Two New Clerodane Diterpenes from Tinospora sagittata

Two new clerodane-type diterpenes, tinosporins C (1) and tinosporins D (2) were isolated from the stems of Tinospora sagittata (Oliv.), together with three known ones, columbin (3), tinophylloloside (4), and tinospinoside D (5). The structures of these compounds were determined on the basis of spectroscopic data interpretation, with that of the absolute configuration of compound 1 was assigned by experimental and calculated ECD spectra. The cytotoxicity and α-glucosidase inhibitory activities of isolated compounds were evaluated in vitro.


Introduction
Tinospora sagittata (Oliv.) Gagnep belonging to the family Menispermaceae, is a perennial indeciduous creeping vine widely distributed throughout the south of China and also found in northern Vietnam [1]. The roots of T. sagittata have been recorded in the traditional Chinese medicine known as "Jinguolan" which has the heat-clearing and detoxifying effects [2,3]. Plants of the genus Tinospora are known to be rich in clerodane diterpenes and the isolation of clerodane diterpenoids from T. sagittata has been reported in many different papers [4][5][6][7][8][9]. Nevertheless, different plant collection locations have yielded diterpenes with a variety of structures, and some of clerodane diterpenoid lactones also showed various bioactivities, such as anticancer [8,10], anti-inflammatory [5], and anti-adipogenic and anti-obesity activities [11]. As part of an ongoing search for novel bioactive compounds, we studied T. sagittata collected in Xiangxi, Hunan Province, China. Herein, this paper reports the isolation and identification of two neo-clerodane diterpenes 1 and 2 from the whole plant, as well as their cytotoxic and α-glucosidase inhibitory activities.
The isolated compounds 1−5 were tested for their α-glucosidase inhibitory activity. Compounds 1 and 5 showed inhibition effects by 48.58%, 47.08% at a concentration of 20 µg/mL, respectively, which is weaker than that of the positive control acarbose (79.09% at 20 µg/mL). Compounds 1 and 2 were further tested for their cytotoxicity against three human tumors HepG2 (human liver hepatocellular The isolated compounds 1−5 were tested for their α-glucosidase inhibitory activity. Compounds 1 and 5 showed inhibition effects by 48.58%, 47.08% at a concentration of 20 µg/mL, respectively, which is weaker than that of the positive control acarbose (79.09% at 20 µg/mL). Compounds 1 and 2 were further tested for their cytotoxicity against three human tumors HepG2 (human liver hepatocellular carcinoma), HCT116 (human colon carcinoma) and SGC-7901 (human gastric carcinoma) cell lines, using a sulforhodamine B (SRB) method. The results indicated that both were inactive at 100 µM.

Plant Material
The whole plants of T. sagittata were collected from Xiangxi, Hunan Province, China, in August 2012, and identified by Prof. Dai-gui Zhang (Key Laboratory of Plant Resources Conservation and Utilization, Jishou University, Jishou, China). A voucher specimen (zdg201208-02) has been deposited in Hunan Agricultural University.

Extraction and Isolation
The fresh whole plant of T. sagittata (4.8 kg) was dried at 40 °C. The dried materials were powdered and extracted three times with EtOH (95% v/v) at room temperature, then the combined extract was evaporated under reduced pressure to obtain a crude residue (347.4 g). The residue was further suspended in H2O (2 L) and then successively extracted with petroleum ether (PE), EtOAc and n-BuOH to yield PE-soluble fraction 93.0 g, EtOAc-soluble fraction 54.7 g and n-BuOH-soluble fraction 60.8 g, respectively. The EtOAc-soluble fraction was subjected to silica gel column chromatography (CC) (100-200 mesh) with elution of CHCl3-MeOH (100:0→60:40, v/v) to give seven fractions (Fr. A-G).

Plant Material
The whole plants of T. sagittata were collected from Xiangxi, Hunan Province, China, in August 2012, and identified by Prof. Dai-gui Zhang (Key Laboratory of Plant Resources Conservation and Utilization, Jishou University, Jishou, China). A voucher specimen (zdg201208-02) has been deposited in Hunan Agricultural University.

Computational methods for ECD of Compound 1
The CONFLEX [17,18] searches based on molecular mechanics with MMFF94S force fields were performed for 1, which gave 8 stable conformers. Selected conformers of 1 with the lowest energy were further optimized by the density functional theory method at the B3LYP/6-31+G (d, p) level in Gaussian 09 program package [19], which was further checked by frequency calculation and resulted in no imaginary frequencies. The ECD of the conformer of 1 was then calculated by the TDDFT method at the B3LYP/6-31G (d, p) level with the CPCM model in methanol solution. The calculated ECD curve was generated using SpecDis 1.51 [20] with σ = 0.20 eV.

α-Glucosidase Inhibition Assay
The α-glucosidase inhibitory activity of 1−5 was determined following the method described in the literature with slight modifications [21]. In brief, α-glucosidase (50 µL, 0.5 U/mL) and a concentration 100 µg/mL of tested compounds (50 µL) in phosphate buffer (pH 6.8) were mixed at room temperature for 10 min. Reactions were initiated by addition of 5.0 mM p-nitrophenylα-D-glucopyranoside (PNPG, 150 µL). The reaction mixture was incubated for 15 min at 37 • C in a final volume of 250 µL. Then, 0.2 M Na 2 CO 3 (100 µL) was added to the incubation solution to stop the reaction. The activities were detected in a 96-well plate, and the absorbance was determined at 405 nm (for p-nitrophenol). The negative blank was set by adding phosphate buffer instead of the sample via the same way as the test. Acarbose was utilized as positive control. The blank was set by adding phosphate buffer instead of the α-glucosidase using the same method. Inhibition rate (%) = [(OD negative control − OD blank ) − (OD test − OD test blank )]/(OD negative blank − OD blank ) × 100%.

Cytotoxicity Assay
The cytotoxicity of the new compounds 1 and 2 against three human cancer cell lines, human gastric carcinoma (SGC-7901), human colon carcinoma (HCT 116) and human liver hepatocellular carcinoma (HepG2) were assayed at the National Center for Drug Screening, Shanghai, China. Sulforhodamine B (SRB) (Sigma-Aldrich Chemie GmbH, Munich, Germany) was used to test the effects of the compounds on cell growth and viability [22]. Adriamycin was used as the positive control.

Conclusions
The phytochemical investigation of whole plant of T. sagittata afforded two new clerodane-type diterpenes, tinosporin C (1) and tinosporin D (2), as well as three known ones, columbin (3), tinophylloloside (4), and tinospinoside D (5). Bioassays showed that compounds 1 and 5 display slight in vitro α-glucosidase inhibitory activities. The discovery of the new compounds expands our knowledge of the structural diversity of the clerodane-type diterpenes produced by the plant T. sagittata.