Complete 1H-NMR and 13C-NMR spectral assignment of five malonyl ginsenosides from the fresh flower buds of Panax ginseng

Yu-Shuai Wang; Yin-Ping Jin; Wei Gao; Sheng-Yuan Xiao; Yu-Wei Zhang; Pei-He Zheng; Jia Wang; Jun-Xia Liu; Cheng-He Sun; Ying-Ping Wang

doi:10.1016/j.jgr.2015.08.003

. 2015 Aug 13;40(3):245–250. doi: 10.1016/j.jgr.2015.08.003

Complete ¹H-NMR and ¹³C-NMR spectral assignment of five malonyl ginsenosides from the fresh flower buds of Panax ginseng

Yu-Shuai Wang ¹, Yin-Ping Jin ¹, Wei Gao ¹, Sheng-Yuan Xiao ^1,², Yu-Wei Zhang ¹, Pei-He Zheng ¹, Jia Wang ¹, Jun-Xia Liu ¹, Cheng-He Sun ¹, Ying-Ping Wang ^1,^∗

PMCID: PMC5005357 PMID: 27616900

Abstract

Background

Ginsenosides are the major effective ingredients responsible for the pharmacological effects of ginseng. Malonyl ginsenosides are natural ginsenosides that contain a malonyl group attached to a glucose unit of the corresponding neutral ginsenosides.

Methods

Medium-pressure liquid chromatography and semipreparative high-performance liquid chromatography were used to isolate purified compounds and their structures determined by extensive one-dimensional- and two-dimensional nuclear magnetic resonance (NMR) experiments.

Results

A new saponin, namely malonyl-ginsenoside Re, was isolated from the fresh flower buds of Panax ginseng, along with malonyl-ginsenosides Rb1, Rb2, Rc, Rd. Some assignments for previously published ¹H- and ¹³C-NMR spectra were found to be inaccurate.

Conclusion

This study reports the complete NMR assignment of malonyl-ginsenoside Re, Rb₁, Rb₂, Rc, and Rd for the first time.

Keywords: flower buds, ginsenoside, malonyl ginsenoside Re, NMR, Panax ginseng

1. Introduction

Panax ginseng Meyer is one of the most widespread traditional drugs used in China for thousands of years to produce various pharmacological and biological effects. The most important components contributing to its multiple medicinal properties are the ginsenosides, a group of triterpenoid saponins. Up to now, > 150 ginsenosides have been isolated from Panax species [1]. Among these known compounds, malonyl ginsenosides (M-Rs; e.g., m-Rb₁, m-Rb₂, m-Rc, and m-Rd) are natural ginsenosides that exist in both fresh and air-dried ginseng and contain a malonyl residue attached at the 6-position of a glucosyl unit of the corresponding neutral ginsenoside [2], [3]. Malonyl ginsenosides are considered an important form of ginsenoside in white ginseng, however, they are unstable and readily demalonylated or decarboxylated to their respective counterparts or acetylates by treatment with hot water or hot methanol [3], [4], [5], [6].

Because malonyl ginsenosides are thermally unstable, their monomeric compounds are hard to obtain, although up to 20 malonyl ginsenosides have been detected by liquid chromatography/quadropole time-of-flight mass spectrometry [7]. Only six malonyl ginsenosides have been isolated and characterized [8], [9], [10], [11]. Kitagawa et al [8] and Yamaguchi et al [9] reported the presence of malonyl ginsenosides Rb1, Rb2, Rc, and Rd in both P. ginseng and P. quinquefolius [8], [9]. Sun et al [10] and Ruan et al [11] isolated malonyl notoginsenoside R₄ and malonyl-ginsenoside Ra₃ from the fresh roots of P. ginseng, respectively [10], [11]. All previously isolated malonyl ginsenosides were derived from protopanoxadiol (PPT)-type ginsenosides [12].

In this study, we isolated five malonyl ginsenosides from the flower buds of P. ginseng and malonyl-ginsenoside Re (M-Re) was obtained as a PPT-type malonyl ginsenoside for the first time. Identification and characterization of ginsenosides are usually conducted using nuclear magnetic resonance (NMR) analyses , but several imperfections and/or inaccuracies existed in the published NMR data of malonyl ginsenosides given the lack of two-dimensional (2D) NMR techniques at the time of characterization. Here, with the help of modern 2D NMR techniques including correlation spectroscopy, rotating frame nuclear Overhauser effect spectroscopy (ROESY), heteronuclear single-quantum coherence (HSQC), and heteronuclear multiple-band coherence (HMBC) experiments, complete NMR assignments of malonyl-ginsenosides Rb₁, Rb₂, Rc, Rd, and Re were determined for the first time.

2. Materials and methods

2.1. General experimental procedure

Medium-pressure liquid chromatography (MPLC) purifications were carried out on a Yamazen YFLC-AI-580 instrument (Yamazen Co., Osaka, Japan) equipped with silica gel columns (Hi-Flash columns, silica gel: 40 μm, 26 mm × 150 mm internal diameter column). Reversed-phase semipreparative high-performance liquid chromatography (HPLC) was performed on an instrument consisting of Prostar/Dynamax system control, a Varian PS-218 pump, and a Prostar 325 UV-Vis detector with a Varian Polaris C18-A semipreparative column (250 mm × 10 mm, 10 μm; Agilent Technologies, Santa Clara, CA, USA). Thin-layer chromatography (TLC) was performed using a silica gel 60 RP-18 F_254S and Kieselgel 60 F₂₅₄, with spots detected by spraying 10% H₂SO₄ in ethanol followed by heating at 105ºC. HPLC was carried out using an Agilent TC-C18 column (5 μm, 250 mm × 4.6 mm; Agilent Technologies) and products were eluted with a step-wise gradient at a flow rate of 1.0 mL/min using solvent A (water containing 0.0005% ammonium hydroxide and 0.02% ammonium acetate) and solvent B (acetonitrile). The elution rate using solvent B was 17.5% for 0–4 min, 17.5–28.9% for 4–9 min, 28.9–40% for 9–19 min, and 40% for 19–24 min.

The ¹H-, ¹³C-, and 2D-NMR spectra were measured using a Bruker AV600 NMR spectrometer (Bruker Co., Karlsruhe, Germany; 600 MHz for ¹H and 150 MHz for ¹³C) with tetramethylsilane as an internal standard. Chemical shifts (δ) are expressed in ppm, with the coupling constants (J) reported in Hertz (Hz). The electrospray ionization mass spectrometry (ESI-MS) and high-resolution electrospray ionization mass spectrometry (HRESIMS) spectra were recorded using an Agilent 1200 HPLC with a 6300 Ion-trap liquid chromatography/mass spectrophotometry (LC/MS; Agilent Technologies; ionization mode, negative; nebulizing gas [N₂] pressure, 35 psi; drying gas [N₂] flow, 8 L/min; temp, 350ºC) and Q-Exactive mass spectrometer (Thermo Scientific, Bremen, Germany), respectively. For the automated MS/MS analysis, the collision energy was optimized automatically from 30% to 200% of 1.0 V and the collision time was 20 ms. Gas chromatography (GC) was performed using the Agilent 7890A GC with flame ionization detector and a HP-5 chiral capillary column (30 m × 0.25 mm; film thickness, 0.25 μm; Agilent Technologies). Column temperatures started at 170ºC and increased to 200ºC at 3ºC/min, then increased further to 220ºC at 0.8ºC/min. Inlet temperature was set to 270ºC, with hydrogen carrier gas and a 1/15 split, and N₂ was used as the carrier gas (1.0 mL/min flow rate). The infrared (IR) spectra were recorded on a Bruker Vertex 70 FT-IR spectrophotometer (Bruker Co., Ettlingen, Germany) using potassium bromide pellets.

2.2. Plant material

The fresh flower buds of P. ginseng were collected from Fu-Song, Jilin, China, in May 2014, and authenticated by one of the authors, Professor Shi-quan Xu. A voucher of the specimen collected (ZYC-RS-20131008) was deposited in the conditions of −20ºC at the Institute of Special Wild Economic Animals and Plants, Chinese Academy of Agricultural Sciences.

2.3. Extraction and isolation

The fresh flower buds of P. ginseng (2.0 kg) were extracted five times with 80% methanol, a 6× dilution of the extracting solution was subjected to a nanofiltration membrane (ESNA1-K1-8040, Hydranautics Corporation, USA) to eliminate most of the pigment, and the filtrate (96.8 g) subjected to column chromatography on a porous polymer polystyrene resin (AB-8). After washing the column with eight column volumes of distilled water, elution was carried out with 30% and 60% aqueous ethanol, and finally with 100% ethanol. The fraction eluted with 30% ethanol (8.9 g) was loaded onto a MPLC system and eluted with CH₂Cl₂-MeOH-H₂O (5:1:0.1-4:1:0.1-3:1:0.1) to yield six fractions (AG1-6). Fraction AG4 (2.2 g) was further separated using semipreparative reversed-phase HPLC and eluted with CH₃CN-H₂O (1:4) at 3 mL/min to yield malonyl-ginsenoside Re (30 mg; t_R 25.2 min). The fraction eluted with 60% ethanol (48.0 g) was processed on a MPLC system using a linear gradient elution (7 mL/min) of 25–45% methanol in CH₂Cl₂ for 250 min in order to collect fraction BG1-9. M-Rb₁, M-Rb₂, M-Rc, and M-Rd were primarily distributed within fraction BG8 through analysis by LC/MS. Fraction BG8 (7.8 g) was then applied to semipreparative reversed-phase HPLC using a linear gradient elution (3 mL/min) of 29–34% acetonitrile in water for 50 min to yield M-Rb₁ (21 mg; t_R 19.7 min), M-Rb₂ (18 mg; t_R 24.0 min), M-Rc (22 mg; t_R 29.8 min), and M-Rd (27 mg; t_R 43.9 min; Fig. 1).

2.4. Characterization of compounds 1–5

Compound 1 was obtained as a white amorphous powder and gave peaks at m/z 1,031.4 [M-H]⁻, 987.6 [M-H-CO₂]⁻, 945.4 [M-COCH₂COOH]⁻, 927.8 [M-COCH₂COOH-H₂O]⁻, 783.7 [M-COCH₂COOH-glu]⁻, 637.5 [M-COCH₂COOH-rha-glu]⁻, and 475.3 [M-CO₂- CH₃COOH-rha- 2glu]⁻ in negative-mode ESI-MS, indicating its molecular weight to be 1,032. HRESIMS: m/z 1,055.5391 [M+Na]⁺ (calculated for C₅₁H₈₄NaO₂₁, 1,055.5397). IR ν_max was 3,408, 2,932, 1,731, 1,636, 1,599, 1,454, 1,385, 1,075, and 1,050 cm⁻¹. Libermann-Buchard and Molish reactions were positive. Eight methyl groups and six quaternary carbons were identified in the analysis of the NMR spectrum (Table 1, Table 2). Molish reaction was used to proof the existence of saccharides, and test of Libermann-Buchard for steroids or triterpenes.

Table 1.

The ¹³C-NMR data of compounds 1–5 (150 MHz, pyridine-d₅, δ_C)

C	Compound 1	Compound 2	Compound 3	Compound 4	Compound 5
1	39.859	39.679	39.654	39.637	39.645
2	28.202	27.087	27.105	27.082	27.239
3	78.880	89.735	89.707	89.713	89.716
4	40.448	40.209	40.187	40.193	40.190
5	61.297	56.945	56.927	56.924	56.922
6	75.122	18.910	18.898	18.903	18.905
7	46.388	35.604	35.596	35.579	35.599
8	41.652	40.500	40.485	40.474	40.487
9	50.027	50.688	50.666	50.645	50.649
10	40.117	37.381	37.370	37.365	37.367
11	31.372	31.276	31.229	31.233	31.335
12	70.593	70.689	70.657	70.716	70.683
13	49.520	49.969	49.926	49.885	49.914
14	51.937	52.090	51.858	51.867	51.890
15	31.166	31.162	31.154	31.153	31.227
16	27.153	27.253	27.244	27.244	27.110
17	51.873	51.852	52.130	52.114	52.117
18	17.742	16.475	16.463	16.434	16.427
19	18.087	16.757	16.747	16.746	16.760
20	83.925	83.918	83.954	83.807	83.783
21	22.483	22.867	22.788	22.822	22.851
22	36.513	36.644	36.617	36.590	36.571
23	23.468	23.674	23.668	23.629	23.691
24	126.501	126.438	126.406	126.484	126.408
25	131.495	131.506	131.552	131.468	131.376
26	26.257	26.261	26.246	26.243	26.225
27	18.272	18.412	18.333	18.325	18.232
28	32.662	28.515	28.510	28.511	28.512
29	17.951	16.977	16.977	16.974	16.978
30	17.769	17.875	17.847	17.824	17.828
	6-O-Glucopyranosyl	3-O-Glucopyranosyl	3-O-Glucopyranosyl	3-O-Glucopyranosyl	3-O-Glucopyranosyl
1'	102.340	105.350	105.337	105.342	105.342
2'	79.083	84.641	84.507	84.512	84.419
3'	79.850	78.549	78.523	78.532	78.501
4'	73.026	72.162	72.263	72.556	72.026
5'	78.756	78.268	78.210	78.215	78.170
6'	63.538	63.270	63.238	63.227	63.261
	2'-O-Rhamnosyl	2'-O-Glucopyranosyl	2'-O-Glucopyranosyl	2'-O-Glucopyranosyl	2'-O-Glucopyranosyl
1''	102.340	106.586	106.535	106.531	106.483
2''	72.867	77.187	77.196	77.185	77.179
3''	72.729	79.720	79.650	79.684	79.670
4''	74.607	71.864	71.843	71.826	71.803
5''	69.951	75.867	75.874	75.867	75.860
6''	19.206	65.624	65.495	65.523	65.432
	20-O-Glucopyranosyl	20-O-Glucopyranosyl	20-O-Glucopyranosyl	20-O-Glucopyranosyl	20-O-Glucopyranosyl
1'''	98.471	98.555	98.592	98.565	98.734
2'''	75.434	75.330	75.365	75.500	75.596
3'''	79.443	78.862	78.910	78.906	78.869
4'''	71.942	71.459	71.444	71.451	71.431
5'''	75.389	77.530	77.196	77.005	78.729
6'''	65.755	70.622	69.671	68.934	63.192
		6'''-O-Glucopyranosyl	6'''-O-Arabinopyranosyl	6'''-O-Arabinofuranosyl
1''''		105.832	105.109	110.587
2''''		75.725	72.602	83.858
3''''		78.813	74.587	79.288
4''''		72.056	69.046	86.464
5''''		78.945	66.072	63.098
6''''		63.270
Malonyl
M1	169.308	169.385	169.72	169.651	169.761
M2	44.401	44.176	44.624	44.486	44.801
M3	171.075	171.039	171.403	171.276	171.398

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C, carbon; NMR, nuclear magnetic resonance.

Table 2.

The ¹H-NMR data of compounds 1–5 (600 MHz, pyridine-d₅, δ_H, J in Hz)

H	Compound 1	Compound 2	Compound 3	Compound 4	Compound 5
1	0.93, 1.63	0.73, 1.51	0.71, 1.52	0.70, 1.50	0.71, 1.49
2	1.74, 1.83	1.80, 2.16	1.81, 2.16	1.81, 2.16	1.79, 2.13
3	3.43 (dd, 4.6, 11.5)	3.24 (dd, 4.3, 11.8)	3.23 (dd, 4.3, 11.8)	3.22 (dd, 4.3, 11.7)	3.23 (dd, 4.3, 11.5)
5	1.36(d, 10.7)	0.67	0.66	0.65	0.66
6	4.65	1.47, 1.36	1.34, 1.46	1.46, 1.34	1.47, 1.36
7	1.96, 2.22	1.13, 1.45	1.20, 1.45	1.14, 1.43	1.14, 1.43
9	1.48	1.33	1.32	1.33	1.34
11	1.47, 2.02	1.53, 1.96	1.52, 1.95	1.48, 1.95	1.52, 1.94
12	4.11	4.30	4.11	4.16	4.10
13	1.92	1.97	1.94	1.95	1.94
15	0.84, 1.44	0.96, 1.53	0.96, 1.52	0.96, 1.34	0.97, 1.53
16	1.21, 1.74	1.36, 1.81	1.34, 1.80	1.34, 1.80	1.34, 1.79
17	2.46	2.56	2.54	2.52	2.52
18	1.15 (s)	0.94 (s)	0.93 (s)	0.92 (s)	0.93 (s)
19	0.93 (s)	0.81 (s)	0.79 (s)	0.79 (s)	0.79 (s)
21	1.54 (s)	1.58 (s)	1.59 (s)	1.61 (s)	1.59 (s)
22	1.73, 2.32	1.81, 2.37	1.80, 2.35	1.80, 2.34	1.80, 2.34
23	2.30, 2.49	2.37, 2.55	2.35, 2.54	2.34, 2.53	2.22, 2.46
24	5.28 (t-like)	5.29 (t-like )	5.28 (t-like)	5.28 (t-like)	5.21 (t, 6.9)
26	1.62 (s)	1.63 (s)	1.61 (s)	1.61 (s)	1.57 (s)
27	1.63(s)	1.63 (s)	1.63 (s)	1.64 (s)	1.59 (s)
28	2.07 (s)	1.32 (s)	1.31 (s)	1.31 (s)	1.30 (s)
29	1.33 (s)	1.14 (s)	1.12 (s)	1.11 (s)	1.11 (s)
30	0.93 (s)	0.92 (s)	0.91 (s)	0.92 (s)	0.91 (s)
	6-O-Glucopyranosyl	3-O-Glucopyranosyl	3-O-Glucopyranosyl	3-O-Glucopyranosyl	3-O-Glucopyranosyl
1'	5.22 (d, 6.8)	4.88 (d, 7.7)	4.87 (d, 7.6)	4.87 (d, 7.6)	4.86 (d,7.6)
2'	4.35	4.13	4.13	4.14	4.12
3'	4.29	4.15	4.23	4.16	4.27
4'	4.19	4.01	4.01	3.95	4.12
5'	3.92	3.90	3.86	4.26	3.85
6'	4.33, 4.47	4.33, 4.51	4.32, 4.51	4.30, 4.51	4.29, 4.43
	2'-O-Rhamnosyl	2'-O-Glucopyranosyl	2'-O-Glucopyranosyl	2'-O-Glucopyranosyl	2'-O-Glucopyranosyl
1''	6.46 (brs)	5.28 (d, 7.3)	5.27 (d, 7.63)	5.27 (d, 7.63)	5.27 (d, 7.6)
2''	4.77 (brs)	4.02	4.03	4.07	4.06
3''	4.64	4.15	4.13	4.16	4.15
4''	4.30	4.20	4.21	4.26	4.18
5''	4.90 (dt, 6.2, 9.3 )	4.03	4.02	4.00	3.98
6''	1.74 (d, 6.1)	4.97	4.96	4.95	4.94
	20-O-Glucopyranosyl	20-O-Glucopyranosyl	20-O-Glucopyranosyl	20-O-Glucopyranosyl	20-O-Glucopyranosyl
1'''	5.04 (d, 7.7)	5.11 (d, 7.8)	5.10 (d, 7.7)	5.11 (d, 7.7)	5.16 (d, 7.7)
2'''	3.93	3.90	3.89	3.94	3.97
3'''	4.13	4.27	4.26	3.87	3.88
4'''	3.95	4.04	4.13	4.13	3.98
5'''	3.96	4.09	3.98	4.09	3.88
6'''	4.71 (dd, 5.6, 11.1),	4.19, 4.70	4.21, 4.65	4.00, 4.63	4.44, 4.51
	4.98 (d, 5.6)
		6'''-O-Glucopyranosyl	6'''-O-Arabinopyranosyl	6'''-O-Arabinofuranosyl
1''''		5.08 (d, 7.7)	4.96 (d,5.94)	5.64 (d, 1.4)
2''''		4.03	4.42	4.84
3''''		4.17	4.19	4.77
4''''		4.26	4.34	4.72
5''''		4.16	4.27,3.77	4.17,4.27,4.33
6''''		4.33, 4.50
Malonyl
M2	3.75 (s)	3.76(s)	3.75	3.75(s)	3.74(s)

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brs, broad singlet; dd, double doublet; H, hydrogen; m, multiplet; NMR, nuclear magnetic resonance; s, singlet; t, triplet; t-like, triplet-like.

Compound 2 was obtained as a white amorphous powder. The molecular formula was determined as C₅₇H₉₄O₂₆ based on HRESIMS data at m/z 1,217.5921 [M+Na]⁺ (calculated for C₅₆H₉₂NaO₂₅, 1,217.5925). Negative-mode ESI-MS (m/z) readings: 1,193.4 [M-H]⁻, 1,149.5 [M-H-CO₂]⁻, 1,107.4 [M-COCH₂COOH]⁻, 1,089.5 [M-COCH₂COOH-H₂O]⁻, 945.5 [M-COCH₂COOH-glu]⁻, 783.2 [M-COCH₂COOH-2glu]⁻, 621.1 [M-COCH₂COOH-3glu]⁻, and 459.3 [M-COCH₂COOH-4glu]⁻. IR ν_max: 3,383, 2,937, 1,724, 1,638, 1,454, 1,383, and 1,076. Libermann-Burchard and Molish reactions were positive. The ¹³C-NMR spectrum and ¹H-NMR data are shown in Table 1, Table 2. The IR spectrum showed absorption bands for hydroxyl (3,383 cm⁻¹), carbonyl (1,724 cm⁻¹), double bond (1,638 cm⁻¹), methyl (1,383 cm⁻¹) and ether moiety (1,076 cm⁻¹).

Compound 3 was obtained as a white amorphous powder. The molecular formula was determined as C₅₆H₉₂O₂₅ based on HRESIMS data at m/z 1,187.5826 [M+Na]⁺ (calculated for C₅₆H₉₂NaO₂₅, 1,187.5820). Negative-mode ESI-MS (m/z) readings: 1,163.4 [M-H]⁻, 1,119.2 [M-H-CO₂]⁻, 1,077.1 [M-COCH₂COOH]⁻, 1,059.1 [M-COCH₂COOH-H₂O]⁻, 945.2 [M-COCH₂COOH-Ara(p)]⁻, 783.2 [M-COCH₂COOH-Ara(p)-glu]⁻, 621.0 [M-COCH₂COOH-Ara(p) -2glu]⁻, and 459.4 [M-COCH₂COOH-Ara(p)- 3glu]⁻. IR ν_max: 3,392, 2,943, 1,729, 1,638, 1,452, 1,385, and 1,077. Libermann-Burchard and Molish reactions were positive. Molish reaction was used to proof the existence of saccharides, and test of Libermann-Buchard for steroids or triterpenes.

Compound 4 was obtained as a white amorphous powder. The molecular formula was determined as C₅₆H₉₂O₂₅ based on HRESIMS data at m/z 1,187.5822 [M+Na]⁺ (calculated for C₅₆H₉₂NaO₂₅, 1,187.5820). Negative-mode ESI-MS (m/z) readings: 1,163.4 [M-H]⁻, 1,119.7 [M-H-CO₂]⁻, 1,077.4 [M-COCH₂COOH]⁻, 1,059.3 [M-COCH₂COOH-H₂O]⁻, 945.2 [M- COCH₂COOH-Ara(f)]⁻, 783.3 [M-COCH₂COOH-Ara(f)-glu]⁻, 621.2 [M-COCH₂COOH-Ara(f)- 2glu]⁻, and 459.1 [M-COCH₂COOH-Ara(f)- 3glu]⁻. IR ν_max: 3,387, 2,942, 1,728, 1,636, 1,452, 1,388, and 1,076. Libermann-Burchard and Molish reactions were positive. The IR spectrum showed absorption bands for hydroxyl (3,387 cm⁻¹), carbonyl (1,728 cm⁻¹), double bond (1,636 cm⁻¹), methyl (1,388 cm⁻¹) and ether moiety (1,076 cm⁻¹). Molish reaction was used to proof the existence of saccharides, and test of Libermann-Buchard for steroids or triterpenes.

Compound 5 was obtained as a white amorphous powder. The molecular formula was determined as C₅₁H₈₄O₂₁ based on HRESIMS data at m/z 1,055.5400 [M+Na]⁺ (calculated for C₅₆H₉₂NaO₂₅, 1,055.5397). Negative-mode ESI-MS (m/z) readings: 1,031.5 [M-H]⁻, 987.4 [M-H-CO₂]⁻, 945.4 [M-COCH₂COOH]⁻, 927.3 [M-COCH₂COOH-H₂O]⁻, 783.3 [M- COCH₂COOH-glu]⁻, 621.2 [M-COCH₂COOH-2glu]⁻, and 459.0 [M-COCH₂COOH-3glu]⁻. IR ν_max: 3,392, 2,944, 1,731, 1,634, 1,453, 1,388, and 1,076. Libermann-Burchard and Molish reactions were positive. Molish reaction was used to proof the existence of saccharides, and test of Libermann-Buchard for steroids or triterpenes.

2.5. Acid hydrolysis of compound 1

Compound 1 (5.0 mg) was hydrolyzed with 3.0N HCl (5 mL) at 100ºC for 2 h. The reaction mixture was extracted with chloroform to afford the aglycone, and the aqueous layer was repeatedly evaporated to dryness with methanol until neutral. The sample was then analyzed by TLC over a silica gel with n-BuOH-AcOH-H₂O (9:4:2) as the developing solvent. The sample spots were detected by spraying diphenylamine-aniline-phosphoric acid reagent (2% aniline in acetone: 2% diphenyl in acetone: 85% phosphoric acid = 5:5:1) and heating at 100ºC [13]. The chromogenic agent was used to react with monosaccharides and appear coloration through heating. β-d-Glucose and α-l-rhamnose were used as authentic samples. Furthermore, the aqueous layer residues mentioned above were dissolved in anhydrous pyridine (2 mL) and stirred with l-cysteine methyl (1.5 mg) for 1 h at 60ºC, then 1.2 mL of hexamethyldisilazane:trimethylchlorosiane (3:1) was added and the mixture was stirred at 60ºC for another 30 min. The precipitate was centrifuged and the supernatant dried under N₂ steam at room temperature [14]. The residue was partitioned between hexane and water, and the hexane layer was analyzed by GC. Identification of d-glucose and l-rhamnose was carried out for compound 1, giving peaks at 9.90 min and 17.08 min, respectively.

3. Results

Compound 1 was obtained as a white amorphous powder. The molecular formula was determined as C₅₁H₈₄O₂₁ based on HRESIMS data at m/z 1,055.5391 [M+Na]⁺ (calculated for C₅₁H₈₄NaO₂₁, 1,055.5397). The IR spectrum showed absorption bands for hydroxyl (3,408 cm⁻¹), carbonyl (1,731 cm⁻¹), and methyl (1,385 cm⁻¹) groups, as well as double bond (1,636 cm⁻¹), and ether moieties (1,075 cm⁻¹). The ¹H-NMR spectrum (Table 2) showed eight methyl groups [δ_H 0.93 (6H, s), H-19, H-30; 1.15 (3H, s), H-18; 1.33 (3H, s), H-29; 1.63 (3H, s), H-27; 1.54 (3H, s), H-21; 1.62 (3H, s), H-26; 2.07 (3H, s), H-28], one olefinic proton [δ_H 5.28 (1H, t), H-24], one oxygen-substituted proton [δ_H 4.65 (1H, m), H-6], and three anomeric protons [δ_H 5.22 (1H, d, J = 6.8), H-1′; 6.46 (1H, brs), H-1′′; 5.04 (1H, d, J = 7.7), H-1‴]. The ¹³C-NMR (Table 2) spectrum showed 51 carbon signals, including a pair of olefinic carbons at C-24 (δc 126.501) and C-25 (δc 131.495), two oxygen-substituted carbons at C-6 (δc 75.122) and C-20 (δc 83.925), and two carbonyl-group signals at C-M1 (δc 169.308) and C-M3 (δc 171.075). These data suggest that compound 1 was a dammarane-type triterpene glycoside with a double bond and a malonyl group [15], [16], [17], [18]. The chemical structure of compound 1 was further elucidated by a HMBC (Figure 2) experiment in which correlations were observed between H-1′ ( δ_H 5.22, d, J = 6.8 Hz ) and the carbon resonance signal at C-6 ( δ_c 75.122 ), H-1′′ ( δ_H 6.46, brs ) and C-2′ ( δ_c 79.083 ), and H-1‴ ( δ_H 5.04, d, J = 7.7 Hz ) and C-20 ( δ_c 83.925 ), which indicated that the C-1_Glc′, C-1_Glc′′, and C-1_Rha′′′ were linked to C-6, C-2′, and C-20, respectively. The malonyl group was assigned to the C20-glc-C-6‴ position based on the correlations of C20-glc-H-6‴ with C-M1 and C-4‴, as shown in Figure 3.

Fig. 2 — Partially enlarged heteronuclear multiple-bond connectivity spectrum of compound 1.

Fig. 3 — Key HMBC and ¹H-¹H COSY correlations for compound 1. COSY, correlated spectroscopy; HMBC, heteronuclear multiplebond correlation.

The ¹H- and ¹³C-NMR spectroscopic data for compound 1 were similar to those of ginsenoside Re, except for the data attributed to a malonyl group (δ_H 3.75, δ_C 169.308, δ_C 44.401, δ_C 171.075). Other carbon shifts included an upfield shift of C-5‴ (δ_C 75.389) and a downfield shift of C-6‴ (δ_C 65.755), as compared to ginsenoside Re [15], [16]. H-5‴ yielded a peak at 3.96, and H-6‴ at 4.98 and 4.71, based on the HSQC spectrum. Acid hydrolysis of compound 1 yielded ginsenoside Re. The absolute configurations of the sugar moieties were further determined to be β-d-glucose and α-l-rhamnose by chiral GC analysis. The relative configuration of 1 was established through analysis of the ROESY experiment. As shown in Figure 1, correlations of H-3 to H-28 and H-5 indicated β-orientation for the 3-OH group. H-17 showed ROESY correlations with H-30 and H-16α, therefore, the side chain at C-17 was β-oriented. ROESY correlation between H-17 and H-21 confirmed assignment of the C-20(S) configuration. Moreover, the chemical shifts of C-17, C-21, and C-22 were 51.873, 22.483, and 36.513, respectively, which corresponded to the NMR data of 20(S)-ginsenoside Re [15], [16], [17].

The structures of compounds 2–5 were identified based on their spectroscopy data and by comparison of their data with literature sources [8], [18]. The NMR spectroscopic data of the malonyl group in the present study showed significant differences with values reported in the literature. The chemical shifts of the methylenes between the two carboxyls of the malonyl group were ∼44.176–44.801, which represented different values than those cited [8], [9], [10], [11]. In the ¹H-NMR spectrum, the chemical shifts of H-5″ and H-6″ were ∼3.98–4.03 and ∼4.94–4.97, respectively, which are reported here for the first time.

By normal-phase silica gel TLC (n-BuOH-CH₃COOH-H₂O = 4:1:5), R_f values were 0.32 for M-Re (1), 0.17 for M-Rb₁ (2), 0.18 for M-Rb₂ (3), 0.20 for M-Rc (4), and 0.27 for M-Rd (5). Reverse-phase ODS TLC (MeOH-H₂O = 2:1) yielded R_f values of 0.72, 0.36, 0.30, 0.35, and 0.27, respectively. Each compound was light purple on TLC when sprayed with 10% H₂SO₄ in ethanol followed by heating at 105ºC. HPLC retention times were 12.8 min for M-Re (1), 18.0 min for M-Rb₁ (2), 19.3 min for M-Rb₂ (3), 18.7 min for M-Rc (4), and 20.9 min for M-Rd (5).

4. Discussion

A phytochemical investigation of the fresh flowers of P. ginseng led to the isolation of a new saponin (20S)-Protopanaxatriol-6-[O-α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl]-20-O-(6-O-malonyl)-β-d-glucopyranoside, along with malonyl-ginsenosides Rb₁, Rb₂, Rc, and Rd. The complete ¹H-NMR data of the malonyl ginsenosides were assigned for the first time.

Malonyl ginsenosides are unstable, not readily available, making them more difficult to analyze by HPLC than their neutral counterparts, and not used as indices for evaluation and quality control of ginseng. However, they are reported to be present in significant quantities in ginseng species, so the conventional evaluation index may not comprehensively reflect all ginseng properties or processed products. This study reports a simple and efficient way to prepare malonyl ginsenosides, as well as their physicochemical and NMR data, which provided scientific basis for the preparation of the standard substance.

Conflicts of interest

All contributing authors declare no conflicts of interest.

Acknowledgments

We are grateful to Gao-Fei Hu (College of Science, Beijing University of Chemical Technology) for measuring the NMR spectra. This work was financially supported by the National Key Technology Research and Development Program of the Ministry of Science and Technology of China (no. 2011BAI03B01 ).

References

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[bib2] 2.Sun G.Z., Liu Z., Li X.G., Zheng Y.N., Wang J.Y. Isolation and identification of two malonyl ginsenosides from the fresh root of Panax ginseng. Chin J Anal Chem. 2005;33:1783–1786. [Google Scholar]

[bib3] 3.Wu W., Sun L., Zhang Z., Guo Y.Y., Liu S.Y. Profiling and multivariate statistical analysis of Panax ginseng based on ultra-high-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry. J Pharmaceut Biomed. 2015;107:141–150. doi: 10.1016/j.jpba.2014.12.030. [DOI] [PubMed] [Google Scholar]

[bib4] 4.Ren G., Chen F. Degradation of ginsenosides in American ginseng (Panax quinquefolium) extracts during microwave and conventional heating. J Agric Food Chem. 1999;47:1501–1505. doi: 10.1021/jf980678m. [DOI] [PubMed] [Google Scholar]

[bib5] 5.Chu C., Xu S.J., Li X.G., Yan J.Z., Liu L. Profiling the ginsenosides of three ginseng products by LC-Q-TOF/MS. J Food Sci. 2013;78:C653–C659. doi: 10.1111/1750-3841.12102. [DOI] [PubMed] [Google Scholar]

[bib6] 6.Kitagawa I., Taniyama T., Yoshikawa M., Ikenishi Y., Nakagawa Y. Chemical studies on crude drug processing. VI Chemical Structures of Malonyl ginsenosides Rb1, Rb2, Rc, and Rd Isolated from the root of Panax ginseng C.A. Meyer. Chem Pharm Bull. 1989;37:2961–2970. [Google Scholar]

[bib7] 7.Sun B.S., Xu M.Y., Li Z., Wang Y.B., Sung C.K. UPLC-Q-TOF-MS/MS analysis for steaming times-dependent profiling of steamed Panax quinquefolius and its ginsenosides transformations induced by repetitious steaming. J Ginseng Res. 2012;36:277–290. doi: 10.5142/jgr.2012.36.3.277. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib8] 8.Kitagawa I., Taniyama T., Hayashi T., Yoshikawa M. Malonyl ginsenosides Rb1, Rb2, Rc and Rd, four new malonylated dammarane-type triterpene oligosaccharides from ginseng radix. Chem Pharm Bull. 1983;31:3353–3356. [Google Scholar]

[bib9] 9.Yamaguchi H., Kasai R., Matsuura H., Tanaka O., Fuwa T. High-performance liquid chromatographic analysis of acidic saponins of ginseng and related plants. Chem Pharm Bull. 1988;36:3468–3473. doi: 10.1248/cpb.36.3468. [DOI] [PubMed] [Google Scholar]

[bib10] 10.Sun G.Z., Li X.G., Liu Z., Wang J.Y., Yang X.W. Isolation and structure characterization of malonyl-notoginsenoside R4 from the root of Panax ginseng. Chin J Anal Chem. 2007;28:1316–1318. [Google Scholar]

[bib11] 11.Ruan C.C., Liu Z., Li X., Liu X., Wang L.J., Pan H.Y., Zheng Y.N., Sun G.Z., Zhang Y.S., Zhang L.X. Isolation and characterization of a new ginsenoside from the fresh root of Panax Ginseng. Molecules. 2010;15:2319–2325. doi: 10.3390/molecules15042319. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[bib14] 14.Hara S., Okabe H., Mihashi K. Gas-liquid chromatographic separation of aldose enantiomers as trimethylsilyl ethers of methyl 2-(phlyhydroxyalkyl)thiazolidine-4- (R)-carboxylates. Chem Pharm Bull. 1987;35:501–506. [Google Scholar]

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[bib17] 17.Yang H., Kim J.Y., Kim S.O., Yoo Y.H., Sung S.H. Complete 1H-NMR and 13C-NMR spectral analysis of the pairs of 20(S) and 20(R) ginsenosides. J Ginseng Res. 2014;38:194–202. doi: 10.1016/j.jgr.2014.05.002. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Complete ¹H-NMR and ¹³C-NMR spectral assignment of five malonyl ginsenosides from the fresh flower buds of Panax ginseng

Yu-Shuai Wang

Yin-Ping Jin

Wei Gao

Sheng-Yuan Xiao

Yu-Wei Zhang

Pei-He Zheng

Jia Wang

Jun-Xia Liu

Cheng-He Sun

Ying-Ping Wang