CHEMICAL CHARACTERIZATION AND BIOACTIVITY OF AYURVEDIC CRUDE DRUGS AND RELATED PREPARATIONS: BOERHAAVIA DIFFUSA L., CONVOLVULUS PLURICAULIS CHOISY AND CURCULIGO ORCHIOIDES

Ayurveda, the traditional Indian medicine, is recognized by the European Union as non-conventional medicine and it includes more than 7000 plants used for therapeutic purposes in complex formulations poorly investigated under a chemical and biological point of view (Guerrini and Sacchetti, 2012). On these premises, preparations (decoction, DEC, and hydro-alcoholic extract, HE) of Boerhaavia diffusa L. (roots), Convolvulus pluricaulis Choisy (whole plant) and Curculigo orchioides Gaertn. (roots) were studied for the first time through a chemical and bioactivity approach. GC-FID, GC-MS, HPLC and HPTLC, and NMR strategies were employed to identify and quantify the main chemical compounds and to verify the repeatability of their fingerprinting. The study of biological activities was driven both by the ethnomedical traditional uses of the species in the Ayurvedic culture (Agrawal et al., 2011; Bhowmik et al., 2012; Chauhan et al., 2010) and by those aspects related to modern western phytotherapic culture: antioxidant activity; genotoxic and anti-genotoxic potential; and cytotoxicity against cancer and normal human cell lines. Ferulic acid and vanillin were identified and quantified in DEC and in HE of B. diffusa. However, HE showed the presence of other two characterising molecules: boeravinone B and eupalitin. β-sitosterol was identified and quantified in both preparations, but resulted more abundant in DEC. Ferulic acid showed antioxidant activity by ABTS assay higher than positive control. Preparations and pure compounds did not show anti-genotoxic activity towards two compounds with mutagenic properties. The study of cytotoxicity using DEC, HE and pure molecules against human cancer cell lines (A549, CaCo-2, MCF7, LoVo and HepG2), showed once more data far from those stated by the American National Cancer Institute (Suffness and Pezzuto, 1991). DEC of Convolvulus pluricaulis was characterised by the presence of phenolic acids (caftaric acid, caffeic acid, p-coumaric acid, iso-ferulic acid and tr-ferulic acid). Stigmasterol, β-sitosterol, lupeol and vanillin isomers and derivatives (2H4MB, 3H4MB and acetovanillone) were also detected in the same preparation. HE showed the presence of p-coumaric acid, vanillin, 2H4MB, acetovanillone, and vanillic acid, but the absence of phytosterols. The antioxidant activity evaluation indicated DEC as the preparation that exhibited always the best efficacy. HE showed instead an higher activity than DEC in the ABTS assay, no activity against DPPH radical and a lower capacity of blocking the radical propagation than DEC in the β-carotene bleaching test. Cytotoxic activity of the considered preparations against A549, CaCo-2, MCF7, LoVo and HepG2 cancer cell lines are not significant. The tests carried out with DEC and HE extracts against two leukaemia cell lines, drug-sensitive (CCRF-CEM) and multi-drug-resistant (CEM/ADR5000), showed, instead, interesting results: DEC CHCl3 extract and the HE soxhlet extract exhibit experimental data in line with the one stated by the American National Cancer Institute against the CCRF-CEM cell line, but respectively about 2 and 3 fold higher in the tests against CEM/ADR5000, showing cross-resistance phenomena. Curculigoside A and orcinol-β-D-glucoside were identified and quantified through HPLC methods in C. orchioides, and chosen as characterising compounds of preparations. β-sitosterol was also identified and quantified using GC-FID. The HE samples showed the highest antioxidant activity in every performed test. Regarding the anti-genotoxic activity, DEC resulted slightly more active than HE, but the highest activity was showed by orcinol-β-D-glucoside, even if it did not reach the 50% inhibition at the maximum concentration tested. The evaluation of the cytotoxic activity against human cancer cell lines exhibited an opposite trend, pointing out DEC as more active than HE for A549, CaCo-2, and HepG2. HE was instead more active against CCRF-CEM and CEM/ADR5000, but with IC50 data far from significant indication given by the American National Cancer Institute. Pure molecules did not exhibit any activity. However, further chemical and biological investigations will be carried out to shed an even more clear light on efficacy and safety of Ayurvedic preparations.