Chinese Chemical Letters  2015, Vol.26 Issue (10): 1298-1302   PDF    
Two new triterpenoid saponins from notoginseng medicinal fungal substance
Wei Xu, Cai-Feng Ding, Xin-Wen Wang, Xiao-Li Liu, Zhi-Dong Qiu     
College of Pharmacy, Changchun University of Chinese Medicine, Changchun 130117, China
Abstract: Two new triterpenoid saponins, named ginsenoside Rh10 (1) and notoginsenoside ST-6 (2), were isolated from notoginseng medicinal fungal substance. Their structures were elucidated by a combination of 1D and 2D NMR, MS and chemical analysis.
Key words: Notoginseng     Triterpenoid ginsenoside     Ginsenoside Rh10     Notoginsenoside ST-6    
1. Introduction

Radix and rhizoma of Panax notoginseng (Burk.) F.H. Chen (San Qi) is a well-known traditional Chinese medicine that has been used in both raw and processed forms due to the different pharmacological activities [1]. Triterpenoid saponins are the main components of San Qi,especially the rare saponins have strong pharmacological activity. In the early study,we got the notoginseng medicinal fungal substance through the interaction of complex enzymes in natural microorganisms forms,thus saponins can be transformed into rare saponins and other new active ingredients [2]. In the course of our continuous research,we studied the EtOH extract of notoginseng medicinal fungal substance and isolated two new dammarane-type triterpenoid saponins,named ginsenoside Rh10 (1) and notoginsenoside ST-6 (2) (Fig. 1). Here,this paper reports the isolation and structure determination of the two new dammarane-type triterpenoid saponins 1 and 2.

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Fig. 1.Structures of compounds 1 and 2.
2. Experimental 2.1. General experimental procedures

ESI-HR-MS was recorded on a Q EXACTIVE (Thermo,America). 1D and 2D NMR spectra were taken on a Bruker AV 500 spectrometer (Bruker,Fllanden,Switzerland) with tetramethylsilane (TMS) as the internal standard. Optical rotations were measured on a Jasco DIP-140 digital polarimeter at 20 ℃. GC analysis was performed on an Agilent GC-7820 A (FID detector) with a HP-5 column (0.32 mm × 30 m,0.25 mm,Agilent,America). High-performance liquid chromatography (HPLC) was performed on Hitachi instrument (pump LC-2130,UV detector LC-2030) equipped with a YMC-Pack ODS-AM column (10 mm × 300 mm,5 mm) with a flow rate 1.2 mL/min. Column chromatography (CC) was performed on silica gel (100-200 mesh,Qingdao Marine Chemical Co.,Ltd.,Qingdao,China). Thin layer chromatography (TLC) was performed on silica gel plates (GF254,Qingdao Marine Chemical Co.,Ltd.,Qingdao,China) and RP-18 F254 (Merck). Fractions were monitored by TLC and spots were visualized by spraying with 10% H2SO4 in EtOH,followed by heating at 105 ℃. CD was recorded on a MOS-450/AF-CD spectrometer (Bio-Logic,French) with the anhydrous DMSO as the reference solvent at room temperature.

2.2. Plant material

Notoginseng were purchased from Ji Lin An Pharmaceutical Co.,Ltd. (Jilin Province,China) on May 2013,and they were identified by professor Li-Li Weng of Changchun University of Chinese Medicine. A voucher specimen (No. SQ03212013) was deposited at College of Pharmacy,Changchun University of Chinese Medicine. The notoginseng medicinal fungal substances were obtained by bio-transformation of the notoginseng.

2.3. Extraction and isolation

The dried notoginseng medicinal fungal substance (5.0 kg) were powdered and extracted with aqueous ethanol (70%,40 L × 2 h × 4) at 80 ℃. After removal of EtOH under reduced pressure,the EtOH extract (980 g) were suspended in H2O (4 L) and then successively partitioned with petroleum ether (PE),EtOAc and n-BuOH. The EtOAc solution was evaporated under reduced pressure to get crude extract (52 g),which was chromatographed on silica gel (2100 g,100 mm × 650 mm) using a step-gradient eluting of CH2Cl2-MeOH (100:0 → 0:100,v/v) to yield 13 fractions (Fr.1-Fr.13). Fr.8 was chromatographed on open ODS (100 g,2.8 mm × 230 mm),using a step-gradient of MeOH-H2O (0:100 → 100:0,v/v) to yield 6 fractions (Fr.8-1-Fr.8-6). Fr.8-3 was purified by semi-preparative HPLC (MeOH-H2O,48:52,v/v) to yield 1 (6.9 mg,tR 71.8 min). Fr.8-4 was purified by semi-preparative HPLC (MeOH-H2O,58:42,v/v) to yield 2 (10.4 mg,tR 79.1 min).

2.4. Determination of absolute configuration of the sugar moieties in compounds 1 and 2

A solution of each saponin (3.0 mg each) in 1.0 mol/L HCl (dioxane/H2O 1:1; 1 mL) was heated at 100 ℃ for 10 h in a H2O bath. After removal of dioxane under vacuum,the solution was extracted with EtOAc (3 × 10 mL). The H2O-soluble part was concentrated on dryness,and subsequently the residue was dissolved in pyridine (each 2 mL). L-Cysteine methyl ester hydrochloride (2.0 mg each) was added to the pyridine solution. The mixture was kept at 60 ℃ for 1.5 h,dried under vacuum and trimethylsilylated with 1-(trimethylsilyl)-1H-imidazole (0.1 mL) at 60 ℃ for 1.0 h. The mixture was suspended in H2O (1 mL),then extracted with hexane (3 × 10 mL). The supernatant was analyzed by GC under the following conditions: capacity column,HP-5 (0.32 mm × 30 m,0.25 mm); column temperature,160 (4 ℃/min),200 ℃ (keep 5 min,10 ℃/min),240 ℃ (keep 10 min); carrier gas,He at 1.0 mL/min,split ratio 1/10; injection temperature,270 ℃; detection temperature,280 ℃; injection volume,1.0 mL. Compounds 1 and 2 gave D-Glc at tR 18.42 min,which is identical to the tR of authentic standard,respectively,and the result proved that the sugar in compounds 1 and 2 was D-glucose (Fig. S1 in Supporting information).

2.5. Determination of absolute configuration of the C-24 of compound 2

According to the literatures [3, 4, 5, 6],the absolute configuration of the C-24 of the compound 2 could be determined by means of a simple and reliable CD method employing dimolybdenum tetracetate [Mo2(OAc)4]. Generally,after the in situ complexation of the vicinal diol with Mo2(OAc)4,a series of bands (250-550 nm) were apparent in the induced circular dichroism (ICD) spectrum. Nevertheless,the sign of band at 310 nm was safely related to the absolute configuration of the vicinal diol [7]. When the vic-diol unit in compound 2 was concerned,it was necessary to avoid the interference from the glucose unit and assign the absolute configuration of the C-24 firstly to fulfill all the requisites of the method.

On the basis of the Mo2(OAc)4 method,to exclude the interference from the glucose unit,compound 2 (5.0 mg) was treated with 1.0 mol/L HCl (dioxane/H2O 1:1) at 100 ℃ for 10 h. After removal of dioxane under vacuum,the solution was extracted with EtOAc,which was then treated by preparative TLC using CHCl3-CH3OH-HOAc (10:1:0.05) as the developing solvent to yield the aglycone 2a (1.0 mg). The 13C NMR (CD3OD,150 MHz) data of aglycone 2a were: d 39.9 (C-1),27.4 (C-2),79.3 (C-3),40.0 (C-4),61.8 (C-5),68.6 (C-6),47.1 (C-7),41.2 (C-8),51.6 (C-9),40.1 (C-10),33.2 (C-11),73.2 (C-12),50.5 (C-13),51.8 (C-14),31.9 (C-15),28.4 (C-16),41.1 (C-17),17.2 (C-18),17.7 (C-19),141.6 (C-20),19.6 (C-21),123.4 (C-22),30.3 (C-23),79.3 (C-24),73.3 (C-25),25.3 (C-26),24.9 (C-27),31.1 (C-28),15.8 (C-29),16.6 (C-30).

Practically,about 1:1 diol (2a)-to-Mo2(OAc)4 mixtures were subjected to CD experiment with 2a prepared at a concentration of 1.81 mg/mL. The first CD spectrum of 2a was recorded. Additionally,the inherent CD of 2a was subtracted. The observed sign of the diagnostic band at 310 nm in the ICD spectrum (Fig. 3) was positive. According to the Snatzke’s method,with the application of transition metal complex Mo2(OAc)4,the conformation of the flexible vic-diols becomes fixed to some extent after ligation to the molybdenum core. The energetically preferred conformation of the vic-diols in the chiral Mo-complex is the one with the antiplanar orientation of both O-C-C-R units. This is due to the fact that only in such a conformation the sterically demanuing R-groups point away from the rest of the complex and do avoid the close interaction with the remaining acetate ligands in the stock complex and thereby the relative configuration of the O-C- C-O moiety is established [8]. In the following step,from the positive sign of the Cotton effect at 310 nm (Fig. 3),the sign of the HO-C-C-OH torsional angle is known,thus,the absolute configuration of C-24 was allowed to be determined as 24 (S).

3. Results and discussion

Compound 1 was obtained as a white amorphous powder. [α]D20 -7.2° (c 0.276,CH3OH). Its molecular formula was determined to be C35H58O10 by negative ESI-HR-MS (m/z 673.3726 [M + Cl]-,calcd. for 673.3724) (Fig. S2 in Supporting information),suggesting 7° of unsaturation. The 1H NMR spectra (Fig. S3 in Supporting information) of 1 showed signals due to seven quaternary methyls,one pair of olefinic H-atoms,three O-bearing CH groups and one anomeric proton. The 13C NMR spectra (Fig. S4 in Supporting information) of 1 revealed 35 C-atom signals including seven Me groups,three O-bearing CH groups,seven CH2 groups,five quaternary C-atoms,one pair of olefins C-atom signals and a carbonyl signal. The 1H NMR data (Table 1) and 13C NMR data of compounds 1 were similar to those of 20 (S)-ginsenoside Rh1 [9],except for side chain. The structure of the side chain moiety of 1 was determined by HMQC (Fig. S5 in Supporting information) and HMBC (Fig. S6 in Supporting information) correlations of H-21 (δH 1.17) with C-17 (δC 54.5),C-20 (δC 74.2) and C-22 (δC 40.1); H-22 (δH 2.38 and 2.57) with C-20,C-21 (δC 27.2),C-23 (δC 147.9) and C-24 (δC 133.9); H-23 (δH 7.09) with C-20,C-22 and C-25 (δC 201.1); H-24 (δH 6.13) with C-22,C-25 and C-26 (δC 26.4); H-26 (δH 2.27) with C-23,C-24 and C-25 (Fig. 2). A pair of olefinic H-atom signals at δH 7.09 (m) and δH 6.13 (d,J = 15 HZ) indicated that the C-23 (24) double bond was of the E form. In addition,the HMBC correlation between δH 4.36 (d,J = 5 HZ,H-10) and δC 80.6 (C- 6) revealed thatthe glucosylunitwas locatedat C-6oftheaglycone (Fig. 2). The absolute configuration of C-20 was suggested as S by comparing the 13C NMR data of C-17 (δC 54.5) and C-21 (δC 27.2) with those of ginsenoside Rg1,while its C-20 R epimer data were δC 50.4 (C-17) and δC 22.5 (C-21) [10]. Taken together with couplingconstant data for the H-atoms and 13C NMR data,the glucosyl unit was identified as β-glucose. The D configuration of glucose units was established after hydrolysis of 1 with 1 mol/L HCl,trimethylsilation and determination of the retention time by GC [11]. From all analysis above,the structure of 1 was determined as 6-O-b-Dglucopyranosyl dammar-23(24)(E)-ene-25-carbonyl-3β,6α,12β,20(S)-tetrol,named ginsenoside Rh10.

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Fig. 2. Key HMBC correlations of compounds 1 and 2.

Table 1
1H NMR (500MHz) and 13C NMR (150MHz) spectral data of compounds 1 and 2 in CD3OD.

Compound 2 was obtained as a white amorphous powder. [α]D20 28.38 (c 0.296,CH3OH). Its molecular formula was determined as to be C36H62O10 by negative ESI-HR-MS (m/z 689.4039 [M + Cl]-,calcd. for 689.4037) (Fig. S7 in Supporting information). NMR spectra,together with 6° of unsaturation,showed that compound 2 was a dammarane-type triterpenoid saponin. The 1H NMR (Fig. S8 in Supporting information) and 13C NMR (Fig. S9 in Supporting information) spectra,sustained by HMQC (Fig. S10 in Supporting information) and HMBC (Fig. S11 in Supporting information) of 2 showed the existence of a glucose unit. The coupling constant data for the glucose anomeric H-atom (d,J = 10.0 HZ) suggested as a b-form. The location of the sugar was affirmed by the HMBC spectrum suggested the correlation of δH 4.35 (H-1')/δC 80.6 (C-6) (Fig. 2). The integration of NMR data (Table 1) of 2 is close to notoginsenoside ST-1 [12]. The only difference is that the record of (C-17) and (C-21). The δC 41.1 (C-17),19.4 (C-21) of compound 2 and δC 51.3 (C-17),13.1 (C-21) of notoginsenoside ST-1 indicated that compounds 2 and notoginsenoside ST-1 were cis-trans isomers. The C-20 (22) double bond of notoginsenoside ST-1 was E form,which indicated that the C-20 (22) double bond of compound 2 was Z form. In the NOESY spectrum (Fig. S12 in Supporting information) of compound 2,the methyl proton at δH 1.73 (s,H-21) was correlated with δH 5.14 (m,H-22),but not with the proton at δH 2.15 and 2.34 (m,H-23). This observation confirmed that the C- 20 (22) double bond was Z form. Moreover,in the NOESY spectrum δH 3.10 (dd,H-3) was correlated with δH 1.33 (s,H- 28a); δH 3.62 (m,H-12) was correlated with δH 0.94 (s,H-30α); δH 4.09 (td,H-6) was correlated with δH 1.00 (s,H-29β),which was demonstrated the orientation of H-3,H-6 and H-12 was α,β and α,respectively. The absolute configuration of C-24 was identified using the Snatzke’s and Frelek’s method. Metal complex of the aglycone 2a-to-Mo2(OAc)4 in anhydrous DMSO gave a positive cotton effect at 310 nm in the induced circular dichroism spectrum (Fig. 3),which identified the absolute configuration of the C-24 was 24 (S). Thus the chemical structure of compound 2 was 3β,6α,12β,24(S),25-pentahydroxydammar-20(22)(Z)-ene-6- O-b-D-glucopyranoside,denominated as notoginsenoside ST-6.

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Fig. 3. Induced circular dichroism spectrum of 2a in DMSO solution of dimolybdenum tetracetate.
4. Conclusion

Two new triterpenoid saponins,ginsenoside Rh10 (1) and notoginsenoside ST-6 (2),were isolated from notoginseng medicinal fungal substance. Their structures were elucidated by a combination of 1D and 2D NMR,MS and chemical analysis. Pharmacological experiments showed weak activity on Hela cells in vitro. We did not do other pharmacological experiment because of their limited amounts. According to the structures of the compounds,they could have antitumor activity on the other tumor cells.

Acknowledgment

This project was financially supported by National Science and Technology Supporting Plan of China (No. 2012BAI29B05).

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version,at http://dx.doi.org/10.1016/j.cclet.2015.07.014.

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