Triterpenoids from Gymnema sylvestre and Their Pharmacological Activities

Because plants are estimated to produce over 200,000 metabolites, research into new natural substances that can be used in the pharmaceutical, agrochemical and agro-industrial production of drugs, biopesticides and food additives has grown in recent years. The global market for plant-derived drugs over the last decade has been estimated to be approximately 30.69 billion USD. A relevant specific example of a plant that is very interesting for its numerous pharmacological properties, which include antidiabetic, anticarcinogenic, and neuroprotective effects is Gymnema sylvestre, used as a medicinal plant in Asia for thousands of years. Its properties are attributed to triterpenoidic saponins. In light of the considerable interest generated in the chemistry and pharmacological properties of G. sylvestre triterpenes and their analogues, we have undertaken this review in an effort to summarise the available literature on these promising bioactive natural products. The review will detail studies on the isolation, chemistry and bioactivity of the triterpenoids, which are presented in the tables. In particular the triterpenoids oxidised at C-23; their isolation, distribution in different parts of the plant, and their NMR spectral data; their names and physico-chemical characterisation; and the biological properties associated with these compounds, with a focus on their potential chemotherapeutic applications.

effects. Additionally, a large percentage of the world's population has no access to conventional pharmacological treatments. The different reasons mentioned above describe a situation in which research has ample space to improve and simplify extraction protocols and increase the levels of metabolites in plants of interest, through classical techniques of genetic improvement or innovative biotechnological approaches aimed at modifying biosynthetic pathways, in addition to testing substances already isolated for the widest possible spectrum of biological activities.

Terpenes and Terpenoids
Among the many biologically active substances of plant origin, a great deal of attention has been paid to terpenes, which are composed of isoprene subunits. More than 30,000 terpenes have been isolated thus far [17]. Terpenes are classified as mono-, sesqui-, di-, ses-, tri-, and tetraterpenes according to the number of isoprenoid units. The carbon skeleton may be acyclic, or they may possess mono-, bi-, tri-, tetra-, and pentacyclic structures [18][19][20][21][22][23][24]. Terpenes are of widespread and can be found in all organisms, both prokaryotic and eukaryotic. However, most bioactive terpenes have been detected in higher plants. Whereas mono-and sesquiterpenes are predominantly found in essential plant oils, the higher terpenes, including triterpenes, are found in balsams and resins [25,26]. Triterpenoids in their free form (sapogenins), bound to glycosides (saponins) or acetylated are particularly important and are ubiquitous throughout the plant kingdom; both in vitro and in vivo studies have shown that they also have important biological functions. Because of their relatively complex structures, terpenoids are usually referred to by trivial names instead of using systematic IUPAC nomenclature.

Classification of Terpenoids and Their Biological Effects
Based on the number of sugars present, saponins are classified as monodesmosides, with a single saccharide chain generally linked to C-3; bidesmosides, with two sugar chains linked to C-3 and C-28; and tridesmosides, with three saccharide chains. The sugars can be linear or branched; the most widespread saccharides in triterpenoid saponins are D-glucose, D-galactose, L-arabinose, L-rhamnose, D-xylose, D-fucose and glucuronic acid [27]. The biological effects of such terpenoids are very different, and can be summarised as follows: antitumor, antiviral, antidiabetic, anti-inflammatory, hepatoprotective, analgesic, antimicrobial, antimycotic, virostatic, immunomodulatory, and tonic activities [17]. A few compounds, such as corosolic acid, a dietary supplement against diabetes, are already on the market, and several others are in clinical trials or are to be launched soon. Many triterpenes exhibit significant biological activity, but several triterpenoids possess haemolytic and cytostatic properties that restrict their pharmaceutical use. To overcome these limitations and to expand the range of usable triterpenes, transformation of the compound by chemical or biotechnological techniques is possible. They are also of extreme interest to the agrochemical field because they are involved in the defence mechanisms of plants. Their biological activities are mainly related to interactions with cell membranes. The main mechanism of action of saponins against fungi appears to be related to their ability to form complexes with sterols in fungal membranes by altering the membrane integrity [28].

Triterpenoids from Gymnema sylvestre and Their Pharmacological Activities
The triterpenoidic saponins also act against insect pests by forming an insoluble complex with cholesterol, which is the precursor in the biosynthesis of the hormone ecdysone. Because cholesterol is the only source of sterols for the majority of the insects and a main energy supply, the cholesterol linked to saponins cannot be used, which leads to insect death [29].
A relevant specific example of a plant that is very interesting for its numerous pharmacological properties, which include antidiabetic, anticarcinogenic, and neuroprotective effects is Gymnema sylvestre, used as a medicinal plant in Asia for thousands of years. Its properties are attributed to triterpenoidic saponins. In light of the considerable interest generated in the chemistry and pharmacological properties of G. sylvestre triterpenes and their analogues, we have undertaken this review in an effort to summarise the available literature on these promising bioactive natural products.
The review will detail studies on the isolation, chemistry and bioactivity of the triterpenoids, which are presented in the following tables: triterpenoids oxidised at C-23 (Table 1 and Figure 1); their isolation, distribution in different parts of the plant, and their NMR spectral data ( Table 2); their names and physico-chemical characterisation (Tables 3 and 4, respectively); and, finally, the biological properties associated with these compounds, with a focus on their potential chemotherapeutic applications (Table 5).

Biological Effects of Triterpenoids
Diabetes mellitus (DM) is a disease caused by a deficiency or diminished effectiveness of endogenous insulin. It is characterised by hyperglycaemia, deranged metabolism and sequelae predominantly affecting the vasculature. There are two main types of diabetes mellitus: Type 1, in which the body does not produce sufficient insulin; Type 2, due to the resistance to the insulin, often initially with normal or increased levels of circulating insulin. In the UK, there were 2.9 million people with diabetes in 2011. It is estimated that 5 million people will have diabetes in the UK by 2025. It is also estimated that there are approximately 850,000 people who have undiagnosed diabetes. The average prevalence of diabetes in the UK is 4.45% of the population, but there are variations between countries and regions. The proportion of people with diabetes increases with age. However, the incidence of diabetes is increasing in all age groups. Type 1 diabetes is increasing in children (especially those < 5 years of age), and type 2 diabetes is increasing, particularly in black and minority ethnic groups. In general, the prevalence of diabetes mellitus and its percentages in different populations is almost the same throughout the world.
Compounds 1−12, 15−20, 39−43 were tested for their antisweet activity. The antisweet activity was tested on a group of volunteers who held a solution of the tested compounds (5 mL of the specific compounds solution in 0.01 M NaHCO 3 ) in their mouth for 3 min, spat, and rinsed with distilled water. The subjects were directed to taste 10 sucrose solutions from 0.1 to 1.0 M. The activity of the compound tested was expressed at the maximum concentration of a sucrose solution whose sweetness was suppressed completely. 0.5 mM of a solution of gymnemic acids I (1) and II (2) led a complete suppression of sweetness induced by 0.4 mM sucrose. More precisely, application of a 1 mM solution of 1 and 2 orally led to a complete suppression of sweetness induced by 0.2 and 0.4 M sucrose, respectively [30]. A 1.0 mM solution of gymnemic acids III (3), IV (4), V (5) and VI (6) led to a complete suppression of sweetness induced by 0.4 mM sucrose [31,32]. The difference between the structures of gymnemic acid III and gymnemic acid IV is only the absence or presence of a double bond in the acyl group. Kurihara et al. [33] have suggested that the acyl groups might play an important role in the generation of the antisweet activity. However, it seems that acyl groups only increase the antisweet activity, but are not essential [34]. A 0.5 mM solution of gymnemic acids VIII (8), IX (9) and X (10) leads to a complete suppression of the sweet taste of 0.2 M sucrose. The results are similar to those for gymnemic acids III and IV, which are acylated at C21 [35].
In general, it has been found that the antisweet activity of these saponins decreases with the decreasing number of acyl groups. In fact, a 0.5 mM solution of each of the gymnemic acids XI (11) and XII (12), as with gymnemic acids I and II, are able to suppress the perception of sweetness due to 0.4 M sucrose. The results suggest the antisweet activity of a triterpenes is directly proportional to the number of acyl groups present in the molecule [35]. A 0.5 M solution of gymnemic acids XV (15), XVI (16), XVII (17), and XVIII (18) completely suppressed the sweet taste of 0.4 M sucrose, showing the same activity of gymnemic acids I and II [36]. Gymnemic acid VII (7), prosapogenin (20) and gymnemagenin (19) were not active at all [30][31][32]. In particular, gymnemic acid IV is a multidirectional antihyperglycaemic agent with antisweet activity [31], glucose uptake inhibitory activity, and gut glycosidase inhibitory action [37]. Moreover, the blood glucose lowering effect of gymnemic acid IV was higher than glibenclamide, which was used as a control in streptozotocin-diabetic mice [29]. Most of these pharmacological effects may synergistically contribute to alleviating type 2 diabetes-related symptoms. Thus, gymnemic acid IV may be used as a prophylactic against diabetes through its different mechanisms of action [38]. A 1.0 mM solution of compounds 41-43 completely reduced the perceived sweetness of 0.1 M sucrose; these results correspond to half of the activity of gymnemic acids I−VI. Compounds 39 and 40 were not active [34].
The literature suggests that the pharmacological effects of G. sylvestre extract or its mixture of triterpenoids occur through mechanisms such as modulation of incretin activity, stimulation of insulin secretion and release, regeneration of β-endocrinocyte Langerhans islets, activation of enzymes responsible for glucose utilisation, reduction of glucose and fatty acid assimilation in the small intestine, and interference with the sensation of sweetness. It is known that hormones that regulate the formation and secretion of hormones by pancreas islets are activated in response to the entry of food into the intestine. Release of specific and well-known gastrointestinal hormones (GIP) into the portal vein in response to the intraduodenal administration of D-glucose in the presence of G. sylvestre extract enriched in gymnemic acids/triterpenoids by inhibitors of certain proposed glucose sensors and transporters in the intestinal lumen has been studied experimentally [39]. Intraduodenal administration of D-glucose caused a dose-dependent increase in the concentration of portal immunoreactive GIP. This suggests that the extract of G. sylvestre leaves or its constituents increases GIP secretion by endocrine k-cells in the small intestine [40]. The literature suggests that the hypoglycaemic activity of G. sylvestre is due to stimulating the release of insulin (and possibly the regeneration of Langerhans islet β-cells) and enzymes responsible for glucose utilisation and inhibition of glucose absorption in the bowel [27,[41][42][43]. This means that the hypertrophy of β-endocrinocytes most likely occurs due to the effect of G. sylvestre on the increased secretion of GIP [44]. An autogenic hormone in the blood that was correlated with the hypoglycaemic effect was observed in experimental animals with an experimental form of immunodependent DM during a study of G. sylvestre activity.
In Russia, tests of mixtures consisting of G. sylvestre extracts of various purities (certified preparations of G. sylvestre dry extracts) in various concentrations in combination with extracts that intensify the antioxidant effect (grape stem) and possess immunomodulating and regenerative properties are underway. More than 30 different combinations have been investigated in detail in vitro and in vivo. Considering these facts, it is obvious that G. sylvestre is a source of biologically active substances.
The very broad spectrum of pharmacological activity for G. sylvestre indicates that the use of its extract or its components at various doses and in various combinations improves the condition of latent forms of DM (prediabetes), treats insulin-independent DM, prolongs the action of hypoglycaemic preparations, and regenerates β-cells for insulin-dependent and insulin-independent DM [45]. Despite the results of these studies, only a few metabolites of G. sylvestre have been tested for their effects on glucose uptake. Among them, gymnemosaponin V (43) and gymnemic acids I-IV increase the amount of insulin in blood plasma in mice with streptozotocin-induced DM after their administration [35,38]. The presence of these compounds, but perhaps not only them, could explain that, when G. sylvestre extract was used for 21 days after streptozotocin intoxication, it reliably reduced the levels of glucose and HbAlc in blood plasma, increased the insulin content, and normalised the concentration of high-density lipoproteins (HDL) [28]. The inhibitory activity of some triterpene glycosides was examined to determine their impact on the increase in serum glucose level in oral glucose-loaded rats. Gymnemoside-b (33) and gymnemic acids III, V, and VII were found to exhibit slight inhibitory activity towards the increase of glucose absorption after a single administration of 100 mg/kg, but gymnemic acid I and gymnemasaponin, lacked this activity at the same dose. Although the above compounds are included in one of three categories (acylated polyhydroxyoleanane 3-O-glucuronide) of glucose absorption inhibitors, their activity is much less than that of their analogues [46]. Gymnemic acids II and III showed potent inhibitory activities on glucose uptake, which were almost equivalent to those of oleanolic acid-3-O-glucuronide and Escin Ia. Gymnemoside-f (37), gymnemic acid IV and gymnemasapoin V were also found to inhibit glucose uptake, while gymenomosides-c (34), -d (35), and -e (36) lacked this activity. Gymnemic acids II and III showed no effect on the serum glucose levels in oral glucose-loaded rats. They exhibited potent inhibitory activity towards glucose uptake in rat small intestine fragments [37]. Gymnemic acid V is normally considered to inhibit glucose absorption in the small intestine at a concentration of 100 mg/kg. The inhibitory effect is particularly marked after 2 h of administration, with values that are quite comparable in absolute terms to those of elatosides, escins, and senegasaponins, which were used as controls. Gymnemic acid I shows an effect almost 50% higher than that of the control after 2 h despite appearing to be less active after 30 min and 1 h. A very similar effect that is only slightly smaller than the control or gymnemic acid V was demonstrated for gymnemic acid VII and gymnemosides-a (32) and -b (33). In order of decreasing activity up to a value of approximately 30% of the control were gymnemic acid II, IV, gymnemasaponins IV and V, gymnesaponin II (40) and gymnemic acid III. However, no apparent structure-activity relationship was observed [47].
In recent years interest in cancer prevention has grown steadily and urgently, therefore it would be particularly important to identify molecules that are able to prevent or avoid the processes of carcinogenesis due to substances of which substantially we can't do without or can't avoid in our daily life. Several anti-inflammatory substances are well known to inhibit the action of tumour promoters in the mouse skin carcinogenesis. The compound 46 was found to inhibit the inflammatory reaction induced by tumour promoters. This anti-inflammatory activity may play an important role in the mechanism of antitumor promotion as it has already been demonstrated [48,49]. The effects of compound 46 on the growth of HepG2 cells were measured. Its inhibitory effect was remarkably effective (ID 50 was 25 µM) as it induced apoptosis at high dose, and with a dose-dependent manner [50,51]. Moreover, the application of antitumor-promoting triterpenoids is highly promising for protection against tumour formation, and many triterpenoids were tested in vitro and in vivo against the action of tumour promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA) induced Epstein-Barr virus (EBV) activation in Raji cells. Recently, ursolic and oleanolic acids have been reported to be inhibitors of TPA and the dose responses of the acids were very similar to those of the antitumor promoters, such as retinoic and glycyrrhetinic acids and their analogues [51,52]. Thus some glycyrrhetinic acid-related compounds were found to be inhibitors of tumour promoter-induced phenomenon in vitro. Among these compounds, the compound 46 also proved to have in vivo antitumor-promoting activity in mouse skin tumour formation induced by 7,12-dimethylbenz[a]anthracene plus tumor promoters TPA [50]. Although its mechanism is unknown, the modulation of phospholipid metabolism appeared to be a very interesting aspect was proved that the inhibitory potency of the triterpenoids for the TPA-enhanced phospholipid synthesis is closely associated with their antitumor-promoting activity. Finally, a sulforhodamine B bioassay was used to determine the cytotoxicity of compound 44. Its cytotoxic activity against four cultured human tumour cells was examined in vitro. The tumour cell lines were A549 (non small cell lung adenocarcinoma), SK-OV-3 (ovarian cancer cells), SK-MEL-2 (skin melanoma), and HCT15 (colon cancer cells) [53,54]. Doxorubicin was used as the positive control. The tested compound was essentially no cytotoxic [55]. A crude extract o mixture of compounds used in traditional medicine frequently contains components that have mutually opposing pharmacological activities beneficial for a specific disease. The higher potency of a crude drug is probably due to a synergistic effect among its component compounds, even though the activity of each compound is weak when used alone. This does not mean that it is not relevant to define a complete picture of the activities or biological properties of each compound. To identify a particular compound or a mixture of those with remarkable properties we should be able to deal with infusions or extracts that may have a different composition for an infinite number of reasons (location and/or harvesting season of the plant, particularly plant species, manner and time of extraction or partial purification etc.). Furthermore, the identification of one or of a few molecules suitable for the purpose would allow us to synthesize them, with a process as much as economically or timely convenient and to obtain pure products or well-defined composition. Table 1. Common name and relative substituents of triterpenes with olean-12-ene skeleton.

No. Activity
Ref.

1
Antisweet activity Glucose uptake in rat small intestinal fragment Increase of serum glucose level in oral glucose-loaded rats Anti-hyperglycemic activity Antisweet activity Increase of serum glucose level in oral glucose-loaded rats [32] [46,47] 10 Antisweet activity [35]

Conclusions
The market of natural substances is very attractive for its economic impact, which, on the other hand grows continuously. Research into new natural substances that can be used in the pharmaceutical, agrochemical and agro-industrial production of drugs, biopesticides and food additives has grown in recent years. Gymnema sylvestre is a relevant specific example of a plant very interesting for its numerous pharmacological properties, which include antidiabetic, anticarcinogenic, and neuroprotective effects, used as a medicinal plant in Asia for thousands of years. Its properties are attributed to triterpenoidic saponins. In light of the considerable interest generated in the chemistry and pharmacological properties of G. sylvestre triterpenes and their analogues, this review summarises the available literature on these promising bioactive natural products. The review shows the results about the isolation, chemistry and bioactivity of the triterpenoids oxidised at C-23, which are schematically presented in few tables, in particular, their isolation, distribution in different parts of the plant, and their NMR spectral data; their names and physico-chemical characterisation; and the biological properties associated with these compounds, with a focus on their potential chemotherapeutic applications.