What Is Ginsenoside Rh2 and Its Derivative?

Ginseng (Panax ginseng C. A. Meyer, Araliaceae) is a traditional precious medicinal herb in China. It has the effects of replenishing vital energy, tonifying the spleen and benefiting the lungs, generating body fluid, calming the mind and improving intelligence. The main active ingredient in ginseng is ginsenoside, which can be divided into protopanaxadiol (PPD), protopanaxatriol (PPT) and oleanane (OA) types according to the aglycone. The ratios of PPD/PPT in the ginseng head, ginseng skin, ginseng leaves, ginseng root, and ginseng beard are 2.5, 1.9, 0.9, 1.2, and 3.8, respectively [1].

The content of ginsenosides of the PPD type is higher than that of the PPT type. For example, ginsenosides Rb1, Rb2, Rc, and Rd are the main ingredients in white ginseng, while ginsenoside Rh2 is a unique ingredient in red ginseng and is almost absent in white ginseng. In 1983, Japanese scholar Isao Kitagawa isolated ginsenoside Rh2 from red ginseng, with a yield of only 0.001%. Nowadays, ginsenoside Rh2 is produced in kilogram quantities. “Jinxing Capsules”, produced by Zhejiang Yaxing Co., Ltd., is already on the market as a health product. Ginsenoside Rh2 has a wide range of pharmacological activities, such as anti-tumor, immune enhancement, anti-allergy, anti-inflammatory, hypoxia tolerance and obesity inhibition. This article reviews the relevant research on ginsenoside Rh2 at home and abroad.

Ⅰ. Structure of ginsenoside Rh2 and its derivatives

The structure of ginsenoside Rh2 and its derivatives is shown in Figure 1 and Table 1.

Ⅱ. Preparation methods for ginsenoside Rh2 and its derivatives

The industrial preparation of ginsenoside Rh2 has always been the focus of research by scholars at home and abroad, mainly focusing on the use of chemical and biotransformation methods to achieve the preparation of ginsenoside Rh2. The possible routes for the preparation of ginsenoside Rh2 are shown in Figure 2.

1. Ginsenoside Rh2 preparation methods

(1) Acid hydrolysis method.

The acid hydrolysis method is simple to operate and not affected by external environmental factors. However, the reaction products are complex and a large amount of waste acid is produced. The natural ginsenoside diol type saponin group C20 position configuration is mainly S configuration. When using acid hydrolysis to hydrolyze diol type saponin to prepare ginsenoside Rh2, the sugar group at the C20 position is first removed, and then a configuration change at the C20 position occurs, generating a mixture of two isomers, with the R configuration being the main one. Ginsenoside Rh2 is converted from ginsenoside Rg3 by acid degradation. The optimal degradation conditions are: 60% acetic acid, 55 °C for 1 h. The total content of ginsenoside Rg3 and Rh2 in the degradation product is 106.7 mg·g-1, and the yield is 71% [8]. The main products were ginsenoside Rg3 and 20(R) -Rh2 [2].

Yu Zhibo et al. [3] hydrolyzed American ginseng stem and leaf diol-type saponins and determined that the optimal conditions for preparing 20(R)-Rh2 were 80°C, 50% H2SO4 (5% by volume of ethanol), and degradation for 4 h. Zhang Lanlan et al. [9] applied for a patent for a ginseng saponin Rh2 extract in 2009, and the preparation method is as follows: step 1, the medicinal materials containing ginseng saponin components are extracted with water, the extract is passed through a macroporous adsorption resin column, eluted with ethanol, the eluate is collected, concentrated to dryness, to obtain the total saponin; step 2, dissolve the total saponin obtained in step 1 in an acid solution and react; after the reaction is complete, adjust the pH to neutral and collect the precipitate; step 3, perform reverse-phase silica gel column chromatography on the precipitate, elute with acetonitrile-water mixture, collect the fraction rich in ginsenoside Rh2, and concentrated to obtain the product.

(2) Alkali hydrolysis method.

Alkali hydrolysis is simple to operate, and the product is relatively simple, but the hydrolysis conditions are harsh, the reaction equipment requirements are high, and a large amount of waste alkali is easily produced. When using the alkali hydrolysis method to prepare ginsenoside Rh2, the sugar group at the C20 position is first removed, and there is no conformational change at the C20 position. The alkali hydrolysis method can be used to prepare 20(S)-Rh2. The main products are 20(S)-Rh2 and PPD [2]. 20(S)-protoginseng diol-type saponin 8.0 g was dissolved in 30 mL of water, and 20 mL of saturated NaOH aqueous solution was added. The mixture was refluxed in a boiling water bath for 6 h, cooled, transferred to a separating funnel, and extracted with n-butanol four times. The n-butanol layer was concentrated, calculated that the conversion rate of 20(S) - Rh2 was 9.64% [10]. Li Xuwen [11] determined that the degradation conditions for the preparation of 20 (S) - Rh2 were: a mass ratio of NaOH to ginseng leaf total saponin of 1.6:1 (w/w), glycerol to ginseng leaf total saponin mass ratio of 15.0:1 (v/w), and 220 ℃ for 40 min, the conversion rate of 55.64%.

(3) microbial transformation method.

The microbial transformation method is dominant in the industrial preparation of ginsenoside Rh2 due to its many advantages, such as low cost and high conversion rate. To prepare ginsenoside Rh2 using the microbial transformation method, ginsenoside diol-type saponins are generally first converted into ginsenoside F2 or ginsenoside Rg3, and then into ginsenoside Rh2. Myrothecium verru- caria, isolated from the soil of ginseng in Changbai Mountain, can convert ginsenoside Rg3 to ginsenoside Rh2 and the diol-type saponin PPD[12]. Fusarium proliferatum ECU2042, isolated from soil, can convert ginsenoside Rg3 to ginsenoside Rh2 under the conditions of 50 °C and 50 mL NaAC/HAC (pH 5.0) for 24 h, with a conversion rate of up to 60% [13]. Zang Yunxia et al. [14] first hydrolyzed the ginseng extract with 1 mol·L-1 HCl, and then used the ginseng extract hydrolyzed by extended Aspergillus fermentation acid, resulting in the conversion of some ginsenosides to ginsenoside Rh2.

Tong Xin et al. [15] took activated Lactobacillus delbrueckii subsp. bulgaricus and inoculated it into MRS medium, added ginsenosides, and fermented at 37°C to 39°C for 240 h to 248 h. The fermentation broth was collected and reacted with saponin glycosidase at 88℃ ~ 92℃ for 240 ~360 h. The reaction solution was collected, filtered, and the filtrate was eluted with an ethanol gradient through a macroporous adsorption resin. The flow fraction was collected to obtain ginsenoside Rh2. This patented preparation has a high conversion rate and can be used for the large-scale preparation of ginsenoside Rh2. Lv Guozhong et al. [16] applied for a patent in 2011 for the use of the fungus Cylindrocarpon didymium and its use in the preparation of ginsenoside Rh2—a ginseng pathogenic fungus Cylindrocarpon didymium, which has the ability to convert ginsenoside Rb1 and Rd into ginsenoside Rh2. The fungus is inoculated onto PDA medium containing ginsenoside Rb1 or Rd and incubated at 25 °C for 5–7 days. Alternatively, the microbial fermentation conversion method can be used, in which the strain is inoculated onto a liquid fermentation medium and incubated at 28°C for 5-7 days. The enzyme solution is collected and mixed with ginsenoside Rb1 or Rd, and the mixture is reacted at 40°C for 24 h. The technical solution of the present invention for producing ginsenoside Rh2 is characterized by high specificity, simplicity and convenience, low cost and few by-products. The purity of the fermentation product Rh2 is above 85%.

(4) Enzyme conversion method.

Ginsenoside Rh2 is prepared in a targeted manner by using enzymes to selectively act on specific glycosidic bonds of ginsenosides. Ginsenoside α-arabinopyranosidase extracted from fresh ginseng roots can convert ginsenoside Rg3 to ginsenoside Rh2. The reaction conditions are as follows: Substrate concentration 10 mg·mL-1, pH 5.0, reaction at 55°C for 24 h, conversion rate up to 60% [17]. A new β-glycosidase purified from Fusarium proliferatum ECU204 can convert ginsenoside Rg3 to ginsenoside Rh2 [18]. Song Zhaohui et al. [19] applied for a patent in 2009 for a ginseng saponin Rh2 extract and a preparation method—extract medicinal materials containing ginseng saponins with water, allow the extract to settle, collect the supernatant, concentrate it to dryness, to obtain total saponins; dissolve the total saponins in a buffer solution with a pH of about 5, add β-glucosidase to react, collect the precipitate; dissolve the precipitate in ethanol, perform silica gel column chromatography, collect the fraction rich in ginsenoside Rh2, and concentrate to obtain. This laboratory has also made important progress in the preparation of ginsenoside Rh2 using industrial enzyme conversion with ginsenoside diol as a substrate.

(5) Chemical synthesis method.

Ginsenoside Rh2 can also be synthesized be synthesized chemically. Hui Yongzheng et al. [20] first selectively protected protopanaxadiol to obtain mono-substituted protopanaxadiol, and then subjected the mono-substituted protopanaxadiol to a glycosidation reaction with a glucose donor under the catalysis of a Lewis acid, and then removed the protective group to obtain 20(S)-Rh2 after separation and purification. This method has mild reaction conditions, low cost, high stereoselectivity of the reaction product, high yield and high purity. The invention is suitable for industrial large-scale production.

Ⅲ. Preparation method of ginsenoside Rh2 derivatives

After structural modification, ginsenoside Rh2 has enhanced water solubility and can be used as a prodrug to enter the body, delay the metabolic process of the drug in the body, and enhance its anti-cancer activity. Liu Jihua et al. [5] carried out a synthetic reaction of 20(S)-Rh2 with Boc-glycine to obtain five monomeric compounds; 20(S)-Rh2 reacted with Boc-alanine, Boc-arginine (Tos), Boc-lysine (Z), Boc-serine, and acetylproline, each resulting in a monomer compound; and the synthesis with acetylphenylalanine resulted in two monomer compounds. Wang Lu et al. [6] used the chlorosulfonic acid-pyridine method in combination with research on the modification of ginsenoside Rb1 by sulfation. The H on the different -OH positions on the Rh2 molecule was replaced with -SO3Na to obtain a pair of isomers. One isomer has the H on the C12 -OH position replaced, and the other has the H on the -OH position on the glc -C6 position replaced. which are abbreviated as S-Rh2 -1 and S-Rh2 -2, respectively. Wei et al. [7] dissolved ginsenoside Rh2 in chloroform, slowly added octyl chloroformate and Et3N, and reacted at room temperature for 15 min to obtain the ester D-Rh2.

Ⅳ. Pharmacological activities of ginsenoside Rh2 and its derivatives

Ginsenoside Rh2 includes two configurations, 20(S) and 20(R), while derivatives of ginsenoside Rh2 include sulfates, amino acid derivatives, esters, etc. The structures of ginsenoside Rh2 and its derivatives are different, and their pharmacological activities also differ greatly.

1. Pharmacological activity of 20(S) ginsenoside Rh2

A large number of literature studies have shown that ginsenoside diol type 20(S)-Rg3 and the aglycone 20(S)-PPD have a strong inhibitory effect on tumor cell proliferation. Compared with the former two, 20(S)-Rh2 has stronger activity in inhibiting glioma cells A172 and T98G, breast cancer cells MCF7 and MDA-MB-468, and lung cancer cells H838, etc., its activity is stronger; while in inhibiting prostate cancer cells LNCaP and PC3, pancreatic cancer cells HPAC and Panc-1, lung cancer cells A549 and H358, etc., its activity is weaker than 20(S)-PPD [21].

20 (S)-Rh2 has an inhibitory effect on the growth of Caco-2 and HT-29 cells. After 20 (S)-Rh2 acts on HT-29 and Caco-2 cells for 48 hours, the half inhibitory concentrations (IC50) were 19.68 and 26.79 μg·mL-1, respectively. The mechanism of action is that 20 (S) -Rh2 can significantly reduce the proportion of HT-29 cells in the G0/G1 phase and G2/M phase, and increase the proportion of S phase cells [22].

2.20(R) ginsenoside Rh2 pharmacological activity

20 (R) -Rh2  plays an important role in inhibiting papilloma and melanoma. Tao Lihua et al. [23,24] found that 20(R)-Rh2 has a significant inhibitory effect on mouse skin papilloma, B16 melanoma and B16-BL6 melanoma metastasis. The mechanism by which it inhibits malignant tumor metastasis may be related to its ability to reduce the invasiveness of cancer cells. Some studies have shown that after cancer cells form, they preferentially metastasize to the bone, and use cytokines in the bone to stimulate osteoclasts, thereby promoting cancer cell growth. Liu et al. [25] studied the in vitro inhibitory effect of 20(S)-Rh2 and 20(R)-Rh2 on osteoclast RAW264, found that only 20 (R) - Rh2 has the activity of inhibiting osteoclastogenesis, indirectly indicating that 20 (R) - Rh2 has the effect of inhibiting tumor cells.

Ⅴ. Comparison of the pharmacological activities of 20 (S)/20 (R) ginsenoside Rh2

Studies have shown that the anti-tumor activity of ginsenoside Rh2 is closely related to its configuration. The same dose of 20(R)-Rh2 and 20(S)-Rh2 was used on human lung adenocarcinoma cells A549. The results showed that both 20(R)-Rh2 and 20(S)-Rh2 promoted apoptosis of A549 cells, and both inhibited A549 cell proliferation in a dose-dependent manner, with inhibition rates of 28.5% and 33.6%, respectively, and IC50 values of 33.4 and 28.5 mg·L-1, respectively. Tip Compared with 20(R)-Rh2, 20(S)-Rh2 has stronger activity in inhibiting A549 cells [26]. In a study on the inhibition of the proliferation of prostate cancer cells (LNCaP, PC3, DU145), the IC50 value of 20(S)-Rh2 was the lowest, 20 (R/S) -Rh2 had the second lowest IC50 value, and 20 (R) -Rh2 had the highest IC50 value. Tung et al. [27] found that 20 (S) -Rh2 was more active than 20 (R) -Rh2 when studying the inhibition of human leukemia HL-60 cells by ginsenoside Rh2. In the study of ginsenoside Rh2's inhibition of different cell lines A-2780, HCT-8, SMMC-7721, and PC-3M, the results showed that the IC50 of 20(S)-Rh2 was nearly twice as small as that of 20(R)-Rh2 [28]. These results show that the 20-position configuration of ginsenoside Rh2 is closely related to its anti-cancer activity, and that 20(S)-Rh2 is more potent than 20(R)-Rh2.

Ⅵ. Pharmacological activity of ginsenoside Rh2 derivatives

After being derivatized, ginsenoside Rh2 can significantly improve its water solubility and has immunostimulatory and antitumor activities. Zhu Wei et al. [29] found that Rh2 sulfates S-Rh2-1 and S-Rh2-2 can significantly inhibit ConA-induced proliferation of mouse splenic lymphocytes when the dosage is lower than that of Rh2, suggesting that Rh2 derivatives have enhanced immunological activity. Wei et al. [7] found that Rh2 esterified with D-Rh2 is significantly less toxic to the liver cell line QSG-7701 in vitro than Rh2, but both have a stronger inhibitory effect on the H22 liver cancer solid tumor in vivo, and the activity of the two is comparable, suggesting that Rh2 esterified with D-Rh2 is a more suitable anti-tumor candidate compound than Rh2.

Ⅶ. Pharmacokinetic study of ginsenoside Rh2

Gu et al. [30] found that the bioavailability of ginsenoside Rh2 in rats and Beagle dogs after oral administration was 5% and 16%, respectively, indicating that the bioavailability of ginsenoside Rh2 varies in different species. Xie Haitang et al. [31] found that the bioavailability of ginsenoside Rh2 in male and female dogs was 17.6% and 24.8%, respectively, after ginsenoside Rh2 was administered to dogs by gavage, indicating that there are also differences in the bioavailability of ginsenoside Rh2 between the sexes. Gu et al. [30] administered ginseng saponin Rh2 to rats by gavage to study its tissue distribution, and the results showed that ginseng saponin Rh2 was mainly distributed in the liver and gastrointestinal tissues. Gu et al. [32] studied the absorption kinetics of 20(R)-Rh2 in different intestinal segments of rats and found that the absorption of 20(R)-Rh2 in the jejunum was the highest, and the absorption rate in the duodenum was the fastest.

Similar to other glycoside components, ginsenoside Rh2 is easily metabolized by intestinal flora after oral administration to produce corresponding aglycones. After ginseng saponin Rh2 was administered to rats by gavage, three metabolites of ginseng saponin Rh2, the de-glycosylated product m1, and the oxidation products m2 and m3, were detected in their feces, and a small amount of ginseng saponin Rh2 was also present in the feces. Note: Under the action of intestinal flora, ginsenoside Rh2 may undergo de-glycosylation and oxidation reactions [33].

Studies have shown that 20(S)-Rh2, when combined with digoxin and fexofenadine, can significantly alter the oral pharmacokinetic behavior of digoxin and fexofenadine [34]. Rats were pre-gavaged with 20(S)-Rh2, and 2 h later, digoxin and fexofenadine, which are P-glycoprotein (P-gp) substrates, were administered separately by gavage. The results showed that the AUC (area under the drug-time curve) of digoxin increased by 1.66 times, Cmax increased by 1.51 times, and the AUC of felodipine increased by 2.62 times, Cmax increased by 3.46 times. Isolated experiments showed that 20(S)-Rh2 can concentration-dependently increase the transport of digoxin A → B and reduce the transport of B → A, decreasing the efflux ratio of digoxin from 6.7 to 1.3. Its inhibitory effect is equivalent to that of the classic P-gp inhibitor verapamil. In addition, 20 (S) -Rh2 can concentration-dependently increase the uptake of rhodamine 123 by Caco-2 cells. It is suggested that 20 (S) -Rh2 is an effective non-competitive P-gp inhibitor.

Ⅷ. Prospects

Ginsenoside Rh2 and its derivatives have attracted the attention of scholars at home and abroad due to their good pharmacological activity. Biotransformation technology has many advantages such as low cost and high yield, and plays an important role in the preparation of ginsenoside Rh2. Based on related research, constructing engineered bacteria with various glycosidases to achieve the targeted preparation of ginsenoside Rh2 will be one of the research directions in the future. At the same time, the preparation of ginsenoside Rh2 and its derivatives using a combination of chemical and biotransformation methods, as well as in-depth studies of their structure-activity relationships, is of great significance for the discovery of drug leads for use in innovative drug research.

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