A new epidioxy-tetracyclic triterpenoid from Poria cocos Wolf

Abstract A new epidioxy-tetracyclic triterpenoid, 3-(2-hydroxyacetoxy)-5α, 8α-peroxydehydrotumulosic acid (10), along with nine known compounds were isolated from Poria cocos Wolf (Polyporaceae). Their structures were determined by extensive spectroscopic analyses and comparison with literature data. The cytotoxicity of these compounds against human cancer cell lines MNK-45 was evaluated by MTT method.


Introduction
During recent years, higher fungi from Polyporaceae have been reported to possess a variety of bioactivities, such as anti-inflammatory, antimicrobial, antioxidant, enzyme inhibition and immuno-enhancing activities (Chen & Wang 2015). Poria cocos Wolf (Polyporaceae), known as Fu-Ling (茯苓) in Chinese, is a well-known medicinal fungus widely used in China and other East Asian countries owing to its various therapeutic effects (Ling et al. 2012). This fungus gives large, edible sclerotia which have been referred as 'tuckahoes' or 'Indian bread' in North America, where the name Wolfiporia cocos is adopted. However, the name Poria cocos is preferred in Asia (Lindner & Banik 2008). Previous studies on this fungus have demonstrated its notable anti-inflammatory activity in different experimental models of acute and chronic inflammation (Ríos 2011). The sclerotia of P. cocos is one of the most important crude drug used as medicinal mushroom in traditional Chinese medicine for treatment of promoting diuresis, excreting dampness, invigorating the spleen and tranquilising the mind (Kobira et al. 2012;Wang et al. 2012).
All the ten isolated compounds were evaluated for their cytotoxicity against human cancer cell lines MNK-45 (gastric carcinoma) by MTT method. 5-Fluorouracil was used as a positive control, and the IC 50 values of all these compounds were examined. As a result, compounds 6 and 10 showed moderate cytotoxic activities against MNK-45 cell lines with respective IC 50 values of 21.7 and 26.2 μM; compounds 2, 7 and 9 showed weak cytotoxic activities with IC 50 values of 41.3, 30.5 and 37.3 μM, respectively; while the other five compounds (1, 3, 4, 5 and 8) exhibited unobvious cytotoxic activities with IC 50 values greater than 50 μM (Table 1).

Plant material
The dried sclerotia obtained from the cultivated P. cocos in Yingshan county, Hubei province, P.

Cytotoxicity assay
The MNK-45 cell lines were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 100 units mL −1 penicillin and 100 μg mL −1 streptomycin. The cell cultures were incubated in the atmosphere of 85% relative humidity, 5% Co 2 and 36.8 °C. Cytotoxicity was measured by MTT assay with minor modification to the reference (Mosmann 1983). Briefly, cells were seeded in 96-well microculture plates with 2 × 10 4 cells per well, and allowed to adhere for 24 h before drug addition. Each tumour cell line was exposed to the test compounds at various concentrations in triplicate for 48 h, with 5-fluorouracil as positive control. After incubation, 20 μL MTT (5 mg mL −1 ) was added to each well, and the incubation continued for 4 h at 36.8 °C. At last, 150 μL DMSo was added to each well to dissolve the formazan crystals of the viable cells. The plates were read at a wavelength of 570 nm using a Microplate Reader Wallac 1420 ARVosx. Three independent experiments were conducted. The data represented the mean of three experiments in triplicate and were statistically analysed with Student's t-test. A difference was considered statistically significant when p < 0.05. The cytotoxic assay results were shown in Table 1.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the National Science and Technology Major Project 'Key New Drug Creation and Manufacturing Program' [grant number 2013ZX09402203] of the People's Republic of China.