Identification of Scoparone from Chinese Olive Fruit as a Modulator of Macrophage Polarization.

Chinese olive (Canarium album L.) has been highlighted for its remarkable health benefits. We previously showed that the ethyl acetate fraction of Chinese olive (COE) is an effective anti-inflammatory agent. In this study, we used a luciferase-based RAW 264.7 cell platform to detect the transcriptional activity of NF-κB, a key mediator of inflammation, and the promoter activity of its downstream target, COX-2. Through functional-oriented screening using these platforms, we further divided COE into several subfractions. Subsequently, we used silica gel column chromatography for purification, and the active compounds were separated and isolated by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The structure of the resulting compound with high anti-inflammatory activity was then identified as scoparone. Our results showed that scoparone not only inhibited lipopolysaccharide (LPS)-induced secretion of nitric oxide and suppressed M1 macrophage markers (iNOS, Il-6, Ccl2, and Tnf-α) but also markedly decreased the production of pro-inflammatory cytokines (IL-6, CCL2, and TNF-α). Treatment with scoparone significantly reduced the protein level of TNF-α in LPS-treated bone-marrow-derived macrophages (BMDMs). In addition, scoparone promoted macrophages toward an M2 anti-inflammatory phenotype, as determined by the significantly increased gene expression of M2 macrophage markers (Arg1, Ym1, Mrc1, Il-10, and Cd206) and the protein level of Arg1. This study indicates that COE fruit has high therapeutic potential for various inflammatory diseases as a result of switching the macrophage phenotype from pro-inflammatory M1 to anti-inflammatory M2.

Discovery of a novel phosphotransferase from Bacillus subtilis that phosphorylates a broad spectrum of flavonoids.

Flavonoids are associated with health benefits, but most of them have poor oral bioavailability due to their extremely low aqueous solubility. Flavonoid O-phosphorylation suggests a potent modification to solve the problems. Here, we isolated, identified and characterized an unprecedented phosphotransferase, flavonoid phosphate synthetase (BsFPS), from B. subtilis. The enzyme catalyzes the ATP-dependent phosphorylation of flavonoid to generate flavonoid monophosphates, AMP and orthophosphate. BsFPS is a promiscuous phosphotransferase that efficiently catalyzes structurally-diverse flavonoids, including isoflavones, flavones, flavonols, flavanones and flavonolignans. Based on MS and NMR analysis, the phosphorylation mainly occurs on the hydroxyl group at C-7 of A-ring or C-4' of B-ring in flavonoid skeleton. Notably, BsFPS is regioselective for the ortho-3',4'-dihydroxy moiety of catechol-containing structures, such as luteolin and quercetin, to produce phosphate conjugates at C-4' or C-3' of B-ring. Our findings highlight the potential for developing biosynthetic platform to obtain new phosphorylated flavonoids for pharmaceutical and nutraceutical applications.

Luteolin Phosphate Derivatives Generated by Cultivating Bacillus subtilis var. Natto BCRC 80517 with Luteolin.

Luteolin (LUT), a plant-derived flavone, exhibits various bioactivities; however, the poor aqueous solubility hampers its applications. Here, we revealed bioconversion of LUT by Bacillus subtilis BCRC 80517, yielding three water-soluble phosphate conjugates. These derivatives were identified as luteolin 4'-O-phosphate (L4'P), luteolin 3'-O-phosphate (L3'P), and luteolin 7-O-phosphate (L7P) by LC-ESI-MS/MS and NMR. Besides, we found that Bacillus subtilis BCRC 80517 was able to convert different levels of LUT but showed a limited conversion rate. By observing bacterial morphology with transmission electron microscopy and confocal fluorescence microscopy, we found that LUT disrupted the bacterial membrane integrity, which explained the incomplete conversion. Additionally, we revealed a spontaneous intramolecular transesterification of L4'P to L3'P, the thermodynamically more stable form, under acidic conditions and proposed the possible mechanism involving a cyclic phosphate as the intermediate. This study provides insight into development of a potent structural modification strategy to enhance the solubility of LUT through biophosphorylation.

Stable carbon isotopic characterization of rice vinegar protein as an intrinsic reference for discriminating the authenticity of brewed rice vinegar.

Rice vinegar plays an important role in daily life. However, some unscrupulous manufacturers may deliberately add synthetic acetic acid in vinegar products to reduce fermentation time and save production costs. To protect the rights and health of consumers, vinegar authenticity must be controlled. The rice vinegar protein was used as an intrinsic reference and its stable carbon isotope ratio (δ13Cprotein) was analyzed by elemental analyzer-isotope ratio mass spectrometry. The stable carbon isotope ratio difference between the acetic acid and the rice vinegar protein (Δδ13Cacetic acid-protein) was calculated to evaluate vinegar authenticity. Sixteen rice vinegar samples were analyzed and a stable carbon isotopic pattern of rice vinegar was established by the 95% confidence interval for Δδ13Cacetic acid-protein (0.27‰-2.10‰). An acetic acid adulteration curve of Δδ13Cacetic acid-protein was also assumed according to the data from rice vinegar samples, and its validity was confirmed by rice vinegar deliberately blended with acetic acid at different ratios (25, 50, and 75%). The Δδ13Cacetic acid-protein values of the adulterated vinegars decreased with increasing amounts blended acetic acid, but the δ13Cprotein values did not, showing that rice vinegar protein could be used as an intrinsic reference for identifying the adulterated rice vinegar. The rice vinegar adulterated with acetic acid at higher than approximately 10% could be detected.

Lower caprylate and acetate levels in the breast milk is associated with atopic dermatitis in infancy.

BACKGROUND: Atopic dermatitis (AD) occurs in exclusively breastfed infants. As fatty acids have some immunomodulatory effect, we aimed to investigate the influence of fatty acid compositions in breast milk (BM) on the development of AD in exclusively breastfed infants. METHODS: We enrolled two- to four-month-old exclusively breastfed infants. The objective SCORing Atopic Dermatitis (objSCORAD) was evaluated. The lipid layer of BM was analyzed by gas chromatography for fatty acid levels. Medical charts were reviewed. RESULTS: Forty-seven AD infants and 47 healthy controls were enrolled. The objSCORAD was 20.5 ± 1.7 (shown as mean ± SEM) in the AD group. The age, sex, parental atopy history, and nutrient intake of mothers were not significantly different between two groups. The palmitate and monounsaturated fatty acid (MUFA) levels in BM positively correlated with objSCORAD, while caprylate, acetate, and short-chain fatty acid (SCFA) levels negatively correlated with objSCORAD (p = .031, .019, .039, .013, .022, respectively). However, the butyrate levels in BM were not significantly different. The caprylate and acetate levels in BM were significantly associated with the presence of infantile AD (p = .021 and .015, respectively) after adjusting for age, sex, parental allergy history, MUFA, palmitate, and SCFA levels in BM. ObjSCORAD in infancy was significantly associated with persistent AD (p = .026) after adjusting for age, sex, parental atopy history, caprylate, palmitate, MUFA, acetate, and SCFA levels in BM. CONCLUSION: Caprylate and acetate levels in BM for exclusively breastfed infants were negatively associated with objSCORAD. Lower caprylate and acetate in BM might be the risk factors for infantile AD, while butyrate in BM was not associated with infantile AD.

Microbial Phosphorylation Product of Hesperetin by Bacillus subtilis BCRC 80517 Improves Oral Bioavailability in Rats.

The flavanoid hesperidin (Hsd) is one of the major polyphenols in citrus fruits. Hsd and its aglycone hesperetin (Hst) have a broad array of bioactivities; however, their low aqueous solubility and low intestinal permeability lead to their limited oral bioavailability. In the present study, we generated two water-soluble derivatives of Hst, namely, Hst 7-O-phosphate and Hst3'-O-phosphate, by a unique bioconversion process of Bacillus subtilis var. natto BCRC80517. The phosphorylated products showed superior aqueous solubility and distinct physicochemical properties compared with the original Hst. The Hst phosphate derivatives (HstPs) remained stable in simulated gastric and intestinal fluids for 240 min and could revert to the original Hst form by alkaline phosphatase treatment in Caco-2 cells, showing enhanced intestinal permeability in vitro. After oral administration in rats, HstPs greatly elevated plasma exposure to Hst and showed better bioavailability than did Hsd. HstPs may be a potential and efficient alternative to Hst.

First Determination of Glycidyl Ester Species in Edible Oils by Reverse-Phase Ultra-Performance Liquid Chromatography Coupled with an Evaporative Light-Scattering Detector.

In this work, a new ultra-performance liquid chromatograph-evaporative light-scattering detector (UPLC-ELSD) method for quantitation of glycidyl esters (GE) contents in edible oils is presented. The method features complete separation of five GE species within 20 min by a C18 column and gradient elution with a mobile phase consisting of 85% and 2.5% methanol aqueous solutions. The coefficients of regression (R2) were all ≥0.9999 for the linear-quadratic regression curves of GE species in a concentration range of 5~80 µg/mL. The intraday and interday recoveries (%) of GE species in solvent were in a range of 81.3~107.3%, and the intraday and interday coefficients of variation (CVs, %) were all ≤8.6%. The average recovery (%) of GE species spiked in extra-virgin olive oil samples ranged from 88.3~107.8% and the intermediate precision (CV, %) of ≤14% indicated acceptable accuracy and precision. The method exhibited limit of quantification (LOQ) for each GE species (0.6 µg glycidol equivalents/g oil). The method was applied to determine GE concentrations of six commercial oil samples, and total glycidol equivalents were consistent with data obtained by GC-MS method. This UPLC-ELSD method could be adopted for precursory screening and research purposes to improve food safety when MS detectors are unavailable.

Determination of glycidyl esters and 3-MCPD esters in edible oils by sample pretreatment with the combination of lipase hydrolysis and modified QuEChERS for GC-MS analysis.

Glycidyl esters (GEs) and 3-chloroprapane-1,2-diol esters (3-MCPDEs) are processing contaminants in refined edible oils that have raised concerns globally owing to their potentially carcinogenic properties. Official analytical methods for GEs and 3-MCPDEs, such as AOCS Cd 29a-13 and AOCS Cd 29b-13, require up to 16 h for chemical hydrolysis. Also, parallel experiments should be conducted to correct for the conversion of analytes during hydrolysis in AOCS Cd 29b-13. For AOCS Cd 29c-13 with the shortest operating time, the reaction time (3.5-5.5 min) and temperature of alkaline hydrolysis should be carefully controlled, implying the accuracy may be influenced by human errors. Here, we propose a novel method based on Candida rugosa lipase hydrolysis and direct detection of free form GEs, glycidol, which was achieved by sample preparation with modified QuEChERS, to prevent side reactions in previous approaches, and also to shorten the overall sample preparation time. Glycidol was directly analyzed without halogenation and derivatization, whereas 3-MCPD required derivatization for analysis by GC-MS. Our method showed good accuracy and precision in terms of repeatability, intermediate precision, and reproducibility (inter-laboratory precision). The limit of detection (LOD) and limit of quantification (LOQ) for glycidol were 0.02 and 0.1 mg/kg, which is sufficient for practical applications. The proposed method was further compared with AOCS Cd 29c-13 by determination of GEs content in commercial oil samples and spiked samples. Our method with a streamlined procedure seems to possess potential advantage of reduced errors from operational factors. This proposed method based on direct detection of glycidol may serve as a simplified alternative for routine analysis of GEs and 3-MCPDEs in edible oils.

Simultaneous Analysis of the Stable Carbon Isotope Ratios of Acetoin and Acetic Acid by GC-C-IRMS for Adulteration Detection in Brewed Rice Vinegar Products.

In this study, we developed a method to simultaneously measure the stable carbon isotope ratio for acetic acid (δ 13Cacetic acid) and acetoin (δ13Cacetoin) in rice vinegar by gas chromatography-combustion-isotope ratio mass spectrometry. The method showed good precision and accuracy. With this method, data from 16 brewed rice vinegars and 10 acetic acid samples were used to evaluate the feasibility of adulteration detection. On the basis that all δ13Cacetoin values of brewed rice vinegars are nearly constant, a characteristic pattern of the stable carbon isotope in rice vinegar was built with the 95% confidence intervals for δ13Cacetic acid (-26.97 to -25.38‰), δ13Cacetoin (-28.14 to -27.09‰), and Δδ13C (0.61 to 2.27‰). An adulteration detection curve of Δδ13C was proposed based on the results of vinegar and acetic acid samples and confirmed by vinegar spiked with different amounts of acetic acid. This method could be useful in estimating the blending ratio of adulterated rice vinegar products. Products containing more than 10% of synthetic acetic acid could be possibly identified.

Isolation of individual isoflavone species from soybean by solvent extraction followed by the combination of macroporous resin and aluminium oxide separation.

Growing interest in the health benefits of soy isoflavones has led to research in the isolation of individual isoflavone species for further application. Herein, we develop a new strategy to isolate daidzein, genistein, daidzin and genistin in soybean. We investigated the impact of solvents used and the extraction time on the extracted isoflavone contents from soybean. A 30-min extraction with 65% aqueous methanol gave a total isoflavone yield of 345 mg/100 g soybean, the highest value among tested conditions. Further, we proposed a two-stage adsorption/desorption chromatography comprising macroporous resin and aluminium oxide to isolate isoflavone. First, HP-20 resin was used to separate the glucosidic and aglyconic forms of isoflavone, then individual species of isoflavone could be isolated using aluminium oxide by specific retention of 5-hydroxy isoflavone. This process achieved overall high recovery (82-97%) and purity (92-95%) of the four isoflavones, which confirms a high separating efficiency for isoflavones from soybean.

Formation of Samin Diastereomers by Acid-Catalyzed Transformation of Sesamolin with Hydrogen Peroxide.

The conversion of sesame lignans is of interest because the derived products may have potential applications. Here, in investigating the transformation of sesamin and sesamolin, main endogenous sesame lignans in sesame seeds, in both acidic aqueous and anhydrous systems, 7R,7'S-samin was identified as one of the major products of sesamolin in both systems catalyzed with common inorganic acids, but sesaminol was not generated. In investigating the effect of different oxidizing agents on the acid-catalyzed conversion of sesame lignans, 7R,7'S-samin was still the major product of sesamolin, whereas sesamolin as well as 7R,7'S-samin stereoselectively rendered 7R,7'R-samin in the presence of hydrogen peroxide. Hydrogen peroxide may play a role in stabilizing the transitional oxonium ions, derived from acid hydrolysis of sesamolin or 7R,7'S-samin by forming a seven-membered ring intermediate through hydrogen bonding, to consequently produce 7R,7'R-samin as the final product.

Complex Tannins Isolated from Jelly Fig Achenes Affect Pectin Gelation through Non-Specific Inhibitory Effect on Pectin Methylesterase.

Jelly fig (Ficus awkeotsang Makino) is used to prepare drinks and desserts in Asia, owing to the gelling capability of its pectin via endogenous pectin methylesterase (PE) catalyzation. Meanwhile, substances with PE inhibitory activity (SPEI) in jelly fig achenes (JFA) residue were noticed to be able to impede the gelation. In this study, we characterized and isolated SPEI from JFA by a series of PE inhibition-guided isolations. Crude aqueous extract of JFA residue was mixed with acetone, and 90% acetone-soluble matter was further fractionated by Diaion HP-20 chromatography. The retained fraction with dominant PE inhibitory activity was collected from 100% methanol eluate. Results from high-performance liquid chromatography mass spectrometry (HPLC/MS) and hydrolysis-induced chromogenic transition revealed the SPEI as complex tannins. Total tannins content was determined in each isolated fraction, and was closely related to PE inhibitory activity. In addition, SPEI in this study could inhibit activities of digestive enzymes in vitro and may, therefore, be assumed to act as non-specific protein binding agent.

Phosphorylation of Isoflavones by Bacillus subtilis BCRC 80517 May Represent Xenobiotic Metabolism.

The soy isoflavones daidzein (DAI) and genistein (GEN) have beneficial effects on human health. However, their oral bioavailability is hampered by their low aqueous solubility. Our previous study revealed two water-soluble phosphorylated conjugates of isoflavones, daidzein 7-O-phosphate and genistein 7-O-phosphate, generated via biotransformation by Bacillus subtilis BCRC80517 cultivated with isoflavones. In this study, two novel derivatives of isoflavones, daidzein 4'-O-phosphate and genistein 4'-O-phosphate, were identified by HPLC-ESI-MS/MS and 1H, 13C, and 31P NMR, and their biotransformation roadmaps were proposed. Primarily, isoflavone glucosides were deglycosylated and then phosphorylated predominantly into 7-O-phosphate conjugates with traces of 4'-O-phosphate conjugates. Inevitably, trace quantities of glucosides were converted into 6″-O-succinyl glucosides. GEN was more efficiently phosphorylated than DAI. Nevertheless, the presence of GEN prolonged the time until the exponential phase of cell growth, whereas the other isoflavones showed little effect on cell growth. Our findings provide new insights into the novel microbial phosphorylation of isoflavones involved in xenobiotic metabolism.

Black garlic: A critical review of its production, bioactivity, and application.

Black garlic is obtained from fresh garlic (Allium sativum L.) that has been fermented for a period of time at a controlled high temperature (60-90°C) under controlled high humidity (80-90%). When compared with fresh garlic, black garlic does not release a strong offensive flavor owing to the reduced content of allicin. Enhanced bioactivity of black garlic compared with that of fresh garlic is attributed to its changes in physicochemical properties. Studies concerning the fundamental findings of black garlic, such as its production, bioactivity, and applications, have thus been conducted. Several types of black garlic products are also available in the market with a fair selling volume. In this article, we summarize the current knowledge of changes in the components, bioactivity, production, and applications of black garlic, as well as the proposed future prospects on their possible applications as a functional food product.

Antimelanogenic Effect of Urolithin A and Urolithin B, the Colonic Metabolites of Ellagic Acid, in B16 Melanoma Cells.

Antimelanogenic agents from natural sources have been widely investigated. Urolithin A (UA) and B (UB), the main gut microflora metabolites of dietary ellagic acid derivatives, have various bioactivities such as anti-inflammatory and antiaging effects. In this study, the metabolites were found to possess depigmentation efficacy by suppressing tyrosinase activity. Both UA and UB could attenuate melanogenesis in B16 melanoma cells to 55.1 ± 3.8 and 76.4 ± 17.4% of control at noncytotoxic dosage, 10 µM, respectively. UA showed comparable efficacy to positive control, 5 µM of kojic acid treatment (51.2 ± 7.8). RT-PCR results revealed that UA and UB inhibited melanin formation by affecting the catalytic activity of tyrosinase rather than its mRNA expression. Kinetics for UA and UB on tyrosinase activity revealed that their inhibition behavior toward cellular tyrosinase involved competitive inhibition. UA and UB may be potent tyrosinase inhibitors and they possess significant antimelanogenesis ability as novel skin-whitening ingredients.

Docosahexaenoic acid increases accumulation of adipocyte triacylglycerol through up-regulation of lipogenic gene expression in pigs.

BACKGROUND: Changing dietary fatty acid composition in modern diet influences the prevalence of obesity. Increasing evidences suggest favorable effects of n-3 PUFA for protecting against obesity and the metabolic syndrome. However, the regulation of n-3 PUFA in adipose is still unclear. Thus, this study addressed metabolism of different dietary fats in the adipose tissue of porcine model. METHODS: Eight-week-old cross-bred pigs were randomly assigned to three groups and fed a 2% fat diet for 30 days from either soybean oil (SBO), docosahexaenoic acid (DHA) or beef tallow. An in vitro experiment was conducted in which linoleic acid (LA), DHA or oleic acid (OA) were added to represent the major fatty acid in the SBO-, DHA- or BT- diets, respectively. Adipocytes size and lipid metabolism related genes were analyzed. RESULTS: Plasma triacylglycerol (TAG) was lower in DHA- than in BT-fed pigs, and the product of lipolysis, glycerol was highest in BT-fed pigs. In addition, expression of the lipolytic genes, adipose triglyceride lipase and hormone sensitive lipase was higher in BT-fed pigs and with OA treatment in vitro. DHA promoted protein kinase A activity in pigs without affecting lipolytic genes. Adipocyte cell sizes, TAG content and expression of lipogenic-related genes including, adipose differentiated related protein (ADRP) and diacylglycerol acyltransferase 1 (DGAT1) were elevated by DHA in vivo and in vitro, indicating DHA promoted adipogenesis to trap TAG in adipose tissue. Fatty acid ß-oxidation genes were increased in the DHA-fed pigs. CONCLUSION: This effect was partly explained by the effect of DHA to promote adipogenesis to trap TAG in adipocytes and also increase expression of genes involved in adipocyte fatty acid oxidation. Therefore, our results suggest a direct effect of DHA on adipocyte metabolism, resulting in TAG turnover and fatty acid dissipation to facilitate plasma lipid uptake from the circulation.

The methanol-ethyl acetate partitioned fraction from Chinese olive fruits inhibits cancer cell proliferation and tumor growth by promoting apoptosis through the suppression of the NF-κB signaling pathway.

Chinese olives (Canarium album L.) have historically been used for medicinal purposes rather than commercially for oil. In this report, we reveal that the methanol-ethyl acetate partitioned fraction from Chinese olive fruits (MEO), of which ellagic acid accounted for 12%, exhibited profound anti-proliferative activities in the human colon cancer cell line, HCT116. Additionally, oral administration of MEO remarkably inhibited the tumor growth of subcutaneously implanted CT26 cells, a mouse colon carcinoma cell line, in BALB/c mice. Treatment with MEO induced a significant increase in the percentage of apoptotic cells and resulted in poly(ADP-ribose) polymerase (PARP) cleavage, suggesting that MEO inhibits cancer cell proliferation by promoting apoptosis. Our study also showed that MEO exerted the most potent effect on the inhibition of NF-κB-mediated signaling among the partitioned fractions from Chinese olives. This process employed the use of reporter-based bio-platforms that are capable of detecting the activation of NF-κB. In addition, phosphorylation of NF-κB signaling-associated proteins, IKKα/ß, IκBα, and p65, was reduced in MEO-incubated cancer cells, indicating that MEO suppresses NF-κB activation. Moreover, MEO treatment significantly suppressed lipopolysaccharide (LPS)-induced cancer cell proliferation, demonstrating that MEO promotes cancer cell apoptosis through the inhibition of the NF-κB signaling pathway. In summary, our findings demonstrate that the methanol-ethyl acetate partitioned fraction from Chinese olive fruits inhibits cancer cell proliferation and tumor growth by promoting apoptosis through the suppression of NF-κB signaling. Therefore, the Chinese olive fruit has promising potential in cancer treatment.

Individual Phosphatidylcholine Species Analysis by RP-HPLC-ELSD for Determination of Polyenylphosphatidylcholine in Lecithins.

Polyenylphosphatidylcholine (PPC), a subgroup of the bioactive agents in phosphatidylcholine (PC), has been indicated to possess liver-protective effects. This study aimed to investigate a promising and feasible method to determine PC molecular species with a reverse phase (RP) high-performance liquid chromatograph (HPLC) equipped with an evaporative light scattering detector (ELSD). Chromatography was achieved using a C30 column and an isocratic mobile phase consisting of acetonitrile/methanol/triethylamine (40/58/2, v/v/v) at a flow rate of 1 mL/min, and ELSD detection was performed using 80 °C for the drift tube and an air flow rate of 1.8 L/min. To identify individual peaks on the chromatogram, MALDI-TOF-MS was employed for initial detection, and then the results were used to investigate the relationship between the retention time and fatty acyl chains of each PC molecule. A linear correlation was observed between the retention time and theoretical carbon number (TCN) of individual PC species. The compositions of PC molecular species in soybean and sunflower lecithins were similar to each other, and the major PC molecular species were 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (LLPC), 1-oleoyl-2-linoleoyl-sn-glycero-3-phosphocholine (OLPC), and 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC). The contents of LLPC in soybean PC and sunflower PC were 40.6% and 64.3%, respectively.

A novel soybean (Glycine max) gene encoding a family 3 ß-glucosidase has high isoflavone 7-O-glucoside-hydrolyzing activity in transgenic rice.

A previous study demonstrated that purified Glycine max ß-glucosidase (GmBGL) could hydrolyze glucosyl isoflavone to the aglyconic form. This study reports the cloning and functional characterization of a soybean cDNA encoding the ß-glucosidase. GmBGL was isolated by use of a purified soybean N-terminal amino acid sequence and conserved sequences of ß-glucosidase genes from other plants. Sequence analysis of GmBGL revealed an open reading frame of 1884 bp encoding a polypeptide of 627 amino acids with a calculated molecular mass of 69 kDa. Phylogenetic analysis classified the GmBGL into the glycosyl hydrolase 3 family. In soybean, the GmBGL transcript was predominantly accumulated in roots and leaves. To examine the enzymatic activity and substrate specificity, GmBGL was ectopically expressed in transgenic rice. Purified GmBGL protein from transgenic rice could catalyze the hydrolysis of genistin and daidzin to produce genistein and daidzein, respectively, which confirmed GmBGL as a functional ß-glucosidase with isoflavone glucoside-hydrolyzing activity. This paper reveals that GmBGL is a key enzyme in transforming glucosyl isoflavones to aglycones in soybean, which may help in genetic manipulation of aglycone-rich soybean seeds.