Chinese Journal of Natural Medicines 2014, 12(4): 0305−0308

Chinese Journal of Natural Medicines

A new saikogenin from the roots of Bupleurum bicaule XU Nan 1, SHI Ya-Nan 2, ZHONG Xu 1, CAO Yue 1,WANG Li 1, JIA Tian-Zhu 1* 1

College of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian 116600, China;

2

Dalian Statute of Drug and Food examination, Dalian 116101, China Available online 20 Apr. 2014 [ABSTRACT] AIM: To study the chemical constituents from the roots of Bupleurum bicaule Helm (Apiaceae). METHOD: Silica gel, Sephadex LH-20, MPLC Rp-C18 column chromatography, and HPLC were used for isolation of compounds. The structures were elucidated on the basis of 1D- and 2D-NMR technology and HRESI-MS. Compounds were evaluated in vitro for their inhibitory ability against the proliferation of rat mesangial cells by the MTT method. RESULTS: Twelve compounds were isolated, and their structures were identified on the basis of their spectroscopic and physico-chemical properties as 13, 28-epoxy-olean-11-en-3-one (1), saikogenin E (2), saikogenin G (3), 11α-methoxy-3β, 16β, 23, 28-tetrahydroxyolean-12-ene (4), saikogenin D (5), prosaikogenin F (6), prosaikogenin A (7), prosaikogenin G (8), prosaikogenin D (9), laccaic acid (10), methyl gallate (11), and ethyl gallate (12). Compounds 1, 2, 7, 8, and 10 were observed to have inhibitory activity against mesangial cell proliferationin to different degrees. CONCLUSION: Compound 1, 8, and 10 exhibit significant inhibitory effects on rat mesangial cell proliferation induced by Ang II.

[KEY WORDS] Bupleurum bicaule; Saikogenins; Triterpenoid saponins; Anti-proliferation

[CLC Number] R284

[Document code] A

[Article ID] 2095-6975(2014)04-0305-04

Introduction Bupleurum is a genus of the Apiaceae, comprising about 200 species [1]. Under the name of Chaihu in Chinese, the roots of several Bupleurum species have been frequently used in the prescriptions of traditional Chinese medicine for the treatment of common cold with fever, influenza, inflammation, hepatitis, malaria, and also for menopausal syndrome in China for 2000 years [2-9]. Saikosaponin triterpenes generally constitute the main class of secondary metabolites in the genus Bupleurum, amounting to up to 7% of the total dry weight in roots [1] . To date, more than 120 glycosylated oleanane-type and ursane-type saponins have been isolated from Bupleurum species [10-12]. Essential oils from European species are characterized by the presence of a high abundance of α/β-pinene, limonene, and 1,8-cineole rather than aliphatic aldehydes [1]. In [Received on] 2-Sep.-2012 [Research funding] This project was supported by the Fund of Program for Traditional Chinese Medicine Scientific Research on Public Health Care (No. 201107007). [*Corresponding author] JIA Tian-Zhu: Prof., Tel: 0411-87586499, E-mail: [email protected] These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved

contrast, β-caryophyllene, β-caryophyllene oxide and spathulenol, in addition to the aforementioned aldehydes, represent the major components of the oil from Chinese species [13] . This difference can be used to distinguish between the oils from Italy or Spain and those from China [14-17]. Present interest is focused on the bioactivity of the isolated triterpene saponins acting as immunomodulatory, anti-inflamatory, and antiviral agents, as well as on the observed anti-ulcer activity of the polysaccharides and the anti-proliferative activity of different lignans [18-21]. Nowadays, Radix Bupleuri used in China is the dried root of B. chinense DC. and B. scorzonerifolium Willd. according to the Chinese Pharmacopoeia (2010 Edition). The requirement of Bupleurum radix for prescriptions and exports is about 8 million kg every year, which makes it difficult to meet demand from wild plants of B. falcatum L. and B. chinense. Consequently, many species of Bupleurum are sourced from different regions of China. New surveys show that “Haile” Bupleurum grows wild in Inner Mongolia with large quantity was for sale as authentic Radix Burpleuri in Hebei Qizhou drug transactions, Three Trees Medicine market and drug store in many province of China [22]. However, there is only one paper reported on the chemical constituents of B. bicaule Helm. In this paper, the isolation and identification of the chemical constituents from B. bicaule were stud-

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ied and their action was evaluated.

Results and Discussion Compound 1 HRESI-MS showed the [M − H]− signal at m/z 453.3335 (Calcd. for 453.3347). The liquid secondary ion mass spectrum (LSI-MS) of 1 exhibited the [M − H]− at m/z 453. The [M − H]−, together with 1H- and 13C NMR data allowed the molecular formula C30H46O3 to be proposed. Compound 1 was obtained as a white crystals mp 241−243 °C. It showed positive Liebermann-Burchard reaction. The signals due to seven angular methyl groups at δ 0.90, 0.95, 0.96, 1.01, 1.10, 1.13, and 1.37 (s, each of 3H） observed in the 1H NMR spectrum suggested that the substance was an oleanane saikogenin. Two olefinic proton signals at δ 5.91 (1H, d, J = 10.3 Hz) and 5.70 (1H, dd, J = 10.3, 2.9 Hz) in the 1H NMR, and the carbon signals at δ 131.5 and 131.6 in the 13C NMR demonstrated that the double bond was located at C-11. The proton signals at 4.39 (1H, d, J = 6.9 Hz) and 3.35 (1H, d, J = 6.9 Hz) in the 1H NMR and the carbon signals at δ 131.5,131.6, 83.3, and 73.0 in the 13C NMR indicated the presence of a 13, 28-epoxy group, which is similar to 13 saikosagenin E [23]. In the C NMR spectrum the signal at δ 215.8 was assigned to a carbonyl group in 1. The direct 1 H-13C correlation spectrum (HMQC) allowed assignment of the methyl carbons δ 34.2, 17.3, 19.1, 20.7, 20.9, 73.0, 34.2, and 23.8, double bond carbons δ 131.5 and 131.6, and hydroxyl group carbons at δ 64.0 and 73.0, respectively. The crosspeaks in the long-range 1H-13C correlation spectrum determined that the carbonyl group was located at C-3 and confirmed the results described above. The comparison of 13C NMR data for 1 with those of saikosagenin E [23] showed that the signals of C-15, C-16, and C-17 is same, however C-2, C-23, and C-4 of 1 undergo an up-field shift (1.5, 5.8 ppm) and downfield shift (2.2 ppm), respectively, between saikosagenin E and 1. These date suggested 1 and saikosagenin E has same configuration for C-16 and furthermore confirmed that the carbonyl group was located at C-3. Therefore, comound 1 was named as 13,28-epoxy-olean-11-en-16-ol-3-one.

4.39 (1H, d, J = 6.9 Hz, H-28a), 3.35 (1H, d, J = 6.9 Hz, H-28b), 0.95 (3H, s, H-29), and 0.90 (3H, s, H-30); 13C NMR (150 MHz, pyridine-d5) δ: 39.0 (C-1), 25.7 (C-2), 215.8 (C-3), 42.0 (C-4), 54.5 (C-5), 19.5 (C-6), 31.0 (C-7), 45.6 (C-8), 52.1 (C-9), 36.2 (C-10), 131.5 (C-11), 131.6 (C-12), 83.8 (C-13), 47.0 (C-14), 36.1 (C-15), 64.0 (C-16), 47.5 (C-17), 52.1 (C-18), 37.8 (C-19), 31.6 (C-20), 34.6 (C-21), 26.2 (C-22), 34.2 (C-23), 17.3 (C-24), 19.1 (C-25), 20.7 (C-26), 20.9 (C-27), 73.0 (C-28), 34.2 (C-29), and 23.8 (C-30). The known compounds saikosagenin E (2) [23], saikosagenin G (3) [23], 11α-methoxyl-3β, 16β, 23, 28-tetrahydroxyolean-12-ene (4) [24-26], saikogenin D (5) [23], prosaikosagenin F (6) [23], prosaikosagenin A (7) [27-28], prosaikosagenin G (8) [23], prosaikogenin D (9) [23], laccaic acid (10) [29], methyl gallate (11) [22] and ethyl gallate (12) [22] were identified by comparing their physical and spectroscopic data with reported data. As the isolated amounts of compounds 3, 4, 5, 6, 9, 11, and 12 were not obtained in an amount sufficient for pharmacological testing, only compounds 1, 2, 7, 8, and 10 were evaluated in the inhibition assay with mesangial cells (MC) using saikosaponin d as positive control. Compared with the blank-control, the test compound did not exhibit the inhibitory activity (P > 0.05), whiles mesangial cells induced by Ang II show significant proliferation (P < 0.05). The result of the cell test (Fig. 1) showed that the test compounds exhibited inhibitory activity to mesangial cell proliferation induced by Ang II to different degrees. Compound 1, 8, and 10 were found to exhibit more significant inhibition than saikosaponin d. These results suggest that compounds 1, 2, 8, and 10 have protective action on the kidney for their down-regulated activity to the MC proliferation induced by Ang II, and did not exhibit injury to the normal MC.

Fig. 2 Inhibitory effects of compounds from B. bicaule on proliferation of rat mesangial cells (mean ± SD, n = 6) *

P < 0.05, #P < 0.01 vs control (Ang II-free)

Fig. 1 Structures of compounds 1 and 4

Compoud 1 White amorphous crystals, ESI-MS m/z: 453 [M − H]−. 1H NMR (600 MHz, pyridine-d5) δ: 5.70 (1H, dd, J = 10.3, 2.9 Hz, H-11), 5.91 (1H, d, J = 10.3 Hz, H-12), 4.53 (1H, m, H-16), 1.13 (3H, s, H-23), 1.01 (3H, s, H-24), 0.96 (3H, s, H-25), 1.37 (3H, s, H-26), 1.10 (3H, s, H-27),

MC located at the center of capillary lobule of kidney glomerulus have many activities, including secretion of extracellular matrix (ECM), generation of cytokine and clearance of macromolecule, which is the inherent cells of kidney [30]. Under normal conditions, MC did not exhibit proliferation. However, if inflammation occurs MC reveal abnormal proliferation. Excessive proliferation of MC could eventually lead to glomerulosclerosis [31]. The occurrence of mesangial hyperplasia, or proliferation of the cells, whose number is

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actually increased, is often encountered in more rapidly evolving diseases, with extensive leukocyte infiltration, necrotizinglesions, and the signs of acute or subacute inflammation. These are features of mesangiocapillary nephritis, lupus nephritis, rapidly progressive glomerulonephritis, or vasculitis [32]. So in present study, mesangial cell proliferation induced by Ang II in the presence or absence of compounds was measured to investigate whether the compounds isolated from B. bicaule inhibited mesangial cell proliferation. Ang II has the action to induce the proliferation of rat mesangial cells and lead to glomerulosclerosis [32]. so in this experiment, Ang II (1 × 10−6 mol·L−1) was used as a positive excitomotor to examine whether the test compounds could inhibit the proliferation of mesangial cells.

Experimental Apparatus and reagents Melting points were measured on Kofler micromelting point apparatus (uncorrected). Negative ion MS: MAT 8500 (Finnigan), matrix glycerol. NMR: 600.13 MHz (1H) and 150.92 MHz (13C), CC: silica gel (145−200 mesh); TLC: silica gel (Qingdao Haiyang Chemical Factroy, China). Mesangial cell was purchased from cell storage center of Wuhan University.

Extraction and isolation B. bicaule was collected in 2011 in Chifeng China and identified by Prof. WANG Bing from Laoning University of Tradition Chinese Medicine. A voucher specimen of the plant is deposited at the College of Pharmacy, Laoning University of Tradition Chinese Medicine. Dried powder of the root of B. bicaule (5.0 kg) was exhaustively refluxed five times with 80% MeOH(25 L/each time) for 1 h each time. After removal of the solvent by evaporation, the residue (780 g) was successively partitioned between H2O and hexane, CHCl3, and n-BuOH. The butanolic fraction was evaporated under reduced pressure at 50°C to obtain a crude saponin mixture (210 g). 200 g of the crud saponin was separated by silica gel eluting with CHCl3− MeOH with increasing amounts of MeOH gave two fractions, I (25 g) and II (10 g). Fr. I was further purified by means of MPLC on Rp-18 eluting with MeOH−H2O (80 : 20), Sephadex LH-20 eluting with MeOH, followed by semipreparative HPLC on Rp-18 eluting with MeOH−H2O (80 : 20) gave the pure saponins 1 (350 mg), 2 (1 g), 3 (100 mg), 4 (25 mg), and 5 (25 mg). Fr. II was further purified on MPLC eluting with MeOH−H2O 70 : 30, followed by semipreparative HPLC on Rp-18 eluting with MeOH−H2O 70 : 0 to give the pure saponin glucosides 6 (30 mg), 7 (50 mg), 8 (40 mg), and 9 (10 mg) and the phenolic compounds 10 (50 mg), 11 (6 mg), and 12 (80 mg). Inhibition of MC proliferation assay The inhibition of proliferation assay was performed using the rat mesangial cell line (MC) [19]. Cells were incubated at 37 °C under a 5% CO2/95% air atmosphere at constant

humidity. MC were seeded to 96-well plates (200 μL, 5 × 104 cells/mL) in cultured medium of DMEM with 10% FBS. After 24 h of incubation, the test material was added and the plates were incubated for 48 h. The test cells (n = 3) were treated with the mixture of AngⅡ1 ×10−6 mol·L−1 of DMEM solution and different concentrations (25 and 50 μmol·L−1) of the test compound dissolved in DMSO, whereas the cells were treated with angiotensin II (AngⅡ1 × 10−6 mol·L−1) of DMEM solution as control, or with culture medium as the blank control. After that, the plates were incubated for 24 h. Cells were washed once before adding MTT solution (20 μL, 5 mg·mL−1) and the incubation was continued for an additional 4 h, the medium was discarded and DMSO (150 μL) was added. Thereafter the optical density was measured at 492 nm. 0.05 % DMSO in culture medium was used as blank and the percentage of cell viability was calculated as follows. Inhibition = (absorbance of Ang II treated cell – absorbance of test compound and Ang II treated cell) /absorbance of Ang II treated cell × 100%

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Cite this article as: XU Nan, SHI Ya-Nan, ZHONG Xu, CAO Yue,WANG Li, JIA Tian-Zhu. A new saikogenin from the roots of Bupleurum bicaule [J]. Chinese Journal of Natural Medicines, 2014, 12(4): 305-308

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