Anti-Inflammatory and Hepatoprotective Effects of the Homogenate of Cucumis Sativus (Cucumber) Fruits

Filed in Articles by on June 20, 2022

Anti-Inflammatory and Hepatoprotective Effects of the Homogenate of Cucumis Sativus (Cucumber) Fruits.

ABSTRACT

Research on inflammation has become the focus of global scientific study because of its implication in virtually all human and animal diseases.

Also, liver diseases have been on increase and of global concern.Cucumis sativus is believed to have anti-oxidant activity, high flavonoid content, anti-inflammatory and analgesic effect, which may be likely of use in the management of these diseases.

The anti-inflammatory and hepatoprotective effects of the homogenate of Cucumis sativus fruit were therefore studied.

The fresh fruit of Cucumis sativus was homogenized and used for all experimental analysis without further dilution. Acute toxicity tests of the homogenate of Cucumis sativusfruit were carried out.

The phytochemical analyses and proximate compositions of the fruit homogenate were carried out. 1, 1-Diphenyl-2-Picryl Hydrazyl (DPPH) radical scavenging activity of the fruit homogenate was determined. The effects of the fruit homogenate on agar-induced paw oedema in rats were investigated.

The effects of the fruit homogenate on liver function enzyme (alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase) activities, total bilirubin concentration and lipid profile (total cholesterol, high density lipoprotein, triacylglycerol and low density lipoprotein concentrations) in rats intoxicated with carbon tetrachloride (CCl4) were evaluated using  standard biochemical methods.

The effects of the fruit homogenate on hypotonicity-induced haemolysis of RBC, phospholipase A2and prostaglandin synthase activitieswere also studied.

Data were analysed using SPSS and two-way ANOVA; the acceptance level of significance was p˂0.05.The qualitative phytochemical tests on the homogenate of Cucumis sativus fruitrevealed the presence of flavonoids, alkaloids, terpenoids, glycosides, resins, steroids, saponins and tannins.

The quantitative phytochemical analysis of the homogenate ofCucumis sativus fruit showed that,reducing sugars (574.36 ± 3.88 mg/g) was highest amount when compared to other phytochemicals, alkaloids (2.22 ± 0.96 mg/g) and flavonoids (2.14 ± 0.56 mg/g) were  moderately present while cyanogenic glycoside (0.21 ± 0.13 mg/g) was the lowest in quantity.

Proximate analysis showed thatCucumis sativus fruit contained the following – fibre (1.30 ± 0.01%), moisture (94.6 ± 0.08%), protein (3.11 ± 0.07%) and ash (1.07 ± 0.24%)contents. The acute toxicity test showed no toxicity up to 5ml/kg (≡ 5000mg/kg) body weight which indicated the possible safety of the fruit to the users.

There was relative increase in the percentage inhibition of DPPH radical scavenging activity with increased amount of the homogenate.

At doses of 2ml and 4ml/kg b.w., the fruit homogenate significantly (p ˂ 0.05) inhibitedagar-induced raw paw oedema relative to control.

Studies on membrane stabilization using hypotonicity-induced red blood cell haemolysis revealed that the fruit homogenate significantly (p˂0.05) inhibited haemolysis when compared to indomethacin (a known standard drug).

The homogenate exhibited a significant (p˂0.05) dose (0.5ml and 1.0ml) related inhibition of prostaglandin synthase activity (79.9% and 81.0% respectively), compared to 0.4mg/ml of indomethacin, standard drug (82.0%). The fruit homogenate like prednisolone significantly (p˂0.05) inhibited phospholipase A2 activity.

Treatment of rats with the homogenate of Cucumis sativus fruits significantly (p˂0.05) decreased CCl4-inducedelevated levels of the liver enzymes ALT, AST and ALP and of total bilirubin in the serum when compared to positive control.

The homogenate also attenuated the CCl4-induced elevation of LDL, total cholesterol and triacylglycerol amounts and ameliorated the induced depletion of HDL.

The results indicated that the homogenate of Cucumis sativus fruits possesses anti-inflammatory activities and hepatoprotective effects.

TABLE OF CONTENTS

Title Page ———- i
Certification —– ii
Dedication ———iii
Acknowledgement- iv
Abstract——- vi
Table of Contents— vii
List of Figures——- xiii
List of Plates — xiv
List of Tables—— xv
List of Abbreviations— xvi

CHAPTER ONE: INTRODUCTION

1.1 Inflammation ——— 1
1.2 Classification of inflammation—— 2
1.2.1 Acute inflammation———- 2
1.2.2 Chronic inflammation ——- 7
1.3. Inflammatory responses ——- 7
1.3.1 Acute vascular response——– 7
1.3.2 Acute cellular response——– 8
1.3.3 Chronic cellular response —– 8
1.3.4 Resolution——— 9
1.4 Inflammatory cells —– 10
1.5 Oxidative damage in inflammation – 11
1.6 Antioxidants ——- 11
1.7 Inflammatory disorders—– 12
1.8 Anti-inflammatory agents— 12
1.8.1 Stabilization of lysosomal membrane —- 13
1.8.2 Phospholipase A2 —– 15
1.8.3 Prostaglandin synthase/Cyclooxygenase — 15
1.9 Anti-inflammatory plants – 18
1.10 Phytochemistry—– 19
1.10.1 Tannins —- 20
1.10.2 Phenols —– 21
1.10.3 Flavonoids— 21
1.10.4 Anthocyanins —- 22
1.10.5 Alkaloids —– 22
1.10.6 Glycosides—— 23
1.10.7 Sterols—- 23
1.10.8 Resins— 24
1.10.9 Terpenoids— 24
1.10.10 Saponins— 25
1.10.11 Reducing sugars— 26
1.11 Hepatotoxicity—— 26
1.11.1 The liver —– 27
1.11.2 Assay associated with hepatotoxicity — 28
1.12 Carbon tetrachloride (CCl4) — 28
1.13Cucumis sativus (Cucumber)—–29
1.13.1Morphology of Cucumis sativus–29
1.13.2Taxonomy and Nomenclature of Cucumis sativus —- 31
1.13.3Nutritional composition of Cucumis sativus —- 31
1.13.4Uses of Cucumis Sativus —- 33
1.14Rationale for the study—— 33
1.15 Aim of the study ———- 33
1.16 Research objectives——- 34

CHAPTER TWO: MATERIALS AND METHODS

2.1 Material—– 35
2.1.1Plant material– 35
2.1.2 Animals ——– 35
2.1.3 Instruments —- 35
2.1.4 Chemicals and reagents — 35
2.2 Methods—– 36
2.2.1Preparation of plant material ———– 36
2.2.2 Qualitative phytochemicals analysis of the homogenate of Cucumis sativus fruit—— 36
2.2.2.1 Test for alkaloids — 36
2.2.2.2 Test for flavonoids—- 37
2.2.2.3 Test for glycoside —-37
2.2.2.4 Test for steroids and terpenoids— 37
2.2.2.5 Test for saponins—- 38
2.2.2.6Test for tannins — 38
2.2.2.7 Test for resins —38
2.2.3 Quantitative phytochemical analysis of the homogenate of Cucumis sativus fruit– 39
2.2.3.1 Determination of tannin conten- 39
2.2.3.2 Determination of phenolcontent —– 39
2.2.3.3 Determination of cyanogenic glycosidecontent — 40
2.2.3.4 Determination of glycosidecontent —- 41
2.2.3.5 Determination of flavonoid content —- 41
2.2.3.6 Determination of saponin content——- 42
2.2.3.7 Determination of alkaloid content — 42
2.2.3.8 Determination of steroid content——– 43
2.2.3.9 Determination of reducing sugars content —– 43
2.2.3.10 Determination of resin content——- 44
2.2.3.11 Determination of terpenoid content —– 45
2.2.3.12 Determination of anthocyanin content– 45
2.2.3.13 Determination of chlorophyll content– 46
2.2.4 Proximate analysis———— 46
2.2.4.1 Determination of crude protein content —- 46
2.2.4.2 Determination of moisture content ——— 47
2.2.4.3 Determination of ash content——— 47
2.2.4.4 Determination of crude fibre content– 48
2.2.5 DPPH radical scavenging activity —-48
2.2.6 Acute toxicity studies——– 49
2.2.7 Anti-inflammatory determination using agar-induced rat paw oedema formation — 50
2.2.8 Biochemical tests —– 50
2.2.8.1 Liver function tests —–51
2.2.8.1.1Assay of serum ALT activity —– 51
2.2.8.1.2 Assay of serum AST activity—- 52
2.2.8.1.3 Assay of serum ALP activity—- 53
2.2.8.1.4 Determination of serum bilirubin concentration– 53
2.2.8.2 Lipid profile tests————- 54
2.2.8.2.1 Determination of serum cholesterol concentration – 54
2.2.8.2.2 Determination of serum HDL concentration ——- 54
2.2.8.2.3 Determination of serum TRIG concentration — 55
2.2.8.2.4 Determination of serum LDL concentration—– 56
2.2.9Hypotonicity-induced haemolysis of RBC –56
2.2.10Assay of phospholipase A2 activity ——- 58
2.2.11Assay of prostaglandin synthase activity — 59
2.2.12 Histopathological examination—— 61
2.2.13 Statistical analysis—- 63

CHAPTER THREE: RESULTS

3.1 Qualitative phytochemical constituents of the homogenate of Cucumis sativusfruit—– 64
3.2 Quantitative phytochemical constituents of the homogenate of Cucumis sativusfruit– 64
3.3 Proximate composition of the homogenate of Cucumis sativusfruit — 64
3.4 Acute toxicity (LD50) test of the homogenate of Cucumis sativus fruit—- 64
3.5 Radical scavenging activity of the homogenate of Cucumis sativus fruit — 69
3.6Effect of the homogenate of Cucumis sativus fruit onagar-induced rat paw oedema —- 71
3.7Effect of the homogenate of Cucumis sativus fruit on serum ALT activity of rats treated with CCl4 — 73
3.8 Effect of the homogenate of Cucumis sativus fruit on serum AST activity ofrats treated with CCl4 – 75
3.9 Effect of the homogenate of Cucumis sativus fruit on serum ALP activity of rats treated with CCl4 —- 77
3.10Effect of the homogenate of Cucumis sativus fruit on serumtotal bilirubin concentration of rats treated with CCl4—– 79
3.11Effect of the homogenate of Cucumis sativus fruit on serumtotal cholesterol concentration of rats treated with CCl4– 81
3.12Effect of the homogenate of Cucumis sativus fruit on serum HDL concentration of rats treated with CCl4—— 83
3.13Effect of the homogenate of Cucumis sativus fruit on serumtriacylglycerol concentration of rats treated with CCl4—– 85
3.14Effect of the homogenate of Cucumis sativus fruit on serum LDL concentration of rats treated with CCl4—- 87
3.15.1 Effect of the homogenate of Cucumis sativus fruit on changes in optical
density of hypotonicity-induced haemolysis of RBC— 89
3.15.2 The concentration of haemoglobin ratio to each of oxy-haemoglobin,
deoxy-haemoglobin and methaemoglobin —— 91
3.16 Effect of the homogenate of Cucumis sativus fruit on phospholipase A2 activity ——— 93
3.17Effect of the homogenate of Cucumis sativus fruit onprostaglandin synthase activity —- 95
3.18Histology of the liver of Cucumis sativus homogenate treated rats– 97

CHAPTER FOUR: DISCUSSION

4.1 Discussion——- 104
4.2 Conclusion —– 114
4.3 Suggestions for further studies —– 114
References— 115
Appendices—– 133

 INTRODUCTION

In most rural communities of developing countries, plant materials are sources of shelter, food and medicinal compounds (Oduolaet al., 2005).

Herbal medicine is fast emerging as an alternative treatment to available synthetic drugs for the treatment of disease possibly due to lower cost, availability, fewer adverse effect and perceived effectiveness (Ubakaet al., 2010).

The World Health Organization (WHO) has shown great interest in plant derived medicines which have been described in the folklore medicines of many countries (Mukherjee, 2002).

However, the historic role of medicinal plants in the treatment and prevention of diseases and their role as catalyst in the development of pharmacology do not assure their safety for uncontrolled use by an uninformed public (Matthew et al., 1999).

It is thus, imperative that plant products, which have been used from ages, have scientific support for their efficacy. Medicinal plants with anti-inflammatory activity are considerably employed in the treatment of several inflammatory disorders (Iwuekeet al., 2006).

Research on inflammation has become the focus of global scientific study because of its implication in virtually all human and animal diseases. Many anti-inflammatory plants and agents modify inflammatory responses by accelerating the destruction or antagonizing the action of the mediators of inflammatory reaction (Anosike et al., 2009).

The inflammatory responses involve a complex array of enzyme activation, mediator release, fluid extravasations, cell migration, tissue breakdown and repair.

These different reactions in the inflammatory response cascade are therapeutic targets which anti-inflammatory agents including medicinal plants interfere with to suppress inflammatory responses usually invoked in such disorders as rheumatoid arthritis, osteoarthritis, in infection or injury (Abebe, 2002).

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