EVALUATION OF AQUEOUS AND METHANOL ROOT EXTRACTS OF CALOTROPIS PROCERA AIT (ASCLEPIADACEAE) FOR ANTI-ASTHMATIC ACTIVITY ON WISTAR RATS AND GUINEA PIG ILEUM


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TABLE OF CONTENT

Title Page
Abstract
Table of contents
Abbrevations, Definitions, Glossary and Symbols

CHAPTER ONE: INTRODUCTION
1.0 INTRODUCTION
1.1 Statementof Research Problem
1.2 Justification
1.3 Theoritical Frame Work
1.4 Aims and Objectives of the Study
1.4.1 Aim
1.4.2 Specific objectives
1.5 Research Hypothesis

CHAPTER TWO: LITERATURE REVIEW
2.0 Literature Review
2.1 Traditional Medicine
2.2 Overview of asthma
2.3 Epidermiology
2.4 Symptoms of Asthma
2.5 Factors Associated with of Asthma
2.5.1 Environmental Factors
2.5.2 Genetics
2.5.3 Socio Economic Factors
2.5.4 Occupation
2.5.5 Population Disparities
2.5.6 Athletics
2.6 Types of asthma
2.6.1 Extrinsic asthma
2.6.2 Intrinsic asthma
2.7 Diagnosis of asthma
2.8 Asthma Inflammation: cells and mediators
2.8.1 Inflammatory cells involved in asthma
2.8.2 Structural cells
2.8.3 Mediators
2.9 Pathophysiology of asthma
2.10 Pathogenesis
2.11 Goals of asthma therapy
2.12 Treatment of bronchial asthma
2.12.1 Non pharmacological interventions to asthma
2.12.2 Pharmacotherapy of bronchial asthma
2.13 Important medicinal plants with anti-asthmatic potential
2.14     The Family,Asclepiadaceae
2.15     Calotropis procera
2.16     Geographical Distribution of the Plant
2.17     Ethnopharmacology
2.18     Some pharmacological properties of the Plant
2.19     Models for Antiasthmatic Study
2.19.1 Spasmolytic activity in isolated guinea pig Ileum preparation
2.19.2 Carrageenan-induced leucocytosis
2.19.3 Passive paw anaphylaxis in rats
2.19.4 Haloperidol-induced catalepsy
2.19.5 Carrageenan-induced rat paw edema

CHAPTER THREE: MATERIALS AND METHOD
3.0       Materials and Method
3.1       Plant Material
3.2       Preparation of Plant Extract
3.3       Experimental Animals
3.4       Drugs and Chemicals
3.5       Equipment and other Materials
3.6       Median Lethal Dose (LD50) Determination in Rats
3.7       Screening for Anti-asthmatic Activity
3.7.1 In vitro study on isolated guinea pig ileum preparation
3.7.2. Carrageenan-induced leucocytosis in rats (Adaptogenic activity)
3.7.3 Haloperidol-induced catalepsy
3.7.4. Passive paw anaphylaxis in rats (Antiallergic activity)
3.7.5. Carrageenan induced rat paw edema (Anti-inflammatory studies)
3.8 Preliminary Phytochemical Screening of Calotropis procera
3.8.1 Test for carbohydrates
3.8.2 Test for flavonoids
3.8.3 Test for cardiac glycosides
3.8.4 Test for cyanogenic glycosides
3.8.5 Test for saponins
3.8.6 Test for tannins
3.8.7 Test for steroids and terpenoids
3.8.8 Test for alkaloids
3.8.9 Test for anthraquinones and their derivatives
3.9 Statistical Analysis

CHAPTER FOUR: RESULTS
4.0 Results
4.1 Percentage Yield
4.2 Chemical Constituents of Roots of Calotropis procera
4.3 Median Lethal Dose (LD50) of the Extracts
4.4 Antiasthmatic Studies
4.4.1 Histamine-Induced Contractions of Isolated Guinea Pig Ileum Chain Preparation
4.4.2 Carrageenan Induced Leucocytosis in Rats
4.4.3 Haloperidol-Induced Catalepsy in Rats
4.4.4 Carrageenan Induced Rat Paw Edema
4.4.5 Passive Paw Anaphylaxis in Rats

CHAPTER FIVE: DISCUSSION
5.1 Discussion

CHAPTER SIX: CONCLUSION AND RECOMMENDATION
6.1 Conclusion
6.2 Recommendations
REFERENCE



ABSTRACT


Asthma, which affects an estimated 300 million people worldwide, is an incurable health disorder of a major public health concern globally. The present orthodox therapy for asthma has several drawbacks including many undesirable side effects and high cost of management (especially challenging for low-income / developing countries). In a bid to develop a cheaper and better antiasthmatic agent with less side effects, this study was carried out to evaluate the effect of aqueous and methanol root extracts of Calotropis procera used in folkloric medicine as an antiasthmatic plant. The study involves In-vitro model on isolated guinea pig ileum preparation. In-vivo models like carageenan-induced leucocytosis in rats, passive paw anaphylaxis, carrageenan induced rat paw edema and haloperidol induced catalepsy. Median Lethal Dose (LD50) determination was conducted using the method as described by Lorke‘s (1983). In vitro studies on isolated guinea pig ileum preparation was carried out to investigate for bronchospasmolytic activity of the extracts. Bioassay of histamine 10 µg/ml in the presence and absence of Calotropis procera extract 10 mg/ml was done. The normalization effects of the extracts were studied in carrageenan-induced total leucocyte count (TLC) after parenteral administration of carrageenan. Thirty five (35) Wistar rats were divided into 7 groups, five aminals per group. Group 1 was not given any treatment, but blood sample was used to establish the reference standard for TLC in rats. Groups 2 – 7, received respectively, distilled water (2 ml/kg), chlorpheniramine maleate (2 mg/kg), Calotropis procera (100 mg/kg), Calotropis procera, CP (200 mg/kg), Calotropis procera (100 mg/kg), and Calotropis procera (200 mg/kg). Immuno-modulatary / antiallergic antiasthmatic activity of the plant was studied using passive paw anaphylaxis model as described by Patil (2010). Wistar rats were sensitized subcutaneously with 100 mg fresh egg albumin for 10 days, after which serum was collected. A fresh set of thirty (30) animals was divided into six (6) groups each containing five (5) rats. Groups 1 – 6 received distilled water 2 ml/kg, dexamethasone 0.27 mg/kg, CP 250 mg/kg aqueous extract, CP 350 mg/kg aqueous extract, CP 250 mg/kg methanol extract and CP 350 mg/kg methanol extract orally respectively. Anti-inflammatory antiasthmatic activity of CP was studied using carrageenan induced rat paw edema model as described by Anita and Babita (2008). Dopaminergic and/or adrenergic antiasthmatic study was carried out using haloperidol-induced catalepsy on wistar rats as described by Patil (2010). Preliminary phytochemical screening of the extracts was carried out as described by Trease and Evans. Extraction of powdered plant gave yields of 15.64% w/w and 13.76%w/w methanol and aqueous extracts respectively. Oral LD50 in Wistar rats for both aqueous and methanol CP extracts were found to be >5,000 mg/kg. Both aqueous and methanolic extracts inhibited histamine induced contraction of isolated guinea pig ileum (p˂0.001), carrageenan-induced leucocytosis (p˂0.05), egg albumin-induced passive paw anaphylaxis (0.05) and carrageenan induced rat paw edema (p˂0.05). However, only methanolic extract inhibited haloperidol-induced catalepsy in wistar rats (p˂0.05). Hence it is concluded that both aqueous and methanolic extracts possess antiasthmatic activity that may be responsible for the antiasthmatic activity and may have potential role in the treatment of asthma.



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