What Is Ginsenoside Rg5?

Ginseng is known as the “king of herbs” and is one of the traditional Chinese precious herbs [1]. It has a significant effect on treating low body immunity [2], neurodegeneration [3], cancer [4, 5], cardiovascular disease [6], etc. Ginsenosides are the main active ingredients of ginseng. More than 80 different types of ginsenoside monomers have been isolated from ginseng roots [7]. In recent years, a large number of studies have been conducted at home and abroad on the pharmacological effects and molecular mechanisms of ginsenosides.

Ginseng saponin Rg5 is one of the main components of red ginseng. It was first isolated by a South Korean research team in 1996 [8]. It is a secondary saponin obtained by selective hydrolysis of the protopanaxadiol group saponins Rb1, Rb2, Rb3, Rc, and Rd (referred to as PPD saponins) in ginseng, and then obtained by stereoselective dehydration of the secondary saponins [9] (see Figure 1). Clinical trials in animals and humans have shown that Rg5 not only has significant effects in reducing cisplatin-induced nephrotoxicity [10], anti-cancer [11], improving lung inflammation [12] and improving memory [13], but also exhibits good safety. In recent years, with the good therapeutic effects of ginsenoside Rg5 in cancer, inflammation, memory and other aspects, research on Rg5 is gradually deepening. This paper mainly reviews the research on this compound at home and abroad from the aspects of preparation, separation and pharmacological activity of ginsenoside Rg5.

1 Preparation method of ginsenoside Rg5

Ginsenoside Rg5 is present in trace amounts in processed products such as ginseng and Panax notoginseng [14]. At present, there is relatively little research on the preparation of the rare ginsenoside Rg5 monomer at home and abroad. The traditional preparation method is to heat ginseng to make processed ginseng, and then extract and isolate ginsenoside Rg5 from it. With the deepening of research on the isolation and preparation techniques and physical and chemical properties of ginsenosides, people have gradually tried to directly extract ginsenoside Rg5 from ginseng powder or prepare ginsenoside Rg5 by modifying the structure of PPD ginsenoside (see Figure 2).

1. 1 Preparation using processed ginseng as raw material

1. 1. 1 Processing methods for ginseng—steaming method

Traditional Chinese medicine processing methods have been developed over thousands of years and include steaming, roasting, boiling, soaking in wine, vinegar or other liquids, etc. The enhanced pharmacological activity of steamed ginseng may be due to the generation of less polar ginsenosides Rg3, Rk1 and Rg5 [15]. Raw fresh ginseng can be dried under different conditions to produce processed ginseng with different amounts of rare ginsenosides, such as white ginseng, red ginseng, black ginseng (also known as purple ginseng), and sun ginseng.

Red ginseng is processed ginseng made by steaming fresh ginseng at 95–100 °C for 3 h and drying at 50–60 °C [16]. KIM et al. [17] used water and methanol solutions with 70% and 100% volume fractions were used for direct ultrasonic extraction or red ginseng was soaked in water for 2 h and then extracted with 100% methanol for 3 h. Ginsenoside Rg5 was not detected in any of the four extraction methods. KWON et al. [18] steamed fresh ginseng at 120 °C for 3 h, then extracted with methanol by reflux for 6 h. The contents of 20 (S)-Rg3, 20 (R)-Rg3, Rk1 and Rg5 in the ginseng extract were significantly increased, and the content of Rg5 reached 3.3% of the ginseng extract. KIM et al. [19] studied the effects of different steaming temperatures on the chemical composition and biological activity of ginseng. fresh ginseng was steamed at 100 °C, 110 °C and 120 °C, respectively. The results showed that the content of ginsenosides Rg3 and Rg5 in ginseng steamed at 120 °C was the highest, accounting for 39% and 19% of the total saponin mass, respectively. The fact that Rg5 was not detected in red ginseng and that its content increased when the processing temperature was raised indicates that, within a certain range, an increase in temperature is conducive to the production of the rare ginsenoside Rg5.

Using fresh ginseng as raw material and drying it at 55 °C for 7 days, white ginseng with a total water content of less than 14% can be obtained. The re-sultant ginseng is obtained by steaming the white ginseng at 98 °C for 30 h, drying at 50 °C for 24 h, and then steaming at intervals of 5 h for 30–45 h, and drying again [20]. Liquid chromatography analysis of the ginsenoside content between the main root and fibrous root of ginseng revealed that steaming the fibrous root produced more Rg3, Rk1, and Rg5 than the main root. In terms of the effect of steaming time, between 60 and 70 h, the content of rare ginsenosides gradually increased with the extension of steaming time, but when the steaming time was further extended, the content of rare ginsenosides decreased slightly. At this time, the content of newly generated ginsenosides 20 (S) -Rg3, 20 (R) -Rg3, Rk1, and Rg5 was 2.398%, 1.501%, 1.136%, and 1.756%, respectively. The experimental results show that, within a certain time frame, long steaming is more conducive to the production of ginsenosides Rg3, Rk1 and Rg5. This steaming method is of great significance for the efficient preparation of ginsenosides.

Black ginseng is processed ginseng that is made by steaming fresh ginseng at 97 °C for 3 h, drying at 60 °C, steaming and drying repeatedly for nine times [21]. SHIN et al. [16] found that the content of the main ginsenosides Rb1, Rb2, Rc, and Rg1 in black ginseng is significantly lower than that in red ginseng. In contrast, the rare ginsenosides Rg3, Rk1 and Rg5 were found to be significantly higher than those in red ginseng, with the total content of Rk1 and Rg5 reaching 14.2% of the total ginsenosides.

Xianshen is a processed ginseng made by heating white ginseng at 120 °C for 3 h [22]. Xianshen was extracted by refluxing with methanol at 70 °C for 2 h. Analysis showed that the total saponin content of the xianshen extract was about 10% by mass, and was mainly composed of saponins Rb1, Rc, Rb2, 20 (S) -Rg3, 20 (R) -Rg3, Rk1, and Rg5. Among these, the low-polarity rare ginseng saponins represented by ginsenoside Rg5 (22.8% of total saponins) account for 70% of the total ginseng saponins.

Due to the differences in the processing methods of fresh ginseng, red ginseng, white ginseng, black ginseng and xianshen produce different distributions of ginsenosides. During the steaming process, appropriately increasing the steaming and drying temperature or extending the time is conducive to the production of more rare ginsenosides Rg3, Rk1 and Rg5.

In addition to ginseng, other ginseng plants such as American ginseng and Panax notoginseng can also be used to prepare ginsenoside Rg5 by steaming. For example, Panax notoginseng roots are steamed at 120 °C for 12 h at a pressure of 0.12 MPa to obtain processed Panax notoginseng. Processed Panax notoginseng root was extracted with an 80% methanol aqueous solution at room temperature, and the content of Rg5 in each gram of Panax notoginseng root powder was 0.287% [14].

The steaming process promoted the conversion of ginsenosides from major ginsenosides to secondary ginsenosides. This is consistent with previous reports of the appearance of new ginsenosides such as Rk1, Rg5, and Rg3 under high temperature conditions [23], but the steaming method has disadvantages such as poor targeting, low efficiency, and long processing times, and further improvement is needed.

1. 1. 2 Processing method for processed ginseng—steaming and puffing

The puffing method is a method of changing the physical and chemical properties of food through heat compression. High-temperature puffing for a short period of time can cause physical and chemical changes in food and Chinese herbal medicines. After puffing, the extraction rate of ginseng and the content of crude saponins are both increased, and it contains more ginsenosides Rg3, Rg5 and Rk1 [24-25]. White ginseng, red ginseng, and black ginseng samples were mixed with rice [m (ginseng): m (rice) = 1:4] and the mixture was heated in a traditional rotary expander. When the chamber pressure reached 490 kPa, the valve was opened to reduce the pressure to 196 kPa, and the chamber was reheated to 784 kPa to obtain the puffed ginseng sample [26]. According to the data, several polar ginsenosides are converted into low-molecular-weight non-polar rare ginsenosides during the roasting process of ginseng. According to the HPLC chromatogram, the contents of rare ginsenosides Rk1 and Rg5 in white ginseng, red ginseng, and black ginseng are 17.2%, 18.5%, and 16.1% of the total ginsenosides, respectively, indicating that the puffing process can greatly change the distribution of ginsenosides in white ginseng, red ginseng, and black ginseng. It is a processing method for ginseng with a high conversion rate of ginsenosides, a short processing time, and low energy consumption [16].

1. 2 Preparation using ginseng powder as raw material

HUANG et al. [27] used ginseng powder as a raw material and prepared ginsenosides Rg5 and Rg3 from protopanaxadiol saponins by tartaric acid induction, and used extended column adsorption chromatography to separate ginsenosides Rg5 and Rg3. The experimental results showed that 10 g of ginseng powder was extracted by ultrasound with 0.8 mol·L-1 D, L-tartaric acid in an aqueous ethanol solution with a volume fraction of 10%, and separated by column chromatography. obtaining 22.2 mg of Rg3 with a purity of 14.6% and 13.3 mg of Rg5 with a purity of 11.9%. Compared with the conventional method, the preparation time is significantly shortened, but the yield of Rg5 is low. Guo et al. [28–29] used ginseng root powder as a raw material, hydrochloric acid as a catalyst, and a microwave-assisted method to directly extract the rare ginsenoside Rg5 from ginseng root powder. A response surface was used to optimize the extraction method of ginsenosides, and the optimal extraction process conditions were obtained as follows: extraction power 500 W, the acid concentration of the extraction solution was 0.12 mol·L-1, the solid-liquid ratio was 1:42 (g·mL-1), and the extraction time was 9 min. At this time, the yield of ginsenoside Rg5 was 3.14% (calculated based on the mass of the ginseng root powder). A recent report by WANG et al. [30] shows that 3.05% Rg5 per gram of black ginseng can be obtained by weighing 10.0 g of black ginseng powder, dissolving it in 1.5 L of 0.1% formic acid solution, and steaming it in an autoclave at 120 °C for 2 h.

Compared with the traditional steaming process for extracting and separating ginsenoside Rg5, the direct preparation of ginsenoside Rg5 from ginseng powder as a raw material significantly shortens the preparation time, improves efficiency, and is highly targeted. It is an ideal preparation method for ginsenoside Rg5.

1.3 Preparation using protopanaxadiol group saponins as raw materials

Guan et al. [31] prepared ginsenoside Rg5 by acid treatment of ginsenoside Rb1. Using 300 mg of ginsenoside Rb1 powder as the raw material, 0.05% acid by volume and 50% ethanol solution by volume were added, reacted under 0.12 MPa for 2 h to obtain rare ginsenosides Rk1 and Rg5 with a yield of 19.88%. SUN et al. [32] used lemon as a catalyst and protopanaxadiol (PPD) as a raw material to prepare rare ginsenoside Rg5 with a yield of 21.53%. Lemon contains citric acid and malic acid, which can be used to catalyze PPD. It has the advantages of low cost, easy operation, and environmental friendliness. In addition, LIU et al. [33] optimized the process conditions for the preparation of the rare ginsenoside Rg5 by microwave-assisted degradation of Panax notoginseng stem and leaf total saponins using a response surface method. When the microwave power was 540 W, the microwave temperature was 153 °C, and microwave time 20 min, the yield of Rg5 can reach 43.07%. This method can quickly prepare ginsenoside Rg5 with a relatively high yield.

The extraction of rare ginsenoside Rg5 from processed ginseng is currently the most commonly used method, but the extraction time is long and the extraction efficiency is low. Compared with this, the ginseng puffing process has the advantages of being time-saving and having a high conversion rate. The direct extraction of ginseng powder to prepare ginsenoside Rg5 can greatly reduce the preparation time, and the yield of Rg5 is relatively high. It is a convenient and efficient method for preparing ginsenoside Rg5. The extraction of Rg5 from protopanaxadiol is simple to operate, has a short reaction time and a high yield. It is currently the best choice for producing ginsenoside Rg5 with a high content.

2. Isolation of ginsenoside Rg5

As research into the pharmacological activity of ginsenoside Rg5 gradually progresses, the primary consideration is how to isolate and purify Rg5 to obtain a high-purity Rg5 monomer. This is of great research significance for the industrial production of ginsenoside Rg5 and its application in various fields.

2.1 Preparative liquid chromatography separation

Preparative liquid chromatography is commonly used in the analysis of biopharmaceuticals and natural products. It is a fast and effective tool for separating organic compounds, and is becoming more and more mature and widely used. KIM [8] and others used preparative liquid chromatography to separate and purify ginsenoside Rg5. 250 g of red ginseng powder methanol extract was taken, and the extract was extracted with a mixture of water and n-butanol, and 114 g of n-butanol extract was obtained by rotary evaporation. The n-butanol extract of red ginseng powder was separated by silica gel column chromatography using CHCl3-MeOH-H2O (10:3:1→9:3:1) and n-BuOH-EtOAc-H2O (10:10:0.5) as eluents, and 250 mg of crude saponin was obtained. and further purified using preparative liquid chromatography [CH3 CN-H2 O (V:V=4:6), flow rate 2.5 mL·min-1] to obtain high-purity Rg5.

In 2017, WANG et al. [30] neutralized the methanol extract of 10 g of black ginseng powder with saturated NaHCO3, extracted with water-saturated n-butanol, removed the solvent at 45 °C, and passed the residue through a silica gel column with CHCl3-MeOH (V:V = 8.5:1.5). obtaining a mixture of ginsenosides Rk1 and Rg5. The mixture was then separated using semi-preparative high-performance liquid chromatography [MeOH: H2 O (V:V = 65:35), flow rate 2.5 mL·min-1] to obtain Rg5 with a purity of 98.0%. Preparative high-performance liquid chromatography can quickly obtain high-purity components. Compared with other methods, it has the advantages of being convenient and fast, and it has a better effect on the separation of Rg5.

2.2 Column chromatography separation

Column chromatography is generally used to purify and separate organic or inorganic substances. Kim et al. [34] extracted the black ginseng root with ethanol, filtered, concentrated the ethanol extract in a rotary evaporator, degreased with ether, extracted with water-saturated n-butanol, and then subjected to column chromatography with CHCl3-MeOH-H2O (70:30:4) as the mobile phase to to obtain subfractions F1-F5. 2.59 g of subfraction F4 was further separated by reverse-phase column chromatography (60% acetonitrile) using 500 g of C-18 packing material to obtain 0.19 g of ginsenoside Rg5. GU et al. [14] used a methanol extract of Panax notoginseng obtained 2. 12 kg of total saponins by chromatographic desugarization on a D101 column. Column chromatography was performed using CHCl3 -MeOH-H2 O (85:15:1→75:25:2) as the eluent to obtain eight components A-H. Component B was separated by RP-18 CC (MeOH H2 O, 1:1→9:1) to obtain five sub-fractions B1-B5. Fraction B1 was purified by RP-18 CC (MeOH-H2 O, V:V=7:3), and recrystallized in MeOH-H2 O (V:V = 75:25) to obtain 43 g of ginsenoside Rg5.

 In 2017, GUO et al. [29] prepared Rg5 from the acid hydrolysis of ginseng root powder, and then subjected to silica gel column chromatography with CHCl3 -MeOH (V:V = 8.5:1.5) as the eluent to obtain a ginsenoside Rg5 product with a purity of 85.31%. This provides a simple and feasible method for preliminary research on Rg5 monomers. As an important separation and analysis method, column chromatography has the advantages of being simple to operate, highly efficient and capable of processing large volumes. With the application and development of chromatography technology, column chromatography is playing an increasingly widespread role.

3 Pharmacological activity

Ginseng is considered to be an herb that prolongs life, and ginseng and Chinese medicinal formulas containing ginseng ingredients are widely used in traditional Chinese medicine. At present, research on the efficacy of ginsenoside Rg5 is still in its infancy. With the gradual maturity of modern separation and analysis techniques, ginsenoside Rg5 has been found to have many pharmacological activities, such as anti-cancer, anti-allergic and anti-inflammatory, improving memory, anti-depression, and promoting cell growth.

3.1 Anti-cancer effect

The role of ginsenosides in preventing cancer has been established. The anti-tumor effect of ginseng is mainly due to ginsenosides, which can induce apoptosis and metastasis of cancer cells. Studies have shown that ginsenoside Rg5 can inhibit the proliferation of various cancer cells, induce cell cycle arrest and apoptosis, and is a promising anti-tumor drug.

Breast cancer is one of the most common malignant tumors in women. According to statistics, breast cancer is the leading cause of cancer death in women. In China, the incidence of breast cancer is increasing year by year and the age of patients is getting younger. In 2014, Kim et al. [34] extracted Rg5 from the root of black ginseng and studied its anti-cancer activity on MCF-7 breast cancer cells. The cell cycle method and western blot method were used to analyze the anti-cancer mechanism of Rg5. To determine whether Rg5 reduced cell viability, the cells were analyzed using flow cytometry after double staining. The results showed that Rg5 stimulated cell cycle arrest and apoptosis in breast cancer cells in the G0/G1 phase by regulating cell cycle proteins and related apoptosis proteins.

LIANG et al. [35] found that ginsenoside Rg5 induces DNA damage in cancer cells. The test results showed that the degree of DNA damage is directly proportional to the concentration of Rg5, and that the increase in Rg5 concentration exacerbates DNA damage. Ginsenoside Rg5 was found to have significant genotoxicity in HeLa and MS751 cervical cancer cells, indicating its potential as a chemotherapeutic agent for cervical cancer cells. In cervical cancer cells, Rg5 blocks the transformation of the cancer cell cycle by reducing cyclin-dependent kinase activity.

ZHANG et al. [36] treated esophageal cancer Eca109 cells with different concentrations of ginsenoside Rg5 for 24 h, and observed a decrease in mitochondrial membrane potential, an increase in cytosolic free calcium levels, and a significant increase in apoptosis. It has been found that the PI3K/Akt signaling pathway is activated in human cancers, Ginsenoside Rg5 inhibits the proliferation of human esophageal cancer cells Eca-109 by inhibiting the activity of the PI3K/Akt signaling pathway. Rg5 induces apoptosis in human esophageal cancer cells through the phosphoinositide-3 kinase and phosphoprotein kinase B signaling pathways, and the apoptosis rate is directly proportional to the duration and concentration of Rg5 action [37-38].

LI et al. [39-40] found that ginsenoside Rg5 inhibited the invasion and migration of gastric cancer BGC-823 cells. The proliferation and apoptosis of gastric cancer BGC-823 cells, apoptosis-related factors and Bcl-2-related protein expression were all regulated by Rg5. Compared with the blank group, the Bcl-2 protein expression in the high-concentration Rg5 group was reduced (P<0.05), indicating that ginsenoside Rg5 can effectively inhibit the proliferation of gastric cancer BGC-823 cells.

The microwave-assisted process effectively improves the structural conversion of ginsenosides in the extracted product, and the contents of Rg3, Rg5, and Rk1 increase significantly. In particular, Rg5 and Rk1 have better medicinal effects [41]. The content of rare ginsenosides Rg5 and Rk1 increases in direct proportion to the microwave time. After 60 minutes of microwave radiation, the amount of ginsenosides Rk1 and Rg5 produced reached a maximum. Treatment with these ginsenosides on five human cancer cell lines showed that an increase in the amount of Rg5 and Rk1 significantly enhanced the inhibition and anti-proliferation of cancer cells.

Multidrug resistance (MDR) to chemotherapeutic drugs remains a major challenge in clinical cancer treatment. When tumor cells become resistant to chemotherapeutic drugs, they kill both cancer cells and normal cells, causing increasingly severe adverse reactions. Today, reducing the resistance of tumor cells to chemotherapeutic drugs has become an urgent challenge in cancer treatment. FENG et al. [42] first found that Rg5 significantly enhanced the antitumor effects of chemotherapeutic drugs such as doxorubicin (DOX), paclitaxel (PTX), docetaxel (TXT), docetaxel (TXT), and doxorubicin (DOX) in MDR cell lines A2780/T and A549/T at non-cytotoxic concentrations, without affecting the toxicity of sensitive cells. Rg5 overcomes ABCB1 transporter-mediated chemoresistance and improves the efficacy of chemotherapeutic drugs, suggesting that Rg5 may be a good candidate for the treatment of MDR.

3. 2 Anti-allergic and anti-inflammatory effects

In 2015, AHN et al. [43] used human keratinocytes and macrophages to evaluate the anti-atopic dermatitis (AD) effects of the rare ginsenosides Rg5/Rk1 in vitro in order to verify the anti-AD effects of the rare ginsenosides Rg5/Rk1. Keratinocytes and macrophages produce different chemokines and cytokines that are considered to be key regulatory factors in the pathogenesis of AD. It was found that the expression of chemokines and cytokines was enhanced when stimulated by TNF-α/IFN-γ and lipopolysaccharide (LPS). Pretreatment with Rg5/Rk1 attenuated NF-κB/p38 MAPK/STAT1 signaling, and reduced lipopolysaccharide-induced nitric oxide and reactive oxygen species (ROS) production in macrophages. In addition, TNF-α/IFN-γ and lipopolysaccharide-induced chemokines and cytokines were significantly inhibited.

Rg5/Rk1 was found to strongly inhibit NF-κB/p38 MAPK/STAT1 signaling and the mRNA and protein expression of keratinocytes and macrophages, suggesting that this compound has potential anti-AD effects. In order to evaluate the anti-allergic effects of ginsenoside Rg5 and its metabolites, ginsenoside Rg5 was orally administered and intraperitoneally injected, and it was found that both were effective in inhibiting the passive cutaneous anaphylaxis (PCA) of mice caused by IgE antigen complexes. In addition, they had an anti-inflammatory effect on TAP-induced mouse ear inflammation. Pharmacological activity studies have shown that oral administration of ginsenoside Rg5 can effectively reduce IgE-induced allergic symptoms such as rhinitis and asthma [44]. Ginsenoside Rg5 can also improve inflammatory skin diseases such as contact dermatitis or psoriasis [45].

LEE et al. [46] studied the anti-inflammatory effect of ginsenoside Rg5 in BV2 microglial cells stimulated by lipopolysaccharide and its molecular mechanism. The results showed that Rg5 inhibited lipopolysaccharide-induced nitric oxide production and promoted TNF-α secretion. In addition, Rg5 inhibited the mRNA expression of iNOS, TNF-α, IL-1 β, COX-2 and MMP-9 mRNA expression, and may provide effective treatment for various neuroinflammatory diseases. LEE et al. [47] prepared rare saponins Rk1 and Rg5 by steaming ginsenoside Rd at 120 °C for 3 h and used them to treat sepsis caused by cecal ligation and puncture. The results showed that they could effectively reduce mortality and tissue damage in the body. The study found that Rk1 and Rg5 may treat sepsis by inhibiting HMGB1 secretion and the expression of HMGB1 receptors. The survival rate of diseased mice was significantly improved after treatment. This indicates that Rk1 and Rg5 can be used to treat severe vascular inflammatory diseases such as sepsis and septic shock.

3.3 Effects on the nervous system

Ginsenoside Rg5 is a major active monomer in ginsenosides that is beneficial for the treatment of Alzheimer's disease [48]. Studies have found that Rg5 can significantly inhibit memory impairment, cognitive dysfunction, and improve learning and memory in mice. In 2017, CHOI et al. [49] found that heat stress can cause cycle arrest in HT22 hippocampal cells, In addition, it can cause HT22 cell damage and activate and reduce memory-related molecules. Rg5, as a natural compound with neuroprotective effects, can effectively control oxidative stress and heat stress-induced cognitive damage by regulation. Data show that scopolamine can cause memory impairment in healthy young people, with symptoms similar to those of Alzheimer's disease. Scopolamine acts on the cholinergic system, which is associated with the functional loss of Alzheimer's disease in patients with dementia, causing damage to it. Ginseng saponins Rg5 and Rh3 may protect against memory deficits by inhibiting acetylcholinesterase activity, increasing neurotrophic factor expression, and activating the binding of cAMP response factor and protein [50]. In order to explore the effects of ginsenoside Rg5 and other ginsenosides on ethanol-induced memory impairment in mice, the passive avoidance crossing method was used to stimulate memory in mice with electric shocks. The results showed that ginsenosides Rg5 and Rk1 and other ginsenosides have a significant effect on improving memory impairment symptoms and have a cognitive enhancing effect [3].

Depression, also known as depressive disorder, is clinically manifested as low mood and slowed movements. In recent years, with the increase in work pressure and the accelerated pace of life, the prevalence of depression has been rising year by year. In 2017, it was found that Rg5 has an antidepressant effect in mice by regulating the activity of the hippocampal brain-derived neurotrophic factor signaling pathway, which provides ideas for the development of new antidepressants with higher efficacy and fewer side effects.

3. 4   Cytotoxic effect

Since the 1980s, ginseng has been used in China to treat cardiovascular disease, but the mechanism of action was not clear at the time. Recent studies have shown [52] that Rg5 acts as a new natural agonist of IGF-1R in the vascular system, improving hypertension, promoting angiogenesis and vasodilation. Rg5 activates multiple signaling pathways by promoting angiogenesis, reduces blood pressure through vasodilation, and has a significant repair effect on new blood vessels and endothelial function. The use of Rg5 to treat cardiovascular diseases caused by endothelial dysfunction (such as ischemia or hypertension) is of positive significance. Osteoblast differentiation markers include alkaline phosphatase (ALP) activity, collagen content, calcium deposition and mineralization. After being stimulated by various growth and differentiation factors, cells differentiate into mature osteoblasts after going through a development cycle. Studies have shown that Rg5 / Rk1 stimulates cell growth and differentiation by increasing ALP activity, collagen synthesis and the formation of mineralized nodules. The marked increase in osteoblast differentiation markers indicates that Rg5/Rk1 promotes osteoblast growth and differentiation in an in vitro system. This suggests that the use of Rg5/Rk1 may enhance bone development and prevent bone metabolic disorders such as osteoporosis, and may help in the development of new therapeutic drugs [53].

3.5 Other effects

In addition, ginsenoside Rg5 has been studied for its effects on diabetes, liver and kidney toxicity, and whitening. PONNURAJ et al. [54] found that Rk1/Rg5 compound treatment can increase the binding site of the IGF-2R receptor, enhance glucose absorption, and improve insulin sensitivity and response through the CHOP-mediated signaling pathway, which plays a role in the treatment of diabetes. Ginsenoside Rg5 can also enhance the protective effect on the kidney by improving the nephrotoxicity induced by cisplatin in mice (including reducing oxidative stress, inhibiting inflammation and apoptosis, etc.) [10], thus broadening the clinical application of platinum compounds in cancer treatment. At the same time, Rg5 also has a certain pharmacological effect on acute liver failure caused by excessive use of acetaminophen (APAP) [30]. In addition to its good pharmacological effects, ginsenoside Rg5 also shows good results in cosmetology. In 2018, JIN et al. [55] evaluated the whitening activity of ginseng. Human skin and zebrafish embryos were used as experimental materials to test the whitening effect of the sample. The results showed that Rg5/Rk1 in ginsenosides achieves whitening effects by activating the MEK-ERK signaling pathway. This study is the first to demonstrate the whitening effect of black ginseng extract.

4 Discussion

Ginseng is a natural herb commonly used in China and Korea. It has been used for thousands of years in Asia as a medicine to prevent disease and prolong life. Ginseng saponins are the main active ingredients isolated from ginseng. As a secondary rare ginseng saponin, ginsenoside Rg5 is present in low concentrations in ginseng, making it difficult to prepare. Research on its pharmacological activity has not been widely conducted. Therefore, more detailed and in-depth research on the preparation, isolation, pharmacological analysis, structure-activity relationship and clinical application of ginsenoside Rg5 monomers is of great reference value for the research and development of new drugs. Ginsenoside Rg5 has a variety of biological activities and pharmacological effects. With the current emphasis on the production and application of natural medicines, the development and utilization of ginsenoside Rg5 will become another hotspot in the research of traditional Chinese medicine.

Reference：

［1 ］ SUN G Z，LIU Z，ZHANG JJ．General situation about the  pharmacological  research  on   Ginsen  Panaxadiol Saponins ［J］．Chinese  Agricultural  Science  Bulletin，2005 (5) : 136 －140.

［2 ］ HONG Y J，KIM  N，LEE K，et al．Korean red ginseng  (Panax ginseng ) ameliorates type  1  diabetes  and  re- stores immune cell compartments［J］．Journal of Eth- nopharmacology，2012，144(2) : 225－233 .

［3 ］ ZHANG J，WANG S  R , CHEN  Q  C，et al．Effects of  ginsenosides Rg3  (R) ，Rg3  ( S) and Rg5 / Rk1  on mem- ory improvement of ethanol treated Mice ［J］．Journal  of Jilin  Agricultural  University，2006，28(3) : 283－284 .

［4 ］ KIM Y J，KWON H C，KO H，et al．Anti-tumor activity of the ginsenoside Rk1  in human hepatocellular car- cinoma cells through inhibition of telomerase activity  and induction of apoptosis［J］．Biological ＆ Pharma- ceutical Bulletin，2008，31 (5) : 826－830 .

［5 ］ LIU T G，HUANG Y，CUI D D，et al．Inhibitory effect  of ginsenoside Rg3  combined with gemcitabine on an- giogenesis and  growth  of lung   cancer  in  mice［J］.   BMC Cancer，2009，9( 1) : 250 .

［6 ］ REN S G．Studies on the nutritional and physicochem- ical  properties  of  micronized  soybean  meal  powder［D］．Ya'an :  Sichuan Agricultural University，2009.

［7 ］ DOU D，CHEN  Y，REN J，et al．Ocotillone-type gin- senoside from  leaves  of  panax   ginseng ［J］．J  Chin  Pharmacol Sci，2002 ; 11 ( 4) : 119 －121.

［8 ］ KIM  S  I，PARK  J  H，RYU J H，et al．Ginsenoside  Rg5 ，a genuine dammarane glycoside from Korean red  ginseng［J］．Archives  of  Pharmacal  Research  ( Se- oul) ，1996，19(6) : 551－553 .

［9 ］ LEE S M ，SHON  H J，CHOI C  S，et al．Ginsenosides  from heat processed ginseng［J］．Chem  Pharm  Bull， 2009，57( 1) : 92－94 .

［10］LI W，YAN M H，LIU Y，et al．Ginsenoside Rg5  amel- iorates    Cisplatin-Induced    nephrotoxicity     in    mice  through  inhibition  of  inflammation，oxidative  stress， and apoptosis［J］．Nutrients，2016，8(9) : 566－583 .［11］ LIANG  L，TAO  H E，TINGWEI D  U，et al．Ginsen- oside-Rg5  induces apoptosis and DNA  damage in hu- man cervical cancer cells［J］．Molecular Medicine Re- ports，2015，11 (2) : 940－946 .

［12］KIM T W，JOH E H，KIM B，et al．Ginsenoside Rg5  a- meliorates lung inflammation in mice by inhibiting the  binding of LPS to toll-like receptor-4 on macrophages  ［J］．International   Immunopharmacology，2012，12  ( 1) : 110 －116.

［13］YAO H，JIN Y  R , YANG J，et al．Conversion rule of rare  ginsenosides  produced from   major  ginsenosides by confined microiwave promoted degradation method ［J］．Chemical Journal of Chinese Universities ，2014，35 ( 11) : 2317－2323 .

［14］ GU  C  Z，LV  J  J，ZHANG  X  X，et  al．Triterpenoids with promoting  effects on the differentiation of PC12 cells  from   the  steamed  roots  of  panax  notoginseng ［J］．Journal   of   Natural   Products，2015，78  ( 8 ) : 1829 －1840.

［15］MIAO X S，METCALFE C D，HAO C，et al．Electrospray   ionization   mass   spectrometry   of   ginsenosides  ［J］．Journal  of  Mass  Spectrometry，2002，37  ( 5 ) :  495－506 .

［16］SHIN J H，PARK Y J，KIM  W，et al．Change of gin- senoside  profiles  in  processed  ginseng s  by  drying， steaming，and  puffing ［J］.Journal   of  Microbiology  and Biotechnology，2019，29(2) ，222－229 .

［17］ KIM  S  N，HA  Y  W，SHIN   H，et  al．Simultaneous quantification  of   14  ginsenosides  in  Panax  ginseng C．A．Meyer  ( Korean  red  ginseng )  by  HPLC-ELSD and its  application  to  quality  control［J］．Journal of Pharmaceutical  and  Biomedical  Analysis，2007，45 ( 1) : 164 －170.

［18］ KWON  S  W，HAN  S  B，PARK  I H，et  al．Liquid  chromatographic  determination  of  less  polar  ginsen- osides in processed  ginseng［J］．Journal  of  Chroma- tography A，2001，921 : 335－339 .

［19］KIM  W  Y，KIM  J  M ，HAN S B，et  al．Steaming of  ginseng at high temperature enhances biological activi- ty［J］．Journal  of  Natural Products，2000，63  ( 12 ) :  1702 －1704.

［20］JO  S  K，KIM  I  S，YOON K S，et  al．Preparation  of ginsenosides  Rg3 ，Rk1 ，and  Rg5 -selectively  enriched ginseng s by  a  simple  steaming  process［J］．European Food   Research   and   Technology，2015，240   ( 1 ) : 251－256 .

［21］LEE J H，SHEN G N，KIM  E K，et al．Preparation of black ginseng and its antitumor activity［J］．Korean J Orient Phy siol Pathol，2006，20:951－956 .

［22］KANG K S，YAMABE N，KIM  H Y，et  al．Effect of  sun ginseng methanol extract on lipopolysaccharide-induced liver injury in rats［J］．Phytomedicine，2007，14 ( 12) : 840－845 .

［23］ KANG  K  S，KIM  H  Y，YAMABE  N，et  al．Ste- reospecificity in hydroxyl radical scavenging activities  of four ginsenosides produced by heat processing［J］.   Bioorganic and Medicinal Chemistry  Letters，2006，16  ( 19) : 5028－5031 .

［24］AN Y E，AHN S C，YANG D C，et al．Chemical con- version  of  ginsenosides  in  puffed  red  ginseng ［J］.   LWT-Food  Science  and  Technology，2011，44  ( 2 ) :  370－374 .

［25］KIM J H，AHN S C，CHOI S W，et al．Changes in ef- fective components of ginseng by puffing［J］．Korean  Soc，2008，51 (3) : 188 －193.

［26］LEE J H，SHEN G N，KIM  E K，et al．Preparation of black ginseng and its antitumor activity［J］．Korean J Orient Phy siol Pathol，2006，20:951－956 .

［27］HUANG D，LI Y，ZHANG M ，et al．Tartaric acid in- duced  conversion  of  protopanaxadiol  to   ginsenosides  Rg3  and Rg5  and their in situ recoveries by  integrated  expanded bed adsorption chromatography［J］．Journal

of Separation Science，2016，39( 15) : 2995－3001 .

［28］GUO  D  D，CHENG  L  Q，LI  L，et  al．Preparation  of ginsenoside  Rg5    with  ginseng   fibrous  root  powder ［J］．Science  and  Technology  of  Cereals  oils  and Foods，2017(3) :70－76 .

［29］GUO D D，CHENG L  Q，LI L，et al．Optimization of  microwave assisted extraction of ginsenoside Rg5  from  ginseng fibrous root powder by response surface meth- odology ［J］．Science and Technology  of Food Indus- try，2017(11) : 229－239 .

［30］WANG Z，HU J，YAN M ，et al．Caspase-mediated anti-apoptotic effect of ginsenoside Rg5 ，a main rare gin- senoside，on  acetaminophen-induced  hepatotoxicity  in  mice［J］．Journal of Agricultural and Food Chemistry， 2017，65 (42) : 9226－9236 .

［31］GUAN D  P，WANG  H，LI  W，et  al．Optimization  of  preparing technology  of  ginsenoside  Rk1   and Rg5   by  high-temperature pyrolysis ［J］．Shanghai  Journal  of  Traditional Chinese  Medicine， 2015 ( 1) : 91－95 .

［32］ SUN  C  P，GAO  W  P，ZHAO  B  Z，et  al．On  conver- sion of  protopanaxadiol  type  saponins  to  ginsenoside  Rg5  by lemon ［J］．Chinese  Traditional Patent  Medi- cine，2013，35 ( 12) : 2694－2698 .

［33］LIU H Y，WANG C X，YANG Y，et al．Optimization  of production of ginenoside  Rg5  by  microwave-assis- tant degradation method of total  saponins from  stems  and leaves of Panax  notoginseng ［J］．Chinese Tradi- tional  and  Herbal  Drugs ，2018 ( 14 ) :  3245－3251 .

［34］KIM S J，KIM A K．Anti-breast cancer activity of Fine Black ginseng   ( Panax  ginseng  Meyer )  and ginsen- oside Rg5 ［J］．J  Ginseng  Res，2015，39  ( 2 ) : 125  -  134.

［35］ LIANG  L，TAO  H E，TINGWEI D  U，et al．Ginsen- oside-Rg5  induces apoptosis and DNA  damage in hu- man cervical cancer cells［J］．Molecular Medicine Re- ports，2015，11 (2) : 940－946 .

［36］ZHANG D M ，WANG A F，FENG J P，et al．Ginsen- oside  Rg5    induces   apoptosis   in   human   esophageal  cancer cells through the phosphoinositide? 3  kinase /  protein  kinase  B   signaling  pathway ［J］．Molecular  Medicine Reports，2019，19 :4019－4026 .

［37］ZHANG D M ，ZHANG Q，LIU L L，et al．Ginsenoside  Rg5  promotes apoptosis of esophageal cancer Eca-109  cells by  down-regulating  Bcl-2  protein ［J］．Chinese  Journal of Laboratory Diagnosis ， 2018，22(4) :700－703 .

［38］ MEI Z  H，DING  Y  M ，LI  F，et  al．Effect  of  ginsen- oside Rg5  on human esophageal cancer cells ［J］．Chi- nese Journal of Laboratory Diagnosis ，2016( 12) : 1982 －1985.

［39］LI W，LIU W，ZOU J J．Effect of ginsenoside Rg5  on  invasion  and   migration  of  gastric  cancer  BGC-823  cells via miR-125b / STARD13 / NEU1  signaling  path- way［J］．Chinese Journal of Experimental Traditional  Medical Formulae，2018，24(22) : 138 －142.

［40］LI W，LIU W，ZOU J J．The effect of ginsenoside Rg5   on proliferation，apoptosis and related factors of apop- tosis of BGC-823 cellin gastric cancer ［J］．Journal of  Emergency in Traditional Chinese Medicine ，2018(6) : 955－958 .

［41］CHOI P，PARK J Y，KIM  T，et al．Improved antican- cer  effect  of  ginseng   extract  by   microwave-assisted  processing   through   the   generation of ginsenosides  Rg3 ，Rg5 and  Rk1［J］．Journal  of  Functional  Foods， 2015，14:613－622 .

［42］ FENG  S  L，LUO  H  B，CAI  L  A，et  al．Ginsenoside Rg5  overcomes chemotherapeutic multidrug  resistance mediated by  ABCB1  transporter : in  vitro  and  in  vivo study［J］．Journal of Ginseng Research，2018，44 (2) : 247－257 .

［43］AHN S，SIDDIQI M  H，ACEITUNO  V  C，et al．Ginsenoside  Rg5 : Rk1   attenuates  TNF-α/ IFN-γ-induced  production of thymus-and activation-regulated chemo- kine  (TARC / CCL17) and  LPS-induced NO  production via ownregulation   of NF-κB /p38   MAPK /  STAT1 signaling  in  human  keratinocytes  and  macro- phages［J］．In  Vitro  Cellular ＆ Developmental  Biolo- gy-Animal，2016，52(3) : 287－295 .

［44］SHIN Y W，BAE E A，HAN M J，et al．Metabolism of ginsenoside Rg5 ，a main constituent isolated from  red  ginseng，by human intestinal microflora and their anti- allergic effect［J］．Journal of  Microbiology  and  Bio- technology，2006，16( 11) : 1791 －1798.

［45］SHIN Y W，BAE E A，KIM  D  H．Inhibitory effect of  ginsenoside Rg5  and its metabolite ginsenoside Rh3   in  an oxazolone-induced mouse chronic dermatitis model ［J］．Archives of Pharmacal Research ( Seoul) ，2006， 29(8) : 685－690 .

［46］LEE Y Y，PARK J S，JUNG J S，et al．Anti-Inflammatory effect of ginsenoside  Rg5  in Lipopolysaccharide- Stimulated  BV2   microglial  cells ［J］．International  Journal of  Molecular  Sciences，2013，14 (5) : 9820  -   9833.

［47］LEE W，CHO S H，KIM J E，et al．Suppressive effects  of  ginsenoside  Rh1   on  HMGB1-mediated  septic  re- sponses［J］．The American Journal of Chinese Medi- cine，2019，47( 1) : 1 －15.

［48］JIN L，JIN X S．Protective effect of red ginseng saponin Rg5  on MPP +  induced oxidative stress injury  in SH-SY5Y cells ［J］．Lishizhen Medicine and Materia Medica Research，2015 (4) : 878 -  880.

［49］CHOI S  Y，KIM  K  J，SONG  J  H，et  al．Ginsenoside  Rg5   prevents  apoptosis  by  modulating  heme-oxygen- ase-1 / Nrf2 signaling and alters the expression of cognitive  impairment-associated  genes  in  thermal  stress-exposed HT22 cells［J］．Journal of Ginseng Research， 2018，42(2) : 225－228 .

［50］ KIM  E  J，JUNG  I  H，VAN  LE  T  K，et  al．Ginsen- osides Rg5  and Rh3  protect scopolamine-induced mem- ory deficits in mice［J］．Journal of Ethnopharmacolo- gy，2013，146( 1) : 294－299 .

［51］XU D，WANG C，ZHAO W，et al．Antidepressant-like  effects of ginsenoside Rg5  in mice: Involving of hippo- campus BDNF  signaling  pathway ［J］．Neuroscience  Letters，2017，645 :97 －105.

［52］CHO Y L，HUR S M ，KIM J Y，et al．Specific activa- tion of  insulin-like  growth  Factor-1  receptor  by  gin- senoside  Rg5   promotes  angiogenesis  and  vasorelax- ation［J］．Journal  of  Biological  Chemistry，2015，290  ( 1) :467－477 .

［53］SIDDIQI M  H，SIDDIQI M Z，AHN S，et al．Stimula tive effect of Ginsenosides Rg5 : Rk1  on murine osteo- blastic M C3T3-E1  cells［J］．Phytotherapy  Research， 2014，28( 10) : 1447 －1455.

［54］PONNURAJ S P，SIRAJ F，KANG S，et al．Ameliora- tion of insulin resistance by Rk1  + Rg5  complex under  endoplasmic reticulum  stress  conditions［J］．Pharmacognosy Research，2014，6(4) : 292－296 .

［55］JIN Y，KIM J H，HONG H D，et al．Ginsenosides Rg5 and Rk1 ，the  skin-whitening  agents in black ginseng ［J］．Journal of Functional Foods，2018，45 :67－74 .