Two new dammarane-type triterpene sapogenins from Chinese red ginseng

Two new dammarane-type triterpene sapogenins were isolated from the Chinese red ginseng. The new sapogenins were named as 24,26-dihydroxy-panaxdiol (1) and 24-hydroxy-panaxdiol (2). Their structures were elucidated by the combined analysis of NMR and mass spectrometry as 20(S),25(R)-epoxydammarane-3β,12β,24β,26-tetraol (1) and 20(S),25-epoxydammarane-3β,12β,24α-triol (2). The complete signal assignments of the two compounds were carried out by 2D NMR spectral and NOE differential spectroscopy analysis.


Introduction
Ginseng belongs to the Araliaceae family and its root has been used as a traditional medicine in Asian countries for over 2000 years (Coon & Ernst 2002). Ginseng has two preparations, and one is air-dried white ginseng and another one is steamed and sun-dried red ginseng. It contains various active components including ginsenosides, polysaccharides, peptides, polyacetylenic alcohols and fatty acids . The red ginseng is made from a ginseng plant going through an intensive process of cleaning, steaming and drying. Heat treatment of ginseng leads to chemical changes of ginsenosides that improve the health benefits of ginseng by inactivating catabolic enzymes and releasing the antioxidant substances from the ginseng. The steamed ginseng has a stiff texture, which not only protects the effective ingredients but also produces new constituents (Kim et al. 2000). Major ginsenosides of red ginseng saponin extract include less polar red ginseng-unique saponins Rg3, Rk1 and Rg5 in a HPLC analysis (Kim et al. 2008).
As supplementation with red ginseng is believed to improve health, numerous studies have been conducted to validate its beneficial effects. They demonstrate that red ginseng has anti-inflammatory (Jung et al. 2012), antidiabetic , anticancer  and arthritis-ameliorating effects (Jhun et al. 2014). At present, Chinese red ginseng and Korean ginseng are both commonly found in a market. There is no significant difference between the quality of Chinese red ginseng and Korean red ginseng according to reports. The contents of ginseng saponins and ginsenoside Rg1, Re, and Rb1 in Tongrentang red ginseng (Chinese red ginseng) are not lower than those in Korean red ginseng (Wu et al. 2007). The Chinese red ginseng is mainly produced in Jilin province. Reported herein are the isolation and the structural elucidation of the two new dammarane-type sapogenins, 1 and 2 from Chinese red ginseng (Panax ginseng C. A. Meyer) by Meyer chemical and spectroscopic methods (1D and 2D NMR, MS). The structure of 1 was determined as 20(S),25(R)-epoxydammarane-3b,12b,24b,26-tetraol and the structure of 2 was determined as 20(S),25-epoxydammarane-3b,12b,24a-triol.

Results and discussion
Repeated column chromatography of the EtOH extract of the Chinese red ginseng led to the isolation of new dammarane-type sapogenins 1 and 2 ( Figure 1 The chemical shifts of 1 showed resemblance with those of protopanaxdiol (Zhao et al. 1996) except the signals of the side chain. Furthermore, compared with 20(S)-panaxadiol (Duc et al. 1994), whose structure is dammar-20S,25-epoxydammarane-3b,6a,12b,24a-tetrol, a 20S,25epoxy group was deduced to exist in compound 1. It can be summarised that the difference of 20 (S) and 20(R) in dammarane-type saponin could be observed from the carbon signal of C-21 (S: d 27; R: d 20) (Fujita et al. 1995). In the 13 C NMR spectrum of compound 1, the chemical shift of C-21 at d 27.4 showed that the configuration of C-20 was S-form. These data can be accommodated on the dammar-20S,25-epoxy-3b,12b-triol triterpene having three secondary hydroxyls and one primary hydroxyl. The location of this functional group at C-24 and C-26 was determined by the heteronuclear multiple-bond connectivity (HMBC) spectrum of 1. In the HMBC spectrum of 1, the correlations between H-26, H-27 with C-24 and H-27, H-24 with C-26 were observed. Combined with 1 H NMR spectrum, and compared with the results from the report (Duc et al. 1994), the location of two hydroxyls was determined to be at C-24 and C-26. HMQC and HMBC experiments showed the correlation between H-21 (d 1.34) with C-17 (d 51.72), C-20 (d 78.09) and C-22 (d 24.01).
The stereochemistry of the side chain was finally determined by nuclear overhauser effect (NOE) differential spectroscopy and 2D NMR spectra. On irradiating the signal of the 21-methyl protons (d 1.34), NOEs were observed at the signals of the H-27-methyl protons (d 1.43), while the irradiation of the signal of the 27-methyl protons showed NOEs at the signals of the H-24 (d 4.01) protons. In addition, the irradiation at the frequency of H-24 gave rise to NOE at the signal of the 27-methyl protons. All the above data pointed to the structure of 1 as 20(S),25(R)epoxydammarane-3b,12b,26,24b-tetraol (Figure 1). Compound 1 was a minor glycoside in the red Panax ginseng C. A. Meyer.
Compound 2 was obtained as a white amorphous solid (MeOH The spectral data of compound 2 showed resemblance with those of compound 1 except the side chain, therefore it also had a 20S,25-epoxy group in compound 2. The location of this functional group at C-24 was determined by the HMBC spectrum of 2, which showed long-range correlations between H-24 and C-25, C-26 and C-27 (d 78.59, 23.28 and 30.12, respectively).
The stereochemistry of the side chain was finally determined by NOE differential spectroscopy and 2D NMR spectra. On irradiating the signal of the 21-methyl protons (d 1.35), NOEs were observed at the signals of the H-26-methyl protons (d 1.53) and the H-17 (d 2.21) protons, while the irradiation of the signal of the 27-methyl protons (d 1.68) showed NOEs at the signals of the H-24 (d 3.87) and H-26 protons. In addition, the irradiation at the frequency of H-24 gave rise to NOE at the signal of the 27-methyl protons. Compared with the report (Duc et al. 1994), all the data pointed to the structure of 2 as 20(S),25-epoxydammarane-3b,12b,24a-triol ( Figure 1). Compound 2 was also a minor glycoside in the red Panax ginseng C. A. Meyer.

Plant material
The Chinese red ginseng was provided by the Jilin HuangFeng group Co., Ltd (No. 110820) had been deposited at the Institute of Frontier Medical Science, Jilin University, China.

Supplementary material
Supplementary material relating to this article is available online at http://dx.doi.org/10.1080/ 14786419.2015.1038538.