Effect of Zingiber Officinale Ethanolic Extract on Liver and Kidney Functions in Rats

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EFFECT OF ZINGIBER OFFICINALE ETHANOLIC EXTRACT ON LIVER AND KIDNEY FUNCTIONS IN RATS

A thesis submitted to the University of Khartoum in partial fulfillment of the requirements for Master of Science in Biochemistry

By Khanssa Ahmed Hamad Hussein B.V.M (2003) University of Khartoum

supervisor DR.Suliman Mohamed EL Hassan

Department of Biochemistry Faculty of Veterinary Medicine January 2010

DEDICATION

To my lovely parents

To my sisters and brothers

To my friends

To all those whom I love

khanssa

ACKNOWLEDGMENTS

I am extremely grateful to my supervisor Dr. Suliman Mohammed El Hassan for his excellent and wise guidance and continuous encouragement during the study period. I am grateful to the staff members of the Medicinal and Aromatic Plants Research Institute (MAPRI). I extend my thanks to the staff members of Laboratory Center in Khartoum teaching hospital especially Susan Mustafa for assistance in analysis of samples. I am highly indebted to the technical assistance of Mohammed Aldo who contributed to this work. Special thanks go to Dr. Shoggy Mohammed Hassan for his assistance in the statistical analysis. Great acknowledgement is extended to my family, friends, teachers and any one donated help and even hopes.

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LIST OF CONTENTS page DEDICATION i ACKNOWLEDGMENTS ii LIST OF CONTENTS iii LIST OF TABLES vi LIST OF FIGURES vii LIST OF ABBREVIATIONS viii ABSTRACT ix ARABIC ABSTRACT x INTRODUCTION 1 CHAPTER ONE: LITERATURE REVEW 1.1 Medicinal plants 2 1.2 Zingiber officinale. 2 1.2.1Taxonomy of Zingiber officinale 2 1.2.2 Ginger 3 1.2.3 General appearance 3 1.2.4 Geographical distribution 4 1.2.5 Nutritional value of ginger root 4 1.2.6 Composition of ginger rhizome 4 1.2.7 Activities and medical uses 5 1.2.8 A current look at ginger use 7 1.2.8.1 Hypolipidemic effect 7 1.2.8.2 Antioxidant effect 7 1.2.8.3 Anti-tumor effect 8 1.2.8.4 Anti-inflammatory effect 9 1.2.8.5 Nausea and emesiss. 9 1.2.8.6 Motion sickness 10 1.2.9 The effect of ginger during pregnancy 11 1.2.10 Precautions 12 CAHPTER TWO: MATERIALS AND METHODS 2.1 Materials 14 2.1.1 Experimental animals 14 2.1.2 Plant material 14

2.1.2.1 Preparation of the ethanolic extract of ginger 14 2.1.3 Experimental design 15 2.1.4 Feeding program 15 2.1.5 Instruments 15 2.1.6 Sample collection 16 2.2 Methods 16 2.2.1 Laboratory analysis 16 2.2.2 Determination of total cholesterol 16 2.2.3 Determination of plasma total protein 18 2.2.4 Determination of plasma albumin 19 2.2.5 Determination of bilirubin 20 2.2.6 Determination of alkaline phoshatase 21 2.2.7 Determination of alanine amino transferase (ALT) 22 2.2.8 Determination of aspartate amino transferase (AST) 23 2.2.9 Determination of creatinine 25 2.2.10 Determination of urea 26 2.2.11 Determination of plasma uric acid 27 2.2.12 Statistical analysis 28 CHAPTER THREE: RESULTS 3.1 The effects of oral administration of Zingiber officinale ethanolic 29 extract on plasma AST, ALT and ALP enzymes in normal rats 3.2 The effects of oral administration of Zingiber officinale ethanolic extract on plasma albumin, total protein, total bilirubin and bilirubin 31 direct in normal rats 3.3 The effects of oral administration of Zingiber officinale ethanolic extract on plasma concentration of urea, creatinine ,uric acid and total 33 cholesterol in normal rats 3.4 The effects of oral administration of Zingiber officinale ethanolic 35 extract on body weight in normal rats CHPTER FOUR: DISCUSSION 37 CONCLUSIONS AND RECOMMINDATIONS 41 REFERENCES 43

LIST OF TABLES

Table (1): The effects of oral administration of Zingiber officinale ethanolic extract on plasma, ALT,AST and ALP enzymes...... 30 Table (2): The effects of oral administration of Zingiber officinale ethanolic on plasma albumin, total protein, total bilirubin and bilirobin direct....32 Table (3): The effects of oral administration of Zingiber officinale ethanolic extract on plasma concentration of urea, creatinine, uric acid and cholesterol …………………………………………………………………………34 Table (4): The effects of oral administration of Zingiber officinale ethanolic extract on body weight of normal rats...... 36

LIST OF FIGURES Figure 1: Ginger plant...... 13 Figure 2: Zingiber officinale rhizome...... 13

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LIST OF ABBREVIATIONS

ALT Alanine amino transferase AST Aspartate amino transferase ALP Alkaline phosphatase Dl Deciliter G Gram Kg Kilogram L Liter Mg Milligram Mmol Millimole NAD+ Nicotinamide adenine dinuclutide (oxidized) P probability SE Standard error

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ABSTRACT The objective of this study was to investigate the effect of ethanolic extract of Zingiber officinale on the level of plasma ALT,AST, ALP, total cholesterol, total albumin, total protein, urea, uric acid, creatinine and bilirubin in nomal Wistar albino rats. The ethanolic extract was prepared using one kilogram of dried powder of Z.officinale and extracted with ethanol (80%).Twenty four healthy Wistar albino rats of either sex were used in this experiment. They were divided into four groups of six rats in each group, the average weight of rats in each group100 g. Group A was kept as control and received only basal diet. Group B, C and D received basal diet and Z.officinale ethanolic extract, which was administrated orally at dose of 200,400 and 800 mg/kg body weight, respectively. Blood samples were collected after 3 weeks and used to estimate some biochemical parameters in plasma. This study showed that Z.officinale decreased numerically the plasma level of ALT, AST, ALP, creatinine, urea and total cholesterol in group B,C and D when compared to the control group. Z.officinale increased numerically the level of plasma total albumin and total bilirubin in group B, C and D when compared to the control group. The level of total protein and direct bilirubin decreased significantly when compared to the control group. However, the level of uric acid was decreased numerically in group B and decreased significantly in group C and D when compared to control group. The rats' body weight was not affected by the administration of Z.officinale ethanolic extract. From the results of this experiment it can be concluded, Z.officinale seems to be not toxic.

ﺍﻟﻤﺴﺘﺨﻠﺹ

ﺍﻟﻬﺩﻑ ﻤﻥ ﻫﺫﻩ ﺍﻟﺩﺭﺍﺴﺔ ﻫﻭ ﻤﻌﺭﻓﺔ ﺘﺎﺜﻴﺭ ﻤﺴﺘﺨﻠﺹ ﺍﻟﻜﺤﻭل ﺍﻻﺜﻴﻠﻲ ﻟﻨﺒﺎﺕ ﺍﻟﺯﻨﺠﺒﻴل ﻋﻠﻲ ﻤﺴﺘﻭﻱ ﺍﻨﺯﻴﻤﺎﺕ (ﺍﻻﻟﻨﻴﻥ ﺍﻤﻴﻨﻭ ﺘﺭﺍﻨﺴﻔﻴﺭﻴﺯ، ﺍﻻﺴﺒﺭﺘﻴﺕ ﺍﻤﻴﻨﻭ ﺘﺭﺍﻨﺴﻔﻴﺭﻴﺯ ﻭﺍﻻﻟﻜﻼﻴﻥ ﻓﻭﺴﻔﺘﻴﺯ)، ﺍﻟﻜﻭﻟﻴﺴﺘﺭﻭل ﺍﻟﻜﻠﻲ، ﺍﻻﻟﺒﻴﻭﻤﻴﻥ ﺍﻟﻜﻠﻲ، ﺍﻟﺒﺭﻭﺘﻴﻨﺎﺕ ﺍﻟﻜﻠﻴﺔ، ﺍﻟﻴﻭﺭﻴﺎ، ﺤﻤﺽ ﺍﻟﻴﻭﺭﻴﻙ، ﺍﻟﻜﺭﻴﺎﺘﻨﻴﻥ ﻭﺍﻟﺒﻠﻴﺭﻭﺒﻥ ﻓﻲ ﺒﻼﺯﻤﺎ ﺠﺭﺫﺍﻥ ﻭﺴﺘﺭ ﺍﻟﺒﻴﻀﺎﺀ.

ﺘﻡ ﺘﺤﻀﻴﺭ ﻤﺴﺘﺨﻠﺹ ﺍﻟﻜﺤﻭل ﺍﻻﺜﻴﻠﻲ ﺒﺎﺴﺘﺨﺩﺍﻡ 1ﻜﻴﻠﻭﺠﺭﺍﻡ ﻤﻥ ﻤﺴﺤﻭﻕ ﺍﻟﺯﻨﺠﺒﻴل ﺍﻟﺠﺎﻑ ﻭ1ﻟﺘﺭ ﻤﻥ ﺍﻻﻴﺜﺎﻨﻭل ﺒﺘﺭﻜﻴﺯ 80%. ﺍﺠﺭﻴﺕ ﺍﻟﺘﺠﺎﺭﺏ ﻓﻲ ﻫﺫﺍ ﺍﻟﺒﺤﺙ ﻋﻠﻲ ﺍﻟﺫﻜﻭﺭ ﻭﺍﻻﻨﺎﺙ ﻤﻥ ﺠﺭﺫﺍﻥ ﻭﺴﺘﺭ ﺍﻟﺒﻴﻀﺎﺀ ﻭﻗﺩ ﺍﺴﺘﺨﺩﻡ 24ﺠﺭﺫ. ﻗﺴﻤﺕ ﺍﻟﺠﺭﺫﺍﻥ ﺍﻟﻲ ﺍﺭﺒﻊ ﻤﺠﻤﻭﻋﺎﺕ ﺘﺤﺘﻭﻱ ﻜل ﻤﺠﻤﻭﻋﺔ ﻋﻠﻲ ﺴﺕ ﺠﺭﺫﺍﻥ ﺒﻤﺘﻭﺴﻁ ﻭﺯﻥ ﺤﻭﺍﻟﻲ 100ﺠﻡ ﻟﻜل ﻤﺠﻤﻭﻋﺔ. ﺍﻟﻤﺠﻤﻭﻋﺔ ﺍﻻﻭﻟﻲ ﻤﺠﻤﻭﻋﺔ ﻀﺒﻁ ﻭﻤﺭﺍﻗﺒﺔ ﻟﻠﻤﺠﻤﻭﻋﺎﺕ ﺍﻻﺨﺭﻱ، ﻭﺍﻟﻤﺠﻤﻭﻋﺎﺕ ﺍﻟﺜﺎﻨﻴﺔ، ﺍﻟﺜﺎﻟﺜﺔ ﻭﺍﻟﺭﺍﺒﻌﺔ ﺠﺭﻋﺕ ﺒﻤﺴﺘﺨﻠﺹ ﺍﻟﻜﺤﻭل ﺍﻻﺜﻴﻠﻲ ﺒﻤﻘﺩﺍﺭ 200، 400 ﻭ800 ﻤل ﺠﺭﺍﻡ ﻟﻜل ﻜﻴﻠﻭﺠﺭﺍﻡ ﻤﻥ ﻭﺯﻥ ﺍﻟﺠﺭﺫﺍﻥ ﻋﻠﻲ ﺍﻟﺘﻭﺍﻟﻲ. ﺒﻌﺩ 3 ﺍﺴﺎﺒﻴﻊ ﻤﻥ ﺒﺩﺍﻴﺔ ﺍﻟﺘﺠﺭﺒﺔ ﺍﺨﺫﺕ ﻋﻴﻨﺎﺕ ﻤﻥ ﺩﻡ ﺍﻟﺠﺭﺫﺍﻥ ﻟﻘﻴﺎﺱ ﻤﻭﺍﺩ ﻜﻴﻤﻭﺤﻴﻭﻴﺔ.

ﻗﻲ ﻫﺫﻩ ﺍﻟﺩﺭﺍﺴﺔ ﻟﻭﺤﻅ ﺍﻥ ﺘﺠﺭﻴﻊ ﻤﺴﺘﺨﻠﺹ ﺍﻟﻜﺤﻭل ﺍﻻﺜﻴﻠﻲ ﻟﻠﺯﻨﺠﺒﻴل ﺍﺩﻱ ﺍﻟﻲ ﺍﻨﺨﻔﺎﺽ ﻋﺩﺩﻱ ﻓﻲ ﺘﺭﻜﻴﺯ ﺍﻨﺯﻴﻤﺎﺕ( ﺍﻻﻟﻨﻴﻥ ﺍﻤﻴﻨﻭ ﺘﺭﺍﻨﺴﻔﻴﺭﻴﺯ، ﺍﻻﺴﺒﺭﺘﻴﺕ ﺍﻤﻴﻨﻭ ﺘﺭﺍﻨﺴﻔﻴﺭﻴﺯ ﻭﺍﻻﻟﻜﻼﻴﻥ ﻓﻭﺴﻔﺘﻴﺯ)، ﺍﻟﻜﺭﻴﺎﺘﻨﻴﻥ، ﺍﻟﻴﻭﺭﻴﺎ ﻭﺍﻟﻜﻭﻟﻴﺴﺘﺭﻭل ﺍﻟﻜﻠﻲ ﻤﻘﺎﺭﻨﺔ ﺒﻤﺠﻤﻭﻋﺔ ﺍﻟﻀﺒﻁ. ﺍﻴﻀﺎ" ﻟﻭﺤﻅ ﺍﺭﺘﻔﺎﻉ ﻋﺩﺩﻱ ﻓﻲ ﺘﺭﻜﻴﺯ ﺍﻻﻟﺒﻴﻭﻤﻴﻥ ﺍﻟﻜﻠﻲ ﻭﺍﻟﺒﻠﻴﺭﻭﺒﻥ ﺍﻟﻜﻠﻲ ﻤﻘﺎﺭﻨﺔ ﺒﻤﺠﻤﻭﻋﺔ ﺍﻟﻀﺒﻁ . ﻜﻤﺎ ﺍﺩﻱ ﺘﺠﺭﻴﻊ ﺍﻟﺯﻨﺠﺒﻴل ﺍﻟﻲ ﺍﻨﺨﻔﺎﺽ ﻤﻌﻨﻭﻱ ﻓﻲ ﺘﺭﻜﻴﺯ ﺍﻟﺒﺭﻭﺘﻴﻥ ﺍﻟﻜﻠﻲ ﻭﺍﻟﺒﻠﺭﻭﺒﻥ ﺍﻟﻤﺒﺎﺸﺭ ﻤﻘﺎﺭﻨﺔ ﺒﻤﺠﻤﻭﻋﺔ ﺍﻟﻀﺒﻁ. ﺒﻴﻨﻤﺎ ﺍﺩﻱ ﺍﻟﻲ ﺍﻨﺨﻔﺎﺽ ﻋﺩﺩﻱ ﻓﻲ ﺘﺭﻜﻴﺯ ﺤﻤﺽ ﺍﻟﻴﻭﺭﻴﻙ ﻓﻲ ﺍﻟﻤﺠﻤﻭﻋﺔ ﺍﻟﺜﺎﻨﻴﺔ ﻭﺍﻨﺨﻔﺎﺽ ﻤﻌﻨﻭﻱ ﻗﻲ ﺍﻟﻤﺠﻤﻭﻋﺘﻴﻥ ﺍﻟﺜﺎﻟﺜﺔ ﻭﺍﻟﺭﺍﺒﻌﺔ. ﻟﻡ ﺘﺘﺄﺜﺭ ﺃﻭﺯﺍﻥ ﺍﻟﺠﺭﺫﺍﻥ ﺒﺘﺠﺭﻴﻊ ﻤﺴﺘﺨﻠﺹ ﺍﻟﻜﺤﻭل ﺍﻻﺜﻴﻠﻲ ﻟﻠﺯﻨﺠﺒﻴل.

ﻤﻥ ﻨﺘﺎﺌﺞ ﻫﺫﻩ ﺍﻟﺘﺠﺭﺒﺔ ﻴﻤﻜﻥ ﺍﺴﺘﻨﺘﺎﺝ ﺍﻥ ﺍﻟﺯﻨﺠﺒﻴل ﻴﺒﺩﻭ ﻏﻴﺭ ﺴﺎﻡ.

INTRODUCTION

Ginger (Zingiber officinale, Roscoe) is a herbaceous rhizomatus perennial plant that is widely cultivated in warm climatic regions of the world such as Nigeria, Taiwan, India, Jamaica and the United States of America. The rhizome contains a spectra of biologically active compounds and several other types of lipids that confers on ginger the characteristic medicinal properties of being pungent and a stimulant (Yoshikawa et al., 1993; Bliddal et al., 2000).

Ginger is a worldwide cultivated rhizome, which is used for various medicinal purposes and as a spice. Some of the undocumented ethno medicinal uses of the peeled rhizome part of the plant, when it is chewed raw, processed or decocted include stimulation of salivary duct secretion, alleviation of toothache, nasal decongestion, cold, cough and asthma. Other uses are as a diuretic, expectorant, anti rheumatic, carminative as well as in infective hepatitis and other forms of liver diseases (Gill, 1992).

Ginger is reported to have various pharmacological activities like antiemetic, antiulcer, anxiolytic, anti-inflammatory and antipyretic activities (Ahmed and Sharma, 1997). Ahmed et al (2000) has shown that addition of ginger (1%) to normal diet of rats prevent formation of free radicals and maintain the integrity of erythrocytes.

Ginger is used in Sudan as spice in coffee and tea, recently ginger has been used in folkloric medicine for treatment of hypercholestermia and hyperlipidemia.

This study aimed to evaluate the effects of Z.officinale ethanolic extract on c liver and kidney functions in Wistar albino rats.

CHAPTER ONE

LITERATURE REVEW

1.1 Medicinal plants:

The current surge of interest in phototherapeutics has led to numerous studies on medicinal plants in many countries. This resulted into a large volume of literature on the taxonomy, biology, chemistry and the recording of the claimed medicine utility of each plant .However, the latter aspect, namely the established clinical value of plant medicines is still lagging in research. Very few plants have been evaluated in systemic manner on medicinal bases and these have largely come from far eastern countries (Toshiyasu et al., 1994; Chen et al., 1985). In the Sudan, numerous plants or plant products are commonly used by rural and urban citizen for the treatment of a wide variety of conditions, often without prior research to prove their claimed efficacy (Bakhiet, 1995; Omer et al., 1992; Ahmed, 1988).

1.2 Zingiber officinale

1.2.1 Taxonomy of Zingiber officinale

Kingdom: Planate

Division: Magnoliphyta

Class: Liliopsida

Order: Zingibeales

Family: Zinigiberaceae

Genus: Zingiber

Specie: Zingiber officinale (ginger)

1.2.2 Ginger:

Ginger is derived from the rhizomes of Zingiber officinale rosc, which is an herbaceous perennial, usually grown as an annual. It originated in south East Asia, but is no known in the wild state. The dried rhizomes may be scraped or peeled before drying and are esteemed for their aroma, flavor and pungency. They may also be used in powdered form (Purseglove et al., 1981).

Ginger is widely used all over the world for culinary purposes in ginger bread, biscuit, cakes, pudding, soups and pickles. It is a frequent constituent of curry powder. It is one of the most widely used spices in Chinese cookery and also used in production of ginger beer, ginger ale and ginger wine. In the west it was formerly much favored for spicing wines, possets and porter, the last being stirred with a red-poker (Purseglove et al., 1981). 1.2.3 General appearance:

Ginger occurs in horizontal, laterally flattened, irregularly branching pieces; 3-16 cm long, 3-4 cm wide, up to 2 cm thick; sometimes split longitudinally; pale yellowish buff or light brown externally.

Longitudinally striated, somewhat fibrous; branches known as "fingers” arise obliquely from the rhizomes, are flattish , obviate, short, about 1-3 cm long; fracture, short and starchy with projecting fibers, internally, yellowish brown, showing a yellow endodermis separating the narrow cortex from the wide stele, and numerous scattered fibro vascular bundles, abundant. Scattered oleoresin cells with yellow contents and numerous lager grayish points, vascular bundles, scattered on the whole surface (Standard of ASEAN Herbal Medicine, 1993, The Japanese PharmacopoeiaX111., 1996).

1.2.4 Geographical distribution:

The plant is probably native to south –East Asia and is cultivated in the tropical regions in both the eastern and western hemisphere. It is commercially grown in Africa, China, India and Jamaica; India is the world's largest producer (African Pharmacopoeia, 1985; Bisset, 1994). 1.2.5Nutritional value of ginger root:

Z.officinale nutritional values per 100g (3.5 oz) are energy 20 kcal carbohydrates 17.77g, sugars 1.7g , dietary fiber 2g, fat 0.75g, protein 1.82g, thiamine(Vit B1) 0.025mg, riboflavin (Vit B2) 0.034mg, niacin (Vit B3) 0.75mg, panothenic acid (B5) 0.203mg, vitamin (B6) 0.16mg, foliate (Vit.B9) 11µg, vitamin C 5mg, calcium 16mg , iron 0.6 mg, magnesium 43 mg, phosphorus 34 mg, potassium 41 mg and zinc 0.34 mg. Z. officinale roscoe contains not less than 2% v/w of volatile oil (Standard of ASEAN Herbal Medicine, 1993), as determinated by the method described in WHO guidelines (Quality Control Methods for Medicinal Plant Material,1998).

Qualitative analysis by thin-layer chromatography; qualitative and quantitative gas chromatography and high performance liquid chromatography analysis of ginger oils showed the presence of gingerols, shogaols, α-zingiberene, β-bisablene, β-sesquiphellandrene, and ar- curcumine (Yoshikawa et al., 1993).

1.2.6 Composition of ginger rhizome:

The rhizome contains 1-4% essential oil and an oleoresin. The composition of the essential oil varies as function of geographical origin, but the chief constituent sesquiterpene hydrocarbons (responsible for the aroma) seem to remain constant. These compounds include (-)-zingiberene, (+)-ar-

4 curcumene,(-)-β-sesquiphellanderene, and β-bisabolene. Monoterpene aldehydes and alcohols are also present. The constituent responsible for the pungent taste of the drug and possibly part of it's anti-emetic properties have been identified as 1-(3'methoxy-4'-hydroxyphenyle)-5hydroxyalkan-3-ones, known as {3-6}-,{8}-,{10}-,and {12}-gingerols (having a side-chain with 7- 10,12,14,or 16 carbon atoms, respectively) and their corresponding dehydration products which are known as shogaols (Awang, 1982).

1.2.7 Activities and medical uses:

Ginger rhizomes are also widely used in foods for their medicinal benefits, especially in Asia. For example, chine women consume ginger vinegar soup following delivery (Chan et al., 2000). According to tradition; the soup provides iron and calcium to the new mother.

Ginger is one of the world's best known spices, and it has also been universally used through out history for its health benefits .The dried extract of ginger contains monoterpenes and sesquterpenes. The main antioxidant active principles in ginger are the gingerol and shogaols and some related phenolic ketone derivatives. Ginger extract possesses antioxidative characteristics, since it can scavenge superoxide anion and hydroxyl radicals (Cao et al., 1993; Krishnakantha and Lokesh, 1993; Reddy and Lokesh, 1992). Ginger also possesses a hepatoprotective activities (Ezeuko et al., 2007; Ajith et al., 2007).

Gingerol from ginger inhibited, at high concentration, ascorbate/ferrous complex induced lipid peroxidation in rat liver microsomes (Reddy and Lokesh, 1992). Gingerol isolated from ginger was shown to inhibit platelet function due to inhibition of thromboxane formation (Guh et al., 1995), and

5 ginger was also suggested to interfere with inflammation processes (Ozaki et al., 1991). It is known that the potency of action of extract may be influenced by the mode of extraction (Stuffness, 1992).

Previous studies indicated that the administration of aqueous extract of ginger to rat orally and intraperitoneally at two different levels of doses did not inflict any histopathological toxicity on the rat (Alnaqeeb et al., 2003). Even though the haemoglobin in the latter route of administration decreased significantly, some serum enzymes activity such as aspartate amino transaminase (AST) alanine amino transaminase (ALT) were affected whereas, the liver acid phosphatase was not (Alnaqeeb et al., 2003). Another study made by Al-Attar and Zari (2007) investigated the effect of ginger oil on diabetic and non-diabetic rats. Ginger oil was administrated orally with different concentrations to the normal and diabetic rats. Ginger oil decreased significantly the level of blood glucose and triglyceride in non- diabetic rats and decreased significantly the level of HDL cholesterol of diabetic rats.

Adanlaw and Dario (2007) investigated the the effect of Zingiber officinale ethanolic extract on some serum enzymes in rats. The rats which were given Zingiber officinale ethanolic extract orally at a dose rat of 100, 200, 300,400,and 500 mg/kg body weight for 28 days, showed that ethanolic extract of ginger root did not have any damage to the heart, brain and the kidney that are vital organs in the body and no change in the enzymes activity.

1.2.8 A current looks at ginger use:

Ginger is used in folk medicine for relief of many ailments, especially nausea motion sickness, and other gastrointestinal disorders (Langer et al.,1998)

1.2.8.1 Hypolipidemic effect:

Ginger acts as a hypolipidemic agent in cholestrol-fed rabbits (Sharma et al., 1996). Feeding rats ginger significantly elevated the activity of hepatic cholestrol-7-hydroxylase, the rate-limiting enzyme in bile acids biosynthesis, thereby stimulating cholesterol conversion to bile acids, resulting in elimination of cholestrol from the body (Srinivasan and Sambaiah, 1991). 1.2.8.2 Antioxidanteffect:

The antioxidant properties of ginger have been shown in various investigations. In one study, the oxidative stress induced by Malathion (a pesticide) into rats was overcomed by introducing ginger into the rats' diet (Ahmed et al., 2000). The antioxidant activity of Zingiber officinale was shown to be as effective as vitamin C in lowering lipid peroxidation in rats by influencing the enzymatic blood level of superoxide dismutase, catalase and glutathione peroxidase (Ahmed et al., 2000). The lipid peroxidation lowering association with ginger consumption was also demonstrated in apolipoprotein E-deficient mice (i.e., mice that are prone to develop atherosclerosis). Mice that consumed ginger (250 mcg of extract/day) in their drinking water showed a significant reduction in their basal concentration of LDL-association lipid peroxides (Fuhrman et al., 2000).

1.2.8.3 Anti-tumor effect:

Ginger is listed among the herbs possessing highest anti-tumor activities. The observed evidence from in vitro experiments warrants further investigations into the potential anti-tumor activity of ginger. This anti- tumor activity was shown to be related to its vanilloids, [6]-gigerol and [6]- paradol. Both compounds were shown to block the epidermal growth factor responsible for cell transformation, thus inhibiting cellular proliferation (Bode et al., 2001; Surh, 1999).

Ginger may also produce its anti-tumor effect by inducing "programmed cell death" also known as apoptosis, in cancer cells (Thatte et al., 2000).

In one study, the cytotoxic effect of [6]-gingerol and [6]-paradol was associated with the induction of apoptosis in human promylocytic leukemia cells (Lee and Surh, 1998).

A third mechanism of ginger's anti-tumor protection is it's modification to the carcinogen-metabolizing enzymes in the liver. Both glutathione s- transferase and aryle-hydrocarbon hydroxylase activity in the liver were elevated following administration of ginger oil to mice for 14 consecutive days (Banerjee et al., 1994).

The application of [6]-gingerol to the shaven backs of mice prior to applying cancer-promotion agents significantly inhibited skin cancer formation (Park et al., 1998).

1.2.8.4 Anti-inflammatory effect:

The Anti-inflammatory effect of [6]-gingerol was studied in a mouse model challenged with aphorol ester. The phenplic compound was capable of suppressing the inflammatory effect of TPA (Park et al., 1998). Male Sprague-Dawley rats with severe arthritis were treated with ginger oil (33 mg/kg per os) for 26 days. The treatment resulted in a significant reduction in joint swelling (Sharma et al., 1994).The oleoresin constituents in Zingiber officinale, as well as the phenolic substances paradol and shogoal, were shown to possess an inhibitory action on cyclooxygenase-2 (COX-2) enzymatic activity, a mechanism important in controlling the inflammatory process (Tjendraputra et al., 2001).

Another mechanism of ginger as an anti-inflammatory herb was proposed to relate to inhibitory effect on leukotriene and prostaglandin biosynthesis, both of which are important in the inflammatory process (Srivastava and Mustafa, 1992; Kiuchi et al., 1992). In a randomized, placebo-controlled, double- blind, crossover study, a ginger extract was compared to ibuprofen in patients with osteoarthritis of the hip and knee. The ibuprofen showed a significant reduction in pain, ginger extract was not significantly different from a placebo when the study was terminated (Bliddal et al., 2000).It appears that issue of using ginger for osteoarthitis or as anti-inflammatory is not supported in human clinical trials.

1.2.8.5 Nausea and emesis:

Some studies (randomized, double-blind, placebo-controlled) have shown that the use of ginger to prevent postoperative nausea and vomiting in women who have undergone gynecological diagnostic laparoscopy was not

9 effective (Visalyaputra et al., 1998; Arfeen et al., 1995). However, another randomized, placebo-controlled, double-blind study involving 60 women who undergone gynecological surgeries shown that the effect of ginger on nausea was similar to that of metoclopramide, both ginger and metoclopramide significantly lowered the incidence of nausea after surgery compared to the placebo group (Bone et al., 1990). In another study, similar results were found involving a larger group of patients (120 women) undergoing the same surgical procedure (Phillips et al., 1993). Evidence from these studies suggests that the usefulness of ginger to alleviate nausea and vomiting is still debatable.

1.2.8.6 Motion sickness:

The uses of ginger root for preventing sea sickness and motion sickness were studied in several studies. In one attempt to prevent sea sickness, ginger root was compared to six other medications in a randomized double- blind of 1.741 tourist volunteers (Schmid et al., 1994). Ginger roots were found to be as effective as five out of the six medications, and superior to scopolamine transdermal therapeutic system in preventing sea sickness. This anti-motion effect of ginger was not shown to be related to its CNS effects (Holtmann et al.,1989). But this anti-motion effect is possibly due to anti- cholinergic and antihistaminic actions (Qian and Liu, 1992). In a separate study, the anti-motion sickness effect of ginger compared to scopolamine or placebo was tested on 28 subjects. Ginger showed no activity against motion sickness in doses as high as 1.000 mg given orally. In contrast, scopolamine (0.6 mg per os) provided significant protection against motion sickness when compared to the control group (Stewart et al., 1991).

Thus the use of ginger for prevention of sea sickness remains controversial. 1.2.9The effect of ginger during pregnancy:

Although ginger is commonly used for morning sickness during pregnancy, no major safety studies of its use has been reported .Scattered studies in animals, however, pointed to ginger's potential harmful effect if used during pregnancy and caution against its use (Wilkinson, 2000).

Ginger tea was given to pregnant Sprague-Dawley rats for 20 days from the day of gestation .The rate of spontaneous loss of fetuses was double in ginger group than in a control group. However, the surviving fetuses were heavier and more developed in the ginger group than those in the control group (Wilkinson, 2000).The potential loss of fetuses after ginger use was also reported in another communication using experimental animal models (Farnsworth et al., 1975). This harmful effect on the fetuses is perhaps linked to blood-thinning properties of ginger, which may facilitate and enhance blood flow. However , other reported experiments failed to show any harmful effect on the developing fetuses in Wistar SPF rats receiving ginger extracts (Weidner and Sigwart, 2001).

In particular, ginger is used for morning sickness during the first trimester of pregnancy. However a recent literature search on ginger concluded that its use during pregnancy has not been shown to be absolutely safe (Wilkinson, 2000). Because of the uncertainty concerning the safety of ginger use, pharmacist should advise women not to include ginger in a self-treatment program before first consulting with their obstetrician /gynecologist's .Until further information on its safety is known, the use of ginger during

11 pregnancy is not recommended. It should be used only after consultation with a healthcare provider.

1.2.10 Precautions:

Although it is not widely recognized by the general public, treatment with herbs can interfere with the action of many drugs, resulting in potentially risk consequences .Ginger can enhance the anti coagulant effect of warfarin, leading to an increased risk of bleeding (Argentor et al., 2000; Miller, 1998). Platelet aggregation in 20 healthy male volunteers was enhanced by the consumption of 100 g of butter per day for one week. Concomitant administration of dry ginger (5g) significantly inhibited platelet aggregation (Verma et al., 1993). In a rat model, however, this interaction between ginger and warfarin was absent (Weidner and Sigwart, 2000). Ginger use is also contraindicated if the patient suffers from gallstones, due to ginger's cholagougue effect (Brinker, 1998).

Figure 1: Ginger plant

Figure 2: Zingiber officinale rhizome

CAHPTER TWO

MATERIALS AND METHODS

This work has been conducted in the Department of Pharmacology in the Medicinal and Aromatic Plants Research Institute (MAPRI), National Center for Research, Khartoum.

2.1 Materials:

2.1.1 Experimental animals:

Twenty four healthy Wistar albino rats of either sex obtained from the National Center for Research were used in this study. The rats were housed identically in cages in a room under suitable conditions. The rats were divided into four groups on the basis of body weight and the average weight of rats in each group was 100g.

2.1.2 Plant material:

Zingiber officinale dried rhizomes were purchased from the local market and authenticated by botanists in the herbarium of the Medicinal and Aromatic Plants Research Institute, National Center for Research, Khartoum.

2.1.2.1 Preparation of the ethanolic extract of ginger:

One kilogram of dried powder of Zingiber officinale was extracted with ethanol (80%) using Soxhelt apparatus for about 16 hours. Extract was then evaporated under reduced pressure using Rota vapor apparatus. Extract was allowed to dry completely and then kept in a refrigerator till used. The extract was dissolved in distilled water before used (Fadoul, 2008).

2.1.3 Experimental design:

The groups were designed as A, B, C and D, six rats were included in each group.

-The rats of group A were kept as normal control.

-The rats of groups B, C and D were served as treated animals.

2.1.4 Feeding program:

Each group was supplied with water and a basal diet of which each kilogram was composed of 725g meat, 240g flour and 1g salt. The feed was available at the rate of 35g per day in each cage. The four groups were fed as follow:

Group A: served as control, received basal diet (BD) only.

Group B: received BD and 200 mg/kg body weight (BWt) Z. officinale ethanolic extract.

Group C: received BD and 400 mg/kg BWt Z. officinale ethanolic extract.

Group D: received BD and 800 mg/kg BWt Z. officinale ethanolic extract.

The ethanolic extract of Z.officinale was administrated orally.

2.1.5 Instruments:

- Heparinized blood containers were used for blood collection.

- Sterile plane containers were used for keeping plasma.

2.1.6 Sample collection:

At day 21, rats were scarified and the blood was collected from them in heparinized containers, the blood was centrifuged at 5000 rpm for 10 minutes. Then the plasma was kept into plane containers and used to estimate some biochemical parameters.

2.2 Methods:

2.2.1 Laboratory analysis:

All plasma parameters were estimated using the Roche diagnostic Hitachi 902 analyzer in Research and Laboratory Unit, Khartoum Teaching Hospital.

Roche diagnostic /Hitachi 902 analyzer:

It is an analyzer to report test results on various body fluid samples for wide range of analysis. It is fully automated, computerized, performs potentiometric and photometric assays, and includes analytical processing unit and luminance crystal display (LCD) touch screen, with a standard printer to print the results. The analyzer is characterized by doing 200 photometric tests/ hour, and refrigerated storage for 40 reagent containers, as well as it has end point, and kinetic and isoenzymes reactions.

2.2.2 Determination of total cholesterol:

Cholesterol was determined according the method designed by (Richmond, 1973).

Principle:

In the presence of cholesterol esterase, the cholesterol esters in the sample are hydrolyzed to cholesterol and free fatty acids. The cholesterol produced is oxidized by cholesterol oxidase to cholesterol-3-one and hydrogen peroxide. Hydrogen peroxide is detected by chromogenic oxygen acceptor, phenol – ampyrone, in the presence of peroxidase. The red quinine formed is proportional to the amount of cholesterol present in the sample.

Reagents and linearity:

Reagent1:

Buffer pH 6.9 50 mmol/L

Phenol 5 mmol/L

Reagent2:

Cholesterol esterase 200 U/L

Cholesterol oxidase 500 U/L peroxidase 3 KU/L

4 – Aminoantipyrine 0.3 mmol/L

Procedure:

The analyzer automatically added an equal quantity from sample, R1 and R2 to the reaction.

Calculations:

The analyzer automatically calculates the analyte concentration of each sample. The result appeared directly on the screen of the diagnostic machine at the end of the reaction between the reagents and samples.

2.2.3 Determination of plasma total protein:

Photometric colorimetric method (Biuret method) was used to determined the total protein in the plasma.

Principle:

Cupric ions in an alkaline solution react with the peptide bonds of proteins and polypeptides containing at least two peptide bonds to produce a violet colored complex. The absorbance of the complex at 546 nm is directly proportional to the concentration of protein in the sample.

OHˉ CU   + Protein Cu-protein complex

Reagents:

Reagents1:

Potassium iodide 30 mmol/L

Potassium sodium tartarat 100 mmol/L

Copper sulphate 30 mmol/L

Sodium hydroxide 3.8 mol/L

Reagen2:

Standard: bovine serum albumin

Procedure:

Procedure is described in 2.2.2

Calculation:

Calculation is described in 2.2.2

2.2.4 Determination of plasma total albumin:

Plasma albumin was determined according to BCG method.

Principle:

At pH= 4.2, albumin bind with bromocresol green to produce a blue – green complex. The change in absorbance at 628 nm correlates with the concentration of albumin.

Reagents:

Reagents1:

Bromocresol green 0.15 mmol /L

Succinate buffer (pH 4.20) 75 mmol / L

Brij 35 7 ml /L

Reagent2:

Albumin standard 50 g/L

Procedure:

Procedure is described in 2.2.2

Calculation:

Calculation is described in 2.2.2

2.2.5 Determination of bilirubin:

Bilirubin concentration in plasma determined according to Van den Berg’s reaction.

Principle:

Bilirubin in the presence of sulphnilic acid reacted with sodium nitrite to form diazotized sulfanilic acid .In the presence of dimethylsulfoxide, total bilirubin reacted with diazotized sulfanilic acid to form azobilirubin .In the absence of dimethylsulfoxide, only the direct bilirubin reacts to give azobilirubin. The absorbance measured at 555nm is proportional to the bilirubin concentration.

Reagents:

Total billirubin

Reagent1:

Sulphanilic acid 3.2 mmol/L

Hydrochloric acid 165 mmol/L

Dimethylsulfoxide 7 mmol/L

Reagent2:

Sodium nitrite 8.6 mmol/L

Bilirubin direct:

Reagent1:

Sulphanilic acid 3.2 mmol/L

Hydrochloric acid 165 mmol/L

Reagent2:

Sodium nitrite 8.6 mmol/L

Procedure:

Procedure is described in 2.2.2

Calculation:

Calculation is described in 2.2.2

2.2.6 Determination of alkaline phoshatase:

The concentration of ALP in the plasma was determined by optimized standard method.

Principle:

Under alkaline condition, p-nitrophenol is converted to 4-nitrophenoxide, which develop a very intense yellow color. The intensity is proportional to the activity of alkaline phosphatase in the sample. p-nitrophenyl phosphate + H2O phosphate + p-nitrophenol

Reagents:

Reagent1:

2-Amino-2-methyl-1-propanol 0.9 mol/l

pH 10.4

Magnesium acetate 1.6 mmol/l

Zinc sulphate 0.4 mmol/l

EDTA 2 mmol/1

Reagent2:

P-nitrophenyl phosphate 16 mmol/l

Procedure:

Procedure is described in 2.2.2

Calculation:

Calculation is described in 2.2.2

2.2.7 Determination of alanine amino transferase (ALT):

The activity of ALT was measured by Reitman’s method.

Principle:

ALT catalyses the transformation of L-alanine and 2-oxoglutarate at optimal pH . The pyruvate released in the reaction is transformed by Lactate dehydrogenase (LDH) in the presence of NADH/NAD coenzyme to L-lactate .while the NADH/NAD oxidoreductive process shows a decrease

22 in absorbance at 340 nm .The change in absorbance correlates with serum ALT activity.

ALT L-Alanine + α-Ketoglutarate pyruvate-L-Glutamate

LDH Pyruvate + NADH L-Lactate-NAD

Reagents:

Reagent1:

Tris buffer, pH: 7.50 110 mmol/L

L-Alanine 600 mmol/L

LDH 1500 u/L

NADH 240 µmol/L

Reagent 2:

α-Ketoglutarate 16 mmol/L

Procedure:

Procedure is described in 2.2.2

Calculation:

Calculation is described in 2.2.2

2.2.8 Determination of aspartate amino transferase (AST):

The activity of AST was measured by Reitman’s method.

Principle:

Two substrates participate in the reaction catalyzed by AST .L- aspartate and oxolglutarate (With the help of NADH coenzyme) .Malate dehydrogenase (MDH) contained in the reagent catalyses the transformation of oxalacetate released in the first reaction .The oxido-reductive process of NADH/NAD is indicated by a decrease in absorbance at 340 nm.The lactate dehydrogenase (LDH) in the medium counteracts the disturbing effect of pyruvate contained in the sample.

AST L-Aspartate + α-Ketoglutarate L-glutamate -Oxalacetate

MDH Oxalacetate + NADH L-Malate -NAD

Reagents:

Reagents1:

Tris buffer, pH: 7.80 88 mmol/L

L-Aspartate 260 mmol/L

LDH 1500 u/L

MDH 900 u/L

NADH 0.24 mmol/L

Reagent 2:

α-Ketoglutarate 12 mmol/L

Procedure:

Procedure is described in 2.2.2

Calculation:

Calculation is described in 2.2.2

2.2.9 Determination of creatinine:

Kinetic method without deproteinisation-jaffe reaction was used to estimate the creatinine concentration in the plasma.

Principle:

Creatinine under alkaline conditions reacts with picrate ions forming a reddish complex. The formation rate of the complex measured through the increase of absorbance in a prefixed interval of time is proportional to the concentration of creatinine in sample.

Reagents:

Picric acid 40 mmol/l

Potassium ferricyanide 40 µmol/l

Phosphate buffer 300 mmol/l

Creatinine Standard 177 mmol/l

Procedure:

Procedure is described in 2.2.2

Calculation:

Calculation is described in 2.2.2

2.2.10 Determination of urea:

Enzymetic colorimetric, endpoint-Berthelot method was used to measure the urea level in the plasma.

Principle:

Urea +2 H2O urease 2 NH4 + 2HCO3

NH4 +2-Oxoglutrate + NADH GLDH LGlutamate + NAD +H2O

Reagents:

Reagent 1:

Tris buffer 120 mmol/l

pH 7.8

2- Oxoglutrate 7 mmol/l

ADP 0.6 mmol/l

Urea 1 mmol/l

Urease > 5 KU/l

Glutamate dehydrogenase > 1 KU/l

Reagent 2:

NADH 0.25 mmol/l

Procedure:

Procedure is described in 2.2.2

Calculation:

Calculation is described in 2.2.2

2.2.11 Determination of plasma uric acid:

The plasma uric acid concentration was measured by method based on the use of the enzyme uricase.

Principle:

Uric acid is converted into allantoin by the action of the enzyme uricase, allantoin by the action of the enzyme peroxidase (POD) in the presence of 4-aminoantipyrine (4-AAP) is converted to chinonimine .The absorbance of which is measured at 540 nm is proportional to uric acid concentration.

Reagents:

Reagent 1:

Phosphate buffer, pH 7 100 mmol/l

Reagent 2:

4-aminoantipyrine 0.3 mmol/l

K4 (Fe (CN) 6) 10 mmol/l

Peroxidase 2 U/L

Uricase 30 U/L

Procedure:

Procedure is described in 2.2.2

Calculation:

Calculation is described in 2.2.2

Quality control:

The precision and accuracy of all methods used in this study were checked each time, a batch was analyzed by including commercially prepared control sera.

2.2.12 Statistical analysis:

The results were analyzed by ANOVA procedure according to SAS

Software. Each test was conducted at 5% level of significance. Means were

Separated by multiple comparisons range test.

CHAPTER THREE

RESULTS

3.1 The effects of oral administration of Zingiber officinale ethanolic extract on plasma AST, ALT and ALP enzymes in normal rats:

Z.officinale ethanolic extract at the different three concentrations when administrated orally to normal rats in this experiment, resulted in mild decrease in plasma AST. Also the plasma concentrations of ALT and ALP decreased numerically when compared to the control rats as shown in Table1.

Table1: The effects of oral administration of Zingiber officinale ethanolic extract on plasma AST, ALT and ALP enzymes:

Groups Parameter A B C D Probability

AST

U/L 167.40a±11.1 159.50a±12.7 152.50a±11.5 166.00a±9.93 0.7566

ALT

U/L 64.60a±6.15 42.25a±4.03 49.17a±4.28 60.00a±7.67 0.0703

ALP

U/L 253.00a±84.4 147.00a±16.3 190.00a±15.30 140.60a±16.80 0.1105

Data are expressed as mean± standard error

Mean values having different superscripts within the same row differ significantly at P≤0.05.

Group A: Normal control rats

Group B: Normal rats dosed with 200 mg/kg Z.officinale ethanolic extract.

Group C: Normal rats dosed with 400 mg/kg Z.officinale ethanolic extract.

Group D: Normal rats dosed with 800 mg/kg Z.officinale ethanolic extract.

3.2 The effects of oral administration of Zingiber officinale ethanolic extract on plasma albumin, total protein, total bilirubin and bilirubin direct in normal rats:

The administration of Z.officinale ethanolic extract showed a mild increase in plasma total albumin concentrations in the treated groups.

The three concentrations of Z.officinale in this experiment decreased significantly plasma total protein in all treated groups when compared to the control group.

Z.officinale increased numerically the levels of total bilirubin in all treated groups. However, the levels of bilirubin direct decreased significantly in all treated groups when compared to the control group as shown in Table2.

Table2: The effects of oral administration of Zingiber officinale ethanolic extract on plasma albumin, total protein, total bilirubin and bilirubin direct:

Groups Parameter A B C D Probability

Total albumin a a a a 3.68 ±0.36 4.05 ±0.17 4.05 ± 0.17 4.02 ± 0.18 0.6255 g/dl

Total protein a b b b 7.16 ±0.71 5.03 ±0.41 5.34 ±0.19 5.08 ±0.22 0.0119 g/dl

Total bilirubin a a a a 0.14 ±0.02 0.25 ±0.03 0.28 ±0.07 0.36 ±0.09 0.1257 mg/dl

Bilirubin direct 0.05a±0.13 0.01b±0 0.01b±0 0.01b±0 0.0003 mg/dl

Data are expressed as mean± standard error

Mean values having different superscripts within the same row differ significantly at P≤0.05.

Group A: Normal control rats.

Group B: Normal rats dosed with 200 mg/kg Z.officinale ethanolic extract.

Group C: Normal rats dosed with 400 mg/kg Z.officinale ethanolic extract.

Group D: Normal rats dosed with 800 mg/kg Z.officinale ethanolic extract.

3.3 The effects of oral administration of Zingiber officinale ethanolic extract on plasma concentration of urea, creatinine, uric acid and total cholesterol in normal rats:

The administration of three concentrations of Z.officinale ethanolic extract decreased numerically the plasma concentrations of urea and creatinine in all treated groups when compared to the control.

Z.officinale also had no significant effect on plasma uric acid of group B. But, in higher concentrations it deceased significantly the plasma uric acid levels of group C and D when compared to the control and also when compared to group B as shown in Table3.

The administration of Z.officinale with different concentrations had no significant effect on plasma total cholesterol. There was a numerical reduction in cholesterol as shown in Table3.

Table (3):The effects of oral administration of Zingiber officinale ethanolic extract on plasma concentration of urea, creatinine, uric acid and cholesterol:

Groups Parameter A B C D Probability

Urea 53.40a±3.67 41.00a±3.00 30.67 a ±8.11 37.60a±2.84 0.0639 mg/dl

Creatinine 0.74a±0.13 0.28a±0.03 0.47a±0.19 0.32a±0.02 0.1174 mg/dl

Uric acid 1.96a±0.23 1.30 ab±0.27 1.03b±0.12 1.14b±0.13 0.0130 mg/dl

Total cholesterol 65.20a±7.08 57.50a±3.93 55.00a±3.10 57.40a±5.77 0.5295 mg/dl

Data are expressed as mean ± standard error

Mean values having different superscripts within the same row differ significantly at P≤0.05.

Group A: Normal control rats

Group B: Normal rats dosed with 200 mg/kg Z.officinale ethanolic extract.

Group C: Normal rats dosed with 400 mg/kg Z.officinale ethanolic extract.

Group D: Normal rats dosed with 800 mg/kg Z.officinale ethanolic extract.

3.4 The effects of oral administration of Zingiber officinale ethanolic extract on body weight in normal rats:

The three concentrations of Zingiber officinale ethanolic extract had no significant effects on rats' body weights in all treated groups as shown in Table 4.

Table (4): The effects of oral administration of Zingiber officinale ethanolic extract on body weight of normal rats:

Groups Parameter A B C D Probability

Pre body weight (g) a a 99.9 ±1.826 100 ±3.873 0,9984 100.1a±2.887 101 a ±3.162

a Post body 103.3 ±1.054 a a 0,9666 1o1.7 ±4.595 103.3 ±3.57 weight (g) a 101.7 ±2.789

Data are expressed as mean ± standard error

Mean values having different superscripts within the same row differ significantly at P≤0.05.

Group A: Normal control rats

Group B: Normal rats dosed with 200 mg/kg Z.officinale ethanolic extract.

Group C: Normal rats dosed with 400 mg/kg Z.officinale ethanolic extract.

Group D: Normal rats dosed with 800 mg/kg Z.officinale ethanolic extract.

CHAPTTER FOUR

DISCUSSION

Common available Zingiber officinale preparations and different extractions are widely used for certain therapeutic purposes including interfering with inflammatory processes (Ozaki et al., 1991) and ginger is listed among the herbs possessing the highest antitumor activities (Bode et al., 2001; Surh, 1999). The protective action of ethanolic ginger extract in cholesterol fed rabbits was examined experimentally (Sharma et al., 1996).

This study aimed to examine the effect of oral adminisration of the ethanolic extract of Zingiber oficinale at different concentrations on plasma ALT, AST, alkline phosphatase, cholesterol, total albumin, total protein, urea, creatinine, uric acid, total bilirubin, bilirubin direct and body weight in normal Wistar albino rats .

In this study, the adminisratoin of Z. officinale ethanolic extract to normal rats had no significant effect on plasma levels of AST, ALT and ALP. This indicated that there were no pathological changes occurred in the liver, kidney or muscles (Coles, 1986). It was noticed in this experiment that there was a mild decrease in ALT and AST levels in all treated groups, this agrees with Al-Attar and Zari (2007) who reported that the administration of ginger oil to normal rats did not affectet the levels of ALT and AST significantly. However, the treatment of streptozotocin-induced diabetic rats with ginger oil noticeably restored the values of blood ALT activity to the control levels and decreased numerically the levels of AST.

Also ALP levels decreased numericaly in this experiment, this agrees with Ajith ,et al.(2007) who found that the aqueous ethanol extract of Z. officinale significantly protected acetaminophen-induced acute hepaotoxiciy in rats. The administration of a single dose of aqueous extract of Z. officinale (200 and 400 mg/kg) prior to acetaminophen significantly declined the activities of serum ALP.

In this study, the administration of Z. officinale ethanolic extract reduced numerically the plasma cholestrol concentrations. Z. officinale stimulating cholestrol conversion to bile acids, resulting in elimination of cholestol from the body (Srinivasan and Sambaiah, 1991). This agrees with Al-Attar and Zari.(2007) who found that ginger oil decreased numerically the cholestrol concentrations of normal rats. Also the treatment of streptozotocin-induced diabetes in rats with ginger oil reduced numerically the cholesterol concentration.

Z.officinale ethanolic extract had no significant effect on plasma total albumin, this also might support that there was no deleterious effects on liver function. Coles (1986) reported that the detections of alteration in the various fractions of plasma proteins may have some value in liver damage.

In this study the plasma total proteins were decreased significantly in all treated groups when compared with the control group. This might not be an indicator for liver damage. Coles (1986) suggested that although the liver plays an important role in protein metabolism, alterations in serum proteins are not specific for liver damage and this hypoproteinemia is most commonly associated with a lack of proper diet or poor absorption of dietary constituents from the intestinal tract. This disagrees with Al-Attar and Zari

(2007) who found that the administration of ginger oil to normal rats did not affect the level of total protein, however, the treatment of streptozotocin- induced diabetic rats with ginger oil decreased numerically total protein levels.

The administration of 200 mg/kg of Z.officinale ethanolic extract decreased the plasma uric acid concentration numerically, while, the administration of 400 and 800 mg/kg decreased plasma uric acid significantly when compared to the control group, this might be associated with the hypoproteinemia. In fact uric acid appears from the degradation of purines (Murry e t al, 2000) and the protein diet is rich in purine. This also disagrees with Al-Attar and Zari (2007) who found that the administration of ginger oil to normal rats showed no alteration in uric acid levels but, the treatment with ginger oil decreased numerically uric acid of streptozotocin-induced diabetic rats.

In this experiment there was mild increased in plasma total bilirubin levels of all treated groups. However, the bilirubin directs decreased significantly in all treated groups when compared to the control group.

The plasma urea concentrations were not affected significantly by Z.officinale ethanolic extract this indicated that Z.officinale probably has no deleterious effect on liver or kidney of normal rats. This agrees with Al- Attar and Zari (2007) who found that the administraion of ginger oil to normal rats showed no alteration in urea concentration but, the adminisration of ginger oil to streptozotocin-induced diabetic rats decreased numerically urea concentrations.

In this study the plasma creatinine levels were decreased numerically in all treated groups, that means Z.officinale had no harmful effect on the kidney

40 function, Creatinine has had a reputation of being a more specific test for the diagnosis and prognosis of progressive renal disease ( Coles, 1986). This agree with Al-Attar and Zari (2007) who found that the administraion of ginger oil to normal rats did not affected creatinine levels, while the treatment of streptozotocin-induced diabetic rats with ginger oil decreased significantly creatinie levels. Moreover, ginger oil noticeably restored the value of blood creatinine to the control levels.

In this experiment rats body weights were not affected significantly by the administration of Z. officinale ethanolic extract. There were mild increase on rats body weight in all treated groups. Ezeuko Vitalis et al (2007) found the supplementation of 10% ginger to the diet of mercuric chloride-induced hepatotoxic rats increased the mean weight of the rats.

CONCLUSIONS AND RECOMMENDATIONS

CONCLUSIONS

From observations and results of these investigations, it can be concluded that:

1- The administration of Z. officinale ethanolic extract to normal rats reduced numerically the plasma cholesterol concentration.

2-The administration of Z. officinale ethanolic extract showed no effect on liver or kidney functions.

3-The administration of Z. officinale ethanolic extract to normal rats had no effect on rats body weights.

4-Thus, it seems ginger has no toxic effect on rats.

RECOMMENDATIONS

Since Zingiber officinale has multiple uses, it is recommended that:

1- The safety of Z .officinale should be examined by administering very high doses for long period of time.

2- Future research should be carried out to determine the effect of Z.oficinale in the absorption and digestive functions of protein.

3- The effect of other extract of Z. oficinale on liver and kidney functions, should be examined.

REFERENCES

Adanlawo, L.G. and Dario,F.A.S. (2007). Nutrient and anti-nutrient constituents of ginger (Zingiber officinale, Roscoe) and the influence of its ethanolic extract on some serum enzymes in Albino rats. International Journal of Biological Chemistry, 1(1):38-46

African Pharmacopoeia. (1985). Organization of African Unity, Scientific, Technical & Research Commission, Vol. 1. lst ed. Lagos.

Ahmed, O.M.M. (1988). Toxicological studies on fruit of Balanites aegyptiaca (Hijlij tree). Ph.D. Thesis, U of. K. Sudan.

Ahmed, R.S. and Sharma, S.B. (1997). Biochemical studies on combined effects of garlic (Allium sativum Linn.) and ginger (Zingiber officinale Rosc.) in albino rats. Ind. J. Exp. Biol., 35: 841-843.

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