Biotransformation of betulinic and betulonic acids by fungi

Betulinic acid (1), a triterpenoid found in many plant species, has attracted attention due to its important pharmacological properties, such as anti-cancer and anti-HIV activities. The closely related, betulonic acid (2) also has similar properties. In order to obtain derivatives potentially useful for detailed pharmacological studies, both compounds were submitted to incubations with selected microorganisms. In this work, both were individually metabolized by the fungi Arthrobotrys, Chaetophoma and Dematium, isolated from the bark of Platanus orientalis as well as with Colletotrichum, obtained from corn leaves; such fungal transformations are quite rare in the scientific literature. Biotransformations with Arthrobotrys converted betulonic acid (2) into 3-oxo-7beta-hydroxylup-20(29)-en-28-oic acid (3), 3-oxo-7beta,15alpha-dihydroxylup-20(29)-en-28-oic acid (4) and 3-oxo-7beta,30-dihydroxylup-20(29)-en-28-oic acid (5); Colletotrichum converted betulinic acid (1) into 3-oxo-15alpha-hydroxylup-20(29)-en-28-oic (6) acid whereas betulonic acid (2) was converted into the same product and 3-oxo-7beta,15alpha-dihydroxylup-20(29)-en-28-oic acid (4); Chaetophoma converted betulonic acid (2) into 3-oxo-25-hydroxylup-20(29)-en-28-oic acid (7) and both Chaetophoma and Dematium converted betulinic acid (1) into betulonic acid (2). Those fungi, therefore, are useful for mild, selective oxidations of lupane substrates at positions C-3, C-7, C-15, C-25 and C-30.     

Triterpenoids from Tripterygium wilfordii

The extract (T(II)) of Tripterygium wilfordii Hook f. afforded four triterpenoids: wilforic acid D (3beta,24-epoxy-2alpha-hydroxy-24R*-ethoxy-29-friedelanoic acid); (E) 3beta,24-epoxy-2-oxo-3alpha-hydroxy-29-friedelanoic acid; (F) 2beta-hydroxy-3-oxo-friedelan-29-oic acid; 29-hydroxy-3-oxo-olean-12-en-28-oic acid and 17 known triterpenoids. Their structures were established on the basis of spectroscopic studies. In a bioactivity analysis, only the known dulcioic acid compound showed a significant inhibitory effect on cytokine production.     

Plant pentacyclic triterpenic acids as modulators of lipid membrane physical properties

Free triterpenic acids (TTPs) present in plants are bioactive compounds exhibiting multiple nutriceutical activities. The underlying molecular mechanisms have only been examined in part and mainly focused on anti-inflammatory properties, cancer and cardiovascular diseases, in all of which TTPs frequently affect membrane-related proteins. Based on the structural characteristics of TTPs, we assume that their effect on biophysical properties of cell membranes could play a role for their biological activity. In this context, our study is focused on the compounds, oleanolic (3β-hydroxy-12-oleanen-28-oic acid, OLA), maslinic (2α,3β-dihydroxy-12-oleanen-28-oic acid, MSL) and ursolic ((3β)-3-hydroxyurs-12-en-28-oic acid, URL) as the most important TTPs present in orujo olive oil. X-ray diffraction, differential scanning calorimetry, (31)P nuclear magnetic resonance and Laurdan fluorescence data provide experimental evidence that OLA, MSL and URL altered the structural properties of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and DPPC-Cholesterol (Cho) rich membranes, being located into the polar-hydrophobic interphase. Specifically, in DPPC membranes, TTPs altered the structural order of the L(β'), phase without destabilizing the lipid bilayer. The existence of a nonbilayer isotropic phase in coexistence with the liquid crystalline L(α) phase, as observed in DPPC:URL samples, indicated the presence of lipid structures with high curvature (probably inverted micelles). In DPPC:Cho membranes, TTPs affected the membrane phase properties increasing the Laurdan GP values above 40°C. MSL and URL induced segregation of Cho within the bilayer, in contrast to OLA, that reduced the structural organization of the membrane. These results strengthen the relevance of TTP interactions with cell membranes as a molecular mechanism underlying their broad spectrum of biological effects.     

Selective oxidation-reduction and esterification of asiatic acid by Pestalotiopsis microspora and anti-HCV activity

Microbial transformation of asiatic acid (AA) by an endophytic fungus, Pestalotiopsis microspora, yielded six metabolites: 2-oxo-3β,15α,23-trihydroxyurs-12-ene-28-oic acid (1); 2-oxo-3β,15α,22α,23-tetrahydroxyurs-12-ene-28-oic acid (2); 2-oxo-3β,15α,23,30-tetrahydroxyurs-12-ene-28-oic acid (3); 2α,3β,15α,23,30-pentahydroxyurs-12-ene-28-oic acid methyl ester (4); 2α,3α,15α,23-tetrahydroxyurs-12-ene-28-oic acid (5); 2α,3α,15α,23,30-pentahydroxyurs-12-ene-28-oic acid (6). The structure elucidation of these products was confirmed based on the spectroscopic data. Compounds 2–6 were new. A possible biotransformation pathway is proposed. The anti-HCV activity of compounds 1–6 was also evaluated.     

锐尖山香圆叶中三萜类成分的研究

从锐尖山香圆(Turpinia arguta (Lindl.) Seem.)叶中分离得到了11个三萜类化合物。通过光谱分析,分别鉴定其结构为熊果酸(1), 3β,6β,23-trihydroxy-12-oleanen-28-oic acid (2), 3β,6β,23-trihydroxyurs-12-en-28-oic acid (3), 3β,6β,19α,23-tetrahydroxyurs-12-en-28-oic acid (4), 1 α, 3β,23-trihydroxy-12-oleanen-28-oic acid (5), arjunglucoside II (6), rosamultin (7), 3β-O-β-D-glucopyranoylcincholic acid (8), cinchonaglycoside C (9), mussaendoside S (10) 和3β-O-β-D-glucopyranosyl quinovic acid 28-O-β-D-glucopyranosyl ester (11)。除化合物1和6,其它化合物均为首次从山香圆叶中分离得到。     

nor-Oleanene type triterpene glycosides from the leaves of Acanthopanax japonicus

Three new (1-3) and two known (4-5) triterpene glycosides were isolated from the leaves of Acanthopanax japonicus (Araliaceae) and elucidated structurally by mass, 1D, and 2D NMR spectroscopy. All the compounds possessed a nor-oleanene triterpene skeleton as the aglycone. The structures of 1-5 were established as 28-O-alpha-L-rhamno-pyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester of 3beta-hydroxy- 30-nor-olean-12,20(29)-diene-23,28-dioic acid, designated as acanjaposide A, 3beta- hydroxy-23-oxo-30-nor-olean-12,20(29)-diene-28-oic acid, named acanjaposide B, 3beta,20alpha-dihydroxy-23-oxo-30-nor-olean-12-en-28-oic acid, named acanjaposide C, and nipponoside E, a known saponin, respectively.     

Anodic electrochemical oxidation of cholic acid

Regioselectivity in the anodic electrochemical oxidation of cholic acid with different anodes is described. The oxidation with PbO(2) anode affords the dehydrocholic acid in quantitative yield after 22 h. 3alpha,12alpha-Dihydroxy-7-oxo-5beta-cholan-24-oic acid (59%) and 3alpha-hydroxy-7,12-dioxo-5beta-cholan-24-oic acid (51%) are obtained stopping the reaction at lower time. The rate of the OH-oxidation is C7 > C12 > C3. The electro-oxidation with platinum foil anode gives selectively the 7-ketocholic acid in 40% yield. On the other hand, the graphite plate anode, varying the reaction conditions, produces selectively the dehydrocholic acid in quantitative yield or the 3alpha,12alpha-dihydroxy-7-oxo-5beta-cholan-24-oic acid (96%) while the 3alpha,7alpha-dihydroxy-12-oxo-5beta-cholan-24-oic acid (34%) is obtained together with the other oxo acids.     

Biotransformation of the antimelanoma agent betulinic acid by Bacillus megaterium ATCC 13368

Microbial transformation of the antimelanoma agent betulinic acid was studied. The main objective of this study was to utilize microorganisms as in vitro models to predict and prepare potential mammalian metabolites of this compound. Preparative-scale biotransformation with resting-cell suspensions of Bacillus megaterium ATCC 13368 resulted in the production of four metabolites, which were identified as 3-oxo-lup-20(29)-en-28-oic acid, 3-oxo-11alpha-hydroxy-lup-20(29)-en-28-oic acid, 1beta-hydroxy-3-oxo-lup-20(29)-en-28-oic acid, and 3beta,7beta, 15alpha-trihydroxy-lup-20(29)-en-28-oic acid based on nuclear magnetic resonance and high-resolution mass spectral analyses. In addition, the antimelanoma activities of these metabolites were evaluated with two human melanoma cell lines, Mel-1 (lymph node) and Mel-2 (pleural fluid).     

Bioactive humulene derivatives from Asteriscus vogelii

The bioactive fractions obtained from the extract of Asteriscus vogelii afforded a new humulene derivative, the 8-oxo-6,7,9,10-tetrahydrohumulen-1,12-olide (1), in addition to the known asteriscunolides A (2), C (3) and D (4), and the acids 8-oxo-alpha-humula-6Z,9Z-dien-12-oic acid (5), 8-oxo-alpha-humula-6E,9Z-dien- 12-oic acid (6) and 8-oxo-alpha-humula-6E,9E-dien-12-oic acid (7), characterized as their methyl esters. Evaluation of their phytotoxic and cytotoxic activities was accomplished. Compounds 3 and 4 gave the highest inhibition of plant cell cultures and of the plant Lemna paucicostata. Compound 4 was also the most active against P-338 lymphoma in mice, A-549 carcinoma of human lung, HT-29 carcinoma of human colon and MEL-28 human melanoma.     

Triterpenoids from enkianthus campanulatus

From the leaves of Enkianthus campanulatus were isolated three new triterpenes, 3-oxo-19,23,24-trihydroxyurs-12-en-28-oic acid, 3β,6β, 19,23-tetrahydroxyurs-12-en-28-oic acid and 3β,6β,23-trihydroxyurs-12-en-28-oic acid.     

Biotransformation of the Antimelanoma Agent Betulinic Acid by Bacillus megaterium ATCC 13368

Microbial transformation of the antimelanoma agent betulinic acid was studied. The main objective of this study was to utilize microorganisms as in vitro models to predict and prepare potential mammalian metabolites of this compound. Preparative-scale biotransformation with resting-cell suspensions of Bacillus megaterium ATCC 13368 resulted in the production of four metabolites, which were identified as 3-oxo-lup-20(29)-en-28-oic acid, 3-oxo-11α-hydroxy-lup-20(29)-en-28-oic acid, 1β-hydroxy-3-oxo-lup-20(29)-en-28-oic acid, and 3β,7β,15α-trihydroxy-lup-20(29)-en-28-oic acid based on nuclear magnetic resonance and high-resolution mass spectral analyses. In addition, the antimelanoma activities of these metabolites were evaluated with two human melanoma cell lines, Mel-1 (lymph node) and Mel-2 (pleural fluid).     

Triterpenes from Periandra dulcis roots

Three oleanane triterpenes were isolated from the roots of Periandra dulcis,and identified as 3β-hydroxy-25-al-olean-18-en-30-oic acid (periandric acid I), 3β-hydroxy-25-al-olean-12-en-30-oic acid (periandric acid II) and 3-oxo-25-hydroxy-olean-12-en-30-oic acid. The former two compounds (periandric acids I and II) were identical with the aglycones obtained by hydrolysis of periandrin I and II, respectively and the latter one was a new triterpene.     

Synthesis of ester-linked lithocholic acid dimers

Four lithocholic acid dimers were synthesised via esterification. The ester-linked dimer, 3-oxo-5beta-cholan-24-oic acid (cholan-24-oic acid methyl ester)-3-yl ester, (3alpha,5beta), was obtained by condensation of methyl lithocholate with 3-oxo-5beta-cholan-24-oic acid. Borohydride reduction of this ester-linked dimer gave 3alpha-hydroxy-5beta-cholan-24-oic acid (cholan-24-oic acid methyl ester)-3-yl ester, (3alpha,5beta), which was acetylated to 3alpha-acetoxy-5beta-cholan-24-oic acid (cholan-24-oic acid methyl ester)-3-yl ester, (3alpha,5beta). Reaction of methyl lithocholate with oxalyl chloride yielded the oxalate dimer, bis(5beta-cholan-24-oic acid methyl ester)-3alpha-yl oxalate.     

Triterpenoid saponins from the aerial parts of Trifolium argutum Sol. and their phytotoxic evaluation

Four triterpenoid saponins (1–4) were isolated from the aerial parts of Trifolium argutum Sol. (sharp-tooth clover) and their structures were elucidated by comprehensive spectroscopic analysis, including 1D and 2D NMR techniques, mass spectrometry and chemical methods. Two of them are new compounds, characterized as 3-O-[α-l-rhamnopyranosyl-(1→2)-β-d-galactopyranosyl-(1→2)-β-d-glucuronopyranosyl]-3β,24-dihydroxyolean-12-ene-22-oxo-29-oic acid (1) and 3-O-[β-d-galactopyranosyl-(1→2)-β-d-glucuronopyranosyl]-3β,24-dihydroxyolean-12-ene-22-oxo-29-oic acid (2). The occurrence of 3β,24-dihydroxyolean-12-ene-22-oxo-29-oic acid (melilotigenin) in its natural form is reported for the first time as a triterpenoid aglycone within Trifolium species. The phytotoxicity of compounds was evaluated on four STS at concentration 1 μM to 333 μM. Compound 1 was the most active, showing more than 60% inhibition on the root growth of L. sativa at the higher dose, with IC₅₀ (254.1 μM) lower than that of Logran^® (492.6 μM), a commercial herbicide used as positive control. The structure–activity relationships indicated that both aglycones and glycosidic parts may influence the phytotoxicity of saponins.     

Clerodane and labdane diterpenoids from Nuxia sphaerocephala

Seven diterpenoids including four clerodane and three labdane derivatives, (13S)-ent-7beta-hydroxy-3-cleroden-15-oic acid (1), ent-7beta-hydroxy-2-oxo-3-cleroden-15-oic acid (2), ent-2,7-dioxo-3-clero-den-15-oic acid (3), ent-18-(E)-caffeoyloxy-7beta-hydroxy-3-cleroden-15-oic acid (4) (13S)-ent-18-(E)-coumaroyloxy-8(17)-labden-15-oic acid (5), ent-18-(E)-caffeoyloxy-8(17)-labden-15-oic acid (6), ent-15-(E)-caffeoyloxy-8(17)-labden-18-oic acid (7), have been isolated from an ethyl acetate extract of the leaves of Nuxia sphaerocephala, together with 17 known compounds. 3-Oxolup-20(29)-en-30-al (3-oxolupenal) (8) and 3beta-hydroxylup-20(29)-en-30-al (3beta-hydroxy-lupenal) (9) showed the best inhibitory activity against Plasmodium falciparum with the IC(50) values between 1.55 and 4.67 microg/ml in vitro, respectively. The structure and the relative stereochemistry of the compounds were established on the basis of their spectroscopic properties. The absolute configuration at C-13 of 1 and 5 was determined by the PGME amide procedure.     

Lanostanes and friedolanostanes from the bark of Garcinia speciosa

The CHCl(3) extract of the bark of Garcinia speciosa contained four 17,14-friedolanostanes and five lanostanes as well as friedelin and common plant constituents. The friedolanostanes were the previously known methyl ester of (24E)-3 alpha,23 alpha-dihydroxy-17,14-friedolanostan-8,14,24-trien-26-oic acid and the methyl esters of three hitherto unknown acids, 3 alpha-hydroxy-16 alpha,23 alpha-epoxy-17,14-friedolanostan-8,14,24-trien-26-oic acid, 3 alpha,23 alpha-dihydroxy-8 alpha,9 alpha-epoxy-17,14-friedolanostan-15-oxo-24-en-26-oic acid and 3 alpha,23 alpha-dihydroxy-17,14-friedolanostan-15-oxo-8(14),24-dien-26-oic acid. New lanostanes were 3 beta,9 alpha-dihydroxylanost-24-en-26-al and the methyl ester of 3 beta-hydroxy-23-oxo-9,16-lanostadien-26-oic acid. Structures were established by analysis of spectroscopic data. In the case of the lanostanes the previously unassigned C-25 stereochemistry was shown to be 25R by X-ray analysis of 3 beta-hydroxy-23-oxo-9,16-lanostadien-26-oic acid. In the case of the friedolanostanes the configuration at C-23 was established as 23R, identical with the absolute configuration at C-23 of mariesiic acids A and B.     

Synthesis of 3 alpha, 7 alpha-dihydroxy-5 beta-cholestan-26-oic acid from 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid: configuration in the bile of Alligator mississippiensis.

Synthesis of 25R- and 25S-diastereoisomers of 3 alpha,7 alpha-dihydroxy-5 beta-cholestan-26-oic acid from 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid is described. The 25S-diastereoisomer of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan- 26-oic acid was obtained by vigorous hydrolysis of the bile of Alligator mississippiensis followed by repeated crystallization of the hydrolysate, and the 25R-diastereoisomer was isolated by hydrolysis of the bile salts in bile of A mississippiensis with rat feces. Acetylation of the 25R- or 25S-diastereoisomer of methyl 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid under controlled conditions yielded the corresponding 3 alpha,7 alpha-diacetate in approximately 70% yield. The diacetate was quantitatively oxidized to methyl 3 alpha,7 alpha-diacetoxy-12-oxo-5 beta-cholestan-26-oate, which was converted into the 12-tosylhydrazone in approximately 58% yield. Reduction of the tosylhydrazone with sodium borohydride in acetic acid yielded the 25R- or the 25S-diastereoisomer of 3 alpha,7 alpha-dihydroxy-5 beta-cholestan-26-oic acid as the major product. Purification via column chromatography yielded the pure diastereoisomers in approximately 25% overall yield. The two diastereoisomers were resolved on thin-layer chromatography and high-performance liquid chromatography. When the bile of A mississippiensis was hydrolyzed with rat fecal bacteria, the 3 alpha,7 alpha-dihydroxy-5 beta-cholestan-26-oic acid isolated via chromatographic purification was shown to be the 25R-diastereoisomer.     

A triterpenoid and its saponin from Phytolacca esculenta.

A new triterpenoid, esculentagenin, and its glycoside, esculentoside M, were isolated from the roots of Phytolacca esculenta and characterized as 11-oxo-3-O-methyloleanata-12-en-2β,3β,23-trihydroxy-28-oic acid and 3-O-[β - -glucopyranosyl (1→4)-β- -Xylopyranosyl]-28-O-β- -glucopyranosyl-11-oxo-30-methyloleanate-12-en-2β,3β,23-trihydroxy-28-oic acid by spectral and chemical evidence.     

Triterpenoids from Viburnum suspensum

Three triterpenoids, 3-oxo-11,13(18)-oleanadien-28-oic acid, 24-hydroxy-3-oxo-11,13(18)-oleanadien-28-oic acid, 6β-hydroxy-3-oxo-11,13(18)-oleanadien-28-oic acid have been isolated together with the previously known virgatic acid, vibsanin B and 3-hydroxyvibsanin E from the leaves of Viburnum suspensum. Their structures were determined by spectroscopic methods and by comparison of their NMR spectral data with those of the previously known 11,13(18)-oleanadien-3β-ol.     

Synthesis of potential cholelitholytic agents: 3 alpha,7 alpha,12 alpha-trihydroxy-7 beta-methyl-5 beta-cholanoic acid, 3 alpha,7 beta,12 alpha-trihydroxy-7 alpha-methyl-5 beta-cholanoic acid, and 3 alpha,12 alpha-dihydroxy-7 xi-methyl-5 beta-cholanoic acid

This report describes the chemical synthesis of six new bile acid analogs, namely, 3 alpha,7 alpha,12 alpha-trihydroxy-7 beta-methyl-5 beta-cholanoic acid (7 beta-methyl-cholic acid), 3 alpha,7 beta,12 alpha-trihydroxy-7 alpha-methyl-5 beta-cholanoic acid (7 alpha-methyl-ursocholic acid), 3 alpha,12 alpha-dihydroxy-7 xi-methyl-5 beta-cholanoic acid (7 xi-methyl-deoxycholic acid), 3 alpha,12 alpha-dihydroxy-7-methyl-5 beta-chol-7-en-24-oic acid, 3 alpha,12 alpha-dihydroxy-7-methyl-5 beta-chol-6-en-24-oic acid, and 3 alpha,12 alpha-dihydroxy-7-methylene-5 beta-cholan-24-oic acid. The carboxyl group of the starting material 3 alpha,12 alpha-dihydroxy-7-oxo-5 beta-cholanoic acid was protected by conversion to its oxazoline derivative. A Grignard reaction of the bile acid oxazoline with CH3MgI followed by acid hydrolysis gave two epimeric trihydroxy-7-methyl-cholanoic acids and three dehydration products. The latter were purified by silica gel column chromatography and silica gel-AgNO3 column chromatography of their methyl ester derivatives. Catalytic hydrogenation of 3 alpha,12 alpha-dihydroxy-7-methyl-5 beta-chol-6-en-24-oic acid and 3 alpha,12 alpha-dihydroxy-7-methylene-5 beta-cholan-24-oic acid gave 3 alpha,12 alpha-dihydroxy-7 xi-methyl-5 beta-cholanoic acid. The configuration of the 7-methyl groups and the position of the double bonds were assigned by proton nuclear magnetic resonance spectroscopy and the chromatographic and mass spectrometric properties of the new compounds. These compounds were synthesized for the purpose of exploring new and potentially more effective cholelitholytic agents. The hydrophilic bile acids 7 beta-methyl-cholic acid and 7 alpha-methyl-ursocholic acid are of particular interest because they should be resistant to bacterial 7-dehydroxylation.