Successive Isolation of Proteinase Hyperproductive Mutants of Aspergillus sojae

Hydrochloric acid treatment of methyl 3-(4-isobutylphenyl)-3-methylglycidate and methyl 2-hydroxy-3-(4-isobutylphenyl)-3-butenoate, a rearrangement product of the former, in acetic acid gave 3-(4-isobutylphenyl)-3-methylpyruvic acid and 2-(4-isobutylphenyl)-pro-panal. The same treatment of 2-hydroxy-3-(4-isobutylphenyl)-3-butenoic acid gave 2-(4-isobutylphenyl)-propanal. Both 3-(4-isobutylphenyl)-3-methylpyruvic acid and 2-(4-iso-butylphenyl)-propanal were oxidized to 2-(4-isobutylphenyl)-propionic acid.     

Hydroxylated jasmonic acid and related compounds from Botryodiplodia theobromae

From the culture filtrate of the fungus Botryodiplodia theobromae five hydroxylated cyclopentane fatty acids of the jasmonic acid type were isolated and identified as (11 S -(-)-hydroxyjasmonic acid; (11R)-(-)-hydroxyjasmonic acid; (-)-12-hydroxyjasmonic acid; (-)-8ξ-hydroxyjasmonic acid; (-)-3-oxo-2-(1ξ-hydroxy-2Z-pentenyl)cyclopent-1-yl-butyric acid; (-)-3-oxo-2(4ξ-hydroxy-2Z-pentenyl)cyclopent-1-yl-butyric acid. In addition, the corresponding hydroxylated iso-jasmonic acid analogues were found as minor constituents. During silica gel chromatography 11,12-didehydrojasmonic acid, 11ξ-acetoxyjasmonic acid, 3-oxo-2-(4ξ-acetoxy-2Z-pentenyl)cyclopent-1-yl-butyric acid 3-oxo-2-(2Z,4-pentadienyl)cyclopent-1-yl-butyric acid were formed as artefacts.     

Triterpenoid saponins from Schefflera arboricola

Nine triterpenoid saponins were isolated from the leaves and stems of Schefflera arboricola. The saponins were characterised, on the basis of chemical and spectral evidence, as 3-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucuronopyranosyl] oleanolic acid, 3-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucuronopyranosyl] echinocystic acid, 3-O-[beta-D-apiofuranosyl-(1-->4)-beta-D-glucuronopyranosyl] oleanolic acid 28-O-beta-D-glucopyranosyl ester, 3-O-alpha-L-ramnopyranosyl-(1-->4)-[alpha-L-arabinopyranosyl-(1-->2)-] beta-D-glucuronopyranosyl oleanolic acid, 3-O-alpha-L-rhamnopyranosyl-(1-->4)-[alpha-L-arabinopyranosyl-(1-->2)-] beta-D-glucuronopyranosyl oleanolic acid 28-O-beta-D-glucopyranosyl ester, 3-O-alpha-L-rhamnopyranosyl-(1-->4)-[beta-D-galactopyranosyl-(1-->2)-] beta-D-glucuronopyranosyl oleanolic acid, 3-O-alpha-L-rhamnopyranosyl-(1-->4)-[beta-D-galactopyranosyl-(1-->2)-] beta-D-glucuronopyranosyl oleanolic acid 28-O-beta-D-glucopyranosyl ester, 3-O-beta-D-apiofuranosyl-(1-->4)-[alpha-L-arabinopyranosyl-(1-->2)-] beta-D-glucuronopyranosyl oleanolic acid and 3-O-beta-D-apiofuranosyl-(1-->4)-[alpha-L-arabinopyranosyl-(1-->2)-] beta-D-glucuronopyranosyl oleanolic acid 28-O-beta-D-glucopyranosyl ester.     

Low-molecular-weight components of olive oil mill waste-waters

A new lignan 1-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-6-(3-acetyl-4-hydroxy-5-methoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane, the secoiridoid 2H-pyran-4-acetic acid,3-hydroxymethyl-2,3-dihydro-5-(methoxycarbonyl)-2-methyl-, methyl ester, the phenylglycoside 4-[beta-D-xylopyranosyl-(1-->6)]-beta-D-glucopyranosyl-1,4-dihydroxy-2-methoxybenzene and the lactone 3-[1-(hydroxymethyl)-1-propenyl] delta-glutarolactone were isolated and identified on the basis of spectroscopic data including two-dimensional NMR, as components of olive oil mill waste-waters. The known aromatic compounds catechol, 4-hydroxybenzoic acid, protocatechuic acid, vanillic acid, 4-hydroxy-3,5-dimethoxybenzoic acid, 4-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid, tyrosol, hydroxytyrosol, 2-(4-hydroxy-3-methoxy)phenylethanol, 2-(3,4-dihydroxy)phenyl-1,2-ethandiol, p-coumaric acid, caffeic acid, ferulic acid, sinapic acid, 1-O-[2-(3,4-dihydroxy)phenylethyl]-(3,4-dihydroxy)phenyl-1,2-ethandiol, 1-O-[2-(4-hydroxy)phenylethyl]-(3,4-dihydroxy)phenyl-1,2-ethandiol, D(+)-erythro-1-(4-hydroxy-3-methoxy)-phenyl-1,2,3-propantriol, p-hydroxyphenethyl-beta-D-glucopyranoside,2(3,4-dihydroxyphenyl)ethanol 3beta-D-glucopyranoside, and 2(3,4-dihydroxyphenyl)ethanol 4beta-D-glucopyranoside were also confirmed as constituents of the waste-waters.     

Efficient production of (R)-3-hydroxycarboxylic acids by biotechnological conversion of polyhydroxyalkanoates and their purification

An efficient method to prepare enantiomerically pure (R)-3-hydroxycarboxylic acids from bacterial polyhydroxyalkanoates (PHAs) accumulated by Pseudomonas putida GPo1 is reported in this study. (R)-3-Hydroxycarboxylic acids from whole cells were obtained when conditions were provided to promote in vivo depolymerization of intracellular PHA. The monomers were secreted into the extracellular environment. They were separated and purified by acidic precipitation, preparative reversed-phase column chromatography, and subsequent solvent extraction. Eight (R)-3-hydroxycarboxylic acids were isolated: (R)-3-hydroxyoctanoic acid, (R)-3-hydroxyhexanoic acid, (R)-3-hydroxy-10-undecenoic acid, (R)-3-hydroxy-8-nonenoic acid, (R)-3-hydroxy-6-heptenoic acid, (R)-3-hydroxyundecanoic acid, (R)-3-hydroxynonanoic acid, and (R)-3-hydroxyheptanoic acid. The overall yield based on released monomers was around 78 wt % for (R)-3-hydroxyoctanoic acid. All obtained monomers had a purity of over 95 wt %. The physical properties of the purified monomers and their antimicrobial activities were also investigated.     

Substrate stereospecificity and active site topography of gamma-aminobutyric acid aminotransferase for beta-aryl-gamma-aminobutyric acid analogues

The substrate and inhibitory properties of (R)- and (S)-4-amino-3-phenylbutanoic acid, (R)- and (S)-4-amino-3-(4-chlorophenyl)butanoic acid (baclofens), (E)-4-amino-3-phenylbut-2-enoic acid, and (E)-4-amino-3-(4-chlorophenyl)but-2-enoic acid are determined and compared with those of 4-aminobutanoic acid, 4-aminobut-2-enoic acid (4-aminocrotonic acid), and the racemic mixtures of 4-amino-3-arylbutanoic acids. All compounds in both series were found to be substrates, except for the R-isomers, which were identified as competitive inhibitors. These results are compared with known pharmacological data regarding the appropriate isomers.     

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.     

The 3-methylaspartase reaction probed using 2H- and 15N-isotope effects for three substrates: a flip from a concerted to a carbocationic amino-enzyme elimination mechanism upon changing the C-3 stereochemistry in the substrate from R to S.

The mechanisms of the elimination of ammonia from (2S,3S)-3-methylaspartic acid, (2S)-aspartic acid and (2S,3R)-3-methylaspartic acid, catalysed by the enzyme L-threo-3-methylaspartase ammonia-lyase (EC 4.3.1.2) have been probed using 15N-isotope effects. The 15N-isotope effects for V/K for both (2S,3S)-3-methylaspartic acid and aspartic acid are 1.0246 +/- 0.0013 and 1.0390 +/- 0.0031, respectively. The natural substrate, (2S,3S)-3-methylaspartic acid, is eliminated in a concerted fashion such that the C(beta)-H and C(alpha)-N bonds are cleaved in the same transition state. (2S)-Aspartic acid appears to follow the same mechanistic pathway, but deprotonation of the conjugate acid of the base for C-3 is kinetically important and influences the extent of 15N-fractionation. (2S,3R)-3-Methylaspartic acid is deaminated via a stepwise carbocationic mechanism. Here we elaborate on the proposed model for the mechanism of methylaspartase and propose that a change in stereochemistry of the substrate induces a change in the mechanism of ammonia elimination.     

Microbial hydroxylation of (+/-)- and (-)-(2Z,4E)-5-(1',2'-epoxy-2',6',6'-trimethylcyclohexyl)-3-methyl-2,4-pentadienoic acid into (+/-)- and (-)-xanthoxin acid by Cunninghamella echinulata

Microbial hydroxylation of (+/-)-(2Z,4E)-5-(1',2'-epoxy-2',6',6'-trimethylcyclohexyl)-3-methyl-2,4-pentadienoic acid (3a) with Cercospora cruenta, a fungus producing (+)-abscisic acid, gave a four-stereoisomeric mixture consisting of (+)- and (-)-xanthoxin acid (4a), and (+)- and (-)-epi-xanthoxin acid (5a) by an HPLC analysis with a chiral column. Screening of the microorganisms capable of oxidizing (+/-)-3a showed that Cunninghamella echinulata stereoselectively oxidized (+/-)-3a to xanthoxin acid (4a) with the some degree of enantioselectivity as (-)-3a to (-)-4a.     

Gas chromatography-mass spectrometry evidence for several endogenous auxins in pea seedling organs

Qualitative analysis by gas chromatography-mass spectrometry (GC-MS) of the auxins present in the root, cotyledons and epicotyl of 3-dold etiolated pea (Pisum sativum L., cv. Alaska) seedlings has shown that all three organs contain phenylacetic acid (PAA), 3-indoleacetic acid (IAA) and 4-chloro-3-indoleacetic acid (4Cl-IAA). In addition, 3-indolepropionic acid (IPA) was present in the root and 3-indolebutyric acid (IBA) was detected in both root and epicotyl. Phenylacetic acid, IAA and IPA were measured quantitatively in the three organs by GC-MS-single ion monitoring, using deuterated internal standards. Levels of IAA were found to range from 13 to 115 pmol g^-1 FW, while amounts of PAA were considerably higher (347–451 pmol g^-1 FW) and the level of IPA was quite low (5 pmol g^-1 FW). On a molar basis the PAA:IAA ratio in the whole seedling was approx. 15:1.Abbreviations IAA 3-indoleacetic acid - 4Cl-IAA 4-chloro-3-indoleacetic acid - IBA 3-indolebutyric acid - IPA 3-indolepropionic acid - PAA phenylacetic acid - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - PFB pentafluorobenzyl ester - PFBBr pentafluorobenzyl bromide - SIM single-ion monitoring - TMSI trimethylsilyl ester     

The effect of 3-sulphation and taurine conjugation on the uptake of chenodeoxycholic acid by rat hepatocytes

The hepatic uptake of chenodeoxycholic acid, taurochenodeoxycholic acid, chenodeoxycholic acid 3-sulphate and taurochenodeoxycholate acid 3-sulphate by isolated rat hepatocytes was examined. Taurochenodeoxycholic acid, taurochenodeoxycholic acid 3-sulphate and chenodeoxycholic acid 3-sulphate uptake occurred by a saturable, energy-dependent process while chenodeoxycholic acid uptake was predominantly non-saturable, possibly simple diffusion. Apparent Km (mumol/l) and Vmax (nmol/mg protein per min) values (mean +/- S.D.), respectively, were: chenodeoxycholic acid (saturable component), 33 +/- 6.4 and 4.8 +/- 0.6; taurochenodeoxycholic acid, 11.1 +/- 2.0 and 3.1 +/- 0.5; chenodeoxycholic acid 3-sulphate, 6.1 +/- 0.9 and 2.3 +/- 0.4; and taurochenodeoxycholic acid 3-sulphate, 5.0 +/- 0.7 and 0.9 +/- 0.15. Both conjugation with taurine and sulphation at the 3 position resulted in a reduction in the values of Km and Vmax. Uptake of each of the bile acids taurochenodeoxycholic acid, taurochenodeoxycholic acid 3-sulphate and chenodeoxycholic acid 3-sulphate was competitively inhibited by the other two, with taurochenodeoxycholic acid a potent inhibitor of both taurochenodeoxycholic acid 3-sulphate and chenodeoxycholic acid 3-sulphate uptake. Other bile acids also inhibited. Uptake was inhibited by albumin in the order chenodeoxycholic acid 3-sulphate greater than taurochenodeoxycholic acid 3-sulphate greater than taurochenodeoxycholic acid and was dependent on the extent of bile acid binding to albumin.     

Detailed structure of lipid A isolated from lipopolysaccharide from the marine proteobacterium Marinomonas vaga ATCC 27119.

The chemical structure of a novel lipid A, the major component of the lipopolysaccharide from the marine gamma-proteobacterium Marinomonas vaga ATCC 27119(T), was determined by compositional analysis, NMR spectroscopy, and MS. It was found to be beta-1,6-glucosaminobiose 1-phosphate acylated with (R)-3-[dodecanoyl(dodecenoyl)oxy]decanoic acid [C10 : 0 (3O-C12 : 0 [3O-C12 : 1])] or (R)-3-(decanoyloxy)decanoic acid [C10 : 0 (3O-C10 : 0)], (R)-3-hydroxydecanoic acid [C10 : 0 (3OH)], and (R)-3-[(R)-3-hydroxydecanoyloxy]decanoic acid (C10 : 0 [3O-[C10 : 0 (3OH)]]) at the 2, 3, and 2' positions, respectively. It showed low lethal toxicity, which is probably related to specific structural attributes. The absence of a fatty acid at the 3' position and a phosphoryl group at the 4' position and also the presence of an amide-linked (R)-3-hydroxyalkanoic acid that is further O-acylated with another (R)-3-hydroxyalkanoic acid, distinguish M. vaga lipid A from other such molecules.     

Glycosides from Roots of Cyathula officinalis Kuan

Abstract: To search for new and bioactive compounds from traditional Chinese medicines, a new glycoside, 3-O-[α- L -rhamnopyranosyl-(1→3)-( n -butyl-β- D -glucopyranosiduronate)]-28-O-β- D -glucopyranosyloleanolic acid ( 1 ), was isolated from the roots of Cyathula officinalis Kuan, along with 3-O-(methyl-β- D -glucopyranosiduronate)-28-O-β- D -glucopyranosyl oleanolic acid ( 2 ), 3-O-β- D -glucopyranosyl oleanolic acid ( 3 ), 3-O-β- D -glucuronopyranosyl oleanolic acid ( 4 ), 3-O-[β- L -rhamnopyranosyl-(1→3)-(β- D -glucuronopyranosyl)] oleanolic acid ( 5 ), 3-O-(β- D -glucuronopyranosyl)-28-O-β- D -glucopyranosyl oleanolic acid ( 6 ), 28-O-β- D -glucuronopyranosyl-(1→4)-β- D -glucopyranosyl hederagenin ( 7 ), 3-O-[β- L -rhamnopyranosyl-(1→3)-β- D -glucuronopyranosyl]-28-O-β- D -glucopyranosyl oleanolic acid ( 8 ), and 3-O-[β- D -glucopyranosyl-(1→2)-α- L -rhamnopyranosyl-(1→3)-β- D -glucuronopyranosyl]-28-O-β- D -glucopyranosyl oleanolic acid ( 9 ). The structures of these compounds were determined based on spectral and chemical evidence. The 50 per cent growth-inhibiting (GI₅₀) of compounds 1 and 5 against MDA-MB-231 (a human breast cancer cell line) was 3.44 × 10^-4 and 4.66 × 10^-4 mol/L, respectively.
(Managing editor: Wei WANG)     

Metabolism of 2-amino-3-phosphonopropionic acid in rats.

The metabolism of 2-amino-3-phosphono-[2-(14)C]propionic acid or 2-amino-3-phosphono-[3-(14)C]propionic acid in rats was studied in vivo and in vitro. The radioactivity in expired CO2 from the [3-(14)C]-labelled compound indicated the cleavage of the carbon-phosphorus (C-P) bond. A small amount of the [2-(14)C]-labelled compound and the [3-(14C]-labelled compound was incorporated into 2-aminoethylphosphonic acid, and polar lipid of the liver and kidney contained the 2-aminoethylphosphonic acid. The 2-amino-3-phosphonopropionic acid was not detected at the lipid level. Incorporation of the [3-(14)C]-labelled compound into a variety of metabolites including 3-phosphonopyruvic acid and 2-phosphonoacetaldehyde suggests the transamination reaction as a decomposition mechanism of 2-amino-3-phosphonopropionic acid in mammals.     

Tyrosine kinase inhibitors from the rainforest tree Polyscias murrayi

A series of 3-(4-hydroxyphenyl) propanoic acid derivatives, which inhibit Itk (interleukin-2 inducible T-cell kinase), a Th2-cell target, were isolated from the Australian rainforest tree Polyscias murrayi. The new compound 3-(4-hydroxyphenyl) propionyl choline and a 2:1 mixture of the new compounds 3,4-di-O-3-(4-hydroxyphenyl) propionyl-1,5-dihydroxycyclohexanecarboxylic acid and 3,5-di-O-3-(4-hydroxyphenyl) propionyl-1,4-dihydroxycyclohexanecarboxylic acid were isolated along with two known compounds 3-(4-hydroxyphenyl) propanoic acid and 3-(3,4-hydroxyphenyl) propanoic acid. Their structures were determined by 1D and 2D NMR spectroscopy. The assay results suggest that both the 3-(4-hydroxyphenyl) propanoate and carboxyl moieties contribute to Itk activity of the compounds.     

Synthesis and thrombolytic activity of pseudopeptides related to fibrinogen fragment

Two kinds of linkers consisting of 3-(S)-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid, ARPAK, GRPAK and QRPAK were synthesized. The thrombolytic activities in vivo indicated that the coupling position of 3-(S)-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid in the peptides effected on the potencies significantly. When the C-terminal of the peptides was amidated by 3-(S)-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid, the thrombolytic potency of the peptides was enhanced or kept. When the N-terminal of the peptides was acylated by 3-(S)-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid, however, the thrombolytic effect of the peptides was banished. The expected specific beta II'-turn conformation and the stability to trypsin in the pseudopeptides with 3-(S)-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid in its C-terminal may be responsible for the enhanced thrombolytic potency.     

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.     

Tetrazolyl isoxazole amino acids as ionotropic glutamate receptor antagonists: synthesis, modelling and molecular pharmacology

Two 3-(5-tetrazolylmethoxy) analogues, 1a and 1b, of (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA), a selective AMPA receptor agonist, and (RS)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA), a GluR5-preferring agonist, were synthesized. Compounds 1a and 1b were pharmacologically characterized in receptor binding assays, and electrophysiologically on homomeric AMPA receptors (GluR1-4), homomeric (GluR5 and GluR6) and heteromeric (GluR6/KA2) kainic acid receptors, using two-electrode voltage-clamped Xenopus laevis oocytes expressing these receptors. Both analogues proved to be antagonists at all AMPA receptor subtypes, showing potencies (Kb=38-161 microM) similar to that of the AMPA receptor antagonist (RS)-2-amino-3-[3-(carboxymethoxy)-5-methyl-4-isoxazolyl]propionic acid (AMOA) (Kb=43-76 microM). Furthermore, the AMOA analogue, 1a, blocked two kainic acid receptor subtypes (GluR5 and GluR6/KA2), showing sevenfold preference for GluR6/KA2 (Kb=19 microM). Unlike the iGluR antagonist (S)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid [(S)-ATPO], the corresponding tetrazolyl analogue, 1b, lacks kainic acid receptor effects. On the basis of docking to a crystal structure of the isolated extracellular ligand-binding core of the AMPA receptor subunit GluR2 and a homology model of the kainic acid receptor subunit GluR5, we were able to rationalize the observed structure-activity relationships.     

Acetylated glucuronide triterpene bidesmosidic saponins from Symplocos glomerata

Nine new bidesmosidic 3-O-glucuronide oleanane triterpenoid saponins were isolated from the stem bark of Symplocos glomerata King along with two known saponins, salsoloside C and copteroside E, and two major lignans, (-)-pinoresinol and (-)-pinoresinol-4'-O-beta-D-glucopyranoside. The structures of the new saponins were established using one- and two-dimensional NMR spectroscopy and mass spectrometry as, 3-O-[beta-D-xylopyranosyl(1-->4)-[2-O-acetyl]-beta-D-glucuronopyranosyl]-28-O-[beta-D-glucopyranosyl]-oleanolic acid, 3-O-[beta-D-xylopyranosyl(1-->4)-[3-O-acetyl]-beta-D-glucuronopyranosyl]-28-O-[beta-D-glucopyranosyl]-oleanolic acid, 3-O-[beta-D-xylopyranosyl (1-->4)-[2,3-O-diacetyl]-beta-D-glucuronopyranosyl]-28-O-[beta-D-glucopyranosyl]-oleanolic acid, 3-O-[alpha-L-arabinopyranosyl(1-->4)-beta-D-glucuronopyranosyl]-28-O-[beta-D-glucopyranosyl]-oleanolic acid, 3-O-[alpha-L-arabinopyranosyl (1-->4)-[2-O-acetyl]-beta-D-glucuronopyranosyl]-28-O-[beta-D-glucopyranosyl]-oleanolic acid, 3-O-[[beta-D-xylopyranosyl (1-->2)]-[beta-D-xylopyranosyl (1-->4)]-[3-O-acetyl]-beta-D-glucuronopyranosyl]-28-O-[beta-D-glucopyranosyl]-oleanolic acid, 3-O-[[beta-D-glucopyranosyl (1-->2)]-[beta-D-xylopyranosyl (1-->4)]-[3-O-acetyl]-beta-D-glucuronopyranosyl]-28-O-[beta-D-glucopyranosyl]-oleanolic acid, 3-O-[[beta-D-glucopyranosyl (1-->2)]-[alpha-L-arabinofuranosyl (1-->4)]-[3-O-acetyl]-beta-D-glucuronopyranosyl]-28-O-[beta-D-glucopyranosyl]-oleanolic acid, and 3beta-O-[beta-D-xylopyranosyl(1-->4)-[2-O-acetyl]-beta-D-glucuronopyranosyl]-28-O-[beta-D-glucopyranosyl]-morolic acid. The EtOH and EtOAc extracts of the stem bark showed no cytotoxic activity. At a concentration of 370 microg/ml, the saponin mixture showed haemolytic activity and caused 50% haemolysis of a 10% suspension of sheep erythrocytes.     

Production of D-(--)-3-hydroxyalkanoic acid by recombinant Escherichia coli

Pathways for extracellular production of chiral D-(-)-3-hydroxybutyric acid (3HB) and D-(-)-3-hydroxyalkanoic acid (mcl-3HA) were constructed by co-expression of genes of beta-ketothiolase (phbA), acetoacetyl-CoA reductase (phbB) and 3-hydroxyacyl-ACP CoA transacylase (phaG), respectively, in Escherichia coli strain DH5alpha. The effect of acrylic acid and glucose on production of both 3HB and mcl-3HA was investigated. It was found that the addition of acrylic acid significantly increased production of 3HB and mcl-3HA consisting of 3-hydroxyoctanoic acid and 3-hydroxydecanoic acid in a ratio of 1:3 from 199 mg x l(-1) to 661 mg x l(-1) and from 27 mg x l(-1) to 135 mg x l(-1), respectively, in shake flask studies when glucose was present in the medium at the very beginning of fermentation. The timing of glucose addition had no effect on 3HB production. In contrast, mcl-3HA production was affected by glucose addition, an mcl-3HA concentration of 193 mg x l(-1) was obtained when glucose was added to the culture at 12 h. A more than seven-fold increase was obtained when compared with that in medium containing glucose at the beginning of fermentation. However, a decrease in production of 3HB and mcl-3HA was found when glucose was added at 12 h to the culture containing acrylic acid. The repressive effect of acrylic acid on acetic acid production was also evaluated and discussed.