(+)-Pinpollitol: A di-O-methyl d-(+)-chiro-inositol from Pinus radiata

(+)-Pinpollitol, a new cyclitol recently isolated from the pollen of Pinus radiata, was found in the needles of this species. (+)-Pinpollitol was found to be a di-O-methyl ether Of d-(+)-chiro-inositol, and tentative isomeric structures have been proposed for the cyclitol. (+)-Pinpollitol is the first di-O-methyl inositol to be found in a gymnosperm and is one of only three di-O-methyl inositols yet found in nature.     

Biosynthesis of (+)-catechin glycosides using recombinant amylosucrase from Deinococcus geothermalis DSM 11300

Amylosucrase (ASase, EC 2.4.1.4) is a glucosyltransferase that hydrolyzes sucrose into glucose and fructose and produces amylose-like glucan polymers from the released glucose. (+)-Catechin is a plant polyphenolic metabolite having skin-whitening and antioxidant activities. In this study, the ASase gene from Deinococcus geothermalis (dgas) was expressed in Escherichia coli, while the recombinant DGAS enzyme was purified using a glutathione S-transferase fusion system. The (+)-catechin glycoside derivatives were synthesized from (+)-catechin using DGAS transglycosylation activity. We confirmed the presence of two major transglycosylation products using TLC. The (+)-catechin transglycosylation products were isolated using silica gel open column chromatography and recycling-HPLC. Two (+)-catechin major transfer products were determined through ¹H and ¹³C NMR to be (+)-catechin-3′-O-α-d-glucopyranoside with a glucose molecule linked to (+)-catechin and (+)-catechin-3′-O-α-D-maltoside with a maltose linked to (+)-catechin. The presence of (+)-catechin maltooligosaccharides in the DGAS reaction was also confirmed via recycling-HPLC and enzymatic analysis. The effects of various reaction conditions (temperature, enzyme concentration, and molar ratio of acceptor and donor) on the yield and type of (+)-catechin glycosides were investigated.     

Preparation of (+)-Trans-Isoalliin and Its Isomers by Chemical Synthesis and RP-HPLC Resolution

Naturally occurring (+)-trans-isoalliin, (R_CR_S)-(+)-trans-S-1-propenyl-L-cysteine sulfoxide, is a major cysteine sulfoxide in onion. The importance of producing it synthetically to support further research is very well recognized. The (+)-trans-isoalliin is prepared by chemical synthesis and reversed-phase (RP)-HPLC. First, S-2-propenyl-L-cysteine (deoxyalliin) is formed from L-cysteine and allyl bromide, which is then isomerized to S-1-propenyl-L-cysteine (deoxyisoalliin) by a base-catalyzed reaction. A mixture of cis and trans forms of deoxyisoalliin is formed and separated by RP-HPLC. Oxidation of the trans form of deoxyisoalliin by H₂O₂ produces a mixture of (−)- and (+)-trans-isoalliin. Finally, RP-HPLC is used successfully in separating (−)- and (+)-trans-isoalliin, and hence, (+)-trans-isoalliin is synthesized for the first time in this study. In addition, the (±) diastereomers of cis-isoalliin are also separated and purified by RP-HPLC.     

Production of the sesquiterpenoid (+)-nootkatone by metabolic engineering of Pichia pastoris

The sesquiterpenoid (+)-nootkatone is a highly demanded and highly valued aroma compound naturally found in grapefruit, pummelo or Nootka cypress tree. Extraction of (+)-nootkatone from plant material or its production by chemical synthesis suffers from low yields and the use of environmentally harmful methods, respectively. Lately, major attention has been paid to biotechnological approaches, using cell extracts or whole-cell systems for the production of (+)-nootkatone. In our study, the yeast Pichia pastoris initially was applied as whole-cell biocatalyst for the production of (+)-nootkatone from (+)-valencene, the abundant aroma compound of oranges. Therefore, we generated a strain co-expressing the premnaspirodiene oxygenase of Hyoscyamus muticus (HPO) and the Arabidopsis thaliana cytochrome P450 reductase (CPR) that hydroxylated extracellularly added (+)-valencene. Intracellular production of (+)-valencene by co-expression of valencene synthase from Callitropsis nootkatensis resolved the phase-transfer issues of (+)-valencene. Bi-phasic cultivations of P. pastoris resulted in the production of trans-nootkatol, which was oxidized to (+)-nootkatone by an intrinsic P. pastoris activity. Additional overexpression of a P. pastoris alcohol dehydrogenase and truncated hydroxy-methylglutaryl-CoA reductase (tHmg1p) significantly enhanced the (+)-nootkatone yield to 208 mg L⁻¹ cell culture in bioreactor cultivations. Thus, metabolically engineered yeast P. pastoris represents a valuable, whole-cell system for high-level production of (+)-nootkatone from simple carbon sources.     

(−)-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.     

(+)-Medioresinol leads to intracellular ROS accumulation and mitochondria-mediated apoptotic cell death in Candida albicans

The phytochemical (+)-Medioresinol, a furofuran type lignan identification and isolation on the stem bark of Sambucus williamsii, which is a folk medicinal plant used in traditional medicine. (+)-Medioresinol is known to possess a lesishmanicidal activity and cardiovascular disease risk reduction but its antifungal effects have not yet been identified. In this study, to confirm (+)-Medioresinol's antifungal properties and mode of action, we observed morphological and physiological change in Candida albicans. In cells exposed to (+)-Medioresinol, arrested the cell cycle and intracellular reactive oxygen species (ROS) which is a major cause of apoptosis were increased. The increase of ROS induced oxidative stress and the mitochondria dysfunction which causes release of pro-apoptotic factors. We investigated a series of characteristic cellular changes of apoptosis by using various apoptosis detection methods. We report here for the first time that (+)-Medioresinol has effects on mitochondria and induced the accumulation of ROS in C. albicans cells. We demonstrated that one of the important features of apoptosis, mitochondrial membrane depolarization is caused by ROS. Substantially, we investigated the release of cytochrome c, which is one of the factors of metacaspase activity. We also show that the effects of (+)-Medioresinol are mediated at an early stage in apoptosis acting on the plasma membrane phosphatidylserine externalization. In addition, (+)-Medioresinol induced apoptotic morphological changes, showing the reduced cell size (low FSC) and enhanced intracellular density (high SSC). In late stage of confirmation of diagnostic markers in yeast apoptosis include the effects of nucleus morphological change, DNA fragmentation and condensation by influence of oxidative stress. These apoptotic phenomena represent that oxidative stress and mitochondria dysfunctions by inducing the phytochemical (+)-Medioresinol must be an important factors of the apoptotic process in C. albicans. These results support the elucidation of the underlying antifungal mechanisms of (+)-Medioresinol.     

Hepatoprotective Effect of C-Phycocyanin: Protection for Carbon Tetrachloride andR-(+)-Pulegone-Mediated Hepatotoxicty in Rats

Effect of C-phycocyanin (fromSpirulina platensis) pretreatment on carbontetrachloride andR-(+)-pulegone-induced hepatotoxicity in rats was studied. Intraperitoneal (i.p.) administration (200 mg/kg) of a single dose of phycocyanin to rats, one or three hours prior toR-(+)-pulegone (250 mg/kg) or carbontetrachloride (0.6 ml/kg) challenge, significantly reduced the hepatotoxicity caused by these chemicals. For instance, serum glutamate pyruvate transaminase (SGPT) activity was almost equal to control values. The losses of microsomal cytochrome P450, glucose-6-phosphatase and aminopyrine-N-demethylase were significantly reduced, suggesting that phycocyanin provides protection to liver enzymes. It was noticed that the level of menthofuran, the proximate toxin ofR-(+)-pulegone was nearly 70% more in the urine samples collected from rats treated withR-(+)-pulegone alone than rats treated with the combination of phycocyanin andR-(+)-pulegone. The possible mechanism involved in the hepatoprotection is discussed.     

Enhanced production of (+)-terrein in fed-batch cultivation of Aspergillus terreus strain PF26 with sodium citrate

(+)-Terrein is a fungal metabolite with multiple biological activities, especially with great value in medicine. However, the mass production of single configuration terrein is still a big challenge. In this study, the effects of acetic acid, sodium acetate, citric acid and sodium citrate on the (+)-terrein production by Aspergillus terreus strain PF26 derived from marine sponge Phakellia fusca were investigated. Sodium citrate was selected for fed-batch cultivation because it showed the best effect on (+)-terrein production among the four regulators tested. As a result, 5.38 g/L (+)-terrein production was achieved by feeding 10 mM sodium citrate on the 3rd day in shake flask, which was 33.8 % higher than the control and represented the highest yield of (+)-terrein. In a 7.5-L stirred bioreactor, 2.58 g/L of (+)-terrein production was achieved by the feeding of 10 mM sodium citrate on the 8th day. The results from this study lay a basis for the high-yield production of (+)-terrein by fermentation.     

(+)-2,3-Trans-pubeschin,the first catechin analogue of peltogynoids from Peltogyne pubescens and P. venosa

The heartwoods of Peltogyne pubescens and P. venosa contain the predominant pair (+)-peltogynol and (+)-mopanol, their 4-epimers, (+)-peltogynol B and (+)-mopanol B, together with the first catechin analogue of peltogynol, (+)-2,3- trans-pubeschin. These are accompanied by ±-2,3-cis- and ±-2,3-trans-3-O-methylfustins, and by α, 2′,3,4,4′-pentahydroxychalcone. Other minor metabolises are 4′,7-dihydroxy- and 3′,4′,7-trihydroxy-flavanones and 5,6-dihydroxyphthalide. (+)-2,3-Trans-pubeschin trimethyl ether was synthesized by reduction of the corresponding (+)-2,3-trans-peltogynone analogue with NaBH₄/BF₃ in diglyme, and its absolute configuration shown to be 2R: 3S.     

Coupling cell growth and biochemical pathway induction in Saccharomyces cerevisiae for production of (+)-valencene and its chemical conversion to (+)-nootkatone

(+)-Nootkatone is a valuable, functional sesquiterpene that is widely used in food, cosmetics, pharmaceutical, agriculture, and other fields. However, only traces of it accumulate in plants, which is insufficient to meet the market demand. Therefore, commercial (+)-nootkatone is currently synthesized from (+)-valencene. Here, we engineered Saccharomyces cerevisiae to achieve high production of (+)-valencene. Employing gene screening, protein engineering and biosynthetic pathway optimization, we achieved 12.4 g/L (+)-valencene production with the mutant strain. This titer was further increased to 16.6 g/L, the highest titer reported to date, by coupling critical factors for cell growth and biochemical pathway induction. Subsequently, (+)-nootkatone was chemically synthesized from bio-fermented (+)-valencene with a yield of 80%. This study achieved efficient microbial synthesis of (+)-valencene, which may be utilized in industrial production and stabilize the supply of (+)-nootkatone.     

(+)-[C-11]-cis-N-benzyl-normetazocine: A selective ligand for sigma receptors in vivo

The in vivo biodistribution profile of the novel sigma (σ) receptor ligand (+)-[C-11]-cis-N-benzyl-normetazocine ([C-11]-(+)-NBnNM) in mouse brain was examined. This radioligand displayed high brain uptake and a distribution consistent with the density of σ receptors. Brain radioactivity levels peaked at 15 min postinjection and were largely maintained (ca. 80% of maximal values) up to 90 min postinjection. Pretreatment with several different σ ligands (haloperidol, (+)-pentazocine, DuP 734, ifenprodil) effectively inhibited [C-11]-(+)-NBnNM binding in a dose-dependent manner in all brain regions. [C-11]-(+)-NBnNM binding sites were shown to be saturable with unlabeled (+)-NBnNM (ED50 = 0.02 mg/kg) and enantioselectively inhibited by the optical isomers of pentazocine. A blocking dose of the dopamine D2 antagonist spiperone (1 mg/kg) did not significantly inhibit [C-11]-(+)-NBnNM binding. Pretreatment with the phencyclidine (PCP) blocker 1-[1-(2-thienyl)cyclohexyl] piperidine (TCP) did not significantly alter total brain tissue radioactivity. Thus, [C-11]-(+)-NBnNM binds with high specificity and selectivity to σ receptors in vivo and offers excellent potential to study σ receptors in living human brain via positron emission tomography.     

Enhanced production of (+)-terrein by Aspergillus terreus strain PF26 with epigenetic modifier suberoylanilide hydroxamic acid

New strategies for improving the fermentation yield of (+)-terrein which is a fungal metabolite with multiple bioactivities are very urgent. In this study, the effect of suberoylanilide hydroxamic acid, one kind of epigenetic modifier, on the biosynthesis of (+)-terrein by Aspergillus terreus strain PF26 isolated from the marine sponge Phakellia fusca was investigated. It was found that suberoylanilide hydroxamic acid exhibited a positive impact on (+)-terrein production, resulting from promoting the biosynthesis of 6-hydroxymellein, the precursor of (+)-terrein. Through optimization of feeding concentration and time of suberoylanilide hydroxamic acid, 5.58 g/L (+)-terrein could be obtained in shake flask cultivation, 29.5% higher than the control. Correspondingly, the fermentation of A. terreus strain PF26 in 7.5-L stirred bioreactor with feeding suberoylanilide hydroxamic acid (900 μM, day 4) yielded 9.07 g/L (+)-terrein, 77.1% higher than the control. These results showed that the epigenetic modifier-suberoylanilide hydroxamic acid could be utilized to enhance the production of (+)-terrein, which laid the foundation of massive production of (+)-terrein by fermentation.     

(+)-5,17-dehydromatrine N-oxide,a new alkaloid in Euchresta japonica

A new lupin alkaloid, (+)-5,17-dehydromatrine N-oxide, was isolated from the fresh aerial parts of Euchresta japonica. Its structure was confirmed by spectrometric data and by direct comparison with a synthetic sample, prepared from (+)-sophoranol ((+)-5-hydroxymatrine). It was also concluded that (+)-5,17-dehydromatrine N-oxide and (+)-matrine N-oxide possess the same configuration with respect to the asymmetric nitrogen by NMR spectra.     

Microbiologically Catalyzed Enantio- and Diastereoselective Oxidation of Chrysanthemol Stereoisomers to Chrysanthemic Acids

The diastereo- and enantioselective microbial oxidation of a mixture of racemic cis/trans-chrysanthemols to the corresponding stereoisomeric chrysanthemic acids by Aspergillus species is described. Of the three microorganisms which were found capable of oxidizing racemic cis/trans-chrysanthemols, A. ochraceus ATCC 18500 showed complete enantioselectivity for (+)-stereoisomers [(+)-trans-chrysanthemol and (+)-cis-chrysanthemol), whereas A. flavipes ATCC 1030 and ATCC 11013 showed complete enantioselectivity for the (+)-cis-chrysanthemol but a time-dependent enantioselectivity during oxidation of trans-chrysanthemol [oxidation of (+)-trans-chrysanthemol prior to (−)-trans-chrysanthemol]. The diastereoselectivity of all three microorganisms was time dependent, in that the trans-stereoisomers were oxidized prior to the cis-isomers.     

Differential Mechanism of Escherichia coli Inactivation by (+)-Limonene as a Function of Cell Physiological State and Drug's Concentration

(+)-limonene is a lipophilic antimicrobial compound, extracted from citrus fruits'' essential oils, that is used as a flavouring agent and organic solvent by the food industry. A recent study has proposed a common and controversial mechanism of cell death for bactericidal antibiotics, in which hydroxyl radicals ultimately inactivated cells. Our objective was to determine whether the mechanism of Escherichia coli MG1655 inactivation by (+)-limonene follows that of bactericidal antibiotics. A treatment with 2,000 μL/L (+)-limonene inactivated 4 log₁₀ cycles of exponentially growing E. coli cells in 3 hours. On one hand, an increase of cell survival in the ΔacnB mutant (deficient in a TCA cycle enzyme), or in the presence of 2,2′-dipyridyl (inhibitor of Fenton reaction by iron chelation), thiourea, or cysteamine (hydroxyl radical scavengers) was observed. Moreover, the ΔrecA mutant (deficient in an enzyme involved in SOS response to DNA damage) was more sensitive to (+)-limonene. Thus, this indirect evidence indicates that the mechanism of exponentially growing E. coli cells inactivation by 2,000 μL/L (+)-limonene is due to the TCA cycle and Fenton-mediated hydroxyl radical formation that caused oxidative DNA damage, as observed for bactericidal drugs. However, several differences have been observed between the proposed mechanism for bactericidal drugs and for (+)-limonene. In this regard, our results demonstrated that E. coli inactivation was influenced by its physiological state and the drug''s concentration: experiments with stationary-phase cells or 4,000 μL/L (+)-limonene uncovered a different mechanism of cell death, likely unrelated to hydroxyl radicals. Our research has also shown that drug''s concentration is an important factor influencing the mechanism of bacterial inactivation by antibiotics, such as kanamycin. These results might help in improving and spreading the use of (+)-limonene as an antimicrobial compound, and in clarifying the controversy about the mechanism of inactivation by bactericidal antibiotics.     

Valencene oxidase CYP706M1 from Alaska cedar (Callitropsis nootkatensis)

(+)-Nootkatone is a natural sesquiterpene ketone used in grapefruit and citrus flavour compositions. It occurs in small amounts in grapefruit and is a major component of Alaska cedar (Callitropsis nootkatensis) heartwood essential oil. Upon co-expression of candidate cytochrome P450 enzymes from Alaska cedar in yeast with a valencene synthase, a C. nootkatensis valencene oxidase (CnVO) was identified to produce trans-nootkatol and (+)-nootkatone. Formation of (+)-nootkatone was detected at 144 ± 10 μg/L yeast culture. CnVO belongs to a new subfamily of the CYP706 family of cytochrome P450 oxidases.     

Catechins with (+)-epi-configuration in nature

(+)-Epicatechin has been isolated from various species of Palmae and (+)-epiafzelechin from Livinstona chinensis. This is the first time that catechins with (+)-epi-configuration have been found in natural sources.     

Inhibitory Activity of (+)-Usnic Acid against Non-Small Cell Lung Cancer Cell Motility

Lichens are symbiotic organisms that produce various unique chemicals that can be used for pharmaceutical purposes. With the aim of screening new anti-cancer agents that inhibit cancer cell motility, we tested the inhibitory activity of seven lichen species collected from the Romanian Carpathian Mountains against migration and invasion of human lung cancer cells and further investigated the molecular mechanisms underlying their anti-metastatic activity. Among them, Alectoria samentosa, Flavocetraria nivalis, Alectoria ochroleuca, and Usnea florida showed significant inhibitory activity against motility of human lung cancer cells. HPLC results showed that usnic acid is the main compound in these lichens, and (+)-usnic acid showed similar inhibitory activity that crude extract have. Mechanistically, β-catenin-mediated TOPFLASH activity and KITENIN-mediated AP-1 activity were decreased by (+)-usnic acid treatment in a dose-dependent manner. The quantitative real-time PCR data showed that (+)-usnic acid decreased the mRNA level of CD44, Cyclin D1 and c-myc, which are the downstream target genes of both β-catenin/LEF and c-jun/AP-1. Also, Rac1 and RhoA activities were decreased by treatment with (+)-usnic acid. Interestingly, higher inhibitory activity for cell invasion was observed when cells were treated with (+)-usnic acid and cetuximab. These results implied that (+)-usnic acid might have potential activity in inhibition of cancer cell metastasis, and (+)-usnic acid could be used for anti-cancer therapy with a distinct mechanisms of action.     

Mono- and sesquiterpenoids of Conocephalum supradecompositum

The liverwort Conocephalum supradecompositum belonging to the Marchantiales is a rich source of the germacranolides (+)-costunolide and (+)-dihydro