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Anti-Tumor Activity of Four Ayurvedic Herbs
Tinospora cordifolia, Curcuma longa, Ocimum sanctum, Zizyphus mauritiana
The anti-tumor activity and chemopreventive potential of four Ayurvedic herbs viz. Curcuma longa L., Ocimum sanctum L., Tinospora cordifolia (Wild) Miers ex Hook.f & Thomas and Zizyphus mauritiana Lam. were evaluated using Dalton Lymphoma ascites (DLA) tumor model in Swiss Albino mice. The outcome was assessed using survival time, peritoneal ascitic fluid (Tumor volume) and hematological indices as parameters. Animals were divided into five groups (n = 6) viz. one DLA control and four Herb + DLA treated groups. All the four herb + DLA groups were pre-treated with respective herbs for 7 days and hematological indices were measured for entire five groups. On day-8 animals were inoculated with 1×106 DLA cells i.p., and Herb + DLA groups were continued with oral herbal treatment for 21-days. Hematological parameters and tumor volume were assessed to find the effects of herbs. Short term in vitro cytotoxicity was determined by Trypan Blue exclusion method and LDH leakage assay using different concentrations of herbal extracts and 5-FU as a positive control and IC50 for each herbal extract and 5-FU were determined. Oral administration of crude herb increased the survival time and decreased the peritoneal ascitic fluid content significantly. Hb, RBCs and total WBC which were altered by DLA inoculation were restored significantly by all the herbs except O. sanctum. All the four herbs showed in vitro cytotoxic activity against DLA cell-line. Moreover inter group comparison of all the four herbs for anti-tumor activity showed efficacy in the following order- T. cordifolia > Z. mauritiana ≥ C. longa > O. sanctum respectively.
Keywords: Curcuma longa, Ocimum sanctum, Tinospora cordifolia, Zizyphus mauritiana, DLA cell line, Cytotoxicity, Anti-tumor activity
Cancer has been a leading cause of death in the age group 45–64 yrs in developed countries. With changing standard of living and food habits and also due to availability of curative treatment for many infectious diseases, cancer is surpassing other illnesses as a principle cause of morbidity and mortality even in developing countries (Noting, 2001). Surgery, radiotherapy and chemotherapy- the established treatment modalities for various cancers are costly, mutilating, having serious side effects and associated with residual morbidity as well as frequent relapses. Ayurveda- a science of health and longetivity has tried many herbal as well as Rasayana remedies with varying degree of success, but its main significance lies in its preventive approach. Hartwell has collected data, about 3000 plants, which possess anti-cancer properties and subsequently been used as potent anti-cancer drugs (Balachandran and Govindrajan, 2005). Among Indian Ayurvedic herbs, some 30 herbs have shown antitumor activities, and the number may rise as more and more herbs are studied (Ramakrishnan et.al., 1984). Searching the Ayurvedic arena for medicinal herbs with probable antitumor activity, there are multitudes of them having various pharmacological actions but many of them are difficult to identify, scarcely available, some have toxic side effects and are used infrequently; so we decided to select a few herbs that are well known, easily available, cheap, whose identity is non-controversial, free from any known toxicity and that are in vogue for centuries. Curcuma longa L., Ocimum sanctum L., Tinospora cordifolia (Wild.) Miers ex Hook. f. & Thoms. and Zizyphus mauritiana Lam. having antioxidant, immunomodulatory and chemopreventive potential, were selected to evaluate their ability and relative efficacy to prevent or modify the course of DLA in Swiss albino mice.
Previous in vivo studies of above herbs for cancer cell lines had been carried out by using some specific extracts in high concentration, that is impossible to achieve in blood, via intraperitoneal root. In present study oral route for administration of herbs was preferred purposefully to directly establish the therapeutic relevance of the herbs tested. The results of ex vivo studies in which the inoculation of tumor cells and administration of herbal extracts both intraperitoneal may not be representative of the actual efficacy of the herbs because absorption, first-pass metabolism, protein binding, serum concentration of actual circulating active compounds and metabolites i.e. the pharmacokinetic effects for oral as well as parenteral route is bypassed. This experiment was designed to put the herbs to an acid test of efficacy to find out the truth about conventional claims and isolated studies of activities contributory to anticancer effects. Though conventionally these herbs have been consumed orally only, to the best of our knowledge nobody has made a study of anti-tumor activity of these herbs by using oral root of administration. Naturally the outcome of this study should be relevant to validate as well as improvise the conventional use.
Curcuma longa (CL) (family Zingiberaceae), known as Haldi in India and Turmeric in English, has demonstrated a wide spectrum of therapeutic effects such as anti inflammatory, antioxidant, anti mutagenic, antitumor, antifungal, antiviral, antibacterial, antispasmodic and hepatoprotective. Recently its potential utility in acquired immune deficiency syndrome (AIDS) was demonstrated (Kohli et.al., 2004).
No acute toxicity in mice was observed on administration of tumeric powder with dose as high as 10g/kg-bw (Sittisomwong et.al., 1990). The lowest published toxic oral dose for mouse is 13650mg/kg for 13-weeks the toxic effect being changes in liver weight (NTPTR., 1993).
Ocimum sanctum (OS) (family Labiatae), known as Tulsi in India and Holy Basil in English is known to have adaptogenic activity (Rege et.al., 1999). OS contains a volatile oil consisting of about 70% eugenol as well as methyl eugenol and caryophyllene. Other constituents with likely pharmacological activity include the triterpenoids ursolic acid, rosmarinic acid, oleanic acid; flavonoids apigenin and luteolin; alkaloids; saponins; phenylpropane glucosides and tannins. The seeds contain a fixed oil containing five fatty acids, including about 17% linolenic acid and just over 50% linoleic acid (Archana and Namasivayam, 2000). It has numerous pharmacological activities like hypoglycemic, antistress, immunomodulatory, analgesic, antipyretic, anti-inflammatory, antiulcerogenic, antihypertensive, CNS depressant, hepatoprotective, chemopreventive, radioprotective, antitumor and antibacterial properties (Samson et.al., 2006, Adhvaryu et. al., 2007).
OS ethanolic extract 200mg/kg-bw for 30-days in rats and 500mg/kg-bw for 15-days in mice did not produce any toxic side effects (Vats, et.al., 2004, Panda and Kar, 1998). Doses upto 4g/kg-bw for 14-days did not produce any toxicity or mortality in rats (Shetty, et.al., 2007). The LD50 of aqueous extract of OS in mice was found to be ≥5gm/kg-bw (Umadevi and Ganasoundari., 1995).
Tinospora cordifolia (TC) (family Menispermaceae); is a large climbing shrub, growing throughout tropical India; and popularly known as Giloya in Hindi and Tinospora in English. It contains tinosporine, tinosporide, tinosporaside, cordifolide, cordifol, heptacosanol, clerodane furano diterpene, diterpenoid furanolactone tinosporidine, columbin and β-sitosterol. The aqueous extract of guduchi stem has shown the presence of arabinogalactan that showed immunological activity. The bitter principle present shows adaptogenic, antispasmodic, anti-inflammatory, antipyretic, anti-neoplastic, hypolipidemic, hypoglycemic, antioxidant, immunopotentiating and hepatoprotective properties (Jagetia and Rao, 2006; Adhvaryu et al., 2007). It is used in general debility, digestive disturbances, loss of appetite and fever in children. It is also an effective immunostimulant (Kapil and Sharma 1997).
400mg/kg-bw of aqueous extract of TC given per oral (PO) to Swiss albino mice for 60 days did not produced any significant toxic effect (Grover et.al., 2000). LD50 for TC PO in Swiss albino mice was found to be 2650 (2209–3901) mg/kg (Atal et.al., 1986).
Zizyphus mauritiana (syn. Zizyphus jujuba L.) (ZM),(family Rhamnaceae) is a plant of very common occurrence. It grows wild in forests and also on wastelands throughout India. In India it is commonly known as ‘Ber’ and in English it is known as Indian berry. Pharmacologically active compounds from seeds include jujubojenin, jujubosides-A1 B and C, acetyljujuboside B1, protojujubosides A, B, & B1 (Yoshikawa et al., 1997; Matsuda et al., 1999). The seeds are traditionally used for insomnia and anxiety. The active compounds are reported to have a potent immunological adjuvant activity (Matsuda et al., 1999) and inhibitory effects on hippocampal formation in vivo and in vitro probably through its anti-calmodulin action (Zhang et al., 2003).
ZM is reported to have very low toxicity when taken orally, in mice and rats; a huge single dose of 50g/kg-bw produced no toxic symptoms and a daily dose of 20g/kg-bw for 30 days did not produced toxic reactions. No side effects were reported (Zhu, 1998).