all cancer patients try CAM, of which herbalism is one major approach (Cassileth et al., 2001; Cassileth and Deng, 2004).
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1 Introduction The incidence of cancer is on the rise with an estimated 15 million new cases, and 10 million deaths expected by the year Conventional treatment strategies for cancer are surgery and radiation therapy, also resulting in increased levels of fatigue, anxiety, depression with consequent effects on physical and mental function, resulting in deterioration of quality-of-life (Kamangar et al., 2006). In the recent times complementary and alternative medicine (CAM) which is defined as any treatment that is used besides conventional treatment, has made significant inroads as an accessory modality in cancer care, providing a feasible option for disease control, increasing the body s ability to fight against the disease, relieve side effects caused by chemotherapy, prevention of recurrence, boosting the immune system, reducing stress/aiding relaxation, and improving quality of life and general well being, or even cure (Munshi et al., 2008; Gratus et al., 2009). New developments of complementary medicine in oncology have emerged out of a general disappointment with the results of more traditional treatment options. Despite several innovative approaches towards cancer control, cancer mortality rates have not been significantly reduced (Beuth and Shierholz, 2007). CAM has become an important component of modern oncology. Despite substantial progress of scientific clinical oncology, the fascination of alternative and especially complementary cancer medicine has not at all declined. In developing countries, plant based drugs are more popular than ever with the patients, which is also related to socioeconomics (Shukla and Pal, 2004). Although one form of CAM i.e., herbal therapy in the management of cancer or its symptoms dates back into ages, modern day usage of herbs is becoming increasingly popular and frequent in cancer patients across the globe; 70 85% of cancer patients have used herbs in conjunction with their cancer treatment. Systematic reviews of surveys from different countries suggests that approximately one-third of Page 1
2 all cancer patients try CAM, of which herbalism is one major approach (Cassileth et al., 2001; Cassileth and Deng, 2004). In addition mounting evidence exists to suggest a substantial impact of complementary phytotherapies as supportive adjuncts. A significant proportion of cancer patients use alternative therapies, however, it is likely that many adopt therapies strictly for complementary purpose. Therefore Integrative Medicine (IM) now encompasses fusion of such alternative treatments complementing modern drug regimen (Diehl, 2009). In this context several herbs which are reported in popular use are Serenoa repens, Panax ginseng (ginseng), Uncaria tomentosa (cat s claw), Echinacea, Oenothera biennis (evening primrose oil), Silybum marianum (milk thistle), Arnica Montana (Arnica), Valeriana officinalis (Valerian), Piper methysticum (Kava kava), Passiflora incarnata (Passion flower), Aloe barbadens (Aloe vera), Dioscorea villosa (Wild yam), Hydrastis canadensis (Golden seal), Viscum album (Mistletoe), Ulmus fulva (Slippery elm) and Cimifuga racemosa (Black cohosh) (Gratus et al., 2009). Saw palmetto is another herb that has been used to treat symptoms of benign prostatic hyperplasia (BPH). Dietary treatment with rosemary was found to inhibit mammary tumor formation in rats. Soy has also generated interest as chemo preventive agent (Lamson and Brignall, 2001). Many Preclinical, Phase-I and Phase-II studies have been undertaken worldwide to evaluate diverse herbal agents, such as berberine and herbs (radiosensitizers in glioma cells), Gingko biloba (gingko) (cancer prevention and stabilization), Scutellaria baicalensis (skullcap) (anti-cancer activity), Morinda citrifolio (Noni) (antitumor), Soy isoflavones (prevention of prostate cancer) (Richardson, 2001). Herbal remedies typically are part of traditional and folk healing processes with long histories of use. Some forms of herbal medicine are found in most areas of the world. The traditional medicines based on Ayurvedic principles which originated in India more than 2000 years ago are most prevalent in south-east Asia. The term Page 2
3 Ayurveda comes from the Sanskrit words Ayur (life) and Veda (knowledge). The hall mark of this traditional healing system is the assumption that an imbalance in the three humors of the body, such as vata, pitta and kapha produce tumors (Arbuda), which eventually infiltrate into the body tissues and increase in size. Three types of arbudas are: `raktarabuda (leukemia), `mamsarbuda (myoma) and `adayarbuda (metastatic tumors) (Chopra and Doiphode, 2005). In many Ayurvedic remedies some of the prominent medicinal plants that are being used are Allium sativum (garlic), Allium cepa (onion), Bacopa monniera (Water Hyssop or moneywort), Ocimim sanctum (Indian holy basil), Curcuma longa (turmeric) and Zingiber officinalis (ginger). Although Ayurvedic treatment regimens which rely mostly on the use of medicinal herbs, and for centuries have remained within the domain of traditional medical practice, these are now increasingly being examined for new sources of anticancer agents in the form of active plant extracts or identified constituents. In fact the role of natural products as a source for remedies has been recognized since ancient times (Cragg et al., 2005). A World Health Organization survey indicated that about 70-80% of the world s population rely on nonconventional medicines, mainly derived from herbal sources, for holistic healthcare. This is specially the case in developing countries where the cost of treatment is beyond the means of most people (Mukinda and Syce, 2007). In this context many pharmaceutical agents have been discovered by screening natural products from plants, animals, marine organism and microorganisms. Plant products contain wide categories of natural products with remarkable biological activities. With the tremendous increase in the global use of medicinal plants, several concerns regarding the efficacy and safety of the herbal medicines have also been raised. The prolonged and apparently uneventful human use of a particular herb usually is considered as a testimony of its safety. However, a history of traditional usage is not always a reliable guarantee of safety since it is difficult for traditional practitioners to detect or monitor delayed effects (e.g. mutagenicity), rare adverse effects, and adverse effects arising from long-term use (Ernst, 1998). An herbal Page 3
4 bioactive entity, may thus have potential for harm, and may produce adverse symptoms. With the result many herbs have come under scrutiny because of safety concerns. The Organization for Economic Cooperation and Development (OECD) guidelines are specifically important to ensure acceptance of toxicity data regulated in thirty member countries of the OECD (OECD, 1996). The OECD guidelines of testing are collection of the most relevant internationally agreed test methods used to determine the safety of chemical and chemical preparations. Toxicological tests such as acute, sub chronic, chronic and special toxicology that are impossible to detect clinically such as immunotoxicity, genotoxicity, carcinogenicity and reproductive toxicity help in the identification of possible target organs involved and the toxic symptoms. Studies of special toxicology such as carcinogenesis are very important if the plants contain compounds with known mutagenic or carcinogenic activities (Chanabra et al., 2003; Remirez, 2006). Withania somnifera Dunal. W. somnifera (`Ashwagandha or Indian ginseng) is one herb which has been used for centuries in Ayurvedic medicine to increase longevity and vitality, as a geriatric tonic and a rasayana (health promoter) to augment resistance of the body against disease, and revitalize the body in debilitated conditions (Bhattacharya and Muruganandam, 2003). W. somnifera is a plant with immense pharmacological potential and many earlier experimental studies have revealed its promising anti-inflammatory, immunemodulating, and anti-stress properties in the whole plant extract and its constituents, which have been found to possess antitumor activity and have been suggested to be used in conjunction with radiation and chemotherapy treatment (Winters, 2006). In recent past, considerable research has been done at the Indian Institute of Integrative medicine (IIIM, CSIR), Jammu to understand the efficacy of W. somnifera as a potential anticancer herb. This has led to the identification of a chemically characterized herbal fraction (designated as WSF), which has been found to possess Page 4
5 anticancer and Th1 immune up regulatory activities (Malik et al., 2009), thus making it to be a unique entity with antitumor and immunomodulatory activity. Since Ayurveda in India is fused into the national health system, and the CAM herbal drug usage among the cancer patients is on the rise, there exists potential use of such entities like WSF with conventional anti-cancer drugs. Whether the novel entity (an herbal extract or a fraction) is used as an alternative medicine or as a complementary medicine, it becomes important to identify it as safe. Therefore the present investigation is focused on the detailed toxicity profiling. Page 5
6 Proposed plan of work and Objective I. Acute toxicity To study adverse effect of WSF on single oral dose administration in Balb/ C mice following gross general observation, behavior, body weight, hematology, serum biochemistry and pathology. II. Chronic toxicity To study adverse effects of WSF on repeated oral administration for 12 months in rats. To study gross general observations, feed and water consumption, body weight changes, hematology, serum biochemistry, urinalysis, gross necropsy and pathology. III. Genotoxicity Study In vivo bone marrow chromosome aberration assay- to study the effect of WSF on structural and numerical changes in metaphases. In vivo COMET assay- to study the potential of WSF for DNA damage. In vivo bone marrow micronucleus assay- to study the effect of WSF on the micronuclei frequency (MNPCE and MNNCE) in bone marrow. In vivo peripheral blood micronuclei- to study the effect of WSF on the frequency of MNRETs in circulatory blood. In vivo sperm abnormality assay- to study the effect of WSF on structural changes in male germinal cells (spermatozoa). In vitro genotoxicity assay- to study the effect of WSF on the potential to cause genotoxicity by micronuclei analysis in CHO cells and CBMN assay. IV. Immunotoxicity Study To study the effect of WSF on the primary and secondary response against SRBC. Page 6
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