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In this study, pulsed electric fields (PEFs) were evaluated as extraction-aiding technology during soymilk manufacturing to improve its isoflavone profile. Low-intensity PEFs were applied at different processing conditions in two stages of the soymilk extraction process, hydrated soybeans (HSB) and soybean slurry (SBS), with the soymilk extracted from the conventional process as control (CSM). Overall, resultant soymilk samples from PEF-HSB and PEF-SBS presented lower concentrations of glucosides isoflavones and greater aglycone content than those in CSM. In contrast to genistin (Gin) and daidzin (Din), which decreased around 18.5-52.6% and 10.9-54.6%, respectively, an increase in genistein (Ge, 12.3-64.4%) and daidzein (Da, 9-55.8%) was observed. The total isoflavone content (TIC) of most soymilk samples prepared from PEF-HSB was lower than that of the CSM. Conversely, when PEF-SBS was used, the TIC of resultant soymilk was not significantly affected or slightly decreased. However, PEF treated HSB at 10 kVcm−1/100 pulses and SBS at 6 kVcm−1/10 pulses led to a significant augment in TIC, of up to 109 ± 2.39 and 110 ± 1.26 μg/g, respectively, in the extracted soymilk samples. These results indicated that low-intensity PEF is a potential technology that could be implemented during soymilk manufacturing processing to modify the isoflavone profile and content of soymilk, mainly increasing its aglycone concentration This research was funded by Tecnologico de Monterrey and the University of Lleida with research funds of FUNFOODEMERTEC Project.

Flavonoids are well known for their extensive health benefits. However, few studies compared the differences between flavonoid O-glycoside and C-glycoside. In this work, flavonoid O-glycoside (isoquercitrin), C-glycoside (orientin), and their aglycones (quercetin and luteolin) were chosen to compare their differences on antioxidant activities and metabolism during in vitro digestion and in vivo. In vitro digestion, the initial antioxidant activity of the two aglycones was very high; however, they both decreased more sharply than their glycosides in the intestinal phase. The glycosidic bond of flavonoid O-glycoside was broken in the gastric and intestinal stage, while the C-glycoside remained unchanged. In vivo, flavonoid O-glycoside in plasma was more elevated than C-glycoside on the antioxidant activity; however, flavonoid C-glycoside in urine was higher than O-glycoside. These results indicate that differences of flavonoid glycosides and their aglycones on antioxidant activity are closely related to their structural characteristics and metabolism in different samples. Aglycones possessed higher activity but unstable structures. On the contrary, the sugar substituents reduced the activity of flavonoids while improving their stability and helping to maintain antioxidant activities after digestion. Especially the C-glycoside was more stable because the stability of the C–C bond is higher than that of the C–O bond, which contributes to the difference between flavonoid O-glycoside and C-glycoside on the absorption and metabolism in vivo. This study provided a new perspective for comparing flavonoid O-glycoside, flavonoid C-glycoside, and their aglycones on their structure–activity relationship and metabolism.

Citrus fruits have been the subject of extensive research over the years due to their impressive antioxidant properties, the health benefits of flavanones, and their potential use in the prevention and treatment of chronic diseases. Grapefruit have been shown in studies to improve overall health, with numerous potential benefits, including improved heart health, reduced risk of certain cancers, improved digestive health, and improved immune system function. The development of cyclodextrin complexes is an exciting approach to increasing the content of flavanones such as naringin and naringenin in the extraction medium while improving the profile of beneficial phenolic compounds and the antioxidant profile. This research aims to optimize the extraction conditions of the flavanones naringin and naringenin with additional compounds to increase their yield from different parts of grapefruit (Citrus × paradisi L.) fruits, such as albedo and segmental membranes. In addition, the total content of phenolic compounds, flavonoids, and the antioxidant activity of ethanolic extracts produced conventionally and with -cyclodextrin was examined and compared. In addition, antioxidant activity was measured using the radical scavenging activity assay (ABTS), radical scavenging activity assay (DPPH), and ferric reducing antioxidant power (FRAP) methods. The yield of naringin increased from 10.53 ± 0.52 mg/g to 45.56 ± 5.06 mg/g to 51.11 ± 7.63 mg/g of the segmental membrane when cyclodextrins (α, β-CD) were used; naringenin increased from 65.85 ± 10.96 μg/g to 91.19 ± 15.19 μg/g of the segmental membrane when cyclodextrins (α, β-CD) were used. Furthermore, the results showed that cyclodextrin-assisted extraction had a significant impact in significantly increasing the yield of flavanones from grapefruit. In addition, the process was more efficient and less expensive, resulting in higher yields of flavanones with a lower concentration of ethanol and effort. This shows that cyclodextrin-assisted extraction is an excellent method for extracting valuable compounds from grapefruit.

The objective of this work was to determine the isoflavones accumulation in soybean pods and grains during the filling period in field conditions. Pods of the same phenological stage (marked in R5, i.e. beginning of grain filling period) were collected at R5, R6 (full grain), and R7 (beginning of physiological maturity) stages. At each analyzed stage, grains always exceed pods isoflavone concentrations. A decreasing tendency of isoflavone concentration was observed in pods towards the late grain filling. In this maternal tissue, no acetyl glucosides at any stage or aglycones at physiological maturity were detected. In grains, glucosides, malonyl glucosides, and total isoflavones increased and acetyl glucosides decreased from R5 to R7, whereas aglycones increased to R6, but then decreased. It is concluded that grains are the main accumulation tissue of these bioactive compounds, whereas pods seem to contribute to their accumulation in advanced stages of grain development. Resumen El objetivo de este trabajo fue determinar la acumulación de isoflavonoides en vainas y granos de soja durante el período de llenado en condiciones de campo. Vainas de la misma fenología (marcadas en R5, es decir, inicio del llenado de granos) fueron colectadas en los estadíos R5, R6 (máximo tamaño de grano) y R7 (inicio de madurez fisiológica). En cada estadio analizado, los granos siempre superaron la concentración de isoflavonoides de las vainas. Se observó una tendencia decreciente de la concentración de isoflavonoides en las vainas hacia el llenado tardío de granos. En este tejido materno no se detectaron acetil glucósidos en ningún estadio ni agliconas a madurez fisiológica. En los granos los glucósidos, los malonil glucósidos y los isoflavonoides totales aumentaron y los acetil glucósidos disminuyeron de R5 a R7, mientras que las agliconas aumentaron a R6, pero luego disminuyeron. Se concluyó que los granos son el principal tejido de acumulación de estos compuestos bioactivos, mientras que las vainas parecen contribuir a su síntesis más tarde en el desarrollo de los granos.

Vicine and convicine may be removed from faba bean by hydrolysis to the corresponding aglycones, divicine and isouramil. For total elimination of their toxicity, further degradation of the aglycones should be shown. The aim of the study was to investigate hydrolysis of vicine and convicine using the enzymatic activity in faba bean in flour suspensions and selected lactic acid bacteria used as starters for faba bean fermentation. In addition, the effect of acidity on the stability of vicine and convicine was investigated. Sourdoughs were used in a baking process to obtain breads as final products. Vicine, convicine, and their aglycones were analyzed using reversed phase high pressure liquid chromatography with UV detection (RP-HPLC-UV). Incubation of the suspensions showed rather small vicine and convicine losses. Acidity itself did not cause losses under the conditions studied, apart from that of convicine at low pH. In sourdough fermentation with strains of Lactobacillus plantarum and Pediococcus pentosaceus, losses of vicine and convicine were dependent on the fermentation temperature and the β-glucosidase activity of the starter. Compared to fermentation at 20 °C, more intense acidification at 25 °C resulted in decrease of vicine up to 85% and convicine up to 47%. Levels of vicine and convicine in breads were comparable to levels in sourdoughs. Furthermore, the aglycones were not detected from breads.

Isoflavones can be found in different chemical forms, but the health beneficial effects mainly appear in their free forms—aglycones. Their yield in red clover (Trifolium pratensis L.) extracts differs due to different extraction and hydrolysis methodologies. The main aim of this study was to obtain the highest yields of daidzein and genistein from red clover blossoms through the various extraction and hydrolysis methods and to increase their quantities using additional excipients. Extracts were obtained by ultrasound-assisted, heat-reflux and maceration methods combining them with acidic, alkaline, and thermal hydrolysis. Using ultrasound-assisted extraction with optimal conditions and heat-reflux method highest yields of isoflavones were obtained in UTE510 (393.23 ± 19.66 µg/g daidzein and 171.57 ± 8.58 µg/g genistein); UTE530 (415.07 ± 20.75 µg/g daidzein and 150.57 ± 7.53 µg/g genistein) and HNE5 (432.30 ± 21.61 µg/g daidzein and 154.50 ± 7.72 µg/g genistein) samples. These conditions were used with excipients: magnesium aluminometasilicate, croscarmellose sodium, sodium carboxymethyl starch and vinylpyrrolidone-vinyl acetate copolymer. This is the first study reporting the ability of the vinylpyrrolidone-vinyl acetate copolymer to promote solubilization and availability of active compounds from a herbal extract, resulting in enhanced isoflavones yield. The results of the present study showing increased solubility and availability provided by the vinylpyrrolidone-vinyl acetate copolymer suggest that this preparation could in principle also reduce variability due to limited water solubility of isoflavones.

Trifolium pratense L. is receiving increasing attention due to the isoflavones it contains, which have been studied for their benefits to human health. A common problem with isoflavone aglycones is a rather low water solubility and limited pharmaceutical applications. The use of excipients, such as cyclodextrins in the production of isoflavone rich extracts, could become one of the new strategies for the extraction of target compounds. The aim of this study was to evaluate an eco-friendly method using the effects of α-, β- and γ-cyclodextrins for isoflavone solubilization in plant extracts in comparison to a standard extract without excipients. Extractions of red clover were prepared using ultrasound-assisted combined with thermal hydrolysis and heat reflux. It was determined that cyclodextrins significantly increased the isoflavones aglycone yields. By increasing cyclodextrins in the extraction media from 1 to 5%, the daidzin concentration increased on average by 1.06 (α-cyclodextrins), 1.4 (β-cyclodextrins) and 1.25 (γ-cyclodextrins) times. Genistein concentration increased using α- and γ-cyclodextrins (1.28 and 1.12 times, α- and γ-cyclodextrins, respectively), but decreased using β-cyclodextrins. The results showed that the cyclodextrin-assisted extraction enhanced the yields of isoflavones from red clover, which suggests using cyclodextrins as a green alternative and a cost-effective method to increase its pharmaceutical application.

While flavanones exist in a variety of chemical forms, their favorable health effects are most prominent in their free form—aglycones. Their concentrations in grapefruit (Citrus × paradisi L.) extracts vary according to the extraction and hydrolysis methods used. The primary aim of this work was to maximize the yields of naringin and naringenin from various parts of fresh grapefruit fruits (flavedo, albedo, and segmental) using different extraction and hydrolysis methods. In addition, we aimed to evaluate the excipient—magnesium aluminometasilicate—and determine its influence on the qualitative composition of grapefruit extracts. Extracts were obtained by heat reflux extraction (HRE), ultrasound-assisted extraction with an ultrasonic homogenizer (UAE*), and ultrasound-assisted extraction with a bath (UAE). Ultrasound-assisted extraction using a bath (UAE) was modulated using acidic, thermal, and alkaline hydrolysis. The highest yield of naringin 8A (17.45 ± 0.872 mg/g) was obtained from an albedo sample under optimal conditions using ultrasound-assisted extraction; a high yield of naringenin 23-SHR (35.80 ± 1.79 µg/g) was produced using the heat reflux method from the segmental part. Meanwhile, ultrasonic combined with thermal hydrolysis significantly increased flavanone extraction from the albedo and segmental parts: naringin from sample 9-A (from 17.45 ± 0.872 mg/g to 25.05 ± 1.25 mg/g) and naringenin from sample 15-S (from 0 to 4.21 ± 0.55 µg/g). Additionally, magnesium aluminometasilicate demonstrated significant increases of naringenin from all treated grapefruit parts. To our knowledge, this is the first report of magnesium aluminometasilicate used as an adsorbent in flavanone extractions.

The objective of this work was to evaluate the effects of the germination conditions of 'BRS 216' soybean (Glycine max) on the length, yield, and isoflavone contents of sprouts. A 23 factorial arrangement was used to evaluate the variables soaking time, irrigation frequency, and germination time. Sprouts that showed better length and yield and higher isoflavone contents were evaluated for their chemical composition and vitamin C content. Soaking and germination time of soybean showed a positive and significant linear effect on sprout length. However, only germination time showed a significant positive linear effect on yield, and a significant negative linear effect on the content of malonylglycosides, aglycones, and total isoflavones. Soybean germination conditions were established at 6 hours of soaking, three days of germination, and 8 hours of irrigation frequency. Under these conditions, the obtained sprouts show a more preserved chemical composition, besides higher contents of β-glycosides, malonylglycosides, aglycones, total isoflavones, and vitamin C than the nongerminated soybean. Under suitable germination conditions, it is possible to produce soybean sprouts with better physical characteristics, higher yield, and higher contents of isoflavones, aglycones, and vitamin C. Resumo: O objetivo deste trabalho foi avaliar os efeitos das condições de germinação da soja (Glycine max) 'BRS 216' sobre o comprimento, o rendimento e o teor de isoflavonas dos brotos. Um delineamento fatorial 23 foi utilizado para avaliar as variáveis tempo de maceração, frequência de irrigação e tempo de germinação. Os brotos que apresentaram melhor comprimento e rendimento e maior teor de isoflavonas foram avaliados quanto à composição química e ao teor de vitamina C. Os tempos de maceração e germinação da soja apresentaram efeito linear positivo e significativo sobre o comprimento dos brotos. No entanto, apenas o tempo de germinação apresentou efeito linear significativo e positivo sobre o rendimento, e efeito linear significativo e negativo sobre o teor de malonilglicosídeos, agliconas e isoflavonas totais. As condições de germinação da soja foram estabelecidas em 6 horas de maceração, três dias de germinação e 8 horas de frequência de irrigação. Nessas condições, os brotos obtidos apresentam composição química mais preservada, além de maior teor de β-glicosídeos, malonilglicosídeos, agliconas, isoflavonas totais e vitamina C do que a soja não germinada. Em condições adequadas de germinação, é possível produzir brotos de soja com melhores características físicas, maior rendimento, e maiores teores de isoflavonas, agliconas e vitamina C.

Glucosinolates are secondary plant metabolites of Brassicaceae. They exert their effect after enzymatic hydrolysis to yield aglycones, which become nitriles and epithionitriles through the action of epithiospecifier (ESP) and nitrile-specifier proteins (NSP). The mechanism of action of broccoli ESP and NSP is poorly understood mainly because ESP and NSP structures have not been completely characterized and because aglycones are unstable, thus hindering experimental measurements. The aim of this work was to investigate the interaction of broccoli ESP and NSP with the aglycones derived from broccoli glucosinolates using molecular simulations. The three-dimensional structure of broccoli ESP was built based on its amino-acid sequence, and the NSP structure was constructed based on a consensus amino-acid sequence. The models obtained using Iterative Threading ASSEmbly Refinement (I-TASSER) were refined with the OPLS-AA/L all atom force field of GROMACS 5.0.7 and were validated by Veryfy3D and ERRAT. The structures were selected based on molecular dynamics simulations. Interactions between the proteins and aglycones were simulated with Autodock Vina at different pH. It was concluded that pH determines the stability of the complexes and that the aglycone derived from glucoraphanin has the highest affinity to both ESP and NSP. This agrees with the fact that glucoraphanin is the most abundant glucosinolate in broccoli florets.

Soybeans offer an abundant source of isoflavones, which confer useful bioactivities when existing in aglycone forms. The conversion of isoflavones into aglycones via fermentation of soybean products is often realized by &beta, glucosidase, an enzyme produced by fungi. In this study, a filamentous fungus, Clerodendron cyrtophyllum, was isolated from root of Clerodendron cyrtophyllum Turcz, which was able to produce the highest activity of &beta, glucosidase up to 33.72 U/mL at 144 h during fermentation on Potato Dextrose Broth (PDB). The obtained fungus was grown on isoflavones-rich soybean extract to produce genistein and daidzein, achieving the conversion rate of 98.7%. Genistein and daidzein were isolated and purified by column chromatography using hexane/acetone (29:1/1:1), reaching purities of over 90% of total isoflavones, as identified and determined by TLC, LC-MS/MS, and 1H and 13C NMR spectroscopy. These results imply that the isolated P. citrinum is a potential fungal strain for industrial-scale production of genistein and daidzein from isoflavones-containing soybean extracts. These products may serve as potential raw materials for manufacture of functional foods that are based on aglycones.

Soybeans offer an abundant source of isoflavones, which confer useful bioactivities when existing in aglycone forms. The conversion of isoflavones into aglycones via fermentation of soybean products is often realized by β-glucosidase, an enzyme produced by fungi. In this study, a filamentous fungus, Clerodendron cyrtophyllum, was isolated from root of Clerodendron cyrtophyllum Turcz, which was able to produce the highest activity of β-glucosidase up to 33.72 U/mL at 144 h during fermentation on Potato Dextrose Broth (PDB). The obtained fungus was grown on isoflavones-rich soybean extract to produce genistein and daidzein, achieving the conversion rate of 98.7%. Genistein and daidzein were isolated and purified by column chromatography using hexane/acetone (29:1/1:1), reaching purities of over 90% of total isoflavones, as identified and determined by TLC, LC-MS/MS, and 1H and 13C NMR spectroscopy. These results imply that the isolated P. citrinum is a potential fungal strain for industrial-scale production of genistein and daidzein from isoflavones-containing soybean extracts. These products may serve as potential raw materials for manufacture of functional foods that are based on aglycones.

The present study was principally aimed at ascertaining the differences in metabolomics profiles and biological activities between non-hydrolyzed (ME) and hydrolyzed methanol extract (HME) of Centaurium erythraea Rafn. UHPLC-MS/MS Orbitrap analysis showed that the enzymatic hydrolysis of the extract caused changes in beta-Dglycoside/aglycone ratio of both flavonoid and secoiridoid compounds. UHPLC/DAD/ + HESI - qqqMS characterization and/or quantification of secoiridoid glucosides (SGs) and their aglycones in both ME and HME revealed gentiopicral and erythrocentaurin as the major aglycones, the same metabolic products which appear after the hydrolysis of pure swertiamarin, the dominant secoiridoid glucoside of C. erythraea. SGs played an antioxidant role only in ABTS assay, whilst the remarkable antioxidant potential of C. erythraea methanol extract is ascribed chiefly to phenolics detected in it. Interestingly, antioxidant activities of swertiamarin and sweroside recorded in ABTS assay increased after the compounds have been hydrolyzed, which highlighted their possible antioxidant role during ingestion. Strong antimicrobial activities of ME and HME against a vast array of pathogens, which exceed the effects of the reference antibiotics and antimycotics, largely depended on the amount of secoiridoids in either of the glycosylation forms. Extracts and pure secoiridoids were especially effective against most of the tested Penicillium species. On the other hand, P. funiculosum has evolved an efficient mechanism of detoxification of sub-lethal concentrations of secoiridoid glucosides, involving their biotransformation and complete digestion. The presented findings will contribute to clarify the fate and role of the SGs after C. erythraea ingestion within in vivo systems, and to further promote this remarkable plant as a food preservation additive with significant health benefits. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3208]

The isoflavonoid composition of an ethanolic extract of fungus-treated soybean sprouts was strongly altered by a combined acid/heat treatment. UHPLC-MS analysis showed that 6a-hydroxy-pterocarpans were completely converted to their respective, more stable, 6a,11a-pterocarpenes, whereas other isoflavonoids, from the isoflavone and coumestan subclasses, were affected to a much lesser extent (loss of ~15%). Subsequently, mixtures enriched in prenylated 6a-hydroxy-pterocarpans (pools of glyceollin I/II/III and glyceollin IV/VI) or prenylated 6a,11a-pterocarpenes (pools of dehydroglyceollin I/II/III and dehydroglyceollin IV/VI) were purified, and tested for activity on both human estrogen receptors (ERa and ERß). In particular, the response toward ERa changed, from agonistic for glyceollins to antagonistic for dehydroglyceollins. Toward ERß a decrease in agonistic activity was observed. These results indicate that the introduction of a double bond with the concomitant loss of a hydroxyl group in 6a-hydroxy-pterocarpans extensively modulates their estrogenic activity.

Hydroxyanthraquinones represent a group of pharmacologically active compounds characteristic for plants of the Rumex and Rheum genera. These compounds in the human intestine act as laxative compounds. As they cause the greatest side effects and are often abused by the public, their accurate analysis in plants and plant-derived laxatives is much needed. To isolate compounds from plants, pressurized liquid extraction (PLE) is frequently applied. The technique has been regarded, so far, as very effective, even in isolation of sensitive compounds for which exposure time in high temperature has a negative impact. This work demonstrates some interesting and surprising results accompanying PLE of hydroxyanthraquinones from the Rumex crispus L. root using methanol/water mixtures as extractant. The presented results demonstrate that glycoside forms of hydroxyanthraquinones (emodin-8-O-β-D-glucopyranoside, chrysophanol-8-O-β-D-glucopyranoside, and physcion-8-O-β-D-glucopyranoside) are hydrolytically unstable even in the short-lasting PLE. The increase of water concentration in the extractant leads to the increase of the transformation degree of the glycoside forms to the corresponding aglycones (emodin, chrysophanol, and physcion), increasing the concentration of the latter. The rise in the PLE temperature accelerates the hydrolytical degradation of the glycoside forms. The extension of the extraction time also intensifies this process. The presented results show that extraction of glycosides using extractants containing water can lead to false conclusions about the chemical composition of plants. Figure Hydrolytical instability of hydroxyanthraquinone glycosides even in the short-lasting PLE Electronic supplementary material The online version of this article (doi:10.1007/s00216-014-7744-5) contains supplementary material, which is available to authorized users.

Equisetum giganteum L., commonly called "giant horsetail", is an endemic species of Latin America. Its aerial parts have been widely used in ethnomedicine as a diuretic and in herbal medicine and food supplements as a raw material. The phenolic composition of E. giganteum stems was studied by liquid chromatography coupled to diode array detection (LC-DAD) and liquid chromatography coupled to electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), which identified caffeic acid derivatives, flavonoids and styrylpyrones. The most abundant glycosilated flavonoids in this sample were kaempferol derivatives. Other rare phenolic components, namely, quercetin-3-O-(caffeoyl)-glucoside and 3-hydroxyhispidin-3,4'-di-O-glucoside, were reported for first time in the Equisetum genus. An LC-UV method for the simultaneous quantification of flavonoid aglycones in E. giganteum obtained after hydrolysis was developed and validated. The method exhibited excellent linearity for all analytes, with regression coefficients above 0.998, LOD ≥ 0.043μg mL(-1), LOQ ≥ 0.158 μg mL(-1) and recovery rates of 96.89-103.33% and 98.22-102.49% for quercetin and kaempferol, respectively. The relative standard deviation for the intra- and inter-day precision was ≤ 3.75%. The hydrolysis process was optimized by central composite rotational design and response surface analysis. The second-order response models for the aglycones contents were as follows: quercetin (μg g(-1))=24.8102+55.2823 × HCl+0.776997 × Time-7.23852 × HCl(2)-7.46528E-04 × Time(2)-0.229167 × HCl × Time; kaempferol (μg g(-1))=-9.66755+974.822 × HCl+11.8059 × Time-130.612 × HCl(2)-0.0125694×Time(2) -3.22917 × HCl × Time, with estimated optimal conditions of 1.18 M HCl and 205 min of hydrolysis. The results obtained with these new methods were compared to those from a spectrophotometric assay used to determine the total flavonoids in the Equisetum arvense monograph (Horsetail, British Pharmacopoeia 2011). For all four species analyzed (E. giganteum, E. arvense, E. hyemale and E. bogotense), the calculated aglycone content was higher using the optimized hydrolysis conditions. Additionally, the LC method was more appropriate and specific for quantitative analysis.

Background : Silk sericin and a few non-protein components isolated from the cocoon layer including two silk proteins in silkworm Bombyx mori has many bioactivities. The dietary sericin possess antinatural oxidation, anticancer, antihyperlipidemic, and antidiabetic activities. The non-protein components surrounding the sericin layer involve in wax, pigments mainly meaning flavonoids, sugars, and other impurities. However, very few investigations have reported the estimation of the total flavonoids derived from the cocoon layer. The flavonoids are commonly present in their glycosylated forms and mostly exist as quercetin glycosides in the sericin layers of silkworm cocoons. Objective : The aim of this study was to find a more accurate method to estimate the level of the total flavonoids in silkworm cocoons. Design : An efficient procedure of hydrolysis-assisted extraction (HAE) was first established to estimate the level of the total flavonoids through the determination of their aglycones, quercetin, and kaempferol. Then, a comparison was made between traditional colorimetric method and our method. In addition, the antioxidant activities of hydrolysis-assisted extract sample were determined. Results : The average contents of quercetin and kaempferol were 1.98 and 0.42 mg/g in Daizo cocoon. Their recoveries were 99.56 and 99.17%. The total sum of quercetin and kaempferol was detected to be 2.40±0.07 mg/g by HAE-HPLC, while the total flavonoids (2.59±0.48 mg/g) estimated by the traditional colorimetric method were only equivalent to 1.28±0.04 mg/g of quercetin. The HAE sample also exhibits that IC 50 values of scavenging ability of diphenyl picryl hydrazinyl (DPPH) radical and hydroxyl radical (HO·) are 243.63 µg/mL and 4.89 mg/mL, respectively. Conclusions : These results show that the HAE-HPLC method is specificity of cocoon and far superior to the colorimetric method. Therefore, this study has profound significance for the comprehensive utilization of silkworm cocoon and also may be applied to the estimation of total flavonoids in other functional foods. Keywords: silkworm cocoon; total flavonoids; aglycones; quercetin; kaempferol; hydrolysis; extraction (Published: 14 March 2016) Citation: Food & Nutrition Research 2016, 60: 30932 - http://dx.doi.org/10.3402/fnr.v60.30932

Phytoalexins from soya are mainly characterised as prenylated pterocarpans, the glyceollins. Extracts of non-soaked and soaked soya beans, as well as that of soya seedlings, grown in the presence of Rhizopus microsporus var. oryzae, were screened for the presence of prenylated flavonoids with a liquid chromatography/mass spectrometry (LC/MS)-based screening method. The glyceollins I–III and glyceollidins I–II, belonging to the isoflavonoid subclass of the pterocarpans, were tentatively assigned. The formation of these prenylated pterocarpans was accompanied by that of other prenylated isoflavonoids of the subclasses of the isoflavones and the coumestans. It was estimated that approx. 40% of the total isoflavonoid content in Rhizopus-challenged soya bean seedlings were prenylated pterocarpans, whereas 7% comprised prenylated isoflavones and prenylated coumestans. The site of prenylation (A-ring or B-ring) of the prenylated isoflavones was tentatively annotated using positive-ion mode MS by comparing the 1,3AR retro-Diels-Alder (RDA) fragments of prenylated and non-prenylated isoflavones. Furthermore, the fragmentation pathways of the five pterocarpans in negative-ion (NI) mode were proposed, which involved the cleavage of the C-ring and/or D-ring. The absence of the ring-closed prenyl (pyran or furan) gave exclusively -H2Ox,yRDA fragments, whereas its presence gave predominantly the common RDA fragments.

The biosynthetic pathway of oleuropein (from 7-ketologanin, oleoside-11-methyl ester, 7-β-1-d-glucopyranosyl-11-methyl oleoside, and ligstroside to oleuropein) was investigated in two fruit species of Oleaceae, namely, Arbequina and Hojiblanca. Main oleuropein precursors and their metabolites, produced by the enzymatic hydrolysis mediated by β-glucosidase, were identified and quantified to establish the oleuropein transformation pathway. Changes in the concentration of these compounds were measured by direct control of in vivo fruit tissue during their ripening. High contents of aglycones at the initial stage of the process were caused by the high activity of β-glucosidase, which supports that oleuropein biosynthesis is coupled with enzymatic hydrolysis, producing its aglycone form. The low oleuropein content at this initial stage was caused by the imbalance between catabolic and anabolic pathways, favoring the former ones. Once the main polyphenol synthesis phase was completed, the biosynthetic capacity diminished and the content of all compounds decreased. Mass balance revealed that precursors of oleuropein, which are rapidly transformed by β-glucosidase and esterases, scarcely contributed to the accumulation of oleuropein. The biosynthetic pathway proposed by Damtoft applies for both varieties, but our study reveals that the β-glucosidase enzyme is involved in oleuropein synthesis. This enzyme shows high substrate specificity to oleuropein, which consequently is degraded to its aglycone form, with diminished efficacy of oleuropein biosynthesis. Different enzymatic activities of varieties will result in oleuropein accumulation and metabolic transformation of phenols., This study was possible by the financial support of the Ministerio de Ciencia e Innovación (Spanish Government, AGL2007-66139-C02).

Aim. This work is devoted a phytochemical study of biologically active substances in herb of Labrador Tea (Lédum palústre L.), as well as investigation of the possibility of complex use of raw materials in order to create on its basis new herbal medicines.Methods. The object of the study served as the herb Labrador Tea. Separation of isolated substances was performed by adsorption and partition chromatography on various adsorbents. The structure of the isolated compounds was determined based on physicochemical methods: paper (PC) and the thin layer (TLC) chromatography, UV, IR and NMR spectroscopy in comparison with the original valid standards of flavonoids. Acid hydrolys was used for the determination of the flavonoid aglycone composition. The content of total flavonoids was determined by differential spectrophotometry at a wavelength of 412 nm from the reaction with aluminum chloride based on the hyperoside-standard (Ukrainian scientific Pharmacopoeial center for quality of medicines (Pharmacopoeial center), Kharkоv).Results. During this study we found more than 40 of phenolic compounds and identified 31compounds. The phytochemical analysis showed the presence of flavonols (11 compounds), catechins (5). Hydroxycinnamic acids represented by caffeic, ferulic, chlorogenic acids. Besides that, it were found phenolic glycoside arbutin, coumarins - coumarin, umbelliferon, scopoletin, esculetin and esculin, tannins – metyl gallate, pyrogallol.Conclusions. The following biologically active substances were defined for the first time: flavonoids – 5-methyl-kaempferol, avicularin, polistahozid, quercitrin; coumarins – esculetin, esculin; as well as hydroxycinnamic acids – ferulic, chlorogenic, neochlorogenic of Labrador Tea. The obtained data justify the prospectivefor creation of new and effective herbal medicines from Labrador Tea, Цели. Данная работа посвящена фитохимическому изучению биологически активных веществ багульника болотного (Lédum palústre L.), а также исследованию возможности комплексного использования этого сырья для создания на его основе новых растительных препаратов.Методы. Объектом изучения служила трава багульника болотного. Разделение выделенных веществ осуществляли с помощью адсорбционной и распределительной хроматографии на различных сорбентах. Структуру выделенных соединений устанавливали с помощью физико-химических методов: данных бумажной (БХ) и тонкослойной (ТСХ) хроматографии, УФ-, ИК- и ЯМР-спектроскопии в сравнении с исходными флавоноидами, их продуктами превращения с достоверными образцами. Для определения агликонового состава флавоноидов использовали кислотный гидролиз. Содержание суммы флавоноидов определяли методом дифференциальной спекторофотометрии в видимой области при длине волны 412 нм по реакции с алюминия хлоридом в пересчете на гиперозид-стандарт (ГП "Украинский научный фармакопейный центр качества лекарственных средств", г. Харьков).Результаты. В результате проведенных исследований обнаружено более 40 компонентов фенольной природы, из которых идентифицировано 31 соединение. Из них больше всего представлены флавоноиды, относящиеся к флавонолам (11 соединений), катехинам (5). Гидроксикоричные кислоты представлены кофейной, феруловой, хлорогеновой кислотами. Кроме того, обнаружены фенологликозид арбутин, кумарины – кумарин, умбеллиферон, скополетин, эскулетин и эскулин. Дубильные вещества представлены метилгаллатом, пирогаллолом.Выводы. Вещества флавоноловой природы – 5-метил-кемпферол, авикулярин, полистахозид, кверцитрин, кумарины – эскулетин, эскулин, а также гидроксикоричные кислоты – феруловая, хлорогеновая, неохлорогеновая из багульника болотного нами выделены впервые. Полученные данные обосновывают перспективность комплексного использования багульника болотного для разработки новых эффективных растительных препаратов

Lactobacillus plantarumBET003 isolated fromMomordica charantiafruit was used to ferment its juice.Momordica charantiafresh juice was able to support good growth of the lactic acid bacterium. High growth rate and cell viability were obtained without further nutrient supplementation. In stirred tank reactor batch fermentation, agitation rate showed significant effect on specific growth rate of the bacterium in the fruit juice. After the fermentation, initially abundant momordicoside 23-O-β-Allopyranosyle-cucurbita-5,24-dien-7α,3β,22(R),23(S)-tetraol-3-O-β-allopyranoside was transformed into its corresponding aglycone in addition to the emergence of new metabolites. The fermentedM. charantiajuice consistently reduced glucose production by 27.2%, 14.5%, 17.1% and 19.2% at 15-minute intervals respectively, when compared against the negative control. This putative anti-diabetic activity can be attributed to the increase in availability and concentration of aglycones as well as other phenolic compounds resulting from degradation of glycosidic momordicoside. Biotransformation ofM. charantiafruit juice via lactic acid bacterium fermentation reduced its bitterness, reduced its sugar content, produced aglycones and other metabolites as well as improved its inhibition ofα-glucosidase activity compared with the fresh, non-fermented juice.

Soy isoflavones (SIF) are present in the systemic circulation as conjugated forms of which the estrogenic potency is not yet clear. The present study provides evidence that the major SIF glucuronide metabolites in blood, genistein-7-O-glucuronide (GG) and daidzein-7-O-glucuronide (DG), only become estrogenic after deconjugation. The estrogenic potencies of genistein (Ge), daidzein (Da), GG and DG were determined using stably transfected U2OS-ERα, U2OS-ERβ reporter gene cells and proliferation was tested in T47D-ERβ cells mimicking the ERα/ERβ ratio of healthy breast cells and inT47D breast cancer cells. In all assays applied, the estrogenic potency of the aglycones was significantly higher than that of their corresponding glucuronides. UPLC analysis revealed that in U2OS and T47D cells, 0.2–1.6% of the glucuronides were deconjugated to their corresponding aglycones. The resulting aglycone concentrations can account for the estrogenicity observed upon glucuronide exposure. Interestingly, under similar experimental conditions, rat breast tissue S9 fraction was about 30 times more potent in deconjugating these glucuronides than human breast tissue S9 fraction. Our study confirms that SIF glucuronides are not estrogenic as such, and that the small % of deconjugation in the cell is enough to explain the slight bioactivity observed for the SIF-glucuronides. Species differences in deconjugation capacity should be taken into account when basing risk–benefit assessment of these SIF for the human population on animal data.

Custard desserts were enriched with a soy germ extract as source of isoflavones and the influence of the thickening agent (starch or carboxymethylcellulose (CMC)) and the presence of fat on the release, partitioning and stability of the isoflavones after mouth, stomach and small intestine in vitro incubations were studied. A suspension in water of the soy germ extract was first analyzed. Mouth incubation did not affect the amount and partitioning of isoflavones. A lower recovery and different partitioning were found after the stomach incubation, which was associated to the low pH. After the intestine incubation, a higher recovery and an effect in the partitioning were found. Regarding the effect of the custard matrix, the starch custards were able to release a significantly higher amount of isoflavones than the CMC ones, due to the higher general enzymatic resistance of the latter one's. The presence of fat significantly increased the bioaccessibility of the aglycone forms, especially of genistein.

We describe a method for the determination of the two major urinary metabolites of vitamin K as the methyl esters of their agyclone structures, 2-methyl-3-(3-3-carboxymethylpropyl)-1,4-naphthoquinone (5C-side-chain metabolite) and 2-methyl-3-(5-carboxy-3-methyl-2-pentenyl)-1,4-naphthoquinone (7C-side-chain metabolite), by HPLC with electrochemical detection (ECD) in the redox mode. Urinary salts were removed by reversed-phase (C18) solid phase extraction (SPE) and the predominately conjugated vitamin K metabolites hydrolysed with methanolic HCl. The resultant carboxylic acid aglycones were quantitatively methylated with diazomethane and fractionated by normal-phase (silica) SPE. Final analysis was by reversed-phase (C18) HPLC with a methanol-aqueous mobile phase. Metabolites were detected by amperometric, oxidative ECD of their quinol forms, which were generated by post-column coulometric reduction at an upstream electrode. The assay gave excellent linearity (r2 typically = 0.999) and high sensitivity with an on-column detection limit of

A liquid chromatographic (LC) method for the separation of both anthraquinone glycosides and aglycones in extracts of Rubia tinctorum was improved. For on-line MS detection atmospheric pressure chemical ionisation as well as electrospray ionisation (ESI) were used. The glycosides were ionised in both positive and negative ionisation (NI) mode, the aglycones only in the NI mode. With ESI ammonia was added to the eluent post-column to deprotonate the compounds. The efficiency of mass detection of the hydroxyanthraquinone aglycones was found to depend on the pK(a) value of the component. LC-diode-array detection and LC-MS provide useful complementary information for the identification of anthraquinones in plant extracts, which was proven with the identification of munjistin and pseudopurpurin. (C) 2002 Elsevier Science B.V. All rights reserved.

A liquid chromatographic (LC) method for the separation of both anthraquinone glycosides and aglycones in extracts of Rubia tinctorum was improved. For on-line MS detection atmospheric pressure chemical ionisation as well as electrospray ionisation (ESI) were used. The glycosides were ionised in both positive and negative ionisation (NI) mode, the aglycones only in the NI mode. With ESI ammonia was added to the eluent post-column to deprotonate the compounds. The efficiency of mass detection of the hydroxyanthraquinone aglycones was found to depend on the pK(a) value of the component. LC-diode-array detection and LC-MS provide useful complementary information for the identification of anthraquinones in plant extracts, which was proven with the identification of munjistin and pseudopurpurin. (C) 2002 Elsevier Science B.V. All rights reserved.

Anthocyanins are large group of water soluble plant pigments responsible for the red, purple, and blue colours of many fruits, vegetables, flowers and grains. Chemically they are subdivided into the sugar-free anthocyanidine aglycons and the anthocyanin glycosides. In nature, there are 6 common anthocyanidins: pelargonidin, cyanidin, peonidin, delphinidin, petunidin, and malvidin. The colour of anthocyanins depends on their structure which can vary by glycosidic substitution at the positions 3 and 5 on the rings A and C as well as by acylation of the sugar groups with organic acids. Anthocyanins are responsible for the colour of red grapes and wine, so different authors have used their profile to classify the grape cultivars and wine authenticity. Thereby, the aim of this study was to investigate the profile of anthocyanidins (malvidin, delphinidin, cyanidin, pelargonidin and peonidin) in grape skin extracts of autochthonous Dalmatian cultivars: Babica, Ljutun and Crljenak. The extracts of anthocyanins were acid-hydrolyzed to release the corresponding anthocyanidins that were analysed by HPLC. HPLC analysis of the acid hydrolysis products of the grape berry skin extracts showed malvidin to be the major anthocyanidin in all extracts (ranged from 52 to 60%). Extract of Ljutun contained also high amounts of delphinidin (22%) while the lowest amounts of peonidin (from 5 to 6%) were found in all extracts. Obtained results on anthocyanidin profile of investigated grape skin extracts could be useful in chemotaxonomy for classifying red grape cultivars and wines.

The objective of this study was to investigate the immobilisation efficiency of soybean β-glucosidase (181.6 U/mL; 23.8 mg protein/mL) on activated chitosan beads. Central Composite Rotational Design (CCDR) 2 was used and the application of immobilised enzyme in commercial soy drink was evaluated. The activation of chitosan beads was achieved with established 2.5% glutaraldehyde, pH 7.5, 8 h incubation time (6 h with agitation and 2 h without agitation) at 37ºC. The highest immobilisation efficiency (%) of soybean β-glucosidase on chitosan beads obtained was 37.74 U/mL and 18.84 mg protein/4 chitosan beads at pH 7.5 and 20 h coupling time of enzyme-matrix (7 h with agitation and 13 h without agitation) at 4ºC. The immobilised enzyme incubated at 50ºC, pH 5.5 resulted in 24% increase in the aglycones content in commercial soy drink after 60 min. Made available in DSpace on 2014-09-17T07:37:21Z (GMT). No. of bitstreams: 1 SoybeanGlucosidaseimmobilisatedonvhitosanbeads.pdf: 179218 bytes, checksum: 7028a8bbb354cd7ef966b91800e7bc46 (MD5) Previous issue date: 2014-09-03

The enzyme beta-glucosidase hydrolyses the isoflavone glucosides developing aglycones, which are compounds with anticancer effects, that are also related with the astringency observed in soybean flavor. Due to the importance of this enzyme, a study was carried out to determine beta-glucosidase activity in soybean (Glycine max (L.) Merrill) cultivars with different contents of isoflavone glucosides (enzyme substrate). The enzyme activity was determined in 51 soybean cultivars sowed in Londrina (latitude 23ºS), in Paraná State, Brazil, and in the cultivar IAS 5 from soybean production regions of different Brazilian states. Among the cultivars, a range of variability of 176.1 to 96.3 units of enzyme activity (cultivars IAC-2 and Embrapa 2, respectively) was observed. A significant variability among cultivars could suggest genetic differences. In the states of Rio Grande do Sul, Paraná and Mato Grosso do Sul, the cultivar IAS 5 presented similar average of beta-glucosidase activity: 132.1, 131.9 and 132.5 units, respectively. Among locations in the states, the cultivar IAS 5 presented a variability for enzyme activity from 138.8 to 124.8 units, which were statistically different. In spite of statistics, the numerical values were not too different to assume that environmental conditions affected enzyme activity. A non-significative correlation for isoflavone glucoside concentrations and enzyme activity was observed among cultivars.

In our research, a phytochemical screening was performed by the method of Cain - Bohmann modified yielding presence of good amount of saponins, which were identified as triterpenoid saponins using qualitative tests that were positive and the test of the foam reaction with the reagent Salwosky, Bouchard Liberman reagent and reaction with sulfuric acid. Three quantitative methods for extracting saponins from a hydroalcoholic extract of the plant and subsequent hydrolysis were developed. The difference of the first two methods was to use a method with the sample defatted with chloroform and the other without scouring . For the third method according to the procedure applied M.E.Wall et al. (1958). Extraction rates for the three methods saponins were 3.90%, 2.40 % and 2.02% respectively. Similarly the corresponding aglycones after acid were obtained by hydrolyzing the crude obtained via., obteniéndose presencia de buena cantidad de saponinas, las cuales fueron identificadas como saponinas triterpenoides utilizando pruebas cualitativas que dieron positivas como la prueba de la espuma, reacción con el reactivo de Salwosky, reactivo de Liberman-Bouchard y reacción con el ácido sulfúrico. Se desarrollaron tres metodologías cuantitativas para la extracción de las saponinas a partir de un extracto hidroalcohólico de la planta y su posterior hidrólisis. La diferencia de los dos primeros métodos consistió en emplear un método con la muestra desengrasada con cloroformo y el otro sin desengrasar. Para el tercer método se aplicó el procedimiento según M.E.Wall et al. (1958). Los porcentajes de extracción de saponinas para las tres metodologías fueron de 3,90%, 2,40% y 2,02% respectivamente. Igualmente se obtuvieron las agliconas correspondientes luego de hidrolizar por vía ácida el crudo obtenido.

The bioavailability of soy isoflavones depends on the composition of the microflora for each subject. Bacteria act on different isoflavones with increased or reduced absorption and cause biotransformation of these compounds into metabolites with higher biological activity. S-equol is the most important metabolite and only 25–65 % of the population have the microflora that produces this compound. The presence of equol-producing bacteria in soy product consumers means that the consumption of such products for prolonged periods leads to lower cardiovascular risk, reduced incidence of prostate and breast cancer, and greater relief from symptoms related to the menopause such as hot flushes and osteoporosis.

Flavonoids from vegetative parts of 29 species of Eurasian Sedum, Sedum meyeri-johannis from central East Africa, 34 species of Sempervivum, and Jovibarba heuffelii have been identified after acid hydrolysis. Ten flavonoid aglycones were detected, i.e. kaempferol, herbacetin, sexangularetin, quercetin, gossypetin, corniculatusin, isorhamnetin, limocitrin, myricetin, and possibly hibiscetin. Quantitative flavonoid data were obtained by gas chromatography after trimethylsilylation and were studied with multivariate data analysis methods. Principal components analysis of the whole data set distinguishes Sedum and Sempervivum as separate groups. Flavonoid variation in Sempervivum is minimal (kaempferol is the principal flavonol of all species), which reflects the morphological uniformity of the genus and similarity in ecological preference of the species. By contrast, Eurasian Sedum contains a much wider range of flavonols and shows a high degree of parallel evolution (notably 8-hydroxylation and 8-O-methylation), which seems to mirror the enormous morphological and cytological variation present in this taxon. The recognition of groups in Sedum by principal components analysis of flavonoid data partially supports the infrageneric classification based on biosystematic data.

Devido à maior concentração de isoflavonas nos hipocótilos dos grãos de soja, a indústria de alimentos tem utilizado esse componente do grão como matéria-prima. No presente trabalho, foram quantificados os teores de isoflavonas nas estruturas formadoras do grão de soja (hipocótilo, cotilédones e casca), analisadas por Cromatografia Líquida de Alta Eficiência (CLAE). Foram avaliadas quatro cultivares de sojas especiais: BRS 184, BRS 216, BRS 257 e BRS 267, desenvolvidas pela Embrapa Soja. Todas as cultivares apresentaram maior teor de isoflavonas totais no hipocótilo (média de 2029,32 mg.100 g-1). No grão inteiro (267,16 mg.100 g-1) e nos cotilédones (209,16 mg.100 g-1), os teores de isoflavonas foram cerca de dez vezes menores do que nos hipocótilos. A cultivar BRS 216 apresentou o teor maior de isoflavonas totais no hipocótilo (2583,20 mg.100 g-1). Embora a casca da soja não apresente isoflavonas, observou-se que esses compostos estavam presentes nessa parte do grão numa quantidade média de 105,58 mg.100 g-1, devido à presença de resíduos do hipocótilo e dos cotilédones provenientes do processo de descasque. Essa composição residual da casca favorece sua utilização como matéria-prima fonte de fibras, porque também fornece isoflavonas e outros compostos da soja. Due to the higher concentration of isoflavones in the soybean hypocotyls, the food industry has used this component of the bean as a raw material. This paper reports the isoflavone contents found in the different parts of the bean (hypocotyl, cotyledon and hull) as analyzed by High Performance Liquid Chromatography (HPLC). Four special soybean cultivars, BRS 184, BRS 216, BRS 257 and BRS 267, developed by Embrapa Soybean, were evaluated. All the cultivars presented higher contents of total isoflavones in the hypocotyls (average of 2029.32 mg.100 g-1). The isoflavone contents of the whole beans (267.16 mg.100 g-1) and the cotyledons (209.16 mg.100 g-1) were about 10 times lower than that of the hypocotyls. Cultivar BRS 216 presented the highest total isoflavone content in the hypocotyl (2583.20 mg.100 g-1). Although soybean hulls do not contain isoflavones, an average of 105.57 mg.100 g-1 was found in this component, due to the presence of hypocotyl and cotyledon residues resulting from the hulling process. This residual composition of the hulls favors their use as a raw material that is a source of fibers, also providing isoflavones and other soybean compounds.

The present study investigates oleuropein metabolism, as well as the involvement of β-glucosidase during the growth, development, and ripening of olive fruit. The results show that in olive fruit the in vivo formation and transformation of oleuropein takes place in three different stages. The first one is characterized by a net accumulation of oleuropein and occurs in the immature fruit. In the second stage, associated with the green and light-green fruits, oleuropein content is maintained practically constant, and finally, a third stage that begins in the green-yellow fruit is characterized by a progressive decline of the oleuropein concentration. Our findings confirm that in the absence of β-glucosidase the Damtoft-proposed pathway is active and that net synthesis of oleuropein is unquestionable. β-Glucosidase activity plays a key role in the oleuropein metabolism catalyzing its in vivo hydrolysis. © 2012 American Chemical Society., This study was possible by the financial support of the Ministerio de Ciencia e Innovacioń (Spanish Government, AGL2007−66139-C02).

0009-3130/11/4701-0105 2011 Springer Science+Business Media, Inc. 1) Laboratoire de Phytochimie et Analyses Physico-Chimiques et Biologiques, Universite Mentouri, Route de Ain El Bey, 25000 Constantine, Algeria, fax: 213 31 81 88 83, e-mail: fbenayache@yahoo.fr; 2) Laboratoire de Valorisation des Ressources Naturelles et Synthese de Substances Biologiquement Actives, Universite Mentouri, Route de Ain El Bey, 25000 Constantine, Algeria; 3) Instituto de Productos Naturales y Agrobiologia, C.S.I.C. Instituto Universitario de Bio-Organica “Antonio Gonzalez”, Universidad de La Laguna, Av. Astrofisico F. Sanchez 3, 38206 La Laguna, Tenerife, Spain. Published in Khimiya Prirodnykh Soedinenii, No. 1, pp. 94–95, January–February, 2011. Original article submitted January 19, 2009. Chemistry of Natural Compounds, Vol. 47, No. 1, March, 2011 [Russian original No. 1, January–February, 2011]

Phytoalexins from soya are mainly characterised as prenylated pterocarpans, the glyceollins. Extracts of non-soaked and soaked soya beans, as well as that of soya seedlings, grown in the presence of Rhizopus microsporus var. oryzae, were screened for the presence of prenylated flavonoids with a liquid chromatography/mass spectrometry (LC/MS)-based screening method. The glyceollins I–III and glyceollidins I–II, belonging to the isoflavonoid subclass of the pterocarpans, were tentatively assigned. The formation of these prenylated pterocarpans was accompanied by that of other prenylated isoflavonoids of the subclasses of the isoflavones and the coumestans. It was estimated that approx. 40% of the total isoflavonoid content in Rhizopus-challenged soya bean seedlings were prenylated pterocarpans, whereas 7% comprised prenylated isoflavones and prenylated coumestans. The site of prenylation (A-ring or B-ring) of the prenylated isoflavones was tentatively annotated using positive-ion mode MS by comparing the 1,3AR retro-Diels-Alder (RDA) fragments of prenylated and non-prenylated isoflavones. Furthermore, the fragmentation pathways of the five pterocarpans in negative-ion (NI) mode were proposed, which involved the cleavage of the C-ring and/or D-ring. The absence of the ring-closed prenyl (pyran or furan) gave exclusively -H2Ox,yRDA fragments, whereas its presence gave predominantly the common RDA fragments.

Among several functional foods available nowadays, isoflavones are important due to beneficial biological effects to human health (estrogenic, antiestrogenic, antioxidant, and antifungal activity among others) particularly because of phytoestrogens, which are the most common isoflavone types and are predominantly found in legumes. They are especially abundant in soybean, its farinaceous products, and subproducts such as soy molasses. In nature isoflavones are predominantly found as glycosides (daidzin, genistin and glycitein) which are not assimilated by our organism due to the presence of sugar in their composition. For the assimilation of isoflavones to occur in the body, it is necessary that they are in their aglycone form (without the presence of sugar molecule). The aim of this work was the bioconversion of glycoside compounds into aglycones using soy molasses as an isoflavone source. The β-glucosidase enzyme used in bioconversion was produced from different filamentous microorganisms of the genre Aspergillus (niger ATCC 16404, oryzae ATCC 1003, niger isolated from soil at the 'Laboratório Ambiental' of 'Itaipu Binacional', niger isolated from indoor atmosphere at the 'Laboratório Ambiental' of 'Itaipu Binacional') using the process of solid state fermentation (SSF), and wheat bran and defatted soy flour as substrates. The crude extract containing the enzyme β-glucosidase was used directly to the bioconversion process without prior purification treatment. The highest enzyme specific activity value, 7.6 U/mg, was obtained using Aspergillus oryzae ATCC 1003 and wheat bran as substrate. The value of 5.9 U/mg enzyme activity was obtained using the Aspergillus niger indoor air employing the substrate with defatted soy flour. The reaction of hydrolysis using 16404 Aspergillus niger, Aspergillus oryzae ATCC 1003, Aspergillus niger and Aspergillus niger soil indoor air for the production of crude enzyme with β-glucosidase at 40 °C for 30 minutes provided 100% bioconversion of all glycoside compounds into aglycones. All enzymes produced in this study using the genre Aspergillus converted glycoside isoflavone compunds (daidzin and genistin) into aglycone isoflavone compounds (daidzein and genistein). Glycoside and aglycone compounds were quantified by high performance liquid chromatography (HPLC). The proposed methodology was efficient for bioconversion of aglycones into glycosides from industrial waste of soy molasses. Dentre os diversos alimentos funcionais disponíveis atualmente, as isoflavonas se destacam pelos efeitos biológicos e benéficos à saúde humana (atividade estrogênica, anti-estrogênica, antioxidante, antifúngica, entre outros) e principalmente pela ação dos fitoestrógenos, que são as formas mais comuns das isoflavonas, sendo predominantemente encontradas em leguminosas. São especialmente abundantes na soja e seus derivados farináceos e subprodutos, como o melaço de soja. Na natureza, as isoflavonas são encontradas predominantemente na forma de glicosídicos (daidzina, genistina e glicitina), os quais não são assimilados pelo organismo, devido à presença deste açúcar em sua composição. Para que ocorra a assimilação das isoflavonas pelo organismo, é necessário que a mesma se encontre em sua forma aglicona (sem a presença da molécula de açúcar). O presente trabalho propôs como objetivo a bioconversão de compostos glicosídicos em agliconas usando o melaço de soja como fonte de isoflavonas. A enzima β-glicosidase utilizada na bioconversão foi produzida a partir de diferentes microrganismos filamentosos do gênero Aspergillus (niger ATCC 16404, oryzae ATCC 1003, niger isolado do solo no Laboratório Ambiental da Itaipu Binacional, niger isolado do ar indoor no Laboratório Ambiental da Itaipu Binacional) empregando o processo de fermentação no estado sólido (FES) contendo os substratos farelo de trigo e a farinha de soja desengordurada. O extrato bruto enzimático contendo a β-glicosidase foi empregado diretamento ao processo de bioconversão sem tratamento prévio de purificação. O maior valor de atividade enzimática determinado, 7,6 U/mg, foi obtido usando o Aspergillus oryzae ATCC 1003, empregando como substrato o farelo de trigo. O valor de 5,9 U/mg de atividade enzimática foi obtido usando o Aspergillus niger ar indoor, empregando o substrato com farinha de soja desengordurada. A reação da hidrólise usando os fungos Aspergillus niger 16404, Aspergillus oryzae ATCC 1003, Aspergillus niger solo e Aspergillus niger ar indoor para a produção de extrato bruto enzimático contento β-glicosidase a 40 °C por 30 minutos proporcionou 100% de bioconversão para todos os compostos glicosídicos em agliconas. Todas as enzimas produzidas neste estudo, utilizando-se fungos do gênero Aspergillus, convertem compostos de isoflavonas glicosídicas (daidzina e genistina) em compostos de isoflavonas agliconas (daidzeína e genisteína). Os compostos glicosídicos e agliconas foram quantificados por cromatografia líquida de alta eficiência (CLAE). A metodologia proposta mostrou-se eficiente para bioconversão de glicosídicos em agliconas a partir do resíduo industrial de melaço de soja. xiii, 58 f

Due to their substitution with an isoprenoid group, prenylated flavonoids have an increased affinity for biological membranes and target proteins, enhancing their potential bioactivity. Although many prenylated flavonoids have been described, there are no methods that specifically screen for their presence in complex mixtures, prior to purification. We describe a method based on ultra-high-performance liquid chromatography (UHPLC) with electrospray ionisation mass spectrometry (ESI-MS) that allows rapid screening for prenylated flavonoids in multi-component plant extracts. Identification of the prenylated flavonoids is based on screening for neutral losses of 42 u and 56 u in the positive-ion mode MS(2) and MS(3) spectra within the MS chromatograms. In addition, this method discriminates between a prenyl chain and a ring-closed prenyl (pyran ring), based on the ratio of the relative abundances of the ions that lose 42 u and 56 u (42:56). The application of this screening method on a 70% aq. ethanol, ethanol and ethyl acetate extract of the roots of Glycyrrhiza glabra indicated the presence of 70 mono- and di-prenylated flavonoids. In addition, of each prenylated flavonoid the type of prenylation, chain or pyran ring was determined.

International audience; The relationship between fruit maturation and volatile contents was investigated in two contrasted Cervil (CER) and Levovil (LEV) tomato (Solanum lycopersicum) lines. As fruits ripened, their volatile contents mainly increased. Although some compounds displayed contrasting patterns, overall, volatiles were clearly more abundant and conferred stronger aromas to CER than to LEV fruits. This intervarietal difference in volatile contents yielding much lower volatile contents in LEV was further investigated to determine whether it is due to a higher capacity of volatile glycosylation within LEV as compared to CER. Again, glycosides mainly increased during fruit maturation and were more abundant within CER than within LEV. Overall glycoside findings were indicative of a superior capacity to biosynthesize rather than an inferior capacity to glycosylate volatiles of CER. Eugenol and 2-methoxyphenol volatiles were exceptional compounds as they remained at higher levels in maturing LEV than in CER. 2-Methylthioacetaldehyde was for the first time identified as putatively related to differences of aroma between lines, as it was abundant in Cervil but absent in Levovil. Considering the described odor value of these three products, they should contribute differently to the particular olfactive features of LEV and CER fruits

The present paper represents the continuation of our research of free (essential oil) and glycosidically bound volatile compounds from Croatian wild-growing aromatic plants. The essential oil and glycosidically bound volatile compounds were isolated from the fresh and dried plant material of Thymus pulegioides L. at three stages of plant development and analysed by gas chromatography-mass spectrometry (GC-MS). The most abundant constituents in essential oils were geraniol and linalool followed by thymol, g-terpinene, thymol methyl ether, borneol and geranyl acetate, all of which showed quantitative variations during the plant development stages and whether fresh or dried plants were used for essential oil isolation. After enzymatic hydrolysis of glycosidic fractions, the GC-MS analysis of volatile aglycones revealed eugenol, geraniol and 1-octen-3-ol as the main aglycones in all samples. Other important aglycones were thymoquinone, 2-phenylethanol, 3-hexenol, 3-octanol, benzyl alcohol and linalool. Quantitative variations were observed when the aglycones were analysed at three different stages of plant development. Because of the high geraniol content in the essential oils and in the aglycone fractions this Thymus pulegioides L. could be interesting as a fragrant material for perfume industry or for food flavouring.

Iberis sempervirens L. belongs to Brassicaceae family. The plants of this family contain glucosinolates. The glucosinolates and their aglycones are biological active compounds. Composition of the obtained aglycones depends on the structure of the side chains in glucosinolates and degradation conditions such as pH-values, temperatures, Fe2+ ions and specific protein (Ikan, 1999.). Up to now, the chemical composition of these aglycones and O-aglycones from candytuft has not been reported. The volatiles of Iberis sempervirens were isolated by hydrodistillation and analyzed using GC and GC/MS. The volatiles were isolated from fresh plant material prior and after the autolysis (Al-Gendy et al., 2003.) and from dried plant material. Isothiocyanates, nitriles aliphatic alcohols, carbonyls, fatty acids, hydrocarbons, terpene compounds, C13-norisoprenoids and phenylpropane derivatives were identified. The major components in all samples were: 3-butenyl isothiocyanate (17.4-63.3%), 5-methylthiopentanonitrile (2.2-9.5%), 4-methylthiobutanonitrile (4.8-6.9%), allyl isothiocyanate (4.0-6.6%). Other compounds were 3-methylthiopropyl isothiocyanate, phenyl isothiocyanate, benzenepropanonitrile, 1-cyano-4, 5-epithiopentane. The volatiles that were isolated after the autolysis are more complex then other two isolates. Some of them were identified in O-aglycones as eugenol, (Z)-3-hexene-1-ol, 1H-indole. Further, from the fresh plant material O-glycosides of volatile compounds were isolated and purified by selective extraction and column chromatography. After hydrolysis of O-glycosides using β -glucosidase liberated volatile O-aglycones were also analysed by GC and GC/MS. Compounds with sulfur and nitrogen (which are characteristic for glucosinolates) were not identified among these compounds. Nineteen O-aglycones were identified. The main O-aglycones were: eugenol (15.2%), 2-phenylethanol (12.9 %), 2-hydroxy-β -ionone (10.6%), 2-tert-buthyl-5-methylphenol (9.2%) and methyl-2, 5-dihidroxybenzoate (4.5%).

Studies were carried out to enhance the development of isoflavone aglycones in soybean. Grains of two soybean cultivars, BR 36 and IAS 5, 115 and 278 mg/100g of total isoflavone, respectively, were treated hydrothermalyl at 45, 60 and 85° C for 5, 30 and 60 minutes. Pre-treatments of grains at 60° C for 60 minutes allowed a considerable increase of the isoflavone aglycones. Non-treated grains of BR 36 and IAS 5 showed 1.2 mg/100g of genistein, after hydrothermal treatments,which increased to 12 and 53 mg/100g, in each variety, respectively. At higher temperature (85 ° C) there was a decrease of the aglycones due to inactivation of beta-glycosidases. Malonyl compounds were also reduced at higher temperatures. In processing functional soybean foods, hydrothermal treatments of the soybean grains, as well as high isoflavone content soybean cultivars will enhance development of aglycone forms.Estudos foram conduzidos para aumentar isoflavonas agliconas (compostos mais biodisponíveis e mais efetivos na prevenção de doenças crônicas) em grãos de soja. Pré-tratamentos hidrotérmicos dos grãos foram conduzidos a 45, 60 e 85 ºC por 5, 30, e 60 minutos. Duas cultivares de soja BR 36 e IAS 5 (115, e 278 mg/100g de isoflavonas totais, respectivamente), foram usadas nos experimentos. Pré-tratamentos dos grãos a 60 ºC por 60 minutos permitiram um considerável aumento das isoflavonas agliconas. Grãos não tratados de BR 36 e IAS 5 apresentaram 1,2 mg/100g de genisteína. Depois dos tratamentos hidrotérmicos, este composto aumentou para 12 e 53 mg/100g, em cada variedade, respectivamente. Em altas temperatures (85 ºC) houve diminuição das agliconas devido a inativação das enzimas beta-glicosidases. Os compostos malonil (térmicamente instáveis), também foram reduzidos sob altas temperaturas. No processamento de alimentos funcionais de soja, pré-tratamentos hidrotérmicos dos grãos, bem como a utilização de cultivares com alto teor de isoflavonas permitirão maior desenvolvimento das formas agliconas.

A liquid chromatographic (LC) method for the separation of both anthraquinone glycosides and aglycones in extracts of Rubia tinctorum was improved. For on-line MS detection atmospheric pressure chemical ionisation as well as electrospray ionisation (ESI) were used. The glycosides were ionised in both positive and negative ionisation (NI) mode, the aglycones only in the NI mode. With ESI ammonia was added to the eluent post-column to deprotonate the compounds. The efficiency of mass detection of the hydroxyanthraquinone aglycones was found to depend on the pK(a) value of the component. LC-diode-array detection and LC-MS provide useful complementary information for the identification of anthraquinones in plant extracts, which was proven with the identification of munjistin and pseudopurpurin. (C) 2002 Elsevier Science B.V. All rights reserved.

LC-MS-MS is becoming a very important tool for the on-line identification of natural products in crude plant extracts. For an efficient use of this technique in the dereplication of natural products, a careful study of the parameters used to generate informative MS-MS spectra is needed. In this paper, the collision-induced dissociation (CED) MS-MS spectra of ubiquitous C-glycosidic flavonoids have been systematically studied using hybrid quadrupole time-of-flight and ion-trap (IT) mass analysers under various CID energy conditions. Efficient differentiation of flavonoid C-glycoside isomers was possible, based on the comparison of CID-MS-MS spectra of particular C-glycoside unit fragments. Striking differences between 6-C and 8-C flavonoid glycosides were especially observed in the product ion spectra of their X-0,2(+) fragments ([M+H-120](+)). Some guidelines for the on-line characterisation of C-glycosidic flavonoids by LC-MS-MS or LC-multiple-stage MS are given.

The enzyme β-glucosidase hydrolyses the isoflavone glucosides developing aglycones,which are compounds with anticancer effects, that are also related with the astringency observed insoybean flavor. Due to the importance of this enzyme, a study was carried out to determineβ-glucosidase activity in soybean ( Glycine max (L.) Merrill) cultivars with different contents of isoflavoneglucosides (enzyme substrate). The enzyme activity was determined in 51 soybean cultivars sowed inLondrina (latitude 23oS), in Parana State, Brazil, and in the cultivar IAS 5 from soybean productionregions of different Brazilian states. Among the cultivars, a range of variability of 176.1 to 96.3 units ofenzyme activity (cultivars IAC-2 and Embrapa 2, respectively) was observed. A significant variabilityamong cultivars could suggest genetic differences. In the states of Rio Grande do Sul, Parana and MatoGrosso do Sul, the cultivar IAS 5 presented similar average of β-glucosidase activity: 132.1, 131.9 and132.5 units, respectively. Among locations in the states, the cultivar IAS 5 presented a variability forenzyme activity from 138.8 to 124.8 units, which were statistically different. In spite of statistics, thenumerical values were not too different to assume that environmental conditions affected enzymeactivity. A non-significative correlation for isoflavone glucoside concentrations and enzyme activitywas observed among cultivars.Index terms: environmental effect, Glycine max , aglycones, soybean flavor.ATIVIDADE DE BETA-GLICOSIDASE E NIVEIS DE ISOFLAVONOIDES GLICOSIDIOSEM CULTIVARES DE SOJA, INFLUENCIADAS PELO AMBIENTERESUMO - A enzima β-glicosidase hidrolisa os isoflavonoides glicosideos desenvolvendo agliconas,compostos com acao anticancerigena, que tambem sao relacionados com a adstringencia observada nosabor da soja. Pela importância dessa enzima, conduziu-se um estudo para determinar a atividade daβ-glicosidase em cultivares de soja ( Glycine max (L.) Merrill) com teores diferenciados de isoflavonoidesglicosideos (substratos da enzima). A atividade enzimatica foi determinada em 51 cultivares de sojasemeadas em Londrina, latitude 23oS, no Estado do Parana, e na cultivar IAS 5, proveniente de regioesprodutoras de soja de diferentes estados do Brasil. Entre as cultivares observou-se uma amplitude navariabilidade de 176,1 a 96,3 unidades de atividade enzimatica nas cultivares IAC-2 e Embrapa 2,respectivamente. Entretanto, a variabilidade significativa observada entre as cultivares pode estar indi-cando diferencas geneticas. Nos estados do Rio Grande do Sul, Parana e Mato Grosso do Sul, a cultivarIAS 5 apresentou atividades similares com valores medios de β-glicosidase iguais a 132,1, 131,9 e 132,5unidades, respectivamente. Entre os locais nos estados, a cultivar IAS 5 apresentou variabilidade naatividade da enzima de 138,8 a 124,8 unidades, as quais foram estatisticamente diferentes. No entanto,apesar da estatistica, os valores numericos nao foram tao diferentes para assumir que houve influenciado ambiente sobre a atividade da enzima. Entre as cultivares, nao houve correlacao significativa entre oteor de isoflavonoides glicosideos e a atividade da enzima.Termos para indexacao: efeito ambiental, Glycine max , agliconas, sabor da soja.

The enzyme beta-glucosidase hydrolyses the isoflavone glucosides developing aglycones, which are compounds with anticancer effects, that are also related with the astringency observed in soybean flavor. Due to the importance of this enzyme, a study was carried out to determine beta-glucosidase activity in soybean (Glycine max (L.) Merrill) cultivars with different contents of isoflavone glucosides (enzyme substrate). The enzyme activity was determined in 51 soybean cultivars sowed in Londrina (latitude 23ºS), in Paraná State, Brazil, and in the cultivar IAS 5 from soybean production regions of different Brazilian states. Among the cultivars, a range of variability of 176.1 to 96.3 units of enzyme activity (cultivars IAC-2 and Embrapa 2, respectively) was observed. A significant variability among cultivars could suggest genetic differences. In the states of Rio Grande do Sul, Paraná and Mato Grosso do Sul, the cultivar IAS 5 presented similar average of beta-glucosidase activity: 132.1, 131.9 and 132.5 units, respectively. Among locations in the states, the cultivar IAS 5 presented a variability for enzyme activity from 138.8 to 124.8 units, which were statistically different. In spite of statistics, the numerical values were not too different to assume that environmental conditions affected enzyme activity. A non-significative correlation for isoflavone glucoside concentrations and enzyme activity was observed among cultivars.

The application of synthetic antioxidants as inhibitors of lipid oxidation is well known in food industry. Widely used artificial antioxidants, such as butylated hydroxytoluene BHT and butylated hydroxyanisole BHA1, 2, are very effective in their role. However, their use in food products have been failing off due to their instability as well as due to suspected action as promoters of carcinogenesis.3, 4 Among the herbs and spices extensively studied, those obtained from the plants of the Lamiaceae (Labiatae) family have been found to possess a significant antioxidant activity. A number of studies on the antioxidant activities of their dried ground leaves, flowers and various extracts were reported in the past. It is now well known that the antioxidative activity of this spices is due to phenolic compounds.5 The antioxidative effect is correlated to their hydroxy groups.6 Regarding the nonvolatile components, oregano7 (Origanum vulgare L.) is one of the most effective aromatic herb. Herrmann et al.8, 9 examined the activity of rosmarinic and caffeic acid, both of which had been isolated from rosemary, but also from oregano and other species such as sage, mint, thyme, marjoram and balm. The study of Banias et al.10 showed that oregano and thyme extracts have strong antioxidant effects in stabilizing lard. Recently, Abdalla and Roozen11 studied effect of different plant extracts on the formation of conjugated diene hydroperoxides in sunflower oil. Very little is known about the extent to which the volatile compounds from aromatic herbs may contribute to the total antioxidant activity.12 In the essential oil of aromatic herbs, eugenol, isoeugenol, thymol, carvacrol and other phenolic volatile components exhibited appreciable antioxidant effect, but they have too strong characteristic odors to use as food additives. However, small amounts of essential oils do not always affect the odor note of food products and contrary may have a favorable effect. Oregano is widely distributed in Mediterranean basin, as well as in Croatia, and is of great economic importance for the spice market. Lagouri et al.13 investigated the chemical composition and antioxidant activity of essential oils from Oregano plants grown wild in Greece by measuring peroxide values of lard stored at 35 oC. In recent studies, Yanishlieva et al.14, 15 examined antioxidant activity of hexane extract from oregano grown in Bulgaria and mechanism of action of pure thymol and carvacrol. Baratta et al.16 investigated chemical composition and antioxidative activity of laurel, sage, rosemary, coriander and oregano essential oil. The potential antioxidant capacity of the oils was examined by modified thiobarbituric acid reactive species using egg yolk and rat liver homogenates as lipid rich media. In the present study, the essential oil of oregano, with thymol and carvacrol as major components, was examined for antioxidant activity on process of autoxidation of lard and sunflower oil. In order to correlate properly the antioxidant activity to the chemical composition, the essential oil was fractionated according to acidity and polarity. Antioxidant effect was examined by peroxide value measurements on lard and sunflower oil samples stored at 60 oC with and without 0.02 or 0.1 % of thymol, complete essential oil or each separated fraction.

The enzyme B-glucosidade hydrolyses the isoflavone glucosides developing aglycones, which are compounds with anticancer effects, that are also related with the astringency observed in soybean flavor. Due to the importance of this enzyme, a study was carried out to determine B-glucosidade activity in soybean (Glycine max (L.) Merrill) cultivars with different contents of isoflavone glucosides (enzyme substrate). The enzyme activity was determined in 51 soybean cultivars sowed in Londrina (latitude 23S), in Parana State, Brazil, and in the cultivar IAS 5 from soybean production regions of different Brazilian states. Among the cultivars, a range of variability of 176.1 to 96.3 units of enzyme activity (cultivars IAC-2 and Embrapa 2, respectively) was observed. A significant variability among cultivars could suggest genetic differences. In the states of Rio Grande do Sul, Parana and Mato Grosso do Sul, the cultivar IAS 5 presented similar average of B-glucosidase activity: 132.1, 131.9 and 132.5 units, respectively. Among locations in the states, the cultivar IAS 5 presented a variability for enzyme activity from 138.8 to 124.8 units, which were statistically different. In spite of statistics, the numerical values were not too different to assume that environmental conditions affected enzyme activit. A non-significative correlation for isoflavone glucoside concentrations and enzyme activity was observed among cultivars. Made available in DSpace on 2011-04-09T17:14:11Z (GMT). No. of bitstreams: 1 pab99012.pdf: 32145 bytes, checksum: 2f924034ad1c975550ec19aa286761cd (MD5) Previous issue date: 2000-08-15