Antioxidative properties of aqueous and aroma extracts of squid miso prepared with Aspergillus oryzae-inoculated koji

To evaluate the antioxidative properties of a newly developed fermented paste (squid miso) prepared from squid mantle flesh with Aspergillus oryzae-inoculated koji, we used in vitro models, including 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl, nitric oxide (NO) and carbon-centered radical-scavenging activity (RSA), reducing power ability (RPA), and linoleic acid oxidation. The antioxidant activity of volatile compounds, which were extracted from squid miso by dichloromethane, was confirmed based on the results obtained by the oxidation models consisting of carotene linoleate and hexanal. The RSA of the miso against several radicals, including DPPH, hydroxyl, nitric oxide and carbon-centered radicals measured by ESR spectrometry increased gradually through the fermentation period. On the other hand, the RPA increased rapidly in early stages of fermentation. Proteins were hydrolyzed to peptides and amino acids during fermentation. Certain peptides of low molecular mass significantly contributed to RSA. 4-Ethylguaiacol in the RSA active fraction, which was obtained by column chromatography, was the most potent antioxidative in the volatile extract. Analysis of phenolics in ethyl acetate fraction clearly suggested that 4-ethylguaiacol in the squid miso is possibly a bioconversion product of ferulic acid derived from koji. However, the aqueous extracts containing antioxidative peptides contributed more remarkably to radical scavenging than heterocyclic volatiles and phenolics.     

Activity-guided isolation and identification of free radical-scavenging components from an aqueous extract of Coleus aromaticus

An activity-directed fractionation and purification process was used to identify the DPPH (l,l-diphenyl-2-picrylhydrazyl) free radical-scavenging components of Coleus aromaticus Benth. Fresh leaves of C. aromaticus were extracted with water and then separated into hexane, ethyl acetate, and water fractions. Among these, only the ethyl acetate phase showed strong DPPH radical-scavenging activity in vitro, when compared with water and hexane phases. The ethyl acetate fraction was then subjected to separation and purification using Sephadex LH-20 chromatography. Three compounds showing strong DPPH radical-scavenging activity were shown, by spectral methods (¹H NMR, ¹³C NMR, and MS) and by comparison with literature values, to be rosmarinic acid, chlorogenic acid and caffeic acid. In addition, HPLC identification and quantification of isolated compounds were also performed. Rosmarinic acid was found as a major component and principally responsible for the radical-scavenging activity of C. aromaticus.     

Metabolomic response of Brassica rapa submitted to pre-harvest bacterial contamination

Plants are continually challenged by natural pathogens. Among a number of environmental factors, pre-harvest contamination of plants with pathogens responsible for enteric diseases in humans is of major international concern. Despite the knowledge of how bacterial attack can affect the biological system of plants, little is known about the effect of the interaction of these bacteria on plant’s metabolome. In order to investigate the metabolic change of Brassica induced by its response to different typically food borne bacteria such as Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Salmonella typhimurium and Shigella flexneri, ¹H NMR and two-dimensional NMR spectra, coupled with principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA) were applied to Brassica rapa which had been subjected to these pathogens during plant growth. The metabolic changes were found to vary according to bacterial species; for example, Gram-positive and Gram-negative bacteria had a different effect on the Brassica metabolome. While threonine and GABA were found to be the discriminating metabolites in Gram-positive bacteria treated plants, those treated with Gram-negative bacteria exhibited a significant increase in sinapoyl-malate, caffeoyl-malate and histidine. The detailed study of the effect of type of bacteria showed that amino acids, alcohols, carbohydrates and phenols were discriminating metabolites. These results prove the potential of NMR-based metabolomics as a tool to study the interaction of these food-borne bacteria with vegetables.     

Studies on the antioxidant potential of flavones of Allium vineale isolated from its water-soluble fraction

The aim of this work was to examine the chemical constituents and antioxidant potential of water-soluble fractions from the commonly consumed vegetable, Allium vineale. The water-soluble fraction, containing phenolic compounds, was extracted with ethyl acetate to obtain flavonoids which were separated and purified by repeated column chromatography over Sephadex LH-20, RP C18 and silica gel. The isolated compounds were identified according to their physicochemical properties and spectral data (UV, HPLC–TOF/MS, ¹H NMR, ¹³C NMR and 2D NMR). Three flavonoids were isolated and identified as chrysoeriol-7-O-[2″-O-E-feruloyl]-β-d-glucoside (1), chrysoeriol (2), and isorhamnetin-3-β-d-glucoside (3). Antioxidant studies of the aqueous extract and three isolated compounds, 1, 2, 3, were undertaken and they were found to have significant antioxidant activity. Antioxidant activities were evaluated for total antioxidant activity by the ferric thiocyanate method, ferric ion (Fe³⁺) reducing antioxidant power assay (FRAP), ferrous ion (Fe²⁺) metal chelating activity, and DPPH free radical-scavenging activity. The water-soluble ethyl acetate and methanol extraction methods were also compared using HPLC–TOF/MS.     

Preparation of a polymethoxyflavone-rich fraction (PRF) of Citrus sunki Hort. ex Tanaka and its antiproliferative effects

The peel of Citrus sunki is a rich source of polymethoxyflavones (PMFs). In this study, a PMF-rich fraction (PRF) was obtained from the peels of C. sunki by hot-water extraction and subsequent fractionation with n-hexane. Six PMFs and two demethylated PMFs were isolated by semi-preparative HPLC, and their structures were identified by ¹H NMR and ¹³C NMR. To define the synergistic effects of PRF, the PRF and its eight individual components were assessed for antiproliferative activity using HL-60 cells. The PRF showed greater growth inhibitory activity (IC₅₀ value = 25.14 μg/ml), compared with its individual components. Flow cytometric analysis confirmed that the PRF significantly increased the percentage of sub-G₁ cells (30.1% at 25 μg/ml). Our study provides an efficient method for preparing a PRF for use as a functional food ingredient.     

Effects of Leuconostoc mesenteroides starter cultures on microbial communities and metabolites during kimchi fermentation

Kimchi fermentation usually relies upon the growth of naturally-occurring various heterofermentative lactic acid bacteria (LAB). This sometimes makes it difficult to produce kimchi with uniform quality. The use of Leuconostoc mesenteroides as a starter has been considered to produce commercial fermented kimchi with uniform and good quality in Korea. In this study, a combination of a barcoded pyrosequencing strategy and a ¹H NMR technique was used to investigate the effects of Leu. mesenteroides strain B1 as a starter culture for kimchi fermentation. Baechu (Chinese cabbage) and Chonggak (radish) kimchi with and without Leu. mesenteroides inoculation were prepared, respectively and their characteristics that included pH, cell number, bacterial community, and metabolites were monitored periodically for 40 days. Barcoded pyrosequencing analysis showed that the numbers of bacterial operational taxonomic units (OTU) in starter kimchi decreased more quickly than that in non-starter kimchi. Members of the genera Leuconostoc, Lactobacillus, and Weissella were dominant LAB regardless of the kimchi type or starter inoculation. Among the three genera, Leuconostoc was the most abundant, followed by Lactobacillus and Weissella. The use of Leu. mesenteroides as a starter increased the Leuconostoc proportions and decreased the Lactobacillus proportions in both type of kimchi during kimchi fermentation. However, interestingly, the use of the kimchi starter more highly maintained the Weissella proportions of starter kimchi compared to that in the non-starter kimchi until fermentation was complete. Metabolite analysis using the ¹H NMR technique showed that both Baechu and Chonggak kimchi with the starter culture began to consume free sugars earlier and produced a little greater amounts of lactic and acetic acids and mannitol. Metabolite analysis demonstrated that kimchi fermentation using Leu. mesenteroides as a starter was completed earlier with more production of kimchi metabolites compared to that not using a starter, which coincided with the decreases in pH and the increases in bacterial cell number. The PCA strategy using all kimchi components including carbohydrates, amino acids, organic acids, and others also showed that starter kimchi fermented faster with more organic acid and mannitol production. In conclusion, the combination of the barcoded pyrosequencing strategy and the ¹H NMR technique was used to effectively monitor microbial succession and metabolite production and allowed for a greater understanding of the relationships between the microbial community and metabolite production in kimchi fermentation.     

Preparative separation of phenylpropenoid glycerides from the bulbs of Lilium lancifolium by high-speed counter-current chromatography and evaluation of their antioxidant activities

Preparative separation of seven phenylpropenoid glycerides from the bulbs of Liliumlancifolium (lily), was conducted by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-0.05% aqueous trifluoroacetic acid (TFA) (3:5:3:5, v/v/v/v). These phenylpropenoid glycerides were identified as 1-O-feruloylglycerol (1), 1-O-p-coumaroylglycerol (2), 1-O-caffeoyl-3-O-p-coumaroylglycerol (3), 1,2-O-diferuloylglycerol (4), 1,3-O-diferuloylglycerol (5), 1-O-feruloyl-3-O-p-coumaroylglycerol (6), and 1,3-O-di-p-coumaroylglycerol (7), respectively, by ESI-MS, ¹H NMR and ¹³C NMR. Their antioxidant activities were evaluated by DPPH radical scavenging activity, ABTS radical cation scavenging activity, and ferric-reducing antioxidant power (FRAP). The trend in antioxidant capacity was similar in all the three assays, with 3 > 1, 4, 5, 6 > 2, 7. This is the first report on simultaneous separation of seven antioxidantive phenylpropenoid glycerides from L. lancifolium by HSCCC.     

Secondary metabolites in a soybean fermentation broth of Paecilomyces militaris

A methanolic extract from a soybean broth fermented by Paecilomyces militaris was analysed by HPLC–PDA–HRMS. It mainly contained mannitol, uracil, adenosine, cordycepin, daizein, genistein, and three new isoflavone glycosides. The isoflavonoids were isolated by preparative HPLC and identified with the aid of HPLC–PDA–HRMS, ¹H NMR, ¹³C, H–H COSY, HMBC, and HMQC NMR analysis as: 7,4′-dihydroxyisoflavone-7-O-(4′′-O-methyl)-β-d-glucopyranoside, 7,4′-dihydroxy-6-methoxyisoflavone-7-O-(4′′-O-methyl)-β-d-glucopyranoside, and 5,7,4′-trihydroxyisoflavone-7-O-(4′′-O-methyl)-β-d-glucopyranoside. The three novel compounds are transformation products of soybean isoflavones. The results of metabolites analysis revealed that the fermentation broth reserved the main functional molecules of soybean and P. militaris or Cordyceps militaris. This suggested that soybean fermented with P. militaris may be able to create a combined healthy food. However, isoflavones are also parts of anti-nutrients of soybean products, and cordycepin is a known antibiotic. This means that the fermentation broth may not suit to be used as normal food. At the same time the safety and bioactivity of the novel methylated glycosides need also further approval.     

Metabolic profiling and aquaculture differentiation of gilthead sea bream by H NMR metabonomics

This work describes a metabolic profiling study of gilthead sea bream, from three different aquaculture systems, using ¹H NMR and chemometrics. A total of 54 samples under two different storage regimens were analysed. The assignment of all major NMR signals of the perchloric extracts was performed. A comprehensive multivariate data analysis proved able to distinguish the fish metabolism amongst the different aquaculture systems and to determine whether a fish was stored or not. The state of energy metabolism of inosine proved a robust biomarker for evaluating storage time. A new multivariate classification tool, iECVA, revealed several metabolites which are important biomarkers for characterising the three different aquaculture systems: glycogen (stress indicator), histidine, alanine and especially glycine for long storage times and mainly betaine for fresh samples. The findings represent a step forward in understanding how in vivo and postmortem processes affect the total quality of the final product.     

Production of enniatins A,A1, B,B1, B4, J1 by Fusarium tricinctum in solid corn culture: Structural analysis and effects on mitochondrial respiration

Enniatins (ENs) are secondary fungal metabolites with hexadepsipeptidic chemical structure and they possess a number of potent biological activities that can contaminate several kind of food and foodstuffs increasing the exposure risk for consumers. ENs are produced by several Fusariun strains including Fusarium subglutinans, Fusarium proliferatum and Fusarium tricinctum. Production of a mixture of ENs was performed by culturing F. tricinctum ITEM 9496 on white corn as substrate. The solid culture components were dried and subsequently extracted with water/methanol (50/50 v/v, 0.5% NaCl), homogenised, filtered, extracted by ethyl acetate and analysed by liquid chromatography with diode array detection (LC-DAD). The crude extract was first separated by low pressure liquid chromatography (LPLC) and then further purified by liquid chromatography (LC), resulting in six compounds with a purity higher than 95% as calculated by ¹H NMR, and with a yield of 30–300 mg per compound. The chemical structures of the ENs were determined by liquid chromatography coupled to mass spectrometry (LC–MS) and nuclear magnetic resonance (NMR). The biological activity of the resulting ENs was determined using a mitochondrial respiration test. We discovered that all the ENs studied induced an increase in the mitochondrial respiration resulting in uncoupling of the oxidative phosphorylation. This effect was most likely due to flux of K⁺ ions into the mitochondrial matrix. The order of potency of the ENs derivatives was: A₁ > B₁ > B > A > B₄ > J₁. These results suggest a correlation between the chemical structures and bioactivity and confirm the severe risks for human associated with consumption of enniatins.     

Anticonvulsant activity of furanocoumarins and the essential oil obtained from the fruits of Heracleum crenatifolium

The anticonvulsant activity of furanocoumarins, coumarin mixture and the essential oil obtained from the fruits of Heracleum crenatifolium was examined against maximal electroshock (MES)-induced seizures in mice. Bergapten showed significant anticonvulsant activity. The furanocoumarins isolated from the fruits of the plant were identified using thin-layer chromatography, melting points and spectroscopic methods (IR, MS, ¹H NMR) as isobergapten (1), pimpinellin (2), bergapten (3), isopimpinellin (4), sphondin (5) and byak-angelicol (6). The essential oil content of the fruits were found as 5.5%. Twenty-two compounds representing 99.3% of the essential oil obtained from the fruits of H. crenatifolium were determined and the major components were identified as octanol and octyl acetate (3.1% and 88.4% respectively) by GC and GC–MS.     

A new benzoquinone and a new benzofuran from the edible mushroom Neolentinus lepideus and their inhibitory activity in NO production inhibition assay

The fruiting bodies or mycelia of mushrooms have been used as food and food-flavoring material for centuries due to their nutritional and medicinal value and the diversity of their bioactive components. The present research is the first to investigate the bioactive secondary metabolites from the solid culture of the edible mushroom Neolentinus lepideus. Two new secondary metabolites, 5-methoxyisobenzofuran-4,7(1H,3H)-dione (1) and 1,3-dihydroisobenzofuran-4,6-diol (2), as well as seven known compounds including one benzoquinone derivative (3) and six cinnamic acid derivatives (4–9) were obtained. Their structures were established by means of spectroscopic methods, including 1D and 2D NMR. The bioactivity on the nitric oxide production in lipopolysaccharide-induced macrophages was evaluated for all metabolites (1–9) isolated. Compound 1 showed strong NO inhibitory activity with the IC₅₀ value of 6.2 μM. Compound 2 displayed moderate NO inhibitory activity with the IC₅₀ value of 88.8 μM. In the DPPH scavenging assay, compound 2 displayed antioxidant activity with IC₅₀ of 68.6 μM. The discovery of new NO production inhibitors from N. lepideus expands its usage as a functional food.     

Commercial extract of the brown seaweed Ascophyllum nodosum enhances phenolic antioxidant content of spinach (Spinacia oleracea L.) which protects Caenorhabditis elegans against oxidative and thermal stress

There is considerable interest to enhance the nutritional quality of fresh produce especially vegetables. The effects of root treatment of spinach with commercial extracts of the brown macro alga, Ascophyllum nodosum (ANE) on antioxidant level of spinach were studied. At the concentration of 1.0 g/L, ANE treatment significantly increased the total phenolics and flavonoids content, total antioxidant activity (as measured by DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging capacity) and Fe²⁺ chelating ability in spinach leaves. The ¹H NMR and LC-MS analyses of spinach extract suggests that the increased antioxidant activity is largely associated with flavonoids. The biological effect of ANE-enhanced polyphenols was tested using the Caenorhabditis elegans nematode model. The extracts from ANE-treated spinach significantly improved the survival of the animals under oxidative stress by 50% and high temperature stress by 61% as compared to the extracts from untreated plants (0% and 38%, respectively). Taken together, the results suggest that ANE stimulate flavonoid synthesis in spinach leaf thus enhancing its nutritional quality. Furthermore, the increased flavonoid content exerts beneficial effects in C. elegans against oxidative and heat stress.     

H NMR-based metabolomic characterization during green tea (Camellia sinensis) fermentation

The metabolic behavior of green tea (Camellia sinensis) during tea fermentation was characterized by ¹H NMR spectroscopy coupled with multivariate statistical analysis to provide comprehensive information on changes in metabolites induced by tea fermentation. Fourteen tea metabolites of epicatechin (EC), epigallocatechin (EGC), epicatechin-3-gallate (ECG), epigallocatechin-3-gallate (EGCG), theanine, alanine, acetate, quinate, glutamate, caffeine, sucrose, glucose, and gallate, as identified by ¹H NMR spectroscopy, were responsible for metabolic differentiation between green tea and fermented tea by principal component analysis. During tea fermentation, levels of EC, EGC, ECG, EGCG, quinate, caffeine, and sucrose were decreased, whereas gallate and glucose levels were increased. In particular, unique changes in caffeine and gallate levels were observed during tea fermentation, which caffeine and gallate levels have been shown to vary after tea fermentation among many reports to date. This study highlights that metabolomics with global profiling and a highly reliable and reproducible ¹H NMR spectroscopic data set can provide a better understanding of unique changes in tea metabolites during tea fermentation.     

Antioxidant activity of a new phenolic glycoside from Lagenaria siceraria Stand. fruits

The antioxidant properties of different extracts of Lagenaria siceraria (bottle gourd) fruit were evaluated. In the process, a new phenolic glycoside (E)-4-hydroxymethyl-phenyl-6-O-caffeoyl-β-d-glucopyranoside (1) was isolated and identified together with 1-(2-hydroxy-4-hydroxymethyl)-phenyl-6-O-caffeoyl-β-d-gluco-pyranoside (2), protocatechuic acid (3), gallic acid (4), caffeic acid (5) and 3,4-dimethoxy cinnamic acid (6). Their structures were elucidated by extensive NMR experiments including ¹H-¹H (COSY) and ¹H-¹³C (HMQC and HMBC) spectroscopy and chemical evidences. The antioxidant potential of the compound 1 and 2 was tested in different in vitro assay systems such as free radical scavenging assay, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay, superoxide scavenging activity, reducing power assay and linoleic acid peroxidation assay.     

Discrimination of young and mature leaves of Melicope ptelefolia using H NMR and multivariate data analysis

The ‘Ulam’, a traditional Malay dish, are plants that can be eaten raw, as a form of local salad. The shoots and young leaves of Melicope ptelefolia are among the popular species, believed to be high in nutritional and medicinal values. The metabolomic fingerprinting analysis of the ethanolic extracts of leaves of M. ptelefolia was carried out using ¹H Nuclear Magnetic Resonance (NMR) spectroscopy and multivariate data analysis in order to differentiate young and mature leaves and to evaluate the variation of their chemical composition. Principle component analysis (PCA) of the ¹H NMR spectra showed a clear discrimination between the young and mature leaves extracts by PC3 and PC4. The compounds responsible for the differentiation were identified by comparison of ¹H NMR chemical shifts and qualitative HPLC. The young leaves were found to be richer in fatty acids and the levels of the three marker compounds, p-O-geranylcoumaric acid, 2,4,6-trihydroxy-3-geranylacetophenone and 2,4,6-trihydroxy-3-prenylacetophenone, were clearly higher. The mature leaves contain higher levels of sugars and glycosidic components.     

Two flavanone compounds from litchi (Litchi chinensis Sonn.) seeds,one previously unreported,and appraisal of their α-glucosidase inhibitory activities

Using a bioactivity-guided method, a new and a previously reported flavanone glycoside were isolated from 50% ethanol extract of litchi (Litchi chinensis Sonn.) seeds. Column chromatography using macroporous resin, silica gel and Sephadex LH-20 was applied during the isolation, and the method of PMP derivatisation was performed to investigate the component sugar of the compounds. The structures of the compounds were elucidated by ¹H, ¹³C, and 2D NMR (HMQC, HMBC) spectroscopy and HR-ESIMS. Compound 1 was identified as (2R)-naringenin-7-O-(3-O-α-l-rhamnopyranosyl-β-d-glucopyranoside), a new, natural product that had been synthesised previously. Compound 2 was (2S)-pinocembrin-7-O-(6-O-α-l-rhamnopyranosyl-β-d-glucopyranoside). The two isolated compounds showed activity in the α-glucosidase inhibition assay.     

Antioxidant properties of a novel flavonoid from leaves of Leucas aspera

The crude methanol extract of Leucas aspera leaves showed strong 1,1-diphenyl-2-picrylhydrazyl (DDPH) and superoxide radical-scavenging activities compared to other polarity-based extracted fractions. It was selected for purification of the bioactive compound. The activity-guided repeated fractionation of the methanol extract by silica gel column chromatography yielded a compound that exhibited strong antioxidant activity. Based on various physicochemical and spectroscopic analyses, e.g. UV, IR, ¹H NMR, ¹³C NMR and mass spectroscopy, the bioactive compound isolated was elucidated as 5,7-dihydroxy-2-[14-methoxy-15-propyl phenyl]-4H-chromen-4-one (leucasin). Radical-scavenging potential and strong inhibition of lipid peroxidation in a liposome model were observed at a leucasin concentration of 40 ppm. These results demonstrate the antioxidant potency of leucasin which could be the basis for its alleged health-promoting potential.     

The production of surfactin during the fermentation of cheonggukjang by potential probiotic Bacillus subtilis CSY191 and the resultant growth suppression of MCF-7 human breast cancer cells

Bacillus subtilis CSY191, the potential probiotics and surfactin-like compound producer, was isolated from doenjang (Korean traditional fermented soybean paste).The survival rate of this strain appeared to be the 58.3% under artificial gastric conditions after 3 h at pH 3.0. Surfactin was purified from the strain CSY191. Three potential surfactin isoforms were detected, with protonated masses of m/z 1030.7, 1044.7, and 1058.71. These different structures were detected in combination with Na⁺, K⁺ and Ca²⁺ ions by MALDI-TOF mass spectrometry. Upon 500 MHz ¹H NMR analysis, the surfactin isoforms had identical amino acids (GLLVDLL) and hydroxy fatty acids (of 13-15 carbons in length). The MTT assay showed that surfactin inhibited growth of MCF-7 human breast cancer cells in a dose-dependent manner, with an IC₅₀ of approximately 10 μg/ml at 24 h. Additionally, the surfactin contents, during cheonggukjang fermentation with strain CSY191, increased from 0.3 to 48.2 mg/kg over 48 h of fermentation, while the level of anticancer activity increased from 2.6- to 5.1-fold.