BRIEF RESUME OF THE INTENDED WORK:
6.1 INTRODUCTION:
Cancer is a major cause of death worldwide and causes serious problems in human life, including mental and physical agony and economic strain. Therefore, many kinds of cancer therapies, including various anticancer agents, have been dev eloped. In all types of cancer, genetic alterations give rise to changes in expression, activation or localization of regulatory proteins in the cells. They then affect the signalling pathways that alter their response to regulatory stimuli and allow the unrestricted cell growth. However, they also have several problems such as serious side effects and drug resistance1.
To resolve these difficulties, development of cancer chemopreventiveagents and improvement of cancer treatment are very important. Accordingly, screening of natural products as potential anticancer agents, in the form of functional foods or nutraceuticals has become an important undertaking2.
The NCI in vitro primary screen consists of a panel of 60 different human tumour cell lines against which compounds are tested over a defined range of concentrations to determine the relative degree of growth inhibition or cytotoxicity against each cell line. The design and operation of the screen is such that, for each compound tested, both the absolute and relative sensitivities of individual cell lines comprising the screen are sufficiently reproducible that a characteristic profile or "fingerprint” of cellular response is generated. Depending upon the extent of differential cellular response, the profile may contain much information which is useful for further research3.
As per World Health organization reports suggests that Cancer is a leading cause of death worldwide, accounted for 7.6 million deaths (around 13% of all deaths) in 2008.It is reported that Lung, stomach, liver, colon and breast cancer cause the most cancer deaths each year, and About 30% of cancer deaths can be prevented. Deaths from cancerworldwide are projected to continue rising, with an estimated over 11 million deaths in 2030.
- It has been studied that only 5-10 % of all cancer cases can be attributed to genetic factors, whereas 90-95 % cases are due to life style and environmental factors, which includes cigarette smoking, diet, alcohol, sun exposure, environmental pollutants, infections, stress, obesity and physical inactivity. Tobacco is the highest cancer causing agent with percentage of 25-30%, followed by diet and infections4.
Withaferin- A is therapeutically active withanolide reported to be present in plant. In animal studies, withaferin-A has shown significant anticancer activity. Majority of the anticancer drugs like Vinblastine, Vincristine, and Taxol have been derived from green flora. Today there is much interest in natural products with anticancer activity. Withanolides are of under research potential as far treatment of cancer is concerned. The scope of studies published in favour of anticancer potential of withaferin-A11.
Curcuma longaLinn. (Turmeric) is used extensively in Indian cuisine and now proved to be useful in treating various types of cancers, diabetic wounds, biliary disorders etc. So the anticancer activity of turmeric was evaluated prophylactically and therapeutically (as pre-induction treatment and post-induction treatment) against the drug induced mammary tumours10.
Administration of turmeric showed anticancer activity in a dose dependent manner and it was more in pre-induction treatment than in-post induction treatment groups. Topical application of turmeric was found to be more effective in pre-induction treatment and topical treatment was more effective when compared to oral treatment. Chemo-preventive role of turmeric was more compared effective than therapeutic role of turmeric.
In India for its medicinal and non-medicinal uses like colouring agent, flavouring agent. Its anti-inflammatory property is known to ancient Indians and Chinese. It is also used for the treatment of sprains and swelling caused by injury , for the treatment of biliary disorders ,anorexia, cough, diabetic wounds, hepatic disorders, rheumatism and sinusitis . It also shows adjuvant chemoprotection in experimental stomach and oral cancer models of Swiss mice and Syrian golden hamsters.Turmeric was already proved beneficial in various types of cancers like duodenal tumours, tongue carcinoma, colon cancer , human breast cancer cells in-vitro ,mammary tumour in-vivo9.
Tulsi is known as “Queen of plants” “The mother medicine of nature”. Tulsi i.e.Ocimum sanctumis a plant with enormous properties for curing and preventing diseases. It is regarded as deity in Indian subcontinent. The genusOcimum sanctumLinn. (Labiateae or Lamiaceae) comprises30 species which are found in tropical and subtropical regions. Leaves and flowering tops are used for extracting essential oil. Decoction of leaves is recommended for cough, malaise and in colds. It is a good mosquito repellent as well. Oil extracted from flowers is used in skin diseases and ring worm infection. Various studies have been performed withOcimum sanctumfor its antibacterial, antioxidant, anti-ulceric, antimalarial, anti-diabetic, anti-inflammatory, anti-lipidemic, anticancer and immunomodulatory properties16.
Ocimum sanctum Linn. (OS, Tulsi), a medicinal herb used in the indigenous system of medicine. OS has been adored in almost all ancient ayurvedic texts for its extraordinary medicinal properties. It is pungent and bitter in taste and hot, light and dry in effect. Its seeds are considered to be cold in effect. The roots, leaves and seeds of Tulsi possess several medicinal properties. Ayurvedic texts categorise OS as stimulant, aromatic and antipyretic. While alleviating kapha and vata, it aggravates pitta. It has a wide range of action on the human body mainly as a cough alleviator, a sweat-inducer and a mitigator of indigestion and anorexia. OS has a variety of biological / pharmacological activities such as antibacterial, antiviral, antifungal, antiprotozoal, antimalarial, anthelmentic, anti-diarrhoeal, analgesic, antipyretic, anti-inflammatory, ant allergic, antihypertensive, cardioprotective, central nervous system (CNS) depressant, memory enhancer, anti hypercholesterolaemic, hepatoprotective, anti-diabetic properties ofOcimumspeciesanti-asthmatic, anti-thyroidic, antioxidant, anticancer, chemopreventive, radioprotective, immunomodulatory, antifertility, anti-ulcer, anti-arthritic, adaptogenic, anti-stress, anti-cataract, antileucodermal and anticoagulant activities. This review will definitely help forth researchers as well as clinicians dealing with O. sanctum to know its proper usage as this herb is seemed to be highly valuable, possessing many pharmacological / medicinal properties16.
In this background a logical formulation (AC-95) containing these ingredients is being evaluated for its possible synergistic anticancer effect.
6.2 REVIEW OF LITERATURE:
- KamaljitKaur et al concluded that the Ashwagandha has been identified as source of anti-tumour activity, anti-inflammatory activity and rejuvenating factors including withanolides that from major constituent of ashwagandha6.
- Oza VP, Parmar PP et al has first reported that the highly purified L-asparginase from Withaniasomnifera L shows an Anticancer property(lymphoblastic leukemia7.
- Dale kieferAshwagandha, an exotic Indian herb, has demonstrated anti-anxiety and neuroprotective effects, and tantalizing evidence suggests that it is also a cancer fighter. Animal toxicity studies indicate that this remarkable plant is safe and well tolerated..
- Mohammad hosseinmirjalil reported that Aswagandha, chemically rich with its varied content of active compounds, such as withanolidess,
- M.Saif islam ,M. Badrulalam was accomplished that crude extract of ocimum sanctum has persuasive antioxidant activity as well as significant reduced tumour growth, viability of tumour cell and normalized haematological profiles and raising life span9.
- Subir Kumar das and D M Vasudevan concluded that tulsi has widely used for curing various ailments due to it great therapeutic potentials.A number of pharmacological effects like hypoglycaemic, immunomodulatory, anti-stress, anti-inflammatory, anti-ulcerogenic, CNS depressant, anti-tumour and antibacterial activity. This help in establishing a scientific basis for therapeutic uses of plant8.
- Gupta sandeep k et al reported that curcumin is a neutraceutical substance with numerous pharmacological activities, some of which have been experimentally and clinically utilize in both man and animals. It is encouraging that it has low toxicity. Despite a plethora of phytochemical, pharmacological, biochemical on curcumin , large well designed clinical trails are warranted to substantiate its usefulness in treatment and/or prevention of cancer12.
- Jasim Hilo Namma et al reported results, it may be concluded that
prevention and cure of cancer13.
- Vijay K, Pramodh K.S concluded thatTurmeric is a natural herbal product which is most commonly use in every house it yellow spice in colour it belong to Zingiberaceae family. The present investigation was an attempt to explore few of the diverse pharmacological activity of turmeric. It produces many multiple activities like anti-inflammatory, anti-oxidant, anticancer, gastrointestinal, Anti-bacterial and anti-fungal anti-HIV. so it is useful for our life because it very chief and effective so it use as natural drugs.The fast growing research on turmeric and its metabolites clearly confirms the versatility and flexibility of curcumin for structural modifications .This review describes various approaches that have been undertaken to prove
Cancer cell lines are extensively used in various cancer researches. The breast cancer cell lines are procured from NCI and the polyherbal drug is evaluated for anticancer activity on suitable laboratory animals.
.
6.3OBJECTIVES OF THE STUDY:
The objective of present study is carry out the in-vivo anticancer effect of polyherbal formulation on breast cancer cell lines
- Sourcing of cell lines
- Induction of cancer
- Treatment
- Evaluation of parameters
- Determination of tumour volume and weight
- Tumour cell count
- Viable/Non-viable cell count
- Determination of median survival time and percentage increase in life span
- Haematological parameters
- Statistical analysis
7. / ENCLOSURE-II
MATERIALS AND METHODS
7.1 SOURCE OF DATA:
Whole work is planned to generate data from laboratory studies i.e. experiments are performed as described in references. Experimental studies in journals and in text books available with college, NCI and other libraries. Literature is searched from various web sites in the internet.
7.2METHOD OF COLLECTION OF DATA:
The polyherbalformulation(AC-95) is to formulate which includes withania Somnifera, Curcuma longa, and Ocimum sanctum and the active ingredient is extracted from each plant for a particular constituent. The cancer cell lines are purchased from the parentrally and the mice is maintained till the growth of the tumour and the experiment animal is treated prophylactically and concurrently and the experiment includes evaluation of following parameters:
a.Determination of tumour volume and weight: The mice were dissected the ascitic fluid was collected from peritoneal cavity. The was measured by taking it in a graduated centrifuge tube and weight immediately.
b.Tumour cell count: The ascitic fluid is taken in WBC pipette and diluted by 100 times. Then a drop of diluted cell suspension was placed on the Neubauers counting chamber and the number of cells in the 64 small square were counted.
c.Viable/Non-viable cell count: The viability and non-viability of the cellwere checked by trypan blue assay. The cell were stained with trypanblue(0.4% in normal saline) dye. The cells that did not take up the dye were viable and those that took the dye were non-viable. These viable and non-viable were counted.
d. Determination of median survival time and percentage increase life span: The mortality was monitored by recording percentage increase in life span and Median survival time.
e. Haematological parameters: Collected blood was used for the estimation of Haemoglobin (Hb) content, Red blood cells (RBC), White blood cells (WBC).
f. statistical analysis: All the data are expressed as Mean+SEM (n= 6 mice per group) statistical significance (p) calculated by student’s T test computed. P<0.001 and P<0.005 were considered to be statistically significant.
7.3Does the study require any investigations or invention to be conducted on patients or other human or animals? If so, please mention briefly.
YES
7.4 Has ethical clearance been obtained from your institution in case of 7.3?
YES
8. / ENCLOSURE-III
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