Recent Progress in Structural Modification and Activities on Panax notoginseng Saponins

Jinna ZHOU, Xiaorong ZHOU, Cheng ZOU, Mu WANG, Hong YU

1. College of Science, Tibet University, Lhasa 850001, China; 2. Department of Pharmacy, Sanming First Hospital, Affiliated Hospital of Fujian Medical University, Sanming 365000, China; 3. School of Pharmaceutical Science & Yunnan Key Laboratory of Pharmacology for Natural Products, Kunming Medical University, Kunming 650500, China; 4. Plant Science College, Tibet Agriculture & Animal Husbandry University, Nyingchi 851418, China; 5. Yunnan Herbal Laboratory, College of Ecology and Environmental Sciences, Yunnan University, Kunming 650106, China

Abstract This paper reviews recent progress in the structural modification and activities on Panax notoginseng saponins (PNS). PNS can not only improve the function of cardio-cerebral system, central nervous system and immune system, but also reveal anticancer, anti-aging and anti-oxidation activities. In order to solve the problem of low bioavailability and poor absorbability of PNS in vivo, usually, the researchers modified the structure of PNS with three methods: glycoside cleavage (including acid hydrolysis, sulfation, etc.), biotransformation method (including enzyme hydrolysis, microbial transformation) and combinatorial chemical method. It was found that the structural modification sites of PNS were single, mainly aimed at C-3, C-6 and C-20, which provided a new perspective for the structural modification of PNS. Therefore, structural modification on PNS with high yield and ready availability are significant in the discovery of new active ingredients and industrialization. Derivatives of PNS are applied to research of structure-activity relationship, which is beneficial to the development of new medicines.

Key words Panax notoginseng saponins, Structural modification, Activities

1 Introduction

Panaxnotoginseng, also known as Tianqi and Jinbuhuan, with over 600 years of pharmaceautical application in China, is a genuine regional medicine in Wenshan, Yunnan Province.P.notoginsenghas been reported to exhibit a variety of pharmacological activities, such as hemostasis, apocatastasis, analgesis. The chemical components ofP.notoginsengcan be divided into two major categories: saponins and non-saponins. Non-saponins are composed of polysaccharides, amino acids, flavonoids, organic acids, sterols and other ingredients[1].P.notoginsengsaponins (PNS) are dammarane-type tetracyclic triterpenoids. According to the substitution group at C-6 of PNS, it can be classified into protopanaxadiol saponins (PPDS) and protopanaxatriol saponins (PPTS), whose aglycones are 20(S)-protopanaxadiol (PPD) and 20(S)-protopanaxatriol (PPT), respectively (see Fig. 1). Different fromP.ginsengandP.quinquefolius, oleanane saponins have not been discovered fromP.notoginsengso far.[2]. Rb1 is the main ingredient of PPD, while Re, Rg1 and R1 are the major components of PPT. To obtain more natural products with higher bioactivities, the structural modification on PNS is usually carried with three methods, such as glycoside cleavage, bioconversion and combinatorial chemistry. Modern pharmacological studies also proved that PNS showed remarkable effects on cardio-cerebral-vascular system, antitumor, central nervous system and immune system.

Fig. 1 Structures of PPD and PPT

PNS are major active components inP.notoginseng. Yoshikawaetal.isolated 12 new saponins from roots ofP.notoginsengsince 1997, such as notoginsenoside A, B, C, D, E, G, H, I, L, M, N, O, P, Q, S and T[4-7]. At present, more than 70 saponins were isolated and identified from the flower, basal part of stem and rhizomes[8-10]. C-3, C-6 and C-20 may be substituted by sugar chain(s).

2 Structural modification

PNS belong to damarane-type tetracyclic triterpenes. Furthermore, a side chain with 5 carbons and 1 double bond (C-24) are located in C-20 position. Meanwhile, there is a hydroxyl group in C-3 and C-12 sites of the A and C rings. On behalf of enhancing their activities, structure-activity relationship is extensively studied.

2.1 Glycoside cleavageThe spatial configuration of glycoside bonds is crucial to the activity of PNS. Some changes in glycosidic bonds may lead to change of bioactivities. C-3, C-6 and C-20 sites of PNS may be acetal (s) of sugar of monomer (s) or polymer(s). Therefore, with various conditions, a series of derivatives can be obtained by fracture of glycoside bonds easily. Via structural identification, the structure-activity relationship and bioactivities were also studied, and derivatives with higher bioavailability, lower toxicity or lower side effects were discovered. Glycoside cleavage methods were discussed as mentioned below.

2.1.1Acid hydrolysis. PNS can be hydrolyzed by acid hydrolysis by dilute hydrochloric acid, sulfuric acid, nitric acid and acetic acid. Hydrolysis is carried out at different temperature and acidity to prepare various compounds. Much works in early time proved that 20 (S)-PPD and 20 (S)-PPT are original aglycones of PNS. The final products of acid hydrolysis are panoxadiol and panoxatriol, which are secondary aglycones formed by rearrangement of side chains from original aglycones under acid catalysis. At the same time, Yang Chongrenetal.also found that the conversion of the side chain of 20 (S)-PPD was dehydrogenation, dehydration and addition reaction under the acid catalysis and the induction of the hydroxyl group at C-12 position, which was realized by rearrangement or cyclization[11]. Generally, the side chain of PNS cannot be cyclized by alkali hydrolysis. The starting material (7) and the cyclization product (8) from acidic hydrolysis were summarized[12].

Under ordinary acid hydrolytic conditions, configuration of C-20 position was easily affected by acid, and both of isomers of 20 (S)-PPD and 20 (R)-PPD were obtained. Chen Yegaoetal.[13]found a mild acid hydrolysis method: PNS were hydrolyzed in boiling 2 mol/L NaOH solution for 8 h, followed by addition of excessive concentrated HCl at room temperature for 7 h. The mixed products were extracted with ethyl acetate, followed by isolation silica gel column chromatography. As a result, only 20 (S) was obtained. Starting from PPD and PPT, Professor Wei Junxianetal.[14]synthetized succinate together with glucosides of PPD and PPT, some of which showed significant anti-cytotoxic activity, and anti-tumor activity. Simultaneously, Zhang Ying[15]performed the aforementioned method to hydrolyze PPD by 36.5% HCl, and to stir the solution for 24 h, finally panaxadiol, PPD, 3-acetyl-25-OH-PPD and 25-OH-PPD were obtained. Then succinic anhydride reaction was carried out with PPD as raw material.

Panoxadiol and panaxatriol were oxidized by pyridinium chlorochromate (PCC) and dichloro dicyanobenzo quinone (DDQ), to obtain the target compounds (16, 18, 20 and 22). With ring-opening reaction of the derivatives with organic acids, the compounds 17, 19, 21 and 23 were obtained. It was detected that the derivatives obtained via panoxadiol oxidation revealed cytotoxicity, whereas the derivatives obtained by panaxatriol oxidation showed no cytotoxicity. In consequence, considering that the introduction of an oxygen-containing substituent at the C-6 position on the compounds may bring about less cytotoxicity[16-17].

Rd and Rb1 are PPDS, and the sugar chain attached to the C-21 site is different; compared with Rd, hydrolyzing the outer sugargroup of Rb1 can achieve higher pharmacological activities. Furthermore, the side chain of PPD was modified via epoxidation and catalytic hydrogenation, which provided a simple method for epoxidation of olefins with non-essential acid to purify compounds conveniently. Besides, in catalytic hydrogenation experiments, hydrogen is absorbed into nickel alloy pores, and active hydrogen atoms are formed to reduce the double bonds.

2.1.2Alkaline hydrolysis. On account of the stable condition with alkali degradation, PNS are hydrolyzed to produce secondary glycosides, probiotic and secondary saponins with good pharmacological activities are obtained. In comparison with acid hydrolysis, alkali degradation is especially suitable for acid-labile saponins, which has the advantages of less hydrolysate, stable products and high yield. Chen Yegao[18]used NaOH solution to heat PNS for 8 h in a water bath to obtain PPD. Chen Yingjieetal.used Rg2 as the raw material, and hydrolyzed that with n-butanol solution of n-butanol or n-butanol solution of sodium hydride to obtain 20 (S)-PPT and Rh1[19]. Gao Lusha hydrolyzed Panax quinquefolius saponins (PQS) via oxidation and obtained 4 compounds[20]. Similarly, Re was degraded by NaOH solution to obtain 20 (S)-PPT, 20(S)-Rh1, and 20 (S)-Rg2[21]. Xu Boetal.reported a method for the conversion of 20 (S)-PPD and 20 (S)-PPT at high temperature and strong alkaline condition. Light derivatives of the side chain of 20 (S)-propane triol were obtained by acetylation, oxidation and saponification with "one-step method"[22].

2.1.3Acetylation and sulfation. Along with acid and alkali hydrolysis, new functional groups linked to compounds are a hot spot in chemical modification in recent years as well. Therefore, chemical modification (acetylation, sulfation, phosphorylation, methylation,etc.) provides new ideas for that. The pyridine-acetic anhydride method is a good example to illuminate particulars. Acetylation is a process in which a mixture of acetic anhydride and Lewis acid or concentrated sulfuric acid reacts with saponins to decompose the glycosides in saponins slowly, and finally to form sugar, secondary glycosides and glycosides. Korean scholars, with 20 (S)-PPD and acetic anhydride, synthesized 20 (S)-PPD-3B, 12B-diacetate with pyridine as the solvent, and then hydrolyzed 12-acetyl group with K2CO3/MeOH synthesis of 20 (S) PPD-3B acetate[23]. Beyond that, Dr. Li acetylated and sulfated Rb1 via the classical acetic anhydride-pyridine method and Wolfrom method[24]. Wang Lu[25]also reported the modification on Rh2 with Wolfrom method to obtain two new compounds (Fig. 2). One of the -SO3Na groups is substituted on the H of the -OH at the C-12, and one is substituted on the H of the -OH at the Glc-C6 one replaced. Meanwhile, studies showed that it increased the immune activity, which provided a basis for improving the activity of carbohydrate in view of antitumor activity and cardio cerebral vasoactivity.

Fig. 2 Structures of 2 new compounds

2.1.4Smith degradation. Smith degradation, which is suitable for the degradation of acid-labile saponins, is one of the mild reactions. This method mainly uses sodium peroxyacid to oxidize two adjacent hydroxide groups of the sugar to produce a dialdehyde which is then reduced to diols with low stability and simple acetal structures. The glycol is hydrolyzed by dilute acid at RT to obtain intact aglycones. Saponins and aglycones, with less sugar, monosaccharide or oligosaccharide, are prepared by smith degradation. Compared with acid and alkali hydrolysis, first of all, smith degradation is mild, and has more complete glycogen. Secondly, the process is divided into three steps[26], in which aglycon and aglycone derivatives can be produced to further study the pharmacological activities of the aglycon and derivatives.

2.1.5Heating hydrolysis. Wang[27]found that Rg3 and Rg5 were the most abundant ginsenosides at 120 ℃. In the process of heating extraction, some saponins inP.notoginsengbroke down and formed secondary saponins. For example, Rg3 and Rg2 were used to prepare Rh2 and Rh1, respectively.

2.1.6Metabolic engineering. Some experiments on the metabolism of PNS in human and rat intestines were carried outinvitro[27], and studies showed that there were two ways to metabolize ginsenosides of different structural types in the intestinal flora of human and rats[28-29]. The main metabolic pathway of PPD with Rb1 as an example: Rb1→Rd→F2 (Rg3)→Compound k (Rh2)→20 (S)-PPD. The primary metabolic pathway of PPT with Re as an example: Re→Rg1→Rh1 (F1)→20 (S)-PPT. At the same time, metabolic engineering is used to successfully produce PPT in transgenic tobacco[30]. Rd, slightly degraded by gastric juice, is one of the main metabolites of PPDS in human intestinal tract and the glycosyl moiety is relatively stable. The metabolites are under the action of enzymes and microorganisms in intestinal fluid. The glycosidic bond is gradually broken, and the sugars attached to the C-3, C-6 and C-20 positions are gradually degraded from the end[31-33].

2.1.7Chemical synthesis. The basic components of PNS are PPT and PPD. Synthetic pathways are mainly divided into the following: PPT→-Rb1→-Rg1→-R1 or PPD→-Rh2→-Rg3→Rd→Rb1. In flowers, PPD is the main pass: PPD→Rh 2→G-Rg3→Rd→Rb2→Fc[34]. The study used "one-pot method" to synthesize Rh2 and bioactive non-natural ginsenosides with PPD as aglycon[35]. In our research group, the glycogen derivatives were obtained directly by Jones oxidation by "one pot method" with PPD as the raw material, and the hexadarane triterpenes of 6 carbon atoms were removed from side chains and was reduced by sodium borohydride[36-37].

In addition, our research group obtained 10 derivatives of PPD and PPT through: Jones oxidation→NaBH4 reduction→ PCC oxidation→NaBH4 reduction, respectively; 4 compounds were new compounds[38]. The study found that C-12 glycosylated saponins produced by semi-synthesis by PPD had higher cytotoxicity than natural saponins[39].

2.2 Biotransformation methodThe biotransformation method is a biochemical process for structurally modifying triterpenoidsaponins via biological systems or enzyme inhibitors. Advantages are as follows: mild conditions, no need for protection or deprotection, high regional and three-dimensional selectivity, ability to complete some reactions that cannot be completed by chemical methods, environmental friendliness, low energy consumption and environmental protection. Currently, biotransformation methods include enzyme cleavage and microbial transformation.

2.2.1Enzyme cleavage. Enzyme cleavage has a high degree of substrate specificity. Different enzyme reactions on glycosidic bonds of various configurations and diverse sugars, so that the structure is transformed to achieve target hydrolysis, and the corresponding products are obtained. In comparison with traditional chemical hydrolysis, enzymatic hydrolysis is mild, selective and efficient, easy to control and to scale up, and can also synthesize drugs which are difficult to be synthesized by chemical methods. Enzyme cleavage is suitable for the industrial production of drugs and is another way to produce some natural medicinal ingredients. In this study, glycosylated PPD ginsenoside was used as the substrate to transform Rc into compound K with prevention of disease and skin anti-aging effect through the mutation enzyme[40]. Different glycosyl groups are linked at C-6 and C-20 loci of PPTS. The glycoside bonds at these two loci break under enzyme cleavage, and one or more sugars are lost to obtain new saponin monomers. The preparation of rare ginsenosides by biotransformation is a very popular and effective technology. The basic conversion pathway for the preparation of rare ginsenosides is used by hydrolysis PPTS. For example, Shinetal.obtained β-xylosidase from the thermoanaeroba cterium thermosaccharolyticum, and hydrolyzed R2 with β-xylosidase to obtain Rh1[41].

It was found that ginsenoside glycosidase IV could hydrolyze the glycosyl groups of panaxatriol saponins C-6-O to form aglycones, but could not hydrolyze the glycosyl groups of C-3-O and C-20-O on panaxadiol saponins[42]. Jiang Binhuietal.transformed notoginsenoside Fe by industrial enzyme (beta-glucanase) and obtained two compounds, namely ginsenoside-Mc and compound C-K[43].

2.2.2Microbial transformation. Microbial transformation is the process of transforming a compound into a more valuable product by using one or more enzymes secreted by microbial cells. Microorganisms are easy to be cultured and observed, and a rich enzyme system is secreted. Leeetal.obtained a recombinant β-glucosidase from the thermophilic archaea, which has the specificity of selectively hydrolyzing the glucosyl bond at the C-6 position and the glucosyl group on the PPTS. The hydrolysis sequence is Rf >Rg1 >Re > R1 >Rh1 > R2[44]. Cheng Leqinetal.used the crude enzyme solution produced by microbacterium esteraromatium GS514 to selectively hydrolyze the β-D-glucopyranose linkage of C-20 on Re to be converted into Rg2. This enzyme can also hydrolyze the glucose on the C-20 locus of Rg1 and convert that into Rh1 and PPT. Chenetal.[45]found that Absidia coerulea AS 3.338 9 had high yield in the transformation of ginsenosides by 42 fungi. With using PPTS as substrates, the strain was able to change the side chain to produce different new saponins. Chenetal.discovered that the microbial SP. GS514 converted Rb1 into 20 (S)-Rg3, and inferred the transformation pathway: Rb1→Rd→Rg3[46], thereby confirming that the production of Rh2 in Saccharomyces cerevisiae could be greatly increased by the inherent hybrid glycosyltransferase UGT51. Furthermore, combination of metabolic engineering can also increase the yield[47]. Dongetal.used 49 microbial strains to biotransform Rg1 and found that only the small filamentous fungusAspergillusniger3.185 8 andAbsidiacoerulea3.353 8 reacted with Rg1 for 6 days, respectively. The glycosylation group at c-20 of Rg1 will be completely hydrolyzed to produce the same metabolite Rh1[48].

2.3 Combinatorial chemistryCombinatorial chemistry is generally defined as the synthesis of a library of compounds containing all possible combinations of reagents. The core step of combinatorial chemistry is to repeatedly join the basic modules of different structures to produce a large number of related compounds, namely compound libraries. Screening is performed, and then the structure of the compound with the target performance is demonstrated. At present, combinatorial chemistry technology is quite mature[49]. According to the principle of combinatorial chemistry, it is an important and effective way to research and develop new drugs by structurally transforming natural products with high yield, which is easy to find new active ingredients and lead compounds[50].

3 Study on activities of PNS

3.1 Cardiovascular systemPNS show a wide range of effects on the cardiovascular system, including protecting the myocardium, improving myocardial ischemia[51-52], improving myocardial remodeling, inhibiting cardiac hypertrophy[53-54], protecting vascular endothelial cells[55]and vascular smooth muscle, and promoting angiogenesis[56]. PNS are mainly used in cardiovascular research, which can improve blood viscosity and reduce blood lipids[57].

3.1.1Protection of myocardium and improvement of myocardial ischemia. The occurrence of cardiovascular diseases such as coronary heart disease and angina pectoris is closely related to high blood viscosity.P.notoginseng, acting like a "vascular scavenger", is to protect myocardium, resist shock and promote thrombolysis[58], which can effectively reduce blood viscosity, because of inhibiting platelet aggregation. It was found that the PPD in PNS could decrease blood pressure in rats, while PPT would increase blood pressure in rats[59]. PNS can protect mice from ischemic brain injury[60]. The studies found that Notoginsenoside R1 plays a role in protecting the myocardium[61-62]. PPDS plays a certain role in protecting myocardium. Rg1, Rd and R1 have the most significant protective effect on myocardium. For example, in the myocardial ischemia reperfusion model, Rg1 can reduce the level of Ca2+, exert myocardial protection through anti-oxidative stress and maintain the balance of Ca2+. What’s more, it can also reduce the myocardial infarction area and enhance myocardial contractility[63-64]. Similarly, in the myocardial ischemia reperfusion model, Rd can reduce serum level of CK, LDH and myocardial infarction area, thereby playing a role in myocardial protection[65]. It is worth mentioning that as the unique component ofP.notoginsengand the main active component of PNS, Notoginsenoside R1 is also important for cardiovascular system[66]. For example, in a rat model of myocardial ischemia reperfusion, Notoginsenoside R1 can reduce the content of myocardial enzymes (CK-MB and LDH), exert myocardial protection[67], and also inhibit inflammatory factors in myocardial tissue, this anti-inflammatory effect can improve myocardial damage in mice[68].

3.1.2Improvement of myocardial remodeling and inhibitant of cardiac hypertrophy. Pharmacological studies have shown that the migration of vascular smooth muscle cells is an important cause of cardiovascular and cerebrovascular diseases. And PNS can block this process, thereby inhibiting myocardial hypertrophy. A large number of pharmacological studies have found that Rg1 can significantly improve myocardial remodeling after myocardial infarction, increase angiogenesis, improve myocardial remodeling, reduce the expression of inflammatory factors, and then effectively improve cardiac function[69-70]. The mechanism of its anti-cardiac hypertrophy effect may be partly attributed to NO pathway[71], or it may be related to the inhibition of myocardial fibrosis by decreasing the expression of mRNA of type I and type III procollagen[72]; Rb1 inhibits intracellular reactive oxygen species (ROS) and protects cardiomyocyte apoptosis induced by ischemia and hypoxia[73]. Rg1 can inhibit myocardial apoptosis by reducing caspase-3 level and increasing bcl-x L level. It was also found that R1 regulated PI3K/AKt signaling pathway and inhibited apoptosis of myocardial cells by activating estrogen receptor[74].

3.1.3Protection of vascular endothelial cells and inhibitant of apoptosis of cardiomyocytes. PNS can promote the proliferation of vascular endothelial cells and inhibit the apoptosis of cardiomyocytes[75]. In the case of PPT, the lack of hydroxide at the C6 position of PPD can effectively reduce cell apoptosis[76-77]. Further studies have shown that Rg1 can protect the vascular endothelium by inhibiting the cascade reaction of mitochondrial apoptosis and activating the ERK signaling pathway[78]. Rb1 inhibits intracellular reactive oxygen species (ROS) and protects the apoptosis of rat cardiac myocytes caused by hypoxia and ischemia[79]. Similarly, the specific component ofP.notoginseng, R1, can significantly improve the activity of myocardial cells and exert myocardial protective effect[80-81].

3.2 AntitumorCurrently, Rg3 has been used as an anticancer drug[82]; "Nano-Ginseng" with a panaxadiol saponin structure has excellent anti-tumor effectsinvivoandinvitro[83]. In the first place, Ma Wenbinetal.firstly used Rh1 and Rh2invivoto prove that Rh2 had a strong anticancer activity, while Rh1 had little effect on inhibiting tumor cells, which indicated that the difference was not related to whether the experiments wereinvivoorinvitro, but was related to the structure of Rh1 and Rh2[84]. A large number of studies have shown that PPD is most widely studied in antitumor, which can accelerate the apoptosis of cultured cells and inhibit the growth of cancer cells; apoptosis activity is superior to that of Rg3 and Rh2, which has been successfully developed as a new class of drugs. 20 (S)-PPD can inhibit the proliferation of lung cancer, the induction of leukemia cells, the growth of myeloma cells,etc.These studies showed that 20 (S)-PPD had a wide range of anti-tumor effects.

PNS can not only increase the number of leukocytes, inhibit the growth of specific tumor cells, induce differentiation and apoptosis of tumor cells, but also prevent the metastasis of tumor cells, and promote intercellular communication. Moreover, PNS can induce apoptosis in breast cancer cells at a certain time and concentration[85]. For example, tumor cells cultured with PNS at a concentration of 120 μg/mL effectively inhibited 92% of the cells, and had a significant inhibitory effect on liver cancer cells, which induced cancer cells to transform into non-cancer cells[86]. Rb1 is an agonist of estrogen receptor β, promoting protein expression and secretion of pigment epithelial-derived factors, thereby inhibiting tubular structure and inhibiting angiogenesis during tumor growth[87]. Leeetal.[88]used rectal cancer HCT-116 cells as a model to confirm that R1 inhibited human colorectal cancer metastasis.

3.3 Central nervous systemA large number of studies showed that[89-92]the active ingredients inP.notoginsenghad a protective effect on the central nervous system. For example, Rb1 and Rb3, as panaxadiol saponin in notoginsenoside, exhibited protective effects against ischemic neuronal death. PNS had a sedative, analgesic and intellectual effect on central nervous system[93]. Clinical trials showed that Rb1, the active ingredient inP.notoginseng, had a sedative effect and inhibited neural autonomic activity by reducing the content of synapse glutamate, which had a good effect on improving sleep quality and could significantly improve nervous insomnia and relieve nerve pain.

In the central nervous system, PNS can exhibit the most obvious effect on improving intelligence; the effective components can not only improve learning and memory, but also prevent Alzheimer’s Disease (AD). DDPU, a propanediol derivative, was reported for the first time as a potential therapeutic agent for AD[94]. In terms of intelligence enhancement, among the metabolites of Rg1, the main metabolite Rh1 and the final metabolite PPT increased the excitability of hippocampus and enhanced the function of learning and memory[95]. The Rhizopus nigricans fermentation product of Rg1 enhanced memory in all three injury models. Rg1 not only reduced AchE, but also enhanced ChAT, showing clinical potential for treating dementia[96]. In addition to Rg1, R1, Rh1 also showed potential effects on improving AD. The experiment confirmed that the learning and memory of AD model mice after intervention in R1 was improved[97-98].

3.4 Immune systemP.notoginsengcan restore the abnormal immune response to normality, and can be used as an immunomodulator. PNS increased the proportion of lymphocytes in the blood and improved the immunity of the body[99]. PNS inhibited the increase of capillary permeability caused by acute inflammation. The mechanism of action may be related to inhibiting the activity of phospholipase A2 in perfusion fluid preventing the increase of free calcium level in inflammatory cells and reducing the release of dinoprostone[100]. In addition, some studies found that the Rb1 had the potential value as an anti-wrinkle agent for skin[101]. F3 has immunopotentiating activity by regulating the production and gene expression of type 1 cytokines and type 2 cytokines in mouse spleen cells[102]. Zhou Xiaolingetal.[103]proved that PNS could significantly enhance the non-specific cellular immune function of the body with detecting the phagocytosis rate of alveolar macrophages, the migration inhibition test of peripheral blood leukocytes, and the rate of splenocyte-specific rosette-forming cells. Zhangetal.[104]found that R1 could reduce intestinal inflammation, debase the activity of peroxidase in colon and reduce the production of cytokines and the expression of pro-inflammatory genes.

3.5 Antioxidation and antifatigueAt the same time, Rg1 protected the function of hematopoietic stem cells in aging mice through a variety of signaling pathways and played an antioxidant role[105-107]. In recent years, the anti-fatigue effect of PNS has been studied increasingly. The main mechanism was that PNS played an anti-fatigue role by accelerating free radical scavenging. For example, Rb1 reduced Ca2+accumulation and exercise fatigue[108].

4 Structure-activity relationship of PNS

Structure activity relationship (SAR) refers to the relationship between the chemical structure of drugs or other physiologically active substances and their physiological activities. Drugs with similar chemical structure can act through the same mechanism and cause similar or opposite effects. There are many differences in the pharmacological effects and clinical therapeutic targeting of PNS. Therefore, the study on the structurally active relationship of PNS is of great significance for the design of drugs and the research and development of new drugs. At present, the structure-activity relationship of PNS for anti-tumor activity has been reported, but there are few reports on the structure-activity relationship of diastolic cardiovascular and cerebrovascular activity.

4.1 SAR of antitumor activityMany anti-tumor results of PPD showed that it could accelerate apoptosis and inhibit the growth of cancer cells. Its activity of inducing apoptosis was better than that of Rg3 and Rh2, which had been successfully developed as a new class of drugs. It has been reported in the literature that most of the 26 saponins have inhibitory effects on a variety of tumor cells, but the effects are different, suggesting that the anti-tumor activity of the saponins may be related to the type of glycosides, sugar chain, and c-20 configuration.

In the first place, modern pharmacological studies have shown that the anti-tumor activity of PNS is closely related to the structure and configuration. The main contents are as following: panaxadiol saponin > panaxtrol saponin; aglycon > monoglycoside > diglycoside > triglycoside > polyglucoside; C20 (R) type saponin > C20 (S) type saponin[109]. Kkodaetal.proved that Rh2 had obvious specific inhibition against tumor cell proliferation, enabling cancer cells to produce an induction effect of so-called redifferentiation in the proliferation process, but Rh1 which was similar to the structure of Rh2, had no such effect[110].

A large number of studies have shown that PPD is most widely studied in antitumor, which can accelerate the apoptosis of cultured cells and inhibit the growth of cancer cells; apoptosis activity is superior to that of Rg3 and Rh2, which has been successfully developed as a new class of drugs. 20 (S)-PPD can inhibit the proliferation of lung cancer, the induction of leukemia cells, the growth of myeloma cells,etc.These studies showed that 20 (S)-PPD had a wide range of anti-tumor effects. What’s more, the antitumor activity was inversely correlated with the number of glycosyl groups, and the antitumor activity was enhanced with the decrease of the number of glycosyl groups in the saponin structure. The antitumor activity varies with the location of glycosyl junction[111].

4.2 SAR of cardiovascular and cerebrovascular activityAt present, PSN preparations have been widely used in the treatment of various cardiovascular diseases, and the Xuesetong is the most commonly used to treat cardiovascular diseases. Xuesetong, with the function of dredging blood vessels is rich in Rg1, Re and Rb1, but less in Rd and other low-polar PPDS[112]. Pharmacokinetic studies indicate that saponins in Xuesetong can be metabolized in the body, and the metabolism is usually gradual de-glycation to obtain aglycone[113-115].

As for the relationship between antioxidant activity and structure of ginsenosides[116], it was proved that both PPDS and PPTS had antioxidant activity when PPDS and PPTS were linked to a single glucose at the C-20 site, such as Rg1, Rb1, Re, Rd and R1[117]. When there was no sugar at the C-20 site, it showed the effect of promoting oxidation, such as Rg3, Rg2 and Rh2. However, there was no sugar at the C-20 site, but there was a glucose saponin at the C-6 site, which still exhibited antioxidant activity, such as Rh1. The antioxidant capacity of Rc was greater than that of Rb1, while the antioxidant capacity of Re was the strongest in the PPTS.

5 Conclusions

P.notoginseng, as a traditional star medicinal plant in China, is still at the low end of the industrial chain at the stage. Agricultural primary processing of raw materials are the main means. The existing products containingP.notoginsengmainly use PNS such as Qiyeshen’an tablets, Xuesaitong, orP.notoginsengextracts such asP.notoginsengGuanxinning, or even directly useP.notoginsengpowder. For example, Compound Danshen tablet has invaluable content and low additional value. At the moment, the extraction technology and identification of effective components inP.notoginsengare very mature. In consideration of the present study on PNS, we can discover that most researchers choose to study the active and high content components, while ignoring some rare saponins. This seems to suggest that the preparation of rare saponins from PNS is a new perspective for researchers. The pharmacokinetics andinvivometabolism of PNS indicate that they have poor absorption, long elimination half-life, low bioavailability, complex components.

Considering the deficiency of saponins inP.notoginseng, it is a wise choice to modify structure. First of all, unlike the total synthesis of PNS, the method of structural modification is convenient and rapid, and can improve the yield and avoid the by-products and impurities formed during the expatiatory reaction. Secondly, the structural modification of PNS mainly focuses on the preparation of compounds with good anti-tumor, diastolic cardiovascular and cerebrovascular activity. The researchers hope that substances with good activity, high bioavailability and good absorption can be found by means of structural modification. The yield of products obtained by various structural modification methods is relatively low, and the products are complex and not easy to separate. Thirdly, the modified parts are mainly concentrated on the glycosides of C-3, C-6 and C-20, however, the structural modification on glycogen is rare, which leads to great difficulty in obtaining raw materials. In consequence, it seems that the glycogen yield is required to be improved in the structural transformation.

In summary, the review described recent advances on the structural modification on PNS, mainly for the structural modification of C-3, C-6, C-20 and other positions of PNS; the research on the relationship between PNS and anti-tumor structure is clear, but there is no clear report on the structure-activity relationship in the cardiovascular system and central nervous system, which seems to suggest that it is necessary to elucidate the pharmacological activities and structure-activity relationship of PNS to provide researchers with excellent ideas for the further study of notoginsenoside.