Identification of anthocyanin compositions in black seed coated Korean adzuki bean (Vigna angularis) by NMR and UPLC-Q-Orbitrap-MS/MS and screening for their antioxidant properties using different solvent systems.

The aim of the present research was to investigate the antioxidant properties and anthocyanin profiles in the black seed coated adzuki bean (Vigna angularis, Geomguseul cultivar). The acidic 60% methanol extract (40 µg/mL) contains the highest total phenolic and flavonoid contents (486 ± 3 mg GAE/100 g; 314 ± 10 mg CE/100 g) with potent antioxidant properties (trolox equivalent 1272 ± 26 and 662 ± 24 mg TE/100 g) against ABTS and DPPH radicals compared to other methanol-water ratios (20, 40, 80, and 100%). Ten anthocyanin components were identified in this extract including delphinidin-3,5-O-digalactoside (1), delphinidin-3,5-O-diglucoside (2), delphinidin-3-O-galactoside (3), delphinidin-3-O-glucoside (4), delphinidin-3-O-rutinoside (5), delphinidin-3-O-(p-coumaroyl)glucoside (6), cyanidin-3-O-glucoside (7), petunidin-3-O-galactoside (8), petunidin-3-O-glucoside (9) and petunidin-3-O-(p-coumaroyl)glucoside (10) via NMR spectroscopy and UPLC-Q-Orbitrap-MS/MS analysis. The key anthocyanins 3 and 4 of delphinidin type were isolated by reversed phase C-18 MPLC. Our results indicate that the anthocyanin profiles as well as the high phenolic and flavonoid contents are important factors determining the antioxidant effects of black adzuki bean.

Isolation and identification of α-glucosidase inhibitory constituents from the seeds of Vigna nakashimae: Enzyme kinetic study with active phytochemical.

The α-glucosidase inhibition effects of the 80% ethanol extracts in the seeds of five Vigna species (V. nakashimae, V. nipponensis, V. umbellate, V. radiate, and V. angularis) were evaluated and their half-maximal inhibitory concentration (IC50) values showed considerable differences (p < 0.05) ranging from 7.3 to >900 µg/ml. V. nakashimae exhibited the most potent inhibition with IC50 value of 7.3 ±â€¯1.1 µg/ml, followed by V. nipponensis (184.0 ±â€¯9.5 µg/ml) and V. umbellata (520.0 ±â€¯8.1 µg/ml). Bioactivity-guided fractionation of V. nakashimae seeds yielded three phenolics by silica gel chromatography and their structures were elucidated as gambiriin D (1), luteoliflavan-7-O-glucopyranoside (2), and catechin-7-O-glucopyranoside (3) using nuclear magnetic resonance (NMR) spectroscopy. In particular, gambiriin D (1) possessed strong inhibition activity with IC50 of 36.8 ±â€¯2.3 µM through simple reversible slow-binding inhibition (kinetic parameters: k4 = 0.0048  µM-1s-1; Kiapp = 48 µM). Furthermore, this compound inhibited recombinant human aldose reductase with IC50 value of 12.0 ±â€¯0.7 µM. Results suggest that V. nakashimae may be a potent α-glucosidase inhibition for health products.

Effects of Mung Bean (Vigna radiata L.) Ethanol Extracts Decrease Proinflammatory Cytokine-Induced Lipogenesis in the KK-Ay Diabese Mouse Model.

Rapid increase in the prevalence of obesity-related metabolic inflammatory diseases has led to research focused on nutraceuticals for their treatment. This study investigated the effects of the ethanol extracts of mung bean testa (MBT) on the metabolic inflammation-induced lipogenesis in gastrocnemius muscle of KK-Ay diabese mice. Ethanol extracts of MBT were orally administered to diabese KK-Ay mice for 4 weeks after diet-induced obesity model was generated by feeding a 60% high-fat diet for 3 weeks. Although there were no changes in body weight gain, MBT treatments decreased total weight of white adipose tissue. MBT also decreased triacylglycerol and total cholesterol levels in the muscle by 30%, which was correlated with suppression of lipogenic genes such as ACC, C/EBP alpha, PGC-1 alpha, and PPAR gamma. In particular, decreased levels of p-ERK1/2, PPAR gamma, and C/EBP alpha in the MBT-treated groups suggest that MBT might inhibit adipogenesis and decrease differentiation via the MEK/ERK pathway. Furthermore, significantly lower amounts of plasma interleukin (IL)-6 and intramuscular tumor necrosis factor (TNF)-alpha and monocyte chemoattractant protein-1 (MCP-1) were detected in MBT groups, confirming the anti-inflammatory effect of mung bean. In addition, our in vitro pilot study with 3T3-L1 cells showed that vitexin, the functional chemical in MBT, inhibited inflammation-induced lipogenesis with significantly lower amounts of IL-6 and MCP-1 after 14 days of vitexin treatment. Thus, the functional compounds in the mung bean ethanol extracts such as vitexin and isovitexin may regulate intracellular lipogenesis and adipogenesis via anti-inflammatory mechanisms and MEK/ERK pathway in the KK-Ay mouse model.

Identification, characterisation, and quantification of phenolic compounds in the antioxidant activity-containing fraction from the seeds of Korean perilla (Perilla frutescens) cultivars.

The present research was the first to investigate phenolic compound profiles and antioxidant properties in the seeds of various perilla (Perilla frutescens) cultivars. The 80% methanol extract (50 µg/ml) of this species showed potent antioxidant activities against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radicals. Phenolic compounds were characterised by nuclear magnetic resonance (NMR) spectroscopy, and ultra performance liquid chromatography with photodiode array detector and electrospray ionisation/mass (UPLC-PDA-ESI/MS) analysis. Nine compounds were elucidated as caffeic acid-3-O-glucoside (1), caffeic acid (2), luteolin-7-O-glucoside (3), apigenin-7-O-glucoside (4), rosmarinic acid-3-O-glucoside (5), rosmarinic acid (6), luteolin (7), apigenin (8), and chrysoeriol (9). The individual and total phenolic contents were remarkably different, especially rosmarinic acid-3-O-glucoside (5) and rosmarinic acid (6) which were the predominant compounds (>95%) in all perilla cultivars. Additionally, Yeupsil cultivar exhibited the highest phenolic content (5029.0 µg/g) and antioxidant activity, whereas the lowest was shown by Dasil (2138.7 µg/g). Therefore, these results suggest that antioxidant effects of perilla seeds are correlated with phenolic contents.

Black soybean anthocyanins inhibit adipocyte differentiation in 3T3-L1 cells.

Anthocyanins are naturally occurring polyphenolic pigments in plants that have been shown to decrease weight gain and insulin resistance in mice-fed high-fat diets. We investigated the effects of anthocyanins on cell growth, differentiation, and lipolysis in 3T3-L1 cells to test our hypothesis that anthocyanins could reduce adipose tissue mass by acting directly on adipocytes. Anthocyanin extracts from black soybeans were used and composed of 3 of the following major anthocyanins: cyanidine-3-O-glucoside (68.3%), delphinidin-3-O-glucoside (25.2%), and petunidin-3-O-glucoside (6.5%). Treatment with 12.5 and 50 µg/mL of black soybean anthocyanins exhibited inhibitory effects on the proliferation of both preconfluent preadipocytes (P < .01) and maturing postconfluent adipocytes (P < .01). In fully differentiated adipocytes, the number of viable cells was reduced by black soybean anthocyanins (P < .01). Treatment with 50 µg/mL of black soybean anthocyanins slightly increased epinephrine-induced lipolysis but decreased the basal lipolysis of fully differentiated adipocytes (P < .05). Black soybean anthocyanins also reduced lipid accumulation and suppressed the expression of the peroxisome proliferator-activated receptor γ, a major transcription factor for the adipogenic gene (P < .01). These results suggest that black soybean anthocyanins inhibit adipocyte differentiation and basal lipolysis, which may contribute to their antiobesity and antidiabetic properties.

Isolation and identification of phenolic compounds from the seeds of Perilla frutescens (L.) and their inhibitory activities against α-glucosidase and aldose reductase.

Five phenolic compounds were isolated from the seeds of Perilla (Perilla frutescens L.) using gradient solvent fractionation, silica gel column chromatography, and preparative high-performance liquid chromatography (HPLC). Their chemical structures were identified as caffeic acid-3-O-glucoside (1), rosmarinic acid-3-O-glucoside (2), rosmarinic acid (3), luteolin (4), and apigenin (5) using NMR spectroscopy and HPLC-ESI/MS analysis. Among them, luteolin (4) inhibited α-glucosidase (EC 3.2.1.20) with IC(50) value of 45.4µM. The inhibition kinetic analysed by Dixon plot indicate that luteolin is a noncompetitive inhibitor, and the inhibition constant K(I) was calculated at 45.0µM. Moreover, rosmarinic acid (3) and luteolin (4) inhibited recombinant human aldose reductase (EC 1.1.1.21) with IC(50) values of 11.2 and 0.6µM, respectively. Notably, the inhibition kinetic of luteolin (4) follows a hyperbolic dependence on aldose reductase inhibition by Dixon plot. Thus, inhibition kinetic indicates that luteolin (4) is a mixed-type inhibitor.

Anthocyanins extracted from black soybean seed coat protect primary cortical neurons against in vitro ischemia.

The present study investigated the neuroprotective effects of anthocyanins extracted from black soybean (cv. Cheongja 3, Glycine max (L.) MERR.) seed coat against oxygen-glucose deprivation (OGD) and glutamate-induced cell death in rat primary cortical neurons. Lactate dehydrogenase (LDH) release and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assays were employed to assess cell membrane damage and viability of primary neurons, respectively. OGD-induced cell death in 7 d in vitro primary cortical neurons was found to be OGD duration-dependent, and approximately 3.5 h of OGD resulted in ≈60% cell death. Treatment with black soybean anthocyanins dose-dependently prevented membrane damage and increased the viability of primary neurons that were exposed to OGD. Glutamate-induced neuronal cell death was dependent on the glutamate concentration at relatively low concentrations and the number of days the cells remained in culture. Interestingly, black soybean anthocyanins did not protect against glutamate-induced neuronal cell death. They did, however, inhibit the excessive generation of reactive oxygen species (ROS) and preserve mitochondrial membrane potential (MMP) in primary neurons exposed to OGD. In agreement with the neuroprotective effect of crude black soybean anthocyanins, purified cyanidin-3-glucoside (C3G), the major component of anthocyanins, also offered dose-dependent neuroprotection against OGD-induced neuronal cell death. Moreover, black soybean C3G markedly prevented excessive generation of ROS and preserved MMP in primary neurons that were exposed to OGD. Collectively, these results suggest that the neuroprotection of primary rat cortical neurons by anthocyanins that were extracted from black soybean seed coat might be mediated through oxidative stress inhibition and MMP preservation but not through glutamate-induced excitotoxicity attenuation.

Anthocyanins from the seed coat of black soybean reduce retinal degeneration induced by N-methyl-N-nitrosourea.

Anthocyanins are known to have antioxidant effects and thus may play an important role in preventing various degenerative diseases. In this study, we examined the effect of anthocyanins extracted from the seed coat of black soybean on an animal model of retinal degeneration (RD), a leading cause of photoreceptor cell death resulting in blindness. RD was induced in rats by an intraperitoneal injection of N-methyl-N-nitrosourea (MNU) (50mg/kg), a DNA-methylating agent that causes photoreceptor damage. Anthocyanins extracted from black soybean seed coat (50mg/kg) were daily administered, orally, for 1, 2, and 4 weeks after MNU injection. Electroretinographic (ERG) recordings and morphological analyses were performed. In control rats with MNU-induced retinal damage, the ERG recordings showed a gradual significant time-dependent reduction in both a- and b-wave amplitudes compared with those of normal animals. In the MNU-induced RD rats given anthocyanins for 4 weeks, ERG responses were significantly increased compared with untreated RD rats, more apparently in scotopic stimulation than in the photopic condition. However, in the MNU-injected rats given anthocyanins for 1 and 2 weeks, the increase in ERG responses was not significant. Morphologically, the outer nuclear layer, where photoreceptors reside, was well preserved in the anthocyanin-treated rat retinas throughout the experimental period. In addition, retinal injury, evaluated by immunolabeling with an antibody against glial fibrillary acidic protein, was markedly reduced in anthocyanin-treated retinas. These results demonstrate that anthocyanins extracted from black soybean seeds can protect retinal neurons from MNU-induced structural and functional damages, suggesting that anthocyanins from black soybean seed coat may be used as a useful supplement to modulate RD.

Antidiabetic effects of Vigna nakashimae extract in db/db mice.

The inhibitory activity of Vigna nakashimae extract against intestinal α-glucosidase was investigated in vitro and in vivo. The extract exerted a significant inhibitory effect against intestinal α-glucosidases. With sucrose-loading, it reduced the peak responses of blood glucose significantly in normal mice. Next, it was administrated to 8-week-old db/db mice for 2 weeks, and then plasma glucose, triglyceride, and total cholesterol levels were measured. The extract significantly suppressed postprandial hyperglycemia and blood glycated hemoglobin in the db/db mice. In addition, it lowered fasting glucose and improved glucose tolerance. Furthermore, it led to significant decreases in plasma triglyceride levels. It reduced endoplasmic reticulum stress in thapsigargin-induced HepG2 cells. Taken together, these results suggest that Vigna nakashimae extract has hypoglycemic and hypolipidemic effects that occur via inhibition of α-glucosidase activity and endoplasmic reticulum stress.

Lipoxygenase inhibitory activity of 6-pentadecanylsalicylic acid without prooxidant effect.

6-Pentadecanylsalicylic acid, referred to as anacardic acid (C15:0), was found to inhibit the linoleic acid peroxidation competitively catalyzed by soybean lipoxygenase-1 (EC 1.13.11.12, Type 1) with an IC50 of 14.3 microM (4.88 microg/mL). This inhibition is a reversible reaction without pro-oxidant effects. The inhibition kinetics analyzed by Dixon plots indicates that anacardic acid (C15:0) is a competitive inhibitor and the inhibition constant, KI, was established as 6.4 microM. The hydrophilic head (salicylic acid) portion first chelates the iron in the active site and then the hydrophobic tail portion begins reversibly interacting with the C-terminal domain where the iron is located. The inhibition of anacardic acid (C15:0) can be explained by a combination of iron ion-chelation and hydrophobic interaction abilities because of its specific structural feature.

Tyrosinase inhibitory effects of 1,3-diphenylpropanes from Broussonetia kazinoki.

Six 1,3-diphenylpropanes exhibiting inhibitory activities against both the monophenolase and diphenolase actions of tyrosinase were isolated from the methanol (95%) extract of Broussonetia kazinoki. These compounds, 1-6, were identified as kazinol C (1), D (2), F (3), broussonin C (4), kazinol S (5) and kazinol T (6). The latter two species (5 and 6) emerged to be new 1,3-diphenylpropanes which we fully spectroscopically characterized. The IC(50) values of compounds (1, 3-5) for monophenolase inhibition were determined to range between 0.43 and 17.9 microM. Compounds 1 and 3-5 also inhibited diphenolase significantly with IC(50) values of 22.8, 1.7, 0.57, and 26.9 microM, respectively. All four active tyrosinase inhibitors (1, 3-5) were competitive inhibitors. Interestigly they all mainfested simple reversible slow-binding inhibition against diphenolase. The most potent inhibitor, compound 4 diplayed the following kinetic parameters k(3)=0.0993 microM(-1)min(-1), k(4)=0.0048 min(-1), and K(i)(app)=0.0485 microM.

Evaluation of lipoxygenase inhibitory activity of anacardic acids.

6-Alkylsalicylic acids inhibit the linoleic acid peroxidation catalyzed by soybean lipoxygenase-1 (EC 1.13.11.12, type 1) competitively and without pro-oxidant effects. This activity is largely dependent on the nature of their alkyl side chains. Inhibitory activities of anacardic acids, viz. 6-pentadec(en)ylsalicylic acids, isolated from the cashew Anacardium occidentale, were initially used for comparison because their aromatic head portions are the same. Consequently, the data should be interpreted to mean that changes in the hydrophobic side chain tail portions of the molecules evaluated correlate with the specific activity determined.

Antibacterial activity of polygodial.

Polygodial was found to possess moderate antibacterial activity against gram-positive bacteria including Bacillus subtilis, Staphylococcus aureus and gram-negative bacteria including Escherichia coli and Salmonella choleraesuis with minimum bactericidal concentrations (MBC) of 100 and 100 microg/mL (0.34 mm) and 100 and 50 microg/mL, respectively. The time kill curve study showed that polygodial was bactericidal against B. subtilis and S. choleraesuis. However, there was a difference in its bactericidal action against endospore-forming B. subtilis and food-borne S. choleraesuis.

Cytotoxic effects of sesquiterpene lactones from the flowers of Hemisteptia lyrata B.

Four guaia-12,6-olide type sesquiterpene lactones, aguerin B (1), 8alpha-acetoxyzaluzanin C (2), cynaropicrin (3), and deacylcynaropicrin (4), were isolated from the flowers of Hemisteptia lyrata Bunge. It is the first report on the isolation of compounds 1-4 from Hemisteptia species. All the isolates (1-4) were examined for their cytotoxic activity against SK-OV-3, LOX-IMVI, A549, MCF-7, PC-3, and HCT-15 human cancer cell lines.