ABSTRACT
The study investigates acute and chronic toxicity studies of indigofera stenophylla on breasts cancer. The active principles of Indigofera Stenophylla were extracted using methanol and ethanol as solvent and studied using gas chromatography coupled with mass spectrometry (GC–MS). We assessed the in vitro antioxidant efficacy by studying the DPPH radical scavenging activity, ferrous reducing antioxidant ca- pacity (FRAC), and ABTS cation radical scavenging activity. Using MTT assay, the cytotoxic activity of SV-ME and EE against human cervical cancer (HeLa) and normal murine fibroblast (L929) cells were evaluated. The toxicity and apoptosis induction was studied by dual acridine orange/ethidium bromide (AO/EB) fluorescent probe and Hoechst dye. Mitochondria membrane potential (Δψm) was assessed using JC1. Ascorbic acid (AA) additive/synergism was studied by co-treating HeLa cells with AA along with SV-ME and SV-EE at IC50 concentration..Our results suggest that adequate use of Indigofera Stenophylla could be a good candidate for developing anticancer agents and administration of Indigofera Stenophylla along with ascorbic acid as adjuvant can be used to manage cervical cancer conditions using Siddha treatment method. The study recommends that Further studies on structural modification and combinatorial effects of the active compounds should be investigated to enhance anticancer activity and reduce toxic effects.
CHAPTER ONE
INTRODUCTION
1.1 Background to the study
Toxicity is the degree to which a substance can harm humans or animals. It can be measured by its effects on the target organism, organ, tissue or cells. The toxic effects of a substance on animal physiology can range from minor changes such as reduced weight gain, small physiological alteration or change in the levels of circulating hormones, to severe effects in organ functional loss leading to death. Intermediate levels of toxicity may cause pain and suffering (Home office, 2004). During toxicity studies, five major aspects are put into consideration. These include organs affected by the chemical, relevance of quantification effect, concentration of chemical to be tested, in vitro markers of toxicity that are relevant to the chemical and how to use the data from in vitro test for risk assessment. Toxicological studies in the pharmaceutical field have been growing exponentially. These developments have been prompted by discovery of teratogenic effects of drugs such as thalidomide, exposure of chemicals to the environment and employees and by conduct and assessment of toxicity studies as part of good manufacturing practice (Traina, 2006).
In the study of herbal remedies, it has been found that toxicity may result from inadvertent substitution of one plant species another. For example, rapidly progressive renal failure resulting in end-stage renal disease has been reported in women who have taken weight-reducing pills containing the Chinese herbs Stephania tetrandra and Magnolia officinalis. This so called
Chinese–herb nephropathy is characterized by a pattern of interstitial fibrosis. The cause of the disease was later noted to be due to inadvertent inclusion of Stephania fangchi containing the nephrotoxic and carcinogenic aristolochic acids instead of S. tetrandra that contains weight reducing tetrandrine (Nortier, 2000; Rotblatt and Zimet, 2002). Another reported case on herbal toxicity involved patients using a dietary supplement containing herbal constituents yohimbine in addition to norephendrine, sodium usiniate and 3,5- diiodothyronine. All patients developed hepatotoxicity within 3 months, recovering spontaneously on withdrawal of the supplement (Flavreau et al., 2002). Another study carried out in Zimbabwe confirmed an increase in incidences of poisoning by herbal remedy since 1971 (Nyanzema, 1986). These facts support the need to have toxicity profiling of all herbal remedies.
Meanwhile, Acute toxicity is caused by an agent when it is administered in one or more doses over a period not exceeding 24 hour and involves harmful effects to the organism through a single or short- term exposure. Acute toxicity studies have also been used during the selection of starting doses for phase-I human and animal studies, and provide information relevant to acute overdosing in humans and animals. The testing is based on the route of substance administration to the animal and therefore it is classified from Class-1 to Class-5 for oral, dermal, gas inhalation, vapor/dust/mist inhalation and injection. Dosing can be repeated during the administration of test material by a variety of routes of exposure, including gavaging which involves stomach intubation or forced feeding, injection, skin, painting and inhalation. The acute toxic class method, a step-wise procedure, involves the use of three animals of a single sex per step. Depending on the mortality and/or moribund status of the animals, on average 2 to 4 steps may be necessary to allow judgment on the acute toxicity of the substance. The OECD Guideline 423 (2001) provides a reproducible method that uses few animals as per appendices 2, 3 and 4.
1.2 Statement of the Problem
The most important aspects during the usage of drugs from all sources are their quality, efficacy and safety. Assumptions have been made that the use of herbal drugs in treatment of parasitic diseases in humans is going on without any noticeable toxic effects (Sushma et al., 2012). The assumption is not based on scientific evidence but on the fact that herbs are often believed to be safe because they are ‘natural’ (Glesler, 1992). Thus they are often used indiscriminately and in unstandardized manner. However, many dangerous or even lethal side effects have been reported including direct toxic effects, allergic reactions, effects from contaminants, and interactions with drugs and other herbs (Ernst, 1998). It is noteworthy that toxicity related to drugs is common in both conventional and herbal therapeutic agents. For example, a study carried out on 548 compounds marketed between 1975 to1999 showed that 10.2 % were withdrawn or acquired a black box warning due to toxicity (Boone et al., 2005). This emphasizes the need to carry out toxicity testing on all drugs intended for human and animal consumption.
Less than 10 % of herbal products in the world are not standardized to known active components, and therefore no strict quality control measures are followed. The active constituents of most herbs or the toxicants are rarely known. Herbs contain complicated mixtures of organic chemicals, the level of which may vary substantially depending upon many factors related to the growth, production, and processing of the herbal product (Stephen and Richard, 2004). Most of the toxic constituents in herbs are secondary metabolites produced by plants as their natural defense to adverse conditions but may end up poisoning the human (Obidike and Salawy, 2013). Deaths have been reported on individuals who have consumed drugs that have been empirically identified via trial and error methods (Pamela et al., 2001). It is very important to put more effort in the study of herbal medicine and especially in the areas of standardization. It is documented that herbs as medicine have not gained enough momentum in the scientific community due to lack of specific standards being prescribed for herbal medicines (Sarika et al., 2006). The challenges faced by herbal formulations arise because of their lack of complete evaluation since evaluation is necessary to ensure quality and purity of the herbal product (Gupta and Amartya, 2012). Besides studies on quality and efficacy of herbal medicines, it is necessary to ensure the safety of a product and this entails toxicity testing.
The empirical process of identifying medicinal agents by trial and error is not efficient and countless individual have died following treatments with plant products that were poisonous and/or ineffective. Thus, the scientific medicine of 20th century should depend on rational codified principles, providing an understanding of why some treatments are effective and others are not. It is very important to ensure that each drug derived from plant products be evaluated for safety and efficacy by methods identical to those used for novel synthetic entities (Rotblatt and Zimet, 2002).
Toxicity testing of herbal drugs has also been found to have a lot of benefits. Notably, it is easy to identify the toxic effects and thus determine the limit of exposure levels especially to sensitive population. Once these toxicants are known they may be discarded or modified via dosage adjustment, chemical group or structural adjustments (Obidike and Salawy, 2013).
From the literature review, there is no prior documented scientific research especially on acute toxicity testing on indigofera stenophylla. Hence the present study seek to investigate Acute and chronic toxicity studies of indigofera stenophylla on breasts cancer.
1.3 Aims and Objective of the study
The aim of this study is to investigate Acute and chronic toxicity studies of indigofera stenophylla on breasts cancer.
Specific Objectives
- To investigate the in vitro antioxidant capacity of Indigofera Stenophylla extracts
- To analyze the Indigofera Stenophylla (SV-ME and SV-EE) exhibits cytotoxic effect on cervical cancer cell line
- To investigate the bioactive compounds in methanol and ethanol extract of Indigofera Stenophylla determined by GC–MS analysis
1.4 Significance of the study
The study was intended to contribute to knowledge of plants used in Kenya for management of cancer. Some of the findings were disseminated through relevant peer reviewed journals so that this information may be accessible by other researchers
The findings provide a baseline for evidence based complementary and alternative medicines and the pharmaceutical industry could use these findings to develop new anticancer drugs. The extracts or fractions would also be standardised and improved by the pharmaceutical industry for commercialization.
The study will equally add to the existing body of knowledge on the subject matter. Students undergoing research work similar to the present study who may wish to use this work as a reference material or a spring board for their own work will find this work really useful.
1.5 Scope of the study
This study is limited to Acute and chronic toxicity studies of indigofera stenophylla on breasts cancer. The researcher chooses this area of study because of its proximity in getting needed data for the study.
1.6 Limitations of the Study
Financial Constraints: The researcher was with limited funds and cannot visit all the areas to get data but was able to get good information concerning the research topic.
Time Constraints: The researcher was involved in other departmental activities like seminars, attendance of lectures et.c which limited the time for the research but the researcher was able to meet up with the time assigned for the completion of the research work.
