Molecular activities, biosynthesis and evolution of triterpenoid saponins

Saponins are bioactive compounds generally considered to be produced by plants to counteract pathogens and herbivores. Besides their role in plant defense, saponins are of growing interest for drug research as they are active constituents of several folk medicines and provide valuable pharmacological properties. Accordingly, much effort has been put into unraveling the modes of action of saponins, as well as in exploration of their potential for industrial processes and pharmacology. However, the exploitation of saponins for bioengineering crop plants with improved resistances against pests as well as circumvention of laborious and uneconomical extraction procedures for industrial production from plants is hampered by the lack of knowledge and availability of genes in saponin biosynthesis. Although the ability to produce saponins is rather widespread among plants, a complete synthetic pathway has not been elucidated in any single species. Current conceptions consider saponins to be derived from intermediates of the phytosterol pathway, and predominantly enzymes belonging to the multigene families of oxidosqualene cyclases (OSCs), cytochromes P450 (P450s) and family 1 UDP-glycosyltransferases (UGTs) are thought to be involved in their biosynthesis. Formation of unique structural features involves additional biosynthetical enzymes of diverse phylogenetic background. As an example of this, a serine carboxypeptidase-like acyltransferase (SCPL) was recently found to be involved in synthesis of triterpenoid saponins in oats. However, the total number of identified genes in saponin biosynthesis remains low as the complexity and diversity of these multigene families impede gene discovery based on sequence analysis and phylogeny.This review summarizes current knowledge of triterpenoid saponin biosynthesis in plants, molecular activities, evolutionary aspects and perspectives for further gene discovery.     

Antiplasmodial and antifungal activities of iridal,a plant triterpenoid

Iridal, a triterpenoidic compound extracted from Iris germanica L., was previously shown to have an interesting activity on two cultured human tumor cell lines (A2780 and K562). In the present work, this same product was tested in vitro on Plasmodium falciparum chloroquine-resistant and -sensitive strains, in vivo on P. vinckei, and on some Candida albicans and C. parapsilosis strains too. The IC(50) obtained in vitro on human malaria strain ranged from 1.8 to 26.0 microg/ml and the ED(50) in vivo is about 85 mg/kg/day by intraperitoneal route. The minimal inhibitory concentrations were higher than to 50 microg/ml, whatever the strain of yeast tested. This product presents an antiplasmodial activity similar to that obtained with extracts from the plant Azadirachta indica classically taken as reference in malaria phytomedicine. Conversely iridal shows no important antifungal activity. The specific activity of iridal on human malaria parasite and on tumor cell lines is discussed.     

A novel triterpenoid 16-hydroxy betulinic acid isolated from Mikania cordata attributes multi-faced pharmacological activities

The aerial parts of extensively used ethnomedicinal plant Mikania cordata (Burm. f.) Robinson growing wild in Bangladesh were investigated to isolate and characterize compounds responsible for the bioactivities of the plant. In the present study, a new derivatives of betulinic acid, 16-hydroxy betulinic acid [3β,16-dihydroxy-lup-20(29)-en-28-oic] was isolated and the structure of the compound was determined by NMR spectroscopic means and comparing with available literature data. The isolated compound was then investigated for different pharmacological activities including antibacterial, antifungal, analgesic, anti-inflammatory and antipyretic potential employing different methods. The compound showed potent antibacterial activity with inhibition zone of diameter ranging from 12.0 to 17.5?mm and antifungal activity with mycelial growth inhibition ranging from 37.6 to 54.5%. The MIC values for antibacterial and antifungal activities ranged from 31.5–125 and 250–1000?μg/mL respectively. The compound (50 and 100?mg/kg body weight) showed potent peripheral and central analgesic activity with 55.19% and 41% of writhing inhibition at 90?min after administration of the compound and the highest 55.98%, 79.18% elongation of reaction time, respectively. In anti-inflammatory activity screening, the compound (100?mg/kg b.w.) revealed the highest 77.08% edema inhibition at 4?h after administration of carrageenan. In antipyretic assay, 16-hydroxy betulinic acid displayed a strong antipyretic effect in yeast-induced rats. From the present study it is apparent that 16-hydroxy betulinic acid might play vital role to establish M. cordata as ethnomedicinal plant to treat wound, cuts and fever.     

Insulin-mimetic and insulin-sensitizing activities of a pentacyclic triterpenoid insulin receptor activator

Five pentacyclic triterpenoids isolated from Campsis grandiflora were tested for insulin-mimetic and insulin-sensitizing activity. The compounds enhanced the activity of insulin on tyrosine phosphorylation of the IR (insulin receptor) beta-subunit in CHO/IR (Chinese-hamster ovary cells expressing human IR). Among the compounds tested, CG7 (ursolic acid) showed the greatest enhancement and CG11 (myrianthic acid) the least. We characterized the effect of CG7 further, and showed that it acted as an effective insulin-mimetic agent at doses above 50 mug/ml and as an insulin-sensitizer at doses as low as 1 mug/ml. Additional experiments showed that CG7 increased the number of IRs that were activated by insulin. This indicates that a major mechanism by which CG7 enhances total IR auto-phosphorylation is by promoting the tyrosine phosphorylation of additional IRs. CG7 not only potentiated insulin-mediated signalling (tyrosine phosphorylation of the IR beta-subunit, phosphorylation of Akt and glycogen synthase kinase-3beta), but also enhanced the effect of insulin on translocation of glucose transporter 4 in a classical insulin-sensitive cell line, 3T3-L1 adipocytes. The results of the present study demonstrate that a specific pentacyclic triterpenoid, CG7, exerts an insulin-sensitizing effect as an IR activator in CHO/IR cells and adipocytes. The enhancement of insulin activity by CG7 may be useful for developing a new class of specific IR activators for treatment of Type 1 and Type 2 diabetes.     

Arjunolic acid, a triterpenoid saponin, ameliorates arsenic-induced cyto-toxicity in hepatocytes

Arsenic is a well-established environmental toxin, which damages various organs of the body. A triterpenoid saponin, arjunolic acid (AA) has been isolated from the bark of Terminalia arjuna. The present study was conducted to investigate the preventive role of AA against arsenic-induced cytotoxicity in isolated murine hepatocytes. Sodium arsenite (NaAsO₂) was chosen as the source of arsenic. Incubation of the hepatocytes with NaAsO₂ (1mM) for 2 h caused reduction in the cell viability and activities of the intracellular enzymatic as well as non-enzymatic antioxidants. Treatment of NaAsO₂ enhanced lipid peroxidation and also increased the activities of the membrane leakage enzymes. Administration of AA (100 μg/ml) before and with the toxin almost normalized the altered activities of antioxidant indices. AA possesses free radical scavenging activity and could enhance the cellular anti-oxidant capability against NaAsO₂-induced cyto-toxicity. The cytoprotective activity of AA was found to be comparable to that of a known antioxidant, vitamin C. Experimental results, therefore, suggest that AA protects arsenic-induced cytotoxicity in murine hepatocytes.     

Anti-inflammatory activities of triterpenoid saponins from Polygala japonica

Bioassay-guided investigation was performed to identify the active constituents from a methanol extract of Polygala japonica, a folk medicinal plant widely used in China to treat inflammatory diseases. The n-BuOH and EtOAc fractions of the P. japonica methanol extract, which show significant anti-inflammatory activity in in vivo test, were further subjected to column chromatography to afford six triterpene glycosides, marked here as saponins 1–6. All compounds were evaluated for their anti-inflammatory activity in the carageenan-induced mouse paw edema test, and saponins 1, 4 and 5 showed significantly anti-inflammatory effects on both phases of carageenan-induced acute paw edema in mice. Saponin 5 was also found to significantly inhibit the production of inflammatory mediators – nitric oxide (NO) in LPS-stimulated RAW264.7 macrophages, with no obvious effects on macrophage viability.     

Jacquinonic acid,an ant-repellent triterpenoid from Jacquinia pungens

The isolation and structure elucidation of jacquinonic acid, an ant-repellent triterpenoid from Jacquinia pungens, are described. This compound is a highly functionalized derivative of the oleanane skeleton, which contains both the uncommon C-13, 28 ether linkage and an α,β-unsaturated 3-keto group.     

Growth inhibitory,apoptotic and anti-inflammatory activities displayed by a novel modified triterpenoid,cyano enone of methyl boswellates

Triterpenoids are pentacyclic secondary metabolites present in many terrestrial plants. Natural triterpenoids have been reported to exhibit anti-inflammatory and anti-carcinogenic activities. Here, we show that modifications of ring A of boswellic acid (2 cyano, 3 enone) resulted in a highly active growth inhibitory, anti-inflammatory, pro-differentiative and anti-tumour triterpenoid compound called cyano enone of methyl boswellates (CEMB). This compound showed cytotoxic activity on a number of cancer cell lines with IC₅₀ ranging from 0.2 to 0.6 μM. CEMB inhibits DNA synthesis and induces apoptosis in A549 cell line at 0.25 μM and 1 μM concentrations, respectively. CEMB induces adipogenic differentiation in 3T3-L1 cells at a concentration of 0.1 μM. Finally, administration of CEMB intra-tumourally significantly inhibited the growth of C6 glioma tumour xenograft in immuno-compromised mice. Collectively, these results suggest that CEMB is a very potent anti-tumour compound.     

A ganoderic acid derivative,a highly oxygenated lanostane-type triterpenoid from Ganoderma lucidum

Ganoderic acid C, a new lanostane-type triterpenoid was isolated from the fruit body of Ganoderma lucidum. The structure of ganoderic acid C was elucidated by spectroscopic data and X-ray analysis of methyl ganoderate C acetate.     

Anti-tumor and anti-viral activities of Galanthus nivalis agglutinin (GNA)-related lectins

Galanthus nivalis agglutinin (GNA)-related lectin family, a superfamily of strictly mannose-binding specific lectins widespread among monocotyledonous plants, is well-known to possess a broad range of biological functions such as anti-tumor, anti-viral and anti-fungal activities. Herein, we mainly focused on exploring the precise molecular mechanisms by which GNA-related lectins induce cancer cell apoptotic and autophagic death targeting mitochondria-mediated ROS-p38-p53 apoptotic or autophagic pathway, Ras-Raf and PI3K-Akt anti-apoptotic or anti-autophagic pathways. In addition, we further discussed the molecular mechanisms of GNA-related lectins exerting anti-viral activities by blocking the entry of the virus into its target cells, preventing transmission of the virus as well as forcing virus to delete glycan in its envelope protein and triggering neutralizing antibody. In conclusion, these findings may provide a new perspective of GNA-related lectins as potential drugs for cancer and virus therapeutics in the future.     

Anti-tumor and immunomodulating activities of a polysaccharide from the root of Sanguisorba officinalis L

A water-soluble polysaccharide, named as SOWP, was extracted and fractioned from the roots of Sanguisorba officinalis L. by DEAE-Sepharose anion exchange and Sepharose 6 Fast Flow column chromatography. The monosaccharide composition of SOWP determined by gas chromatography (GC) identified it was composed of glucose (68.5%), arabinose (13.2%), rhamnose (8.9), xylose (6.2) and galactose (3.0%). At the dose of 50, 100, and 200mg/kg, SOWP not only significantly inhibited the growth of mouse transplantable tumor, but also remarkably increased the spleen index and promoted splenocytes proliferation, macrophage phagocytosis and the production of serum cytokines IL-2, TNF-α and IFN-γ in Sarcoma 180-bearing mice. However, no direct cytotoxic activity against Sarcoma 180 cells was observed. The anti-tumor activity of the polysaccharide from S. officinalis maybe achieved by not directly cytotoxicity but rather immunopotentiation.     

Anti-tumor activities of the angiogenesis inhibitors interferon-inducible protein-10 and the calreticulin fragment vasostatin

Tumor growth depends upon an adequate supply of oxygen and nutrients achieved through angiogenesis and maintenance of an intact tumor vasculature. Therapy with individual agents that target new vessel formation or existing vessels has suppressed experimental tumor growth, but rarely resulted in the eradication of tumors. We therefore tested the combined anti-tumor activity of vasostatin and interferon-inducible protein-10 (IP-10), agents that differently target the tumor vasculature. Vasostatin, a selective and direct inhibitor of endothelial cell proliferation, significantly reduced Burkitt tumor growth and tumor vessel density. IP-10, an "angiotoxic" chemokine, caused vascular damage and focal necrosis in Burkitt tumors. When combined, vasostatin plus IP-10 reduced tumor growth more effectively than each agent alone, but complete tumor regression was not observed. Microscopically, these tumors displayed focal necrosis and reduction in vessel density. Combination therapy with the inhibitors of angiogenesis vasostatin and IP-10 is effective in reducing the rate of tumor growth but fails to induce tumor regression, suggesting that curative treatment may require supplemental drugs targeting directly the tumor cells.     

Isolation and absolute stereochemistry of coussaric acid,a new bioactive triterpenoid from the stems of Coussarea brevicaulis

Coussaric acid (1), a triterpenoid based on an ursane skeleton, and an oleanane-type triterpene acid, 3-epi-spathodic acid (2), as well as four known compounds, barbinervic acid, scutellaric acid, stigmasterol and stigmasterol glucoside, have been isolated from an EtOAc-soluble extract of the stems of Coussarea brevicaulis. The structures of compounds 1 and 2 were elucidated on the basis of spectroscopic investigation, and single-crystal X-ray crystallography was used to confirm the structure of 1. The absolute stereochemistry of 1 was established by chemical transformations and by the Mosher ester procedure. The potential of the isolates and chemical transformation products to induce quinone reductase was evaluated in mouse Hepa lclc7 hepatoma cells.     

Pomolic acid, triterpenoid isolated from Licania pittieri, as competitive antagonist of ADP-induced aggregation of human platelets

Pomolic acid (PA), triterpenoid isolated from Licania pittieri, has previously shown a potent ability to inhibit adenosine diphosphate (ADP)- and epinephrine-induced human platelet aggregation. To investigate whether PA could be an antagonist of ADP-activated receptors of human platelets (P2Y(1) and P2Y(12)), pharmacological studies were conducted to examining its ability to modulate the platelet shape change induced by a selective P2Y(1) receptor agonist MRS2365 and also the nature of its possible interaction with ADP receptors by analyzing the characteristics of log concentration-response curves of ADP constructed in the absence and in the presence of fixed concentrations of PA, using in vitro platelet aggregation assays. PA did not interfere with the activation of P2Y(1) receptor by MRS2365 to induce platelet shape change and displayed a competitive antagonism of ADP-induced platelet aggregation, which most probably involves competition for a single binding site in platelets. The estimated equilibrium dissociate constant (K(b)) of PA as ADP receptor antagonist was 15.4±0.06nM. Together, these findings give indirect evidence for the idea that PA could be a potent competitive antagonist of P2Y(12) receptor, and open the possibility to consider it as new member of the non-nucleotide generation of antiplatelet drugs.     

Lupeol long-chain fatty acid esters and other triterpenoid constituents from Plumeria obtusifolia

From the bark of Plumeria obtusifolia was isolated a series of lupeol fatty esters with the carbon numbers 16, 18,20 and 21–28 in the fatty acid part. Furthermore, lupeol, lupeol acetate, sitosterol, stigmasterol and campesterol were also identified.     

Four new triterpenoid saponins isolated from Schefflera kwangsiensis and their inhibitory activities against FBPase1

Four new triterpenoid saponins, schekwangsiensides H–K (1–4) were isolated from the aerial parts of Schefflera kwangsiensis, together with fifteen esters (5–19) of known saponins. The structures of these compounds were elucidated on the basis of spectroscopic data analysis and chemical evidence. Furthermore, in in vitro assays, compounds 3, 8, 17, and 18 exhibited weak inhibitory activities against fructose-1,6-bisphosphatase (FBPase1).     

The anti-carcinogenic plant compound indole-3-carbinol differentially modulates P450-mediated steroid hydroxylase activities in mice.

Indole-3-carbinol (I3C), a component of cruciferous vegetables, exhibits anti-carcinogenic activity in a variety of model systems. This activity has been attributed in part to the induction of cytochrome P450 CYP1A subfamily members and the resulting increased metabolic inactivation of chemical carcinogens. The present study was undertaken to assess the effects of I3C on several constitutive P450 activities that contribute to both carcinogen and steroid hormone metabolism. Mice were administered I3C in their diet at estimated daily doses of 250, 500 and 750 mg/kg for 1 week. Liver microsomes from treated and untreated mice were subsequently assayed for CYP1A-mediated ethoxy-resorufin O-deethylase (EROD) activity, estradiol 2-hydroxylase activity and seven different testosterone hydroxylase activities. I3C elevated EROD, estradiol 2-hydroxylase and testosterone 6 alpha-hydroxylase activities in a dose-dependent manner. The other six testosterone hydroxylase activities were not significantly affected by in vivo treatment with I3C. In addition to its effects on steroid hydroxylase activities, I3C also elevated NADPH-cytochrome P450 reductase activity, a necessary component to the P450 monooxygenase system. We next examined the direct in vitro effects of I3C and its acid condensation products, as are generated in the stomach following ingestion, on the P450 catalytic activities. Testosterone 6 beta-hydroxylase, the major testosterone hydroxylase activity in untreated mice, was significantly inhibited (IC50 approximately 12 micrograms/ml) by the acid condensation products of I3C. In contrast, all other P450 activities were not appreciably affected by I3C or its acid condensation products. These results indicate that I3C can elicit both inductive and suppressive effects on the constitutive P450s that participate in carcinogen and steroid hormone metabolism. This pleiotropic effect on hepatic catalytic enzymes may contribute to the anti-carcinogenic properties of this compound.     

Anti-tumor activities of the antlered form of Ganoderma lucidum in allogeneic and syngeneic tumor-bearing mice

We investigated the anti-tumor effects of a dry powder preparation of the antlered form of Ganoderma lucidum (G. lucidum AF, rokkaku-reishi in Japanese), a variant type of G. lucidum, not only in allogeneic Sarcoma 180-bearing ddY mice, but also in syngeneic MM 46-bearing C3H/He mice. G. lucidum AF inhibited tumor growth and elongated the life span when orally administered to mice by free-feeding of a 2.5% G. lucidum AF-containing diet. It also showed anti-tumor activity in spite of post-feeding after tumor inoculation. G. lucidum AF significantly countered the depression of splenic CD8+ cells and protected the decrease in interferon-gamma (IFN-gamma) production in regional lymph nodes of MM 46-bearing mice, indicating that the anti-tumor activity of G. lucidum AF might be caused by its immunostimulating action. These results suggest that the ingestion of G. lucidum AF can be useful for the prevention and curing of cancer.