PHYTOCHEMICAL STUDIES AND EFFECT OF METHANOL LEAF EXTRACT OF LEPTADENIA HASTATA(PERS.) DECNE (ASCLEPIADACEAE) ON ACETIC ACID INDUCED WRITHES IN MICE AND VENOM OF ECHIS OCELLATUS


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TABLE OF CONTENT

Title Page
Abstract
Table of Contents
Abbreviations

CHAPTER ONE
1.0       INTRODUCTION
1.1       Brief History of Drug development
1.2       Snake Venom
1.2.1    Effect of Snake venom on Human Health
1.3       Pain
1.3.1    Description of Pain
1.3.2    Causes and Symptoms of Pain
1.4       Analgesic
1.4.1    Herbal Constituents active against Pain
1.5       Statement of Research Problem
1.6       Justification of the Study
1.7       Justification for Plant Selection
1.8       Aim of the Study
1.9       Objective of the Study
1.10     Statement of Research Hypothesis

CHAPTER TWO
2.0       LITERATURE REVIEW
2.1       Literature Review of Leptadeniahastata
2.1.1    Botanical Features of Leptadeniahastata
2.1.2    Local Names of Leptadeniahastata
2.1.3    Ethnomedical Uses of Leptadeniahastata
2.1.4    Phytochemistry of Leptadeniahastata and other Asclepiadaceae species
2.1.5    Pharmacological actions of L.hastata and other Asclepiadaceaespecie
2.2       Literature Review of Echisocellatus
2.2.1    Scientific Classification ofEchisocellatus
2.2.2    Binomal names ofEchisocellatus
2.2.3    Common names of Echisocellantus
2.2.4    Synonyms of Echisocellatus

CHAPTER THREE
3.0       MATERIALS AND METHODS
3.1       Materials
3.1.1    Chemicals and Reagents
3.1.2    Equipment
3.1.3    Experimental Animals
3.1.4    Snake Venom
3.2       Methods
3.2.3    Collection, Identification and Drying of Plant Material
3.2.4    Extraction and Fractionation
3.2.5    Preliminary Phytochemical Screening
3.3       Chromatographic Studies
3.3.3    Column Chromatography of Hexane Fraction
3.3.4    Thin Layer Chromatography
3.3.5    Preparative Thin Layer Chromatography of Hexane Fraction 4
3.3.6    Characterization of Compound M1
3.3.5    Spectral Analysis of M1
3.4       Pharmacological Studies
3.4.3    Acute Toxicity Studies of Leptadeniahastata
3.4.4    Venom Sampling
3.4.5    Lethality Assay of the Venom
3.4.6    Invivo Snake Venom Detoxifying Effect of Methanol Extract
3.4.7    Invitro Snake Venom Detoxifying Effect of Methanol Extract
3.4.8    Analgesic Studies

CHAPTER FOUR
4.0       RESULTS AND DISCUSSIONS
4.1       Yield
4.2       Results of Preliminary Phytochemical Screening
4.3       Results of Thin Layer Chromatography
4.2.1    Result of Thin Layer Chromatography of Crude Methanol Extract
4.2.2    Result of Thin Layer Chromatography of Hexane Fraction
4.2.3    Result of TLC of various HF obtained from Column Chromatography
4.4       Isolation
4.4.1    Isolation of M1
4.4.2    Melting Point Determination
4.4.3    Chemical Test on Compound M1
4.5       Spectral Analysis
4.5.1    Infrared Spectral Analysis of M1
4.5.2    Proton Nuclear Magnetic Resonance Spectral Analysis of M1
4.5.3    13C-NMR and 13C-DEPT NMR Spectral Analysis of M1
4.5.4    HSQC Spectral Analysis of M1
4.5.5    1H-1H COSY Spectral Analysis of M1
4.5.6    HMBC Spectral Analysis of M1
4.5.7    NOESY Spectral Analysis of M1
4.6       Pharmacological Studies
4.6.1    Result of Acute Toxicity Studies
4.6.2    Result of Median Lethal Dose (LD99) of the Venom
4.6.3    Result of Invitro detoxifying effect of ME of Leptadeniahastata against Echisocellatus venom
4.6.4    Result of Invivo detoxifying effect of ME of Leptadeniahastata against Echisocellatus venom
4.6.5    Result of the effect of ME of Leptadeniahastata against acetic acid induced mice

CHAPTER FIVE
5.0       DISCUSSIONS
5.1       Phytochemical Studies
5.2       Pharmacological Studies

CHAPTER SIX
6.0       SUMMARY, CONCLUSION AND RECOMMENDATION
6.1       Summary
6.2       Conclusion
6.3       Recommendation
REFERENCES




ABSTRACT

Leptadeniahastata (Pers.) Decne (Asclepiadaceae) is widely African plant species used in traditional medicine as an anti-venom, antihypertensive, anti-diabetic, analgesic and also used for catarrh and skin disease. The powdered leaves of L. hastatawas extracted with methanol using maceration method and the resulting crude methanol extract (ME) was suspended in water and fractionated into hexane (HF), chloroform (CF), ethylacetate (EF) and n-Butanol (BF). Preliminary phytochemical screening of ME revealed the presence of carbohydrates, alkaloids, flavonoids, anthraquinones, saponins, tannins, glycosides, steroids and triterpenes while (HF) revealed the presence of steroids and triterpenes nucleus. HF was step-wisely eluted in a silica gel packed column to afford ten fractions H1-H10 and fraction H4 was selected for further purification using Preparative Thin Layer Chromatography, with the solvent system of hexane: ethyl acetate (8.5 : 2.5). This resulted in the isolation of a whitish crystalline compound M1 (β-Sitosterol). The structure of the compound was elucidated using chemical test and spectroscopic techniques (IR, 1D & 2D-NMR) and by comparison with reference spectral data. The LD50 of the methanol extract (ME) and the LD99 of the Echisocellatus venom were estimated to be greater than 5000 mg/kg and 2.2 mg/kg respectively. Anti-venom studies suggest that ME possess significant anti-venom activity against Echisocellatusvenom both In vitro and In vivo. Analgesic effect was evaluated using acetic acid induced writhing in mice. The results showed that ME (150 mg/kg) decreased writhing response with 84.6 % inhibition. However, the extract when compared to control showed statistically significant difference at (p˂0.05).




CHAPTER ONE


1.0                                                                         INTRODUCTION

A natural product is a chemical compound or substance produced by a living organism – found in nature that usually has a pharmacological or biological activity for use in pharmaceutical drug discovery and drug design. A natural product can be considered as defined above even if it can be prepared by total synthesis (Newman and Cragg, 2007).


Traditional medicine is the sum total of the knowledge skills, and practices based on the theories, beliefs, and experiences indigenous to different cultures, whether explicable or not, used in the maintenance of health as well as in the prevention, diagnosis improvement or treatment of physical and mental illness.

Traditional use of herbal medicines refers to the long historical use of these medicines. Their use is well established and widely acknowledged to be safe and effective, and may be accepted by national authorities (WHO, 2013).


In recent years, the treatments and remedies used in traditional African medicine have gained more appreciation from researchers in Western science. Developing countries have begun to realize the high costs of modern health care systems and the technologies that are required, thus proving Africa's dependence to it. Due to this, interest has recently been expressed in integrating traditional African medicine into the continent's national health care system.In Africa, the importance of traditional healers and remedies made from indigenous plants play a crucial role in the health of millions......


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