Study on Rare Ginsenoside Rg1 Rb1 Rg3

Ginsenosides are a class of steroid compounds that are triterpene glycosides in the field of natural products. They are mainly derived from Panax ginseng plants and are widely used in health products, functional foods, medicine, cosmetics and other fields due to their unique biological activity and medicinal value. It is generally believed that ginsenosides have structural characteristics such as a relatively high molecular weight, a low lipophilicity coefficient, and a large topological polar surface area.

They have low bioavailability, and after being orally ingested, they need to be metabolized by intestinal flora in the gastrointestinal tract into secondary ginsenosides such as Rg3 and Rh2 before they can be absorbed into the bloodstream and become the active substances that actually exert their medicinal effects. As research has progressed, it has been found that after ginseng saponins are treated using some physical, chemical, and biological transformation methods, some of the sugar chains are degraded or the C-17 side chain is changed, generating secondary saponins that are present in very low or even non-existent amounts in ginseng plants. Rare ginseng saponins have more significant pharmacological activities than the original ginseng saponins, such as anti-tumor [1], liver protection [2], and nervous system protection [3], and therefore show great development prospects.

At present, ginsenoside Rg3, which is the research benchmark for rare ginseng saponins, has been developed into a new type of traditional Chinese medicine monomer anticancer drug, “Ginseng Capsule”; several rare ginsenoside monomers such as ginsenoside Rh2 and C-K have entered clinical trials; a series of traditional Chinese medicine extracts rich in ginsenosides Rg3 and Rh2 are also widely used as raw materials for health food products such as Ginseng Capsule, which are well received by the market.

However, rare ginsenosides are low in natural plants and are particularly difficult to synthesize from simple starting products, which is far from meeting market demand. Therefore, the means of obtaining rare ginsenosides has become a hot topic in recent years. This article summarizes and analyzes the structure of rare ginsenosides and their conversion pathways and methods, in order to provide a scientific basis and theoretical foundation for the further development and application of rare ginsenosides.

1 Introduction to rare ginsenosides

The first rare ginsenosides discovered were products obtained after the transformation or metabolism of protopanaxadiol ginsenosides by the body. Since it is generally difficult to stably and controllably achieve this transformation and metabolic process in vivo, researchers have turned their attention to in vitro studies. In 1980, Kim Bong-seop et al. [4] first used an enzymatic method to prepare the rare ginsenoside Rh2. Later, breakthroughs were made one after the other in the enzymatic conversion of the rare ginsenoside Rg3, and large-scale industrial production was achieved.

Therefore, ginsenosides Rh2 and Rg3 are known as the first generation of rare ginsenosides. Based on pharmacological activity and mechanism of action studies, ginsenosides Rh2 and Rg3 have been developed into drugs and health products with anti-tumor functions. With the progress of modern industrial production technology, rare ginseng saponins have also entered the second generation. The industrialized mass production of ginseng saponins such as Rk2 and Rh3, represented by C-17 side chain polyunsaturated structures, is a landmark achievement. This has led to the widespread use of products with rare ginseng saponins as signature ingredients in pharmaceuticals, health foods, cosmeceuticals and other fields. Compared with the first generation of rare ginsenosides, the second generation of rare ginsenosides has more significant biological activities such as anti-tumor activity, as well as a more reasonable lipophilicity-hydrophilicity coefficient and a smaller relative molecular mass, as shown in Table 1, which also makes their bioavailability better.

According to the conversion relationship between representative rare ginseng saponins and prototypical ginseng saponins, see Figure 1, the parent nucleus structures of the rare ginseng saponins currently discovered (such as ginsenoside Rg3, Rh2, Rh1, etc.) are all dammarane-type tetracyclic triterpenoids, and they can be divided into protopanaxadiol (PPD) type, protopanaxatriol (PPT) type and C-17 side chain multiple double bond type 3 [5]. Compared with protopanaxadiol, which is usually found in high concentrations, the structural difference between the two is mainly the type and number of sugar chains attached to the branched chains at the C-3, C-6 and C-20 positions of the dammarane skeleton structure. Therefore, rare ginsenosides can be obtained by changing the sugar chains attached to the branched chains of the dammarane tetracyclic triterpene [6].

Search CNKI, China Biomedical Literature Database (CBM), Web of Science, PubMed, Embase, China Patent Publication and Announcement Query System, and Yaozhi.com database using search terms such as “minor ginsenoside,” “rare ginsenoside,” “ginsenoside Rg3,” “ginsenoside Rh2,” etc. After sorting, the currently known rare ginsenosides include ginsenoside Rg3, Rh1, Rh2, C-K, F2, and notoginsenoside R2. Their chemical structures and pharmacological effects are shown in Figure 2 and Table 2.

2 Sources and preparation routes of rare ginsenosides

2.1 Sources

Rare ginsenosides can be obtained by converting or heterologous synthesizing prototypical ginsenosides. At present, the prototypical ginsenosides that have been studied more in the conversion pathway mainly include ginsenosides Rb1, Re, Rc, Rd, etc. These ingredients are mainly derived from the roots, leaves, flowers and flower buds of Panax ginseng C. A. Mey. of the genus Panax, the roots and leaves of P. quinquefolium L., and the roots and leaves of P. notoginseng (Burk.) F. H. Chen. Due to factors such as the supply of plant materials, extraction and processing, market prices, etc., this source has a high degree of dependence on the demand for protopanaxadiol ginsenosides.

In order to reduce the dependence on the source, rare ginsenosides can also be synthesized by heterologous synthesis of various key enzymes, starting from source compounds or key intermediates. This method of acquisition includes processes such as the synthesis of methyllactate, the synthesis of 2,3-oxo-squalene, and cyclization reactions, as shown in Figure 3. The source compounds or intermediates involved in this process (such as 2,3-oxo-squalene, cyclized products PPT, PPD, etc.) are all important sources of rare ginsenosides. It should be noted that after the completion of the heterocyclic synthesis reaction, the target ginsenosides must be hydroxylated or glycosylated by genetic engineering to produce the target ginsenosides.

2.2 Preparation route

At present, the main methods for preparing rare ginsenosides using the prototypical ginsenoside conversion method are physical, chemical and biological methods. Physical methods include pyrolysis, chemical methods include acid degradation and base degradation, and biological methods include in vitro enzyme degradation, biotransformation and biosynthesis.

2.2.1 Pyrolysis

Pyrolysis degrades the sugar chains and C-17 side chains of protopanaxadiol ginsenosides through high-temperature treatment, converting them into rare ginsenosides. Sun Baishen et al. [74] obtained rare ginsenosides Rg6, Rs4, and Rs5 from black ginseng processed by the method of steaming and exposing to the air nine times. Qu Wenjia et al. [75] obtained ginsenosides 20(S)-Rg3, 20(R)-Rg3, Rk1, and Rk5. Guan Daping et al. [76] obtained the rare ginsenosides Rk1 and Rg5 by high-temperature heating of ginseng stems and leaves. JEONG S Y et al. [61] isolated and purified the ginsenoside Re from ginseng leaves, and treated it at 120 °C for 6 h, which ultimately converted it into the rare ginsenosides Rh1 and Rh4, as shown in Figure 4.

The advantages of the thermal cracking method are its simplicity and low cost. However, it is time-consuming, not very specific, and has a low conversion rate, which can lead to a waste of resources if the raw materials are not used effectively.

2.2.2 Acid degradation method

Under suitable acidic conditions, some of the sugar chains of ginsenosides are hydrolyzed to form rare ginsenosides. Liu Qian et al. [77] found that under weak acidic conditions, some or all of the sugar chains of ginsenosides are hydrolyzed, but the configuration of the C-20 position changes in a weak acid environment, and ultimately a mixture of two diastereoisomers is obtained. GAO D et al. [59] used citric acid to heat-treat ginseng buds, which converted ginsenoside Rb1 in ginseng flower buds into the rare ginsenosides Rg5 and Rk1, as shown in Figure 5. LI W et al. [78] used citric acid to hydrolyze ginsenoside Re into the rare ginsenosides F4 and Rk3, see Figure 6.

 BAE E A et al. [79] summarized the conditions for acid degradation of ginsenosides. The experimental results showed that ginsenoside Rh2 can be obtained by extracting with ether after hydrolyzing common ginsenosides with 5% hydrochloric acid in methanol and 5% sulfuric acid in ethanol for 4–6 h. It should be noted that strong acids react violently when acting on ginsenosides. not only will the sugar chain of ginsenosides be hydrolyzed and the aglycone be destroyed, but the side chain will also be cyclized or the configuration of the 20th carbon atom will be changed, making it difficult to obtain 20(S)-protopanaxadiol or triol-type saponins. Currently, the acidity of simulated gastric juice can be used to reduce these phenomena. Commonly used acids include tartaric acid, formic acid, acetic acid, citric acid, etc. The overall process of acid degradation to prepare rare ginseng saponins is cumbersome and has many by-products. Therefore, there is still a need to continue exploring efficient acid degradation methods.

2.2.3 Alkali degradation method

Compared with acid degradation, in an alkaline environment formed by reagents such as sodium hydroxide and sodium methoxide, the C-17 side chain of ginsenosides changes less, and the alkali degradation method has fewer side reactions, mild degradation conditions, and easy purification of the product. Chen Yanping et al. [80] found that under mild alkaline conditions, protopanaxatriol can be degraded to obtain the rare ginsenoside Rhl, and protopanaxadiol can be degraded to obtain the rare ginsenoside Rh2.

The disadvantages of the alkaline degradation method mainly lie in the long reaction time and lower yield compared to the acid catalysis method.

2.2.4 In vitro enzymatic degradation method

In vitro enzymatic degradation is a method that uses enzymes to break down the glycosidic bonds of the substrate prototype ginsenoside. Compared with acid degradation and alkaline degradation, enzymatic degradation has the advantages of high efficiency, no pollution and specificity, and is currently the most widely used method of research. Different types of enzymes can be used to modify glycosidic bonds of different types and conformations. Zhao Liya [81], Kim Dong-Sik [82], Xue Lili [83] and others found that using protopanaxadiol-type saponins as substrates and β-glucosidase as a biocatalyst, ginsenosides Rh2, Rg3, Rh3, Rg5, as well as other by-products such as protopanaxadiol-type saponins. With the development of genetic engineering, glycosidases obtained by cloning and expressing bacterial genes have also been used in the transformation of ginseng saponins. ZHENG F et al. [84] used endoglucanase to cleave the glucose at C2 of ginsenosides Rb2, Rb1, Rc, and Rd, and some of them had high selectivity, respectively, which have stronger pharmacological activities, ginseng saponins GypXVII, C-O, C-Mc1, and F2.

In enzymatic degradation methods, phosphate buffer solutions or solutions containing small amounts of organic solvents are often used to dissolve the substrate prototype ginsenoside in order to fully dissolve it. Previous solutions not only affected the activity of the enzyme, but also had a low solubility for the substrate. In response, Fan Yuru et al. [85] developed an assisted enzymatic method using a low eutectic solvent (deep eutectic solvents, DES). using choline chloride and propylene glycol to prepare a DES, dissolving the substrate ginsenoside Rb1 in it, and using snail enzyme for enzymatic conversion to obtain the rare ginsenoside C-K.

The DES used in this method not only increases the solubility of the substrate protosaponin, but also increases the activity of the enzyme, thereby effectively increasing the yield of the rare ginsenoside C-K. However, the DES-assisted enzyme method and previous biological enzyme degradation methods both have the disadvantage that the product and the enzyme cannot be efficiently separated. Therefore, in previous application studies, the key to the enzymatic hydrolysis of ginsenosides is not only the screening of specific and efficient enzymes, but also the continuous optimization of the medium solution required for enzymatic reactions, as well as the exploration of methods that can efficiently separate the product from the enzyme, in order to more efficiently and energy-savingly generate rare ginsenosides industrially.

2.2.5 Microbial transformation method

The microbial transformation method mainly uses microorganisms to specifically hydrolyze ginsenosides to obtain rare ginsenosides through the decomposition of one or more enzymes[86]. SIDDIQI M Z et al.[87] used a mixture of ginsenosides Rb1, Rb2, Rb3, Rc, and Rd, a mixture of ginsenosides, was first obtained by biotransformation technology to obtain a mixture of ginsenosides Rg3, and further conversion can obtain rare ginsenosides Rk1, Rk2, Rg5, and Rh3, as shown in Figure 7. FU Y et al. [58] isolated endophytic bacteria from ginseng that convert ginsenoside Rb1 to rare ginsenoside Rg3, see Figure 8. HASEGAWA H et al. [88-89] studied the specific transformation pathway of rare ginsenoside C-K by intestinal flora and speculated that rare ginsenoside C-K is the form of protopanaxadiol ginsenoside that is most likely to be absorbed through the intestine, see Figure 9.

Guo YP et al. [90] used a sterile rat model to verify that panaxoside can be metabolized into rare ginsenosides such as Rh2 through the intestinal microbiota in rats. Kim KA et al. [91] isolated the genus Bacteroides, Bifidobacterium, and Ruminococcus, which can convert ginsenoside Rb1 into the rare ginsenoside C-K. Compared with physical and chemical conversion methods, bioconversion methods have the unique advantages of being highly efficient, mild reaction conditions, lower cost, and better guarantee of ginsenoside activity. However, bioconversion still requires ginsenosides as substrates, and relying solely on ginseng plants as a source is too limited. Therefore, it is still necessary to continuously explore and develop new technologies (such as plant tissue culture bioreactor technology) to obtain rare ginsenosides.

2.2.6 Bioreactor technology

With the development of biotechnology, the efficient large-scale production of rare ginsenosides using cells and organelles as raw materials has gradually replaced the existing conversion method that requires ginsenosides as substrates. Cells and adventitious roots are cultured in large bioreactors and the accumulation of biomass and ginsenosides is enhanced in a corresponding manner. CAO L et al. [92] induced adventitious roots in a 5 L bioreactor and produced 11 rare ginsenosides, including ginsenoside Rh2. The bioreactor was associated with enzymatic hydrolysis using six β-glycosidases and their combinations, yields 54.32~66.00 mg·L-1 . Further optimization of pH and temperature, immobilization of BglPm and Bgp2, and the yield of ginsenoside Rh7 was further increased by 1%, with a maximum yield of 51.17 mg·L-1 (17.06% of the original ginsenoside mixture). The above method can replace the direct conversion and extraction of rare ginsenosides from Panax plants and can also be used to supplement yeast cell factories.

2.2.7 Biosynthetic method

In recent years, with the continuous development of synthetic biology research, compounds of animal and plant origin can also be synthesized by microorganisms. The method of microbial de novo heterologous synthesis of rare ginsenosides is called the biosynthetic method, also known as the heterologous synthesis method. Biosynthetic methods often use isopentenyl diphosphate produced by the methyloxypivalate pathway to form the precursor compound of ginsenosides, squalene, under the action of various enzymes. Squalene monooxygenase is used to generate the key raw material 2,3-oxidosqualene, and then with the help of the damarenediol synthase to form damarenediol, thus forming the ginsenoside skeleton; and then through genetic engineering technology to hydroxylate or glycosylate to form the target ginsenoside. There are many key rate-limiting enzymes in this method. Genetic and metabolic engineering modifications of the genes of these key rate-limiting enzymes will greatly increase the yield. Lei Jun [93] artificially constructed a four-step enzymatic reaction in tobacco, and for the first time, achieved the heterologous synthesis of ginsenoside F1 in tobacco, as shown in Figure 4g.

Saccharomyces cerevisiae is a food-grade strain and a low-level single-cell eukaryote. Many natural terpene compounds can be synthesized in Saccharomyces cerevisiae by introducing key enzyme genes, thereby constructing a heterologous synthesis pathway, so that the target compounds can be synthesized in Saccharomyces cerevisiae. Chen Qin [94] increased the expression of the key gene UGTPn3 in tobacco cells by plant hormones to promote the synthesis of the ginsenoside Rh2, and the yield could reach 38.67 μg·g-1. ZHUANG Y et al. [95] overexpressed additional genes under the PGK1 and HXT7 promoters into PGM1 and UGP1, causing UGT51 to specifically transfer the glucose moiety to the C-3-OH of PPD converting it into ginsenoside Rh2, and the yield of ginsenoside Rh2 was 36.7 mg·L-1, an increase of 4% in conversion rate.

LI X et al. [96] introduced PgUGT2A71, PgUGT54Q94 and the UDP-xylose biosynthesis pathway into the PPT chassis, and constructed engineered yeast, which resulted in yields of notoginseng saponin R1 and notoginseng saponin R2 of 1.62 and 1.25 g·L-1 , respectively. ZHAO F L et al. [97] integrated a fusion gene Pg PPDS-ATR1 with three copies into the genome of Saccharomyces cerevisiae, which resulted in the conversion of 96.8% of the DM to PPD, yielding 1436.6 mg·L-1 and the engineered yeast was not affected by reactive oxygen species. Lu Wenyu et al. [98] transferred the optimized glycosyltransferase GTK1 gene and the optimized glycosyltransferase UGT1 gene into Saccharomyces cerevisiae, which produces protopanaxadiol PPD, and overexpressed the phosphoglucomutase PGM1 gene and the UDP-glucose pyrophosphorylase UGP1 gene to obtain a ginsenoside F2 yield of 44.83 mg·L-1.

The rare ginsenoside synthesis method successfully developed through biosynthesis technology has solved the problem of low bioconversion efficiency in the past and broken through a major limitation in the large-scale production of rare ginsenosides. However, the key to constructing a heterologous expression system and a de novo synthesis pathway for ginsenosides lies in the optimization of the synthesis pathway, the inhibition of competing pathways, enzyme modification, transcription regulatory factors and other related enzyme genes. This requires the development of omics technologies such as genomics, transcriptomics and proteomics, as well as synthetic biology technology. Further research is needed to achieve more accurate and effective regulation.

3 Analysis of the current situation of the rare ginsenoside industry

Due to their higher bioavailability and stronger biological activity than the prototype ginsenosides, rare ginsenosides have undergone significant development and improvement in research on their application in the fields of pharmaceuticals, health foods, cosmetics, etc., and have shown increasingly broad prospects for development and application [99]. In the past 10 years, medicines and health products containing rare ginseng saponins have been vigorously promoted. Currently, marketed drugs containing rare ginseng saponins include Shenyi Capsules, 20(S)-Ginsenoside Rg3 Eye Ointment, 20(S)-Ginsenoside Rg3 Injection, Jinxing Capsules, and Shenbaiyi Capsules. According to statistics, in the past 20 years, there have been a total of 84 national invention patents related to rare ginsenosides, as shown in Figure 11. Among them, there have been as many as 76 related patents in the past decade, mainly focusing on preparation and processing methods, rare ginsenoside monomer compatibility products, and applications such as anti-cancer and anti-tumor. In comparison, the number of invention patents is much greater than the number of marketed products.

This may be because the existing research and development technology and the limitations of supporting industrial production equipment have not yet resulted in a preparation method that greatly improves the yield, which prevents industrialized production and limits product development. According to statistics, in recent years, sales of rare ginseng saponin products have increased from 406 million yuan in 2017 to 739 million yuan in 2022, with a compound annual growth rate of 12.7%. It is expected to further increase at a higher compound annual growth rate of 16.1% to 156.1 billion yuan in 2027. It can be seen that rare ginsenosides are becoming increasingly popular, and the market value is extremely broad. The related industry has also become a highly promising industry in China. To some extent, the industry can promote the development of the medical industry and reduce the incidence of some diseases. Accelerating the development of the rare ginsenosides industry will certainly have a significant and far-reaching impact on the healthy development of China's industry.

4 Conclusion and outlook

Rare ginsenosides have stronger pharmacological activity than their prototype ginsenosides and are easily absorbed by the human body. At present, most research on rare ginsenosides tends to focus on the development of their medicinal value, but experimental research on their transformation, separation and purification is also of great significance for their development and evaluation. There are many methods for the conversion and isolation of rare ginsenosides, but each method has different advantages and disadvantages. A reasonable preparation method is the key to ensuring a high content of rare ginsenosides. Among them, the biotransformation method and the biosynthesis method are favored for their high efficiency, few by-products, and clear goals.

However, compared to the biosynthesis method, the biotransformation method still cannot do without the prototype ginsenoside as a substrate, and its dependence on enzymes makes it affected by many factors. In recent years, the newly developed biosynthesis method starts with the synthesis of the prototype ginsenoside, which only requires simple basic metabolic pathways in eukaryotes and prokaryotes. However, the gene information of the key enzymes in the metabolic pathway has not yet been fully obtained, Therefore, in the future, efforts should perhaps be focused on developing more efficient, green, economical, and safe standardized biosynthesis conditions, so that more rare ginsenosides can be produced industrially, thereby realizing the industrialization of rare ginsenosides. Combining the development of multi-omics and bioinformatics, we can dig deeper into the information of key enzyme genes in the relevant basic metabolic pathways, improve industrial production to meet market demand, and make a greater contribution to the human pharmaceutical industry and health cause.

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