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Evaluation of the Efficacy of Three Plant Extracts Against Venoms of Five Snake Species in Northern Nigeria

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Evaluation of the Efficacy of Three Plant Extracts Against Venoms of Five Snake Species in Northern Nigeria EVALUATION OF THE EFFICACY OF THREE PLANT EXTRACTS AGAINST VENOMS OF FIVE SNAKE SPECIES IN NORTHERN NIGERIA BY YUNUSA YAHAYA DEPARTMENT OF VETERINARY PUBLIC HEALTH AND PREVENTIVE MEDICINE FACULTY OF VETERINARY MEDICINE AHMADU BELLO UNIVERSITY, ZARIA MARCH 2017 EVALUATION OF THE EFFICACY OF THREE PLANT EXTRACTS AGAINST VENOMS OF FIVE SNAKE SPECIES IN NORTHERN NIGERIA BY YUNUSA Yahaya P15VTPH9009 BSc, (1997), MSc (2006) (ABU, ZARIA) A THESIS SUBMITTED TO THE SCHOOL OF POSTGRADUATE STUDIES, AHMADU BELLO UNIVERSITY, ZARIA IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF DEGREE OF DOCTOR OF PHILOSOPHY IN VETERINARY PUBLIC HEALTH AND PREVENTIVE MEDICINE DEPARTMENT OF VETERINARY PUBLIC HEALTH AND PREVENTIVE MEDICINE, AHMADU BELLO UNIVERSITY, ZARIA March, 2017 i DECLARATION I declare that the work in this thesis entitled “Evaluation of the efficacy of three plant extracts against venoms of five snake species in Northern Nigeria” has been performed by me in the Department of Veterinary Public Health and Preventive Medicine. The information derived from the literature has been duly acknowledged in the text and list of references provided. No part of this dissertation was previously presented for another degree or diploma at this or any other Institution. _____________________ ___________________ ______________ YUNUSA Yahaya Signature Date ii CERTIFICATION The thesis entitled “Evaluation of the efficacy of three plant extracts against venoms of five snake species in Northern Nigeria” by YUNUSA Yahaya meets the regulations governing the award of Doctor of philosophy degree in Veterinary Public Health and Preventive Medicine of Ahmadu Bello University, Zaria and is approved for its contribution to knowledge and literary presentation. Professor J. K. P. Kwaga ____________________________ _____________ ___________ Chairman, Supervisory committee Signature Date Professor M. S. Abubakar __________________________ _____________ ____________ Member, Supervisory Committee Signature Date Professor A.O.Adaudi __________________________ _____________ ____________ Member, Supervisory Committee Signature Date Professor E. C. Okolocha __________________________ ____________ ___________ Head of Department Signature Date Prof S. Z. Abubakar ______________________________ ____________ ___________ Dean School of Postgraduate studies Signature Date iii ACKNOWLEDGEMENTS In the name of Allah the Beneficent the Merciful. Thanks be to God. I really thank my supervisors: Professor J. K. P. Kwaga, Professor M. S. Abubakar and Professor A.O. Adaudi for their patience, advice, and corrections.Special thanks to all those who supported this study, my family, friends and well-wishers. iv ABSTRACT Africa is blessed with abundance of natural resources including both fauna and flora. Nigeria has a lot of medicinal plants that are not yet exploited and some are traditionally known to be used in the prevention and or treatment of snake bites. Screening some of these plants scientifically will prove the traditional claims by the snake charmers and herbalists may lead to the production of single polyvalent antidote for the prevention and treatment of snake bites. The objectives of the study were to: Select, collect and identify plants with potential antivenom properties and five species of poisonous snakes; Determine the phytochemical constituents of the three plants; Carry out acute toxicological studies of the selected plant extracts; Determine the LD50 of venoms of different snake species and the plant extracts; Determine the antivenom potentials of the different fractions of the plant extracts; Carry out interactive studies with various plant extract fractions on the venom in vivo; Determine the protective effects of three plant extracts against the haemorrhagic effect of snake venoms in rats; Carry out studies on the effects of various plant extracts in neutralizing snake venoms on 1kb plasmid DNA migration on agarose gel electrophoresis. Results of the phytochemical analysis of the crude extracts of the three plants (Olax manni, Urginea altissima and Xeromphis nilotica) showed the presence of carbohydrates, glycosides, steroids/triterpens, cardiac glycosides, saponins, glycosides and alkaloids. In addition, Extract of Urginea altissima contained free anthracenes. However, the ferric chloride test failed to detect glycosides in all the extracts, while Bontrager‘s test and modified Bontrager‘s test did not detect free anthracenes and combined anthracenes respectively in the extracts of Olax manni and Xeromphis nilotica. Similarly, Mayer‘s, Dragendoff‘s and Wagner‘s tests failed to detect alkaloids in Olax manni, and Xeromphis nilotica. The in vivo studies revealed that the v aqueous fractions of the 3 plants (A4, B4 and C4) have the highest activity against the snake venoms when administered 30 minutes before, after and when venom and extracts were administered, which showed that the three plants have both curative, prophylactic and neutralization effects against snake venom. Plant B (Urginea altissima) had the highest (100 %) activity with all the rats treated with its aqueous extract surviving envenomation. The prophylactic method of inoculation was found to be the most effective. Toxicity studies showed that the LD50 of the venom of N. haje, N. katiensis, N. nigricollis, E. ocellatus and B. arietans in Wistar rats were found to be 0.22, 0.55, 0.87, 1.24 and 1.80 mg/kg, respectively. Bitis arietans has the highest LD50 followed by Echis ocellatus while Naja haje was having the lowest LD50 follwed by Naja katiensis, found to have the lowest LD50 Toxicity. Anticoagulant activity of the snake venom was measured by the time taken for blood to clot. The clotting time for Naja nigricollis, Naja haje and Naja katiensis were found to be 132, 128 and 115 minutes, respectively. However, the venoms of Bitis arietans and Echis ocellatus did not clot during the period of the experiment. Studies on haemorrhagic effect of venoms in rats indicated that aqueous fraction of Urginia altissima showed antivenom activity against the venoms of Naja nigricollis, Naja haje, Naja Katiensis, Bitis arietans and Echis ocellatus there were no patches observed. The aqueous extract fraction of Olax manni showed protective effect against venoms of Naja nigricollis, Naja haje, Naja katiensis, Bitis arietans and Echis ocellatus while aqueous fraction of Xeromphis nilotica also had protective effect against the venom of Naja nigricollis, Naja haje, Naja katiensis and Bitis arietans. However, there were some patches of blood in the skin of rats inoculated with venom of Echis ocellatus. The results established the aqueous fraction of the three plants the neutralization effects of venom. The interactive study of the aqueous fraction of Urgines altissima, vi venom and the plasmid DNA measured by migration on agarose gelelectrophoresis showed that aqueous fractions had high antivenom activity against the venoms of Naja nigricollis, Naja haje, Naja katiensis, Bitis arietans and Echis ocellatus.1 kb DNA ladder and the plasmid DNA migrated very well on agarose gel electrophoresis. The mixture of plasmid DNA with separate venom of the snakes, showed no migration of the DNA on agarose gel electrophoresis. The plasmid DNA incubated with the aqueous fraction of plant B showed clear migration of the DNA on agarose gel electrophoresis. In conclusion the extract of three plants demonstrated curative, prophylactic and detoxifying effects against the venoms of the five venomous species of snakes. vii TABLE OF CONTENTS TITLE PAGE ........................................................................................................................ i DECLARATION ................................................................................................................ ii CERTIFICATION ............................................................................................................. iii ACKNOWLEDGEMENTS ............................................................................................... iv ABSTRACT ........................................................................................................................... v TABLE OF CONTENTS ................................................................................................. viii LIST OF TABLES ............................................................................................................. xv LIST OF FIGURES ........................................................................................................ xvii LIST OF ABBREVIATION ............................................................................................. xix CHAPTER ONE .................................................................................................................. 1 1.0 INTRODUCTION .......................................................................................................... 1 1.1 Background .................................................................................................................... 1 1.2 Statement of the Research Problem ............................................................................. 4 1.3 Justification ..................................................................................................................... 5 1.4 Aim of the Study ............................................................................................................. 6 1.5 Objectives of the Study ..................................................................................................
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