ANTIHYPERGLYCAEMIC EFFECTS AND ANTIOXIDANT CAPACITY OF ANDROGRAPHIS
PANICULATA (HEMPEDU BUMI) IN
STREPTOZOTOCIN INDUCED DIABETIC RATS
BY
NADIATUL IKMAL ALIAS
A dissertation submitted in fulfilment of the requirement for the degree of Master of Medical Sciences
Kulliyyah of Medicine
International Islamic University Malaysia
SEPTEMBER 2015
ii
ABSTRACT
Diabetes mellitus (DM) is one of the most common health problems worldwide and the prevalence of this chronic illness rises each year. Majority of treatments for diabetes that are available are focusing on controlling the disease. Thus, there are many attempts to develop safe and effective method for treatment of diabetes due to considerable side effects caused by synthetic drugs used as main treatment for diabetes. Therefore, herbal remedies are used as an alternative treatment.
Andrographis Paniculata (AP) or commonly known as hempedu bumi in Malaysia has been used traditionally as diabetes treatment, taken orally by water concoction. In this study, antihyperglycaemic effects and antioxidant capacity of AP towards diabetes rats were investigated and measured. A number of forty male Sprague Dawley rats (SDR) were used. Thirty of them were administered with as a single dose of Streptozotocin (STZ, 70 mg/kg) intraperitoneally to induce experimental diabetes.
Rats was randomized into four groups (n = 10) consist of normal group (non diabetic), diabetic control (untreated), diabetic treated with AP (500mg/kg bodyweight) and diabetic treated with Metformin with the same dosage. The fresh leaves of AP was dried and extracted via cold maceration using 100% ethanol and later evaporated to become crude extract. Subsequently, blood glucose level was observed and monitored on day 0, 5, 10, 20 and 30 while isoprostane (biomarker for oxidative stress) level was measured on the 30th day of the study using commercially available ELISA kit (Elabsience Biotechnology Co., Ltd, China). Diabetic rats treated with ethanolic extracts of AP showed significant (p<0.05) reduction in blood glucose level compared to untreated group. However, diabetic rats treated with AP showed insignificant reduction of isoprostane level in serum when compared to untreated diabetic rats due to several factors. Thus, the study outcome have successfully proved the antihyperglycaemic effects of AP while further investigation need to be done to improve the immunoassay technique using isoprostane to assesses the antioxidant capacity of AP.
iii
ثحبلا صخلم
رلما اذى راشتناو لماعلا ءانحأ عيجم في اعويش ةيحصلا تلاكشلما رثكأ نم دحاو وى يركسلا ضرم لك عفتري نمزلما ض
تلاوالمحا نم ديدعلا كانى ،لياتلابو .ضرلما ىلع ةرطيسلا ىلع زكرت ةرفوتلما يركسلا ضرلم تاجلاعلا ةيبلاغ .ماع ةمدختسلما ةيعانطصلاا يرقاقعلا اهببست ةيربك ةيبناج راثآ ببسب يركسلا ضرم جلاعل ةلاعفو ةنمآ ةقيرط ريوطتل يركسلا ضرم جلاعل ةيسيئر ةليسوك مادختسا تم دقو .ليدب جلاعك ةيبشعلا تاجلاعلا مادختسا متي ،كلذل .
مفلا قيرط نع ذخؤي ،يركسلا ضرلم جلاعك ايديلقت ايزيلام فى ىموب اوديبميى مساب وفورعلما وا سفارقوردنا لاينوتنب دقو مدلا ركسل ضفاخك لاينوتنبلا صئاصخ ةسارد تتم وساردلا هذى فى .ءالما فى هوهط ةطساوب هدسكلال داضمك اتهر
ددع مادختسا تم .ىركسلا ضرم نم نىاعت ناذرج ىلع 04
تتم مهنم نوثلاث .ليواد غابرس عون ناذرلجا روكذ نم
( ينسوتوزوتبيترسلا نم هدحاو وعربج مهتلجاعم 04
تمسق ناذرلجا .ىركس ىضرم اوحبصيل قافسلا ءاشغ لخاد )غلم
( تاعوممج عبرا لىا ايئاوشع 04
عوممج لك فى ابه وعوممج )يركسلاب وباصم يرغ( وميلس ناذرلجا ابه وعوممج :ىىو )و
( لاينوتنبلاب اهتلجاعم تتمو يركسلاب وباصم وعوممج اهتلجاعم متت لمو ىركسلاب وباصم ناذرلجا 044
وليك لكل غلم
ينوتنبلا وعرج سفنب ينمروفتلماب تلجوعو يركسلاب وباصم وعبارلا وعوملمجاو )مسلجا نزو نم مارغ قارولاا فيفتج تم .لا
زيكترب لوناثيلاا مادختساب درابلا عقنلا وطساوب اهنم صلختسلما رضحو لاينوتنبلا نم وجزاطلا 044
جارختسا تم ثم %
فاج صلختسلما حبصيليرخبتلاب لوناثيلاا .
اهتبقارمو ناذرجلل مسلجا نزو فى تايرغتلا و مدلا في ركسلا ىوتسم ظحول
ةفلتمخ ةينمز تاترف في في
موي 400004.,4 و
04 داهجلال ويجولويبلا وملاعلا( ينتسوربوزيلاا ىوتسم سايق تم ينح في
مويلا فى )ىدسكاتلا 04
ترهظا .)ينصلا ،ايجولونكتلل هدودلمحا سنيس بلايا( تك ازيلايا مادختساب وساردلا نم
نخا لاينوتنبلل لىوناثيلاا صلختسلماب ولجاعلماو يركسلا ءادب ةباصلما ناذرلجا مدلا في ركسلا ىوتسم فى ماىو يربك ضاف
ةعرج مادختساب لاينوتنبلاب جلاعلا امنيب .ةلجاعلما يرغ ةعوملمجاب ةنراقم 044
نم مسلجا نزو نم مارغوليك لكل غلم
ةدلم مفلا قيرط نع ةيذغت بوبنأ للاخ 04
لىوناثيلاا صلختسلماب ولجاعلما يركسلا ءادب ةباصلما ناذرلجا ترهظأ ،اموي
بلل يركسلا ءادب ةباصلما ناذرلجا عم ةنراقلماب مدلا لصم في ينتسوربوزيلاا ىوتسم فى ماى يرغو ليئض ضافنخا لاينوتن
لماوع ةدعل ارظن ةلجاعلما يرغ ،مدلا ركسل ضفاخك لاينوتنبلا صئاصخ حاجنب ةساردلا هذى جئاتن تتبثأ دق ،اذكىو .
ا ةيعانلما ةينقتلا تاصوحف نم ديزلما نا ينح فى ةدسكلأل ةداضلما ةردقلا مييقتو ثابثلا ابه مايقلل جاتتح ةمدختسلم
.لاينوتنبلل
iv
APPROVAL PAGE
I certify that I have supervised and read this study and that in my opinion, it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Master of Medical Sciences
…….……….
Rahajoe Imam Santosa Supervisor
…….………
U Aung Gyi
Co-Supervisor ....………..
Wan. M Azizi Wan.Sulaiman Co-Supervisor
………..
Htike Myat Phyu Co-Supervisor
I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Master of Medical Sciences
...………
Nor Zamzila Abdullah Internal Examiner
This dissertation was submitted to the Department of Basic Medical Sciences and is accepted as a fulfilment of the requirements for the degree of Master of Medical Sciences
.……….………
Zunariah Buyong
Head, Department of Basic Medical Sciences
This dissertation was submitted to the Kulliyyah of Medicine and is accepted as a partial fulfilment of the requirements for the degree of Master of Medical Sciences
..…..………..………
Azmi Md Nor
Dean, Kulliyyah of Medicine
v
DECLARATION
I hereby declare that this dissertation is the result of my own investigations, except where otherwise stated. I also declare that it has not been previously or concurrently submitted as a whole for any other degrees at IIUM or other institutions.
Nadiatul Ikmal Alias
Signature ... Date ...
vi
COPYRIGHT PAGE
INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA
DECLARATION OF COPYRIGHT AND AFFIRMATION OF FAIR USE OF UNPUBLISHED RESEARCH
Copyright © 2015 by Nadiatul Ikmal Alias. All copyrights reserved.
ANTIHYPERGLYCAEMIC EFFECTS AND ANTIOXIDANT CAPACITY OF ANDROGRAPHIS PANICULATA (HEMPEDU BUMI)
IN STREPTOZOTOCIN INDUCED DIABETIC RATS
No part of this unpublished research may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without prior written permission of the copyright holder except as provided below.
1. Any material contained in or derived from this unpublished research may be used by others in their writing with due acknowledgement.
2. IIUM or its library will have the right to make and transmit copies (print or electronic) for institutional and academic purposes.
3. The IIUM library will have the right to make, store in a retrieval system and supply copies of this unpublished research if requested by other
universities and research libraries.
Affirmed by Nadiatul Ikmal Alias
……..……..……… ………..
Signature Date
vii
ACKNOWLEDGEMENTS
Alhamdulillah.. all praise to Allah the Almighty and the Most Merciful for His blessings and giving me strength and ease to complete this journey. Only with His will, I can finish my project. May His peace and blessings be upon the noble Prophet Muhammad (Peace be Upon Him). His faithful companions and dutiful follower till the end of time, amin.
First of all, I would like to express my deepest appreciation to my family especially my husband Mazrul Shazli Mamat for being understanding and supporting me throughout this journey and letting me have personal time to complete my studies, my parents Mdm Noramliah Mohd Amin and Mr Alias B Said who raise me up until I can reach this extent, my siblings and also my in laws family.
Secondly, I would like to express my deepest gratitude to my supervisor;
Assoc Prof Dr Rahajoe Imam Santosa for his guidance, consistent supervision and dedication throughout this study until completion. My heartfelt gratitude also goes to my co-supervisor; Assoc Prof Dr Aung Gyi (Basic Medical Sciences Department), Asst Prof Dr Htike Myat Pyu (Community Medicine Department) Asst Prof Dr Wan Mohd Azizi Wan Sulaiman (Kulliyyah of Pharmacy) for the immense knowledge, constructive advice and assistance.
Thirdly, I would like to extend my appreciation towards the endless support from Basic Medical Sciences Department especially the fatherly guidance from Prof Dr Pakeer Oothuman, Head of Department, Dr Zunariah Buyong, technical support from Br Awang and Sr Radziah. Physiology and pharmacology laboratory technologist Hj Suhaimi, Sr Ainirawati, Sr Shafawati, Br Nizam, Br Fadhli, Br Shahriman and Br Shaiful. Not to forget assistance from Biochemistry Laboratory Sr Shafini, Br Aidil, Br Khusyairi and Br Jamil and Sr Aima from Microbiology Laboratory. May Allah repay your kindness.
Thirdly, I would like to thank the Dean of Kulliyyah of Medicine Prof Dr Azmi Md Nor for allowing the Kulliyyah to fund this research, the Postgraduate Office and all the staffs and postgraduate students from Kulliyyah of Pharmacy for their assistance when I‘m doing my laboratory work there.
Last but not least to my collegues, Sr Fazlin, Sr Sayyada, Sr Aayah and Sr Zinab, Dr Soraya, Dr Sandha and everyone who helped me directly or indirectly until completion of my study. Thank very much to all of you. May Allah repay your kindness. Jazakallahu khairan.
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TABLE OF CONTENTS
Abstract ... ii
Abstract in Arabic ... iii
Approval page ...iv
Declaration ... v
Copyright Page ...vi
Acknowledgements ... vii
List of Tables ... x
List of Figures ...xi
List of Abbreviations ... xii
CHAPTER 1: INTRODUCTION ... 1
1.1 Background of the study ... 1
1.2 Statement of the problem ... 4
1.3 Research objectives ... 4
1.3.1 General objectives... 4
1.3.1 Specific objectives ... 4
1.4 Research questions ... 5
1.5 Research hypothesis ... 5
CHAPTER 2: LITERATURE REVIEW ... 6
2.1 Overview of Andrographis paniculata (AP) ... 6
2.1.1 Traditional claims ... 7
2.1.2 Chemical constituents ... 8
2.2 Pharmacology ... 9
2.2.1 Anti-inflammatory activity ... 9
2.2.2 Antifertility activity ... 10
2.3 Overview of antihyperglycaemic effect of AP ... 10
2.3.1 Mechanism of androgapholide as anti-diabetic agent... 11
2.4 Overview of antioxidant capacity of AP ... 12
2.5 Metformin ... 13
2.6 Oxidative stress markers ... 14
2.6.1 Isoprostanes ... 15
2.7 Animal model in DM study ... 17
2.8 Streptozotocin (STZ)... 17
2.8.1 Mechanism of STZ ... 18
2.9 Rationale of the study ... 20
CHAPTER 3: METHODOLOGY ... 21
3.1 Materials ... 21
3.1.1 Plant material ... 21
3.1.2 Experimental animal ... 22
3.1.3 Chemicals ... 22
3.2 Methods ... 23
3.2.1 Preparation of AP ethanol extracts ... 23
3.2.2 Induction of experimental diabetes ... 24
ix
3.2.3 Experimental design... 25
3.2.4 Study variables ... 27
3.2.5 Blood sample collection ... 27
3.3 Laboratory analysis of the study variables ... 29
3.3.1 Effects of AP on blood sugar level ... 29
3.3.1.1 Principles of Glucose Dehydrogenase Pyrroloquinoline- quinone (GDHPQQ) ... 30
3.3.1.2 Determination of the effects of AP on body weight changes ... 30
3.3.2 Determination of the effect of AP leaves on plasma antioxidant ... 31
Concentration using Rat 8-iso-PG (8-iso prostaglandin) ELISA kit 3.3.2.1 Principle of the test ... 31
3.3.2.2 Reagents preparation ... 31
3.3.2.3 Assay procedure ... 32
3.3.2.4 Result calculation ... 33
3.4 Statistical analysis ... 34
CHAPTER 4 : RESULTS ... 36
4.1 Percent yield of plant extraction ... 36
4.2 Antihyperglycaemic effetcs of AP ethanolic extracts ... 36
4.3 Effects of AP ethanolic extracts on body weight changes in rats ... 39
4.4 Effects of AP ethanolic extracts on isoprostane level in diabetic rat‘s serum ... 41
CHAPTER 5 : DISCUSSION AND CONCLUSION ... 42
5.1 Antihyperglycaemic effect of AP ethanolic extracts ... 42
5.2 Effects of AP ethanolic extracts on body weight changes in rat ... 44
5.3 Effects of AP ethanolic extracts on isoprostane level in diabetic rats‘s serum ... 44
5.4 Conclusion ... 46
5.5 Recommendations ... 46
REFERENCES ... 48
APPENDIX ... 56
x
LIST OF TABLES
Figure No. Page No.
3.1 Animal grouping 26
3.2 Standard concentration and OD value 34
4.1 Blood glucose level (mmol/L) at different time intervals in normal, diabetic untreated, AP treated and Metformin treated group
38
4.2 Comparison between mean blood glucose level in AP treatment group with Normal, Diabetic untreated and Metformin treated
group in day 0, 5, 10, 20 and 30 40
xi
LIST OF FIGURES
Figure No. Page No.
2.1 Pressed AP taken from herbarium 8
2.2 Chemical structures of some major compound present in AP
9
2.3 The formation of 8-isoprostane 16
2.4 The mechanism of STZ induced toxic events in β cells of rat pancreas.
19
3.1 AP leaves in powdered form 19
3.2 Sample size calculation using OpenEpi, Version 2, open source calculator—SSMean
24
3.3 The powdered leaves was soaked in ethanol in a biker 23 3.4 The extract was concentrated using rotary evaporator 24 3.5 Workflow of laboratory analysis of study variables 26 3.6 Obtaining blood sample from rat‘s tail vein using mild
prick with needle
28
3.7 Measurement of blood glucose using Glucometer 28
3.8 Administration of Metformin via oral gavage 29
3.9 Standard curve of 8-iso-PG in the rat‘s serum 34
4.1 Mean blood glucose level from day 0 to day day 30 in Normal, Diabetic untreated, AP treated and diabetic treated rats with Metformin.
37
4.2 Effects of administration of AP ethanolic extracts on body
weight changes from day 0 to day 30 in each study group. 40 4.3 Comparison of serum isoprostane level in each study
group using one way ANOVA. 41
xii
LIST OF ABBREVIATIONS
α Alpha
ABTS 2, 2'-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid
ACTH Andrenocorticotrophis hormone
ADP Adenosine diphosphate
AP Andrographis paniculata
ANOVA Analysis of variance
ATP Adenosine Triophosphate
bw Body weight
β Beta
DC Direct current
DPPH 2,2-Diphenyl-1-Picrylhydrazy
DM DM
DNA Deoxyribonucleic Acid
ELISA Enzyme-linked immunosorbent assay
FBS Fasting blood sugar
g Gram
GDH-PQQ Glucose dehydrogenase pyrroloquinolinequinone
GLUT Glucose transporter
GC/MS Gas chromatography- Mass Spectroscopy
H2O2 Hydrogen Peroxide
HRP Avidin-Horseradish Peroxidase
HSD Honestly significance difference
ICRACU Integrated Centre For Research Animal, Care and Use
ip Intraperitoneal
kg Kilogram
L Litre
M Molar
mbar Millibar
MDA Malondealdehyde
mg Milligram
mIU Meter Interface Unit
ml Millilitre
mmol Millimol
mRNA Messenger Ribonucleic Acid
Mut-Q-GDH Mutant variant of quinoprotein glucose dehydrogenase
NAD+ Nicotinamide adenine dinucleotide
nm Nanometre
OD Optical density
O2 Superoxide anion radical
OH Hydroxyl radical
PBS Phosphate Buffered Saline
pH Potential of Hydrogen Ions
pg Prostaglandin
rpm Revolutions per minute
ROS Reactive oxygen species
xiii
SD Standard deviation
SDR Sprague Dawley Rats
SOD Superoxide Dismutase
SPSS Statistical Package for Social Sciences TBARS Thiobarbituric Acid Reactive Substances
TEAC Trolox-Equivalent Antioxidant Capacity
8-iso-PG 8-iso-protaglandin
µL Microlitre
1
CHAPTER ONE INTRODUCTION
1.1 BACKGROUND OF THE STUDY
Diabetes mellitus (DM) is a clinical syndrome that is common throughout the world.
This chronic disease is represented by elevation of blood glucose, weight loss and polyuria. It is classified into two major types that are Type 1 and Type 2 DM. Type 1 DM is caused by body failure to secrete insulin and requires insulin injection. It is an autoimmune disease resulted by severe damage of β cell Langerhan that produce insulin in the pancreas (Kumar 2012; Kirti et al. 2012). In contrary, Type 2 diabetes is characterized by insufficient synthesis of insulin, due to insulin resistance in the liver, muscle and adipose tissue as well as progressive β cell dysfunction causing hyperglycemia (Kumar 2012).
Due to urbanization, increasing numbers of obesity and sedentary lifestyle, the prevalence of diabetes, mostly by type 2 diabetes has increased, becoming global public health threat. The prevalence among adults aged between 20 -70 years old is expected to rise from 285 million in 2010 to 438 million in 2030. There are high prevalence estimates of diabetes and impaired glucose tolerance (IGT) in all Asian countries and is expected to boost in the next two decades (Unwin et al., 2009). In Malaysia, prevalence of type 2 diabetes among adults >18 years old in 2010 is 10.9%, Ginter et al. (2012) and has increased to 15.2% in 2011 (Institute of Public Health 2011).
In order to prevent the prevalence from rising rapidly, there is various treatments available to cure this disease. Hence, majority of treatment for diabetes that is available are focusing on controlling the disease. Thus, there are many attempts to
2
develop safe and effective method of curing diabetes due to considerable side effects such as hypoglycemia, dropsy and weight gain caused by synthetic drugs and insulin (Tehrani et al., 2010). Therefore, since ancient times, herbal remedies have been used as an alternative to treat DM. Among these remedies, Andrographis paniculata (AP) or commonly known as hempedu bumi in Malaysia is chosen for this study because of its antihyperglycaemic effects (Zhang & Tan, 2000; Husen et al., 2004) and antioxidant capacity (Kumar, 2012). Its leaves and roots are used traditionally as diabetic‘s treatment, taken orally by water concoction (Perry & Metzger 1980; Ahmad
& Asmawi, 1993).
Diabetes is a complex disease that involve interaction between many organs such as pancreas, liver and skeletal muscle. Thus, it requires mammalian species such as rats for complete analysis (Fiona & Roger 2011) as animal models are believed to be predictive for development of drugs (Marianne et al., 2012). During the past decades, Streptozotocin (STZ) induced diabetic rats are used as primary model for diabetic research Penn (2008). STZ is used to inflate and ultimately degenerate β cell Langerhans islet resulting in insulin deficiency (Rastellini et al, 1997; Ikebukuro et al., 2002). Streptozotocin/Izostacin/Zanosar (STZ) is an antitumor antibiotic and is a synthetic antineoplastic agent (Akbarzadeh et al., 2007).
Furthermore, hyperglycaemia has become one of leading factors that contribute to oxidative stress and production of ROS in diabetes (Chiasson et al., 2006). Oxygen which is vital for survival of aerobic organism may change frequently into highly reactive form, known as reactive oxygen species (ROS) that has high toxicity towards the cells Willcox et al. (2004). ROS are produced by normal cellular metabolism and is involved in several redox-regulatory metabolisms Watson et al.
(2011) Nutrients, Dietary Supplements, and Nutriceuticals Totowa, NJ: Humana Press
3
2011). It initiates membrane lipid peroxidation resulting in accumulation of lipid peroxidase thus induced oxidative damage to carbohydrate, protein, lipid and nucleic acid.
Recently, the interest in reactive oxygen species (ROS) is increasing due to its involvement in various pathological situations (Song & Yen, 2002). ROS consists of superoxide anion, alkoxyl radical, nitro oxide, hydrogen peroxide, peroxyl radical and hypochloride (Singh et al., 2012) which are also known as free radical (Eun et al., 2011). In the presence of certain transition metal ions, Superoxide anion radical (O2) and Hydrogen Peroxide (H202) can interact to yield hydroxyl radical (OH) (Valentao et al. 2002). The oxidation induced by ROS can caused multiple effects such as disintegration of cell membrane, damage of membrane protein and DNA mutation which play a vital role in aging and can also propagate the progress of various diseases including cancer, diabetes mellitus and coronary heart disease (Moon et al., 2007). Therefore, it was concluded that oxidants are generated in vivo and resulted in significant cell damage.
In order to overcome the threat of oxidant induced damage, the organic systems need to be protected using antioxidant (Venugopal et al., 2007). The function of antioxidant is to prevent propagation of free radicals that can limit the risk of oxidative stress and associated disorder. They inactivate ROS and inhibit oxidative stress at cellular and molecular level by disrupting lipid peroxidation‘s radical reaction (Triantis et al., 2005). As defined by Gutteridge and Halliwell (2000), antioxidants are substances that can compete with other oxidizable substrate in relatively low concentration. It also serves as defensive factor against free radical. Examples of main antioxidant enzymes are superoxide dismutase, catalase and gluthatione peroxidase (Mozafari 2006).
4 1.2 STATEMENT OF THE PROBLEM
Prevalence of DM is uprising each year resulting as one of the most common health problem worldwide. Therefore, there are necessities to develop safe and effective method to cure diabetes. In this study, AP is chosen to evaluate its suitability for diabetes treatment.
Since hyperglycaemia is one of the leading factors that contribute to production of reactive oxygen species (ROS) in diabetes it is essential to assess the antioxidant effects of AP by measuring its ability to inhibit oxidation induced by ROS in order to evaluate its suitability for diabetes treatment.
1.3 RESEARCH OBJECTIVES 1.3.1 General objective
To determine antihyperglycaemic effects and antioxidant capacity of AP in STZ induced diabetic rats.
1.3.2 Specific objectives
The study aimed to achieve the following objectives:
1. To measure and compare blood glucose level in diabetic rats treated with AP to untreated diabetic rats
2. To measure and compare antioxidant capacity between diabetic rats treated with AP and untreated diabetic rats by measuring isoprostane (biomarker of oxidative stress).
5 1.4 RESEARCH QUESTIONS
1. Can extract of AP reduce blood glucose level in STZ induced diabetic rats?
2. Can AP extracts lower the isoprostane level in STZ induced diabetic rats?
1.5 RESEARCH HYPOTHESES
1. AP extracts may reduce blood glucose level in STZ induced diabetic rats.
2. AP extract may inhibit lipid peroxidation by decreasing the concentration of 8-iso-Prostaglandin F2α.
6
CHAPTER TWO LITERATURE REVIEW
2.1 OVERVIEW OF ANDROGRAPHIS PANICULATA (AP)
The genus Andrographis (Acanthaceae), comprising of about 40 species, is essentially distributed in southern India and in tropical Asia. Only a few species are medicinal, of which AP is the most popular. Andrographis Paniculata Nees (AP) is an erect annual herb extremely bitter in taste in all parts of the plant body. The plant is known in north eastern India as Maha-tita, literally "king of bitters", and known by various vernacular names. As an Ayurveda herb it is known as Kalmegh or Kalamegha, meaning "dark cloud". It is also known as Bhui-neem, meaning "neem of the ground", since the plant, though being a small annual herb, has a similar strong bitter taste as that of the large Neem tree (Azadirachta indica) and is commonly known as Creat or King of bitters in English due to its bitter taste. In Malaysia, it is known as hempedu bumi, which literally means 'bile of earth' since it is one of the bitterest plants that are used in traditional medicine. It is also known as Fah Tah Lai in Thailand, Chuan-Xin-Lin in China and Senshinren in Japan. AP is an annual - branched, erect-running ½ to 1 meter in height in moist shady place. The aerial parts of the plant (leaves and stems) are used to extract the active phytochemicals. It grows abundantly in south-eastern Asia: India, Sri Lanka, Pakistan and Indonesia (Kiritikar & Basu, 1975).
2.1.1 Traditional claims
Traditionally the leaves and roots have been used for different medicinal purpose in Asia. The primary medicinal component of AP is known chemically as ―diterpene lactone‖ based on its ring like structure, having a very bitter taste and is colourless
7
crystalline. Research conducted in ‗80‘s and ‗90‘s has confirmed that AP administration has a surprising broad range of pharmacological effects. It is used to treat various diseases such as liver disorder, heart diseases, stimulates gall bladder contraction and act as antioxidant agent (Zhang & Tan 2000, Valdiani et al., 2012).
In China, the herb derived from the leaves or aerial parts of is known as Chuanxinlian, Yijianxi or Lanhelian. It was described as bitter and cold and was considered to be antipyretic, anti-inflammatory, and detumescent, and is thought to remove ―pathogenic heat‖ from the blood. AP is used for the treatment of pharyngolaryngitis, diarrhea, dysentery, and cough with thick sputum, carbuncle, sores, and snake bites. Various preparations and compound formulas of the herb have been used to treat infectious and non-infectious diseases, with significant effective rates reported for conditions such as epidemic encephalitis B, neonatal subcutaneous annular ulcer, vaginitis, neurodermatitis, eczema, and burns. A primary modern use of AP is for the prevention and treatment of the common cold. It appears to have antithrombotic actions, suggesting a possible benefit in cardiovascular disease.
Pharmacological and clinical studies suggest the potential for beneficial effects in diseases like cancer and HIV infections (Arukamjorn & Nemoto, 2008
8
Figure 2.1 Pressed AP taken from herbarium
2.1.2 Chemical Constituents
Andrographolide (C20H3O5) was the major active component identified in AP and was found in aerial parts. It is known as diterpene lactone and was colourless, crystalline and bitter in taste (Abishek et al., 2008). Other reported compounds include Neandrographolide, 14-Deoxy-11, 12-didehydroandrographolide, 14-Deoxy-11- oxoandrographolide, 14 deoxyandrographolide, 5-Hydroxy 7, 8, 2‘, 3‘-tetramethoxy flavone, andrograpanin, isoandrographolide, skullcaflavone, andrographoneo and
9
andrographoside (Wu et al. 2008). Structures of the compounds is as shown in figure 2.2.
Figure 2.2 Chemical structures of some major compounds present in AP (Niranjan et al. 2010)
2.2 PHARMACOLOGY
2.2.1 Anti-inflammatory activity
The anti-inflammatory activities of andrographolide have been reported by many studies associated with chemical induced inflammation. A study done by Deng (1978) concluded that inflammation caused by histamine, dimethyl benzene and adrenaline
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was significantly reduced by dehydroandrographolide, neoandrographolide and andrographolide. Moreover, Madav et al. (1995) suggested in his study that andrographolide produced a moderate anti-inflammatory activity in different category that is acute, subacute and chronic models of inflammation without adverse effects on gastric mucosa. Recent studies have indicated that andrographolide has anti- inflammatory activity due to modulation of macrophages and neutrophil activity. A study done by Yin and Guo (1993) shows that the anti-inflammatory action of dehydroandrographolide was due to its effects on elevating the synthesis and release of andrenocorticotropic hormone (ACTH) released from the pituitary gland of brain.
2.2.2 Antifertility activity
Several studies done on experimental animals, both male and female have proven that AP has clear anti fertility effects. A study done by Akbarsha and Murugian (2000) found that AP leaves in powder form stopped the development of spermatogenesis in male rats. They also demonstrate the possible mechanism of anti – inflammatory activity by administrating the compound into 3 month‘s old Wistar albino rats at two levels for 48 days. The result from the study showed a decrease in sperm counts along with several abnormal spermatozoa.
2.3 OVERVIEW OF ANTIHYPERGLYCAEMIC EFFECT OF AP
For the past few decades, a number of studies have been published and reported that extracts of AP for both aqueous and ethanolic, and its active components andrographolide possessed antihyperglycaemic effects towards induced diabetic rats.
For instance, Anil Kumar et al (2012) stated that aqueous extracts of AP have significantly reduced fasting serum glucose by 52.9% in STZ induced diabetic rats
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when administered with a dose of 50mg/kg orally twice a day in 14 days duration while ethanol extracts of AP (400mg/kg) give a significant reduction of the same analyte. Similar result was obtained by Jie Xu et al (2012). He reported that administration of 100mg/kg AP twice daily for two weeks shows potent reduction of serum blood glucose. (p<0.01). In addition, Zhang and Tan (2000) concluded that ethanolic extracts of AP exhibit significant difference in fasting serum glucose in day 15 by approximately 10.8% in STZ induced diabetics rats compared to correspondent level in day 1. He used the dosage of 400 mg/kg administered orally twice a day for 14 days.
Similar studies were done in Malaysia by Husen et al. (2004) and Ahmad et al.
(2007). Both of them indicated that treatment of AP extracts to STZ induced diabetics rats have significantly reduced (p<0.05) the fasting blood glucose and insulin level in normal and diabetics rats compared to pre- treatment level. In the study, Ahmad used three different amounts of ethanol extracts that is 85%, 50% and 20%. On the other hand, Husen et al (2004) revealed that administration of 50mg/kg aqueous AP showed the most significant reduction (52.9%) in fasting blood glucose level when compared to Phyllantus niruri Lin, Zingiber zerumbet and Eurycoma Longifolia.
Meanwhile, a study done by Rao (2006) had found that treatment of 50, 100 and 150 mg/kg chloroform extracts of AP roots to STZ induced diabetic rats have significantly reduced fasting blood glucose level by 59.15 % (P<0.001).
2.3.1 Mechanism of andrographolide as anti-diabetic agent
Andrographolide is the main component found in different parts of AP, particularly in the leaves. The mechanism of andrographolide as anti-diabetic agent was studied by Yu et al. (2003) using isolated muscle of STZ induced diabetics rats. From his study,
