Phytochemical analysis and cytotoxic activity of Petiveria alliacea (Phytolaccaceae)

Petiveria alliacea (Phytolaccaceae) is a small shrub which grows in Africa and tropical America, where it is popularly known as “tipim”, “tipi” and “guiné”. The leaves are used in folk medicine as diuretic, sedative or analgesic. Phytochemical investigation led to the identification of eight compounds, six of which are being described for the first time in this species: stigmasterol, stigmastenol, stigmastanol, loliolide, 3-hydroxy-5,6-epoxy-β-ionone and benzyl-β-glucopyranoside. The hexane and dichloromethane fractions presented good antitumor activity besides low toxicity. The compounds isolated from the above mentioned fractions were also tested and showed good antitumor activity, which confirms a significant potential of steroids as antitumor agents. These results indicate the potential of P. alliacea as source of antitumor agents.


INTRODUCTION
Petiveria alliacea (Phytolaccaceae) is an herbaceous plant with a typical height of about 1m found in Africa and tropical America. In Brazil, this species occurs in north, south and southeast regions, being popularly known as "tipim", "tipi", "guiné" and "erva-de-alho" ( Other studies also report inhibitory effect against Trypanosoma cruzi, insecticidal activity, acaricide activity, besides antimicrobial activity against some This paper presents the identification of eight compounds, six of which are described for the first time occurring in this species: stigmasterol, stigmastenol, stigmastanol, loliolide, 3-hydroxy-5,6epoxy-β-ionone and benzyl β-glucopyranoside. Moreover, antitumor activity of the extracts and substances against histiocytic lymphoma (U937) were evaluated, besides cytotoxicity on peripheral blood mononuclear cells (PMBC).

GENERAL EXPERIMENTAL PROCEDURE
High performance countercurrent chromatographs (HPCCC) were acquired using a Dynamic Spectrum HPCCC apparatus (Berkshire, United Kingdom) equipped with a pair of preparative columns with 142 mL capacity and a pair of analytical columns with 27.5 mL capacity. The β-value ranges from 0.52 to 0.86 and the revolution speed was set up to 1600 rpm. The system comprises two Knauer Smartline 100 V5010 pumps (Berlin, Germany), a Buchi C-660 fraction collector and a 5 mL manual injection valve (Hichrom -Bellefonte, USA).

EXTRACTION AND ISOLATION
Dried and ground leaves (1735.89 g) were submitted to Soxhlet extraction with methanol for 24h. The methanolic extract (70 g) was fractionated by liquidliquid partition with different organic solvents yielding four fractions: n-hexane (32.71 g), dichloromethane (7.53 g), ethyl acetate (6.67 g) and n-butanol (9.29 g).
The hexane fraction (3 g) was chromatographed on silica gel with n-hexane: ethyl acetate gradients. TLC analysis of fraction 7 revealed the presence of impurities that were removed by solubilization in ethyl acetate yielding a white solid 1 (46.4 mg). Fraction 10 also revealed the presence of impurities that were removed by solubilization in methanol yielding a white solid 2 (37.6 mg).
The aqueous lower phase (143.1 mg) was submitted to HPCCC using a solvent system comprising acetonitrile: ethyl acetate: iso-butanol: H2O (0.5:3:2:5; v/v). The lower mobile phase was eluted by flow 1.5 mL/min from the head-to-tail, bloodletting of 24 mL and sample elution time 02.30 min. Compound 5 (8.3 mg) was obtained as a yellow solid.

CYTOTOXICITY AGAINST TUMOR CELL LINES
The MTT assay used to evaluate the antitumor potential of the leaves extracts (Mosmann, 1983) against human cancer cell line U937 (histiocytic lymphoma) was purchased from American Type Culture Collection (ATCC, Rockiville, MD, USA) and that against human normal cell line (PBMC) peripheral blood mononuclear cells was obtained from the venous blood of healthy volunteers (Bennett and Breit, 1994). Cytotoxicity analysis of the fractions and compounds were quantified by assessing the ability of living cells to reduce the yellow dye 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide (MTT) to a purple formazan product. All cell lines were maintained in DMEM-F12 medium (Gibco ® ) supplemented with 10% fetal bovine serum and 20 μg/mL gentamicin (Gibco ® ) at 37ºC with 5% CO2 and controlled humidity. Cells were plated in 96-well plates 100 μL/well (1x10 6 cells/mL) and incubated with various concentrations of fractions and compounds. After 48 h of incubation, 20 µL of MTT (10 mg/mL) was added to each well plate. Plates were maintained at 37ºC with 5% CO2 and controlled humidity for 4 h. After this period, 150 μL of the supernatant were removed from each well and 100 μL of a 0.0014% solution of HCl in isopropanol, was added and homogenize until complete dissolution of the purple formazan. After centrifugation for 10 min, at 1500 rpm, 100 μL of the supernatant was transferred to another 96-well culture plate in order to measure absorbance at 570 nm (Epoch™, BioTek® Instruments, Inc.). Doxorubicin (Sigma Aldrich) was used as positive control for the tested cell lines.

STATISTICAL ANALYSIS
Cell viability was evaluated by two-way ANOVA (analysis of variance). The means and respective standard errors were analyzed using the Graph Pad software Prism version 5.0 (GraphPad Software Incorporated, San Diego, USA). Significant difference was p < 0.5, <0.01 and <0.001.
According to the American National Cancer Institute, IC50 of 30 µg.mL -1 is the upper limit for an crude extract or fraction there of to be considered a potential antitumor drug (Suffness and Pezzuto 1990). The best antitumor activity was found on the achieved by hexane and dichloromethane fractions, IC50 29.91 µg/mL ± 1.09 and 24.89 µg/mL ± 1.16, respectively. These fractions also showed low cytotoxicity in PBMC, 94.61 ± 1.22 µg/mL and IC50 117.2 ± 1.24 µg/mL. Thus, the phytochemical investigation was focused on these fractions and the isolated compounds were also evaluated against U937 and PBMC (

CONCLUSION
Eight compounds were identified in this study, namely, one triterpene, four steroids, two norsesquiterpenoids and one phenolic glycoside. Six of the eight substances are described here for the first time in the species P. alliacea (stigmasterol, stigmastenol, stigmastanol, loliolide, 3-hydroxy-5,6epoxy-β-ionone and benzyl-β-glucopyranoside). In the cytotoxic activity assay we found that the hexane and dichloromethane fractions presented excellent antineoplastic activity and low toxicity. The tests performed with the compounds obtained by these fractions confirm the antineoplastic potential of the steroid class.