A new triterpenoid saponin from Clinopodium chinense (Benth.) O. Kuntze

Abstract A new triterpene saponin, 3β,16β,23α,28β,30β-pentahydroxyl-olean-11,13(18)-dien-3β-yl-[β-d-glucopyranosyl-(1→2)]-[β-d-glucopyranosyl-(1→3)]-β-d-fucopyranoside, was named Clinoposaponin D (1), together with six known triterpene saponins, buddlejasaponin IVb (2), buddlejasaponin IVa (3), buddlejasaponin IV (4), clinopodisides D (5), 11α,16β,23,28-Tetrahydroxyolean-12-en-3β-yl-[β-d-glucopyranosyl-(1→2)]-[β-d-glucopyranosyl-(1→3)]-β-d-fucopyranoside (6) and prosaikogenin A (7), and two known triterpenes, saikogenin A (8) and saikogenin F (9) were isolated from Clinopodium chinense (Benth.) O. Kuntze. Their structures were elucidated on the basis of 1D, 2D NMR and MS analysis. Meanwhile, the effects of all compounds on rabbit platelet aggregation and thrombin time (TT) were investigated in vitro. Compounds 4 and 7 had significant promoting effects on platelet aggregation with EC50 value at 53.4 and 12.2 μM, respectively. In addition, the highest concentration (200 μM) of compounds 2 and 9 shortened TT by 20.6 and 25.1%, respectively. Graphical abstract


Introduction
The genus Clinopodium comprises nearly 20 species and is mainly distributed in Asia and Europe. Clinopodium chinense (Benth.) O. Kuntze (Lamiaceae), a traditional Chinese Medicine (TCM) is popularly used for the treatment of haematuria, allergic dermatitis, dysentery, influenza, heliosis and trauma, etc. (Wang et al. 2013;Zhong et al. 2014). Furthermore, C. chinense is one of two medicinal herbs in 'Duan Xueliu' , which has long been used as a TCM with an astrictive remedy property, and is officially listed in the Chinese Pharmacopoeia (Pharmacopeia Committee of P. R. China 2010). In recent years, its aqueous or ethanol extract has been found to exhibit haemostatic activities (Zhong et al. 2012). In the course of our research for bioactive constituents from C. chinense, one new triterpene saponin named Clinoposaponin D (1), together with six known triterpene saponins (2-7) and two triterpenes (8-9) were obtained. In the present study, we reported the isolation and structural identification of compounds 1-9 and their effects on platelet aggregation and thrombin time (TT) in vitro.

Results and discussion
Compound 1 was isolated as a white amorphous powder. Its molecular formula was established as C 48 (Tsuyoshi et al. 2004;Liu et al. 2013 [δ C 127.4, 126.2, 137.1 and 133.6], which were characteristic olefinic carbons of Δ 11,13(18) -Olean diene. The data above suggested that 1 was a highly oxygenated oleanolic triterpenoid saponin derivative with three sugar moieties Liu et al. 1995;Bi et al. 2012;Zhang et al. 2012). The assignment of 1 H-NMR and 13 C-NMR spectroscopic data of 1 (Table S1) was based on 1 H-1 H COSY ( Figure S7), HSQC ( Figure S8) and HMBC spectrum ( Figure S9). The 1 H-NMR and 13 C-NMR features of 1 were similar to those of the known compound buddlejasaponin IVb (2) (Table S1), except for the proton signal (H-30) of methylene group that connected to hydroxy group in compound 1. The correlation between δ H 6.13 (1H, brs, 30-OH) and δ H 3.67 (2H, s, H-30) in the 1 H-1 H COSY spectrum confirmed their connection. In the HMBC spectrum, the correlations between 30-OH and C-30, H-29 and C-20 proved the linkage of C-30/C-20/C-29. Specific HMBC correlations were given in Figure S10.
Further correlations between 30-OH/H-28 and H-29/H-22α in the ROESY spectrum ( Figure  S11) proved that the C-30 hydroxy group was β-oriented and C-29 methyl group was α-oriented. Specific ROESY correlations are given in Figure S12.
Blood platelets play an essential role in haemostasis by involving in the formation of thrombi to prevent blood loss and maintain vascular integrity (Huang et al. 2014). The previous study showed that total saponins of C. chinense significantly increased the rat platelet aggregation (Liu et al. 1985). Therefore, the platelet aggregation effect of isolated compounds was evaluated. The results showed that compounds 1, 2, 4, 6, 7 and 9 exhibited varying degrees to promote rabbit platelet aggregation at concentrations up to 100 μM. Additionally, compounds 4 and 7 exhibited potent platelet aggregation activity and their EC 50 value were 53.4 and 12.2 μM, respectively. Interestingly, their effects on promoting platelet aggregation activity were stronger than polyphyllins II at the concentration of 100 μM (Table S2). To determine the effect of the isolated compounds on haemostasis in vitro, TT were measured. As shown in Table S3, 200 μM of compounds 2 and 9 remarkably shortened TT by 20.6 and 25.1%, respectively. Similarly, thrombin (0.006 u) remarkably shortened TT by 22.0%.

Plant material
The herbs of C. chinense (Benth.) O. Kuntze were collected from Putian city, Fujian province, China, and identified by Prof. Sheban Pu from Department of Pharmacognosy, School of Traditional Chinese Pharmacy, China Pharmaceutical university, Nanjing, China. The voucher specimen (CPu-CC20100820) was deposited in the Herbarium of China Pharmaceutical university.

Acid hydrolysis of compound 1
Compound 1 (1.4 mg) was heated in 3 mol L −1 HCl-CH 3 OH (5 mL) for 3 h in a sealed tube at 90 °C. The mixture was then extracted with EtOAc. The aqueous layer was evaporated to dryness with ethanol in vacuo at 50 °C until neutral. The residues were determined in comparison with authentic monosaccharides using HPTLC (CHCl 3 :CH 3 OH:H 2 O = 3:2:0.2, visualisation with ethanol-5% H 2 SO 4 spraying). For the sugars of compound 1, the R f values of glucose and fucose by TLC were 0.32 and 0.40, respectively.

Effect of compounds on platelet aggregation
The blood platelet aggregation test was performed according to the method of Born et al. (1963). Briefly, whole blood samples were collected from the carotid of urethaneanesthetised rabbits and mixed with 3.8% sodium citrate (9:1 v/v), followed by centrifuging at 1000 rpm for 10 min. The supernatants were obtained and used as platelet rich plasma (PRP). While the remaining blood sample was centrifuged at 3000 rpm for10 min and the supernatants were collected as platelet poor plasma (PPP). The effect of individual compounds on the platelet aggregation was measured by the Born's Light Transmission Aggregometry method using Agg-RAM-Aggregometer (Helena Laboratories, Beaumont, Texas). Briefly, the amount of platelet in PRP with PPP was adjusted to 3 × 10 11 L −1 . The light transmission was set at 0% with PRP and at 100% with PPP. PRP (225 μL) was incubated with 25 μL of vehicle (final concentration 0.3% DMSO) or different concentrations of individual compounds in turbidimetric cup at 37 °C for 5 min. The changes in the light transmittance of the reaction mixture were continuously recorded for 5 min and the maximal aggregation was recorded. Polyphyllins II and ADP were used as positive control.

TT assay
PPP was prepared referring to the method in Section 3.5. TT was determined by a coagulation-analysis instrument (LG-PABER-I, Steellex Co., China) (Liu et al. 2010). A reagent kit according to the manufacturer's recommendations was used to assay TT. Briefly, 50 μL of PPP and 10 μL of individual compounds solution (100, 200 μM) was incubated for 3 min at 37 °C, followed by addition of 100 μL of thrombin solution. Shortly after adding thrombin solution, the coagulation-analysis instrument was started up to record TT.

Statistical analysis
All data are expressed as mean ± SD. Statistical differences among groups were analysed by the one-way analysis of variance (ANOVA) and two-tailed Student's t-test. A value of p < 0.05 was considered significant.

Conclusions
In this study, seven triterpenoid saponins and two triterpenes including a new triterpenoid saponin were isolated and elucidated from C. chinense (Benth.) O. Kuntze. Among them, compounds 4 and 7 markedly promoted rabbit platelet aggregation and compounds 2 and 9 significantly shortened plasma TT, which indicated that compounds 4 and 7 could accelerate the thrombosis through promoting platelet aggregation so as to stop the bleeding and compounds 2 and 9 could affect the function of fibrinogen in plasma. Such results suggest that compounds 2, 4, 7 and 9 might be the main haemostatic ingredients of C. chinense. Furthermore, the Δ 11,13(18) -Olean diene saponins and 13, 28-epoxy-Olean-11-ene saponins exhibited better haemostatic activity when compared to the Δ 12 -Oleanene saponins in C. chinense (Benth.) O. Kuntze.