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Cacalia delphiniifolia and Cacalia hastata are edible wild plants in Japan. We found that these plants have anti-melanogenic activity in B16F10 mouse melanoma cells. Three furanoeremophilanes, cacalol (from C. delphiniifolia), dehydrocacalohastin, and cacalohastin (from C. hastata), were identified as the main active components. The genus Cacalia may be a good source of beneficial materials with anti-melanogenic effects.

Dihydromaniwamycin E (

Ramaria botrytis is a popular mushroom in Asian countries, particularly known for its crispy texture and rich nutrient content. In this study, we found potent lipid peroxidation-inhibiting activity in this mushroom and identified the active compound associated with this activity as pistillarin. To our knowledge, this is the first report of the presence of pistillarin in R. botrytis. Our study is the first to investigate the inhibitory effects of pistillarin against physiological reactive oxygen species such as 1O2, •OH, and O2- and lipid peroxidation, which damages living tissues. We also clarified its characteristic antioxidant activities, with no 1O2 and •OH quenching activity (IC50100 μМ), moderate O2- quenching activity (IC50 of 10.2 μМ), and potent lipid peroxidation-inhibiting activity (IC50 of 0.66 μМ), for the first time. Furthermore, we found a new pistillarin-related compound (pistillarin B) in salted R. botrytis. We isolated pistillarin B, determined its structure, and examined its lipid peroxidation-inhibiting activity (IC50 of 6.88 μМ).

Recombinant Escherichia coli cells, which express the higher-plant CaZEP (zeaxanthin epoxidase) and AtCYO1 (molecular chaperone) genes and an NADPH regeneration system in addition to the IDI, crtE, crtB, crtI, crtY, and crtZ genes for zeaxanthin biosynthesis, were cultured in 2 × YT medium for the efficient production of antheraxanthin and violaxanthin. The carotenoids accumulated in the cells were purified by two-phase partition, silica gel column chromatography, and preparative silica gel HPLC. Mass spectrometry and nuclear magnetic resonance analyses of these carotenoids revealed that a novel carotenoid, determined to be (3R,6′S)-3,6′-dihydroxy-β,e-caroten-3′-one and named as 6′-hydroxy-3′-didehydrolutein, was produced in addition to zeaxanthin, antheraxanthin and violaxanthin. It is likely that the new carotenoid was generated from an unstable epoxy-carotenoid antheraxanthin that was synthesized from zeaxanthin with CaZEP. Its antioxidant activities were also examined.

Angelica keiskei (ashitaba) is an edible plant belonging to the Apiacea family. We focused on sesquiterpenes in the leaves eaten by humans (specifically, in the Japanese population), and confirmed the presence of several sesquiterpenes by GC-MS. Thus, total RNA was extracted from the ashitaba leaves, reverse transcribed, and the resultant cDNAs were used for degenerate PCR followed by rapid amplification of cDNA ends. Consequently, we were able to isolate two full-length Tps genes (designated AkTps1 and AkTps2). Functional analysis of these two genes was carried out with Escherichia coli cells that expressed mevalonate pathway genes to increase the substrate (farnesyl diphosphate) amount of sesquiterpene synthase, revealing that AkTps1 encodes germacrene D synthase, and AkTps2 codes for an enzyme that catalyzes the generation of germacrene B and smaller amounts of germacrene D (a germacrene B and D synthase). We proposed biosynthetic routes of these two sesquiterpenes from farnesyl diphosphate (FPP) via farnesyl cation.

Mono-(5Z)-, -(9Z)-, and -(13Z)-lycopenes are found in food containing processed tomato products, while tetra-Z-(7Z, 9Z, 7'Z, 9'Z)-lycopene (prolycopene) is found in tangerine-strain tomatoes. We prepared pure mono-Z-lycopenes from all-E-lycopene via chemical reaction (heating in CH2Cl2 at 80℃ for 1 h) followed by purification using preparative silica gel HPLC, while prolycopene was isolated from tangerine tomatoes by partitioning with n-hexane and 90% MeOH followed by silica gel column chromatography. A simple method of distinguishing the mono-Z-lycopenes using the 13C NMR chemical shifts of their Z-methyl carbons is proposed. Additionally, the 1O2 quenching and 3T3-L1 cell differentiation activities of the compounds were then compared with all-E-lycopene for the first time. All the evaluated Z-isomers showed 1O2 quenching activities that were equal to or slightly lower than that of all-E-lycopene, with the IC50 values for the 1O2 quenching activities of (all-E)-, (5Z)-, (9Z)-, (13Z)-, and (7Z, 9Z, 7'Z, 9'Z)-lycopene being 4.4±0.36, 4.0±1.44, 5.3±1.08, 6.9±1.67, and 8.7±0.34 µM, respectively. The mouse 3T3-L1 cell differentiation activities followed the order: (all-E) > (9Z) > (5Z) ≈ (9Z) ≈ (13Z) ≈ (7Z, 9Z, 7'Z, 9'Z).

Background Members of the genus Planococcus have been revealed to utilize and degrade solvents such as aromatic hydrocarbons and alkanes, and likely to acquire tolerance to solvents. A yellow marine bacterium Planococcus maritimus strain iso-3 was isolated from an intertidal sediment that looked industrially polluted, from the Clyde estuary in the UK. This bacterium was found to produce a yellow acyclic carotenoid with a basic carbon 30 (C30) structure, which was determined to be methyl 5-glucosyl-5,6-dihydro-4,4′-diapolycopenoate. In the present study, we tried to isolate and identify genes involved in carotenoid biosynthesis from this marine bacterium, and to produce novel or rare C30-carotenoids with anti-oxidative activity in Escherichia coli by combinations of the isolated genes. Results A carotenoid biosynthesis gene cluster was found out through sequence analysis of the P. maritimus genomic DNA. This cluster consisted of seven carotenoid biosynthesis candidate genes (orf1–7). Then, we isolated the individual genes and analyzed the functions of these genes by expressing them in E. coli. The results indicated that orf2 and orf1 encoded 4,4′-diapophytoene synthase (CrtM) and 4,4′-diapophytoene desaturase (CrtNa), respectively. Furthermore, orf4 and orf5 were revealed to code for hydroxydiaponeurosporene desaturase (CrtNb) and glucosyltransferase (GT), respectively. By utilizing these carotenoid biosynthesis genes, we produced five intermediate C30-carotenoids. Their structural determination showed that two of them were novel compounds, 5-hydroxy-5,6-dihydro-4,4′-diaponeurosporene and 5-glucosyl-5,6-dihydro-4,4′-diapolycopene, and that one rare carotenoid 5-hydroxy-5,6-dihydro-4,4′-diapolycopene is included there. Moderate singlet oxygen-quenching activities were observed in the five C30-carotenoids including the two novel and one rare compounds. Conclusions The carotenoid biosynthesis genes from P. maritimus strain iso-3, were isolated and functionally identified. Furthermore, we were able to produce two novel and one rare C30-carotenoids in E. coli, followed by positive evaluations of their singlet oxygen-quenching activities.

Carnosic acid (CA) and carnosol (CS) are components of rosemary reported to possess antioxidative and peroxisome proliferator-activated receptor gamma (PPARγ) transcriptional activities that are useful for preventing metabolic disorders. The aims of this study were to identify new bioactive compounds in heated rosemary and examine their effects on antioxidative stress and PPARγ activation. In heated rosemary, we found five CA-and CS-related compounds and determined their structures by mass spectrometry and nuclear magnetic resonance analyses. Three compounds were identified as dehydromiltirone (1), miltiodiol (2), and miltirone (4), whereas two compounds (3 and 5) were newly discovered molecules. We examined their antioxidant activities and found that 2 and 3 showed strong activity. Furthermore, in PPARγ−transactivation and adipocyte-differentiation assays, compounds 3 and 5 showed more potent PPARγ agonistic activity than CA.

Mono-(5Z)-, -(9Z)-, and -(13Z)-lycopenes are found in food containing processed tomato products, while tetra-Z-(7Z, 9Z, 7'Z, 9'Z)-lycopene (prolycopene) is found in tangerine-strain tomatoes. We prepared pure mono-Z-lycopenes from all-E-lycopene via chemical reaction (heating in CH

Bacteria must survive harsh environmental fluctuations at times and have evolved several strategies. "Collective" behaviors have been identified due to recent progress in single-cell analysis. Since most bacteria exist as single cells, bacterial populations are often considered clonal. However, accumulated evidence suggests this is not the case. Gene expression and protein expression are often not homogeneous, resulting in phenotypic heterogeneity. In extreme cases, this leads to bistability, the existence of two stable states. In many cases, expression of key master regulators is bimodal via positive feedback loops causing bimodal expression of the target genes. We observed bimodal expression of metabolic genes for alternative carbon sources. Expression profiles of the frlBONMD-yurJ operon driven by the frlB promoter (PfrlB), which encodes degradation enzymes and a transporter for amino sugars including fructoselysine, were investigated using transcriptional lacZ and gfp, and translational fluorescence reporter mCherry fusions. Disruption effects of genes encoding CodY, FrlR, RNaseY, and nucleoid-associated protein YlxR, four known regulatory factors for PfrlB, were examined for expression of each fusion construct. Expression of PfrlB-gfp and PfrlB-mCherry, which were located at amyE and its original locus, respectively, was bimodal; and disruption of ylxR resulted in the disappearance of the clear bimodal expression pattern in flow cytometric analyses. This suggested a role for YlxR on the bimodal expression of PfrlB. The data indicated that YlxR acted on the bimodal expression of PfrlB through both transcription and translation. YlxR regulates many genes, including those related to translation, supporting the above notion. Depletion of RNaseY abolished heterogenous expression of transcriptional PfrlB-gfp but not bimodal expression of translational PfrlB-mCherry, suggesting the role of RNaseY in regulation of the operon through mRNA stability control and regulatory mechanism for PfrlB-mCherry at the translational level. Based on these results, we discuss the meaning and possible cause of bimodal PfrlB expression.

Angelica keiskei (ashitaba) is an edible plant belonging to the Apiacea family. We focused on sesquiterpenes in the leaves eaten by humans (specifically, in the Japanese population), and confirmed the presence of several sesquiterpenes by GC-MS. Thus, total RNA was extracted from the ashitaba leaves, reverse transcribed, and the resultant cDNAs were used for degenerate PCR followed by rapid amplification of cDNA ends. Consequently, we were able to isolate two full-length Tps genes (designated AkTps1 and AkTps2). Functional analysis of these two genes was carried out with Escherichia coli cells that expressed mevalonate pathway genes to increase the substrate (farnesyl diphosphate) amount of sesquiterpene synthase, revealing that AkTps1 encodes germacrene D synthase, and AkTps2 codes for an enzyme that catalyzes the generation of germacrene B and smaller amounts of germacrene D (a germacrene B and D synthase). We proposed biosynthetic routes of these two sesquiterpenes from farnesyl diphosphate (FPP) via farnesyl cation.

Terpene synthase (TPS) genes were isolated and functionally characterized from three traditional edible plants, Acanthopanax sciadophylloides ("Koshiabura") and Acanthopanax sieboldianus ("Himeukogi"), belonging to the family Araliaceae, and Curcuma zedoaria (zedoary, "Gajutsu"), belonging to the family Zingiberaceae. These plants emit characteristic fragrances and are used for traditional foods and folk medicines. From their fragrant tissues, i.e., sprouts of Araliaceae plants and developing rhizomes of zedoary, total RNAs were extracted and reverse transcribed. The resultant cDNAs were used for degenerate PCR followed by rapid amplification of cDNA ends. From the contig sequences obtained, full-length Tps genes were amplified by PCR with newly synthesized primer sets. The isolated full-length genes were introduced into engineered Escherichia coli cells, which can utilize acetoacetate to synthesize farnesyl diphosphate, the substrate for TPSs, through the mevalonate pathway. TPS products synthesized in the transformed E. coli cells were analysed by gas chromatography-mass spectrometry, nuclear magnetic resonance, and optical rotation. Consequently, the isolated Tps genes were found to encode β-caryophyllene synthase, germacrene D synthase, linalool/(3S)-(+)-nerolidol synthase, β-eudesmol synthase, and germacrene B synthase. These results lead us to expect that some of the effective ingredients in folk medicines are volatile terpenes and that intake of traditional foods including these edible plants would have some positive effects on our health.

In this study, we investigated the antioxidant activities of antheraxanthin-related carotenoids. Antheraxanthin and 9-cis-antheraxanthin were prepared from persimmon and orange fruit, respectively, and converted to other carotenoids under acidic conditions. Resulting carotenoids were purified using preparative silica gel HPLC, and their structures were analyzed in detail by NMR spectra. Both antheraxanthin and 9-cis-antheraxanthin were found to be converted to (8R)- and (8S)-mutatoxanthin at an approximate ratio of 3:2. High antioxidant activities were observed for antheraxanthin, 9-cis-antherxanthin, (8R)-mutatoxanthin, and (8S)-mutatoxanthin, with potent lipid peroxidation inhibitory and moderate 1O2-quenching activities.

Antioxidant compounds in the mushroom Boletopsis leucomelas (PERS.) FAYOD were isolated using chromatographic methods, and their structures were determined via detailed analyses using high-resolution atmospheric pressure chemical ionization mass spectrometry and nuclear magnetic resonance. We identified five known p-terphenyl compounds (Bl-I, Bl-II, Bl-III, cycloleucomelon-leukopentaacetat, and Bl-IV) and one p-terphenyl new compound (Bl-VI); we determined the complete structure of cycloleucomelon-leukopentaacetat in this study. All these compounds possess potent lipid peroxidation-inhibiting activities. We further investigated changes in their chemical structures and antioxidant activities by applying heat (grilling, boiling, and microwave heating), and proved the production of two known p-terphenyl compounds (BI-V and boletopsin A) and one new p-terphenyl compound (BI-VII) via deacetylation of the original p-terphenyl compounds for the first time. We also found that DPPH radical scavenging activity was enhanced upon moderate heat cooking (boiling and microwave heating) due to changes in p-terphenyl compounds.

Violaxanthin and 9-cis-violaxanthin (major epoxycarotenoids in fruit) were prepared from mango fruit, purified, and converted to other carotenoids under acidic conditions. The resulting carotenoid structures were then analyzed in detail. Not only violaxanthin but also 9-cis-violaxanthin were found to be converted to (8S,8'S)-, (8S,8'R)-, and (8R,8'R)-auroxanthin at an approximate ratio of 4:6:1. Antioxidant activities of violaxanthin, 9-cis-violaxanthin, (8S,8'S)-auroxanthin, and (8S,8'R)-auroxanthin were examined. They possessed potent lipid peroxidation inhibitory and very weak 1O2 quenching activities.

The production of methylated C41-C43 15-cis-phytoene, lycopene, β-carotene, and zeaxanthin using recombinant Escherichia coli that express the genes encoding isopentenyl pyrophosphate, C4-methyltransferase, and carotenoid biosynthetic enzymes, as well as the detection of their chemical structures using high-pressure liquid chromatography-mass spectroscopy (HPLC-MS) analyses have been previously reported. However, the positions of the methyl groups were not fully elucidated due to multiple possible sites that could be methylated. In this study, we have isolated the methylated carotenoids using chromatographic methods and unambiguously determined their structures as 16-methyl derivatives of 15-cis-phytoene, lycopene, β-carotene, and zeaxanthin via detailed MS, 1D, and 2D NMR analyses. These compounds were all identified as novel C41 carotenoids.

Improving and maintaining memory function is effective in preventing cognitive decline and dementia. Previously, we demonstrated that iso-α-acids, the hop-derived bitter components in beer, prevent cognitive impairment in an Alzheimer’s disease mouse model. In this report, we investigated the effects of matured hop bitter acids (MHBA) containing components of oxides derived from α- and β-acids, and structurally similar to iso-α-acids, on cognitive function using behavioral pharmacological procedures. MHBA and the representative components of MHBA, 4′-hydroxyallohumulinone (HAH) and 4′-hydroxy-cis-alloisohumulone (HAIH) improved spatial working memory in scopolamine-induced amnesia mice. MHBA also enhanced episodic memory in the novel object recognition test (NORT). The administration of MHBA increased the amount of norepinephrine (NE) and NE release into cerebrospinal fluid (CSF) in hippocampus. The MHBA activity in improving memory function was attenuated by treatment with a β-adrenergic receptor inhibitor. In addition, vagotomized mice did not display the memory improvement induced by MHBA. Together, our results suggest that MHBA improves memory function via stimulation of the vagus nerve and enhancement of NE release in the hippocampus. Vagus nerve activation by the intake of food materials including MHBA may be a safe and effective approach for improving cognitive function.

Seven cDNA clones encoding terpene synthases (TPSs), their structures closely related to each other, were isolated from the flower of Camellia hiemalis (‘Kantsubaki’). Their putative TPS proteins were phylogenetically positioned in a sole clade with the TPSs of other Camellia species. The obtained Tps genes, one of which was designated ChTps1 (ChTps1a), were introduced into mevalonate-pathway-engineered Escherichia coli, which carried the genes for utilizing acetoacetate as a substrate, and cultured in a medium including lithium acetoacetate. Volatile products generated in the E. coli cells transformed with ChTps1 were purified from the cell suspension culture, and analyzed by NMR. Consequently, the predominant product with ChTPS1 was identified as valerianol, indicating that the ChTps1 gene codes for valerianol synthase. This is the first report on a gene that can mediate the synthesis of valerianol. We next synthesized a Tps ortholog encoding ChTPS1variant R477H (named CsiTPS8), whose sequence had been isolated from a tea tree (Camellia sinensis), carried out similar culture experiment with the E. coli transformant including CsiTps8, and consequently found valerianol production equally. Furthermore, GC-MS analysis of several teas revealed that valerianol had been an unknown ingredient in green tea and black tea.

The bitter taste of beer originates from resins in hops (Humulus lupulus L.), which are classified into two subtypes (soft and hard). Whereas the nature and reactivity of soft-resin-derived compounds, such as α-, β-, and iso-α-acids, are well studied, there is only a little information on the compounds in hard resin. For this work, hard resin was prepared from stored hops and investigated for its compositional changes in an experimental model of beer aging. The hard resin contained a series of α-acid oxides. Among them, 4'-hydroxyallohumulinones were unstable under beer storage conditions, and their transformation induced primary compositional changes of the hard resin during beer aging. The chemical structures of the products, including novel polycyclic compounds scorpiohumulinols A and B and dicyclohumulinols A and B, were determined by HRMS and NMR analyses. These compounds were proposed to be produced via proton-catalyzed cyclization reactions of 4'-hydroxyallohumulinones. Furthermore, they were more stable than their precursor 4'-hydroxyallohumulinones during prolonged storage periods.

During the course of screening for antioxidative carotenoids from bacteria, we isolated two novel C30 carotenoids, 4-[2-O-11Z-octadecenoyl-β-glucopyranosyl]-4,4′-diapolycopene-4,4′-dioic acid (1) and 4-[2-O-9Z-hexadecenoyl-β-glucopyranosyl]-4,4′-diapolycopene-4,4′-dioic acid (2), from Methylobacterium strains. These compounds possessed the common structure ‘β- d -glucosyl 4,4′-diapolycopene-4,4′-dioic acid’, while the fatty acid attached at the C-2 position of glucose differed. In the singlet oxygen (1O2) quenching model, both 1 and 2 exhibited potent antioxidative activities (IC50: 3.2 and 3.6 μM, respectively).

There are many reports about carotenoid-producing bacteria and carotenoid biosynthesis genes. In databases for Pseudomonas genome sequences, there are genes homologous to carotenoid biosynthesis genes, but the function of these genes in Pseudomonas has not been elucidated. In this study, we cloned the carotenoid biosynthesis genes from a Pseudomonas sp. strain, named Akiakane, which was isolated from the excrement of the Autumn Darter dragonfly. Using an Escherichia coli functional expression system, we confirmed that the idi, crtE, crtB, crtI, and crtY gene products of the Akiakane strain show predictable catalytic activities. A cluster of six genes was also found, which was comparable to other carotenoid-producing bacteria that belong to the α-Proteobacteria or γ-Proteobacteria class.

A simple method to purify volatile sesquiterpenes from recombinant Escherichia coli was developed using the cells that carried known sesquiterpene synthase (Tps) genes ZzZss2 (ZSS2) and ZoTps1. This method was applied for the purification and structural analyses of volatile sesquiterpenes produced by E. coli cells that carried unidentified Tps genes, which were isolated from the Aralia-genus edible plants belonging to the family Araliaceae. Recombinant cells carrying each Tps gene were cultured in the two-layer medium (n-octane/TB medium), and volatile sesquiterpenes trapped in n-octane were purified through two-phase partition, silica gel column chromatography, and reversed-phase preparative high-performance liquid chromatography, if necessary. Further, their structures were confirmed by nuclear magnetic resonance, [α]D, and gas chromatography–mass spectrometry analyses. Herein, the products of E. coli cells that carried two Tps gene (named AcTps1 and AcTps2) in Araria cordata “Udo” and a Tps gene (named AeTps1) in Aralia elata “Taranoki” were studied resulting in identifying functionalities of these cryptic Tps genes.

Tolerogenic dendritic cells (DCs) have the ability to induce regulatory T cells and play an important role in preventing chronic inflammatory and autoimmune diseases. We have identified a novel compound, 14-dehydroergosterol, from Koji, a Japanese traditional food material fermented with fungi. 14-dehydroergosterol is an ergosterol analogue with a conjugated double bond, but the activity of 14-dehydroergosterol is much higher than that of ergosterol. 14-dehydroergosterol induces the conversion of murine bone marrow (BM)-derived DCs and differentiated DCs into tolerogenic DCs, in which the production of IL-12 is suppressed and that of IL-10 is increased. In a co-culture experiment, DCs treated with 14-dehydroergosterol induced the conversion of naïve CD4-positive T cells into regulatory T cells. In a murine model of multiple sclerosis, experimental autoimmune encephalopathy, 14-dehydroergosterol suppressed the clinical score and inflammatory responses of myeloid DCs and T cells to myelin oligodendrocyte glycoprotein. 14-dehydroergosterol-treated human DCs induced from PBMCs also showed a tolerogenic phenotype. This is the first report to identify a novel compound, 14-dehydroergosterol, that induces DCs to convert to a tolerogenic type. 14-dehydroergosterol is contained in various fermented foods based on Koji, so 14-dehydroergosterol might be a helpful aid to prevent chronic inflammatory and autoimmune diseases.

The transformation of α-acids [in hops (Humulus lupulus L.)] to iso-α-acids (in beer) during the brewing process is well known, but the occurrence and structure of the oxidized α-acids during hop storage are not well documented. Because an understanding of these oxidized compounds is essential to optimize the effects of oxidized hops on the quality of beer, we investigated the autoxidation products of humulone (a representative congener of α-acids) using a simplified autoxidation model. Among the oxidation products, tricyclooxyisohumulones A (1) and B (2), tricycloperoxyisohumulone A (3), deisopropyltricycloisohumulone (4), and the hemiacetal 5 of tricycloperoxyhumulone A (5') were isolated, and their structures were elucidated for the first time. The occurrence of compounds 1-4 in stored hops was verified using LC/MS/MS analysis. We also monitored the levels of compounds 1-4 during hop storage using LC/MS/MS analysis.

Recombinant Escherichia coli, carrying the crtE, crtB, crtI, crtY, and crtZ genes from the bacterium Pantoea ananatis in addition to the Haematococcus pluvialis idi gene, can dominantly produce zeaxanthin. Vigorous rotary-shaking culture of the recombinant cells to promote strong aeration led to the production of two structurally similar polar carotenoids [1 (3.7%) and 2 (2.9%)] and eschscholtzxanthone (2.3%), along with zeaxanthin (72.6%), β-cryptoxanthin (7.3%), β-carotene (7.1%), and rubixanthin (2.1%). Carotenoids 1 and 2 were purified, and their structures were determined as (6E)-9′-apo-rohodoxanthinone and (6Z)-9′-apo-rhodoxanthinone, respectively, based on UV–vis, MS, 1H NMR, and 13C NMR spectral data. These novel compounds were shown to exhibit excellent singlet oxygen-quenching activity by the methylene blue-sensitized photooxidation system.

The plastid genome of lettuce (Lactuca sativa L.) cv. Berkeley was site-specifically modified with the addition of three transgenes, which encoded β,β-carotenoid 3,3′-hydroxylase (CrtZ) and β,β-carotenoid 4,4′-ketolase (4,4′-oxygenase; CrtW) from a marine bacterium Brevundimonas sp. strain SD212, and isopentenyl diphosphate isomerase from a marine bacterium Paracoccus sp. strain N81106. Constructed transplastomic lettuce plants were able to grow on soil at a growth rate similar to that of non-transformed lettuce cv. Berkeley and generate flowers and seeds. The germination ratio of the lettuce transformants (T0) (98.8 %) was higher than that of non-transformed lettuce (93.1 %). The transplastomic lettuce (T1) leaves produced the astaxanthin fatty acid (myristate or palmitate) diester (49.2 % of total carotenoids), astaxanthin monoester (18.2 %), and the free forms of astaxanthin (10.0 %) and the other ketocarotenoids (17.5 %), which indicated that artificial ketocarotenoids corresponded to 94.9 % of total carotenoids (230 μg/g fresh weight). Native carotenoids were there lactucaxanthin (3.8 %) and lutein (1.3 %) only. This is the first report to structurally identify the astaxanthin esters biosynthesized in transgenic or transplastomic plants producing astaxanthin. The singlet oxygen-quenching activity of the total carotenoids extracted from the transplastomic leaves was similar to that of astaxanthin (mostly esterified) from the green algae Haematococcus pluvialis.

Reverse cholesterol transport (RCT) removes excess cholesterol from macrophages to prevent atherosclerosis. ATP-binding cassette, subfamily A, member 1 (ABCA1) is a crucial cholesterol transporter involved in RCT to produce high density lipoprotein-cholesterol (HDLC), and is transcriptionally regulated by liver X receptor alpha (LXRα), a nuclear receptor. Quercetin is a widely distributed flavonoid in edible plants which prevented atherosclerosis in an animal model. We found that quercetin-3-O-glucuronide (Q3GA), a major quercetin metabolite after absorption from the digestive tract, enhanced ABCA1 expression, in vitro, via LXRα in macrophages. In addition, leaf extracts of a traditional Asian edible plant, Nelumbo nucifera (NNE), which contained abundant amounts of quercetin glycosides, significantly elevated plasma HDLC in mice. We are the first to present experimental evidence that Q3GA induced ABCA1 in macrophages, and to provide an alternative explanation to previous studies on arteriosclerosis prevention by quercetin.

Escherichia coli cells that express the full six carotenoid biosynthesis genes (crtE, crtB, crtI, crtY, crtZ, and crtX) of the bacterium Pantoea ananatis have been shown to biosynthesize zeaxanthin 3,3'-β-D-diglucoside. We found that this recombinant E. coli also produced a novel carotenoid glycoside that contained a rare carbohydrate moiety, quinovose (chinovose; 6-deoxy-D-glucose), which was identified as 3-β-glucosyl-3'-β-quinovosyl zeaxanthin by chromatographic and spectroscopic analyses. The chirality of the aglycone of these zeaxanthin glycosides had been shown to be 3R,3'R, in which the hydroxyl groups were formed with the CrtZ enzyme. It was here demonstrated that zeaxanthin synthesized from β-carotene with CrtR or CYP175A1, the other hydroxylase with similar catalytic function to CrtZ, possessed the same stereochemistry. It was also suggested that the singlet oxygen-quenching activity of zeaxanthin 3,3'-β-D-diglucoside, which has a chemical structure close to the new carotenoid glycoside, was superior to that of zeaxanthin.

A variant of P450 BM3 with an F87V substitution [P450 BM3 (F87V)] is a substrate-promiscuous cytochrome P450 monooxygenase. We investigated the bioconversion of various flavonoids (favanones, chalcone, and isoflavone) by using recombinant Escherichia coli cells, which expressed the gene coding for P450 BM3 (F87V), to give their corresponding hydroxylated products. Potent antioxidative activities were observed in some of the products.

Isoflavone glucosides (2'-hydroxy,5-methoxy genistein-7-O-glucoside (1), 2'-hydroxy genistein-7-O-gentibioside (2), 5-methoxy genistein-7-O-glucoside (3), 3',5-dimethoxy genistein-7-O-glucoside (4), 2'-hydroxy genistein-7-O-glucoside (5), genistein-7-O-gentibioside (6), 2'-hydroxy,5-methoxy genistein-4',7-O-diglucoside (7), and 2'-hydroxy genistein-4',7-O-diglucoside (8)) were isolated from the groundnut of Apios americana Medik. Their structures were elucidated on the basis of HR-ESI-MS and 1D- and 2D-NMR analyses. Compounds 1, 2, 4, and 7 are new compounds presented here for the first time. Compounds 2 and 5 were proven to be androgen receptor antagonists due to their binding activities for androgen receptors (IC50 280 and 160 μM, respectively) and the inhibitory activity of androgen-induced expression of prostate-specific antigen (PSA) mRNA in LNCaP (prostate adenocarcinoma) cells (IC50 20 and 18 μM, respectively).

Recombinant Escherichia coli, carrying the crtE, crtB, crtI, crtY, and crtZ genes from the bacterium Pantoea ananatis in addition to the Haematococcus pluvialis idi gene, can dominantly produce zeaxanthin. Vigorous rotary-shaking culture of the recombinant cells to promote strong aeration led to the production of two structurally similar polar carotenoids [1 (3.7%) and 2 (2.9%)] and eschscholtzxanthone (2.3%), along with zeaxanthin (72.6%), β-cryptoxanthin (7.3%), β-carotene (7.1%), and rubixanthin (2.1%). Carotenoids 1 and 2 were purified, and their structures were determined as (6E)-9′-apo-rohodoxanthinone and (6Z)-9′-apo-rhodoxanthinone, respectively, based on UV–vis, MS, 1H NMR, and 13C NMR spectral data. These novel compounds were shown to exhibit excellent singlet oxygen-quenching activity by the methylene blue-sensitized photooxidation system.

Activation of the transcriptional factor Nrf2 is a powerful strategy for protecting oxidative stress-related disease. To find novel compounds from mushrooms, the NAD(P)H:quinone oxidoreductase 1 (NQO1)-inducing activity of 43 ethyl-acetate extracts of mushrooms were examined using Hepa1c1c7 cells. In this screening, an extract of Mycoleptodonoides aitchisonii significantly induced NQO1. This extract also increased ARE reporter activity and inhibited H 2 O 2 -induced cell death in RAW264.7 cells. The induction of anti-oxidative and phase II enzymes was attenuated in peritoneal macrophages from Nrf2-deficient mice, so these activities were probably mediated by Nrf2 activation. Oral administration of the extract upregulated expression of these genes in the liver and small intestinal epithelium of mice. We purified 11 active compounds (including three novel ones), and dihydro-4-phenyl-2(3H)-furanone was the most effective NQO1-inducer. These results suggest that dietary intake of M. aitchisonii may contribute to the prevention of oxidative stress-related diseases by inducing anti-oxidative and phase II enzymes.

The aim of this study was to produce rare β-carotene-modified carotenoids possessing 2-O (-H or -glu) and/or 3-O (-H or -glu) functionalities in their β-ionone ring(s) using a recombinant Escherichia coli approach. This involved expressing seven carotenoid biosynthesis genes (crtE, crtB, crtI, crtY, crtZ, crtX and crtG). From the cells of the recombinant E. coli, caloxanthin (β,β-carotene-2,3,2',3'-tetrol)-3'-β-D-glucose, zeaxanthin (β,β-carotene-3,3'-diol) 3,3'-β-D-diglucoside, and nostoxanthin (β,β-carotene-2,3,3'-triol) (rare carotenoids) were isolated and identified. Caloxanthin 3'-β-D-glucose displayed potent (1)O(2) quenching activity (IC(50) 19 μM).

We performed combinational bioconversion of substituted naphthalenes with PhnA1A2A3A4 (an aromatic dihydroxylating dioxygenase from marine bacterium Cycloclasticus sp. strain A5) and prenyltransferase NphB (geranyltransferase from Streptomyces sp. strain CL190) or SCO7190 (dimethylallyltransferase from Streptomyces coelicolor A3(2)) to produce prenyl naphthalen-ols. Using 2-methylnaphthalene, 1-methoxynaphthalene, and 1-ethoxynaphthalene as the starting substrates, 10 novel prenyl naphthalen-ols were produced by combinational bioconversion. These novel prenyl naphthalen-ols each showed potent antioxidative activity against a rat brain homogenate model. 2-(2,3-Dihydroxyphenyl)-5,7-dihydroxy-chromen-4-one (2',3'-dihydroxychrysin) generated with another aromatic dihydroxylating dioxygenase and subsequent dehydrogenase was also geranylated at the C-5'-carbon by the action of NphB.

Tractable plasmids (pAC-Mv-based plasmids) for Escherichia coli were constructed, which carried a mevalonate-utilizing gene cluster, towards an efficient functional analysis of cytochromes P450 involved in sesquiterpene biosynthesis. They included genes coding for a series of redox partners that transfer the electrons from NAD(P)H to a P450 protein. The redox partners used were ferredoxin reductases (CamA and NsRED) and ferredoxins (CamB and NsFER), which are derived from Pseudomonas putida and cyanobacterium Nostoc sp. strain PCC 7120, respectively, as well as three higher-plant NADPH-P450 reductases, the Arabidopsis thaliana ATR2 and two corresponding enzymes derived from ginger (Zingiber officinale), named ZoRED1 and ZoRED2. We also constructed plasmids for functional analysis of two P450s, α-humulene-8-hydroxylase (CYP71BA1) from shampoo ginger (Zingiber zerumbet) and germacrene A hydroxylase (P450NS; CYP110C1) from Nostoc sp. PCC 7120, and co-transformed E. coli with each of the pAC-Mv-based plasmids. Production levels of 8-hydroxy-α-humulene with recombinant E. coli cells (for CYP71BA1) were 1.5- to 2.3-fold higher than that of a control strain without the mevalonate-pathway genes. Level of the P450NS product with the combination of NsRED and NsFER was 2.9-fold higher than that of the CamA and CamB. The predominant product of P450NS was identified as 1,2,3,5,6,7,8,8a-octahydro-6-isopropenyl-4,8a-dimethylnaphth-1-ol with NMR analyses.

The cytochrome P450 CYP153 family has been isolated from alkane-assimilating bacteria. CYP153 has been shown to mediate terminal hydroxylations of linear alkanes or alkyl aromatics. We here performed the biotransformation of various aromatic compounds by Escherichia coli cells that expressed the CYP153A13a (P450balk) gene, which was isolated from an alkane-degading marine bacterium Alcanivorax borkumensis . Aromatic compounds including a short alkyl moiety or methyl ether moiety, and phenolic compounds were converted to their respective hydroxylated products, whose structures were determined by HRMS and NMR analyses. The present study revealed that the catalytic function of CYP153A13a is multifunctional, i.e., it can hydroxylate not only the terminal of short alkyl groups that attached to aromatic rings but also the p -position of phenolic compounds substituted with a halogen or the acetyl group. CYP153A13a was also shown to demethylate methylether-including aromatic compounds.

While screening for antioxidative carotenoids from marine bacteria, we isolated and identified sarcinaxanthin and its glucosylated compounds (sarcinaxanthin monoglucoside and sarcinaxanthin diglucoside) from a moderately halophilic bacterium-Micrococcus yunnanensis strain AOY-1. In the singlet oxygen ((¹O₂) quenching model, the IC(50) values of the antioxidative activities of these carotenoids were as follows: sarcinaxanthin , 57 µM; sarcinaxanthin monoglucoside, 54 µM; and sarcinaxanthin diglucoside, 74 µM. In addition, the complete proton nuclear magnetic resonance (¹H NMR) assignments of sarcinaxanthin monoglucoside pentaacetate and sarcinaxanthin diglucoside octaacetate, and fast atom bombardment mass spectrometry/mass spectrometry (FAB-MS/MS) analyses of sarcinaxanthin and sarcinaxanthin monoglucoside are reported for the first time.

The elevation of such dicarbonyl compounds as glyoxal and the depletion of GSH occur simultaneously in diabetic patients. Enabling a nonenzymatic glycation reaction with GSH and glyoxal is therefore proposed. However, the reaction mechanism for GSH and glyoxal has not been precisely defined. We isolated in this study the major products obtained by the reaction of GSH and glyoxal under physiological conditions, and clarified the chemical structure of these compounds by MS and NMR analyses for the first time. We identified the major product after 24 h as N-[3-(2,5-dioxomorpholin-3-yl)propanoyl]cysteinylglycine, and the one after 30 min as N-glycoloyl-gamma-glutamylcysteinylglycine (the intermediate of the former compound). Our results suggest that GSH reacted with glyoxal at the alpha-NH(2) group of the glutamate residue, but not at the SH group of the cysteine residue.

Two vectors, pT7NScamAB and pRED, have been used for the functional expression of bacterial class I cytochrome P450 (P450) genes in Escherichia coli, which utilize putidaredoxin reductase (CamA) and putidaredoxin (CamB), and the reductase domain of a self-sufficient P450RhF respectively, for electron transfer from NAD(P)H to a P450 protein. We here compared the efficiency of bioconversion with the two vectors towards n-octane, cyclohexane, n-butylbenzene, and 2-n-butylbenzofuran using two well-characterized CYP153A genes, aciA and CYP153A13a (P450balk). As for n-octane bioconversion, aciA and pT7camAB was the best combination for the production of 1-octanol and 1,8-octanediol. As for the bioconversion of cyclohexane, n-butylbenzene and 2-n-butylbenzofuran, CYP153A13a with pRED achieved the most efficient bioconversion, as compared by conversion ratio per active CYP153A protein content. It was also found that 2-n-butylbenzofuran is biotransformed into 4-benzofuran-2-yl-butyric acid via 4-benzofuran-2-yl-butan-1-ol with E. coli cells expressing CYP153A.

Methyl Glucosyl-3,4-dehydro-apo-8′-lycopenoate, a Novel Antioxidative Glyco-C 30 -carotenoic Acid Produced by a Marine Bacterium Planococcus maritimus