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.     

Polar lipid and fatty acid profiles – Re-vitalizing old approaches as a modern tool for the classification of mycoplasmas?

A set of 20 Mollicutes strains representing different lines of descent, including the type species of the genus Mycoplasma, Mycoplasma mycoides, Acholeplasma laidlawii and a strain of Mesoplasma, were subjected to polar lipid and fatty acid analyses in order to evaluate their suitability for classification purposes within members of this group. Complex polar lipid and fatty acid profiles were detected for each examined strain. All strains contained the polar lipids phosphocholine-6'-alpha-glucopyranosyl-(1'-3)-1, 2-diacyl-glycerol (MfGL-I), 1-O-alkyl/alkenyl-2-O-acyl-glycero-3-phosphocholine (MfEL), sphingomyelin (SphM), 1-O-alkyl/alkenyl-glycero-3-phosphocholine (lysoMfEL), the unknown aminophospholipid APL1 and the cholesterol Chol2. A total of 19 strains revealed the presence of phosphatidylethanolamine (PE) and/or phosphatidylglycerol (PG), and the presence of diphosphatidylglycerol (DPG) was detected in 13 strains. The unknown aminolipid AL1 was found in the extracts of 17 strains. Unbranched saturated and unsaturated compounds predominated in the fatty acid profiles. Major fatty acids were usually C16:0, C18:0, C18:1 omega9c and 'Summed feature 5' (C18:2 omega6, 9c/C18:0 anteiso). Our results demonstrated that members of the M. mycoides cluster showed rather homogenous polar lipid and fatty acid profiles. In contrast, each of the other strains was characterized by a unique polar lipid profile and significant quantitative differences in the presence of certain fatty acids. These results indicate that analyses of both polar lipid and fatty acid profiles could be a useful tool for classification of mycoplasmas.     

Structural analysis of the carbohydrate backbone of Vibrio parahaemolyticus O2 lipopolysaccharides

A structural investigation has been carried out on the carbohydrate backbone of Vibrio parahaemolyticus O2 lipopolysaccharides (LPS) isolated by dephosphorylation, O-deacylation and N-deacylation. The carbohydrate backbone is a short-chain saccharide consisting of nine monosaccharide units i.e., 1 mol each of D-galactose (Gal), D-glucose (Glc), D-glucuronic acid (GlcA), L-glycero-D-manno-heptose (L,D-Hep), D-glycero-D-manno-heptose (D,D-Hep), 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (NonlA), and 2 mol of 2-amino-2-deoxy-D-glucose (D-glucosamine, GlcN). Based on the data obtained by NMR spectroscopy, fast-atom bombardment mass spectrometry (FABMS) and methylation analysis, a structure was elucidated for the carbohydrate backbone of O2 LPS. In the native O2 LPS, the 2-amino-2-deoxy-D-glucitol (GlcN-ol) at the reducing end of the nonasaccharide is present as GlcN. The lipid A backbone is a beta-D-GlcN-(1-->6)-D-GlcN disaccharide as is the case for many Gram-negative bacterial LPS. The lipid A proximal Kdo is substituted by the distal part of the carbohydrate chain at position-5. In the native O2 LPS, D-galacturonic acid, which is liberated from LPS by mild acid treatment or by dephosphorylation in hydrofluoric acid, is present although its binding position is unknown at present.     

Glucuronide triterpene saponins from Bersama engleriana

Five 3-O-glucuronide triterpene saponins (1-5) were isolated from the stem bark of Bersama engleriana Gurke along with two known saponins, polyscias saponin C and aralia saponin 15, and one major C-glycoside xanthone, mangiferin. The structures of the saponins were established mainly by means of spectroscopic methods (one- and two-dimensional NMR spectroscopy as well as FAB-, HRESI-mass spectrometry) as 3-O-[beta-D-glucopyranosyl-(1-->2)-beta-D-glucuronopyranosyl]-28-O-[beta-D-glucopyranosyl]-betulinic acid (1), 3-O-[beta-D-glucopyranosyl-(1-->2)-[beta-D-galactopyranosyl-(1-->3)]-beta-D-glucuronopyranosyl]-oleanolic acid (2), 3-O-[beta-D-glucopyranosyl-(1-->3)-beta-D-glucuronopyranosyl]-28-O-[beta-D-xylopyranosyl-(1-->6)-beta-d-glucopyranosyl]-oleanolic acid (3), 3-O-[beta-D-galactopyranosyl-(1-->3)-beta-D-glucuronopyranosyl]-28-O-[beta-D-glucopyranosyl-(1-->4)-beta-D-glucopyranosyl]-oleanolic acid (4), and 3-O-[beta-d-glucopyranosyl-(1-->3)-beta-D-galactopyranosyl-(1-->3)-beta-D-glucuronopyranosyl]-28-O-[beta-d-xylopyranosyl-(1-->6)-beta-D-glucopyranosyl]-oleanolic acid (5).     

Antifungal constituents of the stem bark of Bridelia retusa

Antifungal activity guided fractionation of solvent extracts of the stem bark of Bridelia retusa of the family Euphorbiaceae against Cladosporium cladosporioides, furnished new bisabolane sesquiterpenes, (E)-4-(1,5-dimethyl-3-oxo-1-hexenyl)benzoic acid, (E)-4-(1,5-dimethyl-3-oxo-1,4-hexadienyl) benzoic acid, (R)-4-(1,5-dimethyl-3-oxo-4-hexenyl)benzoic acid and (-)-isochaminic acid, together with the known (R)-4-(1,5-dimethyl-3-oxohexyl)benzoic acid (ar-todomatuic acid), 5-allyl-1,2,3-trimethoxybenzene (elemicin), (+)-sesamin and 4-isopropylbenzoic acid (cumic acid). All these compounds showed fungicidal activity on TLC bioautography method at very low concentrations except elemicin.     

Anti-babesial ellagic acid rhamnosides from the bark of Elaeocarpus parvifolius

Bioassay-guided investigation of the bark of Elaeocarpus parvifolius led to the isolation of three new ellagic acid derivatives, 4-O-methylellagic acid 3'-alpha-rhamnoside (2), 4-O-methylellagic acid 3'-(3'-O-acetyl)-alpha-rhamnoside (3), and 4-O-methylellagic acid 3'-(4'-O-acetyl)-alpha-rhamnoside (4) in addition to the known ellagic acid derivative, 4-O-methylellagic acid 3'-(2',3'-di-O-acetyl)-alpha-rhamnoside (1). Their structures were elucidated on the basis of analysis of 1H NMR, 13C NMR, HMQC, HMBC and MS spectroscopic data. Compounds 1-4 were evaluated for their growth-inhibitory effect on Babesia gibsoni in vitro. Compounds 2 and 4 showed very weak activity, while compounds 1 and 3 showed moderate activity, with IC50 values of 28.5 and 52.1 microg/ml, respectively.     

A chloro amino acid from Amanita solitaria

The mushroom Amanita solitaria contains in excess of 1000 ppm 2(S)-amino-4,5-hexadienoic acid (I), 300 ppm trans-2-amino-5-chloro-4-hexenoic acid (II), and a chloride ion concentration (2000 ppm) significantly greater than that found in other basidiomycetes. I can be converted into II in hydrochloric acid, but II is not an artifact of isolation.     

Chemotaxonomic evaluation of Turkish species of Salvia: Fatty acid compositions of seed oils

Fatty acid composition of nine species of Salvia, naturally growing in Turkey was determined: Salvia syriaca, Salvia potentillifolia, Salvia candidissima ssp. occidentalis, Salvia macrochlamys, Salvia poculata, Salvia tomentosa, Salvia recognita, Salvia virgata and Salvia ceratophylla. The main compounds were found to be linoleic acid (18:2; 24.3–69.2%), linolenic acid (18:3; 0.6–40.8%), oleic acid (18:1; 8.3–31.0%), palmitic acid (16:0; 3.8–21.0%) and stearic acid (18:0; 1.8–5.2%). Fatty acid composition of Salvia seed oils could be used as a chemotaxonomical marker.     

27-Nor-triterpenoid glycosides from Mitragyna inermis

From the bark of Mitragyna inermis, two 27-nor-triterpenoid glycosides, named inermiside I (1) and II (2), were isolated and their structures determined based on extensive 2D-NMR and MS spectral analysis as 6-deoxy-beta-D-glucopyranosyl-[3-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl]-pyrocincholate and 6-deoxy-beta-D-glucopyranosyl-pyrocincholate, respectively. In addition, the known quinovic acid (6), 3-O-[beta-D-glucopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl]-quinovoic acid (3),beta-D-glucopyranosyl-[3-O-(beta-D-glucopyranosyl)]-quinoviate (4) and cytotoxic 3-O-(beta-D-6-deoxy-glucopyranosyl)-quinovic acid (5) were also isolated.     

cis-Jasmone induces accumulation of defence compounds in wheat, Triticum aestivum

Liquid phase extraction (LPE) and vapor phase extraction (VPE) methodologies were used to evaluate the impact of the plant activator, cis-jasmone, on the secondary metabolism of wheat, Triticum aestivum, var. Solstice. LPE allowed the measurement of benzoxazinoids, i.e. 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), 2-hydroxy-7-methoxy-1,4-benzoxazin-3-one (HMBOA) and 6-methoxy-benzoxazolin-2-one (MBOA), and phenolic acids such as trans-p-coumaric acid, syringic acid, p-hydroxybenzoic acid, vanillic acid and cis- and trans-ferulic acid. Using LPE, a significantly higher level of DIMBOA was found in aerial parts and roots of T. aestivum following treatment with cis-jasmone, when compared with untreated plants. Similar results were obtained for phenolic acids, such as trans-ferulic acid and vanillic acid in roots. Using VPE, it was possible to measure levels of 2-hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (HBOA), benzoxazolin-2(3H)-one (BOA), ferulic acid, syringic acid and coumaric acid. The levels of HBOA in aerial parts and roots were significantly greater in cis-jasmone treated plants compared to untreated plants. cis-Jasmone is known to be a plant activator in terms of production of defence-related volatile semiochemicals that repel aphids and increase the foraging activity of aphid parasitoids. These results show, for the first time, that cis-jasmone also induces selective production of secondary metabolites that are capable of directly reducing development of pests, diseases and weeds.     

A phosphopantetheinyl transferase gene essential for biosynthesis of n-3 polyunsaturated fatty acids from Moritella marina strain MP-1

A phosphopantetheinyl transferase (PPTase) gene (pfaE), cloned from the docosahexaenoic acid (DHA)-producing bacterium Moritella marina strain MP-1, has an open reading frame of 861 bp encoding a 287-amino acid protein. When the pfaE gene was expressed with pfaA-D, which are four out of five essential genes for biosynthesis of eicosapentaenoic acid (EPA) derived from Shewanella pneumatophori SCRC-2738 in Escherichia coli, the recombinant produced 12% EPA of total fatty acids. This suggests that pfaE encodes a PPTase required for producing n-3 polyunsaturated fatty acids, which is probably involved in the synthesis of DHA in M. marina strain MP-1.     

Cyclization of natural allene oxide in aprotic solvent: formation of the novel oxylipin methyl cis-12-oxo-10-phytoenoate

Allene oxide, (9Z,11E)-12,13-epoxy-9,11-octadecadienoic acid (12,13-EOD), was prepared by incubation of linoleic acid (13S)-hydroperoxide with flaxseed allene oxide synthase (AOS) and purified (as methyl ester) by low temperature HPLC. Identification of pure 12,13-EOD was substantiated by its UV and (1)H NMR spectra and by GC-MS data for its methanol trapping product. The methyl ester of 12,13-EOD (but not the free carboxylic acid) is slowly cyclized in hexane solution, affording a novel cyclopentenone cis-12-oxo-10-phytoenoic acid. Free carboxylic form of 12,13-EOD does not cyclize due to the exceeding formation of macrolactone (9Z)-12-oxo-9-octadecen-11-olide. The spontaneous cyclization of pure natural allene oxide (12,13-EOD) into cis-cyclopentenone have been observed first time.     

Characterization of cross-linked hydroxycinnamic acid amides isolated from potato common scab lesions

Four feruloyl amides, N-trans-feruloyloctopamine (1), N-cis-feruloyloctopamine (2), N-trans-feruloyltyramine (3), N-cis-feruloyltyramine (4), a cross-linked N-trans-feruloyltyramine dimer (5), and a cross-linked N-cis-feruloyltyramine dimer (6) were isolated from potato common scab lesions. The compounds were purified by TLC and characterized by a combination of (1)H and (13)C NMR spectroscopic techniques. The presence of an accompanying minor complex of cross-linked dimers containing both feruloyltyramines and feruloyloctopamines was also demonstrated. This is the first characterization of cross-linked hydroxycinnamic acid amides associated with wound healing in potato (Solanum tuberosum) tubers.     

Analysis of monosaccharides, fatty constituents and rare O-acetylated sialic acids from gonads of the starfish Asterias rubens

A previous study (Bergwerff et al., Biochimie 74 (1992) 25-37) reported that sialic acids present in Asterias rubens gonads were essentially composed of 8-methyl-N-glycolylneuraminic acid (Neu5Gc8Me), a large part of it being acetylated in position 9. Using GC/MS of heptafluorobutyrate derivatives (Zanetta et al., Glycobiology 11 (2001) 663-676) on the chloroform/methanol soluble and insoluble fractions, we showed that most sialic acids were found in the latter and demonstrated that all sialic acids were derived from N-glycolylneuraminic acid, most of them being 8-methylated, but that the majority were also acetylated in position 4 or 7 (or both positions). GC/MS analyses of the constituents liberated using acid-catalysed methanolysis verified that major glycoprotein-bound glycans were N-linked and of the gluco-oligomannosidic type. Major fatty acids were poly-unsaturated (especially C20:4) and long-chain bases were C22:1 phytosphingosine and C22:2 6-hydroxysphingenine. Major monosaccharides found in the chloroform/methanol extract (quinovose and fucose) were derived from steroidal saponins.     

Antioxidant constituents of Nymphaea caerulea flowers

As part of an ongoing search for antioxidants from medicinal plants, 20 constituents were isolated from the Nymphaea caerulea flowers, including two 2S,3S,4S-trihydroxypentanoic acid (1), and myricetin 3-O-(3'-O-acetyl)-alpha-L-rhamnoside (2), along with the known myricetin 3-O-alpha-L-rhamnoside (3), myricetin 3-O-beta-D-glucoside (4), quercetin 3-O-(3'-O-acetyl)-alpha-L-rhamnoside (5), quercetin 3-O-alpha-L-rhamnoside (6), quercetin 3-O-beta-D-glucoside (7), kaempferol 3-O-(3'-O-acetyl)-alpha-L-rhamnoside (8), kaempferol 3-O-beta-D-glucoside (9), naringenin (10), (S)-naringenin 5-O-beta-D-glucoside (11), isosalipurposide (12), beta-sitosterol (13), beta-sitosterol palmitate (14), 24-methylenecholesterol palmitate (15), 4alpha-methyl-5alpha-ergosta-7,24(28)-diene-3beta,4beta-diol (16), ethyl gallate (17), gallic acid (18), p-coumaric acid (19), and 4-methoxybenzoic acid (20). The structures were determined by spectroscopic means. Compounds were tested for antioxidant activity and nine compounds 2-7, 11, 12 and 18 were considered active with IC(50) of 1.16, 4.1, 0.75, 1.7, 1.0, 0.34, 11.0, 1.7 and 0.95 microg/ml, respectively, while 1 was marginally active (IC(50)>31.25 microg/ml). The most promising activity was found in the EtOAc fraction (IC(50) 0.2 microg/ml). This can be attributed to the synergistic effect of the compounds present in it.     

Non-natural cinnamic acid derivatives as substrates of cinnamate 4-hydroxylase

Cinnamate 4-hydroxylase (C4H), a monooxygenase in the plant phenylpropanoid pathway, was assayed for its ability to hydroxylate 29 substrate analogues. Nine of the tested analogues with various aromatic side chains, including 3-coumaric acid, were metabolized by C4H. Seven products from these reactive analogues were characterized using LC/MS, 1H NMR and 13C NMR spectroscopic analysis. For example, caffeic acid was the product of 3-coumaric acid. The products 4-hydroxy-2-chlorocinnamic acid and 4-hydroxy-2-ethoxycinnamic acid are novel compounds that have not been previously reported. The kinetic parameters of C4H towards these analogues were determined.     

Triterpenoid saponins with N-acetyl sugar from the bark of Albizia procera

Three (1,2,4) and one known (3) triterpenoid saponins were isolated from the bark of Albizia procera. The saponins were characterized as 3-O-[beta-D-xylopyranosyl-(1-->2)-alpha-L-arabinopyranosyl-(1-->6)-2-acetamido-2-deoxy-beta-D-glucopyranosyl] echinocystic acid (1), 3-O-[alpha-L-arabinopyranosyl-(1-->2)-beta-D-fucopyranosyl-(1-->6)-2-acetamido-2-deoxy-beta-D-glucopyranosyl] echinocystic acid (2) and 3-O-[beta-D-xylopyranosyl-(1-->2)-alpha-L-arabinopyranosyl-(1-->6)-2-acetamido-2-deoxy-beta-D-glucopyranosyl] acacic acid lactone (4). Their structures were elucidated by 1D and 2D NMR experiments, FABMS as well as chemical means. Saponins 1 and 3 exhibited cytotoxicity against HEPG2 cell line with IC50 9.13 microg/ml and 10 microg/ml, respectively.     

Construction and application of a mass spectral and retention time index database generated from plant GC/EI-TOF-MS metabolite profiles

The non-supervised construction of a mass spectral and retention time index data base (MS/RI library) from a set of plant metabolic profiles covering major organs of potato (Solanum tuberosum), tobacco (Nicotiana tabaccum), and Arabidopsis thaliana, was demonstrated. Typically 300-500 mass spectral components with a signal to noise ratio > or =75 were obtained from GC/EI-time-of-flight (TOF)-MS metabolite profiles of methoxyaminated and trimethylsilylated extracts. Profiles from non-sample controls contained approximately 100 mass spectral components. A MS/RI library of 6205 mass spectral components was accumulated and applied to automated identification of the model compounds galactonic acid, a primary metabolite, and 3-caffeoylquinic acid, a secondary metabolite. Neither MS nor RI alone were sufficient for unequivocal identification of unknown mass spectral components. However library searches with single bait mass spectra of the respective reference substance allowed clear identification by mass spectral match and RI window. Moreover, the hit lists of mass spectral searches were demonstrated to comprise candidate components of highly similar chemical nature. The search for the model compound galactonic acid allowed identification of gluconic and gulonic acid among the top scoring mass spectral components. Equally successful was the exemplary search for 3-caffeoylquinic acid, which led to the identification of quinic acid and of the positional isomers, 4-caffeoylquinic acid, 5-caffeoylquinic acid among other still non-identified conjugates of caffeic and quinic acid. All identifications were verified by co-analysis of reference substances. Finally we applied hierarchical clustering to a complete set of pair-wise mass spectral comparisons of unknown components and reference substances with known chemical structure. We demonstrated that the resulting clustering tree depicted the chemical nature of the reference substances and that most of the nearest neighbours represented either identical components, as judged by co-elution, or conformational isomers exhibiting differential retention behaviour. Unknown components could be classified automatically by grouping with the respective branches and sub-branches of the clustering tree.     

A lipoxygenase-divinyl ether synthase pathway in flax (Linum usitatissimum L.) leaves

Incubation of linoleic acid with an enzyme preparation from leaves of flax (Linum usitatissimum L.) led to the formation of a divinyl ether fatty acid, i.e. (9Z,11E,1'Z)-12-(1'-hexenyloxy)-9,11-dodecadienoic [(omega5Z)-etheroleic] acid, as well as smaller amounts of 13-hydroxy-9(Z),11(E)-octadecadienoic acid. The 13-hydroperoxide of linoleic acid afforded the same set of products, whereas incubations of alpha-linolenic acid and its 13-hydroperoxide afforded the divinyl ether (9Z,11E,1'Z,3'Z)-12-(1',3'-hexadienyloxy)-9,11-dodecadienoic [(omega5Z)-etherolenic] as the main product. Identification of both divinyl ethers was substantiated by their UV, mass-, (1)H NMR and COSY spectral data. In addition to the 13-lipoxygenase and divinyl ether synthase activities demonstrated by these results, flax leaves also contained allene oxide synthase activity as judged by the presence of endogenously formed (15Z)-cis-12-oxo-10,15-phytodienoic acid in all incubations.