DISSERTATION ON
“IN VITRO ANTIMUTAGENIC ACTIVITY OF ECLIPTA ALBA USING AMES TEST”
Dissertation submitted to
THE TAMILADU DR. M.G.R. MEDICAL UNIVERSITY In partial fulfillment of the regulations
For the award of the degree of M.D. PHARMACOLOGY – BRANCH VI
CHENNAI MEDICAL COLLEGE HOSPITAL AND RESEARCH CENTRE, IRUNGALUR, TRICHY – 621 105
THE TAMILNADU DR. M.G.R. MEDICAL UNIVERSITY CHENNAI – 600 032
APRIL 2018
CERTIFICATE
This is to certify that the dissertation entitled “IN VITRO ANTIMUTAGENICACTIVITY OF ECLIPTA ALBA USING AMES TEST” by Dr.
LEENA RANJINI V., Postgraduate in Pharmacology (2015 – 2018), is a bonafide research work carried out under our direct supervision and guidance and is submitted to The Tamilnadu Dr. M.G.R. Medical University, Chennai for M.D. degree Examination in Pharmacology, Branch IV, to be held in April 2018. The period of study was from 2015 – 2018.
Dr. S. Vijayarangan, M.D., Dr. K. Vasanthira, M.D., Associate Professor Professor and Head
Department of Pharmacology Department of Pharmacology Chennai Medical College Hospital Chennai Medical College Hospital
and Research Centre, and Research Centre,
Irungalur, Trichy Irungalur, Trichy
Dr. Sukumaran Annamalai, M.D., D.H.H.M., Dean
Chennai Medical College Hospital and Research Centre, Irungalur, Trichy
DECLARATION
I, Dr. Leena Ranjini V. solemnly declare that the dissertation title “IN VITRO ANTIMUTAGENICACTIVITY OF ECLIPTA ALBA USING AMES TEST” was done by me at Chennai Medical College Hospital and Research Centre, Irungalur, Trichy, under the supervision and guidance of Dr. S. Vijayarangan, Associate Professor of Pharmacology
This dissertation is submitted to the Tamilnadu Dr. M.G.R. Medical University, towards the partial fulfillment of requirement for the award of M.D. Degree (Branch-VI) in Pharmacology.
Place: Irungalur, Trichy Dr. LEENA RANJINI V.
Date: Postgraduate Student
Department of Pharmacology Chennai Medical College Hospital and Research Centre (SRM group) Irungalur, Trichy
GUIDE CERTIFICATE GUIDE
Dr. S. Vijayarangan, M.D., Associate Professor
Department of Pharmacology
Chennai Medical College Hospital and Research Centre Irungalur, Trichy
CO-GUIDE
Dr. N. Prabhusaran, Ph.D., (D.Sc.,)
Associate Professor of Research and Microbiology Chennai Medical College Hospital and Research Centre Irungalur, Trichy
Remarks of the Guide:
The work done by Dr. Leena Ranjini V. on titled “IN VITRO ANTIMUTAGENICACTIVITY OF ECLIPTA ALBA USING AMES TEST” is under my supervision and I assure that this candidate has abide the rules of the Ethical committee.
Guide: Dr. S. Vijayarangan, M.D., Associate Professor
Department of Pharmacology
Chennai Medical College Hospital
and Research Centre,
Irungalur, Trichy
CERTIFICATE – II
This is to certify that this dissertation work titled INVITRO ANTIMUTAGENIC
PROPERTIES OF ECLIPTA ALBA USING AMES TEST of the candidate DR.V.LEENA
RANJINI with register number 201516502 for the award of M.D PHARMACOLOGY
in the branch of VI. I personally verified the urkund.com website for the purpose
of plagiarism check. I found that the uploaded thesis file contains from
introduction to conclusion pages and results shows 7% percentage of plagiarism
in the dissertation.
Guide & Supervisor sign with seal
ACKNOWLEDGEMENT
First and foremost, I pray god almighty and my Parents for showing their blessings on me in the successful completion of this research work.
I express my thanks to Honourable Chairman Dr. R. Shivakumar, MD., Ph.D., Dean Dr. A. Sukumaran Annamalai, MD., DHHM., Medical Superintendent Dr. P.
Anusuya, MD., and Vice Principal Dr. Gurudatta S. Pawar., MD., CMCH&RC, Tiruchirapalli, for providing opportunity to mould a stone for my path.
I express my sincere thankfulness to my research guide Dr. S. Vijayarangan, M.D., Associate Professor, Department of Pharmacology, Chennai Medical College Hospital and Research centre (CMCH&RC) (SRM Group), Tiruchirapalli, for his steadfast, generous, scrupulous, valuable guidance and motivation for successful completion of my research.
I am very thankful to Co-guide Dr. N. Prabhusaran, Associate Professor of Research and Microbiology, Institutional Research Board, CMCH&RC, Tiruchirapalli, for his scrupulous guidance, help, encouragement and suggestions.
I express my heartfelt thanks to Dr K. Vasanthira, MD., Professor and Head of Pharmacology, CMCH&RC, Tiruchirapalli, for her motivation, enthuciasm and timely support.
I record my thanks to my department teachers Dr. P. Revathi, MD., Former Associate Professor, Dr. S. Kanaga Santhosh, MD., Assistant Professor, Dr. T.
Nivethitha, MD., Assistant Professor, Tutors, and co-postgraduate friends, Department of Pharmacology for their continuous encouragement and motivation during the study.
I also acknowledge all the Technicians and Attenders of Department of Pharmacology and Institutional Research Board for their unending support and secretarial assistance.
Dr. V. Leena Ranjini
ABBREVIATIONS 2-AF - 2-aminofluorene
AFB1 - Aflatoxin B
CC - Cytosine-cytosine
DNA - Deoxyribonucelic acid
EEEA - ethanolic extract of Eclipta alba EMS - ethyl methane sulphonate
GC-MS - Gas chromatography – mass spectroscopy
his - histidine
MMC - Mitomycin C
NaN3 - Sodium azide
NPD - O-nitro phenelene diamine NQO - Nitroquinoline 1-oxide
OECD - Organization for Economic Co-operation and Development rfa - rough characters
RNA - Ribonucleic acid
RNS - Reactive nitrogen species ROS - Reactive oxygen species
TC - Thymidine-cytosine
TT - Thymidine-thymidine
UVR - Ultra violet radiation WHO - World Health Organization
CONTENTS
Chapter No. Particulars Page No.
1 INTRODUCTION 1
2 AIM AND OBJECTIVES 10
3 REVIEW OF LITERATURE 11
4 MATERIALS AND METHODS 41
5 RESULTS 57
6 DISCUSSION 71
7 CONCLUSION 77
REFERENCES
INTRODUCTION
World Health Organization (WHO), defined the Cancer as a generic term for various diseases and disorders that are characterised by abnormal growth of cells compared to their boundaries that can leads to multiorgan dysfunction (MoD) to multiorgan failure (MoF). Among the mortality description, 8.8 million death were accounting for 1in 6 of all global deaths whereas, 70% of deaths are from developing countries. Lung, prostate and stomach cancers are common among males and breast, cervix, colorectal, stomach are common among female population.
In general, cancer is a result of interaction of genetic factors with three main components
Physical carcinogen including ionising and Ultraviolet radiation (UVR)
Chemical carcinogen including tobacco, aflatoxin (food contaminant) and asbestos
Biological carcinogen including viral, bacterial and parasitic infections
The defects of innate metabolism occur in the cellular system are mainly caused by mutations that leads in elevating morbidity and mortality. Mutation is considered as one of the important factors in carcinogenesis. Thus, the rate of cancer prevalence may be minimized by reduced rate of mutation. The most effective preventive measures to cut down the rate of mutation induced by mutagens and carcinogens is to avoid the exposure to them1.
Mutagen and Carcinogen
Mutagen is an agent that activates the genetic material in cells whereas
carcinogen is a substance that enhances the undifferentiated growth of cells leads to cancer. Eventhough mutagen and carcinogen are not same in origin, chemistry and action, the cancer induction rate are equally and strongly linked. Mutagenesis is a process that involved in modifications and also induces the tumor formation. DNA- adducts represented the mutagenicity of a chemical substance as well as increase in cancer risk2.
The specificity and sensitivity of mutagens in eukaryotic system is directly relative to the inferences of certain environmental mutagens that cause human cancers.
Ultraviolet light and aflatoxin B1 (AFB1) have long been suspected of causing skin cancer and liver cancer, respectively. Recent research suggested that the DNA based
sequencing of mutations in a human cancer gene has provided scientific evidences. The important mechanism takes place in the cancer patients are well defined to mutate tumor-suppressor genes (p53 that encodes proteins)3.
The prevalence of liver cancer in developing countries like India is mainly due to the high exposure to AFB1. While analyzing tumour suppressor gene p53 mutations in cancer patients, Guanine to Thymidine transversions and the fingerprint of AFB1- induced mutations, were documented in liver cancer patients from developing counties
like India but not found among lung, colon, or breast cancer. The observation of p53 mutations in liver cancer patients from areas of low AFB1 exposure did not result from G → T transversions. Thus the results from the mutagenic specificity studies of AFB1 provide us AFB1-induced mutations play an important role in liver cancer.
UVR mutagenesis
The inter-relationship of exposure to ultraviolet rays (UVR) and human skin cancers are identified by sequencing p53 mutations especially among invasive human squamous cell carcinomas. Further in UVR induced mutations at the sites of dipyrimidine authenticated the role of Cytosine to Thymidine substitutions when the C is the 3′ pyrimidine of a TC dimer. Studies highlighted that several tumours have p53 mutations that results in CC → TT double base change, which is largely observed among UV-induced mutagenesis4.
A wide range of chemicals in the form of pharmaceuticals, cosmetics, agricultural and industrial and environmental pollutants are highly responsible for taking place of mutagenesis in cellular system5, and chemicals have been confirmed as carcinogenic and mutagenic [Eg. Food preservatives (AF-2), the food fumigant (ethylene dibromide), the antischistosome drug (hycanthone), several hair-dye additives and the industrial compound (vinyl chloride) are very active and dangerous to humans while they expose to it6.
Reactive Oxygen Species (ROS)
The generation of reactive oxygen species (ROS) by mutagens and carcinogens are widely identified as inducing oxidative damage to cellular and biomolecular structures including proteins, lipids and nucleic acids7,8. The crucial step in carcinogenesis is DNA mutation that elevates the levels of oxidative DNA lesions in many tumours leads to cancer resulting multiorgan dysfunction to failure. The DNA oxidation are highly mutagenic, that prevents oxidative DNA lesions leads to control the processes of cytostasis, cytotoxicity and mutagenesis; thereby mutation-related diseases are reduced9,10.
The chemicals detected in these processes can be defined as the major sources of somatic mutations and mutations in germinal cells. For detecting such mutational molecules, the Ames test is routine and cumbersome due to its simple and cost effective manner, thus routinely performing this test, large numbers for detecting potentially hazardous compounds for mutagenicity and potential carcinogenicity11.
The proved, authenticated and well documented test systems have been devised to analyze for carcinogenicity and most of them are time consuming, typically requiring laborious research with small mammals. More rapid tests do exist that make use of microbes and test for mutagenicity rather than carcinogenicity due to its similarity in methods and results.
Ames test and its description
In1970s, Bruce Ames developed a working method with Salmonella typhimurium that named as Ames test having two auxotrophic histidine mutations, which revert by
different molecular mechanisms. Later the method was modified and developed for the performance of genetically engineering methods by using S. typhimurium to make it suitable for mutagen detection with the following descriptions
1. Carry a mutation that inactivates the excision-repair system.
2. Carry a mutation that eliminates the protective lipopolysaccharide coating of wild-type Salmonella to facilitate the entry of many different chemicals into the cell12,13.
The usage of bacteria for mutagenic studies has real advantages in identifying chemicals that are carcinogenic to humans. The chemical and genomic complex of DNA is found similar in all living organisms, thus it was proved that compound acting
as a mutagen in one organism is having same mutagenic effects in other organisms12. For this purposes, Ames devised a path to induce and study the human metabolism in the bacterial system. In mammals, the ingestion of chemicals and its processing takes place in the liver, where further the chemicals are detoxified. In certain study, the action of liver enzymes can emerge a toxic or mutagenic compound from a substance that was not considered as dangerous. Thus Ames used mammalian liver enzymes (rat liver) for this bacterial test system12,13.
Genotoxicity
Apart from various genotoxic physical and biological agents, the synthetic (hair dyes, preservatives, persticides) and natural (some phytotoxins) are known to act as
mutagenic, co-carcinogenic and/or carcinogenic agents. In general, agents that cause mutagenesis are largely involved in the initiation and promotion of various human diseases including cancer, the importance of novel bioactive compounds from medicinal herbs in counteracting the promutagenic and anticarcinogenic and are collectively called as antimutagens. These agents have been first reported almost four decades ago, and still the research is continuously carried out to explore novel compounds, which might protect humans against DNA-damage and its complex consequences. Antimutagenic and anticarcinogenic properties of dietary constituents and plant secondary metabolites have been reported in several studies14,15,16,17,18
. Plant as a source of medicine
The major phytoconstituents like flavonoids and phenolic compounds present in foods are having high degree of antimutagenic and protective effects. Also it has been reported to exhibit a wide range of other biological activities such as antimicrobial, anti-inflammatory, antiallergic, antioxidant and free radical scavenging of the same phytocontituents19. Naturally available antimutagens from edible and medicinal plants are of particular importance because they may be useful in preventing human cancer and have no undesirable xenobiotic effects on living organisms17. The rich diversity of Indian medicinal plants have not yet systematically screened for antimutagenic activity20. Thus the exhibitions of such novel molecules are not yet come to clinical practice.
Around one thousand plants are used in the treatment of various ailments in the traditional systems of Indian medicine. The preparations of polyherbal complex are also documented. Based on the chemical diversity of known active antimutagens from plant origin, many traditionally used Indian medicinal plants may explore such need based properties due to similarity in the major class of phytoconstituents.
The authenticity of scientific information related to medicinal herbs used in folk medicine and their effect on human health or on genetic material has been the subject of many different types of investigation. The explorations of such medicinal herbs, which are having a great source of biologically active compounds whose effect on human health or genetic materials are very limited. The usage of traditional phytocontituents for the treatment of different types of illness is very common in Indian system of medicine and is considered as the substitute for modern medicines. Now a days, Scientist has been shown greater interest in evaluating compounds originating from plants and their effects on DNA21.
Other in vitro assays
The method of determining antimutagenic effect is done with many different types of assays employing different organisms. The actions of these compounds may be studied in maintaining the balance between the consumption of mutagenic and antimutagenic substances, thus contributing to increases or reductions in the incidence of cancer in the population. Compounds from plants could act as protective agents with respect to human carcinogenesis, acting against the initiation, promotion or progression
stages of this process22 or, perhaps, destroying or blocking the DNA-damaging mutagens which is found externally leads to inhibition of cell mutations23. Mutagenic and antimutagenic activities have been correlated with the presence of certain phytochemical substances including flavonoids24,25,26,27,28,29,30,31,32,33.
A relationship has been reported between structure and activity, both for mutagenic activity26,32,34 and for protection of the genetic material22. Flavonoids are consumed naturally through the intake of beverages such as beer, coffee and wine (on average ∼1 mg/l flavonoids) and may reach 25 mg/l in black tea25 and are relatively stable compounds, resistant to heat, light and oxygen, and are moderately acidic34.
Next to flavanoids, the antimutagenic activity has also been attributed by using tannins15,34,35,36 that are considered as the major compounds and are widely distributed in medicinal herbs. Studies in cell cultures revealed that tannins have an antimutagenic effect at low concentrations in assays in the presence of S9 mix, promoting increased excision repair. In contrast, a co-mutagenic effect was observed in the absence of S9 mix and in the presence of high concentrations of tannin metabolites35.
Chemo preventive phytoconstituents
Recently, considerable attention has been focused on identifying naturally occurring chemo-preventing substances capable of inhibiting, retarding or reversing the multistage carcinogenesis (usually initiated by mutation)37. Eclipta alba (L.) Hassk.
(also known as Eclipta prostrata Roxb.) belongs to the Asteraceae family and is commonly known as false daisy in English and bhringoraj or bhringraj in Bangladesh
and India. It is regarded as a weed of ethnomedicinal significance. It is known in the three major forms of traditional medicinal systems in the Indian subcontinent, namely, Ayurveda, Unani, and Siddha, as bhringoraaja, bhangraa, and karissalaankanni, respectively38,39.
The available ethnomedicinal reports indicate that although there are a variety of diseases treated with the plant or plant parts, the major uses are limited to treatment of gastrointestinal disorders, respiratory tract disorders (including asthma), fever, hair loss and greying of hair, liver disorders (including jaundice), skin disorders, spleen enlargement, and cuts and wounds40.
AIM AND OBJECTIVES
In this study, we have critically evaluated the antimutagenic activities of Eclipta alba leaf extracts using ethanolic extract by Salmonella typhimurium strains (Ames test). The mechanism on reversing the mutation with sodium azide, ethidium bromide, hydroxyl amine and nicotine content in tobacco leaves and cigarette in S. typhimurium strains TA 98 and TA 1535 were studied. The objectives of the study are
To determine in vitro antimicrobial activity of effectiveness of various extracts of E. alba by in vitro method including Salmonella typhimurium strains TA98 and TA 1535.
To confirm the genotype of the strains of S. typhimurium TA98 and TA 1535 using histidine requirement, rfa mutation, UVrB mutation and R factor
To determine the toxicity of E. alba extracts to S. typhimurium strains
To understand the antimutagenic effect of ethanolic and acetone extracts of E. alba using TA98 and TA 1535 strains of S. typhimurium against direct acting
mutagens such as sodium azide, ethidium bromide and hydroxyl amine.
To analyze the antimutagenic activity of ethanolic and acetone extracts of E.
alba against mutagens including nicotine in tobacco and cigarette by using S9 mix essentially.
REVIEW OF LITERATURE Epidemiology of cancer
Various research institutions in India highlighted to have over seventeen lakhs and thirty thousand (17.3 lakh) new cases of cancer and over eight lakh and eighty thousand (8.8 lakh) deaths die to cancer by 2020 with the major types of breast, lung, cervix etc and the same was also supported by Indian Council of Medical Research (ICMR), New Delhi.
Breast Cancer is considered as the top among various cancers with estimated 1.5 lakh new cases during 2016 followed by lung cancer with estimated 1.14 lakh where, 83,000 among males and 31,000 among females. The third common is Cervix Cancer with estimated one lakh new cases in 2016. Further interestingly it was ound out that cancers associated with the usage of tobacco accounting 30% of all cancers in both the genders. The data collected from 2012 to 2014 from various population based cancer
registeries (PBCR) and registers from medical institutions highlighted that there was a noteworthy elevation of rectum and colon cancers in male population in Bangalore, Chennai, and Delhi and in females in Barshi and Bhopal.
Further, there was also an increase in the cases of cancers of colon, rectum and prostate in Bangalore, Chennai and Delhi among male population while among females there was a significant increase in the rate of breast, uterus, ovary and lung cancers.
Among paediatric population, Delhi rate top in the documentation of cancer. In western states of India, mouth cancer is leading in registry among males whereas East Khasi hills in Meghalaya recorded the highest number of cases of mouth cancer among women. The geographical distribution is depicted in Figure 1.
Figure 1: Geographic distribution of cancer types in Indian continent
Even though there are certain improvements in living standards and human development index rankings (HDIRs), but there are typically linked to increases in the occurrence of various cancer studies.
Examples
1. Sex hormone exposure related cancers and its epidemiologically associated with reduced average family sizes
2. The positive gains that economic and social development bring 3. Improved food quality - normally far outweigh any such costs.
The International Agency for Research on Cancer (IARC) has predicted that cancer burden in India would be double in the next twenty years, leads to more than 1·7 million by 2035 where these projections indicated that the absolute number of mortality of cancer will also leads to 1·2 million in the same period (Figure 2). The observation of cancer related disability and mortality will elevate partly due to the role of technical, advancement and political decisions made in future, improvement in cancer research, awareness of cancer harm-reduction and on environmental and societal changes may reduce the new incidence and outcomes related to severe morbidity and mortality.
Figure 2: National profile of non-communicable diseases
Etiopathology of cancer
Mutations are caused by permanent transmissible changes in the DNA structure and have been implicated in the etiopathology of cancer, neurodegenerative diseases and other degenerative diseases14. These changes may involve individual genes, blocks of genes or whole chromosomes, and are heritable. DNA damage alters the genetic message carried by genes involved (Figure 3).
Figure 3: Intrinsic and Extrinsic factors of cancer etiology
Agents or substances that cause alteration of DNA are termed mutagens and can range from chemicals to radiation and sunlight41. All mutagens elicit a genotoxic response, hence they are also known as genotoxins. DNA damage can be in the form of single and double strand breaks, point mutations and structural and numerical chromosomal aberrations42.
Oxidative stress and DNA damage
Chronic degenerative diseases such as cancer, cardiovascular diseases, atherosclerosis etc share common risk factors and common pathogenic determinants such as DNA damage, oxidative stress and chronic inflammation43. For instance, frame- shift mutations are apparent in severe genetic diseases such as Tay-Sachs disease and cystic fibrosis44. Mutations are also involved in the inception of degenerative diseases including hepatic disorders, neurodegenerative disorders, cardiovascular disorders, diabetes, arthritis, chronic inflammation and the process of aging. These diseases are the leading causes of diseases in developed countries43,45.
DNA damage is present both in the circulating cells of patients with atherosclerosis and atherosclerotic plaques46. DNA strand breaks, oxidized pyridines and altered purines (related to environmental exposure to genotoxic chemicals) are higher in patients with coronary artery disease47. There is considerable evidence that gene and chromosomal mutations are important factors in carcinogenesis48,49. Even though only certain mutations lead to cancer, most mutagens that were tested and identified by Ames et al., (1983) are classified as potential carcinogens14. The field of genetic toxicology remains an important tool in the development of new pharmaceuticals50. As a consequence of mutations, several in vitro and in vivo tests
have been developed to assess the potential DNA damaging effects of chemicals (Figure 4).
Figure 4: An overview of DNA damage
Mutagens and Antimutagens
Antimutagens are chemical agents that reduce or counteract the mutagenicity of physical and chemical mutagens, either by in activating the mutagen or by preventing the reaction between a mutagen and DNA51,52. Since mutagens are involved in the initiation and promotion of several human diseases, research focusing on the identification of novel bioactive phytocompounds that reduce mutagenicity and counteract mutagenesis has gained credence in recent years53,54. It is generally accepted that antimutagenic compounds have chemopreventive properties.
Antimutagens play a major role in the primary prevention of mutations and cancer development by lowering the frequency and/or rate of mutations, or blocking initiation
of carcinogenesis, a chemopreventive role. The use of antimutagens and anticarcinogens in everyday life may be effective in the prevention of human cancers and chronic diseases that share common pathogenetic mechanisms such as DNA damage, oxidative stress and chronic inflammation43. It is thus evident that cancer and other mutation-related diseases can be prevented not only by avoiding exposures to recognised risk factors but also by favouring intake of protective factors.
Cancer prevention
Chemoprevention was first defined as the inhibition or reversal of carcinogenesis by the use of non-cytotoxic nutrients or pharmacological compounds that protect against the development and progression of mutant clones of malignant cells55. Chemoprevention with respect to mutations is the pharmacological approach that uses either natural or synthetic chemical agents to inhibit, reverse, suppress or prevent and arrest mutagenesis32,33.
Most chemopreventive agents have antioxidant activity and detoxifying properties33 (Figure 5). In addition to their antimutagenic activity and anticarcinogenic properties, they exert additional health benefits including antiproliferative and anti- inflammatory properties26,29.
Antimutagenic agents have different mechanisms of action including but not limited to antioxidant potency, inhibition and deactivation of mutagens, and blocking interaction of mutagens with DNA, while others possess multiple mechanisms of action54.
Figure 5: Cancer chemoprevention Strategies
The major mechanisms of antimutagens broadly include chemical or enzymatic inactivation, prevention of formation of active species and antioxidant and free radical scavenging14. Based on their mechanism of action, antimutagens are divided into two major groups, namely bioantimutagens and desmutagens15,17.
An overview of biomutagens
Bioantimutagens are antimutagens that act as modulators of DNA replication and repair processes in cells. This group of antimutagens act by preventing fixation of premutagenic lesions into mutations, resulting in a decline in mutation frequency.
Bioantimutagens are considered to be “true” antimutagens.
Desmutagens – a newer molecule
Desmutagens are antimutagens that inactivate mutagens or prevent their interaction with DNA. They may be antimutagens as they indirectly fully or partially inactivate the mutagen. Among desmutagenic agents, antioxidants are of special interest because they are implied in inhibition of all stages of carcinogenesis. The mechanism of inhibition of mutagenesis and initiation of carcinogenesis by antioxidants include scavenging of reactive oxygen species and inhibition of mutagen/carcinogen binding to DNA.
The possibility of moderating the response of cells to a particular mutagen by phytomedicines opens new horizons in cancer prevention. On this basis, the search for antimutagens presents many possibilities for the discovery of new anticarcinogenic compounds. Determination of the antimutagenic potential of plant extracts is an important step in the discovery of new effective cancer chemopreventive agents.
In recent years there has been greater interest in investigating compounds originating from plants and their effects on DNA. This is done with many different
types of assays employing different organisms. The actions of these compounds may be involved in maintaining the balance between the consumption of mutagenic and antimutagenic substances, thus contributing to increases or reductions in the incidence of cancer in the population21.
Flavanoids and Phenolics
The antimutagenic or protective effect has been attributed to many classes of phytocompounds mainly flavonoids and phenolic compounds present in foods.
However, such compounds have also been reported to exhibit a wide range of other biological activities such as antimicrobial, anti-inflammatory, antiallergic, antioxidant and free radical scavenging. Natural antimutagens from edible and medicinal plants are of particular importance because they may be useful for human cancer prevention and have no undesirable xenobiotic effects on living organisms. The rich diversity of Indian medicinal plants have not yet systematically screened for antimutagenic activity. More than 800 plants are used in the treatment of various ailments in the traditional systems of Indian medicine mainly polyherbal preparations sometime with other minerals (Figure 6). Based on the chemical diversity of known active phytoantimutagens, many traditionally used Indian medicinal plants may exhibit such desired properties due to similarity in the major class of phytocompounds53.
Figure 6: Natural antimutagenic properties in Indian spices
Many mutagens act through generation of reactive oxygen species (ROS) which induce oxidative stress in living cells. Many mutations related to oxidative stress, or DNA damage and repair, have been identified in human disease syndromes56. Oxidative stress is involved in more than 100 common diseases including cancer, all inflammatory diseases (arthritis, vasculitis lupus etc.), autoimmune diseases, diabetes, emphysema, catactogenesis and macular degeneration, gastric ulcers, hemochromatosis, hypertension, heart diseases, and neurologic diseases (multiple sclerosis, Alzheimer‟s disease, Parkinson’s disease, amyotrophic lateral sclerosis, muscular dystrophy etc.)57,58.
Oxygen free radicals or more generally reactive oxygen species (ROS) and reactive nitrogen species (RNS) are products of cellular metabolism and are common in biological systems (Figure 7). ROS and RNS harms living systems by inducing oxidative damage to cell structures and biomolecules such as lipids, nucleic acids and proteins. Normally there is a balance between the amount of free radicals generated in
the body and the defence systems that scavenge or quench these free radicals, preventing them from causing deleterious effects in the body. When there is a shift or imbalance in the pro-oxidation and antioxidation homeostatic phenomena resulting from excessively high levels of these oxidative species in the body, either due to environmental conditions or being produced within the body, these free radicals, increase the burden in the body leading to oxidative stress which results in tissue injury and subsequent diseases59,60.
Figure 7: Repair mechanism by antioxidants
In recent years, there has been an increased interest in the application of antioxidants in the health sector61. Widespread attention is currently being given to the identification of novel potent antioxidant compounds. Antioxidants are chemical
substances that are capable of slowing or preventing the oxidation of other molecules.
They have a wide application in the health sector due to the pathological role of free radicals in a variety of diseases and in the food sector because free radicals result in deterioration of food products62. Antioxidants are widely used as ingredients in dietary supplements in the hope of preventing diseases such as cancer and coronary heart disease. Prevention of cancer and cardiovascular disease has been linked to the intake of vegetables, fruits and teas rich in natural antioxidants63.
Polyphenols – a effective polyphenols
Kaur et al, (2006) reviewed the antimutagenic and anticarcinogenic potential of polyphenols and concluded that polyphenolic compounds have a major place in the chemoprotection against cancer64. They conclude that it is worth investigating what place these compounds have in the prevention of cancer (Figure 8).
Figure 8: Polyphenolic preparations – an overview
Plant phenolic compounds such as those occurring in wine could protect against degenerative diseases involving oxidative damage due to their antioxidant action. The role of phenolic compounds from food and beverages in the prevention of free radical- mediated diseases has become more important. The emphasis placed by the European Commission on enhancing the nutrient content of food crops through traditional plant breeding as well as food-processing technologies confirms the importance of phenolic compounds in terms of health benefits to the international community65,66.
Mutagenesis and Carcinogenesis
The application of new, sensitive techniques of analytical chemistry has confirmed the importance of endogenous oxidative DNA damage in the etiology of many human cancers. Permanent modification of genetic material resulting from “oxidative damage”
incidents represents the first step in mutagenesis, carcinogenesis, and ageing. DNA
damage can result in either arrest or induction of signal transduction pathways, replication errors, and genomic instability, all of which are associated with carcinogenesis. Since oxidative DNA damage can play a significant role in mutagenesis, the decrease of oxidative stress seems to be the best strategy for the prevention of development of mutation related diseases10.
Natural product research continues to provide a variety of lead structures which are used as templates for the development of new drugs by the pharmaceutical industry67. Numerous useful drugs have been developed from lead compounds discovered from medicinal plants68. To date, plants still remain an essential route for the discovery of new pharmaceuticals. There is growing research interest in the use of medicinal plants as dietary supplements and for the development of new medicinal products69.
Plants offer excellent opportunities for the discovery of new therapeutic products70,71. They are considered to be a rich source of medicines as they produce a host of pharmacologically active compounds recognized by pharmacologists to have reactions towards sickness72. The enormous chemical diversity of plant secondary metabolites presents a valuable resource for possible development of new pharmaceuticals (Figure 9).
The use of plants and medicinal plants has been recommended to combat the effect of free radicals/mutagens because they can induce phase II enzymes reducing the action of initiation, promotion or progression stages of cancer and other degenerative diseases. Also the plants are rich source of secondary metabolites such as flavonoids,
phenolics, carotenoids, coumarins, anthraquinones, tannins, terpenoids, saponins that play a prominent role in inhibiting human carcinogenesis and repair the cell mutations73.
Figure 9: Metabolites from phytoconstituents – a medicinal approach
Biomolecular analysis
Approximately 80% of populations in developing countries use medicinal plants to help meet their health care needs74. In South Africa alone, a large percentage of the population relies fully on medicinal plants for their health care needs and food
security75. The use of plants for medicinal purposes is a worldwide practice and is recognized by the World Health
Organization as an essential component of health care72,74.
Drug discovery from medicinal plants led to the isolation of numerous useful drugs76,77. Farnsworth and Soejarto (1985) listed a detailed
summary of drugs derived from plants that are currently used70. A few of the drugs listed in their study are simple synthetic modifications of naturally obtained substances.
Below are examples of important plant compounds developed for the benefit of human health68.
Opium alkaloids- Isolated from Papaver somniferum, from which morphine, codeine, noscapine and papaverine were derived.
Salicin from Salix species resulted in the production of Aspirin, Albyl and Disprilused as pain killers, fever reducing agents and as an anti-coagulant.
Atrakurium is a registered medicine used as a muscle relaxant developed from tubocurarine and strychnine. Tubocurarine and strychnine were isolated from Chondrodendron tomentosum and Strychnos nux-vomica respectively.
Atropine, hyscyamine and scopolamine- All these compounds are present in Atropa belladonna, Hyoscyamus niger and Datura stramonium and are used in various medical conditions including treatment of asthma and ophthalmological disorders.
Cardiac glycosides- Digitoxin isolated from Digitalis purpurea is one of the cardiac glycosides that are still used to treat certain heart conditions.
Artemisia annua and Cinchona species are the sources of the well-known remedies for malaria, artemisinin and quinine. Quinine was isolated from Cinchona species and has been in use for a long time in the treatment of malaria, and artemisinin from Artemisia annua is used both in the prophylaxis and treatment of malaria.
Podophyllotoxin- a compound isolated from the roots of Podophyllum peltatum served as a lead compound for the development of cancer chemotherapeutic agents teniposide and eposide.
Vinblastin and vincristin are successful antineoplastic agents developed from vinblastine and vincristine isolated from Catharanthus roseus.
Mainly antimutagenic activity has also been attributed to the tannins15,35,36,78. These are phenolic compounds and are widely distributed in plants. Studies in cell cultures performed by Imanishi et al. (1991) revealed that tannins have an antimutagenic effect at low concentrations in assays in the presence of S9 mix, promoting increased excision repair35. In contrast, a co-mutagenic effect was observed in the absence of S9 mix and in the presence of high concentrations of tannin metabolites.
The mechanisms and the types of active compounds involved in the protective effects of plants against mutations have not been clearly identified. A common factor in the pathogenesis of chronic degenerative diseases is the involvement of oxidative stress.
Plant compounds may reduce oxidative stress, thereby reducing the risk of diseases79. To minimize the detrimental genotoxic effects of mutagens caused by exposure to free radicals, chemical compounds, air pollutants or metabolic processes, the use of natural antimutagens is a good alternative. Antimutagens that complement DNA repair systems and those that have antioxidant properties may be found in plants80. It is
certainly worth investigating what place these compounds have in the prevention of mutations (Figure 10).
Figure 10: DNA repair system
Phenolics – effect on free radical-mediated diseases
Recently the role of phenolics in the prevention of free radical-mediated diseases has become more important. Due to their antimutagenic/anticarcinogenic activities, phenolic compounds (simple phenols, phenolic acids, naphthoquinones, xanthones, stilbenes, flavonoids, lignans, lignins and condensed tannins) have a major role in the chemoprevention of cancer.
Higher plants are known to synthesize structurally varied biologically active secondary metabolites that have shown various therapeutic potential as well as antimutagenic and anticarcinogenic properties51,81. Much attention and research has
been focused on screening of higher plants for the presence of antimutagenic compounds82. Antimutagens can play a major role as chemopreventive agents52.
In order to detect the various mechanisms of mutations, different mutagens and different in vitro assays were used to investigate the antimutagenic effects of the selected plants. Three mutagens (4-nitroquinoline 1-oxide, mitomycin-C and ethyl methane sulphonate) were used. 4-Nitroquinoline 1-oxide (4-NQO) and mitomycin C (MMC) were used in both the Ames test and micronucleus/cytome assay whereas ethyl methane sulphonate (EMS) was used in the comet assay.
Today, bacteria are being used for the assessment of antimutagenic activities of different compounds in a short-time with excellent results. One of the methods used for assessing the mutation prevention properties of a compound in bacteria is the Ames test (Figure 11). Ames test is a worldwide short-term bacterial reverse mutation test specifically designed for screening a variety of new chemical substances and drugs that can produce genetic damage that leads to gene mutations83. The Salmonella strains used in the test have different mutations in various genes in the histidine operon, each of these mutations is designed to be responsive to mutagens that act via different mechanisms and the same is impregnated in the table 1.
Table 1: Salmonella typhimurium strains and its applications in Ames test
Strain Amino acid marker Other relevant
mutations Histidine Type of Main Cell DNA
mutation mutation DNA target
wall repair
S. typhimurium TA97
hisD6610 Frame shift rfa GC uvrB
S. typhimurium TA98
hisD3052 Frame shift rfa GC uvrB
S. typhimurium TA100
hisG46 Base pair substitution
rfa GC uvrB
S. typhimurium TA102
hisG428 Frame shift rfa AT Nil
Figure 11: An overview of Ames test
The Salmonella mutagenicity test is designed to detect chemically induced mutagenesis. With minor modifications, this assay can be used to detect substances with
antimutagenic activity. The Ames test is a widely accepted short term bacterial reverse mutation assay designed to detect a wide range of chemical substances that can produce genetic damage that leads to gene mutations. The test employs several Salmonella strains with pre-existing mutations in various genes in the histidine operon that renders the bacteria histidine dependant.
These mutations act as hot spots for mutagens that cause DNA damage via different mechanisms. In genotoxicity/mutagenicity testing, Salmonella tester strains are grown on a minimal media agar plate containing a trace of histidine. Only those bacteria that revert to histidine independence are able to form colonies. When a mutagen is added to the plate, the number of revertant colonies per plate is increased, usually in a dose-related manner84,85. Some study highlighted the importance of histidine requirements for the completion of Ames test to determine the in vitro antimutagenic properties among the tested phytoconstitutents from various sources.
The number of spontaneously induced revertant colonies per plate is relatively constant. For antigenotoxicity/antimutagenicity testing, a variation of the Ames test is
used where the mutagen in combination with the presumed antimutagen are added to the plate. When a mutagen is incubated with a presumed antimutagen the number of revertant colonies in the plates with mutagen alone vs number of revertant colonies in the plate with a combination of the mutagen and test sample (presumed antimutagen) provides a measure of antimutagenicity (Figure 12).
Figure 12: Spontaneous revertants
[a – Spontaneous revertants using TA 100 and b – revertant colonies after treatments with phytoconstitutents]
In essence, in the presence of an antimutagenic substance, the number of colonies will decrease when compared to the number of revertant colonies in the plate with mutagen alone. In this study, Salmonella typhimurium tester strains TA98, TA100 and TA102 were used for both mutagenicity and antimutagenicity studies. Strain TA98 gives an indication of frame-shift mutations, TA100 indicates base-pair substitutions and TA102 indicates transitions/transversions85.
Plant medicine – antimutagenic effect
Plant extracts with good antimutagenic effects in S. typhimurium TA98 in general
had good antioxidant activity and relatively higher phenolic content. Plant extracts with good antimutagenic effects in S. typhimurium TA100 generally had higher antioxidant activity but with lower phenolic content (Table 2).
Table 2: Genotypic analysis of histidine mutations
It is clear that there is a direct correlation between antioxidant activity and antimutagenicity. There is also a direct correlation between antioxidant activity and total phenolic contents of the extracts tested. The relationship between antioxidant activity and total phenolics in all plant extracts will be analyzed for exploring newer biomolecules to treat various carcinogenic complications84,86.
Eclipta alba is a plant species that has been used medicinally for many years in traditional Asian medicine and is widely used in Ayurvedic medicine for a variety of ailments especially related to the liver. Eclipta alba is a member of the sunflower family and is also known as false daisy and bhringraj as well as its scientific name Eclipta prostrata.
The plant is believed to have originated on the Indian subcontinent and now grows in various other warm parts of the world such as Brazil, Thailand and China. The plant bears a small flower that can be red, blue or yellow but it is the white species that is most commonly harvested for its medicinal benefits.
Supplementation in Ayurveda
White Eclipta alba has a long tradition if use in Ayurvedic medicine where it is synonymous with the treatment of liver complaints. Nowadays it is used for a wider range of complaints from pain relief to hair health. Both the leaves and flowers of the plant are used to make supplements and it can be used either internally or topically depending on the condition (Figure 13).
Figure 13: Phenotypic parts of E. alba
Eclipta alba – a herbal evaluation
Eclipta alba (L.) is small branched annual herbaceous plant with a long history of traditional medicines uses in many countries especially in tropical and subtropical
regions. The herb has been known for its curative properties and has been utilized as antimytotoxic, analgesic, antibacterial, antihepatotoxic, antihaemorrhagic, antihyperglycemic, antioxidant, immunomodulatory properties and it is considered as a good rejuvenator too.
Recent studies showed an antivenom property & corrosion pickling inhibitor action on mild steel in hydrochloric acid. A wide range of chemical compounds including coumestans, alkaloids, thiopenes, flavonoids, polyacetylenes, triterpenes and their glycosides have been isolated from this species. Extracts and metabolites from this plant have been known to possess pharmacological properties. This contribution provides a comprehensive review on ethnomedicinal uses, chemical composition, and the pharmacological profile as medicinal plant. Particular attention is given to antihepatotoxic, analgesic, antioxidant, antihyperglycemic, antiaggresive, wound healing properties and insecticidal effects (Table 3).
Table 3: Medicinal properties of Eclipta alba87
Phytoconstituents Medicinal properties Hepatoprotective compounds –
Wedelolactone, coumeston
Liver sickness including jaundice and Hepatitis
Flavanoids, Saponins, tannins, terpenoids
Antimicrobial activity
Fresh leaves, glycosides Pain relief
Antioxidant materials and saponins Digestive complaints Expectorant materials, alkaloids Respiratory relief
Diuretic compounds Bladder infections Soothening compounds, volatile oil Hair health
Free radicals Ophthalmic complications
Cardiac strengthening compounds Heart health Antioxidant molecules, cytotoxic
compounds
Cancer prevention
Tannin, flavanoids Insecticides
Eclipta alba (L.) contains wide range of active principles which includes coumestans, alkaloids, flavonoids, glycosides, polyacetylenes, triterpenoids. The leaves contain stigmasterol, a-terthienylmethanol, wedelolactone, demethylwedelolactone, luteolin, Echinoacetic acid, apigenin and demethylwedelolactone-7-glucoside. The roots give hentriacontanol and heptacosanol. The roots contain polyacetylene substituted thiophenes.The aerial part is reported to contain a phytosterol, P-amyrin in the n-hexane extract and luteolin-7-glucoside, P-glucoside of phytosterol, a glucoside of a triterpenic acid and wedelolactone in polar solvent extract. The polypeptides isolated from the plant yield cystine, glutamic acid, phenyl alanine, tyrosine and methionine on hydrolysis. Nicotine and nicotinic acid are reported to occur in this plant87.
The major coumestan isolated from Eclipta alba includes wedelolactone 0.5- 0.55% and desmethylwedelolactone. Taraxastane triterpene glycosides, named eclalbasaponins VII-X were isolated, along with four oleanane glycosides eclalbasaponins I-VI. The structures of eclalbasaponins VII-X were characterized as 3β, 20β, 16β and 3β, 20β, 28β trihydroxytaraxastane glycosides, and their sulphated saponins. Contemporary clinical tests showed that the herb contains the alkaloid ecliptine. Bioassay-guided fractionation of the MeOH extract of Eclipta alba using three yeast strains (1138, 1140, and 1353) resulted in the isolation of eight bioactive steroidal alkaloids (1-8), six of which are reported for the first time from nature. The major alkaloid was identified as (20S)(25S)-22,26-imino-cholesta-5,22(A)-dien-3P-ol (verazine, 3), while the new alkaloids were identified as 20-epi-3-dehydroxy-3-oxo-5,6- dihydro-4,5-dehydroverazine, ecliptalbine [(20R)-20-pyridyl-cholesta-5-ene-3P,23-
diol], (20R)-4P-hydroxyverazine, 4P-hydroxyverazine, (20R)-25P-hydroxyverazine, and 25β-hydroxyverazine. Ecliptalbine, in which the 22,26-imino ring of verazine was replaced by a 3-hydroxypyridine moiety, had comparable bioactivity to verazine88.
The volatile components were isolated from the aerial parts of this plant by hydrodistillation and analysed by GC-MS. A total of 55 compounds, which were the major part (91.7%) of the volatiles, were identified by matching mass spectra with a mass spectrum library (NIST 05.L). The main components were as follows:
heptadecane (14.78%), 6,10,14-trimethyl-2-pentadecanone (12.80%), w-hexadecanoic acid (8.98%), pentadecane (8.68%), eudesma-4(14),11-diene (5.86%), phytol (3.77%), octadec-9-enoic acid (3.35%), 1,2-benzenedicarboxylic acid diisooctyl ester (2.74%), (Z,Z)-9,12-octadecadienoic acid (2.36%), (Z)-7,11-dimethyl-3- methylene-1,6,10- dodecatriene (2.08%) and (Z,Z,Z)-1,5,9,9-tetramethyl-1,4,7-cycloundecatriene (2.07%)89.
From the whole plant of Eclipta alba, a new triterpene saponin, named eclalbatin, together with alpha-amyrin, ursolic acid and oleanolic acid were isolated. The structure of eclalbatin has been established as 3-O-beta-D-glucopyranosyl-3-beta-hydroxy-olean- 12-en-28-oic acid, 28-O-beta-D-arabinopyranoside (1) on the basis of chemical and spectral data. Dasyscyphin C was isolated from Eclipta alba which were studied on the HeLa cells for the anticancer activity90. To keep all the above mentioned wide medicinal properties of Eclipta alba, it was further analyzed the effect of E. alba as antimutagenic properties in this study.
MATERIALS AND METHODS Ethical Approval
This study was done after obtaining clearance from Institutional ethical committee (IEC) from Institutional Research Board, Chennai Medical College Hospital and Research Centre (Affiliated to The Tamilnadu Dr. M.G.R. Medical University, Chennai), Irungalur, Tiruchirapalli (IEC approval letter was enclosed)
Study Design
This study is an in vitro experimental analysis, no animals were directly involved;
thus Institutional Animal Ethical Clearance (IEAC) is not necessary to proceed this work.
Study Centre
This study is conducted in Chennai Medical College Hospital and Research Centre (SRM Group), Affiliated to The Tamilnadu Dr. M.G.R. Medical University Irungalur, Tiruchirapalli.
Study Duration
The IEC was approved on August 2016, thus the present work was initiated on September 2016 and completed by August 2017. It is an experimental design work and the duration is one year (12 months).
Method used
The method used for this in vitro antimutagenic activity is “Ames test” where Salmonella typhimurium strains are used.
Bacterial strain used
The Salmonella typhimurium strains used in this study are a. S. tyhimurium TA-98
b. S. tyhimurium TA-1535 List of chemicals
1. Magnesium sulphate heptahydrate 2. Sodium citrate
3. Di-potassium hydrogen phosphate 4. Potassium dihydrogen phosphate 5. Sodium dihydrogen phosphate 6. Di-sodium dihydrogen phosphate 7. Potassium chloride 8. Magnesium chloride 9. Sodium chloride
10. Ammonium sulphate 11. Crystal violet 12. Acetone
13. Chloroform
14. Ethanol
15. Petroleum ether Media and its components
1. Nutrient broth 2. Nutrient Agar
3. Agar-agar
4. Histidine 5. Biotin 6. Glucose
7. S9 fraction (obtained from Veterinary College, Namakkal) of Wister rats Mutagens
1. Sodium azide 2. Ethidium bromide
3. Hydroxyl amine
Antibiotics - Ampicillin Equipments
1. Autoclave
2. Deep freezer 3. Incubator 4. Hot air oven 5. Cooling centrifuge 6. Laminar flow unit
7. Precision balance 8. Hot plate 9. Cyclo mixer 10. Soxhlet Apparatus
Plant used
Figure 14: Leaves of Eclipta alba
Various extracts of Eclipta alba and was dissolved in minimum quantity of hexane and used for the experiment. The plant descriptions of Eclipta alba are
Botanical Name Eclipta alba
Common Name Bhringraj
Classification
Kingdom: Plantae
Subkingdom: Viridaeplantae Division: Tracheophyta Class: Magnoliopsida Subclass: Asteridae Order: Asterales Family: Asteraceae Genus: Eclipta Species: E. alba
1. Extraction of crude compounds from leaves of Eclipta alba
The leaves of Eclipta alba was collected from the paddy field with the help of identifying initially by the experienced farmer and further the plant was botanically authenticated by the Botanist from Bharathidasan University, Tiruchirapalli. The collected leaves were surface cleaned by water and shadow dried for 10 to 15 days.
Further the dried leaf saplings were grinded coarsely using kitchen blender. The jar of the kitchen blender was surface sterilized with ethanol before starting coarse grinding of the leaves (Figure 15). The coarse powdered leaves were kept in air tight bottles in a dark condition until use (not necessary to refrigerate for 30 days).
Figure 15: Coarse powder of leaves of Eclipta alba
The extraction procedure was done using Soxhlet extraction apparatus. The grinded leaves were packed in the whatman No. 1 filter paper and placed in the Soxhlet apparatus (Figure 16,17 & 18) and the set up as run using 200ml of acetone, ethanol, chloroform, petroleum ether and water.
Figure 16: Crude solvent exposure of E. alba before Soxhlet extraction
Figure 17: Preliminary filtration procedures
Figure 18: Crude compound extraction of E. alba leaves by Soxhlet apparatus
The extraction was continued for 4 to 5 hours and was collected in a separate petridish. The petridishes were kept open for few hours for complete evaporation of the solvent in an aseptic environment (Laminar air flow) without providing air flow (Figure 19 & 20).
Figure 19: Evaporation of crude extract in Laminar air flow
Figure 20: Evaporation of solvents after crude extract preparation
2. In vitro antimicrobial activity of various extracts of leaves of Eclipta alba
The crude leaf extracts of Eclipta alba using various solvents including acetone, ethanol, petroleum ether and water. Using Mueller Hinton agar medium, the in vitro
antibacterial activity of various crude leaf extracts were performed by plate inhibition method.
The bacterial pathogens including in this study are Acinetobacter baumanii, Bacillus cereus, Citrobacter species, Escherichia coli, Enterbacter faecalis, Klebsiella pneumonia, Micrococcus species, Pseudomonas aeruginosa, Salmonella typhi, Salmonella paratyphi A, Salmonella paratyphi B, Shigella dysentriae, Staphylococcus aureus, Streptococcus pneumoniae and Vibrio cholerae. The bacterial isolates were obtained from Clinical Microbiology department of Chennai Medical College Hospital and Research Centre after confirmed by the Clinical Microbiologists.
Various concentrations of different solvent extracted crude compounds were dispensed in the Mueller Hinton agar and the test bacterial pathogens were inoculated by surface plate method and incubated at 37oC for 18 to 48 hours. By using the extracts, the test bacterial strains used for the antimutagenic assay also subjected for antimicrobial activity using Salmonella typhimurium TA163 and TA96. The extract that showed resistance to S. typhimurium TA163 and TA96 strains are subjected further to mutagenicity test.
3. Salmonella mutagenicity test (Ames test)
Salmonella typhimurium strains TA1535, and TA98 were kindly supplied by Lady Hardinge Medical College, New Delhi and were used for antimutagenicity studies.
TA1535 detected mutagens that caused base-pair substitutions; TA98 detected various frameshift mutagens Frozen cultures of the tester strains were stored at -20oC. A fresh nutrient broth culture was grown to a density of 1-2 X 109 cells /ml and for each 1ml of culture 0.09ml of dimethyl sulphoxide was added as a cryoprotective agent. The bacterial culture was inoculated in fresh nutrient broth and grown for 12 hours at 37oC before each experiment.
3.1. Preparation of reagents and media
The solutions and media were prepared according to the method of Ames.
3.1.1. Spizizen’s salt solution (10x)
Magnesium sulphate (MgSO4. 7H2O) - 0.02g Sodium citrate - 1 g Dipotassium hydrogen phosphate (K2HPO4) - 14g Potassium dihydrogen phosphate (KH2PO4) - 6g Diammonium sulphate ((NH4)2SO4) - 2g
Distilled water - 100ml
pH - 6.5
This solution was sterilized by autoclaving at 121oC for 20 min.
3.1.2. Histidine / biotin solution (0.5 mM)
D-Biotin - 12.4mg
L-Histidine.HCL - 9.6mg
Distilled Water - 100ml
pH - 6.5
Biotin was dissolved by heating the water and to this solution histidine was added and autoclaved for 20 minutes at 121oC and stored in a glass bottle at 4oC.
3.1.3. Histidine biotin plates
Agar - 1.5 g for 100ml Distilled water - 83.4ml
10 X Spizizen’s salt - 10ml 40% glucose - 5ml Sterile histidine.HCl.H2O - 1ml Sterile 0.5 mM biotin - 0.6ml
pH - 6.5
The agar and water were autoclaved for 20 min at 121oC and 10ml of sterile Spizizen’s salt solution, 5ml sterile 40% glucose and histidine were added to the hot agar solution. After the solution was cooled slightly the sterile biotin was added and poured into petri plate.
3.1.4. Nutrient broth
Nutieint broth - 1.3 gm Distilled water - 100 ml
pH - 6.5
Nutrient broth was dissolved in 100 ml distilled water and was autoclaved before using.
3.1.5. Minimal agar plates
Agar - 1.5 g for 100ml Distilled water - 85ml
10 X Spizizen’s salt - 10ml 40% glucose - 5ml
pH - 6.5
The agar and water were autoclaved for 20 min at 121oC and 10ml of sterile Spizizen’s salt solution and 5ml sterile 40% glucose were added and this solution was poured into petri dishes and used for the experiment.
3.1.6. Ampicillin Plate
Agar - 1.5 g for 100ml Distilled water - 83ml
10 X Spizizen’s salt - 10ml 40% glucose - 5ml Sterile histidine.HCl.H2O - 1ml Sterile 0.5 mM biotin - 0.6ml Sterile ampicillin solution
(8mg/ml 0.02N NaOH) - 0.4ml
pH - 6.5
