Stimulating effect of a new triterpene derived from Anoectochilus elwesii on glucose uptake in insulin-resistant human HepG2 cells

A new triterpene (1), 3-β-O-olean-11,13 (18)-diene-23,28-dioic acid, together with five known compounds (2–6), was isolated from Anoectochilus elwesii and their structures were elucidated by extensive spectroscopic methods and comparison with the literature data. Compound 1 was the first example of highly oxygenated triterpene obtained from Anoectochilus genus. The isolated compounds were evaluated on insulin-resistant human HepG2 cells for stimulating glucose uptake activity and the new compound displayed highly potent effect on the stimulation of glucose uptake in human HepG2 cells.


Introduction
Anoectochilus elwesii (Clarke ex Hook. f.) King et Pantl. has been used in Chinese folk medicine in diabetes and nephropathy ailments (Anonymous 1999). Pharmacological evaluation of the antihyperglycemic activity of the ethanol extract of this plant in rats confirmed the folk information (Cai et al. 2014). A previous phytochemical study on A. elwesii has revealed the occurrence of sterols, triterpenes and flavonoids (Zhu et al. 2013). As part of our ongoing investigation for the chemistry of A. elwesii growing in Yunnan Province, the south-west of China, here we report on the isolation and characterisation from the herbs of A. elwesii of a novel triterpene, 3-b-O-olean-11,13 (18)-diene-23,28-dioic acid (Figure 1).
Metabolic syndrome is a complex and chronic disease associated with adverse functioning of many organs. Among the metabolic syndrome components, hyperglycemia caused by insulin resistance is the main contributor to the associated disorders (Zaid et al. 2008). Thus, regulating glucose uptake is important for the treatment of diabetes and metabolic syndrome. A. elwesii is one of the commonly used Chinese folk medicinal herbs and has been reported to have several pharmacological effects (Zhang & Li 2010;Cai et al. 2012). However, the pharmacological effects of individual components are largely unknown. Hence, in this study, we obtained six compounds from A. elwesii and examined their effects on glucose uptake in insulin-resistant HepG2 cells under high glucose conditions. We found that, of the six compounds, the novel compound showed the most potent stimulatory effects on glucose uptake. It significantly increased glucose uptake in a good dose-dependent manner, which increased more than 50% over basal level during 20-100 mM. Metformin brought about an increase in glucose uptake, and was utilised as a positive control, which the glucose uptake increased about 50% over the basal level in response to metformin (Table 1). Compounds 2-6 also showed moderate effects on the stimulation of glucose uptake. Meanwhile, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tests revealed the tested six compounds did not show any cellular toxicity up to 100 mM concentration.

Cell culture
Human HepG2 cells were maintained in DMEM supplemented with 10% foetal bovine serum (FBS), 100 U mL 21 penicillin, 100 mg mL 21 streptomycin, 2 mM L-glutamine (Invitrogen, CA, USA), and kept at 378C; in a humidified atmosphere of 5% CO 2 in air. Cells were grown to 70% confluence and then preincubated in serum-free medium for 24 h before treatment.

Insulin-resistant HepG2 cell model
The HepG2 cells were seeded into 96 multi-well plates in DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin. The cells were cultured in a humidified incubator (5% CO 2 ) at 378C, and were allowed to attach for 24 h. Insulin-resistant cell model was induced according to the previous method (Chen et al. 2006) with a slight modification. In brief, HepG2 cells were incubated with fresh medium containing 1% FBS and 5 £ 10 27 mol/L bovine insulin for 24 h. Subsequently, the medium was exchanged with medium containing 10 29 mol/L insulin and test compounds, or metformin, and then incubation was conducted for 12 h.

Cell viability assay
Cells were seeded into 96-well plates at a density of 2 £ 10 3 cells/well and cultured for 24 h. After incubation of the cells with the isolated compounds 1 -6 at different concentrations for 48 h, the cytotoxicity of them was determined by the MTT assay as previously described (Mosmann 1983). Percentage cell viability was calculated based on the absorbance measured relative to the absorbance of control cells exposed to the vehicle alone.

Statistical analysis
Data are presented as the means^SD.

Conclusions
In conclusion, our study on isolation of triterpenes and their evaluation for stimulating glucose uptake activities are indicative of the potential of the oxygenated triterpene for improving the cells' resistant state, which may be further developed to be good proponents for the treatment of diabetes.

Supplementary material
Supplementary material relating to this article is available online.