Profiling of volatile and non-phenolic metabolites-Amino acids, organic acids, and sugars of green tea extracts obtained by different extraction techniques.

Volatile compounds and non-phenolic metabolites (amino acids, organic acids, and sugars) of aqueous green tea extracts obtained by ultrasonic extraction (UE), agitation extraction (AE), hot water extraction (HWE), and conventional extraction (CE) were determined using SPME-GC-MS and HPLC, respectively. Significantly higher (P < 0.05) yields of volatiles and non-phenolic metabolites were obtained via UE and AE than via HWE and CE. UE, AE, HWE, and CE released 212, 201, 103, and 65 volatiles, respectively. Sum total of amino acid and organic acid in extracts was 54.57, 54.35, 27.11, and 12.67 (mg/100 g), and 5.96, 6.19, 3.81, and 1.68 (mg/100 g) for UE, AE, HWE, and CE, respectively. Volatiles except nitrogen-containing compounds had higher positive correlations with l-theanine, sucrose, malic acid, and catechins yields. Findings of the current study suggest that an efficient extraction technique may significantly increase volatile and non-phenolic metabolite yields in aqueous green tea extract.

Carbon stable isotopic compositions of citric acid and malic acid in Japanese apricot liqueur decrease as the fruit ripens.

The carbon stable isotopic composition (δ13C) is often analyzed to quantify the addition of acidulants to Japanese apricot liqueur, but little is known about the variation in the δ13C values of the main organic acids arising from differences in the ripeness of Japanese apricots. We show that in Japanese apricot liqueur prepared using fruits at different stages of ripeness, the δ13C values of citric acid and malic acid ranged from -25.1‰ to -23.7‰ and from -22.3‰ to -19.7‰, respectively, and the δ13C values decreased as the fruit ripened. The average δ13C value of citric acid from liqueurs was 0.7‰ higher than that from fresh fruits, whereas the δ13C values of malic acid showed no isotope discrimination. The variation in δ13C values of the main organic acids in Japanese apricot liqueurs will help detect acidulant addition and control authenticity.

p-Coumaroyl Malate Derivatives of the Pandanus amaryllifolius Leaf and Their Isomerization.

A novel p-coumaroyl dimethyl malate (1) was isolated from the Pandanus amaryllifolius leaf in addition to three known analogs of p-coumaroyl dimethyl malate (2-4), and their structures were elucidated by analysis of the spectroscopic data. The p-coumaroyl malate derivatives were isolated as a mixture of E and Z isomers. To determine the cause of isomerization, the p-coumaroyl malate isolated in this study was synthesized. We concluded that the Z isomer might be an artifact generated from the E isomer through purification steps.

1H NMR Metabolic Profiling of Earthworm (Eisenia fetida) Coelomic Fluid, Coelomocytes, and Tissue: Identification of a New Metabolite-Malylglutamate.

Earthworm metabolism is recognized as a useful tool for monitoring environmental insults and measuring ecotoxicity, yet extensive earthworm metabolic profiling using 1H nuclear magnetic resonance (NMR) spectroscopy has been limited in scope. This study aims to expand the embedded metabolic material in earthworm coelomic fluid, coelomocytes, and tissue to aid systems toxicology research. Fifty-nine metabolites within Eisenia fetida were identified, with 47 detected in coelomic fluid, 41 in coelomocytes, and 54 in whole-worm samples and tissue extracts. The newly detected but known metabolites 2-aminobutyrate, nicotinurate, Nδ,Nδ,Nδ-trimethylornithine, and trigonelline are reported along with a novel compound, malylglutamate, elucidated using 2D NMR and high-resolution MS/MS. We postulate that malylglutamate acts as a glutamate/malate store, chelator, and anionic osmolyte and helps to provide electrolyte balance.

Phenolic compounds from Bletilla striata.

Two new malic acid derivatives, namely eucomic acid 1-methyl ester (2) and 6'''-acetylmilitaline (7), together with ten known compounds (1, 3-6, 8-12), were isolated from the dry tubers of Bletilla striata (Thunb.) Reichb. F., a perennial traditional Chinese medicinal herb, which was used for the treatment of pneumonophthisis, pneumonorrhagia, tuberculosis, and hemorrhage of the stomach or lung. Their structures were elucidated by spectroscopic analyses, including 1D-, 2D-NMR, and HR-ESI-MS.

Economic co-production of poly(malic acid) and pullulan from Jerusalem artichoke tuber by Aureobasidium pullulans HA-4D.

BACKGROUND: poly(L-malic acid) (PMA) is a water-soluble polyester with many attractive properties in medicine and food industries, but the high cost of PMA fermentation has restricted its further application for large-scale production. To overcome this problem, PMA production from Jerusalem artichoke tubers was successfully performed. Additionally, a valuable exopolysaccharide, pullulan, was co-produced with PMA by Aureobasidum pullulans HA-4D. RESULTS: The Jerusalem artichoke medium for PMA and pullulan co-production contained only 100 g/L hydrolysate sugar, 30 g/L CaCO3 and 1 g/L NaNO3. Compared with the glucose medium, the Jerusalem artichoke medium resulted in a higher PMA concentration (114.4 g/L) and a lower pullulan concentration (14.3 g/L) in a 5 L bioreactor. Meanwhile, the activity of pyruvate carboxylase and malate dehydrogenas was significantly increased, while the activity of α-phosphoglucose mutase, UDP-glucose pyrophosphorylase and glucosyltransferase was not affected. To assay the economic-feasibility, large-scale production in a 1 t fermentor was performed, yielding 117.5 g/L PMA and 15.2 g/L pullulan. CONCLUSIONS: In this study, an economical co-production system for PMA and pullulan from Jerusalem artichoke was developed. The medium for PMA and pullulan co-production was significantly simplified when Jerusalem artichoke tubers were used. With the simplified medium, PMA production was obviously stimulated, which would be associated with the improved activity of pyruvate carboxylase and malate dehydrogenas.

Method for the isolation of citric acid and malic acid in Japanese apricot liqueur for carbon stable isotope analysis.

A method for detecting the undeclared addition of acidic ingredients is required to control the authenticity of Japanese apricot liqueur. We developed an analytical procedure that minimizes carbon isotope discrimination for measurement of the δ(13)C values of citric and malic acid isolated from Japanese apricot liqueur. Our results demonstrated that freeze-drying is preferable to nitrogen spray-drying, because it does not significantly affect the δ(13)C values of citric acid and results in smaller isotope discrimination for malic acid. Both 0.1% formic acid and 0.2% phosphoric acid are acceptable HPLC mobile phases for the isolation of citric and malic acid, although the δ(13)C values of malic acid exhibited relatively large variation compared with citric acid following isolation using either mobile phase. The developed procedure allows precise δ(13)C measurements of citric and malic acid isolated from Japanese apricot liqueur.

Isolation and characterization of two new 2-isobutylmalates from Bletilla striata.

The present study isolated 17 compounds from the tubers of Bletilla striata (Orchidaceae), using various chromatographic techniques. Their structures were identified based on their physical-chemical properties and spectroscopic analyses. Among them, two new 2-isobutylmalates, named bletimalates A (1) and B (2), together with other fifteen known compounds (3-17), were isolated and identified. Additionally, compounds 3, 4, and 8 were isolated from this plant for the first time.

Production of citramalate by metabolically engineered Escherichia coli.

Citramalic acid (citramalate) is a five carbon hydroxy-dicarboxylic acid and potential precursor for the production of methacrylic acid from renewable resources. We examined citramalate production in Escherichia coli expressing the citramalate synthase gene cimA. Although, knockouts in ldhA coding lactate dehydrogenase and glcB/aceB coding malate synthase did not benefit citramalate accumulation, knockouts in gltA coding citrate synthase, and ackA coding acetate kinase significantly increased citramalate accumulation compared to the control strain. A fed-batch process in a controlled fermenter using a glucose feed resulted in 46.5 g/L citramalate in 132 h with a yield of 0.63 g/g, over 75% of the theoretical maximum yield from glucose of 0.82 g/g. Biotechnol. Bioeng. 2016;113: 2670-2675. © 2016 Wiley Periodicals, Inc.

Purification of vandaterosides from Vanda teres (Orchidaceae) by stepwise gradient centrifugal partition chromatography.

Vandaterosides are polar glucosyloxybenzyl eucomate derivatives found in Vanda teres (Orchidaceae), which display biological activities that slow the skin ageing process. In order to obtain larger quantities to allow us to go further in the bioassays, the hydroalcoholic extract of aerial parts (leaves and stems) of V. teres were fractionated by centrifugal partition chromatography, combining isocratic, gradient, and dual elution modes. The first fractionation was performed on the extract maintained in the stationary phase as water saturated in butanol, while increasing the polarity of the mobile phase by changing the proportions of ethyl acetate/1-butanol/water, in order to obtain two enriched fractions. Vandateroside I was then purified by isocratic mode with ethyl acetate/ethanol/water (46:14:40), while vandateroside II was obtained by combining isocratic elution with ethyl acetate/isopropanol/water (30:20:50) followed by a multiple dual mode with ethyl acetate/ethanol/water (46:14:40). In this manner, hundreds of milligrams of vandateroside I and II were recovered from 10 g of V. teres extract.

[Studies on glucosyloxybenzyl 2-isobutylmalates of Pleione bulbocodioides].

Ten glucosyloxybenzyl 2-isobutylmalates and one benzyl alcohol glycoside were isolated from the dry tuber of Pleione bulbocodioides, which is a specie of Orchidaceae family and its dry tuber is one of the main sources of traditional Chinese medicine "shanci-gu", by a combination of various column chromatographic methods, including ODS, macroporous adsorbent resin, Sepheadex LH-20, and preparative HPLC. Their structures were identified on the basis of chemical evidences and spectroscopic analysis asloroglossin (1), grammatophylloside A (2), cronupapine (3), (-)-(2R, 3S)-1-(4-ß-D-glucopyranosyloxybenzyl)-4-methyl-2-isobutyltartrate (4), vandateroside II (5), grammatophylloside B (6), bis [4-(ß-D-glucopyranosyloxy) -benzyl] (S) -2-isopropylmalate (7), gymnoside I (8), militarine (9), dactylorhin A (10), gastrodin (11). Compounds 1-7 were isolated from this genus for the firt time.

Characterization of four popular sweet cherry cultivars grown in Greece by volatile compound and physicochemical data analysis and sensory evaluation.

Volatile compounds, physicochemical and sensory attributes of four sweet cherry cultivars (Canada giant, Ferrovia, Lapins and Skeena) grown in Northern Greece were determined. Eighteen volatile compounds were identified and semi-quantified in cherries using solid phase micro extraction in combination with gas chromatography/mass spectrometry (SPME-GC/MS). Carbonyl compounds were the most abundant in sweet cherry aroma, followed by alcohols, esters and hydrocarbons/terpenes. Cherry cultivars in order of increasing amounts of volatiles were: Lapins < Canada giant < Ferrovia < Skeena. Physicochemical parameters determined included: titratable acidity (TA), pH, total soluble solids (TSS), maturity index (MI) and total phenolic content (TPC). TA ranged between 0.21 and 0.28 g malic acid/100 g fresh weight (FW). The pH ranged between 3.81 and 3.96. TSS ranged between 13.00 and 16.00 °Brix. MI ranged between 51.8 and 75.0. TPC ranged between 95.14 and 170.35 mg gallic acid equivalents (GAE)/100 g FW. Sensory evaluation showed that cherry colour, in order of increasing intensity, was: Canada giant < Ferrovia < Lapins < Skeena. Respective order for cherry firmness was: Canada giant < Lapins ≤ Ferrovia < Skeena and for flavour: Lapins < Canada giant < Skeena ≤ Ferrovia. Correlation of volatiles to physicochemical and sensory attributes showed varying trends.

New neolignan glycoside and an unusual benzoyl malic acid derivative from Maytenus senegalensis leaves.

Further investigation of the methanol leaf extract of Maytenus senegalensis led to the isolation of six compounds, including mayselignoside (1) and an unusual benzoyl malic acid derivative, benzoyl R-(+)-malic acid (2). Two known lignan derivatives (+)-lyoniresinol (3) and (-)-isolariciresinol (4), a known neolignan derivative dihydrodehydrodiconiferyl alcohol (5) and the triterpenoid, ß-amyrin (6) were also isolated. The structures of these compounds were elucidated by a combination of 1D and 2D NMR and mass spectroscopy. All compounds were tested for cytotoxicity against mouse lymphoma cell line (L5178Y) and for antimicrobial activity against strains of bacteria and fungi. None of the compounds showed promising cytotoxic and/or antimicrobial activities.

Aromatic acid derivatives from the lateral roots of Aconitum carmichaelii.

Seven new aromatic acid derivatives (1-7), together with five known analogs, were isolated from the lateral roots of Aconitum carmichaelii. Structures of the new compounds were determined by spectroscopic and chemical methods as 4-methyl ( - )-(R)-hydroxyeucomate (1), 4-butyl ( - )-(R)-hydroxyeucomate (2), 4-butyl-1-methyl (+)-(R)-2-O-(4'-hydroxy-3'-methoxybenzoyl)malate (3), 1-butyl-4-methyl (+)-(R)-2-O-(4'-hydroxy-3'-methoxybenzoyl)malate (4), dimethyl (+)-(R)-2-O-(4'-hydroxy-3'-methoxybenzoyl)malate (5), dimethyl (+)-(R)-2-O-(4'-hydroxybenzoyl)malate (6), and methyl ( ± )-3-(4'-hydroxy-3'-methoxyphenyl)-3-sulfopropionate (7), respectively. Compounds 1 and 2 are 2-benzylmalates (eucomate derivatives), 3-6 belong to 2-O-benzoylmalates, and 7 is a rare phenylpropionate containing a sulfonic acid group. The absolute configurations of eucomate derivatives were confirmed by X-ray crystallographic analysis of 4-methyl eucomate (11).

Assessment the levels of tartrate-resistant acid phosphatase (TRAP) on mice fed with eggshell calcium citrate malate.

Optimized conditions were obtained by one-factor-at-a-time test (OFAT) and ternary quadratic regression orthogonal composite design (TQROCD) respectively. By pulse electric fields (PEF) technology, the process of eggshell calcium citrate malate (ESCCM), eggshell calcium citrate (ESCC) and eggshells calcium malate (ESCM) were comprehensive compared. The levels of tartrate-resistant acid phosphatase (TRAP) and the bioavailability on mice fed with eggshell calcium citrate malate (ESCCM) treated by pulsed electric field (PEF) were evaluated. Results showed that the rates of calcium dissolution of the different acids studied can be arranged as ESCCM (7.90 mg/mL)>ESCC (7.12 mg/mL)>ESCM (7.08 mg/mL) from highest to lowest rate of dissolution. At the same dose 133.0 mg kg(-1) d(-1), the levels of TRAP in the ESCCM treatment groups were significantly lower than those in ESCM and ESCC (P<0.05). Bone calcium content in the mice fed with ESCCM was generally higher than fed with ESCM and ESCC.

Oxaloacetate and malate production in engineered Escherichia coli by expression of codon-optimized phosphoenolpyruvate carboxylase2 gene from Dunaliella salina.

A new phosphoenolpyruvate carboxylase (PEPC) gene of Dunaliella salina is identified using homology analysis was conducted using PEPC gene of Chlamydomonas reinhardtii and Arabidopsis thaliana. Recombinant E. coli SGJS115 with increased production of malate and oxaloacetate was developed by introducing codon-optimized phosphoenolpyruvate carboxylase2 (OPDSPEPC2) gene of Dunaliella salina. E. coli SGJS115 yielded a 9.9 % increase in malate production. In addition, E. coli SGJS115 exhibited two times increase in the yield of oxaloacetate over the E. coli SGJS114 having identified PEPC2 gene obtained from Dunaliella salina.

Malic acid production from thin stillage by Aspergillus species.

The ability of Aspergillus strains to utilize thin stillage to produce malic acid was compared. The highest malic acid was produced by Aspergillus niger ATCC 9142 at 17 g l(-1). Biomass production from thin stillage was similar with all strains but ATCC 10577 was the highest at 19 g l(-1). The highest malic acid yield (0.8 g g(-1)) was with A. niger ATCC 9142 and ATCC 10577 on the stillage. Thus, thin stillage has the potential to act as a substrate for the commercial production of food-grade malic acid by the A. niger strains.

Hydroxycinnamoylmalic acids and their methyl esters from pear (Pyrus pyrifolia Nakai) fruit peel.

Two novel caffeoylmalic acid methyl esters, 2-O-(trans-caffeoyl)malic acid 1-methyl ester (6) and 2-O-(trans-caffeoyl)malic acid 4-methyl ester (7), were isolated from pear (Pyrus pyrifolia Nakai cv. Chuhwangbae) fruit peels. In addition, 5 known hydroxycinnamoylmalic acids and their methyl esters were identified: 2-O-(trans-coumaroyl)malic acid (1), 2-O-(cis-coumaroyl)malic acid (2), 2-O-(cis-coumaroyl)malic acid 1-methyl ester (3), 2-O-(trans-coumaroyl)malic acid 1-methyl ester (4), and 2-O-(trans-caffeoyl)malic acid (phaselic acid, 5). The chemical structures of these compounds were determined by spectroscopic data from ESI MS and NMR. Of all the isolated compounds, five hydroxycinnamoylmalic acids and their methyl esters (2-4, 6, 7) were identified in the pear for the first time.

Glucosyloxybenzyl eucomate derivatives from Vanda teres stimulate HaCaT cytochrome c oxidase.

Eucomic acid [(2R)-2-(p-hydroxybenzyl)malic acid)] (1) and three new glucopyranosyloxybenzyl eucomate derivatives, vandaterosides I (2), II (3), and III (4), were isolated and identified from the stems of Vanda teres. Their cellular antiaging properties were evaluated in a human immortalized keratinocyte cell line (HaCaT) by monitoring their effect on cytochrome c oxidase activity, implicated in mitochondrial respiratory function and cellular energy production. Eucomic acid (1) and vandateroside II (3) increased cytochrome c oxidase activity and/or expression, without enhancing cellular mitochondrial content. These two V. teres biomarkers apparently contributed to stimulate respiratory functions in keratinocytes. Since aging and its pathologies may be ascribed to a decline in mitochondrial functions, these biomarkers have the potential to become new natural ingredients for antiaging preparations to remedy age-related disorders such as skin aging.

1-methylmalate from camu-camu (Myrciaria dubia) suppressed D-galactosamine-induced liver injury in rats.

To evaluate the protective effects of fruit juices against D-galactosamine (GalN)-induced liver injury, lyophilized fruit juices (total 12 kinds) were fed to rats for 7 d, and then we evoked liver injury by injecting GalN. The juice of camu-camu (Myrciaria dubia) significantly suppressed GalN-induced liver injury when the magnitude of liver injury was assessed by plasma alanine aminotransferase and aspartate aminotransferase activities, although some other juices (acerola, dragon fruit, shekwasha, and star fruit) also tended to have suppressive effects. An active compound was isolated from camu-camu juice by solvent fractionation and silica gel column chromatography. The structure was determined to be 1-methylmalate. On the other hand, malate, 1,4-dimethylmalate, citrate, and tartrate had no significant effect on GalN-induced liver injury. It is suggested that 1-methylmalate might be a rather specific compound among organic acids and their derivatives in fruit juices in suppressing GalN-induced liver injury.