Two new polyhydroxylated triterpenoids from Salvia urmiensis and their cytotoxic activity

Abstract Two new polyhydroxylated triterpenoids were isolated from the acetone extract of the aerial parts of Salvia urmiensis Bunge. Their structures were elucidated by 1D and 2D NMR and HR-ESI-MS analyses as olean-12-ene-1β,3β,11α,22α-tetraol (1) and urs-12-ene-1β,3β,11β,22α-tetraol (2). The effect of these compounds on cell viability of MCF-7 cells was investigated by the MTT assay. Compounds 1 and 2 showed weak cytotoxicity with IC50 values of 110.23 ± 0.12 and 88.35 ± 0.09 μM, respectively. Graphical abstract


Introduction
The genus Salvia is one of the largest members of the family Lamiaceae, comprising more than 900 species, large widespread all over the world. Salvia species have been used in traditional medicine all around the world since the ancient times. Their essential oils and extracts have shown antimicrobial, antioxidant, antidiabetic, antitumour, antiplasmodial and anti-inflammatory activities (Sivropoulou et al. 1997;Dorman & Deans 2000;Ulubelen 2003;Tepe et al. 2004;Kamatou et al. 2008;Şenol et al. 2010;Chan et al. 2011;Loizzo et al. 2014;Bahadori et al. 2015). Many Salvia species are used as herbal tea and in food, cosmetics, ABSTRACT Two new polyhydroxylated triterpenoids were isolated from the acetone extract of the aerial parts of Salvia urmiensis Bunge. Their structures were elucidated by 1D and 2D NMR and HR-ESI-MS analyses as olean-12-ene-1β,3β,11α,22α-tetraol (1) and urs-12-ene-1β,3β,11β,22α-tetraol (2). The effect of these compounds on cell viability of MCF-7 cells was investigated by the MTT assay. Compounds 1 and 2 showed weak cytotoxicity with IC 50 values of 110.23 ± 0.12 and 88.35 ± 0.09 μM, respectively. perfumery and the pharmaceutical industry, and some species are grown in gardens as ornamental plants . A diverse spectrum of secondary metabolites has been identified in Salvia species (Wu et al. 2012). In Iran, there is a high diversity of Salvia species and accessions which includes 61 species, 17 of them are endemic (Jamzad 2012). Some Iranian Salvia species have been investigated from a phytochemical viewpoint, and their biological activities (Moridi Farimani et al. 2008, 2012Gandomkar et al. 2011;Rafatian et al. 2012;Farjam et al. 2013;Habibi et al. 2013;Ebrahimi et al. 2014;Esmaeili & Moridi Farimani 2014;Moridi Farimani & Mazarei 2014;Moridi Farimani & Miran 2014;Akaberi et al. 2015;Salimikia et al. 2016). Salvia urmiensis Bunge is an aromatic perennial herb that is found in the West Azerbaijan province of northwestern Iran. We recently identified several triterpenoids with rare carbon skeletons from the n-hexane and acetone extracts of this species (Moridi Farimani et al. 2013;Moridi Farimani, Bahadori, et al. 2015;Moridi Farimani, Mohammadi, et al. 2015). Further examination of the acetone extract of S. urmiensis led to the isolation of two new triterpenoids, with polyhydroxylated oleanane and ursane skeletons (1, 2) on the basis of extensive spectroscopic data ( Figure 1). Also, cytotoxic activity of the purified compounds was determined on MCF-7 cells.
Compound 2 was isolated as an amorphous powder. It had the same molecular formula C 30 H 50 o 4 (m/z 497.3605 [M + Na] + , Calcd 497.3601), as 1. The NMR spectroscopic data of 2 showed strong similarities to those of 1. In particular, rings A, B, C and D appeared to be identical. However, in the 1 H and 13 C NMR spectrum of 2, the signals of two methyl singlets at δ H /δ C 0.84/33.0 and 0.86/23.9 in 1 were replaced by two methyl doublets (δ H /δ C 0.85/17.2 and 0.86/20.9). Also, the resonances of a methylene (δ H 1.01 and 1.65; δ C 45.3) and a quaternary carbon (δ C 45.3) in 1 were replaced by those of two methine at δ H /δ C 1.32/38.8 and 1.35/31.6. Careful inspection of the resonances in the E-ring region revealed the ursane skeleton of 2 with the hydroxyl group at C-22, as in 1. Analysis of the NoESY spectrum confirmed the relative configuration of all stereochemically centres as obtained for 1. Thus, the structure of compound 2 was elucidated to be urs-12-ene-1β,3β,11α,22α-tetraol (Figure 1).

General experimental procedures
HR-ESI-MS was carried out on a Bruker 147 microToF-ESI-MS system. NMR spectra were recorded on Bruker AVANCE III-500 spectrometer operating at 500.13 MHz for 1 H NMR and 125.77 MHz for 13 C NMR with TMS as an internal standard. IR spectra were recorded using a Bruker Tensor 27 spectrometer. optical rotations were measured using a Perkin Elmer-341 automatic digital polarimeter. Silica gel (70-230 mesh) was used for column chromatography (CC), and precoated silica gel F 254 (20 cm × 20 cm) plates for TLC, both supplied by the Merck.

Plant material
The aerial parts of S. urmiensis were collected at full flowering stage in May 2012 in Takab, West Azarbaijan province in Iran. A voucher specimen (MPH-1220) has been deposited in the herbarium of Medicinal Plants and Drugs Research Institute of Shahid Beheshti University, Tehran, Iran.

Cell culture and treatment
The human breast adenocarcinoma (MCF-7) cell line was purchased from National Cell Bank of Iran, Pasteur Institute (Tehran, Iran), and maintained in DMEM medium supplemented with 10% fetal bovine serum and 100 U/mL penicillin and 100 μg/mL streptomycin. This cell was kept at 37 °C in a humidified atmosphere containing 5% Co 2 . Compounds 1 and 2 were dissolved in DMSo to make a stock of 1 mM and further diluted to final concentrations of 10-1000 μM with the serum free culture medium.

Cell viability assay
Cell viability was determined using the MTT assay. Briefly, 2.5 × 10 4 cells were seeded in 96-well plates at 37 °C with 5% Co 2 for overnight incubation and treated with appropriate concentrations of compounds of 1 and 2 for 24 h. The cells were then incubated with a serumfree medium containing MTT at a final concentration of 0.5 mg/mL for 4 h. The dark formazan crystals formed were dissolved in DMSo and the absorbance was measured at 570 nm.

Conclusion
Two new polyhydroxylated triterpenoids were isolated from the acetone extract of aerial parts of S. urmiensis Bunge. All previously reported triterpenoids of this plant were rare in nature having a ε-lactone E-ring or a C 17 -C 22 E-seco-ursane skeletons (Moridi Farimani et al. 2013, Farimani, Mohammadi, et al. 2015. While compounds 1 and 2 have usual skeletons as pentacyclic triterpenoids with four hydroxyl group at C-1, C-3, C-11 and C-22. Several polyhydroxylated ursane and oleanane type triterpenoids were previously found in different Salvia species (e.g. Salvia kronenburgii, Salvia argentea L. and Salvia argentea var. aurasiaca (Pomel) Batt. & Trab.) (Bruno et al. 1987;Topçu et al. 2004;Lakhal et al. 2014). All of these compounds possess some hydroxyl or acetoxy group at different situations. However, the most distinguished feature of the isolated compounds from S. urmiensis is their hydroxylation at C-22.

Supplementary material
1D and 2D NMR spectra of compounds 1 and 2 can be found as Supporting Information.