20(22) Z and 20(22) E Dammarane Saponins From the Roots of Panax pseudoginseng Wall.

Abstract

Panax pseudoginseng Wall. is famous as a traditional Vietnamese medicinal plant used to promote health and aid in cancer treatment. From the roots of this plant, 1 new (1) and 4 known (2-5) Δ²⁰⁽²²⁾-dammarane-type triterpenoid glycosides were isolated by various chromatographic methods. Their chemical structures were determined as 3β,6α,12β-trihydroxydammarane-(Z)-20(22),24-diene 6-O-β-D-glucopyranoside (1), 3β,6α,12β-trihydroxy-dammar-(E)-20(22),25-diene 6-O-β-D-glucopyranoside (2, ginsenoside Rh4), ginsenoside Rg5 (3), 3β,12β-dihydroxydammarane-(E)-20(22),24-diene 6-O-β-D-xylopyranosyl-(1→2)-β-D-glucopyranoside (4), and 3β,12β-dihydroxydammarane-(E)-20(22),24-diene 6-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside (5). The E/Z-Δ²⁰⁽²²⁾ configurations in the dammarane compounds were further evidenced by 1D and 2D NMR data.

Keywords

Araliaceae dammarane glycoside ginsenoside Rh4 ginsenoside Rg5

Introduction

Panax pseudoginseng Wall. (Araliaceae), one of the best-known herbs belonging to the family of ginseng plants, is widely distributed in Nepal, China, and northern Vietnam. The roots and flowers of this plant have been used in Vietnam as medicinal herbs to strengthen immunity, promote health, relieve pain, reduce swelling, cholesterol, blood pressure, muscle aches, osteoarthritis, poliomyelitis, and to treat cancer.¹ The main chemical components of the roots and flowers of this plant are dammarane-type triterpenoid saponins, a class of substances very typical of the genus Panax.^1–5 Most of these triterpenoids have a double bond at C-24/C-25 and some have another at C-20/C-22. Interestingly, the configuration of the C-20/C-22 double bond of most of the compounds is (E) and only very rarely (Z). This paper reports on 5 Δ²⁰⁽²²⁾-dammarane-type triterpenoids, including one new compound, with a Δ²⁰⁽²²⁾-(Z)-configuration. The (E) and (Z) configurations of Δ²⁰⁽²²⁾-dammarane compounds were further determined and noted in this report.

Results and Discussion

A phytochemical study of the methanolic extract of the roots of P. pseudoginseng led to the isolation of 5 dammarane saponins, one of which is new. The 4 known compounds were identified as 3β,6α,12β-trihydroxy-dammar-(E)-20(22),25-diene 6-O-β-D-glucopyranoside (2),⁶ ginsenoside Rg5 (3),⁷ 3β,12β-dihydroxydammarane-(E)-20(22),24-diene 6-O-β-D-xylopyranosyl-(1→2)-β-D-glucopyranoside (4),⁸ and 3β,12β-dihydroxydammarane-(E)-20(22),24-diene 6-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside (5)⁹ by analyzing their NMR data and comparison of these with those reported in the literature; they matched perfectly (Table 1). Furthermore, the (E)-Δ²⁰⁽²²⁾-configuration of compound 2 was clearly confirmed by the NOESY spectrum. The above evidence, together with the previous reported data,^10–12 indicated that the carbon chemical shifts of C-21 for dammarane compounds with an (E)-Δ²⁰⁽²²⁾-configuration were between δ 12.9 to 13.5, which were very different from that of the (Z)-Δ²⁰⁽²²⁾-configuration (δ_C−21 ∼20.4).¹²

Table 1.

NMR Spectroscopic Data for 1 to 5 and 2a (in CD₃OD).

Pos.	1		2a	2	3	4	5
Pos.	δ_C	δ_H (mult, J in Hz)	δ_C	δ_C	δ_C	δ_C	δ_C
1	40.3	1.08 (m)/1.74 (m)	40.3	40.2	40.3	40.3	40.4
2	28.9	1.46 (m)/1.90 (m)	27.6	29.4	29.4	29.4	29.4
3	79.9	3.17 (dd, 12.0, 4.8)	79.8	79.8	91.3	80.1	79.8
4	40.5	–	40.4	40.5	41.2	40.5	40.5
5	61.8	1.12 (d, 10.8)	61.8	61.8	57.6	61.9	61.5
6	80.9	4.11 (ddd, 10.8, 10.8, 3.6)	80.9	80.9	19.2	80.7	74.9
7	45.6	1.65 (m)/2.07 (dd, 10.8, 3.6)	45.5	45.5	36.1	45.4	46.3
8	42.1	–	42.1	42.0	40.6	42.2	42.2
9	51.4	1.49 (dd, 13.2, 3.0)	51.3	51.3	51.4	51.3	51.1
10	40.5	–	40.5	40.4	38.9	40.5	40.3
11	32.6	1.20 (m)/1.80 (m)	32.3	32.3	32.3	32.3	32.3
12	74.0	3.61 (ddd, 10.8, 10.8, 5.4)	74.0	74.0	74.1	74.0	74.0
13	51.0	1.87 (dd, 10.8, 12.0)	51.0	51.0	51.3	51.1	51.1
14	52.2	–	51.8	51.8	51.9	51.9	51.9
15	33.3	1.20 (m)/1.71 (m)	33.3	33.3	33.4	33.3	33.5
16	27.6	1.60 (m)/1.69 (m)	29.4	27.6	27.2	27.5	27.6
17	41.6	3.10 (m)	51.3	51.2	51.8	51.4	51.3
18	17.9	1.13 (s)	17.5	17.5	16.7	17.5	17.3
19	17.9	1.01 (s)	17.9	17.9	16.8	17.9	17.8
20	140.2	–	140.3	140.3	140.4	140.4	140.4
21	19.6	1.72 (s)	12.9	12.9	12.9	12.9	12.9
22	124.9	5.11 (t, 6.6)	124.6	124.6	124.2	124.2	124.2
23	27.5	2.78 (dd, 6.6, 6.6)	29.7	27.9	27.9	27.9	27.9
24	124.9	5.11 (t, 6.6)	124.1	124.2	124.6	124.6	124.6
25	131.7	–	132.1	132.1	132.1	132.2	132.2
26	25.9	1.69 (s)	25.9	25.9	25.8	25.8	25.8
27	17.6	1.67 (s)	17.8	17.8	17.8	17.8	17.8
28	31.4	1.35 (s)	31.4	31.4	28.4	31.4	31.9
29	16.1	1.03 (s)	16.1	16.1	16.1	16.5	17.2
30	17.1	1.00 (s)	17.1	17.1	17.2	17.1	17.3
1′	105.6	4.38 (d, 7.8)	105.5	105.5	104.5	103.9	101.6
2′	75.5	3.23 (dd, 9.0, 7.8)	75.5	75.4	81.1	79.7	79.2
3′	79.1	3.37 (dd, 9.0, 9.0)	79.0	79.0	78.3	79.3	79.2
4′	71.7	3.30 (dd, 9.0, 9.0)	71.7	71.6	71.6	71.7	71.9
5′	77.7	3.28 (m)	77.6	77.6	77.7	78.3	78.1
6′	62.9	3.68 (dd, 12.0, 5.4) 3.84 (dd, 12.0, 1.8)	62.9	62.9	62.8	62.9	63.1
1′′					105.4	103.9	101.6
2′′					76.3	75.6	72.2
3′′					78.5	77.6	72.5
4′′					71.9	71.3	74.0
5′′					77.8	66.9	69.7
6′′					63.1		18.0

2a: 20(22)Z-Ginsenoside Rh4.¹³

The molecular formula of 1 was determined as C₃₆H₆₀O₈ by HRESIMS (Supplemental Figure S1). The NMR spectra of compound 1 were very similar to those of 2 suggesting a dammarane-type triterpenoid glycoside with one sugar moiety (Supplemental Figures S2 to S7).⁶ Detailed comparison of the NMR spectral data of 1 and 2 (Table 1) showed that they mostly the same, but there were marked difference at C-21 (δ_C 19.6 for 1 and 12.9 for 2-5) suggesting the (Z)-Δ²⁰⁽²²⁾-configuration for 1 (Figure 1). The NMR assignments of 1 were taken by analyzing ¹H, ¹³C NMR, HSQC, ¹H-¹H COSY, and HMBC spectra (Figure 2). HMBC correlations were observed from H-28 (δ_H 1.35) and H-29 (δ_H 1.03) to C-3 (δ_C 79.9), from H-21 (δ_H 1.72) to C-17 (δ_C 41.6)/C-20 (δ_C 140.2)/C-22 (δ_C 124.6), from H-26 and H-27 to C-24 (δ_C 124.9)/C-25 (δ_C 131.7), from the anomeric proton (δ_H 4.38) to C-6 (δ_C 80.9), and from H-13 (δ_H 1.87)/H-17 (δ_H 3.10) to C-12 (δ_C 74.0) confirming the hydroxy group at C-3, 2 double bonds at C-20(22) and C-24(25), the sugar moiety attached to C-6, and the other hydroxy group at C-12 with a β-configuration, as in all the Panax dammarane compounds. In the NOESY spectrum of 1, the cross peaks of H₃-21 (δ_H 1.17) and H-22 (δ_H 5.11), H-17 (δ_H 3.10) and H₂-23 (δ_H 2.78), H-17 and H₃-30 (δ_H 1.03), H₃-28 (δ_H 1.35) and H-3 (δ_H 3.11), and H₃-29 (δ_H 1.35) and H-6 (δ_H 4.11) were clearly observed (Supplemental Figures S8 and S9). This evidence indicated the (Z)-configuration of the C-20(22) double bond, protons H-3, H-17, H-28, and H-30 in an α-orientation, and H-29 and H-6 in a β-orientation. In addition, the appearance of H-12 with 2 large coupling constants (10.8 and 10.8 Hz) suggested the axial orientation of this proton. The β-form of the glycosyl linkage of 1 was evidenced by the large J_{H-1′′/H-2′′} value (7.8 Hz). Acid hydrolysis of 1 obtained D-glucose, which were identified by the positive sign of its optical rotation and TLC analysis in comparison with the authentic monosaccharide.^{14, 15} Thus, compound 1 was determined to be 3β,6α,12β-trihydroxydammarane-(Z)-20(22),24-diene 6-O-β-D-glucopyranoside. In the previous report,¹³ the NMR data did not match those of 20(22)Z-ginsenoside Rh4 (2a), but did with 20(22)E-ginsenoside Rh4 (Table 1).⁶ Especially, the carbon chemical shift value of δ_C-21 = 12.9 ppm of ginsenoside Rh4 in the previous report¹³ corresponded to the 20(22)E-configuration,¹² but was quite different from the value of δ_C-21 = 19.6 ppm in the 20(22)Z-configuration. Both (E)- and (Z)-configurations were isolated and reported herein, which were further clearly determined by their NOESY spectra. The above data evidenced that 1 was 20(22)Z-ginsenoside Rh4, a previously unpublished compound.

Figure 1.

Chemical structures of 1 to 5.

Figure 2.

Key ¹H-¹H COSY and HMBC correlations of 1 and 2.

The NMR spectra for compounds 2 to 5 are shown in Supplemental Figures S10 to S24.

Materials and Methods

General Experimental Procedures

The used characterization equipment is the same as described in our previous work.^16,17 Refer to Supplemental Material.

Plant Materials

The roots of P. pseudoginseng Wall. were collected in Si Ma Cai district, Lao Cai province, Vietnam during September 2021 and identified by Dr Nguyen The Cuong (Institute of Ecology and Biological Resources, VAST). The voucher specimen (NCCT-P147) has been deposited at IMBC, VAST.

Extraction and Isolation

Dried roots of P. pseudoginseng were powdered (3.8 kg) and then sonicated with methanol (each 15.0 L, room temperature, 4 h). After removal of solvent, the MeOH extract (212 g) was suspended in water (5 L) and then partitioned with n-hexane, and ethyl acetate to give the corresponding residues, PP1 (52.2 g), PP2 (17.5 g), and the water layer. The water layer was chromatographed on a Diaion HP-20 column eluting with water to remove sugar, and then with increasing concentrations of methanol in water (25%, 50%, 75%, and 100%) to obtain 4 fractions, PPA (37.2 g), PPB (51.0 g), PPC (23.4 g), and PPD (37.6 g). PPD (37.0 g) was chromatographed on a silica gel column eluting with dichloromethane/methanol (v/v) 20:1, 10:1, 5:1, and 2.5:1, and 1/1 (each 2 L) to give 5 fractions, PPD1 (4.5 g), PPD2 (4.2 g), PPD3 (6.9 g), PPD4 (7.4 g), and PPD5 (8.2 g). PPD2 (4.2 g) was chromatographed on a YMC column eluting with acetone/water (3/1, v/v, 3.5 L) to give 4 smaller fractions, PPD2A-PPD2D. Fraction PPD2C (243 mg) was chromatographed by HPLC (J'sphere H-80 column, 250 mm length × 20 mm ID, eluting with 82% methanol in water, at a flow rate of 3.0 mL/min) to give compounds 1 (t_R 40.78, 14.5 mg) and 2 (t_R 42.96, 16.7 mg). Fraction PPD3 (4.2 g) was chromatographed on an YMC column eluting with methanol/water (2/1, v/v, 3.5 L) to give 11 smaller fractions, PPD3A-PPD3L. Fraction PPD3G (273 mg) was chromatographed by HPLC eluting with 40% ACN in water, at a flow rate of 3.0 mL/min to give compounds 4 (t_R 57.84, 12.3 mg) and 5 (t_R 63.21, 11.8 mg). Fraction PPD3L was chromatographed by HPLC using the same conditions to obtain compound 3 (t_R 52.67, 16.2 mg).

3β,6α,12β-Trihydroxydammarane-(Z)-20(22),24-Diene 6-O-β-D-Glucopyranoside (1)

Colorless powder, $[{α]}_{D}^{25}$ : + 18.5 (c 0.1, MeOH); HRESIMS m/z 621.4330 [M + H]⁺ (calcd. for [C₃₆H₆₁O₈]⁺: 621.4361, Δ = −5.0 ppm); m/z 643.4188 [M + Na]⁺ (calcd. for [C₃₆H₆₀O₈Na]⁺: 643.4180, Δ = + 1.2 ppm). ¹H NMR (CD₃OD, 600 MHz) δ (ppm), ¹³C NMR (CD₃OD, 150 MHz) δ (ppm) data: see Table 1.

Acid Hydrolysis of Compound 1

Refer to Supplemental Material.^14,15

Conclusions

From the MeOH extract of the roots of P. pseudoginseng, 5 Δ^20(22),24-dammarane-type triterpenoid glycosides were isolated, including one new compound, 3β,6α,12β-trihydroxydammarane-(Z)-20(22),24-diene 6-O-β-D-glucopyranoside (1), together with 4 known ones, 3β,6α,12β-trihydroxy-dammar-(E)-20(22),25-diene 6-O-β-D-glucopyranoside (2), ginsenoside Rg5 (3), 3β,12β-dihydroxydammarane-(E)-20(22),24-diene 6-O-β-D-xylopyranosyl-(1→2)-β-D-glucopyranoside (4), and 3β,12β-dihydroxydammarane-(E)-20(22),24-diene 6-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside (5). Their chemical structures were elucidated by HR-ESI-MS, and 1D and 2D NMR spectroscopic analysis. The results indicated that the carbon chemical shifts of C-21 are around 13.0 ppm for (E)-Δ²⁰⁽²²⁾-dammarane, and 20.0 ppm for (Z)-Δ²⁰⁽²²⁾-dammarane compounds. In addition, the majority of Δ²⁰⁽²²⁾-dammarane compounds have the (E)-configuration, while the (Z)-configuration is very rare.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X221099055 - Supplemental material for 20(22)Z and 20(22)E Dammarane Saponins From the Roots of Panax pseudoginseng Wall.

Supplemental material, sj-docx-1-npx-10.1177_1934578X221099055 for 20(22)Z and 20(22)E Dammarane Saponins From the Roots of Panax pseudoginseng Wall. by Hoang Van Hung, Luc Quang Tan, Nguyen Huy Hoang, Bui Huu Tai and Phan Van Kiem in Natural Product Communications

Footnotes

Author Contribution

Research idea, HV Hung, PV Kiem; Isolation, LQ Tan, NH Hoang; Collect sample: LQ Tan; Structure elucidation and writing, BH Tai, PV Kiem, HV Hung.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Thai Nguyen University of Technology (grant no. ĐHTN2021-TN11-03.).

ORCID iDs

Luc Quang Tan

Phan Van Kiem

Supplemental Material

Supplemental material for this article is available online.

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