Advances in Saponin Diversity of Panax ginseng.

Ginsenosides are the major bioactive constituents of Panax ginseng, which have pharmacological effects. Although there are several reviews in regards to ginsenosides, new ginsenosides have been detected continually in recent years. This review updates the ginsenoside list from P. ginseng to 170 by the end of 2019, and aims to highlight the diversity of ginsenosides in multiple dimensions, including chemical structure, tissue spatial distribution, time, and isomeride. Protopanaxadiol, protopanaxatriol and C17 side-chain varied (C17SCV) manners are the major types of ginsenosides, and the constitute of ginsenosides varied significantly among different parts. Only 16 ginsenosides commonly exist in all parts of a ginseng plant. Protopanaxadiol-type ginsenoside is dominant in root, rhizome, leaf, stem, and fruit, whereas malonyl- and C17SCV-type ginsenosides occupy a greater proportion in the flower and flower bud compared with other parts. In respects of isomeride, there are 69 molecular formulas corresponding to 170 ginsenosides, and the median of isomers is 2. This is the first review on diversity of ginsenosides, providing information for reasonable utilization of whole ginseng plant, and the perspective on studying the physiological functions of ginsenoside for the ginseng plant itself is also proposed.

1. Introduction

Panax ginseng Meyer (P. ginseng), known as the king of all herbs, has been frequently used as traditional medicine and healthy food in China, Korea, and Japan. In 2012, P. ginseng was approved as a new food resource by Chinese government, and it has been widely used as the raw material of healthcare products [1]. Ginseng contains a large amount and number of ginsenosides. More than 289 saponins were reported from eleven different Panax species [2]. In addition, at least 123 ginsenosides have been identified in different P. ginseng species, and these include both naturally occurring compounds and those from steaming and biotransformation [3]. In addition, 112 saponins were reported from raw or processed ginseng, including hydrolysates, semisynthetic, and metabolites [4]. Ginsenosides are known to possess a lot of biological activities including regulatory effects on immunomodulation, protection functions in the central nervous and cardiovascular systems, anti-diabetic, anti-aging, anti-carcinogenic, anti-fatigue, anti-pyretic, anti-stress, boosting physical vitality, and promotion of DNA, RNA, and protein synthesis activities [5,6,7,8,9]. In addition, the biosynthesis of triterpenoid is an important factor of saponin diversity. Consequently, biosynthetic mechanisms for the backbone synthesis [4] and structural diversification and genes/enzymes involved in the biosynthesis [10] were reviewed in the cited references. Therefore, ginsenosides are recognized as the main bioactive components and a key index for quality evaluation of ginseng.

Due to the complexity of the ginsenosides and their structures, multi-platform analytical techniques are used in the detection of ginseng products, such as thin layer chromatography (TLC), high performance thin layer chromatography (HPTLC), gas chromatography (GC), high performance liquid chromatography (HPLC), ultra performance liquid chromatography (UPLC) [3,11,12]. However, these methods detect only small numbers of ginsenosides and lack in provision of structural information. Liquid chromatography coupled with tandem mass spectrometry can provide structural information with high sensitivity, specificity, and versatility in characterizing complex natural product samples. It has been successfully used as a powerful tool for ginsenoside analysis with high throughput [1]. In recent years, a number of novel ginsenosides have been detected in aerial parts of the ginseng plant using the HPLC-MS/MS method, such as stems, leaves, rhizomes, flowers, and flower buds, which enlarged the number of ginsenoside family members [13,14,15]. Several reviews have summarized the progress from a viewpoint of structural features, and conclude that ginsenosides are generally classified into four groups: protopanaxadiol type (PPD), protopanaxatriol type (PPT), C17 side-chain varied type (C17SCV), and oleanolic acid type (OA) [2,16,17,18]. However, spatial distribution of ginsenoside in different parts of P. ginseng is not yet summarized. This information will make better use of the whole ginseng plant and provide clues for studying the biological function of saponins. This review updates the ginsenoside list (from P. ginseng) to 170 by the end of 2019, and aims to highlight the diversity of ginsenosides in multiple dimensions, including chemical structure, tissue spatial distribution, time, and isomeride.

2. History of Saponins Isolated from P. ginseng

The history of ginsenoside isolation can be divided into three periods (before 1980 for Period I, 1980–2000 for Period II, after 2000 for Period III) based on the development of analytical techniques. The study on ginsenoside started in 1854. A ginsenoside-containing constituent was firstly isolated from American ginseng by American scholar Garriques [19], and subsequently, Japanese chemists reported panaquilon, panacon, panaxasapogenol, and ginsenin preliminarily separated from P. ginseng. For almost 100 years since the middle of the nineteenth century, it was difficult to obtain a pure ginsenoside due to the under development of separation techniques. In the early 1950s, with the development of separation technology and the invention of modern analytical instruments, such as GC, TLC, etc., the studies on the chemical ingredient of ginseng made remarkable progress. In 1963, for the first time, Shibata et al. reported the chemical property and structure of the panaxadiol separated from ginseng root [20]. In the 1970s, 17 ginsenosides were detected in ginseng, named as ginsenoside Ro, Ra, Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg2, Rg3, F1, F2, F3, Rb3, Rh, and 20-glucoginsenoside-Rf [21,22,23,24,25,26]. The second period began when the 13C NMR technique was introduced into the structure analysis of ginsenosides. By comparison of the measured 13C NMR spectroscopic data with known compounds, the accurate structure of new ginsenosides (G-Rh1, Rh2, Rh3, Rg4, Ra1, Ra2, Ra3, La, Rf2, Rs3, Ia, Ib, etc.) could be resolved from different parts of ginseng (root, steamed root, flower bud, stem, and leaf). In this period, more and more scientists focused on ginsenoside isolation, and most of ginsenosides were found in the aerial parts of ginseng [27,28,29,30,31,32,33,34,35,36]. The third period was defined by high-efficiency separation methods, as methods such as high-speed counter current chromatography (HSCCC), high performance centrifugal partition chromatography (HPCPC), and 2D NMR spectroscopic techniques were used for separating and identifying ginsenosides. The application of these powerful new techniques helps to identify the complex chemical structure, for instance, C17 side-chain variation and malonyl group. More than 50 new ginsenosides were isolated from 2000 to 2019, among which most of those possessed variations in the C17 side-chain, besides a part of malonyl ginsenosides [37,38,39,40,41].

3. Classification of Saponins Identified from P. ginseng

Although most ginsenosides have a rigid four-trans-ring steroid skeleton, they produce multiple pharmacological and biological effects that are different from one another due to minor variations on: (1) Type of sapogenins; (2) number, type, and site of glycosyl units; and (3) modification of C17 side-chains [11,42,43]. Therefore, the study of ginsenoside structure will help to elucidate the mechanism of multiple functions of ginsenosides. The reported ginsenosides are classified into protopanaxadiol type (PPD), protopanaxatriol type (PPT), oleanolic acid type (OA), and C17 side-chain varied (C17SCV) subtypes according to their determined sapogenin structures (Figure 1). The glycosyl components of saponin were mainly β-d-glucopyranosyl group, followed by α-l-rhamnopyranosyl group, a few binding α-l-arabinopyranosyl group and β-d-xylopyranosyl group, and the β-d-glucopyranosiduronyl group only appears in saponins with oleanolic acid-type (OA) sapogenin. In dammarane-type triterpenoid saponins, β-d-glucopyranosyl group (2→1)-β-d-glucopyranosyl oligosaccharide chains occur more frequently, and are mostly bound to C-3 of sapogenin to generate oxyglycoside; β-d-glucopyranosyl group (2→1)→α-l-rhamnopyranosyl group oligosaccharide chains are mostly bound to C-6 of sapogenin to form oxyglycoside. The tetracyclic parent nucleuses are relatively stable, whether they are PPT and/or PPD type. Moreover, the substituents that occur in the C17 side-chains often undergo oxidation, reduction, cyclization, and epimerization, contributing to diversity in chemical structure [12,16]. Table 1 displays the molecular formulas, molecular masses, and structural categories of 170 ginsenosides, isolated from different parts of P. ginseng. As a result, four ginsenosides are OA type, 59 ginsenosides are PPD type, 42 ginsenosides are PPT type, and 65 ginsenosides are C17CSV type. Among them, four PPD-type ginsenosides (Rb1, Rb2, Rc, Rd), three PPT-type ginsenosides (Re, Rf, Rg1), and one OA-type ginsenoside Ro (the structures are shown in Figure 2) are the most abundant in P. ginseng, and account for more than 70% of the total saponins [5].

Figure 1

Structures of PPD, PPT, OA, and C17SCV sapogenins. The typical glycosylation sites for these sapogenins are marked in blue frame. (a) 20(S)-PPD: Protopanaxadiol type; (b) 20(R)-PPD: Protopanaxadiol type; (c) 20(S)-PPT: Protopanaxatriol type; (d) 20(R)-PPT: Protopanaxatriol type; (e) OA: Oleanolic acid type; (f) C17SCV: C17 side-chain variation type. R1 in C17SCV: -H, -OH, -OR. R2 in C17SCV: The variations in the C17 side-chain mainly comprise H2O-addition, hydroxylation, methoxylation, peroxidization, dehydration at C-20, carbonylation, dehydrogenation, cyclization, oxidation (at the double bond), and degradation. The stereochemistry of chiral centers are shown in (a) and (b).

Table 1

The 170 ginsenosides isolated from P. ginseng.

No.	Subtype	Saponins	Formula	Molecular Mass	Plant Part	Refs
1	OA 1	Polyacetylene ginsenoside Ro	C65H100O21	1216.6757	Root	[44]
2	OA	Ginsenoside Ro methyl ester	C49H78O19	970.5137	Root(steamed)	[45]
3	OA	Calenduloside B	C48H78O18	942.5188	Root	[46]
4	OA	Ginsenoside Ro	C49H80O18	956.5345	Root, flower, fruit, leaf	[16,47]
5	PPD	Ginsenoside Ra1	C58H98O26	1210.6346	Root	[48]
6	PPD	Ginsenoside Ra2	C58H98O26	1210.6346	Root	[49]
7	PPD	Ginsenoside Ra3	C59H100O27	1240.6452	Root	[50]
8	PPD	Ginsenoside Rs1	C55H92O23	1120.6029	Root(steamed)	[51]
9	PPD	Ginsenoside Rs2	C55H92O23	1120.6029	Root(steamed)	[51]
10	PPD	Malonyl-ginsenoside Ra3	C62H102O30	1326.6456	Root(fresh)	[52]
11	PPD	Malonyl-notoginsenoside R4	C62H102O30	1326.6456	Root	[52]
12	PPD	Ginsenoside Ra4	C62H102O27	1278.6608	Root	[53]
13	PPD	Ginsenoside Ra5	C60H99O27	1251.6373	Root	[53]
14	PPD	Ginsenoside Ra6	C58H96O24	1176.6292	Root	[53]
15	PPD	Ginsenoside Ra7	C57H93O23	1145.6108	Root	[53]
16	PPD	Ginsenoside Ra8	C57H94O23	1146.6186	Root	[53]
17	PPD	Ginsenoside Ra9	C57H94O23	1146.6186	Root	[53]
18	PPD	20(S)-ginsenoside Rg3	C42H72O13	784.4973	Root(steamed), fruit, leaf	[54]
19	PPD	Ginsenoside Rs3	C44H74O14	826.5079	Root(steamed)	[55]
20	PPD	Ginsenoside IV	C58H96O24	1176.6292	Root	[47]
21	PPD	Ginsenoside V	C54H92O24	1124.5979	Root	[47]
22	PPD	Gypenoside-V	C54H92O22	1092.6080	Root	[46]
23	PPD	20(R)-ginsenoside Rs3	C44H74O14	826.5079	Root(steamed)	[45]
24	PPD	Acetyl-ginsenoside Rd	C50H84O19	988.5607	Root(mountain ginseng)	[56]
25	PPD	Ginsenoside F2	C42H72O13	784.4973	Root, fruit, leaf	[57]
26	PPD	Pseudoginsenoside Rc1	C50H84O19	988.5607	Fruit	[57]
27	PPD	Gypenoside XVII	C48H82O18	946.5501	Fruit, leaf	[57]
28	PPD	Gypenoside IX	C47H80O17	916.5396	Fruit, leaf	[57]
29	PPD	Quinquenoside L10	C47H80O17	916.5396	Fruit	[57]
30	PPD	25-Hydroxyprotopanaxadiol	C30H54O4	478.4022	Fruit	[58]
31	PPD	20(S)-protopanaxadiol	C30H52O3	460.3916	Fruit, leaf	[41,59]
32	PPD	20(R)-protopanaxadiol	C30H52O3	460.3916	Fruit	[59]
33	PPD	Notoginsenoside Fd	C47H80O17	916.5396	Fruit	[60]
34	PPD	Ginsenoside Rd2	C47H80O17	916.5396	Leaf	[61]
35	PPD	20(R)-ginsenoside Rg3	C42H72O13	784.4973	Root(steamed), fruit, leaf	[62,63]
36	PPD	20(S)-ginsenoside Rh2	C36H62O8	622.4445	Root(steamed), fruit, leaf	[64]
37	PPD	20(R)-ginsenoside Rh2	C36H62O8	622.4445	Fruit, leaf	[65]
38	PPD	Notoginsenoside Fe	C47H80O17	916.5396	Fruit, leaf	[61]
39	PPD	Acetyl-ginsenoside Rb1	C56H96O24	1152.6292	Root(mountain ginseng), leaf	[56]
40	PPD	Acetyl-ginsenoside Rc	C55H92O23	1120.6029	Root(mountain ginseng), leaf	[56]
41	PPD	Acetyl-ginsenoside Rb3	C55H92O23	1120.6029	Root(mountain ginseng), leaf	[56]
42	PPD	Ginsenoside compound O	C47H80O17	916.5396	Root, fruit, leaf	[16,66]
43	PPD	Malonyl-ginsenoside Rb2	C56H92O25	1164.5928	Root, flower, fruit, leaf	[16]
44	PPD	Ginsenoside Mc	C41H70O12	754.4867	Leaf	[16,66]
45	PPD	Ginsenoside compound Y	C41H70O12	754.4867	Leaf	[16]
46	PPD	Ginsenoside compound K	C36H62O8	622.4445	Root, fruit, leaf	[16]
47	PPD	Ginsenoside Rb1	C54H92O23	1108.6029	Root, flower, fruit, leaf	[16,67]
48	PPD	Malonyl-ginsenoside Rb1	C57H94O25	1178.6084	Root, flower, fruit, leaf	[16,67]
49	PPD	Ginsenoside Rc	C53H90O22	1078.5924	Root, flower, fruit, leaf	[16,67]
50	PPD	Malonyl-ginsenoside Rc	C56H92O25	1164.5928	Root, flower, fruit, leaf	[16,67]
51	PPD	Ginsenoside Rb2	C53H90O22	1078.5924	Root, flower, fruit, leaf	[16,67]
52	PPD	Ginsenoside Rb3	C53H90O22	1078.5924	Root, flower, fruit, leaf	[16,67]
53	PPD	Malonyl-ginsenoside Rb3	C56H92O25	1164.5928	Root, flower, leaf	[16,67]
54	PPD	Ginsenoside Rd	C48H82O18	946.5501	Root, flower, fruit, leaf	[16,67]
55	PPD	Malonyl-ginsenoside Rd	C51H84O21	1032.5505	Root, flower, fruit, leaf	[16,67]
56	PPD	Malonyl-floralginsenoside Rd2	C51H84O21	1032.5505	Flower	[68]
57	PPD	Malonyl-floralginsenoside Rd3	C51H84O21	1032.5505	Flower	[68]
58	PPD	Malonyl-floralginsenoside Rd4	C51H84O21	1032.5505	Flower	[68]
59	PPD	Malonyl-floralginsenoside Rd5	C51H84O21	1032.5505	Flower	[68]
60	PPD	Malonyl-floralginsenoside Rd6	C54H87O24	1119.5587	Flower	[68]
61	PPD	Malonyl-floralginsenoside Rc2	C56H92O25	1164.5928	Flower	[68]
62	PPD	Malonyl-floralginsenoside Rc3	C56H92O25	1164.5928	Flower	[68]
63	PPD	Malonyl-floralginsenoside Rc4	C56H92O25	1164.5928	Flower	[68]
64	PPT	20(S)-ginsenoside Rg2	C42H72O13	784.4973	Root, fruit, leaf	[54,69]
65	PPT	Koryoginsenoside R1	C46H76O15	868.5184	Root	[36]
66	PPT	Ginsenoside Re6	C46H76O15	868.5184	Root	[70]
67	PPT	Ginsenoside Re2	C48H82O19	962.5450	Root	[70]
68	PPT	Ginsenoside Re3	C48H82O19	962.5450	Root	[70]
69	PPT	Ginsenoside Re4	C47H80O18	932.5345	Root	[70]
70	PPT	Notoginsenoside Rt	C44H74O15	842.5028	Root	[46]
71	PPT	Majoroside F6	C48H82O19	962.5450	Root	[46]
72	PPT	Pseudoginsenoside Rt3	C42H70O13	782.4816	Root	[46]
73	PPT	Vinaginsenoside R15	C42H72O15	816.4871	Root	[46]
74	PPT	20(R)-ginsenoside Rf	C42H72O14	800.4922	Root	[45]
75	PPT	20(R)-notoginsenoside R2	C41H70O13	770.4816	Root	[45]
76	PPT	Ginsenoside Ia	C42H72O14	800.4922	Fruit	[71]
77	PPT	Chikusetsusaponin LM1	C41H70O13	770.4816	Fruit	[57]
78	PPT	25-Hydroxyprotopanaxatriol	C30H54O5	494.3971	Fruit	[58]
79	PPT	20(S)-protopanaxatriol	C30H52O4	476.3866	Fruit, leaf	[59]
80	PPT	20(R)-protopanaxatriol	C30H52O4	476.3866	Fruit	[59]
81	PPT	Notoginsenoside R3	C48H82O19	962.5450	Fruit	[60]
82	PPT	20-glucoginsenoside Rf	C48H82O19	962.5450	Root, flower, leaf	[16]
83	PPT	Saponin IIb	C36H62O9	638.4394	Leaf	[72]
84	PPT	Saponin IIIc	C37H62O10	666.4343	Leaf	[72]
85	PPT	20(S)-ginsenoside Rh1	C36H62O9	638.4394	Leaf	[62]
86	PPT	20(R)-ginsenoside Rh1	C36H62O9	638.4394	Root(steamed), leaf	[21]
87	PPT	Acetyl-ginsenoside Rg1	C44H74O15	842.5028	Root(mountain ginseng), leaf	[56]
88	PPT	Acetyl-ginsenoside Re	C50H84O19	988.5607	Root(mountain ginseng), leaf	[56]
89	PPT	Notoginsenoside R2	C41H70O13	770.4816	Root, fruit, leaf	[16]
90	PPT	Notoginsenoside R1	C47H80O18	932.5345	Root, flower, fruit, leaf	[16,67]
91	PPT	Ginsenoside Rg1	C42H72O14	800.4922	Root, flower, fruit, leaf	[16,67]
92	PPT	Ginsenoside Re	C48H82O18	946.5501	Root, flower, fruit, leaf	[16,67]
93	PPT	Malonyl-ginsenoside Rg1	C45H74O17	886.4926	Root, flower, leaf	[16,67]
94	PPT	Malonyl-ginsenoside Re	C51H84O21	1032.5505	Root, flower, fruit, leaf	[16,67]
95	PPT	Ginsenoside Rf	C42H72O14	800.4922	Root, flower, fruit, leaf	[16,67]
96	PPT	20(R)-ginsenoside Rg2	C42H72O13	784.4973	Root(steamed), flower, fruit, leaf	[16,67]
97	PPT	Ginsenoside Rf3	C41H70O13	770.4816	Flower	[67]
98	PPT	Floralginsenoside M	C53H90O22	1078.5924	Flower, leaf	[73]
99	PPT	Floralginsenoside N	C53H90O22	1078.5924	Flower, leaf	[73]
100	PPT	Floralginsenoside P	C53H90O23	1094.5873	Flower	[73]
101	PPT	Ginsenoside F1	C36H62O9	638.4394	Flower, fruit, leaf	[74]
102	PPT	Ginsenoside F3	C41H70O13	770.4816	Flower, fruit, leaf	[74]
103	PPT	Ginsenoside F5	C41H70O13	770.4816	Flower, fruit, leaf	[74]
104	PPT	Malonyl-floralginsenoside Re2	C51H84O21	1032.5505	Flower	[68]
105	PPT	Malonyl-floralginsenoside Re3	C51H84O21	1032.5505	Flower	[68]
106	C17SCV	Koryoginsenoside R2	C54H92O24	1124.5979	Root	[36]
107	C17SCV	Ginsenoside Re5	C42H72O15	816.4871	Root	[70]
108	C17SCV	Ginsenoside Rs4	C44H72O13	808.4973	Root(sun cured)	[75]
109	C17SCV	Dehydroprotopanaxadiol I	C30H50O2	442.3811	Root(steamed)	[2]
110	C17SCV	Ginsenoside Rg5	C42H70O12	766.4867	Root(steamed)	[76]
111	C17SCV	Dehydroprotopanaxatriol I	C30H50O3	458.3760	Root(steamed)	[2]
112	C17SCV	Ginsenoside Rs6	C38H62O9	662.4394	Root(sun cured)	[75]
113	C17SCV	Ginsenoside Rz1	C42H70O12	766.4867	Root(steamed)	[77]
114	C17SCV	Dehydroprotopanaxadiol II	C30H50O2	442.3811	Root(steamed)	[2]
115	C17SCV	Ginsenoside Rs5	C44H72O13	808.4973	Root(sun cured)	[75]
116	C17SCV	Dehydroprotopanaxatriol II	C30H50O3	458.3760	Root(steamed)	[2]
117	C17SCV	Ginsenoside Rg6	C42H70O12	766.4867	Root(steamed)	[78]
118	C17SCV	Ginsenoside Rk3	C36H60O8	620.4288	Root(steamed)	[76]
119	C17SCV	Ginsenoside Rs7	C38H62O9	662.4394	Root(sun cured)	[75]
120	C17SCV	Ginsenoside Rg9	C42H70O13	782.4816	Root(steamed)	[79]
121	C17SCV	12-O-glucoginsenoside Rh4	C42H70O13	782.4816	Root(steamed)	[80]
122	C17SCV	Ginsenoside Rg10	C42H69O13	781.4738	Root(steamed)	[79]
123	C17SCV	Ginsenoside Rh10	C36H62O8	622.4445	Root(steamed)	[80]
124	C17SCV	Ginsenoside Rg11	C42H70O14	798.4766	Root(steamed)	[80]
125	C17SCV	Vinaginsenoside R8	C48H82O19	962.5450	Fruit	[57]
126	C17SCV	Ginsenoside Rh4	C36H60O8	620.4288	Root(steamed), fruit	[4,57]
127	C17SCV	Ginsenoside Rh5	C36H60O9	636.4237	Root(steamed), fruit	[4,57]
128	C17SCV	Isoginsenoside-Rh3	C36H60O7	604.4339	Fruit	[81]
129	C17SCV	Ginsenoside Rf2	C42H72O14	800.4922	Fruit	[82]
130	C17SCV	Ginsenoside Rk2	C36H60O7	604.4339	Root(steamed), fruit	[76,83]
131	C17SCV	Pseudoginsenoside RT5	C36H62O10	654.4343	Fruit	[83]
132	C17SCV	Ginsenoside Rh3	C36H60O7	604.4339	Root(steamed), fruit	[76,83]
133	C17SCV	Ginsenoside Rg4	C42H70O12	766.4867	Root, fruit	[16]
134	C17SCV	Ginsenoside F4	C42H70O12	766.4867	Root, fruit, leaf	[16]
135	C17SCV	Ginsenoside Rg7	C36H60O9	636.4237	Leaf	[39]
136	C17SCV	Ginsenoside Rh6	C36H62O11	670.4292	Fruit, leaf	[39]
137	C17SCV	Ginsenoside Ki	C36H62O10	654.4343	Leaf	[39]
138	C17SCV	Ginsenoside Km	C36H62O10	654.4343	Leaf	[84]
139	C17SCV	Ginsenoside Rh9	C36H60O9	636.4237	Leaf	[39]
140	C17SCV	12,23-Epoxyginsenoside Rg1	C42H70O14	798.4766	Leaf	[85]
141	C17SCV	Ginsenoside Rh7	C36H60O9	636.4237	Leaf	[39]
142	C17SCV	Ginsenoside Rh8	C36H60O9	636.4237	Leaf	[39]
143	C17SCV	Hexanordammaran	C24H40O4	392.2927	Leaf	[86]
144	C17SCV	Floralginsenoside A	C42H72O16	832.4820	Flower	[87]
145	C17SCV	Ginsenoside La	C42H70O13	782.4816	Leaf	[35]
146	C17SCV	Vinaginsenoside R4	C48H82O19	962.5450	Root, fruit, leaf	[16]
147	C17SCV	Ginsenoside Rk1	C42H70O12	766.4867	Root(steamed), fruit, leaf	[16]
148	C17SCV	Floralginsenoside H	C50H84O21	1020.5505	Flower	[88]
149	C17SCV	Floralginsenoside Tc	C53H90O24	1110.5822	Flower	[89]
150	C17SCV	Floralginsenoside Td	C53H90O24	1110.5822	Flower	[84]
151	C17SCV	Ginsenoside I	C48H82O20	978.5400	Flower	[90]
152	C17SCV	Ginsenoside II	C48H82O20	978.5400	Flower	[90]
153	C17SCV	Floralginsenoside C	C41H70O15	802.4715	Flower	[74]
154	C17SCV	Floralginsenoside J	C48H82O20	978.5400	Flower	[88]
155	C17SCV	Floralginsenoside Ka	C36H62O11	670.4292	Flower	[91]
156	C17SCV	Floralginsenoside La	C48H82O19	962.5450	Flower	[88]
157	C17SCV	Floralginsenoside Lb	C48H82O19	962.5450	Flower	[88]
158	C17SCV	Floralginsenoside Ta	C36H60O10	652.4187	Flower	[89]
159	C17SCV	Floralginsenoside E	C42H72O15	816.4871	Flower	[74]
160	C17SCV	Floralginsenoside F	C42H72O15	816.4871	Flower	[74]
161	C17SCV	Floralginsenoside G	C50H84O21	1020.5505	Flower	[88]
162	C17SCV	Floralginsenoside K	C48H82O21	994.5349	Flower	[88]
163	C17SCV	Floralginsenoside O	C53H90O22	1078.5924	Flower	[73]
164	C17SCV	Floralginsenoside B	C42H72O16	832.4820	Flower	[74]
165	C17SCV	Floralginsenoside D	C41H70O15	802.4715	Flower	[74]
166	C17SCV	Floralginsenoside I	C48H82O20	978.5400	Flower	[88]
167	C17SCV	Floralginsenoside Kb	C45H76O19	920.4981	Flower	[91]
168	C17SCV	Floralginsenoside Kc	C45H76O20	936.4930	Flower	[91]
169	C17SCV	Floralginsenoside Tb	C35H62O11	658.4292	Flower	[89]
170	C17SCV	Ginsenoside III	C48H80O19	960.5294	Flower	[92]

1 OA: Oleanolic acid; PPD: Protopanaxadiol; PPT: Protopanaxatriol; C17SCV: C17 side-chain varied.

Figure 2

Structures of eight high-abundance saponins in P. ginseng. (a) PPD-type ginsenoside Rb1; (b) PPD-type ginsenoside Rb2; (c) PPD-type ginsenoside Rc; (d) PPD-type ginsenoside Rd; (e) PPT-type ginsenoside Re; (f) PPT-type ginsenoside Rf; (g) PPT-type ginsenoside Rg1; (h) OA-type ginsenoside Ro.

4. Spatial Distribution of Ginsenosides in Different Parts

The Venn diagram (Figure 3) shows the number of ginsenosides commonly and separately shared by the following four groups: R&S (roots, rhizomes, and steamed roots), L&S (leaves and stems), F&P (fruits and fruit pedicels), and F&B (flowers and flower buds). Among them, the number of unique ginsenosides in group R&S, F&P, L&S, and F&B are 52, 15, 14, and 36, respectively, accounting for 30.6%, 8.8%, 8.2%, and 21.2% of the number of total ginsenosides, respectively. The result gives some explanation why ginseng root is designated as medicinal parts rather than the other parts. Sixteen ginsenosides are commonly existed in all tissues, and among them, there are nine PPD type (Rc, Rd, Rb2, Rb1, Rb3, m-ginsenoside Rb1, m-ginsenoside Rc, m-ginsenoside Rb2, m-ginsenoside Rd), six PPT type (Re, Rg1, Rf, 20(R)-ginsenoside Rg2, Notoginsenoside R1, m-ginsenoside Re), one OA type (Ro), and none of C17SCV type. Numbers of ginsenosides shared by R&S and F&P, F&P and L&S, L&S and F&B, R&S and F&B were 32 (18.8%), 37 (21.7%), 24 (14.1%), and 19(11.2%), respectively. In addition, 13 malonyl-ginsenosides were existing specifically in flowers and buds; however, none of them was observed in fruit. This implies that these malonyl-ginsenosides show not only spatial specificity, but also temporal specificity. Here in, we speculate that malonyl-ginsenosides may play a physiological role during tissue development.

Figure 3

Venn diagram of ginsenosides according to different parts of P. ginseng. R&S: Roots, rhizomes, and steamed roots; L&S: Leaves and stems; F&P: Fruits and fruit pedicels; F&B: Flowers and flower buds.

As indicated by Figure 4, the numbers of PPD-type ginsenosides (blue bar) are highest in R&S, F&P, and L&S, while the C17SCV-type ginsenoside is highest in F&B. Interestingly, C17SCV-type ginsenosides exhibit significant variation among different groups. Only nine C17SCV-type ginsenosides are shared by more than two groups, whereas the other 58 C17SCV-type ginsenosides are unique to a particular group. For the OA-type ginsenoside, three are specific to group R&S (Polyacetyleneginsenoside-Ro, Ginsenoside Ro methyl ester, Calenduloside-B) and one (Ginsenoside Ro) is commonly shared by all parts.

Figure 4

Structural categories of ginsenosides in different parts of P. ginseng. R&S: Roots, rhizomes, and steamed roots; F&P: Fruits and fruit pedicels; L&S: Leaves and stems; F&B: Flowers and flower buds; OA: Oleanolic acid; PPD: Protopanaxadiol; PPT: Protopanaxatriol; C17SCV: C17 side-chain varied.

5. Isomers of Ginsenosides

The total 170 ginsenosides are divided into 69 molecular formula groups. Therefore, it is common that one molecular formula corresponds to several ginsenosides. (Table 2). The molecular formula with the largest number of isomers is C48H82O19 (molecular weight 962.5450), with a total of nine isomers; followed by C51H84O21 (molecular weight 1032.5505) with a total of eight isomers, and C41H70O13 (molecular weight 770.4816) with a total of seven isomers. The isomers median of 69 molecular formulas is 2, which means that one molecular formula corresponds to two isomers equally. Optical and position isomerism are the dominant types of ginsenoside isomers, whilst cis-trans isomerism and tautomerism are detected occasionally.

Table 2

Isomers of 170 ginseng saponins.

No.	Formula	Molecular Mass	No. of Isomers	No.	Formula	Molecular Mass	No. of Isomers
1	C24H40O4	392.2927	1	36	C46H76O15	868.5184	2
2	C30H50O2	442.3811	2	37	C47H80O17	916.5396	6
3	C30H50O3	458.3760	2	38	C47H80O18	932.5345	2
4	C30H52O3	460.3916	2	39	C48H78O18	942.5188	1
5	C30H52O4	476.3866	2	40	C48H80O19	960.5294	1
6	C30H54O4	478.4022	1	41	C48H82O18	946.5501	3
7	C30H54O5	494.3971	1	42	C48H82O19	962.5450	9
8	C35H62O11	658.4292	1	43	C48H82O20	978.5400	4
9	C36H60O10	652.4187	1	44	C48H82O21	994.5349	1
10	C36H60O7	604.4339	3	45	C49H78O19	970.5137	1
11	C36H60O8	620.4288	2	46	C49H80O18	956.5345	1
12	C36H60O9	636.4237	5	47	C50H84O19	988.5607	3
13	C36H62O10	654.4343	3	48	C50H84O21	1020.5505	2
14	C36H62O11	670.4292	2	49	C51H84O21	1032.5505	8
15	C36H62O8	622.4445	4	50	C53H90O22	1078.5924	6
16	C36H62O9	638.4394	4	51	C53H90O23	1094.5873	1
17	C37H62O10	666.4343	1	52	C53H90O24	1110.5822	2
18	C38H62O9	662.4394	2	53	C54H87O24	1119.5587	1
19	C41H70O12	754.4867	2	54	C54H92O22	1092.6080	1
20	C41H70O13	770.4816	7	55	C54H92O23	1108.6029	1
21	C41H70O15	802.4715	2	56	C54H92O24	1124.5979	2
22	C42H69O13	781.4738	1	57	C55H92O23	1120.6029	4
23	C42H70O12	766.4867	6	58	C56H92O25	1164.5928	6
24	C42H70O13	782.4816	4	59	C56H96O24	1152.6292	1
25	C42H70O14	798.4766	2	60	C57H93O23	1145.6108	1
26	C42H72O13	784.4973	5	61	C57H94O23	1146.6186	2
27	C42H72O14	800.4922	5	62	C57H94O25	1178.6084	1
28	C42H72O15	816.4871	4	63	C58H96O24	1176.6292	2
29	C42H72O16	832.4820	2	64	C58H98O26	1210.6346	2
30	C44H72O13	808.4973	2	65	C59H100O27	1240.6452	1
31	C44H74O14	826.5079	2	66	C60H99O27	1251.6373	1
32	C44H74O15	842.5028	2	67	C62H102O27	1278.6608	1
33	C45H74O17	886.4926	1	68	C62H102O30	1326.6456	2
34	C45H76O19	920.4981	1	69	C65H100O21	1216.6757	1
35	C45H76O20	936.4930	1

6. Mass Spectrometry-Based Metabolomics Analysis on P. ginseng

Recently, MS and its hyphenations with chromatographic separation techniques have emerged as an instrumental trend in ginsenoside analysis [93,94]. HPLC/MS can overcome the problems related to ginsenoside pre-analysis derivatization and the low abundance of molecular ions [95,96]. The use of on-line MS detection shows superior sensitivity and specificity compared with conventional UV and ELSD detection [97,98]. The sensitivity of MS detection can surpass 1000 times that of UV absorbance [99]. In addition, the possible matrix effects encountered with many Panax ginseng formulations may be compromised by MS [100]. Despite these advantages, MS remains costly for use in routine analysis. With the development of soft ionization techniques, HPLC/MS has been successfully applied for the qualitative and quantitative analyses of Panax ginseng [101]. Among the various mass spectrometry ionization techniques, electrospray mass spectrometry (ESI-MS) is the approach that is most commonly coupled with HPLC [15,102,103]. While ESI-MS suffers from matrix-induced ionization suppression difficulties [104], atmospheric pressure chemical ionization (APCI) can offer itself as one possible alternative [105]. Quadrupole time-of-flight mass spectrometry (QTOF-MS), a powerful tool for the identification of analytes, provides several advantages in structural analysis, such as a higher resolution and accuracy in mass measurements. Coupled with QTOF-MS, UPLC has been introduced for metabolite profiling and metabolomics purposes [99]. In recent years, orbitrap technology has achieved great breakthrough in resolution and scanning speed and realized the high-resolution detection of multi-stage mass spectrometry by combining the linear ion trap and quadrupole mass spectrometry, which can be widely applied in the development of new drugs [106].

According to the available literature, Wang et al. in 1999 [97] firstly identified ginsenosides by LC/MS/MS and differentiated P. ginseng and P. quinquefolius based on the ginsenoside Rg1/Rf and Rc/Rb2 ratios. A liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was developed to distinguish Asian ginseng and North American ginseng. The method is based on the baseline chromatographic separation of two potential chemical markers: Rf and 24(R)-pseudo ginsenoside F11 [107]. Z X. et al. 2000 developed a similar LC/MS/MS method to determine ginsenoside in ginseng. Nine ginsenosides were determined, among which five of them were identified according to molecular weight [108]. In the late 1990s and early 2000s, the resolution of mass spectrometry was low and the number of identified ginsenosides was limited, which could be used for distinguishing Asian ginseng and American Ginseng, and identifying ginsenosides.

Chen et al. [109] established a chemical finger-print metabolomics approach using ultra-high-performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS). The method was successfully used to authenticate and evaluate Panax Ginseng of various commercial grades. Using UPLC-QTOF-MS/MS, Zhang et al. evaluated the overall quality of commercially available white ginseng and red ginseng, and investigated their characteristic chemical composition indicators. Fifty-one major chromatographic peaks of white ginseng and red ginseng samples were separated within 24 min [110]. By means of UPLC-DAD-QTOF-MS/MS, Wang et al. conducted qualitative and quantitative analysis of ginsenosides of cultivated ginseng and mountain ginseng. A total of 131 ginsenosides were detected in cultivated ginseng and mountain ginseng, and all the components were completely separated within 10 min, among which contents of 19 typical ginsenoside were accurately quantified. This method has been validated for quality evaluation of ginseng and identification of cultivated ginseng and mountain ginseng [13]. Zhang et al. Quickly and comprehensively identified the ginsenosides using high-resolution time-of-flight mass spectrometry, electrospray dual-spray ion source, and negative ion mode. A total of 95 saponins in suncured ginseng were identified within 11 min, providing a feasible basis for the quality control of suncured ginseng [111]. With the emergence of high-resolution mass spectrometry and the development of high-throughput screening technologies, several time-saving methods were established for commercial ginseng product evaluation.

Since 2015, Orbitrap mass spectrometer had been applied in ginsenoside detection. In 2017, a total of 101 malonyl-ginsenosides were firstly systematic analyzed by hybrid LTQ-Orbitrap mass spectrometer after UHPLC separation, and ten potential malonyl-ginsenoside markers were discovered for the discrimination of P. ginseng, P. quinquefolius, and P. notoginseng [112]. Shi et al. established an untargeted profiling strategy on a linear ion-trap/Orbitrap mass spectrometer coupled to ultra-high performance liquid chromatography to analyze malonyl-ginsenosides in several Panax species. Finally, 178 malonyl-ginsenosides were characterized from roots, leaves, and flower buds of P. ginseng, P. quinquefolius, and P. notoginseng [113]. To investigate the variation of ginsenosides among different processed red ginseng, Zhong et al. tested steamed, vinegared and dried red ginseng samples by UPLC-Q-Orbitrap MS. In total, 32 ginsenosides were identified and ginsenosides m-Rb1, Rh1, F1, 20(R)-Rh1, Rg5, and Rs5 were only found in red ginseng processed by vinegar [114]. With the development of Orbitrap and multi-mass spectrometry techniques, ginsenosides with complex structures, such as malonyl and C17 side-chain variation, have been increasingly detected, and the types of ginsenosides have been greatly extended.

7. Conclusions

In this review, we summarized the existing studies related to saponin analysis of P. ginseng, and sorted out the information of structural characteristic, spatial distribution, and isomer of 170 ginsenosides. There are 16 common ginsenosides present in all parts of P. ginseng. In contrast, each part has unique ginsenosides, and ginsenosides in different parts show obvious structural diversity. It should be emphasized that ginseng aerial parts can regenerate every year, and there is a large amount of rare ginsenosides in stems, leaves, and flower buds. In light of previous research results of the rare ginsenoside bioactivity in red ginseng, it seems that the aerial parts of P. ginseng are highly worth developing and utilizing. A conclusion can also be drawn that C17SCV-type ginsenosides and malonyl-ginsenoside are rich in flowers and buds. Therefore, a hypothesis that ginsenosides have physiological roles in ginseng plant development is proposed. The rapid development of high-performance liquid chromatography and mass spectrometry techniques significantly raise the throughput and accuracy of ginsenoside determination.

In the future, (1) with the continuous advancement of detection and identification technology, the analysis method of ginsenosides will develop in the direction of being more sensitive, convenient, and environmentally-friendly, with high-throughput and high-precision. By leveraging these technologies, more monomer compounds will be separated and identified from ginseng, which will develop the knowledge of the diversity of chemical structure of ginsenosides. (2) It is necessary to conduct further research on spatial distribution of ginsenosides in different parts of ginseng, and multidisciplinary collaborations among genomics, proteomics, metabonomics, and transcriptomics could be used to study the physiological functions of ginsenosides. (3) With increasing separation of ginsenosides possessing a complex structure, such as malonyl and C17 side-chain variation, the pharmacological action and pharmacokinetics of these ginsenosides would be further studied to clarify the efficacy of ginseng.