crude aqueous a E) form. The CAE was freshly prepareed right extract (CAE before the biooassay was staarted. For the targeted t speccies of worm ms, the samplle was obtained from m the researchh centre was inn mixture of species. s Thus, the speccies differentiaation must be done to differrentiate the species annd the percenttage of each sppecies was reccorded. Then, each sppecies was disttributed (n=500) in each welll in the 96-multiwellss plate respecttively, togetheer with positiive and negative control. The vehhicle solutionn which is distilled d w for the positive p water was used as a negattive control while t larvae (L3 3) were control, PBS solution was used. After the will be mixed up with the teest and inserted into the well, it w on. At every ttime of trial, tw wo sets of weell were control solutio Figure 2. Perceentage of L3 reduction after incubaation with CAE of o T. caatappa IV. D DISCUSSION w collectedd and After larvaee culture wass done, L3 were obbserved underr the microscoope to identiffy their speciees. T. coolubriformis iss the most dom minant speciees at 50% folloowed 166 by C. curticei and H. contortus at 30% and 20% of total population of this sample (Fig. 1). The reduction percentages of L3 are shown in Fig. 2. After 3hrs of incubation, the reduction were 70%, 63%, and 73% respectively while for 5hrs of incubation, the reduction were 77%, 67% and 80% respectively. The reduction percentages were slightly different between both incubation periods. Decreasing trend can be seen in both periods; with the number of motile L3 at 5hrs incubation is less than motile L3 after 3hrs incubation. Significant difference (P>0.05) was determined for all three targeted species compared to the control when analyzed with Simple T-Test. Longer incubation period will reduce more number of motile L3. These results proved that there is anthelmintic activity in T. catappa; by inhibiting the motility and survivality of larvae as mentioned previously in ethnoveterinary reports. V. REFERENCES  Wahab A. R. 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Improved Bioassay for Estimation of Inhibitory Effects of Ovine Gastrointestinal Mucus and Anthelmintics on Nematode Larval Migration. International Journal of Parasitology 24. 1994. 671- 676.  Makkar, H. P. S., Bluemmel, M., Borowy, N. K. and Becker, K. Gravimetric determination of tannins and their correlations with chemical and protein precipitation methods. J. Sci. Food Agric. 61, 1993. 161-165. CONCLUSSION As a conclusion, this preliminary modification can be use to evaluate the in vitro anthelmintic efficacy of this potential plant. By observing the motility and survival of the larvae following incubation process, the anthelmintic efficacy of T. catappa can be evaluated. For future study, the parameters of this assay should be optimized to obtain better results so that it can be widely used and promoted. ACKNOWLEDGMENT This work is supported by Fundamental Research Grant Scheme (Vot no. 59164), under Ministry of Science, Technology and Innovation of Malaysia. The authors wish to thank Dr. P. Chandrawathani, Mdm. Nurulaini and Ms. Imelda from Veterinary Research Institute of Ipoh for their help and valuable advices during this study. A million thanks to these particular person namely Ms. Rawaidah Anas, Ms. Khadijah Saad and Mr. Muhammad Embong for helping me during the plant sampling and data analysis. 167