Anti-Inflammatory Effects of an Extract from Pseudomonas aeruginosa and Its Purified Product 1-Hydroxyphenazine on RAW264.7 Cells.

The purpose of this study was to discuss the effects of an extract from the culture medium of Pseudomonas aeruginosa (P. aeruginosa) 2016NX1 (chloroform extract of P. aeruginosa, CEPA) and its purified product 1-hydroxyphenazine on RAW264.7 cell inflammation. Cell viability was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) method. TNF-α production was determined by an ELISA method. The effects of CEPA and its purified product 1-hydroxyphenazine on cell morphology were investigated using an inverted microscope. Quantitative real-time PCR was performed to determine mRNA expression levels. CEPA and 1-hydroxyphenazine had no obvious toxicity to cells when their concentrations were no more than 20 µg ml-1 and 5 µg ml-1, respectively. Both CEPA and 1-hydroxyphenazine suppressed the secretion of TNF-α and significantly reduced the mRNA expression levels of TNF-α, IL-1ß, and IL-6. Both CEPA and 1-hydroxyphenazine inhibited M1 cell polarization after lipopolysaccharide (LPS) stimulation. The results in this article lay a good foundation for the biopharmaceutical applications of CEPA and 1-hydroxyphenazine in the future. CEPA and 1-hydroxyphenazine had certain anti-inflammatory activity, and inhibited LPS-induced RAW264.7 cell inflammation. Our findings suggest that CEPA and 1-hydroxyphenazine are potential chemicals with anti-inflammatory activity.

Epidemiology of intussusception among children less than 2 years of age; findings from baseline surveillance before rotavirus vaccine introduction in Myanmar.

BACKGROUND: Rotavirus vaccine was planned to be introduced in the National Immunization Program of Myanmar in 2020. Reported potential association of a small increased risk of intussusception after rotavirus vaccination in some countries is a major safety concern and it is mandatory to collect baseline information before vaccine introduction. METHODS: Retrospective study reviewed medical records of intussusception cases for past 3 years (2015-2018) and prospective, active study was conducted from August 2018 to January 2020 at three tertiary children hospitals where pediatric surgical facility is present. Brighton Level 1 Criteria was used for confirmation of intussusception among children <2 years of age admitted to surgical wards. Demographic, clinical, diagnostic and treatment practices data were collected and descriptive data analysis was performed. RESULTS: A total of 697 (421 in retrospective and 276 in prospective) confirmed intussusception cases were identified. Majority of intussusception cases (550/697, 78.9%) were observed in the first year of life and most frequent between 5-7 months of age (292/697, 41.9%) with a peak at 6 months (114/697, 16.4%). The most common clinical presentations were vomiting and bloody diarrhea accounting 82.1% and 77.5% respectively. Regarding diagnosis and treatment, 458/697 (65.7%) required surgical intervention either manual reduction or intestinal resection and 34.4% by either air or barium enema. Overall mortality was 0.7% (5/697) and four out of five children died needed intestinal resection. Late arrival to hospital (>3days after onset) is significantly associated with requirement of surgery (61/85, 71.8%), which in turn is significantly associated with longer hospital stay (296/452, 65.5%) (p < 0.05). CONCLUSIONS: Intussusception occurrence is most frequent between 5-7 months age group which is old enough to be vaccinated under the schedule that has now been introduced in Myanmar. More than half of the cases were treated by surgery and late arrival to hospital enhances requirement of surgery and poor outcome. Findings of this baseline surveillance provide important facts for public health officials in balancing risks and benefits of rotavirus vaccine introduction, defining targeted age and dosage scheduling and facilitate monitoring system in post-vaccination.

Production of optically pure (-)-borneol by Pseudomonas monteilii TCU-CK1 and characterization of borneol dehydrogenase involved.

(-)-Borneol is a bicyclic plant secondary metabolite. Optically pure (-)-borneol can only be obtained from plants, and demand exceeds supply in China. In contrast, chemically synthesized borneol contains four different stereoisomers. A strain of Pseudomonas monteilii TCU-CK1, isolated in Hualien, Taiwan, can accumulate (-)-borneol in growth culture and selectively degrades the other three isomers when chemically synthesized borneol is used as sole carbon source. This (-)-borneol production method can be scaled-up for production of large quantities in the future. More importantly, laborious plant cultivation and harvest is no longer required. The main enzyme that appears in this degradation pathway, borneol dehydrogenase (BDH), and the genome sequence of TCU-CK1 are reported. The kcat/Km values of TCU-CK1 BDH on (+)- and (-)-borneol are 538.4 ± 38.4 and 17.7 ± 1.1 (s-1 mM-1), respectively. About ∼30 fold difference in the kcat/Km value between (+)-borneol and (-)-borneol was observed, in good agreement with the fact that TCU-CK1 prefers to degrade (+)-borneol, rather than (-)-borneol. A BDH isozyme was identified in a strain in which the primary BDH gene had been knocked out. (-)-Camphor can work as an inhibitor of BDH with a Ki of 1.03 ± 0.11 mM at pH 7.0, leading to the accumulation of (-)-borneol in culture. (Patent pending).

Extreme neonatal hyperbilirubinaemia in refugee and migrant populations: retrospective cohort.

OBJECTIVE: To describe neonatal survival and long-term neurological outcome in neonatal hyperbilirubinaemia (NH) with extreme serum bilirubin (SBR) values. DESIGN: Retrospective chart review, a one-off neurodevelopmental evaluation. SETTING: Special care baby unit in a refugee camp and clinics for migrant populations at the Thailand-Myanmar border with phototherapy facilities but limited access to exchange transfusion (ET). PATIENTS: Neonates ≥28 weeks of gestational age with extreme SBR values and/or acute neurological symptoms, neurodevelopment evaluation conducted at 23-97 months of age. MAIN OUTCOME MEASURES: Neonatal mortality rate, prevalence of acute bilirubin encephalopathy (ABE) signs, prevalence of delayed development scores based on the Griffiths Mental Development Scale (GMDS). RESULTS: From 2009 to 2014, 1946 neonates were diagnosed with jaundice; 129 (6.6%) had extreme SBR values during NH (extreme NH). In this group, the median peak SBR was 430 (IQR 371-487) µmol/L and the prevalence of ABE was 28.2%. Extreme NH-related mortality was 10.9% (14/129). Median percentile GMDS general score of 37 survivors of extreme NH was poor: 11 (2-42). 'Performance', 'practical reasoning' and 'hearing and language' domains were most affected. Four (10.8%) extreme NH survivors had normal development scores (≥50th centile). Two (5.4%) developed the most severe form of kernicterus spectrum disorders. CONCLUSION: In this limited-resource setting, poor neonatal survival and neurodevelopmental outcomes, after extreme NH, were high. Early identification and adequate treatment of NH where ET is not readily available are key to minimising the risk of extreme SBR values or neurological symptoms.

Clinical Impact of the Introduction of Pediatric Intussusception Air Enema Reduction Technology in a Low- to Middle-Income Country Using Low-Cost Simulation-Based Medical Education.

INTRODUCTION: Pediatric intussusception is a common cause of bowel obstruction in infants. Air enema (AE) reduction is routine first-line management in many countries; however, there is a high rate of operative intervention in low- and middle-income countries. The aims of the study were to use simulation-based medical education with an intussusception simulator to introduce AE reduction to Myanmar and to assess its effect on provider behaviors and the resulting clinical care. METHODS: Clinical evaluation was conducted by comparing clinical outcomes data for children with intussusception 12 months before implementation with that from 12 months subsequent to implementation. These included the following: AE success rates, recurrence rates, length of stay, intestinal resection, and operative intervention rates. An educational workshop was developed that used a low-cost mannequin to facilitate practice at the reduction of intussusception using AE. Curriculum evaluation was performed through 5-point rating scale self-assessment in several domains. Data analysis was performed with Mann-Whitney U test, Student t test, or Wilcoxon signed-ranks test as appropriate; a P value of less than 0.05 was considered to be significant. RESULTS: After implementation, there was a significant reduction in the overall operative intervention rates [82.5% (85/103) vs. 58.7% (44/75), P = 0.006]. Intestinal resection rates increased [15.3% (13/85) vs. 35.9% (14/39), P = 0.02]. The success rate with attempted AE reduction was 94.4% (34/36), with a recurrence rate of 5.6% (2/36). The simulation-based medical education workshop was completed by 25 local participants. There was a significant difference in the confidence of performing (1.9 vs. 3.6, P ≤ 0.0001) or assisting (2.8 vs. 3.7, P = 0.018) an AE reduction before and after the workshop. CONCLUSIONS: Simulation-based educational techniques can be successfully applied in a low- and middle-income country to facilitate the safe introduction of new equipment and techniques with significant beneficial impact on provider behaviors and the resulting clinical care.

Xanthone suppresses allergic contact dermatitis in vitro and in vivo.

Xanthone is a phenolic compound found in a few higher plant families; it has a variety of biological activities, including antioxidant, anti-inflammatory, and anticancer properties. However, the molecular and cellular mechanisms underlying the activity of xanthone in allergic contact dermatitis (ACD) remain to be explored. Therefore, this study aimed to investigate the regulatory effects of xanthone in ACD in human keratinocytes (HaCaT cell), and human mast cell line (HMC-1 cell) in vitro and in an experimental murine model. The results demonstrated that treatment with xanthone reduced the production of pro-inflammatory cytokines and chemokines including interleukin (IL)-1ß, IL-6, IL-8, and expression of chemokines thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC) in tumor necrosis factor (TNF)-α and interferon (IFN)-γ-stimulated HaCaT cells. Xanthone also suppressed the production of pro-inflammatory cytokines, chemokines, and allergic mediators in phorbol myristate acetate/A23187 calcium ionophore (PMACI)-stimulated HMC-1 cells. Xanthone significantly suppressed the phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) and activation of caspase-1 signaling pathway in vitro model. Additionally, xanthone administration alleviated 2,4-dinitrofluorobenzene (DNFB)-induced atopic dermatitis like-skin lesion by reducing the serum levels of immunoglobulin E (IgE), histamine, and pro-inflammatory cytokines and suppressing MAPKs phosphorylation. Xanthone administration also inhibited mortality due to compound 48/80-induced anaphylactic shock and suppressed the passive cutaneous anaphylaxis (PCA) reaction mediated by IgE. Collectively, these results suggest that xanthone has a potential for use in the treatment of allergic inflammatory diseases.

Characterization of bacterial endophytes from Myanmar medicinal plants for antimicrobial activity against human and plant pathogens

This research aimed to investigate the antagonistic activity of the bacterial endophytes from Myanmar medicinal plants. Thirty-one bacterial isolates were isolated from Myanmar medicinal plants: Tinospora cordifolia (Wild.) Miers., Catharanthus roseus G. Don., Tectona hamiltoniana Wall. and Boscia variabilis Collett & Hemsl. (Capparaceae). Dual culture and agar well diffusion methods were used for antimicrobial assay. One endophyte from Catharanthus roseus and two bacterial isolates from Boscia variabilis Collett & Hemsl. (Capparaceae) had not only the antibacterial activity towards the human pathogenic bacteria but also the antifungal activity against the plant pathogenic fungi. From 16S rRNA sequencing, one strain from Catharanthus roseus G. Don. was Bacillus amyloliquefaciens DSM7 and two antagonistic strains from Boscia variabilis Collett & Hemsl. (Capparaceae) were Bacillus subtilis subsp. subtilis str. 168 and Bacillus amyloliquefaciens DSM7, respectively. The best medium for the maximum production of the bioactive compounds was Bacillus medium supplemented with the 4% of starch and 0.3% of peptone for B. amyloliquefaciens DSM7 and B. subtilis subsp. subtilis str. 168 showed the maximum antimicrobial compounds production when it was incubated in the medium amended with 3% of starch and 2% of peptone. The optimum conditions for the the maximum production of the antimicrobial compound were the medium pH of 6 at 35 ºC after two days of incubation for B. amyloliquefaciens DSM 7 and B. subtitlis subsp. subtilis str. 168 secreted the maximum concentration of the bioactive compounds at pH 7.5 and 35˚C on second day incubation period. In conclusion, the isolated endophytic bacteria showed the strong antimicrobial activity towards the pathogenic microbes and they could be used in medicine and agriculture as well.

Cyanidin 3-Rutinoside, an Anthocyanin Pigment of Schisandra chinensis Baill, Inhibits Allergic Inflammation.

The occurrence of allergy-mediated inflammatory diseases such as asthma and atopic dermatitis have increased, but comprehensive treatment remains difficult. Previous studies have shown that Schisandra chinensis Baill has antioxidant, antidiabetic, and antitumorigenic effects. Cyanidin 3-rutinoside (CR) is the major anthocyanin pigment of S. chinensis. However, the biological effects of CR have been rarely studied to date. Therefore, the aim of this study was to investigate the regulatory effects of CR on phorbol-12-myristate-13-acetate (PMA)/A23187-induced allergic inflammation in vitro. CR inhibited the secretion of inflammatory cytokines such as interleukin-6 and tumor necrosis factor-α, and it also suppressed the phosphorylation of nuclear factor-kappa B. These results show that CR ameliorated PMA/A23187-induced allergic inflammation via the suppression of inflammatory cytokines in HMC-1 cells. Therefore, CR has potential as a therapeutic agent for allergic diseases.

Resolution of isoborneol and its isomers by GC/MS to identify "synthetic" and "semi-synthetic" borneol products.

Borneol is a plant terpene commonly used in traditional Chinese medicine. Optically pure (+)-borneol and (-)-borneol can be obtained by extraction from the plants Dipterocarpaceae and Blumea balsamifera, respectively. "Synthetic borneol" is obtained from the reduction of (±)-camphor to lead to four different stereoisomers: (+)-isoborneol, (-)-isoborneol, (+)-borneol, and (-)-borneol. In contrast, "semi-synthetic borneol" is produced from the reduction of natural camphor, (+)-camphor, to afford two isomers: (-)-isoborneol and (+)-borneol. We established a convenient method to identify them by treating the four stereoisomers with two chiral reagents, (R)-(+)-α-methoxy-α-trifluoromethylphenylacetyl chloride ((R)-(+)-MTPA-Cl) and (1S)-(-)- camphanic chloride. The resulting derivatives from the above mentioned method were analyzed by gas chromatography. The enantiomers of (+)- and (-)-isoborneol were successfully separated from (+)- and (-)-borneol isomers in this study to make this a useful method in the identification of "synthetic" and "semi-synthetic" borneols. Furthermore, we also examined five different commercial borneols. During this course, a novel and unprecedented partial epimerization from isoborneol-camphanic ester to borneol-camphanic ester was observed. However, this phenomenon did not occur in isoborneol-MTPA esters epimerization to borneol-MTPA case under the same conditions. The DFT calculation of activation energies for both reactions was in a good agreement with the results obtained from GC analysis.

Borneol Dehydrogenase from Pseudomonas sp. Strain TCU-HL1 Catalyzes the Oxidation of (+)-Borneol and Its Isomers to Camphor.

Most plant-produced monoterpenes can be degraded by soil microorganisms. Borneol is a plant terpene that is widely used in traditional Chinese medicine. Neither microbial borneol dehydrogenase (BDH) nor a microbial borneol degradation pathway has been reported previously. One borneol-degrading strain, Pseudomonas sp. strain TCU-HL1, was isolated by our group. Its genome was sequenced and annotated. The genome of TCU-HL1 consists of a 6.2-Mbp circular chromosome and one circular plasmid, pTHL1 (12.6 kbp). Our results suggest that borneol is first converted into camphor by BDH in TCU-HL1 and is further decomposed through a camphor degradation pathway. The recombinant BDH was produced in the form of inclusion bodies. The apparent Km values of refolded recombinant BDH for (+)-borneol and (-)-borneol were 0.20 ± 0.01 and 0.16 ± 0.01 mM, respectively, and the kcat values for (+)-borneol and (-)-borneol were 0.75 ± 0.01 and 0.53 ± 0.01 s-1, respectively. Two plant BDH genes have been reported previously. The kcat and kcat/Km values of lavender BDH are about 1,800-fold and 500-fold lower, respectively, than those of TCU-HL1 BDH. IMPORTANCE: The degradation of borneol in a soil microorganism through a camphor degradation pathway is reported in this study. We also report a microbial borneol dehydrogenase. The kcat and kcat/Km values of lavender BDH are about 1,800-fold and 500-fold lower, respectively, than those of TCU-HL1 BDH. The indigenous borneol- and camphor-degrading strain isolated, Pseudomonas sp. strain TCU-HL1, reminds us of the time 100 years ago when Taiwan was the major producer of natural camphor in the world.

Predictive Factors for BRCA1 and BRCA2 Genetic Testing in an Asian Clinic-Based Population.

PURPOSE: The National Comprehensive Cancer Network (NCCN) has proposed guidelines for the genetic testing of the BRCA1 and BRCA2 genes, based on studies in western populations. This current study assessed potential predictive factors for BRCA mutation probability, in an Asian population. METHODS: A total of 359 breast cancer patients, who presented with either a family history (FH) of breast and/or ovarian cancer or early onset breast cancer, were accrued at the National Cancer Center Singapore (NCCS). The relationships between clinico-pathological features and mutational status were calculated using the Chi-squared test and binary logistic regression analysis. RESULTS: Of 359 patients, 45 (12.5%) had deleterious or damaging missense mutations in BRCA1 and/or BRCA2. BRCA1 mutations were more likely to be found in ER-negative than ER-positive breast cancer patients (P=0.01). Moreover, ER-negative patients with BRCA mutations were diagnosed at an earlier age (40 vs. 48 years, P=0.008). Similarly, triple-negative breast cancer (TNBC) patients were more likely to have BRCA1 mutations (P=0.001) and that these patients were diagnosed at a relatively younger age than non-TNBC patients (38 vs. 46 years, P=0.028). Our analysis has confirmed that ER-negative status, TNBC status and a FH of hereditary breast and ovarian cancer (HBOC) are strong factors predicting the likelihood of having BRCA mutations. CONCLUSIONS: Our study provides evidence that TNBC or ER-negative patients may benefit from BRCA genetic testing, particularly younger patients (<40 years) or those with a strong FH of HBOC, in Asian patients.

Accumulation of phenylpropanoids and correlated gene expression in hairy roots of tartary buckwheat under light and dark conditions.

Differential expression patterns of flavonoid biosynthetic pathway genes in the hairy roots of tartary buckwheat cultivars "Hokkai T8" and "Hokkai T10" were studied over a time course of the light-dark cycle. The Agrobacterium rhizogenes-mediated transformation system was applied for inducing hairy roots. Further, a total of six phenolic compounds and two anthocyanins were analyzed in the hairy roots which were exposed to both light and dark conditions, and their amounts were estimated by HPLC. The gene expression levels peaked on day 5 of culture during the time course of both dark and light conditions. Notably, FtPAL, Ft4CL, FtC4H, FtCHI, FtF3H, FtF3'H-1, and FtFLS-1 were more highly expressed in Hokkai T10 than in Hokkai T8 under dark conditions, among which FtPAL and FtCHI were found to be significantly upregulated, except on day 20 of culture. Significantly higher levels of phenolic compound, rutin, along with two anthocyanins were detected in the hairy roots of Hokkai T10 under both conditions. Furthermore, among all the phenolic compounds detected, the amount of rutin in Hokkai T10 hairy roots was found to be ∼5-fold (59,01 mg/g dry weight) higher than that in the control (12.45 mg/g dry weight) at the respective time periods under light and dark conditions.

Accumulation of astragalosides and related gene expression in different organs of Astragalus membranaceus Bge. var mongholicus (Bge.).

Astragalus membranaceus is one of the most important traditional Korean and Chinese medicinal herbs because it contains triterpenoid saponins (astragaloside I, II, III, and IV), which have beneficial and pharmacological effects on health. In this study, we analyzed 10 mevalonate pathway genes that are involved in astragaloside biosynthesis using the Illumina/Solexa HiSeq2000 platform. We determined the expression levels of the 10 genes using quantitative real-time PCR, and analyzed the accumulation of astragalosides in different organs using high-performance liquid chromatography. Genes related to the mevalonate pathway were expressed in different levels in different organs. Almost all genes showed high transcript levels in the stem and leaf, with the lowest transcript levels being recorded in the root. In contrast, most astragalosides accumulated in the root. In particular, the astragaloside IV content was distributed in the following order: root (0.58 mg/g DW) > flower (0.27 mg/g DW) > stem (0.23 mg/g DW) > leaf (0.04 mg/g DW). In the root, astragaloside II exhibited the highest content (2.09 mg/g DW) compared to astragaloside I, III, and IV. Notably, gene expression did not follow the same pattern as astragaloside accumulation. We suggest carefully that astragalosides are synthesized in the leaves and stem and then translocated to the root. This study contributes towards improving our understanding of astragaloside biosynthesis in A. membranaceus.

Accumulation of flavonoids and related gene expressions in different organs of Astragalus membranaceus Bge.

Astragalus membranaceus is one of the important medicinal plant in China and Korea. It is used to increase metabolism and digestion, enhance the immune system, and promote the healing of wounds and injuries. In the present study, we used quantitative real-time PCR to investigate the expression of genes related to the biosynthesis of flavonoids, in addition to high-performance liquid chromatography to assess calycosin and calycosin-7-O-ß-D-glucoside accumulation, in the different plant organs of A. membranaceus. The transcript levels of all genes (AmPAL, AmC4H, Am4CL, AmCHS, AmCHR, AmCHI, AmIFS, AmI3'H, and AmUCGT) involved in calycosin and calycosin-7-O-ß-D-glucoside biosynthesis were the highest in the flower. Calycosin content was ordered as follows: leaf (145.56 µg/g dry weight [DW]) > stem (18.3 µg/g DW) > root (1.64 µg/g DW) > flower (0.09 µg/g DW), whereas calycosin-7-O-ß-D-glucoside content was ordered as follows: root (4.88 µg/g DW) > stem (3.86 µg/g DW) > leaf (2.0 µg/g DW) > flower (not detected). All genes exhibited the highest transcription levels in the flower, whereas calycosin and its glycoside content were the highest in the leaf and root, respectively. Our results indicate that the enhancement of calycosin-7-O-ß-D-glucoside in the roots may originate from high calycosin accumulation in the stem and leaf. Thus, the mechanisms regulating calycosin and calycosin-7-O-ß-D-glucoside content differ in the different organs of A. membranaceus. The results are expected to provide baseline information from which the mechanism of flavonoid biosynthesis in the different organs of A. membranaceus may be elucidated.

Effects of light-emitting diodes on expression of phenylpropanoid biosynthetic genes and accumulation of phenylpropanoids in Fagopyrum tataricum sprouts.

Buckwheat sprouts are a popular food item in many countries. The effects of light-emitting diodes (LEDs) on sprout growth and development, changes in mRNA transcription, and accumulation of phenylpropanoid compounds were studied in tartary buckwheat 'Hokkai T8' sprouts. The highest transcript levels were observed after 2 days of LED exposure for all genes, especially FtPAL and FtF3'H, which showed higher expression in sprouts grown under blue and white light than in those grown under red light. Catechin content in sprouts grown under red light increased dramatically throughout the 10 day time course. Maximum rutin content (43.37 mg/g dry weight (DW)) was observed in sprouts at 4 days after exposure (DAE) to blue light. Similarly, the highest cyanidin 3-O-rutinoside content (0.85 mg/g DW) was detected at 10 DAE to blue light. On the basis of these results, blue LED light is recommended as a light source for enhancing the content of phenolic compounds in tartary buckwheat sprouts.

Transcripts of anthocyanidin reductase and leucoanthocyanidin reductase and measurement of catechin and epicatechin in tartary buckwheat.

Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) play an important role in the monomeric units biosynthesis of proanthocyanidins (PAs) such as catechin and epicatechin in several plants. The aim of this study was to clone ANR and LAR genes involved in PAs biosynthesis and examine the expression of these two genes in different organs under different growth conditions in two tartary buckwheat cultivars, Hokkai T8 and T10. Gene expression was carried out by quantitative real-time RT-PCR, and catechin and epicatechin content was analyzed by high performance liquid chromatography. The expression pattern of ANR and LAR did not match the accumulation pattern of PAs in different organs of two cultivars. Epicatechin content was the highest in the flowers of both cultivars and it was affected by light in only Hokkai T8 sprouts. ANR and LAR levels in tartary buckwheat might be regulated by different mechanisms for catechin and epicatechin biosynthesis under light and dark conditions.

Effects of white, blue, and red light-emitting diodes on carotenoid biosynthetic gene expression levels and carotenoid accumulation in sprouts of tartary buckwheat (Fagopyrum tataricum Gaertn.).

In this study, the optimum wavelengths of light required for carotenoid biosynthesis were determined by investigating the expression levels of carotenoid biosynthetic genes and carotenoid accumulation in sprouts of tartary buckwheat (Fagopyrum tataricum Gaertn.) exposed to white, blue, and red light-emitting diodes (LEDs). Most carotenoid biosynthetic genes showed higher expression in sprouts irradiated with white light at 8 days after sowing than in those irradiated with blue and red lights. The dominant carotenoids in tartary buckwheat sprouts were lutein and ß-carotene. The richest accumulation of total carotenoids was observed in sprouts grown under white light (1282.63 µg g(-1) dry weight), which was relatively higher than that in sprouts grown under blue and red lights (940.86 and 985.54 µg g(-1), respectively). This study might establish an effective strategy for maximizing the production of carotenoids and other important secondary metabolites in tartary buckwheat sprouts by using LED technology.

Metabolomic analysis and differential expression of anthocyanin biosynthetic genes in white- and red-flowered buckwheat cultivars (Fagopyrum esculentum).

Red-flowered buckwheat ( Fagopyrum esculentum ) is used in the production of tea, juice, and alcohols after the detoxification of fagopyrin. In order to investigate the metabolomics and regulatory of anthocyanin production in red-flowered (Gan-Chao) and white-flowered (Tanno) buckwheat cultivars, quantitative real-time RT-PCR (qRT-PCR), gas chromatography time-of-flight mass spectrometry (GC-TOFMS), and high performance liquid chromatography (HPLC) were conducted. The transcriptions of FePAL, FeC4H, Fe4CL1, FeF3H, FeANS, and FeDFR increased gradually from flowering stage 1 and reached their highest peaks at flowering stage 3 in Gan-Chao flower. In total 44 metabolites, 18 amino acids, 15 organic acids, 7 sugars, 3 sugar alcohols, and 1 amine were detected in Gan-Chao flowers. Two anthocyanins, cyanidin 3-O-glucoside and cyanidin 3-O-rutinoside, were identified in Gan-Chao cultivar. The first component of the partial least-squares to latent structures-discriminate analysis (PLS-DA) indicated that high amounts of phenolic, shikimic, and pyruvic acids were present in Gan-Chao. We suggest that transcriptions of genes involved in anthocyanin biosynthesis, anthocyanin contents, and metabolites have correlation in the red-flowered buckwheat Gan-Chao flowers. Our results may be helpful to understand anthocyanin biosynthesis in red-flowered buckwheat.

Molecular characterisation and the light-dark regulation of carotenoid biosynthesis in sprouts of tartary buckwheat (Fagopyrum tataricum Gaertn.).

Seven partial-length cDNAs and 1 full-length cDNA that were involved in carotenoid biosynthesis and 2 partial-length cDNAs that encoded carotenoid cleavage dioxygenases were first isolated and characterised in 2 tartary buckwheat cultivars (Fagopyrum tataricum Gaertn.), Hokkai T8 and Hokkai T10. They were constitutively expressed at high levels in the leaves and flowers, where carotenoids are mostly distributed. During the seed development of tartary buckwheat, an inverse correlation between transcription level of carotenoid cleavage dioxygenase and carotenoid content was observed. The light-grown sprouts exhibited higher levels of expression of carotenoid biosynthetic genes in T10 and carotenoid content in both T8 and T10 compared to the dark-grown sprouts. The predominant carotenoids in tartary buckwheat were lutein and ß-carotene, and very abundant amounts of these carotenoids were found in light-grown sprouts. This study might broaden our understanding of the molecular mechanisms involved in carotenoid biosynthesis and indicates targets for increasing the production of carotenoids in tartary buckwheat.

Metabolomic analysis and phenylpropanoid biosynthesis in hairy root culture of tartary buckwheat cultivars.

Buckwheat, Fagopyrum tataricum Gaertn., is an important medicinal plant, which contains several phenolic compounds, including one of the highest content of rutin, a phenolic compound with anti-inflammatory properties. An experiment was conducted to investigate the level of expression of various genes in the phenylpropanoid biosynthetic pathway to analyze in vitro production of anthocyanin and phenolic compounds from hairy root cultures derived from 2 cultivars of tartary buckwheat (Hokkai T8 and T10). A total of 47 metabolites were identified by gas chromatography-time-of-flight mass spectrometry (GC-TOFMS) and subjected to principal component analysis (PCA) in order to fully distinguish between Hokkai T8 and T10 hairy roots. The expression levels of phenylpropanoid biosynthetic pathway genes, through qRT-PCR, showed higher expression for almost all the genes in T10 than T8 hairy root except for FtF3'H-2 and FtFLS-2. Rutin, quercetin, gallic acid, caffeic acid, ferulic acid, 4-hydroxybenzoic acid, and 2 anthocyanin compounds were identified in Hokkai T8 and T10 hairy roots. The concentration of rutin and anthocyanin in Hokkai T10 hairy roots of tartary buckwheat was several-fold higher compared with that obtained from Hokkai T8 hairy root. This study provides useful information on the molecular and physiological dynamic processes that are correlated with phenylpropanoid biosynthetic gene expression and phenolic compound content in F. tataricum species.