Sterols and Terpenoids from Viburnum odoratissimum

A new stigmasterol type natural product, viburodorol A (1), along with eleven known sterols and terpenoids (2–12), were isolated from the aerial parts of Viburnum odoratissimum. The structure of 1 was elucidated on the basis of comprehensive spectroscopic analysis. It’s noteworthy that compound 2, the major constituent of this plant, can significantly stimulate glucose absorption in insulin resistant HepG2 cells without affecting cell viability. Furthermore, this compound can also restore the glucose absorption in DXMS-induced insulin resistant 3T3-L1 cells.


Introduction
Diabetes mellitus (DM) is a heterogeneous group of disorders characterized by hyperglycemia [1], resulting from absolutely insulin deficiency, insulin resistant and/or abnormal secretion [2]. Over 90 % of diabetes cases around the world belong to Type 2 diabetes [3], a heterogeneous, polygenic disorder resulting from interaction between susceptibility genes and lifestyle/environmental factors [4]. About 4.8 million people died and 471 billion USD were spent due to DM in 2012 globally. According to a latest survey, the number of people living with DM has reached up to 371 million; unfortunately, the number is keep on rising [5]. DM may lead to a series of complications, such as blindness, renal failure, nerve damage, stroke, and limb amputation [6]. Currently available therapies for DM include some main classes of agents such as sulphonylureas, biguanides, a-glucosidase inhibitors, and thiazolidinediones [7]. However, they may show different kinds of adverse effects in varying degrees, like gastrointestinal disturbances, and hypoglycemia [4]. Thus, more effective and safer agents need to be found.
Insulin resistance is the main character of type 2 diabetes which leads to decreased glucose absorption by fat, liver, skeleton muscle, etc. A potential anti-diabetic drug should be highly active in restoring glucose absorption and none or less toxic to body tissues. Recently, we found that the extract of Viburnum odoratissimum significantly stimulated glucose absorption in insulin resistant HepG2 cells at concentrations that did not affect cell viability. At the concentration of 25 lg/mL, the extract increased glucose consumption of insulin resistant HepG2 cells by 7.5 %, showing ability to stimulate glucose absorption and restore insulin sensitivity of insulin resistant HepG2 cells.
V. Odoratissimum Ker-Gawl was an evergreen shrub or dungarunga distributed in southeast of China and used in folk as a medicine to diminish inflammation, relieve pain, and treat rheumatism [8]. Several diterpenes, triterpenes, flavones, lignans, and coumarins have been reported as the main secondary metabolites of this plant [9]. With the aim of searching for structural interesting metabolites with insulin sensitizing activities, the chemical constituents of this plant was investigated, and a new stigmasterol type natural product, viburodorol A (1), along with eleven known sterols and terpenoids (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12), were isolated ( Fig. 1). It's noteworthy that compound 2, the major constituent of this plant in this study, can also significantly stimulate glucose absorption in insulin resistant HepG2 cells without affecting cell viability. Furthermore, this compound can also restore the glucose absorption in DXMS-induced insulin resistant 3T3-L1 cells. Reported herein, was the isolation and structural elucidation of these natural products as well as insulin sensitizing activity of 2.
Compound 1 was obtained as white powder. , and C-25 (d H 2.86, d C 28.5), two quaternary signals for C-10 (d C 37.1) and C-13 (d C 43.8), together with the seven typical methyls mentioned above, 1 could be ascribed to be a steroid type metabolite related closely to stigmasterol with thirty carbons in the skeleton [21,22].
Detailed comparison of the NMR spectral data of 1 with those of 4a-methyl-24S-ethyl-5a-cholestan-3b-ol indicated that they are similar to one another [21]. Comparatively, the oxygenated methine at C-3 of the known analogue was replaced by a carbonyl group (d C 213.3) in 1, which can be confirmed by its HMBC correlations with H- . Thus, the planar structure of 1 was elucidated and can be confirmed by the undoubted HMBC correlations of the seven methyls (Fig. 2), as well as the six proton systems obtained by 1  The relative configuration of 1 was deduced based on ROESY experiment conjugated with the biogenetic can not be defined. Therefore, the structure of 1 was deduced as shown and named viburodorol A. In addition, insulin resistance in HepG2 cells was induced by dexamethasone (DXMS) treatment and the new (1) and major constituent (2) were assessed the activity in enhancing glucose consumption of the cells. The result exhibited that compound 2 can significantly stimulate glucose uptake in insulin resistant HepG2 cells (P \ 0.05-0.001, Table 2) without affecting cell viability, showing ability to restore the impaired insulin sensitivity of cells. At the concentrations of 10, 5, 2.5, 1.25 lmol/L, compound 2 increased glucose consumptions of insulin resistant HepG2 cells from 71.6 % to 168.7, 155.9, 128.3 and 90.1 % respectively, showing excellent activity in stimulating glucose uptake. Further investigation on the activity in murine fat cells of 2 was also carried out. DXMS was also employed for induction of insulin resistant as described above. Rosiglitazone which is a typical insulin sensitizer was used as positive drug control. Table 3 showed that, unlike rosiglitazone which strongly prompted glucose uptake in both insulin sensitive and resistant 3T3-L1 adipocytes, (P \ 0.001 and 0.01), Compound 2 at 10 or 5 lmol/L did not obviously increase glucose consumption in sensitive cells (P [ 0.05) but completely restored the impaired glucose uptake in DXMS-induced insulin resistant cells (P \ 0.05). These results suggested that 2 might be a promising candidate as insulin sensitizer for treatment of type 2 diabetes.

General Experimental Procedures
Optical rotations were measured with a HORIBA SEPA-300 high-sensitive polarimeter. IR spectra were determined on a Bruker Tensor-27 infrared spectrophotometer with KBr disks. UV spectra were recorded on a Shimadzu  UV2401A ultraviolet-visible spectrophotometer. HRTOFMS analysis was carried out on an API QSTAR time-of-flight spectrometer and on a Waters Auto Spec Premier P776 mass spectrometer. NMR spectra were recorded on Bruker AV400, DRX-500 and DRX-600 spectrometers at 25°C, using TMS as an internal standard. Chemical shifts were reported in units of d (ppm) and coupling constants (J) were expressed in Hz. Column chromatography (CC) were carried out over silica gel (200-300 mesh, Qingdao Haiyang Chemical Co. Ltd), Rp-18 (40-63 lm, Merck), and Sephadex LH-20. Pre-coated silica gel plates (Qingdao Haiyang Chemical Co. Ltd.) were used for TLC. Detection was done under UV light (254 and 365 nm) and by spraying the plates with 10 % sulfuric acid followed by heating. An Agilent series 1100 (Agilent Technologies) were used for HPLC. An Agilent ZORBAX SB-C 18 column 5 lm 143 Å column (250 9 9.4 mm) were used for semi-preparative HPLC separations.

Plant Material
The aerial parts of V. odoratissimum were collected in Kunming Botany Garden, Yunnan Province, P. R. China, in October 2008. The plant was identified by Dr. Xiao Cheng, Kunming Institute of Botany, Kunming, P. R. China. A voucher specimen was deposited at Kunming Institute of Botany with identification number 200810V02.

Cell Line and Cell Culture
Human hepatoma cell line HepG2 was purchased from the Laboratory Animal Center of Sun Yat-Sen University (Guangzhou, China) and was maintained in DMEM containing 2.0 g/L of glucose and 10 % of FBS. Murine preadipocyte cell line 3T3-L1 was a gift from Prof. WF Fong of Hong Kong Baptist University and was maintained in DMEM containing 4.5 g/L of glucose and 10 % of FBS. Both cell lines were incubated at 37 8C, 5 % CO 2 , and saturate humidity.

Glucose Consumption in HepG2 Cells
Effect of test samples on glucose absorption in insulin resistant HepG2 cells were performed in 96-well format. In brief, 8 9 10 3 HepG2 cells suspended in 100 lL of growth medium (DMEM ? 10 % FBS, Gibco products) were seeded in 96-well plates and incubated for 24 h to let cells to adhere to the well bottom. Then the cells were replaced the medium with 100 lL of fresh growth medium containing 1 lmol/L of DXMS and different concentrations of test samples. After 48 h incubation, 5 lL per well of the medium was taken for measurement of glucose concentration using Glucose Assay kits (Changchun Huili Biotech Co., Ltd, China). Cells treated with 1 lmol/L of DXMS alone were set as insulin resistant control while cells without any drug treatment were set as insulin sensitive control, respectively. Glucose consumed by cells of different treatments was expressed as relative glucose consumption (%) over to insulin sensitive control cells whose relative glucose consumption was set as 100 % [23].
3.4.4 Glucose Consumption in 3T3-L1 Adipocytes 5 9 10 3 3T3-L1 preadipocytes suspended in 100 lL of growth medium were seeded in 96-well plates and let cells grow to confluence. Then cells were induced for adipocyte differentiation in growth medium containing 10 lg/mL of insulin (Sigma-Aldrich), 1 lmol/L of DXMS (Sigma-Aldrich), and 0.5 mmol/L of 3-isobutyl-1-methylxanthine (Sigma-Aldrich) for 48 h, followed in growth medium containing 10 lg/mL of insulin for 48 h, and lastly in fresh growth medium. The induction lasted for 8 days and the media were refreshed every 2 days.
The differentiated adipocytes were replaced the medium with 100 lL fresh growth medium containing different concentrations of test samples. Glucose left in medium after 48 h of incubation was measured by Glucose Assay kits as described above. To investigate the action of test samples on insulin resistant adipocytes, 1 lmol/L of DXMS was added to the medium together with the test sample [24].

Cell Viability Assay
After taking 5 lL per well of the medium for glucose consumption assay, the 96-well plate was added 10 lL of CCK-8 (DOJINDO LABORATORISE, Japan) and further incubated at 37°C for 1.5 h. Optical density (OD) positively correlated with the number of living cells was read at 450 nm. Effects of test samples on cell viability were evaluated according to OD value [25].

Statistical Analysis
Relative glucose consumption after 48 h of incubation was expressed as Mean value ± Standard deviation. Statistical analyses were performed by One-way ANOVA. Differences were considered significant when the P value was less than 0.05.