Evaluation of Phyto-Chemical and Medicinal Properties of Cannabis Sativa Linn. (Family Moraceae).

Abstract

This work studied the various delivery systems (oral and parental formulations) of crude Cannabis. The stability, pharmacological and toxicological profiles of Cannabis resin in the formulations was also studied. Dried powdered leaves of Cannabis sativa L. were extracted with chloroform, the extract was concentrated to obtain an oil-based tar (resin) and its phytochemical composition determined. The Cannabis crude resin was formulated as syrup, capsule and suppository dosage forms. The stability of the resin in the formulations was studied by accelerated stability technique. The in vivo release profile of the capsules was also studied. The antimicrobial activity of Cannabis crude resin was investigated using clinically isolated Pseudomonas aeruginosa and Staphylococcus aureus. The minimum inhibitory concentration (MIC) and killing rate constant against the organisms were determined by agar diffusion and viable count methods respectively. The antiinflammatory effect of Cannabis crude resin and syrup formulations was studied using egg-albumin-induced paw oedema in rats. The acute toxicity (LDSo) of the crude extract was determined by the brine-shrimp method while effects on biochemical parameters (glutamate-pyruvate-transaminase, glutamate-oxaloacetate-transaminase and urea) haematological parameters (eosinophile, monocyte, neutrophiles and lymphocyte counts, packed-cell volume and red blood cells) were determined in Wistar rats after 1, 2 and 3 weeks of treatment. The, internal, acgans~(liver, heart, kidney, spleen, lungs and the brain) from the treated rats were examined histopathologically to ascertain degeneration on chronic administration of Cannabis crude resin. Results were analysed statistically using the Analysis of Variance (SPSS) and Student's t-test. Means that differed significantly were identified by the least significant difference (LSD) Post-hoc test at 95 % confidence interval i.e. (Pc0.05). The result showed that the extraction process yielded 10 % (wlw) of the Cannabis crude resin. The presence of alkaloids, glycosides, saponins, tannin, terpenes and steroids was detected in the crude resin and dried plant. Cannabis crude resin syrup was stable even without the addition of EDTA with a shelf-life of 50 days. The degradation of Cannabis crude resin syrup formulation followed a first-order kinetic with the degradation rate constant of 0.0039 min-I. At a minimum inhibitory xxi concentration (MIC) of 12.5 mglml, the chloroform, methylene chloride, ethanol, nhexane: chloroform (1:l) and n-hexane extracts of Cannabis sativa showed significantly (Pc0.05) higher antimicrobial activity against S. aureus and Ps. aeruginosa than Penicillin G (MIC 18.0 mgfml). The crude resin syrup formulation exhibited a significant (Pc0.05) dose-related inhibition of paw edema in rats. The crude resin caused changes in biochemical and haematological parameters in a dosedependent manner with no significant increase in the first two weeks of treatment, which markedly changed at week 3. The histopathological results of the various organs showed lesions of varied intensity suggesting possible toxicity in chronic use of Cannabis crude resin. These findings suggest that crude Cannabis resin can be used for its antimicrobial and antiinflamatory effect and that its formulation into syrup favours its pharmacological activity and have good stability.

TABLE OF CONTENTS

Title page .......................................................................................... ,.i

Certification ...................................................................................... ..II

Dedication ......................................................................................... ,111

Acknowledgement .............................................................................. ..iv

Table of contents.. ................................................................................. vi

List of tables ......................... .... ................................................. .XII

List of figures ................................................................................... .xiv

List of abbreviations ..................... ................................................ XVII

List of plates .................................................................................. ..XVIII

Abstract ...................... ............................................................... XXII

CHAPTER ONE .................................................................................. I

INTRODUCTION ...................................................................... 1

Historical Background of Marihuana (Cannabis). .................................. ..2

Cultivation ................................................................................ .3

Chemistry ................................................................................ ..3

Structure - Activity Relationships (SAR) ............................................ .7 

Structure - Activity Relationships (SAR) in Man ................................... .7

Structure - Activity Relationships (SAR) in Animals ............ .............,. ..... 12

Pharmacology (Therapeutic potential in man and laboratory animals) ......... .28

Health Aspect of Cannabis ............... : .............................................. 28 ,I - .-

Acute and Chronic Effects of Cannabis in Humans/Animals ..................... 28

1.6.2.1 Acute studies ........................................................................... ..28

1.6.2.2 Chronic Studies ....................................................................... ..30

1.6.3 Possible Adverse Effects of Cannabis on Health .................................... 30

1.6.3.1 Immunity ................................................................................. 30

1.6.3.2 Chromosomal Damage ................................................................ .3 1

1.6.3.3 Pregnancy and Foetal Development ................................................ ..32

Animal Studies .................................................................. 32

Human Studies .................................................................. 33

Cell Metabolism ................................................................ 33

Psychopathology ................................................................ 33

Acute Panic Reaction .......................................................... 34

Toxic delirium ................................................................. 34

Psychoses ........................................................................ 35

Flash backs ..................................................................... -35

Violence ......................................................................... 36

Amotivational Syndrome ...................................................... 36

Residual Psychomotor Impairment ........................................... 37

Tolerance and Dependence ................................................... 37

Physical dependence /Withdrawal ........................................... 38

Lung Problems .................................................................. 39

Bronchial and Pulmonary Damage ......................................... -39

Bronchial Tissue changes .................................................... 39

Macrophages ................................................................... 40

Cardiovascular Problems ...................................................... 40

Therapeutic uses ....................... .. ................................. 41

Multiple SclerosisMeuC~:'ology.~~.:". ............................................ 41

Antiemetic Effects ............................................................. 42

Alcoholism ..................................................................... -43

Antiglaucoma Effects .......................................................... 43 ..... 

Prostaglandin synthesis ........................................................ 44

Anticonvulsant .................................................................. 45

Analgesic ......................................................................... 45

Neurotransmitter receptors ..................................................... 46

Opioid receptors ............................................................... -46

Prostaglandins .................................................................. 46

Anti inflammatoiy Activity ................................................... 47

Anti-asthmatic ................................................................... 48

1.6.4.10 Antibiotic ....................................................................... -48

1.6.4.1 1 Appetite Stimulant ............................................................ 48

1.6.4.12 Miscellaneous Uses ............................................................ 49

1.6.4.12.1 Antineoplastic and Immunosuppressant Activity .......................... 49

1.6.4.12.2 Anorexia ........................................................................ -49

1.6.4.12.3 Antifertility Activity ........................................................... 49

1.6.4.12.4 Gastrointestinal effects ......................................................... 50

The Legal . Illegality of Clinical Cannabis use in Modern Therapy ............. 50

In The Netherlands ..................................................................... -51

In Germany ............................................................................... 51

In Spain ....................... ....................................................... 52

In Italy .................................................................................... 52

In Australia ............................................................................... 52

In The USA .............................................................................. 53

In Nigeria ................................................................................. 54

In United kingdom ...................................................................... 54

AIM AND SCOPE OF STUDY ..................................................... -55

CHAPTER TWO

2 . 0 Experimental ............... .;,.+...........A. ................................................. 56

2.1 Materials .................................................................................. 56

2.1 . 1 Chemicals and Reagents ................................................................ 56

2.1.2 Instruments1 Equipment ................................................................. 58

2.2 Source and Identity of plant materials.". .... : .:": .......................................... 59

2.3 Preparation of plant extract ................................................................. 59

Preparation of n-Hexane: Chloroform extract ....................................... 60

Ultraviolet (UV) Absorption Spectra ............................................... -60

Phytochemcal Tests ...................................................................... 60

Test for the presence of reducing sugar .............................................. 60

Test for the Presence of Flavonoids ................................................... 60

Test for the Presence of Glycosides ................................................... 61

2.5.3.1 Tests for 0 . and C - glycosides ....................................................... 61

2.5.3.2 Borntagger's test for Anthracene glycosides ........................................ 61

2.5.3.3 Test for cyanogenic glycosides ........................................................ 62

2.5.4 Test for the presence of alkaloids ..................................................... 62

2.5.5 Test for Starch ........................................................................... 62

2.5.6 Test for the Presence of Proteins ...................................................... 62

2.5.7 Test for Tannin ........................................................................... 63

2.5.8 Test for Saponins ........................................................................ 63

2.5.8.1 Frothing Test ............................................................................. 63

2.5.8.2 Emulsion Test ........................................................................... -63

2.5.9 Test for Flavonoid ........................................................................ 64

2.5.10 Test for Carbohydrate ................................................................. -64

2.6 Microbiological Evaluations ........................................................... 64

2.6.1 Isolation and characterisation of test microorganisms .............................. 64

2.6.2 Maintenance and standardisation of stock cultures ................................. 64

2.6.3 Preliminary Sensitivity Tests .......................................................... 65

2.6.3.1 Sensitivity test of Cannabis Crude Resin Extracts ................................. 65

2.6.4 Minimum Inhibitory Concentrations (MICs) of Extracts ........................... 65

2.6.5 Determination of Rate of Kill of the Crude Extracts ................................ 66

2.7 Toxicity Tests .................... ,',.c.q ...>.... i. .............................................. 66

2.7.1 Cytotoxicity Assay (CDS0) .............................................................. 66

2.8 Administration of Cannabis Suspension ............................................. 67

2.9 Determination of Haematological Parameters ....................................... 67

2.9.2 Sample Collection ....................................................................... 67

2.9.3 Procedure for the haematological determination .................................... 68

2.9.4 Data Analysis ........................................................................... -69

2.10 Preparation of Cannabis Formulations ................................................ 69

2.10.2 Materials .................................................................................. 69

2.10.2.1 Preparation of Suppositories .................................................. 69

2.10.3 Evaluation of Suppositories ............................................................ 69

2.10.3.1 Appearance ..................................................................... -69

2.10.3.2 Uniformity of Weight .......................................................... 69

2.10.3.3 Liquefaction time ............................................................... 70

2.10.3.4 Construction of Calibration Curve (Beer's plot) ........................... 70

2.10.3.5 Release studies .................................................................. 70

2.1 1 Anti inflammatory test .................................................................. 71

2.12 Determination of Biochemical Parameters: .......................................... 72

2.12.1 Determination of the serum glutamate-oxaloacetate-transaminase (SGOT) .... 72

2.12.1.1 Preparation of standard curve for SGOT .................................... 73

2.12.2 Determination of the serum glutamate-pyruvate-transaminase (SGPT) .......... 73

2.12.2.1 Preparation of standard curve for SGPT ..................................... 74

2.12.3 Determination of urea levels ........................................................... 74

2.13 Histopathological determination ...................................................... 75

2.13.1 Isolation of some vital organs of rats .................................................. 75

2.14 Stability studies of Cannabis crude resin formulation .............................. 77

2.14.1 Preparation of stock solutions ......................................................... 77

2.14.2 Exposure of the Samples to various Temperatures ................................ -79

2.14.3 TLC determination ...................................................................... 79

2.14.4 Determination of Percentage Degradation ........................................... 80

2.14.4.1 Determination of Shelf Life ........................................................... 81

CHAPTER THREE ............................................................................. -82

3.0 Results and Discussion ................................................................. 82

3.1 Yield .......................... ... ...................................................... 82 ......

3.2 Phytochemical Analysis of Cannabis sativa leaves ................................. 82

3.3 Cytotoxicity Assay LCSo .............................................................. -82

3.4 Pharmacological Parameters ........................................................... 85

3.4.1 Effects of formulation of Cannabis resin on rat paw edema: ..................... 85

3.5 Antimicrobial Assay .................................................................... 95

3.6 effect of crude Cannabis resin extract on Haematological Parameters ....................................... 117

3.7 Cannabis Suppositories formulation and analysis ................................. 125

3.7.1 Appearance of the suppositories ...................................................... 125

3.8 Stability studies ........................................................................ 138

3.9 BiochemicalParameters ............................................................... 148

3.9.1 Effect of Cannabis Crude Resin on Glutamate-Oxaloacetate ................... 148

3.9.2 Effect of Cannabis Crude Resin on Glutamate-Pyruvate Transaminase Activity in Rats ............................................................................................ 148

3.9.3 Effect of Cannabis Crude Resin on Urea Activity in Rats ....................... 148

3.10 Histopathological effects of Cannabis crude resin ................................. 154

3.10.1 Effects of Cannabis Crude Resin on the Liver of Rats ............................ 154

3.10.2 Effects of Cannabis Crude Resin on the Brain of Rats ........................... 160

3.10.3 Effects of Cannabis Crude Resin on the Kidney of Rats ......................... 160

3.10.4 Effects of Cannabis Crude Resin on the Spleen of Rats ......................... 169

CONCLUSION ................................................................................ 173

REFERENCES ................................................................................. 174

APPENDIX .................................................................................... -199