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Triterpenoids are widely distributed among plants of the legume family. However, most studies have focused on triterpenoids and their biosynthetic enzymes in model legumes. We evaluated the triterpenoid aglycones profile of the medicinal legume tree Bauhinia forficata by gas chromatography-mass spectrometry. Through transcriptome analyses, homology-based cloning, and heterologous expression, we discovered four oxidosqualene cyclases (OSCs) which are responsible for the diversity of triterpenols in B. forficata. We also investigated the effects of the unique motif TLCYCR on α-amyrin synthase activity. B. forficata highly accumulated α-amyrin. We discovered an OSC with a preponderant α-amyrin-producing activity, which accounted for at least 95% of the total triterpenols. We also discovered three other functional OSCs (BfOSC1, BfOSC2, and BfOSC4) that produce β-amyrin, germanicol, and cycloartenol. Furthermore, by replacing the unique motif TLCYCR from BfOSC3 with the MWCYCR motif, we altered the function of BfOSC3 such that it no longer produced α-amyrin. Our results provide new insights into OSC cyclization, which is responsible for the diversity of triterpenoid metabolites in B. forficata, a non-model legume plant.

The functions of Val483, Trp534, and Met729 in Euphorbia tirucalli β-amyrin synthase were revealed by comparing the enzyme activities of site-directed mutants against that of the wild type. The Gly and Ala variants with a smaller bulk size at position 483 predominantly afforded monocyclic camelliol C, which suggested that the orientation of the (3S)-2,3-oxidosqualene substrate was not appropriately arranged in the reaction cavity as a result of the decreased bulk size, leading to failure of its normal folding into the chair-chair-chair-boat-boat conformation. The Ile variant, with a somewhat larger bulk, afforded β-amyrin as the dominant product. Intriguingly, various variants of Trp534 exhibited significantly decreased enzymatic activities and provided no aberrantly cyclized products, although the aromatic Phe and Tyr residues were incorporated and the steric sizes of the aliphatic residues were altered. Therefore, the Trp534 residue does not stabilize the transient cation through a cation-π interaction. Furthermore, the Trp residue, with the largest steric bulk among all natural amino acids, is essential for high enzymatic activity. Robust CH-π complexation between the Val483 and Trp534 residues is proposed herein. Altering the steric bulk at the Met729 position afforded the pentacyclic skeletons. Thus, Met729 is positioned at the E-ring formation site. More detailed insights into the functions of the Val483, Trp534, and Met729 residues are provided by homology modeling.

Conyza blinii H.Lev. is a widely used medicinal herb in southwestern China. The main pharmacological components of C. blinii are a class of oleanane-type pentacyclic triterpene glycosides known as conyzasaponins, which are thought to be synthesized from β-amyrin. However, no genes involved in the conyzasaponin pathway have previously been identified. Here, we identify an oxidosqualene cyclase (OSC), a β-amyrin synthase, which mediates cyclization of 2,3-oxidosqualene to yield β-amyrin. Ten OSC sequences were isolated from C. blinii transcript tags. Phylogenetic analysis was used to select the tag Cb18076 as the putative β-amyrin synthase, named CbβAS. The open reading frame of CbβAS is 2286 bp and encodes 761 amino acids. Its mature protein contains the highly conserved motifs (QXXXGXW/DCTAE) of OSCs and (MWCYCR) of β-amyrin synthases. Transcription of CbβAS was upregulated 4-24 h after treatment of the seedlings of the C. blinii cultivar with methyl jasmonate. Furthermore, expression of CbβAS in Saccharomyces cerevisiae successfully yielded β-amyrin. The chemical structures and concentrations of β-amyrin were confirmed by GC-MS/MS. The target yeast ultimately produced 4.432 mg·L-1 β-amyrin. Thus, CbβAS is an OSC involved in conyzasaponin biosynthesis.

The medicinal plant sweet basil (Ocimum basilicum) accumulates bioactive ursane- and oleanane-type pentacyclic triterpenes (PCTs), ursolic acid and oleanolic acid, respectively, in a spatio-temporal manner; however, the biosynthetic enzymes and their contributions towards PCT biosynthesis remain to be elucidated. Two CYP716A subfamily cytochrome P450 monooxygenases (CYP716A252 and CYP716A253) are identified from a methyl jasmonate-responsive expression sequence tag collection and functionally characterized, employing yeast (Saccharomyces cerevisiae) expression platform and adapting virus-induced gene silencing (VIGS) in sweet basil. CYP716A252 and CYP716A253 catalyzed sequential three-step oxidation at the C-28 position of α-amyrin and β-amyrin to produce ursolic acid and oleanolic acid, respectively. Although CYP716A253 was more efficient than CYP716A252 for amyrin C-28 oxidation in yeast, VIGS revealed essential roles for both of these CYP716As in constitutive biosynthesis of ursolic acid and oleanolic acid in sweet basil leaves. However, CYP716A253 played a major role in elicitor-induced biosynthesis of ursolic acid and oleanolic acid. Overall, the results suggest similar as well as distinct roles of CYP716A252 and CYP716A253 for the spatio-temporal biosynthesis of PCTs. CYP716A252 and CYP716A253 might be useful for the alternative and sustainable production of PCTs in microbial host, besides increasing plant metabolite content through genetic modification.

β-Amyrin synthase incubation experiments have been conducted to determine the influence of steric effects at the 6-, 10- and 15-positions of 2,3-oxidosqualene on the polycyclisation pathway. Nor- and ethyl-substituted oxidosqualene analogues were synthesised. Cyclisation of the ethyl-substituted analogues did not occur, but the nor analogues underwent a polycyclisation cascade to yield fully cyclised products with 6/6/6/6/6-fused pentacyclic scaffolds generated via a final oleanyl cation. Previously, we reported that 19- and 23-ethyl-substituted analogues underwent polycyclisation reactions. Therefore, the catalytic domain involved in earlier cyclisation steps is notably compact. In contrast, the catalytic domain in the later cyclisation steps is more loosely packed (less compact) to accommodate the bulky ethyl group. The reaction cavities for recognising branched methyl groups are discussed by comparing β-amyrin synthase with other triterpene cyclases such as lanosterol and hopene synthases.

Centella asiatica is a herbaceous plant of Asian traditional medicine. Besides wound healing, this plant is recommended for the treatment or care of various skin conditions such as dry skin, leprosy, varicose ulcers, eczema, and/or psoriasis. Triterpene saponins, known as centellosides, are the main metabolites associated with these beneficial effects. Considering the interest in these high value active compounds, there is a need to develop biosustainable and economically viable processes to produce them. Previous work using C. asiatica plant cell culture technology demonstrated the efficient conversion of amyrin derivatives into centellosides, opening a new way to access these biomolecules. The current study was aimed at increasing the production of centellosides in C. asiatica plant cell cultures. Herein, we report the application of a new elicitor, coronatine, combined with the addition of amyrin-enriched resins as potential sustainable precursors in the centelloside pathway, for a positive synergistic effect on centelloside production. Our results show that coronatine is a powerful elicitor for increasing centelloside production and that treatments with sustainable natural sources of amyrins enhance centelloside yields. This process can be scaled up to an orbitally shaken CellBag, thereby increasing the capacity of the system for producing biomass and centellosides.

Oxidosqualene cyclases catalyze the transformation of oxidosqualene (1) into numerous cyclic triterpenes. Enzymatic reactions of 24-noroxidosqualene (8) and 30-noroxidosqualene (9) with Euphorbia tirucalli β-amyrin synthase were conducted to examine the role of the branched methyl groups of compound 1 in the β-amyrin biosynthesis. Substrate 8 almost exclusively afforded 30-nor-β-amyrin (>95.5 %), which was produced through a normal cyclization pathway, along with minor products (

The function of the active-site residues of oxidosqualene cyclases (OSCs) has been presumed mainly in light of the product distribution; however, not much research has been performed into the enzymatic activity of mutated OSCs. β-Amyrin, which is widely found in the plant kingdom, is classified as an OSC; mutational studies on β-amyrin cyclase are very limited. Six site-specific mutations targeted at the Phe728 residue of Euphorbia tirucalli β-amyrin synthase (EtAS) were constructed to inspect the function of this aromatic residue. We developed a simple method to evaluate the in vivo enzymatic activity; the expression levels of EtASs and the quantities of the cyclic triterpenes produced were determined by use of western blot and GC analyses, respectively. Measurement of the relative in vivo activity of the mutants versus that of the wild-type enzyme showed that the Ala, Met, His, and Trp variants had significantly decreased activity, but that the Tyr mutant had a high activity, which was nearly the same as that of the wild-type enzyme. In contrast to Tyr, Ala and Met possess no π-electrons; thus, the role of Phe728 is to stabilize the cationic intermediates, resulting in facilitation of the ring-expansion processes, especially by stabilizing the secondary cations. The decreased activity of the Trp mutant is ascribed to the introduction of a large steric bulk, leading to looser binding of oxidosqualene in the Trp variant. The His mutant afforded germanicol as the main product, indicating that the Phe residue is located near the D/E-ring-formation site. Changes in the steric bulk gave some cationic intermediates, resulting in the formation of 13 cyclic triterpenes, including an unnatural triterpene, (17E)-dammara-17(20),24-dien-3β-ol, and isoursenol, which has rarely been found in nature. In this study, we provide the first experimental evidence that cation-π interactions play a key role in the catalytic action of OSCs.

β-Amyrin, a natural triterpene, is widely distributed in the plant kingdom, and its pentacyclic skeleton is produced by oxidosqualene cyclase (OSC). OSC enzymes are classified as membrane proteins, and they catalyze the polycyclization reaction of (3S)-2,3-oxidosqualene to yield nearly 150 different cyclic triterpene skeletons. To date, no report has described the successful purification and characterization of plant β-amyrin synthase. The β-amyrin synthase from Euphorbia tirucalli (EtAS) was expressed as a polyhistidine-tagged protein in Saccharomyces cerevisiae GIL77, which lacks the lanosterol synthase gene. The expression yield, determined by western blotting analysis, was 5-7 mg. By Ni(2+) -nitrilotriacetic acid affinity column chromatography and careful selection of the proper imidazole concentration during the purification processes of washing and elution, a single band was successfully obtained on SDS/PAGE. We then tested the effects of four detergents on the enzyme activity. Supplementation with Triton X-100 at a concentration of 0.05% yielded the highest activity. The optimal pH and temperature were 7.0 and 30 °C, respectively. The kinetic parameters, K(m) and k(cat) , were determined to be 33.8 ± 0.53 μm and 46.4 ± 0.68 min(-1), respectively. To the best of our knowledge, there are no reports describing both K(m) and k(cat) for OSCs except for two examples of rat and bovine lanosterol synthases. The β-amyrin synthase purified in this study showed a significantly higher catalytic efficiency (k(cat)/K(m)) (~ 10(3)-fold) than those of the two reported lanosterol synthases. Gel-filtration HPLC indicated that the OSC exists as a monomer, and the eluted OSC retained its activity. Furthermore, the inhibition constants K(i) and IC(50) and types of inhibition by iminosqualene, Ro48-8071 and U18666A were determined, and indicated that iminosqualene and Ro48-8071 are potent inhibitors. Additionally, this is the first report of the kinetic data of the mutated enzymes targeted for the DCTAE(485-489) motif, which is a putative initiation site for the polycyclization reaction. No activity of the D485N variant and significantly decreased activity of the C564A variant were found, definitively demonstrating that the acidic carboxyl residue Asp485 serves as a proton donor to initiate the polycyclization reaction, and that Cys564 is involved in hydrogen bond formation with the carboxyl residue Asp458 to enhance the acidity. The CD spectrum is the first to be reported for OSCs, and the CD spectra of the wild-type and the mutated EtASs were almost the same, indicating that the protein architecture was not altered by these mutations.

BACKGROUND AND PURPOSE Pharmacological activation of cannabinoid CB1 and CB2 receptors is a therapeutic strategy to treat chronic and inflammatory pain. It was recently reported that a mixture of natural triterpenes α- and β-amyrin bound selectively to CB1 receptors with a subnanomolar Ki value (133 pM). Orally administered α/β-amyrin inhibited inflammatory and persistent neuropathic pain in mice through both CB1 and CB2 receptors. Here, we investigated effects of amyrins on the major components of the endocannabinoid system. EXPERIMENTAL APPROACH We measured CB receptor binding interactions of α- and β-amyrin in validated binding assays using hCB1 and hCB2 transfected CHO-K1 cells. Effects on endocannabinoid transport in U937 cells and breakdown using homogenates of BV2 cells and pig brain, as well as purified enzymes, were also studied. KEY RESULTS There was no binding of either α- or β-amyrin to hCB receptors in our assays (Ki > 10 µM). The triterpene β-amyrin potently inhibited 2-arachidonoyl glycerol (2-AG) hydrolysis in pig brain homogenates, but not that of anandamide. Although β-amyrin only weakly inhibited purified human monoacylglycerol lipase (MAGL), it also inhibited α,β-hydrolases and more potently inhibited 2-AG breakdown than α-amyrin and the MAGL inhibitor pristimerin in BV2 cell and pig brain homogenates. CONCLUSIONS AND IMPLICATIONS We propose that β-amyrin exerts its analgesic and anti-inflammatory pharmacological effects via indirect cannabimimetic mechanisms by inhibiting the degradation of the endocannabinoid 2-AG without interacting directly with CB receptors. Triterpenoids appear to offer a very broad and largely unexplored scaffold for inhibitors of the enzymic degradation of 2-AG. LINKED ARTICLES This article is part of a themed section on Cannabinoids. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.167.issue-8

Th e morphology, histochemistry and ultrastructure of the glandular trichomes on Empetrum nigrum leaves have been studied and more than a third of the metabolites were identifi ed. Samples of the leaves were fi xed and processed for light and electron microscopy. Glandular trichomes are situated on the inner surface of the rolled leaves. Th ey have a clavate head and a short stalk. Histochemical tests and fl uorescent microscopy demonstrate diff erentiated staining of the various cell types in the glandular trichome for proteins, pectins, lipids, tannins and phenylpropanoids. During secretion, the secretory cells contain rough and smooth endoplasmic reticulum, Golgi stacks with large vesicles, diversiform leucoplasts in contact with a reticular sheath and opaque deposits in the vacuoles. Th ere are ultrastructural and functional diff erences between the secretory cells in the trichome head: synthesis of hydrophilic substances predominately occurs in the upper and middle secretory cells, whereas synthesis of lipophilic compounds takes place in the middle and lower secretory cells. Gas chromatography – mass spectrometry was used to determine the content of metabolites in the methanol extracts from the leaves. Many phenolic compounds (phenolic acids, bibenzyls, catechins, fl avanones and fl avan-3-ols) as well as several terpenoids were found. Two chalcones (2 ′ ,4 ′ -dimethoxydihydrochalcone and 2 ′ ,4 ′ ,6 ′ -trihydroxydihydrochalcone), one bibenzyl (batatasin III), one fl avanone (7-hydroxyfl avanone) and 8 terpenoids (including phytol, α -tocopherol, s -sitosterol, α -amyrin, uvaol, oleanolic acid, ursolic acid and dehydroursolic acid) were identifi ed in E. nigrum leaf extracts. Th e total yield of phenolic compounds is fi ve to six times higher than the yield of terpenoids. It has been established that chalcones have no hydroxyl groups in ring B whereas bibenzyls have a hydroxyl group in the 3-position in ring B. On the basis of the histochemistry, fl uorescent microscopy, ultrastructure and chemical analysis, it may be concluded that synthesis and accumulation of phenolic substances and terpenoids takes place in the clavate glandular trichomes. Secondary metabolites synthesized in the trichomes protect leaf tissues from viruses, bacteria and pathogenic fungi.

The pentacyclic triterpenes, in particular ursolic acid and oleanolic acid and their derivatives, exist abundantly in the plant kingdom, where they are well known for their anti-inflammatory, antitumour and antimicrobial properties. α-Amyrin and β-amyrin are the precursors of ursolic and oleanolic acids, respectively, formed by concerted cyclization of squalene epoxide by a complex synthase reaction. We identified three full-length expressed sequence tag sequences in cDNA libraries constructed from apple (Malus × domestica 'Royal Gala') that were likely to encode triterpene synthases. Two of these expressed sequence tag sequences were essentially identical (> 99% amino acid similarity; MdOSC1 and MdOSC3). MdOSC1 and MdOSC2 were expressed by transient expression in Nicotiana benthamiana leaves and by expression in the yeast Pichia methanolica. The resulting products were analysed by GC and GC-MS. MdOSC1 was shown to be a mixed amyrin synthase (a 5 : 1 ratio of α-amyrin to β-amyrin). MdOSC1 is the only triterpene synthase so far identified in which the level of α-amyrin produced is > 80% of the total product and is, therefore, primarily an α-amyrin synthase. No product was evident for MdOSC2 when expressed either transiently or in yeast, suggesting that this putative triterpene synthase is either encoded by a pseudogene or does not express well in these systems. Transcript expression analysis in Royal Gala indicated that the genes are mostly expressed in apple peel, and that the MdOSC2 expression level was much lower than that of MdOSC1 and MdOSC3 in all the tissues tested. Amyrin content analysis was undertaken by LC-MS, and demonstrated that levels and ratios differ between tissues, but that the true consequence of synthase activity is reflected in the ursolic/oleanolic acid content and in further triterpenoids derived from them. Phylogenetic analysis placed the three triterpene synthase sequences with other triterpene synthases that encoded either α-amyrin and/or β-amyrin synthase. MdOSC1 and MdOSC3 clustered with the multifunctional triterpene synthases, whereas MdOSC2 was most similar to the β-amyrin synthases.

Dendropanax morbifera Leveille (Araliaceae) is well known in Korea traditional medicine for a variety of diseases. The methanol extract of the lower stem parts of D. morbifera was investigated for its activity against chloroquine-sensitive strains of Plasmodium falciparum using the parasite lactate dehydrogenase assay method. Two cycloartane-type glycosides oleifoliosides A (1) and B (2), and dendropanoxide (3), β-amyrin (4), α-amyrin (5) have been isolated from the stem parts of D. morbifera. All five compounds were evaluated for in vitro antiplasmodial activities as well as their cytotoxic potential on SK-OV-3 cancer cell lines. Compounds 2 and 3 showed notable growth inhibitory activity against chloroquine-sensitive strains of Plasmodium falciparum with IC50 values of 6.2 and 5.3 µm. This compound showed no significant cytotoxicity (IC50 > 150 µm) evaluated using SK-OV-3 cancer cell lines. This is the first report on the antiplasmodial activity of the compounds from D. morbifera. Copyright © 2009 John Wiley & Sons, Ltd.

Using a degenerate primer designed from triterpene synthase sequences, we have isolated a new gene from the medicinal plant Artemisia annua. The predicted protein is highly similar to beta-amyrin synthases (EC 5.4.99.-), sharing amino acid sequence identities of up to 86%. Expression of the gene, designated AaBAS, in Saccharomyces cerevisiae, followed by GC/MS analysis, confirmed the encoded enzyme as a beta-amyrin synthase. Through engineering the sterol pathway in S. cerevisiae, we explore strategies for increasing triterpene production, using AaBAS as a test case. By manipulation of two key enzymes in the pathway, 3-hydroxy-3-methylglutaryl-CoA reductase and lanosterol synthase, we have improved beta-amyrin production by 50%, achieving levels of 6 mg.L(-1) culture. As we have observed a 12-fold increase in squalene levels, it appears that this strain has the capacity for even higher beta-amyrin production. Options for further engineering efforts are explored.

Triterpenes exhibit a wide range of structural diversity produced by a sequence of biosynthetic reactions. Cyclization of oxidosqualene is the initial origin of structural diversity of skeletons in their biosynthesis, and subsequent regio- and stereospecific hydroxylation of the triterpene skeleton produces further structural diversity. The enzymes responsible for this hydroxylation were thought to be cytochrome P450-dependent monooxygenase, although their cloning has not been reported. To mine these hydroxylases from cytochrome P450 genes, five genes (CYP71D8, CYP82A2, CYP82A3, CYP82A4 and CYP93E1) reported to be elicitor-inducible genes in Glycine max expressed sequence tags (EST), were amplified by PCR, and screened for their ability to hydroxylate triterpenes (beta-amyrin or sophoradiol) by heterologous expression in the yeast Saccharomyces cerevisiae. Among them, CYP93E1 transformant showed hydroxylating activity on both substrates. The products were identified as olean-12-ene-3beta,24-diol and soyasapogenol B, respectively, by GC-MS. Co-expression of CYP93E1 and beta-amyrin synthase in S. cerevisiae yielded olean-12-ene-3beta,24-diol. This is the first identification of triterpene hydroxylase cDNA from any plant species. Successful identification of a beta-amyrin and sophoradiol 24-hydroxylase from the inducible family of cytochrome P450 genes suggests that other triterpene hydroxylases belong to this family. In addition, substrate specificity with the obtained P450 hydroxylase indicates the two possible biosynthetic routes from triterpene-monool to triterpene-triol.

Reports on the methylsterol fractions of hazelnut oils are scarce. The objectives of this study were to characterize methylsterols in hazelnut and virgin olive oils and to study the possibility of detection of adulteration of virgin olive oils. In hazelnut oils, 4-desmethylsterols were present in higher proportions (86 to 91%) than in virgin olive oils where this fraction was ca. 50% of the total sterol. In the 4-monomethylsterol fraction, citrostadienol was the major component in both kinds of oils followed by cycloeucalenol and obtusifoliol in virgin olive oils, and obtusifoliol in hazelnut oils. 24-Methylenecycloartanol was predominant in both kinds of oils in the 4,4′-dimethylsterols. For the first time, δ-amyrin was tentatively identified by comparing published mass spectral data in the analyzed samples of both kinds of oils. An unknown compound X (containing a lupane skeleton) and lupeol were detected only in the 4,4′-dimethylsterols fraction of hazelnut oils at a level of 2–8 and 6–10%, respectively. GC-MS analysis showed that adulteration of virgin olive oil by hazelnut oil could be detected at a level less than 4% by using these two compounds as possible potential markers.

Six oleanane-type triterpenoid esters were isolated from the golden flowers of Tagetes erecta. Spectral studies characterized their structures as 3-O-[(9Z)-hexadec-9-enoyl]erythrodiol (1), 11α,12α:13β,28-diepoxyoleanan-3β-yl (9Z)-hexadec-9-enoate (2), 13β,28-epoxyolean-11-en-3β-yl (9Z)-hexadec-9-enoate (3), 28-hydroxy-11-oxoolean-12-en-3β-yl (9Z)-hexadec-9-enoate (4), 3-O-[(9Z-hexadec-9-enoyl]-β-amyrin (5), and 11-oxoolean-12-en-3β-yl (9Z)-hexadec-9-enoate (6). Compounds 1–4 and 6 are new natural products, while the known 5 was isolated for the first time from the genus Tagetes, from which only one triterpenoid has earlier been obtained. Aerial oxidation (autoxidation) converted amyrin 1 into 2–4 and transformed amyrin 5 into 6. The configuration of 1–6 and an autoxidation mechanism (Scheme) involving the formation of the intermediate 11α-hydroxyolean-12-ene derivatives 1b and 5b on thermal decomposition of the labile 11α-OOH derivatives 1a and 5a, respectively, under neutral conditions are discussed. For the first time, the reactivity of the allylic HC(11) bond of triterpenoids of type 1 and 5 toward aerial oxidation was observed. The long-chain ester group at C(3) of 1 and 5 may be responsible for their labile nature, as β-amyrin (7), erythrodiol (8), and ursolic acid were found to be inert toward autoxidation.

Naturally occurring gums and resins with beneficial pharmaceutical and nutraceutical properties were tested for their possible protective effect against copper-induced LDL oxidation in vitro. Chiosmastic gum (CMG) (Pistacia lentiscus var. Chia resin) was the most effective in protecting human LDL from oxidation. The minimum and maximum doses for the saturation phenomena of inhibition of LDL oxidation were 2.5 mg and 50 mg CMG (75.3% and 99.9%, respectively). The methanol/water extract of CMG was the most effective compared with other solvent combinations. CMG when fractionated in order to determine a structure-activity relationship showed that the total mastic essential oil, collofonium-like residue and acidic fractions of CMG exhibited a high protective activity ranging from 65.0% to 77.8%. The other natural gums and resins (CMG resin 'liquid collection', P. terebinthus var. Chia resin, dammar resin, acacia gum, tragacanth gum, storax gum) also tested as above, showed 27.0%-78.8% of the maximum LDL protection. The other naturally occurring substances, i.e. triterpenes (amyrin, oleanolic acid, ursolic acid, lupeol, 18-a-glycyrrhetinic acid) and hydroxynaphthoquinones (naphthazarin, shikonin and alkannin) showed 53.5%-78.8% and 27.0%-64.1% LDL protective activity, respectively. The combination effects (68.7%-76.2% LDL protection) of ursolic-, oleanolic- and ursodeoxycholic- acids were almost equal to the effect (75.3%) of the CMG extract in comparable doses.

A practical synthesis of α-amyrin (1), β-amyrin (2), and lupeol (3) was accomplished in total yields of 32, 42, and 40% starting from easily available ursolic acid (4), oleanolic acid (5), and betulin (6), respectively. Remarkably, these three natural pentacyclic triterpenes exhibited potential inhibitory activity against human oxidosqualene cyclase.

1. 5-Lipoxygenase (5-LOX) products from endogenous arachidonic acid in ionophore-stimulated peritoneal polymorphonuclear leukocytes (PMNL) and from exogenous substrate (20 microM) in 105,000 g supernatants were measured. 2. The effects of natural pentacyclic triterpenes and their derivatives on 5-LOX activity were compared with the inhibitory action of acetyl-11-keto-beta-boswellic acid (AKBA), which has been previously shown to inhibit the 5-LOX by a selective, enzyme-directed, non-redox and non-competitive mechanism. 3. The 5-LOX inhibitory potency of AKBA was only slightly diminished by deacetylation of the acetoxy group or reduction of the carboxyl function to alcohol in intact cells (IC50 = 1.5 vs. 3 and 4.5 microM, respectively) and in the cell-free system (8 vs. 20 and 45 microM). 4. beta-Boswellic acid (beta-BA), lacking the 11-keto function, inhibited 5-LOX only partially and incompletely, whereas the corresponding alcohol from beta-BA, as well as amyrin, acetyl-11-keto-amyrin, 11-keto-beta-boswellic acid methyl ester had no 5-LOX inhibitory activity up to 50 microM in either system. 5. beta-BA only partially prevented the AKBA-induced 5-LOX inhibition, whereas the non-inhibitory compounds, amyrin and acetyl-11-keto-amyrin, almost totally antagonized the AKBA effect and shifted the concentration-inhibition curve for the incomplete inhibitor beta-BA to the right. In contrast, the non-inhibitory 11-keto-beta-BA methyl ester exerted no antagonizing effect. 6. The results demonstrate that the pentacyclic triterpene ring system is crucial for binding to the highly selective effector site, whereas functional groups (especially the 11-keto function in addition to a hydrophilic group on C4 of ring A) are essential for 5-LOX inhibitory activity.

Phytochemical inves tigation o f the whole plants of Artemisia arenaria affords one new ClIdcSIllL1n e sesqlliterpene, eudesm-5-ene-I~, 4a-dioll and ten known compounds, a-amyrin 2, ~ -amyrin 3 , ~s itostero l 4 , 4-hydroxyacetophcnone S, 5 , 7-dihydroxy-4 '-mcthoxyfl avanone 6 , 5-hydroxy-7 , 4'-d imcthoxy ll ava nonc 7 , 5. 6, 3'-trimethoxy fl avanone 8 , 5-hydroxy-7, 4' -dimethoxyfl avo ne 9 , 5-hydroxy-6, 7, 4' -trimcthoxy l'lava nnne 10 and sti gmasterol 11 . Their structures have been elucidated on the basis of spectroscopic mcthods.

A new and very direct enantioselective total synthesis of members of the β-amyrin family of pentacyclic triterpenes has been developed starting with acylsilane 5, 2-propenyllithium, and cyclohexenylmethyl bromide 6, which were assembled to form tetraene 7. Cationic cyclization of 7 and silylation afforded 8, which after vinyl triflate formation was cyclized via a Cu(I) intermediate (Scheme 2) to form the TBS ether of aegiceradienol 10, a versatile intermediate that is readily converted into natural β-amyrins such as β-amyrin (1) and oleanolic acid (2). The C(14)-diastereomer (13) of aegiceradienol was also synthesized from the C(14)-diastereomer of 8 using an intramolecular Stille reaction for the closure of ring D (Scheme 4).

Cyclization of oxidosqualene into tetra- and pentacyclic carbon skeleton of sterols and triterpenes, catalyzed by oxidosqualene cyclases (OSCs), is one of the most complex and fascinating reactions found in nature. OSCs generate multiple stereogenic centers in a single reaction, and are responsible for the diverse triterpene skeletons. In order to investigate the origin of structural diversity of triterpene skeletons, cDNA cloning of OSCs and analysis of their product specificity were carried out. From triterpene producing plants, over twenty-five OSC clones were obtained, and their enzyme function established by expression in yeast. They included cycloartenol, cucurbitadienol, lupeol, β-amyrin, isomultiflorenol and mixed amyrin synthases. Studies of chimeric proteins between β-amyrin synthase and lupeol synthase, and mutant proeins constructed by site directed mutagenesis identified the amino acid residues responsible for their product specificity. Trp 259 of β-amyrin synthase (PNY) was identified to be the critical residue controlling β-amyrin formation. In further mutation studies, PNY Y 261 H mutant produced dammara-18, 21-dien-3β-ol (as a 3 : 5 mixture of E/Z isomer at Δ18) together with minor amount of dammara-18 (28), 21-dien-3β-ol. These triterpenes have not been reported from nature, and therefore, could be categorized as “unnatural” natural products. The results of this study opened up the possibility of generating new triterpene synthases with additional novel functions through point mutations.

Summary In an approach to understand the mechanism(s) of resistance in raspberry to infestation by the aphid Amphorophora idaei, progeny plants segregating for the A. idaei resistance gene, A10, were bioassayed and dichloromethane extracts from the leaf surface were examined by capillary column gas chromatography (GC). No single GC peak was detected that was present in only the resistant progeny plants. Nevertheless, thirteen compounds present in all samples were quantified and identified by mass spectrometry. They were of four major classes; straight chain hydrocarbons, acetic acid esters of long chain alcohols, tocopherols and triterpenoid compounds, including α and β amyrin. Several of these compounds were not recorded previously in raspberry leaves. Linear discriminant analysis, applied to the standardised chromatographic data in an attempt to relate chemical composition to resistance, successfully partitioned 24 of the 26 plants into resistant and susceptible types as determined by bioassay. These data provide further evidence that resistance in raspberry to A. idaei is related to the chemical composition of the leaf surface.

Tritiated β-amyrin (olean-12-en-3β-ol, 2) was synthesized in six steps, from epikatonic acid (3), with a specific activity of 550 GBq/mmole. The protracted incubation of this triterpene in a pond mud led by biodegradation to labelled aromatic tetracyclic hydrocarbons, identical with known sedimentary biomarkers.

With a view to obtain new compounds bearing potential applications in the biogenetic type synthesis of natural products, acetylmethyl ursolic acid ( 1 ) was subjected to prolonged treatment with H 2 O 2 /AcOH on boiling water bath. Several such products ( 2 , 3 , 5 & 7 ) have thus been obtained. A new one-pot partial synthesis of 15α-hydroxy Δ 12 -analogues of pentacyclic triterpenes of amyrin series has been achieved. 13 C-NMR spectral data of the major product are also described.

When cultures ofPhytophthora cactorum were incubated on solid medium for 3 weeks in the dark at 20 C with [14C] cholesterol or [14C]β-amyrin, the [14C] cholesterol was assimilated into the advancing mycelium to a greater extent than the [14C]β-amyrin. Examination of the mycelium and medium for radioactive metabolites showed significant differences in the metabolism of the 2 labeled compounds. Cholesterol was converted mainly to esters and to some extent to glycosides, whereas β-amyrin was only slightly converted to esters and not at all to glycosides.

Unsaponifiable matter (UNS) of lipids from cashew nuts either roasted, unroasted or unroasted in shell was fractionated on a Florisil column. Gas-liquid chromatography (GLC) of the hydrocarbons showed that squalene was the major component of the 21 peaks detected. There were three homologous hydrocarbon series: normal, iso and/or anteiso and branched chain. GLC of the alcohol fraction silyl derivatives indicated the presence of three homologous series: normal, iso and/or anteiso and multiple branched chain alcohols. Beta and/or gamma tocopherol was found to be the major tocopherol. This component decreased during nut shell removal and roasting. Cycloartenol, amyrin (alpha-beta) and 24-methylene cycloartanol were the triterpenoids identified. GLC of the sterol fraction silyl derivatives showed beta-sitosterol to be the major sterol followed by campesterol.

Die jahrliche Produktion an schwarzem Tee in der Turkei betragt etwa 50 000 t. Man schatzt, das die Turkei jahrlich 14 000 t Teesamen und hieraus etwa 3000 t ol produzieren kann. Der Samen besteht zu 70% aus Kern und zu 30% aus Schale. Der Olgehalt der Kerne betragt 33%. Das turkische Teesamenol hat folgende Kennzahlen: Spez. Gew. bei 25° C: 0.9180; Brechungsindex bei 25° C: 1.4692; VZ: 192.8; JZ (Hanus): 90.9; RMZ: 1.0; OHZ: 5.25; Unverseifbares: 1.05%. Die gaschromatographische Bestimmung der Fettsauren fuhrte zu folgenden Durchschnittswerten: Spuren von Myristinsaure, 16.0% Palmitinsaure, 1.67% Stearinsaure, 59.4% Olsaure, 21.8% Linolsaure und 1.23% Arachinsaure. Der Olkuchen enthalt 14% Saponin und hat deshalb einen bitteren Geschmack. Teesaponin ist ein Triterpensaponin vom Typ Amyrin. Der Aglykon-Teil besteht aus funf verschiedenen Sapogeninen. Der Kohlenhydrat-Teil des Teesaponins enthalt folgende Zucker: Xylose, Galaktose, Glukose und Galakturonsaure. Turkish Teaseed Oil and Tea Saponin The annual production of black tea in Turkey is about 50 000 t. It is estimated, that Turkey can produce annually 14 000 t tea seed and 3000 t teaseed oil. The seed consists of 70% kernel and 30% hull. The oil content of the kernel is 33%. The Turkish teaseed oil has following characteristics: Specific weight at 25° C: 0.9180; refractive index at 25° C: 1.4692; saponification value: 192.8; iodine value (Hanus): 90.9; RM-value: 1.0; hydroxyl value: 5.25; unsaponifiable: 1.05%. Gas chromatographic determination of fatty acids gave following average values: myristic acid traces, palmitic acid 16.0%, stearic acid 1.67%, oleic acid 59.4%, linoleic acid 21.8% and arachidic acid 1.23%. The oilcake contains 14% saponin, therefore, has a bitter taste. Tea saponin is a triterpenic saponin of the type of amyrin. The aglycone consists of five different sapogenins. The carbohydrate part of the tea saponin contains following sugars: xylose, galactose, glucose and galacturonic acid.

Die Darstellungen von 20oz-Homo-β-amyrin (Id) und seines 20s-Isomeren (Ib) erfolgen ausgehend von der Glycyrrhetinsaure (Ia).

Die Inkubation des Titelepoxids (I) in einem Cyclase-System aus Pisum sativum fuhrt zu einer nach Acetylierung isolierten cyclischen Verbindung, die aufgrund von massenspektroskopischen und chromatographischen Vergleichen sowie unter Verwendung markierter Substanzen als Acetat (IIb) identifiziert wird.