Protective activity of andrographolide and arabinogalactan proteins from Andrographis paniculata Nees. against ethanol-induced toxicity in mice
Introduction
Alcohol toxicity is one of the world's major health problems as the significant numbers of peoples are affected due to several fatal diseases caused by alcohol (Fleming et al., 1998). Herbal alternatives are one of the best ways to minimize these disease conditions. For this we have chosen ethanol-induced toxicity in mice model to study the effects of ethanol on liver and kidney. As a herbal alternatives, we have used Andrographis paniculata Nees. (Ap). It is also known as “King of Bitters” of Acanthaceae family. This plant has been used through out the centuries against different diseases (Barilla, 1999) especially as hepatoprotective agent (Khare, 2004). But the targeted study against the ethanol toxicity in mice with the metabolites present in the plant in acute conditions is lacking and thus it is essential to investigate the role of major metabolites of Ap against ethanol-induced toxicity.
The beneficial claims have to some extent been correlated with the different bitter principles present in different parts of the plant with varied concentrations. Of the different bitter principles present, andrographolide, a diterpenoid lactone, is the most medicinally active phytochemical. Analysis of the whole plant also revealed the presence of andrographolide (C20 H30 O5, mp: 230–239 °C), 14-deoxy-11-oxoandrographolide (DA, C20 H28 O5, mp: 98–100 °C), 14-deoxy-11,12-didehydroandrographolide (DDA, C20 H30 O4, mp: 175 °C) and neoandrographolide (C26 H40 O8, mp: 167–168 °C) (Balmain and Connolly, 1973). Andrographolide was also tested for its protective action against carbon tetrachloride-induced liver toxicity in mice (Kapil et al., 1993) and as well as the Ap extract was found to be effective against carbon tetrachloride-induced liver damage which was evident by morphological, biochemical and functional parameters (Handa and Sharma, 1990a, Rana and Avadhoot, 1991). Besides the aqueous extract appears to be better inhibitors of hepatic microsomal enzymes under in vivo short term and in vitro conditions in compare to andrographolide (Choudhury et al., 1987). Thus, these findings suggests that there may be several other ill-defined component(s) present along with andrographolide in the crude or aqueous extract of Ap leaf. Hence, it is important to isolate and characterize the compounds present in aqueous or crude extract of the title plant (Fig. 1).
We have done the metabolite profiling and isolated andrographolide (ANDRO) and arabinogalactan proteins (AGPs) and reported its antimicrobial activity against pathogenic microorganisms (Singha et al., 2003). In the present study, an attempt has been made to minimize the ethanol-induced toxicity in acute conditions by treating the mice prior to exposure of 25% ethanol (7.5 g/kg of body weight) with the purified and quantified ANDRO and AGPs of the title plant at 62.5 mg/kg of body weight, 125 mg/kg of body weight, 250 mg/kg of body weight and 500 mg/kg of body weight of mice, respectively. Also we have used silymarin, which is a standard hepatoprotective agent (Salmi and Sarna, 1982) to compare the degree of hepato-renal activity. The study was performed by analyzing the biochemical parameters in liver and kidney tissues as these two tissues are affected due to ethanol toxicity (Wallace, 1997).
Section snippets
Plant materials
Andrographis paniculata Nees. (Acanthaceae) air-dried herbs were collected from the campus of IIT, Kharagpur, West Bengal, India in July–August. A voucher specimen has been deposited to the CAL herberium, Botanical Survey of India, Howrah, India under the accession number IIT-VU/Ap-1.
Extraction of ANDRO
Extraction of ANDRO was done according to Harborne (1998). Ap powder was homogenized in methanol and water mixtures and filtrate was first acidified with sulphuric acid and then extracted with chloroform. The
Isolation of ANDRO
Isolation of ANDRO was carried out by HPTLC (Fig. 2a and b). The band parallel to reference was eluted out and used for further analysis as well as mice experiments.
Detection of ANDRO by FTIR
Isolated andrographolide was detected FTIR. The FTIR spectra of the Ap showed several intense bands in the region between 4000 and 1200 cm−1 (Fig. 3a and b) and three notable features are noted: (i) a broad band near 3400 cm−1 due to ν(–OH) stretching, (ii) a strong band near 1700 cm−1 due to ν(–COOR, ester) stretching frequency, (iii)
Discussion and conclusion
Study of any herbal molecules becomes more significant when it ameliorates some diseases conditions. In the present study, the protective effects of the partially characterized metabolites have been carried out from Ap in ethanol-induced liver and kidney tissues. For this we have characterized two major metabolites viz.; ANDRO and AGPs (Singha et al., 2003). ANDRO was isolated by HPTLC, FTIR and quantified by HPLC. AGPs were isolated by β-glucosyl Yariv reagent, which is red dye, prepared by
Acknowledgements
We acknowledge the help provided by Dr. Sibaprasad Mukherjee, Department of Chemistry, Indian Institute of Chemical Biology, Kolkata, West Bengal, India for providing the standard andrographolide and Prof. Basudham Adhikari, Material Science Center, IIT, Kharagpur, India for helping to do FTIR analysis. We are also grateful to Anchrom, Mumbai for doing HPTLC analysis.
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