CYTOTOXIC CONSTITUENTS FROM AERIAL PARTS OF HELICTERES HIRSUTA COLLECTED IN BINH PHUOC PROVINCE

Helicteres hirsuta is a member of Helicteres genus of the plant family Sterculiaceae and widely found in countries of South Asia, such as Vietnam, Lao, and Thailand. In recent years, it is known as a new folk medicine protecting and securing people against human lung carcinoma, hormone-dependent human prostate carcinoma, and human liver. In this work, three compounds were isolated and structurally elucidated from the aerial parts of Helicteres hirsuta collected in Binh Phuoc province (October 2016). They are -stigmasterol (1), protosta-17(20),24-dien-3-ol (2), and icosanoic acid (3). Compound 2 has a remarkable cytotoxic activity against SK-LU-1, Hep-G2, and Hela cell lines with IC50 values from 32.86 to 77.31 g/mL. Meanwhile, compound 3 shows a moderate cytotoxic activity against SKMel-2, AGS, SK-LU-1, Hep-G2, and Hela cell lines with IC50 values from 59.02 to 80.87 g/mL.


Introduction
Helicteres hirsuta, called "An Xoa" in Vietnamese, has been used as a folk medicine for curing lung carcinoma, human liver, etc. [1,2].Previous studies on Helicteres species have disclosed that flavonoids, neolignans, quinines, and triterpenoids [3][4][5][6] are the major chemical constituents.There have been no reports on the biological activity or phytochemical investigation of Helicteres hirsuta in Vietnam.In this paper, we describe our preliminary phytochemical study on the chemical constituents of the aerial parts of Helicteres hirsuta collected in Binh Phuoc province.
constant J (by Hz) for 1 H NMR spectrum.ESI-LC-MS spectra were recorded on an Agilent LC mass spectrometer.Silica gel (Merck Co., Germany) was used for flash chromatography.Thinlayer chromatography (TLC) was carried out on precoated Si gel GF254 (Merck Co., Germany), and TLC spots were viewed at 254, 302 and 366 nm and visualized by spraying with a vanillin -10% H2SO4 solution.

Plant material
The aerial parts of Helicteres hirsuta were collected in the Ta
In this assay, 200 L of cells at the concentration of 3 × 10 4 cells/mL were inoculated into a 96well plate in the RPMI 1640 medium.Pure compounds 1-3 were applied at final concentrations 128, 32, 8, 2 and 0.5 µg/mL, and the cultures were incubated for 3 days at 37 °C with 5% CO2.
Then, 50 L of MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide prepared at 1 mg•mL -1 in FBS was added to the microculture well.After 4 h of incubation, 250 L of the supernatant was removed from each well and 100 L of DMSO was added and thoroughly mixed.The absorbance was measured at 540 nm in the Genios TECAN spectrophotometer.The IC50 value was calculated on the basis of the percentage of growth inhibition (ODcontrol-ODsample)/ODcontrol.Ellipticine was used as a referential compound.

Structural identification of isolated compounds Compound 1
Compound 1 was isolated as a white crystal, well soluble in chloroform and DMSO.The 1 H NMR spectrum of compound 1 shows the presence of two methyl singlets at  0.67 and 0.96 ppm, three methyl doublets at  0.80, 0.82 and 0.90 ppm, and a methyl multiplet at  0.84 ppm.
The 1 H NMR spectrum together with 13 C NMR and HSQC spectra also shows the presence of three olefinic protons at  5.07, 5.21, and 5.25 ppm, suggesting that they belonged to two double bonds: >C=CH-and -CH=CH-.The proton that appears as a multiplet at  3.53 ppm was assigned to a methine proton, bonded to the carbinol carbon (-CHOH-).From the analysis above, we suggested that compound 1 should belong to the group of sterols.and C-20/C-21 with six methyl groups (Table 1).Thus, compound 1 was assigned as the known -stigmasterol (Fig. 1).The physical and spectral data are consistent with the reported literature values [8].This sterol is very popular in plants.Compound 2 was isolated as a white amorphous powder, well soluble in chloroform and DMSO.The 1 H NMR spectrum of compound 2 shows the presence of six methyl singlets at  0.77, 0.79, 0.85, 0.87, 0.93, and 0.98 ppm.The 1 H NMR spectrum also shows the presence of one olefinic proton at  5.26 ppm, suggesting that it belongs to double bond >C=CH-.The proton that appears as a double of doublet at  3.22 ppm was assigned to the methine proton bonded to carbinol carbon C-3 (-CHOH-).The α-orientation of this H-3 (or the β-orientation of the 3-OH) was deduced from the coupling constants, J = 11.0 and 4.5 Hz, between axial H-3 and equatorial H-28, axial H-5, and from reference [9].From the analysis above, we suggested that compound 2 should belong to the group of sterols.

Compound 3
Compound 3 was isolated as a white amorphous powder, well soluble in chloroform and DMSO.Its molecular formula was deduced to be C20H40O2 on the basis of the pseudo-molecular ion [M+H] + peak at m/z 313.1 in the ESI-LC-MS spectrum and on the basis of its 1 H NMR and 13 C NMR spectral data.
The 1 H NMR spectrum of compound 3 shows the presence of only saturated protons in the high field: one methyl triplet at  0.88 ppm, one methylene triplet at  2.35 ppm, and one multiplet with 34 protons at  1.25-1.30ppm.This information enables us to suggest that compound 3 should be an aliphatic acid.The 13 C NMR spectrum of compound 3 shows the presence of twenty carbons: one methyl carbon ( 14.1 ppm), eighteen methylene carbons with  from 22.7 to 33.7 ppm

The 13 C
NMR spectrum of compound 1 shows the presence of six methyl( 11.4, 11.5,   18.7, 19.2, 20.3, and 22.5), nine methylene, eleven methine, and three quaternary carbons.Four carbon signals at  141.1 and 119.8, and 137.0 and 128.7 were assigned to two double bonds: a cyclic >C=CH-and an acyclic -CH=CH-, respectively.The carbon signal at  69.8 was assigned to a cyclic carbinol carbon of a sterol (C-3).The spectral data above support the presence of a sterol skeleton having a hydroxyl group at the C-3 position with two double bonds at C-5/C-6