Plant Triterpenoid Research Articles

In vitro effect of ursolic acid on the inhibition of Mycobacterium tuberculosis and its cell wall mycolic acid

Mycobacterium tuberculosis is a dangerous intracellular pathogen. In order to protect against mycobacterium infection, novel agents with anti-mycobacterial activity should be given emergency priority for evaluation. Ursolic acid (UA), a plant triterpenoid, shows promising bioactivities, including anti-mycobacterial potency. In this study, the action of UA against Mycobacterium tuberculosis H37Ra was evaluated, and the inhibitory concentration was found to range between 10 and 20 μg/ml in a resazurin assay and MGIT 960 instrument. The total mycolic acid in UA-treated H37Ra was detected and compared with INH-treated and non-treated bacterium by LC-MS/MS. Quantitative LC-MS/MS data confirmed that both UA and INH decreased mycolic acid biosynthesis in a dose-dependent manner. Thin-layer chromatogram (TLC) analysis showed that all mycolic acid subtypes were affected by UA treatment in the wild type but not in strains resistant to UA. Electron microscopy images also confirmed that UA treatment affected both H37Ra cell and intracellular content of H37Ra. Altogether, these data confirmed the promise of the inhibitory action of UA in mycolic acid, which might further delineate the mechanistic pathway of mycobacterial inhibition by UA.

Biosynthesis of Plant Triterpenoid Saponins: Genes, Enzymes and their Regulation

Saponins are ubiquitous plant natural products, essentially involved in plant defense against biotic stresses, with numerous pharmaceutical and agricultural applications. The common precursor for triterpenoid saponins is squalene (30 carbon molecule) which, via cationic intermediates, is oxidized to 2,3-oxidosqualene. After cyclization, the basic triterpenoid cyclic structure undergoes oxidation by monoxygenases and glycosylations of hydroxyl groups. Chemical synthesis of saponins essentially recapitulates the main biosynthetic steps. However, to date, plants are the most viable source of these molecules. Jasmonic or salicylic acid, as well as their respective methylated derivatives, are important signaling molecules in the responses culminating in triterpenoid saponin production. The current challenges to improve triterpenoid saponin production include a better understanding of the signal transduction pathways leading to their accumulation (with emphasis on late enzymes and “master” regulatory transcription factors), isolation and heterologous expression of biosynthetic genes, and structural and modeling studies of biosynthetic enzymes and their catalytic mechanisms.

Ameliorative effects of oleanolic acid on fluoride induced metabolic and oxidative dysfunctions in rat brain: Experimental and biochemical studies

Beneficial effects of oleanolic acid on fluoride-induced oxidative stress and certain metabolic dysfunctions were studied in four regions of rat brain. Male Wistar rats were treated with sodium fluoride at a dose of 20mg/kgb.w./day (orally) for 30days. Results indicate marked reduction in acidic, basic and neutral protein contents due to fluoride toxicity in cerebrum, cerebellum, pons and medulla. DNA, RNA contents significantly decreased in those regions after fluoride exposure. Activities of proteolytic enzymes (such as cathepsin, trypsin and pronase) were inhibited by fluoride, whereas transaminase enzyme (GOT and GPT) activities increased significantly in brain tissue. Fluoride appreciably elevated brain malondialdehyde level, free amino acid nitrogen, NO content and free OH radical generation. Additionally, fluoride perturbed GSH content and markedly reduced SOD, GPx, GR and CAT activities in brain tissues. Oral supplementation of oleanolic acid (a plant triterpenoid), at a dose of 5mg/kgb.w./day for last 14days of fluoride treatment appreciably ameliorated fluoride-induced alteration of brain metabolic functions. Appreciable counteractive effects of oleanolic acid against fluoride-induced changes in protein and nucleic acid contents, proteolytic enzyme activities and other oxidative stress parameters indicate that oleanolic acid has potential antioxidative effects against fluoride-induced oxidative brain damage.

Antiatherogenic effects of oleanolic acid in apolipoprotein E knockout mice

Oleanolic acid (OA) is a plant triterpenoid steroid with potentially antiatherogenic properties. We investigated whether OA affected atherosclerosis development and vascular function in apolipoprotein E knockout (ApoE(-/-)) mice. ApoE(-/-) mice were fed a high cholesterol Western-type diet in combination with OA (100 mg/kg/day), fluvastatin (5 mg/kg/day) or vehicle, with wild type (WT) mice serving as controls. After 8 weeks of treatment atherosclerotic plaque areas in the aortic arch and plasma lipid concentrations were determined. Vasoconstriction and relaxation of the proximal part of aorta were investigated in vitro. Inducible nitric oxide synthase (iNOS) was visualized using immunoblotting. As opposed to WT and fluvastatin- and vehicle-treated mice, OA-fed ApoE(-/-) mice gained no weight during the treatment period. Plasma concentrations of total-cholesterol and triglyceride were not significantly reduced by OA- or fluvastatin treatment. Plaque area of vehicle-treated mice was 25%, but only 14% in OA- and 19% in fluvastatin-treated mice. As compared to WT, vasoconstriction to phenylephrine was attenuated in ApoE(-/-) mice. The NOS inhibitor asymmetric dimethylarginine (ADMA) enhanced phenylephrine constriction, but significantly more so in vehicle- and fluvastatin-treated than in OA-treated and WT mice. Relaxation to acetylcholine was only slightly attenuated in ApoE(-/-) mice and not affected by OA or fluvastatin treatment. ADMA abolished acetylcholine relaxation almost completely. In ApoE(-/-) mice iNOS expression was reduced by OA treatment. In conclusion OA exerts potent antiatherogenic effects independent of plasma lipid levels and without major changes in eNOS-mediated acetylcholine relaxation. However, OA reduced iNOS expression possibly altering vascular reactivity to phenylephrine.

Ursolic acid is a PPAR-α agonist that regulates hepatic lipid metabolism

In this study, we confirmed that ursolic acid, a plant triterpenoid, activates peroxisome proliferator-activated receptor (PPAR)-α in vitro. Surface plasmon resonance and time-resolved fluorescence resonance energy transfer analyses do not show direct binding of ursolic acid to the ligand-binding domain of PPAR-α; however, ursolic acid enhances the binding of PPAR-α to the peroxisome proliferator response element in PPAR-α-responsive genes, alters the expression of key genes in lipid metabolism, significantly reducing intracellular triglyceride and cholesterol concentrations in hepatocytes. Thus, ursolic acid is a PPAR-α agonist that regulates the expression of lipid metabolism genes, but it is not a direct ligand of PPAR-α.

Avicin D, a Plant Triterpenoid, Induces Cell Apoptosis by Recruitment of Fas and Downstream Signaling Molecules into Lipid Rafts

Avicins, a family of triterpene electrophiles originally identified as potent inhibitors of tumor cell growth, have been shown to be pleiotropic compounds that also possess antioxidant, anti-mutagenic, and anti-inflammatory activities. We previously showed that Jurkat cells, which express a high level of Fas, are very sensitive to treatment with avicins. Thus, we hypothesized that avicins may induce cell apoptosis by activation of the Fas pathway. By using a series of cell lines deficient in cell death receptors, we demonstrated that upon avicin D treatment, Fas translocates to the cholesterol- and sphingolipid-enriched membrane microdomains known as lipid rafts. In the lipid rafts, Fas interacts with Fas-associated death domain (FADD) and Caspase-8 to form death-inducing signaling complex (DISC) and thus mediates cell apoptosis. Interfering with lipid raft organization by using a cholesterol-depleting compound, methyl-β-cyclodextrin, not only prevents the clustering of Fas and its DISC complex but also reduces the sensitivity of the cells to avicin D. Avicin D activates Fas pathways independent of the association between extracellular Fas ligands and Fas receptors. A deficiency in Fas and its downstream signaling molecules leads to the resistance of the cells to avicin D treatment. Taken together, our results demonstrate that avicin D triggers the redistribution of Fas in the membrane lipid rafts, where Fas activates receptor-mediated cell death.

Oleanolic Acid, a plant triterpenoid, significantly improves survival and function of islet allograft.

BACKGROUND.: Oleanolic acid (OA) is a ubiquitous triterpenoid, with potent antioxidant and anti-inflammatory properties. Here, we tested whether these combined properties of OA can prevent nonimmunologic primary nonfunctioning and immunologic phenomena ascribed to graft rejection hence prolong islet allograft survival. METHODS.: Islet transplants were performed under kidney capsule of streptozotocin-induced diabetic C57BL/6 mice with BALB/c islets. Recipients were treated with 0.5 mg/day of OA intraperitoneally, and serum samples were collected once in 2 days and used for luminex, ELISA, and donor-specific antibody screening. Transplanted mice were killed at different time intervals to obtain splenocytes and kidney samples for ELISPOT, mixed leukocyte reaction, and immunohistochemical studies. RESULTS.: After transplantation, the decrement of blood glucose was significantly faster in mice receiving OA less than 2+/-1 days compared with untreated (4+/-2 days). OA prolonged survival of transplanted islets up to 23+/-3 days and reversed diabetes even with 250 islets. Treatment group showed increased serum interleukin (IL)-10 (twofold) and decreased inducible protein-10 and IL-4 (threefold) in luminex. Significantly reduced frequency of interferon-gamma (4.5-fold), IL-4 (3.5-fold), IL-2 (2.3-fold), and IL-17 (fourfold) producing T-cell populations were found in ELISPOT. OA-treated grafts had significant reduced and delayed infiltration of CD4+ and CD8+ T cells. OA also delayed donor-specific antibody generation up to 19 days after transplantation. Combined treatment with cyclosporine A, OA further prolonged the islet allograft survival to 34+/-3 days. CONCLUSIONS.: In conclusion, OA is an attractive, dietary nontoxic plant triterpenoid, which suppresses the production of proinflammatory cytokines and delays graft-specific immune responses to prolong islet allograft survival.

Anabolic activity of ursolic acid in bone: Stimulating osteoblast differentiation in vitro and inducing new bone formation in vivo

In the field of osteoporosis, there has been growing interest in anabolic agents that enhance bone mass and improve bone architecture. In this study, we demonstrated that the ubiquitous plant triterpenoid, ursolic acid, enhances differentiation and mineralization of osteoblasts in vitro. We found that ursolic acid induced the expression of osteoblast-specific genes with the activation of mitogen-activated protein kinases, nuclear factor-κB, and activator protein-1. Additionally, noggin, an antagonist of bone morphogenetic proteins (BMPs), inhibited ursolic acid-induced osteoblast differentiation. Noggin also inhibited the activation of Smad and the induction of BMP-2 mRNA expression by ursolic acid in the late stage of osteoblast differentiation. Importantly, ursolic acid was shown to have bone-forming activity in vivo in a mouse calvarial bone formation model. A high proportion of positive immunostaining of BMP-2 was found in the nuclear region of woven bone formed by ursolic acid. These results suggested that ursolic acid has the anabolic potential to stimulate osteoblast differentiation and enhance new bone formation.

Morphological manifestations for the protective effect of miliacin in organs of immunogenesis after treatment with methotrexate

Morphological changes in lymphoid organs induced by antitumor drug methotrexate were studied in 219 male (CBAxC57B1/6)F1 mice. Plant triterpenoid miliacin attenuated the lymphotoxic effect of this drug.

A plant triterpenoid, avicin D, induces autophagy by activation of AMP-activated protein kinase

Avicins, a family of plant triterpene electrophiles, can trigger apoptosis-associated tumor cell death, and suppress chemical-induced carcinogenesis by its anti-inflammatory, anti-mutagenic, and antioxidant properties. Here, we show that tumor cells treated with benzyloxycarbonylvalyl-alanyl-aspartic acid (O-methyl)-fluoro-methylketone, an apoptosis inhibitor, and Bax(-/-)Bak(-/-) apoptosis-resistant cells can still undergo cell death in response to avicin D treatment. We demonstrate that this non-apoptotic cell death is mediated by autophagy, which can be suppressed by chloroquine, an autophagy inhibitor, and by specific knockdown of autophagy-related gene-5 (Atg5) and Atg7. Avicin D decreases cellular ATP levels, stimulates the activation of AMP-activated protein kinase (AMPK), and inhibits mammalian target of rapamycin (mTOR) and S6 kinase activity. Suppression of AMPK by compound C and dominant-negative AMPK decreases avicin D-induced autophagic cell death. Furthermore, avicin D-induced autophagic cell death can be abrogated by knockdown of tuberous sclerosis complex 2 (TSC2), a key mediator linking AMPK to mTOR inhibition, suggesting that AMPK activation is a crucial event targeted by avicin D. These findings indicate the therapeutic potential of avicins by triggering autophagic cell death.

Triterpenoid miliacin inhibits stress-induced lipid peroxidation

A plant triterpenoid miliacin prevented stress-induced activation of LPO and accumulation of LPO products in the blood. The inhibitory effect of miliacin was not related to direct inhibition of reactive oxygen species generation.

4,4‘-Dimethyldinaphtho[a,d]cycloheptane, a Naturally Occurring Polyaromatic Derivative Related to Triterpenoids of the Serratane Series

[reaction: see text] The polyaromatic hydrocarbon 4,4'-dimethyldinaphtho[a,d]cycloheptane (1a) has been identified by NMR studies after isolation from an Oligocene sediment. The original symmetrical structure of 1a, which bears a central seven-membered ring, is closely related to higher plant triterpenoid precursor(s) of the serratane series and is believed to have been formed in the subsurface by a microbially mediated aromatization process.

Antiplasmodial and Antifungal Activities of Iridal (I), a Plant Triterpenoid.

Antiplasmodial and Antifungal Activities of Iridal (I), a Plant Triterpenoid.

Novel plant triterpenoid drug amooranin overcomes multidrug resistance in human leukemia and colon carcinoma cell lines.

Amooranin (AMR), a plant terpenoid, isolated from Amoora rohituka, was investigated for its ability to overcome multidrug resistance in human leukemia and colon carcinoma cell lines. AMR IC(50) values of multidrug-resistant leukemia (CEM/VLB) and colon carcinoma (SW620/Ad-300) cell lines were higher (1.9- and 6-fold) than parental sensitive cell lines (CEM and SW620). AMR induced G(2)+M phase-arrest during cell cycle traverse in leukemia and colon carcinoma cell lines and the percentage of cells in G(2)+M phase increased in a dose-dependent manner. Coincubation of tumor cells with both DOX and AMR reversed DOX resistance in 104-fold DOX-resistant CEM/VLB and 111-fold DOX-resistant SW620/Ad-300 cell lines with a dose modification factor of 50.9 and 99.6, respectively. Flow cytometric assay showed that AMR causes enhanced cellular DOX accumulation in a dose-dependent manner. AMR inhibits photolabeling of P-glycoprotein (P-gp) with [(3)H]-azidopine and the blocking effect enhanced with increasing concentrations of AMR. Our results show that AMR competitively inhibits P-gp-mediated DOX efflux, suggestive of a mechanism underlying the enhanced DOX accumulation and reversal of multidrug resistance by AMR.

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 μg/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 μg/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.

Biosynthesis of triterpenoid saponins in plants.

Many different plant species synthesise triterpenoid saponins as part of their normal programme of growth and development. Examples include plants that are exploited as sources of drugs, such as liquorice and ginseng, and also crop plants such as legumes and oats. Interest in these molecules stems from their medicinal properties, antimicrobial activity, and their likely role as determinants of plant disease resistance. Triterpenoid saponins are synthesised via the isoprenoid pathway by cyclization of 2,3-oxidosqualene to give primarily oleanane (beta-amyrin) or dammarane triterpenoid skeletons. The triterpenoid backbone then undergoes various modifications (oxidation, substitution and glycosylation), mediated by cytochrome P450-dependent monooxygenases, glycosyltransferases and other enzymes. In general very little is known about the enzymes and biochemical pathways involved in saponin biosynthesis. The genetic machinery required for the elaboration of this important family of plant secondary metabolites is as yet largely uncharacterised, despite the considerable commercial interest in this important group of natural products. This is likely to be due in part to the complexity of the molecules and the lack of pathway intermediates for biochemical studies. Considerable advances have recently been made, however, in the area of 2,3-oxidosqualene cyclisation, and a number of genes encoding the enzymes that give rise to the diverse array of plant triterpenoid skeletons have been cloned. Progress has also been made in the characterisation of saponin glucosyltransferases. This review outlines these developments, with particular emphasis on triterpenoid saponins.

Dose-Response Susceptibility of Pest Aphids (Homoptera: Aphididae) and their Control on Hydroponically Grown Lettuce with the Entomopathogenic FungusVerticillium lecanii,Azadirachtin, and Insecticidal Soap

The objective of our research was to identify alternatives to synthetic pesticide treatments to prevent aphid outbreaks in greenhouse lettuce crops. In the laboratory, we determined the susceptibility of three lettuce-infesting aphid species, Macrosiphum euphorbiae (Thomas), Myzus persicae (Sulzer), and Nasonovia ribisnigri (Mosley), to the hyphomycete Verticillium lecanii (Viegas) (strain Vertalec), the plant triterpenoid molecule azadirachtin (BioNeem), and an insecticidal soap (Safer’s). Estimated LC50 and LT50 obtained in the laboratory indicated that the three aphid species were susceptible to the entomopathogenic fungus, the plant extract, and the soap. Under greenhouse conditions, we assessed the potential of the three pesticides to reduce aphid populations and compared it with that of a synthetic insecticide, the organophosphate Malathion. Greenhouse experiments demonstrated that all three pesticides significantly reduced the population of each aphid species compared with the untreated plants. This study also revealed differences in aphid susceptibility between aphid species and between laboratory bioassays and greenhouse trials. The high initial aphid densities, difficulties to reach the aphids on the undersurface of leaves, stains left by Vertalec on the harvested lettuce, and high cosmetic standards for lettuce mitigated the performance of the insecticides. The potential of using Vertalec, BioNeem, and Safer’s soap for the control of lettuce aphids is discussed in relation to aphid species and crop management.

Organic geochemistry of lacustrine sediments: a record of the changing trophic status of Rostherne Mere, U.K.

The elemental, molecular (lipid) and isotopic ( 13C/ 12C) composition of organic matter in samples from the eutrophic lake, Rostherne Mere, U.K. were examined at high resolution in a single sediment core from a deep part of the lake. The distributions of lipids indicate an important contribution of allochthonous organic matter to the Rostherne sediments. However, increases in concentrations of unsaturated and branched chain fatty acids, phytol and C 27 and C 28 sterols toward the surficial sediments, and changing carbon isotope composition and C/N ratios, suggest that the autochthonous contribution to sedimentary OM has increased as a result of recent eutrophication. The elevated supply of labile OM to the sediments has influenced some key diagenetic processes, notably those of phytol degradation and higher plant triterpenoid degradation and aromatisation.