Search

Abstract: Traditional Chinese medicine (TCM) is a huge treasure trove for the discovery and development of modern pharmaceuticals. Indeed, numerous biologically and pharmacologically significant molecules have been identified from TCM; among them, a considerable portion belongs to glycosides. These glycosides are extremely diverse in structures, consisting of a large variety of aglycones, glycans, as well as glycosidic linkages. In addition, these glycosides occur frequently as a complicated mixture of congeners in the nature sources. This micro‐heterogeneity leads to the isolation of a homogeneous glycoside in an appreciable amount a formidable task. Therefore, development of synthetic approaches toward these nature glycosides has received great attention. In this account, we summarize our twenty‐year efforts spent on this topic. Fifty five representative TCM‐relevant glycosides synthesized by our group are highlighted. Based on the aglycone structure, these glycosides are categorized into three groups, i.e., steroid glycosides, triterpene glycosides, and flavonoid and phenolic glycosides. Typical synthetic approaches are discussed, showing the demanding tactics for regio‐ and stereo‐controlled installation of glycans and overall manipulation of protecting groups. These expedient and scalable synthetic approaches have provided practical alternatives to the accessibility of these TCM components and thus opportunities for their in depth biological and pharmacological studies. [ABSTRACT FROM AUTHOR]

Zearalenone (ZEN), a mycotoxin with estrogenic activity, can exert adverse endocrine effects in mammals and is thus of concern for humans. ZEN is found in cereal crops and grain-based foods, often along with modified (‘masked’) forms usually not detected in routine contaminant analysis, e.g., ZEN-O-β-glucosides and ZEN-14-sulfate. These contribute to mycoestrogen exposure, as they are cleaved in the gastrointestinal tract to ZEN, and further metabolized in animals and humans to α- and β-zearalenol (α-ZEL and β-ZEL). ZEN and its metabolites are mainly excreted as conjugates in urine, allowing to monitor human exposure by a biomarker-based approach. Here, we report on a new study in German adults (n = 60) where ZEN, α-ZEL, and β-ZEL were determined by LC-MS/MS analysis after enzymatic hydrolysis and immunoaffinity column clean-up of the aglycones in urines. Biomarkers were detected in all samples: ZEN ranges 0.04-0.28 (mean 0.10 ± 0.05; median 0.07) ng/mL; α-ZEL ranges 0.06-0.45 (mean 0.16 ± 0.07; median 0.13) ng/mL, and β-ZEL ranges 0.01-0.20 (mean 0.05 ± 0.04; median 0.03) ng/mL. Notably, average urinary levels of α-ZEL, the more potent estrogenic metabolite, are higher than those of ZEN, while β-ZEL (less estrogenic than ZEN) is found at lower levels than the parent mycotoxin. Similar results were found in ten persons who collected multiple urine samples to gain more insight into temporal fluctuations in ZEN biomarker levels; here some urines had higher maximal concentrations of total ZEN (the sum of ZEN, α-ZEL, and β-ZEL) with 1.6 and 1.01 ng/mL, i.e., more than those found in the majority of other urines. A preliminary approach to translate the new urinary biomarker data into dietary mycotoxin intake suggests that exposure of most individuals in our cohort is probably below the tolerable daily intake (TDI) of 0.25 µg/kg b.w. set by EFSA as group value for ZEN and its modified forms while that of some individuals exceed it. In conclusion, biomonitoring can help to assess consumer exposure to the estrogenic mycotoxin ZEN and its modified forms and to identify persons at higher risk. [ABSTRACT FROM AUTHOR]

Spiny restharrow root ( Ononis spinosa L.) and its preparations are mainly used for the treatment of urinary infections or bladder stones in numerous countries. Spiny restharrow root is rich in isoflavonoids (formononetin, calycosin and pseudobaptigenin), pterocarpans (medicarpin and maackiain) and dihydroisoflavonoids (onogenin and sativanone), which metabolites are present as glucosides, glucoside malonates, glucoside acetates and free aglycones in the root. The in-depth analysis of tandem mass spectrometric (MS) and high-resolution MS (HR-MS) data revealed the presence of nitrogen-containing compounds in the root extracts. An ion-exchange-based purification and a preparative-scale reversed phase chromatographic isolation procedure was developed for the characterization of these new natural products. For the unambiguous identification of the isolated compounds NMR experiments were carried out. The thorough characterization confirmed the presence of six piperidin-2-yl-acetic acid (homopipecolic acid) esters of isoflavonoid glucosides. This is the first report of homopipecolic acid esters isolated from higher plants. [ABSTRACT FROM AUTHOR]

Autophagy dysfunction has been implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Natural compounds present in bergamot polyphenol fraction (BPF) prevent NAFLD and induce autophagy in rat livers. Here, we employed HepG2 cells expressing DsRed-LC3-GFP, a highly sensitive model system to screen for proautophagic compounds present in BPF. BPF induced autophagy in a time- and dose-dependent fashion and the effect was amplified in cells loaded with palmitic acid. Autophagy was mediated by the hydrophobic fraction of acid-hydrolyzed BPF (A-BPF), containing six flavanone and flavone aglycones as identified by liquid chromatography-high-resolution mass spectrometry. Among them, naringenin, hesperitin, eriodictyol and diosmetin were weak inducers of autophagy. Apigenin showed the strongest and dose-dependent proautophagic activity at early time points (6 h). Luteolin induced a biphasic autophagic response, strong at low doses and inhibitory at higher doses. Both flavones were toxic in HepG2 cells and in differentiated human liver progenitors HepaRG upon longer treatments (24 h). In contrast, BPF and A-BPF did not show any toxicity, but induced a persistent increase in autophagic flux. A mixture of six synthetic aglycones mimicking A-BPF was sufficient to induce a similar autophagic response, but it was mildly cytotoxic. Thus, while six main BPF flavonoids fully account for its proautophagic activity, their combined effect is not sufficient to abrogate cytotoxicity of individual compounds. This suggests that a natural polyphenol phytocomplex, such as BPF, is a safer and more effective strategy for the treatment of NAFLD than the use of pure flavonoids. [ABSTRACT FROM AUTHOR]

Gardenia blue is widely used in Eastern Asia as a natural food colorant. To evaluate the genotoxic potential of gardenia blue, as well as genipin, the natural starting material from which it is produced, a GLP-compliant test battery was conducted according to OECD guidelines. No evidence of mutagenicity of gardenia blue was detected in a 5-strain bacterial reverse mutation assay, with or without metabolic activation; an equivocal response for genipin occurred in S. typhimurium TA97a without metabolic activation. In in vitro micronucleus and chromosome aberration assays, genipin tested positive under some test conditions; however, gardenia blue tested negative in both assays. In combined micronucleus/comet assays conducted in male and female B6C3F1 mice, exposure to genipin at doses reaching maximal toxicity (74 and 222 mg/kg bw/day for males and females, respectively) or gardenia blue tested up to the limit dose (2000 mg/kg bw/day) did not induce micronuclei in peripheral blood or DNA damage in several examined tissues. Modified (“reverse”) comet assays showed no evidence of DNA crosslinking potential of either genipin, known to form crosslinks with other macromolecules, or gardenia blue. Our results indicate that consumption of gardenia blue in food products does not pose a significant genotoxic concern for humans. [ABSTRACT FROM AUTHOR]

Both protective and pre-spacer features of 4-(2-chloroethoxy)phenyl (CEP) aglycon, which belong to the class of Janus aglycons, were engaged in a benzyl-free synthesis of oligosaccharide fragments of polysaccharides from rhizobacterium Azospirillum brasilense sp7. Introduction of α-1,4-linked L-fucose residue was performed using 3,4-di- O -benzoyl-2- O -triisopropylsilyl-α-L-fucopyranosyl N -phenyltrifluoroacetimidate in excellent stereoselectivity and high yields. The obtained deprotected di-, tri- and tetrasaccharides contain 4-(2-azidoethoxy)phenyl (AEP) spacer aglycon, which allows straightforward preparation of neoglycoconjugates that will be used for the study of the role of lipopolysaccharide of rhizobacterium A. brasilense sp7 in plant–microbe symbiosis. The intermediate protected oligosaccharide building blocks with cleavable CEP/AEP aglycons have a strong potential for further application in the synthesis of more complex oligosaccharides. [ABSTRACT FROM AUTHOR]

Genipin, an aglycone derived from the iridoid glycoside, geniposide, is isolated and characterized from the extract of Gardenia jasminoides Ellis fruit (family Rubiaceae). It has long been used in traditional oriental medicine for the prevention and treatment of several inflammation driven diseases, including cancer. Genipin has been shown to have hepatoprotective activity acting as a potent antioxidant and inhibitor of mitochondrial uncoupling protein 2 (UCP2), and also reported to exert significant anticancer effects. It is an excellent crosslinking agent that helps to make novel sustained or delayed release nanoparticle formulations. In this review, we present the latest developments of genipin as an anticancer agent and briefly describe its diverse mechanism(s) of action. Several lines of evidence suggest that genipin is a potent inhibitor of UCP2, which functions as a tumor promoter in a variety of cancers, attenuates generation of reactive oxygen species and the expression of matrix metalloproteinase 2, as well as induces caspase-dependent apoptosis in vitro and in in vivo models. These finding suggests that genipin can serve as both a prominent anticancer agent as well as a potent crosslinking drug that may find useful application in several novel pharmaceutical formulations. [ABSTRACT FROM AUTHOR]

Six undescribed pentacyclic triterpenoids including four triterpenoid aglycones, 1 β ,2 a ,3 β ,23-tetrahydroxyurs-12-en-28-ursolic acid, 2 a ,3 a ,6 β ,19 α ,23-pentahydroxyurs-12-en-28-ursolic acid, 2 α ,3 α ,20 β ,23-tetrahydroxyurs-12-en-28-ursolic acid and 1 β ,2 a ,3 β ,23-tetrahydroxyurs-12,20(30)-dien-28-ursolic acid, and two triterpenoid glucosides, 2 a ,3 a ,23-trihydroxy-12,20(30)-dien-28-ursolic acid 28- O - β - d -glucopyranoside and 1-oxo-3 β ,23-dihydroxyolean-12-en-28-oic acid 28- O - β - d -xylopyranoside, along with 5 known triterpenoids were isolated from a CH 3 Cl-soluble extract of the leaves of Cyclocarya paliurus . Their structures were established on the basis of chemical and spectroscopic approaches. These compounds were assessed for their antioxidant effects on FFA-induced hepatic steatosis in HepG2 cells. The results revealed that three saponins and two aglycones markedly increased SOD activity and reduced MDA level. [ABSTRACT FROM AUTHOR]

The complex mixtures of colorants present in different madder species can provide significant information about which plant species or technique was used to dye the fibres of historical textile artefacts, hence, when extracting and analysing colorants from textile artefacts as much of this information as possible should be preserved. Historical textiles are most commonly extracted with 37% hydrochloric acid: methanol: water (2:1:1, v/v/v ), but this solvent system hydrolyses dye glycosides and may also induce chemical reactions. One of the primary components in Dyers' madder ( Rubia tinctorum L.) is lucidin primeveroside, but it is rarely seen in artefacts, nor is the corresponding aglycon lucidin. It has been demonstrated that the hydrochloric acid method causes hydrolysis of anthraquinone glycosides to their aglycon counterpart. Herein it is demonstrated that lucidin is not stable in such acidic conditions and degrades rapidly to xanthopurpurin. This is confirmed by HPLC, LC-MS and 1 H NMR, which also provide evidence of the mechanism of degradation being a retro-aldol process. [ABSTRACT FROM AUTHOR]

Endometriosis is an estrogen-dependent disease, and isoflavones interact with estrogen receptors. The purposes of this study are to investigate the in vitro and in vivo effects of daidzein-rich isoflavone aglycones (DRIAs), dietary supplements, on cellular proliferation in endometriosis. Stromal cells isolated from ovarian endometrioma (OESCs) and normal endometrium (NESCs) were cultured with DRIAs, i.e. , each of the DRIA components (daidzein, genistein, or glycitein), or isoflavone glycosides (IG; DRIA precursors). A mouse model of endometriosis was established by transplanting donor-mouse uterine fragments into recipient mice. Our results showed that DRIAs (0.2–20 μM) inhibited the proliferation of OESCs ( P  < 0.05 for 0.2 μM; P  < 0.01 for 2 and 20 μM) but not of NESCs. However, daidzein, genistein, glycitein, and IG did not inhibit their proliferation. DRIA-induced suppression was reversed by inhibition of the estrogen receptor (ER)β by an antagonist, PHTPP, or by ERβ siRNA ( P  < 0.05), but not by MPP, an ERα antagonist. In OESCs, DRIAs led to reduced expression of IL-6, IL-8, COX-2, and aromatase, as well as reduced aromatase activity, serum glucocorticoid-regulated kinase levels, and PGE 2 levels ( P  < 0.05). Western blot and immunofluorescence assays revealed that DRIAs inhibited TNF-α-induced IκB phosphorylation and p65 uptake into the nuclei of OESCs. In the mouse model, a DRIA-containing feed significantly decreased the number, weight, and Ki-67 proliferative activity of endometriosis-like lesions compared to in mice fed with an IG-containing feed and the control feed ( P  < 0.01). In conclusion, DRIAs inhibit cellular proliferation in endometriosis, thus representing a potential therapeutic option for the management of endometriosis. [ABSTRACT FROM AUTHOR]

β-D-xylosides with cytotoxic aglycones have augmented cytotoxicity towards animal cells because β-D-xyloside-primed glycosaminoglycans further enhance the aglycone's cytotoxicity. In this study, we designed and synthesized different 4-anilinequinazoline β-D-xylosides and found that compounds 7–10 possessing 3-chloro-4-((3-fluorobenzyl)oxy)aniline group as in anticancer drug lapatinib also primed glycosaminoglycans and were highly cytotoxic to cancer cells. [ABSTRACT FROM AUTHOR]

Hypoxia-inducible factor-1 (HIF-1) and vascular endothelial growth factor (VEGF) play important roles in cancer progression in various cancer cell lines. Although genipin, a constituent of Gardenia fruit, has been shown to have anti-tumor activity, its role in the suppression of HIF-1 and its downstream target genes is not well understood. We examined the effect of genipin on the intracellular level of HIF-1α and extracellular level of VEGF using the colon cancer cell line HCT116. We observed that genipin suppressed the accumulation of HIF-1α under hypoxia in various cancer cell lines, including HCT116, via the modulation of protein degradation. Genipin also suppressed the expression of VEGF and the invasion of colon cancer cells by blocking the extracellular signal-regulated kinase signaling pathway. Taken together, our results provide new insights into the potential role of genipin in suppressing colon cancer progression. [ABSTRACT FROM AUTHOR]

Biotransformation of isoflavones glycosides into the aglycone form is essential to attain the maximum bioavailability. The factors affecting deglycosylation of genistin in soy flour using commercial β-glucosidase enzyme were evaluated. The presence of genistin in soy flour was confirmed by isolation through chromatographic fractionation and identification by spectral method. Two-levels Plackett-Burman design was applied and effective variables for genistein production were determined. Agitation rate, enzyme concentration, and reaction time, owing to their significant positive effect, and pH, owing to its significant negative effect, were further evaluated using Box-Behnken model. Accordingly the optimal combination of the major reaction affecting factors was "enzyme concentration, 1 IU; agitation speed, 250 rpm; reaction time, 5 h and pH 4. The concentration of genistein can be increased by 9.91 folds (from 0.8 mg/g in the non biotransformed soy flour to 7.93 mg/g in the biotransformed one) using the determined optimal combination of major reaction affecting factors. The antioxidant activity of the non biotransformed and biotransformed soy flour extracts was determined by DPPH method. It was found that biotransformation increase the antioxidant activity by two folds. The concentration causing a 50% reduction of DPPH absorbance (EC50) were 10 and 5 mg/mL for the non biotransformed and biotransformed soy flour extracts, respectively. [ABSTRACT FROM AUTHOR]

In the feed industry, β-glucosidase has been widely used in the conversion of inactive and bounded soybean isoflavones into active aglycones. However, the conversion is frequently inhibited by the high concentration of intestinal glucose in monogastric animals. In this study, a GH1 β-glucosidase (AsBG1) with high specific activity, thermostability and glucose tolerance (IC50 = 800 mM) was identified. It showed great glucose tolerance against substrates with hydrophobic aryl ligands (such as pNPG and soy isoflavones). Using soybean meal as the substrate, AsBG1 exhibited higher hydrolysis efficiency than the GH3 counterpart Bgl3A with or without the presence of glucose in the reaction system. Furthermore, it is the first time to find that the endogenous β-glucosidase of soybean meal, mostly belonging to GH3, plays a role in the hydrolysis of soybean isoflavones and is highly sensitive to glucose. These findings lead to a conclusion that the GH1 rather than GH3 β-glucosidase has prosperous application advantages in the conversion of soybean isoflavones in the feed industry. [ABSTRACT FROM AUTHOR]

Exposure to exogenous and endogenous chemicals and subsequent cellular and molecular changes has been linked to enhanced cell proliferation and restricted apoptosis phenomenon. Though in the past decades numerous anticancer drugs inducing programmed cell death in cancer cells by targeting specific apoptotic markers have reached the market, they have been allied with unwanted side effects, ranging from mild to severe toxicity. With further understanding on the functional mechanism of p53 and MDM2 in apoptosis and in our continuous search for new and potent multi-target anticancer lead compounds, we have carried out molecular docking and inhibition studies of the selected aglycones along with selected anticancer leads, against the specific apoptotic and cell cycle markers using AutoDock Tools 4.0 and other computational softwares. The docking results have been analyzed in terms of binding energies (kcal/mol) and inhibition constant (µM). The study clearly proposes our aglycones [solanidine (Solanid-5-en-3β-ol), solasodine (Solasod-5-en-3β-ol), and tomatidine (5α-Tomatidan-3β-ol)] induce apoptosis by inhibiting the p53-MDM2 complex, p21Waf1/Cip1, and Bcl-2 proteins, which were even found comparable with the anticancer drugs nutlin and/or halofuginone. The work further emphasizes that the individual molecular targets such as BAX and Bcl-2 may result in misleading data at any level; however, ratio of responses to BAX and Bcl-2 shall be considered for better clue about a compound to be pro- or anti-apoptotic. [ABSTRACT FROM AUTHOR]

Abstract: Phenolic compounds are useful markers to control olive oil technological processes, including the virgin olive oil (VOO)/water separation after olive oil extraction. In this investigation, VOO extracted from olives of cv. Coratina using a mild oil/water separator called the hydrocyclone sedimentation system (Hydroil) was compared with VOO obtained using a conventional vertical centrifuge separator (Cenoil), which is mostly used in the modern olive oil industry. Secoiridoid aglycones were selected, among phenolic compounds, as markers and analyzed using reversed‐phase liquid chromatography coupled to linear quadrupole ion‐trap mass spectrometry with electrospray ionization in the negative mode. VOO samples obtained using the Hydroil system were found to contain significantly higher levels of secoiridoid aglycones, compared to the Cenoyl‐type samples. In particular, the total content of the aglycones of decarboxymethyl oleuropein, decarboxymethyl ligstroside, ligstroside, and oleuropein, expressed in terms of oleuropein, was estimated as 35.40 ± 0.80 mg kg−1, compared to 8.06 ± 0.41 mg kg−1 in the Cenoil samples (n = 3). Since no significant difference in residual water (P < 0.05) was found between the two types of VOO samples, the higher amount of secoiridoids obtained for Hydroil‐type ones was explained by the lower extent of oxidation occurring during the mild oil/water separation achieved using the Hydroil system. [ABSTRACT FROM AUTHOR]

To develop a new healthy functional tofu product, the pH, total cell number, SDS-PAGE patterns, contents of reducing sugar, peptide, isoflavones, antioxidant activity and digestibility of freeze-thawed tofu, fermented with Bacillus subtilis for various time periods, were investigated. In SDS-PAGE patterns, the band intensities of 7S and 11S globulins were slightly decreased and new protein bands of lower molecular weight appeared by fermentation for 12 h. After 18 h, the bands of 7S globulin and the acidic subunit of 11S globulin had almost entirely disappeared and the basic subunit band of 11S globulin was fainter. An 18 h of fermentation was thought to be the most desirable, because the contents of reducing sugar, total peptide, and isoflavone aglycones (daidzein and genistein), as well as antioxidant activities and digestibility of freeze-thawed tofu were more increased by the fermentation for 18 h. [ABSTRACT FROM AUTHOR]

A series of arylnaphthalene lignan lactones based on the structure of the phyllanthusmins, a class of potent natural products possessing diphyllin as the aglycone, has been synthesized and screened for activity against multiple cancer cell lines. SAR exploration was performed on both the carbohydrate and lactone moieties of this structural class. These studies have revealed the importance of functionalization of the carbohydrate hydroxy groups with both acetylated and methylated analogues showing increased potency relative to those with unsubstituted sugar moieties. In addition, the requirement for the presence and position of the C-ring lactone has been demonstrated through reduction and selective re-oxidation of the lactone ring. The most potent compound in this study displayed an IC 50 value of 18 nM in an HT-29 assay with several others ranging from 50 to 200 nM. In an effort to elucidate their potential mechanism(s) of action, the DNA topoisomerase IIa inhibitory activity of the most potent compounds was examined based on previous reports of structurally similar compounds, but does not appear to contribute significantly to their antiproliferative effects. [ABSTRACT FROM AUTHOR]

The purpose of the study is to develop the new methodology of strategic ultrasound treatment on lactic acid bacteria (LAB) to induce stress response for the enhancement of β-glucosidase activity that can be used for the biotransformation of glucosides into aglycones isoflavones in soymilk. Among the five LAB tested, Lactobacillus acidophilus BCRC 10695 showed the best ability to release β-glucosidase for further ultrasonic stimulation to induce proper stress response. With ultrasound (20 kHz, amplitude at 20%) to irradiate on L. acidophilus BCRC 10695 at stationary phase of growth for 2 min and 24 h of re-incubation, the β-glucosidase activity was enhanced to 3.91 U/ml, which was 1.82 times of that without ultrasound treatment. Using the ultrasound-treated L. acidophilus BCRC 10695 to ferment soymilk, the fraction of aglycones in total isoflavones in soymilk was effectively increased from 21.8% initially to 97.9% in 24 h. The strategic ultrasound treatment on L. acidophilus BCRC 10695 demonstrated promotion of β-glucosidase activity, and this methodology had the potential to be applied in the production of functional soymilk by adding probiotics LAB to increase the bioactive isoflavones and nutritional values for human health. [ABSTRACT FROM AUTHOR]

Quercetin is a major flavonoid, present as its glycosidic forms in plant foods. In this study, quercetin-3-glucoside (Q3G) was administered intraduodenally to thoracic lymph-cannulated rats, and its lymphatic transport was investigated. The resulting lymphatic and plasma metabolites were identified with LC-MS/MS and compared with those after administration of quercetin aglycone. The total concentration of quercetin metabolites in the lymph was about four times lower than that in the plasma, and quercetin and its methylated form isorhamnetin were detected as their glucuronides, sulfates and diglucuronides both in the lymph and the plasma after Q3G and quercetin administrations. The lymph levels of the glucuronides after Q3G administration were lower than those after quercetin administration, whereas those in the plasma showed the opposite pattern. Both the lymph and plasma levels of the sulfates after Q3G administration were lower than those after quercetin administration. Some of the intestinal metabolites like quercetin monoglucuronides were transported directly into the lymph and the hepatic metabolites like the diglucuronides were eventually transferred from the plasma into the lymph. These results indicate that the absorbed Q3G is partly transported into the intestinal lymph as quercetin metabolites. Deglycosylation in the enterocyte is also suggested to affect the subsequent metabolic pathways. [ABSTRACT FROM AUTHOR]

Roots of Desmostachya bipinnata Stapf are used in the treatment of jaundice and asthma in Indian traditional medicine. Phytochemical investigation of the methanol extract of the roots of this plant led to isolation of three flavonols. The structures of these compounds were assigned on the basis of elemental analysis and spectroscopic evidences as apigenin, luteolin and quercetin, of which apigenin and luteolin were isolated for the first time. The yield of these compounds was 0.0034, 0.0026 and 0.0042 % w/w, respectively. These findings suggested the possibility that the three flavonoids isolated from the roots of Desmostachya bipinnata could be responsible for the benefits to human health offered by these roots. [ABSTRACT FROM AUTHOR]

Xylanases Tx-xyn10 and Tx-xyn11 were compared for their transxylosylation abilities in the presence of various acceptors. Tx-xyn10 exhibited a broad specificity for various acceptors, whereas xylanase Tx-xyn11 catalysed transxylosylation reactions only in presence of polyphenolic acceptors. A modelling approach was developed to study the molecular bottlenecks into the active site of the enzyme that could be responsible for this restricted specificity. The glycosyl-enzyme intermediate of Tx-xyn11 was modelled, and a rotamer of the Y78 residue was integrated. In silico mutations of some residues from the (+1) and (+2) subsites were tested for the deglycosylation step in the presence of non-polyphenolic acceptors. The results indicated that the mutant W126A was able to use aliphatic alcohols and benzyl alcohol as acceptors for transxylosylation. Experimental validation was tested by mutating the xylanase Tx-xyn11 at position W126 into alanine. The specific activity and catalytic efficiency of the W126A mutant during the hydrolysis of xylans decreased by 2-fold and 4-fold, respectively, compared to wild-type xylanase. Among tested acceptors, transxylosylation catalysed by mutant W126A was improved with benzyl alcohol leading to a 2-fold higher concentration of benzyl xylobioside, as predicted by in silico mutation. This improved transxylosylation in the presence of benzyl alcohol leading to higher synthesis of benzyl xylobioside could likely be explained by lowest steric hindrance in the aglycone subsite of the mutated xylanase. No secondary hydrolysis of benzyl xylobioside occurred for both wild-type and mutant xylanases. Finally, our results demonstrated that the modelling approach was limited and that accounting for protein dynamics can lead to improved models. [ABSTRACT FROM AUTHOR]

Binding kinetics of α-galactopyranoside homologs with fluorescent aglycones of different sizes and shapes were determined with the lactose permease (LacY) of Escherichia coli by FRET from Trp151 in the binding site of LacY to the fluorophores. Fast binding was observed with LacY stabilized in an outward-open conformation (kon = 4-20 µM-1⋅s-1), indicating unobstructed access to the binding site even for ligands that are much larger than lactose. Dissociation rate constants (koff) increase with the size of the aglycone so that Kd values also increase but remain in the micromolar range for each homolog. Phe27 (helix I) forms an apparent constriction in the pathway for sugar by protruding into the periplasmic cavity. However, replacement of Phe27 with a bulkier Trp does not create an obstacle in the pathway even for large ligands, since binding kinetics remain unchanged. High accessibility of the binding site is also observed in a LacY/nanobody complex with partially blocked periplasmic opening. Remarkably, E. coli expressing WT LacY catalyzes transport of α- or β-galactopyranosides with oversized aglycones such as bodipy or Aldol518, which may require an extra space within the occluded intermediate. The results confirm that LacY specificity is strictly directed toward the galactopyranoside ring and also clearly indicate that the opening on the periplasmic side is sufficiently wide to accommodate the large galactoside derivatives tested here. We conclude that the actual pathway for the substrate entering from the periplasmic side is wider than the pore diameter calculated in the periplasmic-open X-ray structures. [ABSTRACT FROM AUTHOR]

The effects of olive ripeness, areas of production and their interaction on the chemical and sensory characteristics of cv. Bosana oil were assessed. The study was carried out in three areas of the Sassari province, Sardinia (Italy), at three stages of maturation. The results indicated the independence of the two factors: ripeness influenced saturated fatty acids, pigment content and deacetoxy oleuropein aglycone (DAOA) content and didn’t affect the sensory characteristics, while production area influenced unsaturated fatty acids, content of vanillic acid and some sensory characters. In order to verify the interdependency of the two factors, statistical analyses (two-way ANOVA) were performed. Our study showed that a thoughtful planning of harvest times and production area could allow to obtain Bosana virgin olive oil of the highest quality. Furthermore, utilizing cultivars that maintain the properties of their oils even at late dates of harvest, it would be possible to optimize harvest times. [ABSTRACT FROM AUTHOR]

Most β-d-xylosides with hydrophobic aglycones are nontoxic primers for glycosaminoglycan assembly in animal cells. However, when Ebselen was conjugated to d-xylose, d-glucose, d-galactose, and d-lactose (8A-D), only Ebselen β-d-xyloside (8A) showed significant cytotoxicity in human cancer cells. The following facts indicated that the aglycone Ebselen and β-d-xyloside primed glycosaminoglycans co-contributed to the observed cytotoxicity: 1. Ebselen induced S phase cell cycle arrest, whereas 8A induced G2/M cell cycle arrest; 2. 8A augmented early and late phase cancer cell apoptosis significantly compared to that of Ebselen and 8B-D; 3. Both 8A and phenyl-β-d-xyloside primed glycosaminoglycans with similar disaccharide compositions in CHO-pgsA745 cells; 4. Glycosaminoglycans could be detected inside of cells only when treated with 8A, indicating Ebselen contributed to the unique property of intracellular localization of the primed glycosaminoglycans. Thus, 8A represents a lead compound for the development of novel antitumor strategy by targeting glycosaminoglycans. [ABSTRACT FROM AUTHOR]

Processing may affect the bioactivity of soy food products, due to the conversion between isoflavones’ chemical forms. We investigated the effect of dry-heating or fermentation by Saccharomyces cerevisiae on soybean meal (SBM) chemical composition, with emphasis on isoflavone glycosides to aglycones conversion. Biscuits were prepared with aglycone-rich SBM and evaluated regarding their technological, nutritional, functional and sensory properties. Fermentation yielded more isoflavone aglycones (7.8-fold) than dry-heating (2.5-fold). Fermentation improved nutritional quality (50% increase in fiber, and 32%, 83% and 69% decreases in carbohydrate, lipid and trypsin inhibitors, respectively) and increased antioxidant activity (up to 206%) of SBM. Soybean meal biscuits showed adequate technological properties and improved nutritional and functional qualities in comparison to wheat biscuits (up to 3.7-, 4.6-, 2.8- and 4.7-fold increases in protein, dietary fiber, ash and antioxidant activity, respectively; 2.1-fold decrease in carbohydrate). SBM biscuits showed good sensory acceptance, whereas fermented SBM biscuits showed low sensory scores. [ABSTRACT FROM AUTHOR]

α-L-Rhamnosyl-β-D-glucosidase (rutinosidase) hydrolyzes the glycosidic linkage between the disaccharide 6-O-α-L-rhamnosyl-β-D-glucoside (rutinose) and the aglycone. We identified a hypothetical protein (annotated as AO090012000917) encoded in the Aspergillus oryzae genome that exhibits sequence similarity with Aspergillus niger rutinosidase. The recombinant enzyme was expressed in Pichia pastoris GS115 and purified as a glyco-protein with apparent molecular mass of 65-75 kDa by SDS-PAGE. After N-deglycosylation, we observed a 42- and 40-kDa band, representing proteins before and after N-terminal signal peptide processing, respectively. Optimal enzymatic activity was observed at pH 4.0 and temperature of 45 °C. This enzyme is also significantly thermo-stable, with 90% activity retained after 1 h at 45 °C and 70% activity retained after 4 h, even at 50 °C. Biochemical characterization revealed that the enzyme has higher substrate specificity for 3-O-linked flavonoid β-rutinosides like rutin and kaempferol-3-O-rutinoside, than for 7-O-linked flavonoid β-rutinoside like hesperidin. However, no activity was found with naringin, diosmin, monoglycosylated chromogenic substrates, and polymeric laminarin substrate. Kinetic analyses showed that Km value toward rutin was higher than those toward hesperidin and kaempferol-3-O-rutinoside. Meanwhile, kcat value toward hesperidin was lower than those toward kaempferol-3-O-rutinoside and rutin. Overall, the catalytic efficiency (kcat/Km) was highest for kaempferol-3-O-rutinoside. [ABSTRACT FROM AUTHOR]

Abstract 1 1 Preliminary results were presented as a poster at the EMBO conference series. Cellular Signaling & Cancer Therapy. May 27–31, 2016 | Cavtat-Dubrovnik, Croatia. Abstract was published in the Abstracts of poster presentations. Abnormally high levels of CK2 and PIM-1 serine/threonine kinases have been documented in many cases of cancer. The elevation of CK2 and PIM-1 in cells entails suppression of apoptosis and implies a protective role for the kinases against cell death. Downregulation of these enzymes by chemical methods promotes apoptosis in cells. The aim of the present study was to explore the anticancer activity of inhibitors of protein kinases CK2 and PIM-1 on neoplastic cell lines in vitro. We studied a series of deoxynucleosides with various tetrahalobenzimidazoles as aglycone moiety. Cytotoxicity, induction of apoptosis by the tested inhibitors, mitochondrial membrane potential, activity of caspases, changes in cell cycle progression, as well as a mechanism of action were determined by flow cytometry and other methods. The results indicate that the studied compounds, e.g., 1-(β-D-2′-deoxyribofuranosyl)-4,5,6,7-tetrabromo-1 H -benzimidazole called K164 (also termed TDB), showed diverse cytotoxicity and proapoptotic efficacy in cell lines. Our results showed that the tested compounds are potential anticancer agents for targeted therapy, particularly in the treatment of myeloid leukaemia and androgen-responsive prostate cancer. [ABSTRACT FROM AUTHOR]

Selectively protected mono-, di- and trisaccharide thioglycoside building blocks with unprotected primary hydroxy group at the non-reducing end, available in only one step from 3- O -benzoyl β- d -arabinofuranose 1,2,5-orthobenzoate, were used in the synthesis of linear α(1 → 5)-linked oligoarabinofuranosides up to octasaccharide. The obtained oligosaccharides contain 4-(2-chloroethoxy)phenyl (CEP) or 4-(2-azidoethoxy)phenyl (AEP) pre-spacer aglycons that allow preparation of neoglycoconjugates. [ABSTRACT FROM AUTHOR]

Kalopanax septemlobus, commonly named the castor aralia tree, is a highly valued woody medicinal tree belonging to the family Araliaceae. Kalopanax septemlobus contains approximately 15 triterpenoid saponins primarily constituted of hederagenin aglycones. Hederagenin is a representative precursor for hemolytic saponin in plants. In the present study, transcriptome analysis was performed to discover genes involved in hederagenin saponin biosynthesis in K. septemlobus. De novo assembly generated 82,698 unique sequences, including 17,747 contigs and 64,951 singletons, following 454 pyrosequencing. Oxidosqualene cyclases (OSCs) are enzymes that catalyze the formation of diverse triterpene skeletons from 2,3-oxidosqualene. Heterologous expression of an OSC sequence in yeast revealed that KsBAS is a β-amyrin synthase gene. Cytochrome P450 genes (CYPs) make up a supergene family in the plant genome and play a key role in the biosynthesis of sapogenin aglycones. In total, 95 contigs and 110 singletons annotated as CYPs were obtained by sequencing the K. septemlobus transcriptome. By heterologous expression in yeast, we found that CYP716A94 was β-amyrin 28-oxidase involved in oleanolic acid production from β-amyrin, and CYP72A397 was oleanolic acid 23-hydroxylase involved in hederagenin production from oleanolic acid. Engineered yeast co-expressing KsBAS, CYP716A94 and CYP72A397 produced hederagenin. Kalopanax septemlobus CYP72A397 is a novel CYP enzyme that synthesizes hederagenin aglycone from oleanolic acid as a single product. In conclusion, we characterized three genes participating in sequential steps for hederagenin biosynthesis from β-amyrin, which are likely to play a major role in hederagenin saponin biosynthesis in K. septemlobus. [ABSTRACT FROM AUTHOR]

The first total synthesis of (−)-dendroside C aglycon, consisting of a 1,4,5-tri-cis-guaiane skeleton, from a versatile hydroazulene intermediate has been accomplished. The key features of the syntheses include the stereoselective preparation of the unusual cis-hydroazulene core via a sequence of a unique Dieckmann condensation of the bicyclic lactone system, which was concisely prepared by the tandem conjugate addition and intramolecular allylic alkylation of a butenolide precursor, and construction of the characteristic tricyclic skeleton by a carbene-mediated cyclopropanation. [ABSTRACT FROM AUTHOR]

Fisetin (FIS) is a flavonoid aglycone which is widely distributed in several fruits and vegetables. Based on its complex biochemical action in the body, FIS seems a promising candidate for the treatment of neurodegenerative diseases, diabetes, or cancer. During the 3′- O -methylation of FIS by catechol- O -methyltransferase geraldol (GER) is formed which is also pharmacologically active. Similarly to other flavonoids, FIS and GER are poorly soluble in water. Microcapsulation of flavonoids can enhance their aqueous solubility and may improve their pharmacokinetic properties. Since both FIS and GER are fluorescent molecules, the interaction of these flavonoids by native and chemically modified β-cyclodextrins was investigated by fluorescence spectroscopy. Both FIS-CD and GER-CD complexes show considerable emission with maxima at 475 and 540 nm reflecting the two conformers of the complexes. Thereafter, fluorescence enhancement of each tautomer by cyclodextrins and binding constants of flavonoid-cyclodextrin complexes were determined. Chemically modified cyclodextrins were stronger fluorescence enhancers of flavonoids and formed more stable complexes with FIS and GER than the native β-cyclodextrin. Finally, influence of cyclodextrins on the cellular impact of FIS and GER was tested on HepG2 tumor cell line. [ABSTRACT FROM AUTHOR]

Hydrolysis of arabinoxylan (AX) by glycoside hydrolase family 10 (GH10) xylanases produces xylo- and arabinoxylo-oligosaccharides ((A)XOS) which have shown prebiotic effects. The thermostable GH10 xylanase Rm Xyn10A has shown great potential to produce (A)XOS. In this study, the structure of Rm Xyn10A was investigated, the catalytic module by homology modelling and site-directed mutagenesis and the arrangement of its five domains by small-angle X-ray scattering (SAXS). Substrate specificity was explored in silico by manual docking and molecular dynamic simulations. It has been shown in the literature that the glycone subsites of GH10 xylanases are well conserved and our results suggest that Rm Xyn10A is no exception. The aglycone subsites are less investigated, and the modelled structure of Rm Xyn10A suggests that loop β 6 α 6 in the aglycone part of the active site contains a non-conserved α-helix, which blocks the otherwise conserved space of subsite +2. This structural feature has only been observed for one other GH10 xylanase. In Rm Xyn10A, docking revealed two alternative binding regions, one on either side of the α-helix. However, only one was able to accommodate arabinose-substitutions and the mutation study suggests that the same region is responsible for binding XOS. Several non-conserved structural features are most likely to be responsible for providing affinity for arabinose-substitutions in subsites +1 and +2. The SAXS rigid model of the modular arrangement of Rm Xyn10A displays the catalytic module close to the cell-anchoring domain while the carbohydrate binding modules are further away, likely explaining the observed lack of contribution of the CBMs to activity. [ABSTRACT FROM AUTHOR]

Aglycon protopanaxatriol (APPT) has valuable pharmacological effects such as anti-inflammatory and anti-stress activities. However, the complete conversion of all typical glycosylated protopanaxatriol ginsenosides to APPT has not been achieved to date. β-Glycosidase from the hyperthermophilic bacterium Dictyoglomus turgidum (DT-bgl) hydrolyzes the glucose residues at C-6 and the inner glucose at C-20 in protopanaxatriol (PPT), but not the outer rhamnose residues at C-6. In contrast, β-glycosidase from the hyperthermophilic bacterium Pyrococcus furiosus (PF-bgl) hydrolyzes the outer rhamnose residue at C-6 but not the inner glucose residues at C-6 and C-20 in PPT. Thus, the combined use of DT-bgl and PF-bgl resulted in the complete the conversion of all typical glycosylated PPT ginsenosides, including R1, R2, Re, Rg1, Rg2, Rh1, Rf, F1, F3, and F5, to APPT. DT-bgl combined with PF-bgl completely hydrolyzed 1.0 mg ml R1 and 1.0 mg ml total PPT-type ginsenosides in Panax notoginseng root extract to 0.5 and 0.63 mg ml APPT for 4 and 3 h, with molar conversions of 100% and productivities of 125 and 210 mg l h, respectively. To the best of our knowledge, this is the first report of the complete conversion of all typical glycosylated PPT ginsenosides to APPT and the highest productivity of APPT obtained from ginseng extract achieved to date. [ABSTRACT FROM AUTHOR]

Anthocyanins are ubiquitous pigments in the human diet, but their metabolic fate is poorly understood. Absorption of these compounds may occur in the stomach, where acidic pH favors anthocyanin stability. The NCI-N87 cell line gastric model was used to examine the effects of anthocyanin chemical structure on their transport and uptake at pH 3, representative of the gastric environment. Chokeberry, containing different cyanidin derivatives, was used to determine the effects of different sugar substitutions; red grape was used as a source of monoglucosylated derivatives of different aglycones. The type of sugar substitution on cyanidin affected both cellular uptake and transport to the basolateral chamber, with cyanidin-3-arabinoside showing the greatest transport and cyanidin-3-glucoside the highest uptake by the cell. The aglycone structure also affected uptake and transport. Anthocyanins bearing B-ring di-substitution, cyanidin-3-glucoside and peonidin-3-glucoside, were transported and taken up the most by cells. Transport through NCI-N87 cells was most efficient for cyanidin-3-glucoside followed by peonidin, delphinidin, pelargonidin, malvidin, and petunidin-3-glucosides, in that order. Cyanidin-3-glucoside was also more efficiently taken up by the cells, followed by peonidin, pelargonidin, delphinidin, petunidin, and malvidin-3-glucosides. Although the metabolism of anthocyanins requires much more study, further evidence of the role of chemical structure is provided. [ABSTRACT FROM AUTHOR]

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 β- d -glycoside/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. [ABSTRACT FROM AUTHOR]

Mulberry leaves have been widely used to produce various health products. In this paper, an efficient procedure of ultrasound-assisted hydroalcohol-acid extraction (UAHAE) was established to estimate the total flavonoids content in mulberry leaves by quantifying their resulting aglycones (quercetin and kaempferol) using HPLC-DAD. Effective hydrolysis of glycosides to aglycones was achieved in an ethanol-HCl-water (7/2/1, v/v/v) solution at 75 °C by ultrasound (40 kHz) for 60 min. The average contents of quercetin and kaempferol were 7.12 mg/g and 2.13 mg/g, respectively, in the variety of cultivar MC308, and their recoveries were 105.48% and 105.81%, respectively. The total flavonoid content of most varieties was between 4-10 mg/g, accounting for 80% in 86 species of mulberry leaves. With the increase in leaf maturity, the general trend of the change in flavonoid content was a decrease at first and then an increase. The highest total flavonoid content in commercial mulberry tea (CT 2) is 4.76 mg/g, and the DPPH radical scavenging activities were similar to the distribution of total flavonoids measured by UAHAE in three types of mulberry tea. These finding provide a basis for industrial-scale manufacturing of edible medicinal mulberry leaf products. [ABSTRACT FROM AUTHOR]

MS/MS spectrum matching currently serves as a favored means to identify the concerned metabolites attributing to the accessibility of several famous databases. However, the rule that takes the entire structure into account frequently leads to "0 hit" when inquiring MS/MS (usually MS2) spectrum in the databases. Conjugation plays an important role for the high-level structural diversity of metabolites in all organisms, and a given conjugate usually consists of two or more substructures. If MS3 spectra participate in database retrieval, the structural annotation potential of those databases should be dramatically expanded via identifying substructures. Attributing to the ubiquitous distribution pattern, flavonoid glycosides were deployed as the representative family to justify whether the primary fragment ion termed as Y 0 +, resulted from neutral loss of glycosyl residue(s), generated identical MS3 spectrum with MS2 spectrum of the aglycone cation namely [A+H]+. Because of owning unique ability to measure MS/MS spectrum with the exactly desired exciting energy, linear ion trap chamber of Qtrap-MS was responsible for generating the desired MS3 and MS2 spectra. When taking both m/z and ion intensity features into consideration, the findings included: 1) glycosides sharing identical aglycones produced the same MS3 spectra for Y 0 +; 2) different MS3 spectra for Y 0 + occurred amongst glycosides bearing distinct, even isomeric, aglycones; 3) isomeric aglycones generated different MS2 spectra; and 4) MS3 spectra for Y 0 + agreed with MS2 spectra of [A+H]+ when comparing paired glycoside and aglycone. Together, fingerprint comparison between MS3 and MS2 spectra could structurally annotate the substructures and further advance MS/MS spectrum matching towards the identification of, but not limited to, aglycones for flavonoid glycosides. [Display omitted] • Fingerprint comparison between MS3 and MS2 spectra advance MS/MS spectrum matching. • The MS3 spectrum of Y 0 + was well-aligned with the MS2 spectrum of [A+H]+. • Forty-eight authentic flavonoids including glycosides and aglycones were used. • Spectrum matching helps the annotation of substructures for numerous metabolites. [ABSTRACT FROM AUTHOR]

• Two-step and highly efficient method to synthesize a series of novel C 21 -steroidal aglycone 3β-nitrogenous heterocyclic ester derivatives. • Most of synthetic derivatives showed moderate to significant cytotoxicities. • Compounds 1s and 2c exhibited the highest cytotoxicity with IC 50 values ranging from 3.30 to 6.73 μM and from 3.07 to 6.37 μM, respectively. • Compound 2c induced HepG2 cells apoptosis and cell cycle arrest at S phase in a dose dependent manner. To investigate the effect of introduction of the nitrogenous heterocyclic ester group on the antitumor activities of naturally occurring C 21 -steroidal aglycone, a series of novel 3 β -nitrogenous heterocyclic ester derivatives of caudatin, kidjoranin, qingyangshengenin, and rostratamin were synthesized. The results showed that most of synthetic derivatives showed moderate to significant cytotoxic activities against four human cancer cells (MCF-7, HCT-116, HeLa, and HepG2), in which compounds 1s and 2c with piperidine-4-acetyl substituent exhibited the highest cytotoxic activities against the four human cancer cells with IC 50 values ranging from 3.30 to 6.73 μM and from 3.07 to 6.37 μM, respectively. Further antitumor mechanism studies displayed that compound 2c was effective in inducing apoptosis, arresting the cell cycle in the S phase in a dose- dependent manner in vitro. Overall, these present studies showed that introduction of nitrogenous heterocyclic ester group into C-3 position is one of effective strategy to improve anticancer activity for naturally occurring C 21 -steroidal aglycones deserved further investigation. [ABSTRACT FROM AUTHOR]

β-glucosidases hydrolyse glycosidic bonds to release non-reducing terminal glucosyl residues from glycosides and oligosaccharides via catalytic mechanisms. It is very well known that the β-glucosidase enzyme is used in biorefineries for cellulose degradation, where β-glucosidases is the rate-limiting enzyme for the final glucose production from cellobiose. The β-glucosidase enzyme is used as a catalyst in other industrial sectors, including pharmaceuticals, breweries, dairy, and food processing. With the aid of β-glucosidase enzymes, cyanogenic glycosides and plant glycosides are transformed into sugar moiety and aglycones. These aglycone compounds are employed as aromatic compounds in the food processing and brewing industries. They are also used as medications and dietary supplements based on their pharmacological qualities. Applications of aglycones and the microbiological sources of β-glucosidase in aglycone production have been discussed in this review. [Display omitted] • β-glucosidase is crucial for hydrolysing glycosides and releasing value-added compounds in biotechnological sectors. • It is possible to obtain a potential catalyst with high stability by optimising conditions for microbial sources. • NGS technology helps to recreate desired products by exploring the functional roles of genes coding for β-glucosidase. [ABSTRACT FROM AUTHOR]

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) 23 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.

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 enzyme, β-glucosidase (β-Glu; EC 3.2.1.21), is a commercially important enzyme. First, β-Glu enzyme and specific activity were screened in total of 54 strains. In these strains, β-Glu-specific enzyme activity was assessed varying from 0.250 to 3.000 U/mg. Next, β-Glu enzyme belonging to the strains (Lactobacillus rhamnosus EA1 and Lactobacillus casei SC1) that exhibited high β-Glu-specific enzyme activity optimization were done. In these strains, optimum pH was 7.5, optimum temperature was 30°C, and optimum buffer was potassium phosphate. Then, the strains that displayed high β-Glu-specific enzyme activity were decided to hydrolyze the isoflavone glucosides using high-pressure liquid chromatography. Finally, following the partial purification of β-Glu enzyme, its molecular weight was detected to be approximately 78 K. Ultimately, in the present study, β-Glu enzyme acquired from L. casei SC1 strain became prominent because its activity was higher. It did not lose its activity under different environmental conditions and demonstrated high hydrolyzing the isoflavone. [ABSTRACT FROM AUTHOR]

Soymilk is a rich source of isoflavones (phytoestrogens). Lactic acid bacteria particularly, lactobacilli cultures produce β-glucosidase enzyme and converts into aglycone forms of isoflavones during the fermentation of soy milk. In the study, Lactobacillus rhamnosus MTCC 5945 (NS4) and Lactobacillus helveticus MTCC5463 (V3) produced highest β-glucosidase activity (0.93 and 0.91 U/ml, respectively) compared to other lactic cultures after supplementing whey protein concentrate (WPC70) at 2% in soy milk. Similarly, L. helveticus MTCC 5463 (V3) and L. rhamnosus MTCC 5945 (NS4) also biotransformed aglycones from glycone forms during fermentation of soy milk supplemented with WPC70 at 2%. These two cultures could be exploited for the development of functional soy-fermented products with higher bioavailability of aglycones. Practical applications The beneficial effects of soy Isoflavones are the protection against breast and prostate cancers, lowering of hematic cholesterol and lipids, antioxidant activity, prevention and treatment of osteoporosis, cardiovascular protection and alleviation of menopausal symptoms. Soy-derived isoflavones in Asian populations can lower the incidence of osteoporosis, mortality from cardiovascular disease and menopausal symptoms. L. rhamnosus MTCC 5945 (NS4) and L. helveticus MTCC5463 (V3) produced active form of isoflavones by producing β-glucosidase enzyme and converted into aglycone forms of isoflavones during the fermentation of soy milk supplemented with WPC70 at 2%. Soy-fermented foods added with whey protein concentrate could be prepared with these two cultures which may have various health beneficial effects like antioxidative, anticancer and anti-osteoporosis. [ABSTRACT FROM AUTHOR]

Benzoic acids (BAs) are important structural elements in a wide variety of essential compounds and natural products, and play crucial roles in plant fitness. BA is a precursor of diverse benzenoid compounds, including the hormone salicylic acid (SA) and the aglycone moiety of salicin, which is particularly important in the Salicaceae family. The biosynthetic pathways leading to BA formation in plants are largely unknown. Recently, the CoA-dependent β-oxidative BA biosynthesis pathway, which occurs in peroxisomes, has been characterized in petunia. The core of this pathway is cinnamic acid → cinnamoyl-CoA → 3-hydroxy-3-phenylpropanoyl-CoA → 3-oxo-3-phenylpropanoyl-CoA → benzoyl-CoA. Here, we used 454 pyrosequencing to analyze the transcriptome of Populus davidiana and isolate putative genes involved in BA biosynthesis. De novo assembly generated 57,322 unique sequences, including 15,217 contigs and 42,105 singletons. From the unique sequences, we selected six genes exhibiting high similarity to genes encoding L-phenylalanine ammonia lyase, cinnamate:CoA ligase, cinnamoyl-CoA hydratase-dehydrogenase, 3-ketoacyl-CoA thiolase, benzoyl-CoA:benzyl alcohol O-benzoyltransferase, and benzaldehyde dehydrogenase. Each of these enzymes might be involved in BA biosynthesis. Real-time PCR (qPCR) analysis revealed that these six genes were highly transcribed in the aerial organs of P. davidiana, particularly in leaves. Treating the leaves of in vitro cultured plants with methyl jasmonate (MeJA) strongly enhanced the mRNA accumulation of all 6 genes, and this treatment also clearly enhanced the accumulation of BA, SA, salicyl alcohol, benzyl alcohol, benzyl benzoate, and benzaldehyde but not salicin. Our study shows that P. davidiana may possess a CoA-dependent β-oxidative BA synthesis pathway. We also identified a relationship between the transcription of these genes and the accumulation of benzenoids, including BA and SA, which are highly responsive to the defense signaling molecule (MeJA). [ABSTRACT FROM AUTHOR]

The composition of terpen lactones and flavonol glycosides of commercial preparation series based on Ginkgo biloba extracts was investigated by 1H NMR spectroscopy. The content of individual terpen lactones was determined using DMSO-d6 and acetone-d6 solvents. The effect of the structure of flavonol glycosides on the signal of the hydroxyl proton at a position 5 of the ring A was examined. A new approach was proposed for semiquantitative determination of the total amount of flavonol glycosides by the integral intensity of this signal, which is a superposition of the singlets in the region of 12.5–12.65 ppm of individual flavonoids in DMSO-d6. Since the corresponding signals of aglycones (quercetin, kaempferol, isorhamnetin), which are minor components of the Ginkgo biloba extracts, appear separately in a slightly different region (12.45–12.48 ppm), the proposed method can also be used for detecting adulteration of Ginkgo biloba extracts by means of the addition into them of relatively cheap aglycones or rutin as well as for assessment of the content of flavonoids of similar structure in some types of plant raw materials. [ABSTRACT FROM AUTHOR]

The activity of the GH1 β-glucosidase from Humicola insolens (Bglhi) against p-nitrophenyl-β-D-glucopyranoside (pNP-Glc) and cellobiose is enhanced 2-fold by glucose and/or xylose. Kinetic and transglycosylation data showed that hydrolysis is preferred in the absence of monosaccharides. Stimulation involves allosteric interactions, increased transglycosylation and competition of the substrate and monosaccharides for the -1 glycone and the +1/+2 aglycone binding sites. Protein directed evolution has been used to generate 6 mutants of Bglhi with altered stimulation patterns. All mutants contain one of three substitutions (N235S, D237V or H307Y) clustered around the +1/+2 aglycone binding sites. Two mutants with the H307Y substitution preferentially followed the transglycosylation route in the absence of xylose or glucose. The strong stimulation of their pNP-glucosidase and cellobiase activities was accompanied by increased transglycosylation and higher monosaccharide tolerance. The D237V mutation favoured hydrolysis over transglycosylation and the pNP-glucosidase activity, but not the cellobiase activity, was stimulated by xylose. The substitution N235S abolished the preference for hydrolysis or transglycosylation; the cellobiase, but not the pNP-glucosidase activity of the mutants was strongly inhibited by xylose. Both the D237V and N235S mutations lowered tolerance to the monosaccharides. These results provide evidence that the fine modulation of the activity of Bglhi and mutants by glucose and/or xylose is regulated by the relative affinities of the glycone and aglycone binding sites for the substrate and the free monosaccharides. [ABSTRACT FROM AUTHOR]

Pectolinarin and pectolinarigenin have been reported to be major compounds in Cirsium setidens . In the present study, we demonstrated inhibitory effects of pectolinarin and pectolinarigenin from C. setidens on melanogenesis. Melanin synthesis was decreased in both pectolinarin- and pectolinarigenin-treated melan-a cells and in a reconstructed human skin model. However, pectolinarigenin treatment showed more potent inhibitory activity of melanin synthesis than did pectolinarin treatment. The concentrations of pectolinarin and pectolinarigenin in C. setidens water extracts were determined by HPLC. Unfortunately, the amount of pectolinarigenin of C. setidens water extract was lower than that of pectolinarin. To increase the pectolinarigenin content in C. setidens water extract, several component conversion methods were studied. Consequently, we identified that microwave irradiation under 1% acetic acid was an optimum sugar elimination method. [ABSTRACT FROM AUTHOR]