High Rabdosiin and Rosmarinic Acid Production in Eritrichium sericeum Callus Cultures and the Effect of the Calli on Masugi-Nephritis in Rats

During an investigation of plant cell cultures that might be useful in the treatment of renal disorders, we established a vigorously-growing E-4 callus culture of Eritrichium sericeum that produced large amounts of caffeic acid metabolites, (?)-rabdosiin (1.8% dry wt) and rosmarinic acid (4.6% dry wt). Elicitation of the calli by methyl jasmonate induced a 38% increase in total polyphenol production. The most efficient method of eliciting (?)-rabdosiin biosynthesis was through the treatment of E-4 calli with cuprum glycerate, which induced an increase in (?)-rabdosiin production of as much as 4.1% dry wt. Oral administration of E-4 callus biomass (100 mg/kg/d for 30 d) to rats with induced Masugi-nephritis caused an increase in diuresis and lowered creatinine excretion and proteinuria levels as compared with Masugi-nephritis untreated rats. While all of the Masugi-nephritis untreated rats began to suffer, near a quarter of the E-4 treated rats remained in good health. This result indicates that the E-4 culture has the potential to alleviate the symptoms associated with nephritis.     

咖啡酸代谢物的抗肾炎活性分析

在研究治疗肾功能紊乱植物的细胞培养时, 建立了一个Eritrichium sericeum的E-4愈伤组织株系, 发现此株系可产生大量的咖啡酸代谢物、(–)-rabdosiin (1.8%干重)和迷迭香酸(4.6%干重), 通过诱导(–)-rabdosiin的含量提高至4.1% (干重)。将E-4愈伤组织喂服Masugi肾炎大鼠, 结果发现, 与对照组(未喂服E-4愈伤组织的Masugi肾炎大鼠)相比, 处理组(喂服E-4愈伤组织)中的大鼠出现如下症状: 尿多、排泄物中肌氨酸酐降低、尿蛋白水平降低; 当对照组中所有大鼠都出现疼痛症状时, 处理组中仍有约/4的大鼠表现出健康状况良好。以上结果表明, E-4株系具有缓解肾炎症状的潜在功能。此外, 利用富含多酚的rolC转基因的细胞株系, 研究了愈伤组织中咖啡酸代谢物的诱导合成机制。结果发现,在rloC转基因的E. sericeum愈伤组织中,咖啡酸代谢物的高产与迷迭香酸生物合成中的关键基因CYP98A3的高表达有关。     

High rabdosiin and rosmarinic acid production in Eritrichium sericeum callus cultures and the effect of the calli on masugi-nephritis in rats

During an investigation of plant cell cultures that might be useful in the treatment of renal disorders, we established a vigorously-growing E-4 callus culture of Eritrichium sericeum that produced large amounts of caffeic acid metabolites, (-)-rabdosiin (1.8% dry wt) and rosmarinic acid (4.6% dry wt). Elicitation of the calli by methyl jasmonate induced a 38% increase in total polyphenol production. The most efficient method of eliciting (-)-rabdosiin biosynthesis was through the treatment of E-4 calli with cuprum glycerate, which induced an increase in (-)-rabdosiin production of as much as 4.1% dry wt. Oral administration of E-4 callus biomass (100 mg/kg/d for 30 d) to rats with induced Masugi-nephritis caused an increase in diuresis and lowered creatinine excretion and proteinuria levels as compared with Masugi-nephritis untreated rats. While all of the Masugi-nephritis untreated rats began to suffer, near a quarter of the E-4 treated rats remained in good health. This result indicates that the E-4 culture has the potential to alleviate the symptoms associated with nephritis.     

The chemotaxonomic and medicinal significance of phenolic acids in Arctopus and Alepidea (Apiaceae subfamily Saniculoideae)

The occurrence of (R)-3′-O-β-d-glucopyranosylrosmarinic acid, rosmarinic acid and caffeic acid in two important South African medicinal plants is reported for the first time. (R)-3′-O-β-d-Glucopyranosylrosmarinic acid and rosmarinic acid were isolated and identified in several samples from three species of the genus Arctopus L. (sieketroos) and three species of the genus Alepidea F. Delaroche (ikhathazo), both recently shown to be members of the subfamily Saniculoideae of the family Apiaceae. The compounds occur in high concentrations (up to 15.3 mg of (R)-3′-O-β-d-glucopyranosylrosmarinic acid per g dry wt) in roots of Arctopus. Our results provide a rationale for the traditional uses of these plants, as the identified compounds are all known for their antioxidant activity, with rosmarinic acid further contributing to a wide range of biological activities. Furthermore, we confirm the idea that (R)-3′-O-β-d-glucopyranosylrosmarinic acid is a useful chemotaxonomic marker for the subfamily Saniculoideae.     

Formation of (-⊃-1′,2′-epi-2-cis-xanthoxin acid from a precursor of abscisic acid

Tomato shoots and avocado mesocarp supplied with (±)-[2-¹⁴C]-5-(1,2-epoxy-2,6,6-trimethylcyclohexyl)-3-methylpenta-cis-2-trans-4-dienoic acid metabolize it into (+)-abscisic acid and a more polar material that was isolated and identified as (?)-epi-1′(R),2′(R)-4′(S)-2-cis-xanthoxin acid. The (+)-1′(S),2′(S)-4′(S)-2-cis-xanthoxin acid recently synthesized from natural violaxanthin, has the 1′,2′-epoxy group on the opposite side of the ring to that of the 4′(S)-hydroxyl group and the compound is rapidly converted into (+)-abscisic acid. The 1′,2′-epoxy group of (?)-1′,2′-epi-2-cis-xanthoxin acid is on the same side of the ring as the 4′(S) hydroxyl group: the compound is not metabolized into abscisic acid. The configuration of the 1′,2′-epoxy group probably controls whether or not the 4′(S) hydroxyl group can be oxidized. (+)-2-cis-Xanthoxin acid is probably not a naturally occurring intermediate because a ‘cold trap’, added to avocado fruit forming [¹⁴C]-labelled abscisic acid from [2-¹⁴C]mevalonate, failed to retain [¹⁴C] label.     

(−)-Epilamprolobine and its N-oxide,lupin alkaloids from Sophora tomentosa

From the fresh leaves of Sophora tomentosa, three new lupin alkaloids, (?)-epilamprolobine, (+)-epilamprolobine N-oxide and 5-(3′-methoxycarbonylbutyroyl)aminomethyl-trans-quinolizidine N-oxide, have further been isolated along with (+)-matrine, (+)-matrine N-oxide, (+)-sophocarpine N-oxide, (?)-anagyrine, (?)- baptifoline, (?)-cytisine, (?)-N-methylcytisine, (?)-N-formylcytisine, (?)-N-acetylcytisine and (±)-ammodendrine. The absolute configurations of (+)-epilamprolobine N-oxide (1R:5R:6S) and (?)-epilamprolobine (5R:6S) have also been established by spectroscopic data and by comparison with synthetic (+)-epilamprolobine (5S:6R)derived from (?)-lupinine (5R:6R). (?)-Epilamprolobine is a diastereomer of (+)-lamprolobine (5R:6R) in Lamprolobium fruticosum and 5-(3′-methoxycarbonylbutyroyl) aminomethyl-trans-quinolizidine N-oxide is presumed to be an artefact. A biosynthetic pathway for the formation of (?)-epilamprolobine is also proposed.     

Highly enantioselective oxidation of racemic phenyl-1,2-ethanediol to optically pure (R)-(−)-mandelic acid by a newly isolated Brevibacterium lutescens CCZU12-1

Enantioselective oxidation of racemic phenyl-1,2-ethanediol into (R)-(?)-mandelic acid by a newly isolated Brevibacterium lutescens CCZU12-1 was demonstrated. It was found that optically active (R)-(?)-mandelic acid (e.e.p?>?99.9 %) is produced leaving the other enantiomer (S)-(+)-phenyl-1,2-ethanediol intact. Using fed-batch method, a total of 172.9 mM (R)-(?)-mandelic acid accumulated in the reaction mixture after the seventh feed. Moreover, oxidation of phenyl-1,2-ethanediol using calcium alginate-entrapped resting cells was carried out in the aqueous system, and efficient biocatalyst recycling was achieved as a result of cell immobilization in calcium alginate, with a product-to-biocatalyst ratio of 27.94 g (R)-(?)-mandelic acid g^?1 dry cell weight cell after 16 cycles of repeated use.     

Leucoflavonine,a new bioactive racemic flavoalkaloid from the leaves of Leucosceptrum canum

A new flavoalkaloid racemate, leucoflavonine (1), together with its flavonoid precursor pectolinarigenin (2), was isolated from the leaves of Leucosceptrum canum collected from Tibet. Its structure was established by comprehensive spectroscopic analysis. Chrial separation of the enantiomers of 1 was achieved, and their absolute configurations were determined as S-(+)- and R-(?)-leucoflavonines ((+)-1a and (?)-1b) by comparison of their computational and experimental optical rotations. Biological assays indicated that both (+)-1a and (?)-1b exhibited inhibitory activity against acetylchlorinesterase (AChE) in vitro (IC₅₀?=?68.0?±?8.6 and 18.3?±?1.8?μM, respectively). Moreover, (?)-1b displayed cytotoxicity against human hepatoma cells HepG2 (IC₅₀?=?52.9?±?3.6?μM), and inhibited the production of interleukelin-2 (IL-2) in Jurkat cells (IC₅₀?=?16.5?±?0.9?μM), while (+)-1a showed no obvious activity in these assays.     

Highly selective biotransformation of (+)-(1S)- and (−)-(1R)-camphorquinone by Aspergillus wentii

Abstract

To clarify the structures of biotransformation products and metabolic pathways, the biotransformation of monoterpenoids, (+)- and (?)-camphorquinone (1a and b), has been investigated using Aspergillus wentii as a biocatalyst. Compound 1a was converted to (?)-(2S)-exo-hydroxycamphor (2a), (?)-(2S)-endo-hydroxycamphor (3a), (?)-(3S)-exo-hydroxycamphor (4a), (?)-(3S)-endo-hydroxycamphor (5a), and (+)-camphoric acid (6a). Compound 1b was converted to (+)-(2R)-exo-hydroxycamphor (2b), (+)-(2R)-endo-hydroxycamphor (3b), (+)-(3R)-exo-hydroxycamphor (4b), (+)-(3R)-endo-hydroxycamphor (5b), and (?)-camphoric acid (6b). Compound 1a mainly produced 2a (65.0%) with stereoselectivity, whereas 1b afforded 3b (84.3%) with high stereoselectivity. These structures were confirmed by gas chromatography–mass spectrometry, infrared, ¹H nuclear magnetic resonance (NMR), and ¹³C NMR spectral data. The products illustrate the marked ability of A. wentii for enzymatic oxidation and ketone reduction.     

Structure and functions of γ-dodecalactone isolated from Antrodia camphorata for NK cell activation

The preserved fungal species Antrodia camphorata has diverse health-promoting effects and has been popularly used in East Asia as a traditional herb. We isolated a volatile compound from the culture medium of A. camphorata and identified it as γ-dodecalactone (γ-DDL). Cytomic screening for immune-modulating activity revealed that γ-DDL can activate human NK cells to express the early activation marker CD69. Further experiments showed that γ-DDL not only can induce NK cells to express CD69 but also stimulate NK cells to secrete cytotoxic molecules (FasL and granzyme B) and Th1 cytokines (TNF-α and INF-γ).Measuring the distribution of γ-DDL in the subcellular compartments of NK cells revealed that γ-DDL has been converted to 4-hydroxydodecanoic acid (an acyclic isomer of γ-DDL) in a time-dependent manner in the cytoplasm.Synthetic (R,S)-4-hydroxydodecanoic acid activated NK cells to express CD69 mRNA within 10 min, in contrast to γ-DDL, which activated NK cells to express CD69 within 50 min. This faster activation suggests that γ-DDL has converted to 4-hydroxydodecanoic acid and to stimulate the NK cells to express CD69.Optically pure (R)-(+)-4-hydroxydodecanoic acid and (S)-(?)-4-hydroxydodecanoic acid were obtained via: (1) synthesis of its diastereomeric esters of (R,S)-4-hydroxydodecanoic (R)-(?)-2-phenylpropionate; (2) separation of diastereomers via preparative HPLC, and (3) subsequent hydrolysis of the obtained optical pure ester of (R)-(+)-4-hydroxydodecanoic acid (R)-(?)-2-phenylpropionate and (R)-(?)-4-hydroxydodecanoic acid (R)-(?)-2-phenylpropionate, respectively. Further assays of NK cells activation using each enantiomer showed that only the (R)-(+)-4-hydroxydodecanoic acid can activate NK cells.     

Biosynthesis of pisatin: Experiments with enantiomeric precursors

Feeding experiments in cupric chloride-treated Pisum sativum pods and seedlings have demonstrated the preferential incorporation of (+)-(6aS,11aS)-[³H]maackiain over (?)-(6aR, 11aR)-[¹⁴C]maackiain into (+)-(6aR, 11aR)-pisatin, establishing that the 6a-hydroxylation of pterocarpans proceeds with retention of configuration. (+)- (6aR,11aR)-6a-hydroxymaackiain was similarly incorporated much better than (?)-(6aS,11aS)-6a- hydroxymaackiain. Where (?)-isomers were incorporated, optical activity measurements on the pisatin produced indicated significant synthesis of (?)-pisatin as well as the normal (+)-pisatin. 7,2′-Dihydroxy-4′,5′- methylenedioxyisoflav-3-ene and both enantiomers of 7,2′-dihydroxy-4′,5′-methylenedioxyisoflavan were poor precursors of pisatin.     

Metabolism of nicotine in Nicotiana glauca

A mixture of (?)-nicotine-[2′-³H] and (±)-nicotine-[2′-¹⁴C] was administered to Nicotiana glauca plants for 3 days, resulting in the formation of radioactive nornicotine (49·5% incorporation) and myosmine (2·05% incorporation). Negligible activity was detected in anabasine, cotinine, or 3-acetylpyridine, the last two compounds being added as carriers to the harvested plants. The radioactive nornicotine consisted of 48% (?)-nornicotine-[2′-¹⁴C,³H] and 52% (+)-nornicotine-[2′-¹⁴C]. Thus if (+)-nornicotine is formed from (?)-nicotine the transformation must involve loss of the hydrogen from C-2′. Myosmine is presumably formed from nicotine via nornicotine. However by feeding myosmine-[2′-¹⁴C] to N. glauca it was shown that the dehydrogenation is not reversible, no activity being detected in nornicotine. Nicotinic acid (0·14% incorporation) was a metabolite of myosmine-[2′-¹⁴C]. Essentially all the activity of the nicotinic acid was located on its carboxyl group, indicating that myosmine was a direct precursor.     

Alkaloids of Thermopsis Lupinoides

Ammodendrine, together with seven other known lupin alkaloids, was isolated from Thermopsis lupinoides. (+)-Lupanine (+)-17-oxolupanine occurred together with (?)anagyrine, (?)-baptifoline, (?)-cytisine, (?)-N-methylcytisine (?)N-formylcytisine. These alkaloids have the opposite stereochemistry to that of (+)-lupanine and (+)-17-oxolupanine. The distribution of alkaloids in fresh flowers, leaves, stems roots of this plant was also examined.     

Determination of absolute configuration and binding efficacy of benzimidazole-based FabI inhibitors through the support of electronic circular dichroism and MM-GBSA techniques

We have previously reported benzimidazole-based compounds to be potent inhibitors of FabI for Francisella tularensis (FtFabI), making them promising antimicrobial hits. Optically active enantiomers exhibit markedly differing affinities toward FtFabI. The IC₅₀ of benzimidazole (?)-1 is ～100× lower than the (+)-enantiomer, with similar results for the 2 enantiomers. Determining the absolute configuration for these optical compounds and elucidating their binding modes is important for further design. Electronic circular dichroism (ECD) quantum calculations have become important in determining absolute configurations of optical compounds. We determined the absolute configuration of (?)/(+)-1 and (?)/(+)-2 by comparing experimental spectra and theoretical density functional theory (DFT) simulations of ECD spectra at the B3LYP/6-311+G(2d, p) level using Gaussian09. Comparison of experimental and calculated ECD spectra indicates that the S configuration corresponds to the (?)-rotation for both compounds 1 and 2, while the R configuration corresponds to the (+)-rotation. Further, molecular dynamics simulations and MM-GBSA binding energy calculations for these two pairs of enantiomers with FtFabI show much tighter binding MM-GBSA free energies for S-1 and S-2 than for their enantiomers, R-1 and R-2, consistent with the S configuration being the more active one, and with the ECD determination of the S configuration corresponding to (?) and the R configuration corresponding to (+). Thus, our computational studies allow us to assign (?) to (S)- and (+) to (R)- for compounds 1 and 2, and to further evaluate structural changes to improve efficacy.     

Enantiotopically Selective Oxidation of 1,5-Diols with Gluconobacters Preparation of (S)-Mevalonolactone

(±)-(2Z,4E)-α-Ionylideneacetic acid (2) was enantioselectively oxidized to (?)-(l′S)-(2Z,4E)-4′-hydroxy-α-ionylideneacetic acid (3), (+)-(1′R)-(2Z,4E)-4′-oxo-α-ionylideneacetic acid (4) and (+)-abscisic acid (ABA) (1) by Cercospora cruenta IFO 6164, which can produce (+)-ABA and (+)-4′-oxo-α-acid 4. This metabolism was confirmed by the incorporation of radioactivity from (±)-(2-¹⁴C)-(2Z,4E)-α-acid 2 into three metabolites. (?)-4′-Hydroxy-α-acid 3 was a diastereoisomeric mixture consisting of major 1′,4′-trance-4′-hydroxy-α-acid 3a and minor 1′,4′-cis-4′-hydroxy-α-acid 3b. These structures, 3a and 3b, were confirmed by ¹³C-NMR and ¹H-NMR analysis. Also, the enantioselectivity of the microbial oxidation was reexamined by using optically pure α-acid (+)-2 and (?)-2, as the substrates.     

Caffeic acid oligomers in Lithospermum erythrorhizon cell suspension cultures

Lithospermum erythrorhizon cells cultured in pigment production (M-9) medium produced lithospermic acid B, a dimerized caffeic acid ester derivative, in quantities similar to the production of shikonin. The cells also produced a related dimer, (+)-rabdosiin. In Linsmaier-Skoog liquid medium, which suppresses shikonin production, both lithospermic acid B and (+)-rabdosiin were still formed. Lithospermic acid, a caffeic acid-rosmarinic acid conjugate, was isolated as a main constituent in Lithospermum hairy root cultures. In the aerial parts of L. erythrorhizon, the content of these phenylpropanoid oligomers was relatively low compared to that of rosmarinic acid.     

Eudesmane alcohols from jasonia glutinosa

Three new sesquiterpene alcohols have been isolated from Jasonia glutinosa. Their structures were elucidated by spectroscopic methods and chemical correlations as (?)-[11R]-4α,14-epoxyeudesm-11,12-diol, (?)-[11R]-eudesm-4(14)-en-5β,11,12-triol and (+)-[11R]-eudesm-4(14)-en-5α,11,12-triol and they are called α-epoxy kudtdiol, 5-epi-kudtriol and kudtriol respectively.     

Enhanced production of α-cyclodextrin glycosyltransferase in Escherichia coli by systematic codon usage optimization

Enhancing the production of α-cyclodextrin glycosyltransferase (α-CGTase) is a key aim in α-CGTase industries. Here, the mature α-cgt gene from Paenibacillus macerans JFB05-01 was redesigned with systematic codon optimization to preferentially match codon frequencies of Escherichia coli without altering the amino acid sequence. Following synthesis, codon-optimized α-cgt (coα-cgt) and wild-type α-cgt (wtα-cgt) genes were cloned into pET-20b(+) and expressed in E.?coli BL21(DE3). The total protein yield of the synthetic gene was greater than wtα-cgt expression (1,710?mg?L^?1) by 2,520?mg?L^?1, with the extracellular enzyme activity being improved to 55.3?U?mL^?1 in flask fermentation. ΔG values at -3 to +50 of the pelB site of both genes were ?19.10?kcal?mol^?1. Functionally, coα-CGTase was equally as effective as wtα-CGTase in forming α-cyclodextrin (α-CD). These findings suggest that preferred codon usage is advantageous for translational efficiency to increase protein expression. Finally, batch fermentation was applied, and the extracellular coα-CGTase enzyme activity was 326?% that of wtα-CGTase. The results suggest that codon optimization is a reasonable strategy to improve the yield of α-CGTase for industrial application.     

Sphingosine derivatives from red algae of the ceramiales

(+)-2(S)-N-Acetamido-3(R)-acetoxyoctadecan-1-ol, a diacetate of dihydrosphingosine, and fatty acid amides of (?)-2(S)-amino-3(R)-hydroxyoctadec-4(E)-en-1-ol (sphingosine) have been isolated from extracts of Hawaiian Laurencia nidifica and Amansia glomerata, respectively. Although well known as constituents of nerve tissue hydrolyzates throughout the animal kingdom, these compounds have not been previously found in plants.     

Absolute configuration of the isomer of fluorocitrate that inhibits aconitase

The absolute configuration of the isomer of fluorocitrate that is a strong inhibitor and activator of aconitase has been determined to be 2R, 3R, as shown in the Fischer and perspective diagrams below.

Fischer diagram Perspective diagramThis is the isomer described by Dummel and Kun (1969, J. Biol. Chem.244, 2966) and defined as (?)-erythro-fluorocitrate by them. We report here structural studies of the mirror image (enantiomer) of this isomer. Crystals of the complex of the diethyl ester of (+)-erythro-fluorocitrate with (?)-methylbenzylamine were studied by X-ray diffraction techniques. Since the absolute configuration of (?)-methylbenzylamine has already been determined experimentally, the absolute configuration of the (+)-erythro isomer of fluorocitrate is thereby established. This isomer is shown to be noninhibitory with the enzyme aconitase, while its racemate is a powerful inhibitor. Thus it is proved that the absolute configuration of the isomer of fluorocitrate that is formed from fluoroacetyl-CoA by the enzyme citrate synthase, and that inhibits aconitase, is the 2R, 3R, isomer.