Triterpenoids from the Roots of Sanguisorba tenuifolia var. Alba

The ethyl acetate soluble fraction from the roots of Sanguisorba tenuifolia was found to have a hypoglucemic effect in alloxan-induced diabetic rats. Two new triterpenoids, identified as 2-oxo-3β,19α-dihydroxyolean-12-en-28-oic acid β-D-gluco-pyranosyl ester (1) and 2α,19α-dihydroxy-3-oxo-12-ursen-28-oic acid β-D-glucopyranosyl ester (4) were isolated from this fraction, along with thirteen known triterpenoids. Their structures were elucidated by chemical and spectroscopic methods. All these compounds demonstrated inhibitory activities against α-glucosidase with IC50 values in the 0.62-3.62 mM range.


Introduction
Diabetes mellitus (DM), considered a lifestyle related diseases, is a metabolic disease with hyper-glycemia as a symptom and causes many complications [1]. Recently, DM is becoming a serious problem around the World, and according to World Health Organization, it affects approximately 171 million people worldwide and the number is expected to reach to 366 million over the next 20 years [2]. Many researchers have enthusiastically studied the development of antidiabetic OPEN ACCESS agents, however, many potential therapeutics have a number of serious adverse effects [3,4], therefore there is a growing trend toward using natural products as treatment [5]. China has a long history of using herbs for the treatment of human diseases and several medicinal plants are used for the treatment of diabetes. S. tenuifolia is one such plant.
S. tenuifolia (Rosaceae) is a perennial herb, which is widely distributed in China's Heilongjiang, Liaoning, and Jilin provinces and Inner Mongolia. The residents in Northeast China regard S. tenuifolia as a substitute for S. officinalis, and apply its roots for the treatment of diarrhea, chronic intestinal infections, duodenal ulcers, diabetes mellitus and bleeding [6,7]. Our studies indicated that ethyl acetate fraction of a S. tenuifolia root ethanol extract contains plenty of triterpenes, which can inhibit plasma glucose levels in alloxan-induced diabetic rats. α-Glucosidase inhibitors are oral anti-diabetic drugs used for diabetes mellitus type 2. They can significantly delay the absorption of carbohydrates from the small intestine and thus have a lowering effect on postprandial blood glucose and insulin levels [8]. Based on a bioassay-guided isolation, a phytochemical study of S. tenuifolia was performed and two new triterpenoids were isolated from its ethyl acetate fraction, along with thirteen other known triterpenoids. The new compounds were identified as 2-oxo-3β,19α-dihydroxy-olean-12en-28-oic acid β-D-glucopyranosyl ester (1) and 2α,19α-dihydroxy-3-oxo-12-ursen-28-oic acid β-D-glucopyranosyl ester (4), respectively. In the present report, we describe the structural elucidation of 1 and 4, together with the α-glucosidase inhibitory activity data of all the compounds 1-15 ( Figure 1).

Results and Discussion
Compound , and an olefinic proton signal at δ H 5.45 (br s), which were characteristic of the oleanolic acid skeleton. Comparison of the data 1 with those of oleanolic acid [9][10], suggested that the aglycone of 1 was an oleanolic acid derivative with one hydroxyl group at the ring E portion, as well as one ketone carbonyl group. The proton signal at δ H 3.50 showed long-rang correlations with C-13, C-17, and C-28 in the HMBC spectrum, and was assigned to the H-18 ( Figure 2). This proton had a proton spin-coupling correlation with the signal at δ H 3.54, which was associated with the carbon signal at δ C 81.0 (CH) in the HSQC spectrum. Thus, the presence of a hydroxyl group at C-19 was  The 13 C-NMR spectrum shows seven methyl, nine methylene, eleven methine, and nine quaternary carbon signals, including one ester carbonyl at δ C 177.0, a quaternary olefinic carbonyl at δ C 139.5, one anomeric carbon signal at δ C 95.9, a ketone carbonyl at δ C 216.6. Its 1 H-NMR spectrum shows the presence of a hydroxymethine proton at δ H 4.82 (1H, dd, J = 12.5, 6.3 Hz), one trisubstituted olefinic proton at (δ H 5.50, br s), six singlets at δ H 1.19, 0.99, 1.18, 1.15, 1.59, 1.37 for six tertiary methyl groups, one secondary methyl group (δ H 1.05, d, J = 6.6Hz), one methine proton characteristic of H-18 of pomolic acid (δ H 2.91, s), and one anomeric proton (δ H 6.30 d, J = 8.0 Hz). The secondary methyl signal on ring E provides a most useful indicator for the presence of an urs-12-ene skeleton [10].

General
Open column chromatography (CC) was carried out using silica gel (200-300 mesh, Qingdao Marine Chemical Co., Qingdao, China) or octadecyl silica gel (ODS, 25-40 μm, Fuji) as stationary phases. TLC employed precoated silica gel plates (5-7 μm, Qingdao Marine). Preparative HPLC was carried out on a Waters 600 instument equipped with a Waters UV-2487 detector. A Waters Sunfire prep C18 OBD (19 × 250 mm i.d.) column was used for this purpose. The IR spectra were recorded as KBr pellets on a Jasco 302-A spectrometer. Optical rotation was recorded on a Jasco P-2000 polarimeter. HRESIMS were measured on a FTMS-7 instrument (Bruker Daltonics). Melting points were determined on a Gallenkemp apparatus and are uncorrected. The 1 H-, 13

Plant Material
The roots of S. tenuifolia were collected in October 2008 from Fangzheng of Heilongjiang Province, China, and identified by Zhenyue Wang, of Heilongjiang University of Chinese Medicine.
A voucher specimen (20081023) was deposited at the herbarium of Heilongjiang University of Chinese Medicine, Harbin, China.

Extraction and Isolation
The dried roots of S. tenuifolia (5.0 kg) were extracted with 70% EtOH (3 × 10 L) to afford the EtOH extract (1.3 kg) which was then suspended in water (10 L) and then extracted with petroleum ether and ethyl acetate (EtOAc) (3 × 10 L each), yielding petroleum ether (10.2 g) and ethyl acetate (222.5 g) extracts. The EtOAc fraction (222.5 g) was subjected to silica gel column with a stepwise CH 2 Cl 2 -MeOH gradient (30:1; 20:1; 10:1; 5:1, v/v), and finally with MeOH alone, to give five fractions I-V. Fraction I (40.8 g) was separated using silica gel CC eluting with CH 2 Cl 2 -MeOH (50:1, 30:1, 10:1, v/v) to obtain three sub-fractions, I 1 -I 3 . Sub-fraction I 2 (10.6 g) was further separated by ODS silica gel CC with MeOH-H 2 O (9:1, v/v) and to 11 (33.2 mg), 12 (37.5 mg) and 15 (25.5 mg); Fraction II (38.3 g) was subjected to silica gel CC eluting with CH 2 Cl 2 -MeOH (30:1, 20:1, 10:1, v/v) to afford four sub-fractions, II 1 -II 4 . Sub-fraction II 1 (13.  After the dioxane was removed, the solution was extracted with EtOAc (3 mL × 3) to remove the aglycone. The aqueous layer was neutralized by passing through an ion-exchange resin column (Amberlite MB-3, Organo, Tokyo, Japan) and concentrated to dryness under reduced pressure to give the sugar fraction. The residue was dissolved in pyridine (0.1 mL) to which 0.1 M L-cysteine methyl ester hydrochloride in pyridine (0.1 mL) was added. The mixture was heated at 60 °C for 1 h. After the reaction mixture was dried in vacuo, the residue was trimethylsilylated with l-trimethylsilylimidazole (0.2 mL) for 2 h. The mixture was partitioned between hexane and H 2 O (0.6 mL, each), and the hexane extracted was analyzed by GC under the following conditions: capillary column, DM-5 (0.25 mm × 30 m × 0.25 μm); detector, FID; injector temperature, 280 °C, detector temperature, 280 °C; initial temperature was maintained at 160 °C for 2 min and then raised to 195 °C at a rate of 10 °C/min; carrier gas, N 2 . In the acid hydrolysate of 1, D-glucose was confirmed by comparison of the retention time of their derivatives with those of D-glucose and L-glucose derivatives prepared in a similar way, which showed retention times of 28.56 and 27.72 min, respectively. The sugar from 4 (30 mg) was also identified by the same method.

α-Glucosidase Inhibition Assay
α-Glucosidase (EC.3.2.1.20) enzyme inhibition assay has been performed according to the literature [19]. α-Glucosidase (25 μL, 0.2 U/mL), various concentrations of samples (25 μL), and 67 mM phosphate buffer (pH 6.8, 175 μL) were mixed at room temperature for 10 min. Reactions were initiated by the addition of 23.2 mM p-nitrophenyl-α-D-glucopyranoside (25 μL). The reaction mixtures were incubated at 37 °C for 15 min in a final volume of 250 μL, and then 1 M Na 2 CO 3 (50 μL) was added to the incubation solution to stop the reaction. The activities of glucosidase were detected in a 96-well plate, and the absorbance was read at 405 nm by a microplate spectrophotometer (Spectra Max, Molecular Devices, USA). The negative control was prepared by adding phosphate buffer instead of the sample in the same way as the test. Acarbose was utilized as the positive control. The blank was prepared by adding phosphate buffer instead of α-glucosidase using the same method. The inhibition rates (%) were calculated from the following formula: [(OD negative control − OD blank ) − (OD test − OD test blank )] / (OD negative blank − OD blank ) × 100%