Pterocarpan profiles for soybean leaves at different growth stages and investigation of their glycosidase inhibitions

Soybean leaves are eaten as seasonal edible greens in Korea. Analysis of the ethyl acetate extract of these leaves showed that it exhibited potent and selective neuraminidase inhibition, which began at the R3 stage and peaked at R7. Ten pterocarpans, including the new 6a-hydroxypterocarpan 10, were isolated from soybean leaves and their inhibition activities tested against a range of glycosidases. The relationship between structure and enzyme inhibition was investigated: 6a-hydroxypterocarpans exhibited much higher inhibition against neuraminidase (IC(50) = 2.4-89.4 μM) than α-glucosidase (IC(50) = 90.4-?>100 μM). Glyceollin VII (7) displayed 40-fold greater activity (IC(50) = 2.4 μM) against neuraminidase than α-glucosidase (IC(50) = 90.4 μM). On the other hand, coumestanes (1-3) were good α-glucosidase inhibitors (IC(50) = 6.0-42.6 μM). In kinetic analysis, the most potent neuraminidase inhibitors (5-10) were noncompetitive. HPLC analysis indicated that most pterocarpan synthesis began from the R3 stage, and a rapid change of pterocarpan concentrations was observed between the R4 and R7 stages.     

Synthesis and antityrosinase mechanism of benzaldehyde thiosemicarbazones: novel tyrosinase inhibitors

p-Hydroxybenzaldehyde thiosemicarbazone (HBT) and p-methoxybenzaldehyde thiosemicarbazone (MBT) were synthesized and established by (1)H NMR and mass spectra. Both compounds were evaluated for their inhibition activities on mushroom tyrosinase and free-cell tyrosinase and melanoma production from B(16) mouse melanoma cells. Results showed that both compounds exhibited significant inhibitory effects on the enzyme activities. HBT and MBT decreased the steady state of the monophenolase activity sharply, and the IC(50) values were estimated as 0.76 and 7.0 μM, respectively. MBT lengthened the lag time, but HBT could not. HBT and MBT inhibited diphenolase activity dose-dependently, and their IC(50) values were estimated as 3.80 and 2.62 μM, respectively. Kinetic analyses showed that inhibition type by both compounds was reversible and their mechanisms were mixed-type. Their inhibition constants were also determined and compared. The research may supply the basis for the development of new food preservatives and cosmetic additives.     

Bioactivity-guided fractionation for the butyrylcholinesterase inhibitory activity of furanocoumarins from Angelica archangelica L. roots and fruits

Isolation and identification of the inhibitors of butyrylcholinesterase (BChE), obtained from the extracts of roots and fruits of Angelica archangelica L., are reported. Our results confirmed the weak inhibitory effect of Angelica roots on acetylcholinesterase activity. BChE inhibition was much more pronounced at a concentration of 100 μg/mL for hexane extracts and attained a higher rate than 50%. The TLC bioautography guided fractionation and spectroscopic analysis led to the isolation and identification of imperatorin from the fruit's hexane extract and of heraclenol-2'-O-angelate from the root's hexane extract. Both compounds showed significant BChE inhibition activity with IC(50) = 14.4 ± 3.2 μM and IC(50) = 7.5 ± 1.8 μM, respectively. Only C8-substituted and C5-unsubstituted furanocoumarins were active, which could supply information about the initial structures of specific BChE inhibitors.     

HIV-1 integrase and neuraminidase inhibitors from Alpinia zerumbet

AIDS and influenza are viral pandemics and remain one of the leading causes of human deaths worldwide. The increasing resistance of these diseases to synthetic drugs demands the search for novel compounds from plant-based sources. In this regard, the leaves and rhizomes of Alpinia zerumbet, a traditionally important economic plant in Okinawa, were investigated for activity against HIV-1 integrase (IN) and neuraminidase (NA). The aqueous extracts of leaves and rhizomes had IN inhibitory activity with IC(50) values of 30 and 188 μg/mL, whereas against NA they showed 50% inhibition at concentrations of 43 and 57 μg/mL, respectively. 5,6-Dehydrokawain (DK), dihydro-5,6-dehydrokawain (DDK), and 8(17),12-labdadiene-15,16-dial (labdadiene) were isolated from the rhizomes and were tested for enzyme inhibitions. DK and DDK strongly inhibited IN with IC(50) of 4.4 and 3.6 μg/mL, respectively. Against NA, DK, DDK, and labdadiene exhibited mixed type of inhibition with respective IC(50) values of 25.5, 24.6, and 36.6 μM and K(i) values ranging from 0.3 to 2.8 μM. It was found that DDK is a slow and time-dependent reversible inhibitor of NA, probably with a methoxy group as its functionally active site. These results suggest that alpinia could be used as a source of bioactive compounds against IN and NA and that DK and DDK may have possibilities in the design of drugs against these viral diseases.     

Inhibition strength of short peptides derived from an ACE inhibitory peptide

A series of peptides, derived from an ACE inhibitory peptide (VTVNPYKWLP) found in our previous work, were synthesized. Their half maximal inhibition concentrations (IC(50)) for ACE inhibition have been determined. The effect of amino acid sequence on ACE inhibition was discussed on the basis of IC(50) of the synthetic peptides, and the following characteristics of the ACE inhibitory peptide have been clarified. First, the active portion of this peptide for ACE inhibition is KW. Second, the amino acid sequences near this dipeptide (KW) have a strong effect on the inhibitory activity. Especially, the proline residue in the C-terminal end strongly enhanced ACE inhibition. It should be noted that the IC(50) value of KWLP (5.5 μM) is the same as the ACE inhibitory peptide (VTVNPYKWLP) and that the IC(50) value of KW is 7.8 μM. The stability and absorption efficiency in vivo would be significantly improved by shortening the peptide length from 10 amino acids to four amino acids or two amino acids.     

NMR, HPLC-ESI-MS, and MALDI-TOF MS analysis of condensed tannins from Delonix regia (Bojer ex Hook.) Raf. and their bioactivities

The structures of the condensed tannins isolated from leaf, fruit, and stem bark of Delonix regia (Bojer ex Hook.) Raf. have been investigated with (13)C nuclear magnetic resonance ((13)C NMR) and high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS) coupled with thiolysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses. The results showed that these condensed tannins from D. regia possessed structural heterogeneity in monomer units and degree of polymerization. Propelargonidin (PP) and procyanidin (PC) were found in the leaf, fruit, and stem bark of D. regia, while prodelphinidin (PD) was found only in the leaves. The polymer chain lengths of condensed tannins from leaf and fruit organs were detected to be trimers to hexadecamers but from trimers to tridecamers for stem bark. B-type linkages were present in all these compounds. Condensed tannins from different parts of D. regia can be explored as tyrosinase inhibitors and food antioxidants because of their potent antityrosinase and antioxidant activities. The inhibitor concentration leading to 50% enzyme activity (IC(50)) was estimated to be 38 ± 1, 73 ± 2, and 54 ± 1.5 μg/mL for the condensed tannins of leaf, fruit, and stem bark. Condensed tannins extracted from stem bark exhibited the highest antioxidant activity; the DPPH scavenging activity (IC(50)) and the FRAP values were 90 ± 2 μg/mL and 5.42 ± 0.09 mmol AAE/g, respectively.     

Synthesis and antifungal activities of carabrol ester derivatives

Thirty-eight new ester derivatives of carabrol were designed, synthesized, and characterized by (1)H and (13)C NMR and HR-ESI-MS. Their antifungal activities against the fungal pathogen Colletotrichum lagenarium were evaluated using a spore germination assay. Of these 38 ester derivatives, 16 showed higher antifungal activity than that of carabrol and 7 showed higher antifungal activity than that of carabrone. It was found that the C-4 position of carabrol was a key position involving its antifungal activity, which showed the variation of 50% inhibition concentration (IC(50)) from 2.70 to 52.33 μg/mL. When substituted by the phenyl ring, the ester derivatives with electron-attracting groups showed higher activity than those with electron-donating ones. Two ester derivatives, carabryl 4-cynaobenzoate (II-17, IC(50) 2.70 μg/mL) and carabryl 4-isopropylbenzoate (II-27, IC(50) 2.82 μg/mL), showed only slightly lower antifungal activity than that of the positive control chlorothalonil (IC(50) 0.87 μg/mL) and have been identified as promising leads for development of new environmentally friendly fungicides.     

α-Glucosidase and aldose reductase inhibitory activities from the fruiting body of Phellinus merrillii

The inhibitory activity from the isolated component of the fruiting body Phellinus merrillii (PM) was evaluated against α-glucosidase and lens aldose reductase from Sprague-Dawley male rats and compared to the quercetin as an aldose reductase inhibitor and acarbose as an α-glucosidase inhibitor. The ethanol extracts of PM (EPM) showed the strong α-glucosidase and aldose reductase activities. α-Glucosidase and aldose reductase inhibitors were identified as hispidin (A), hispolon (B), and inotilone (C), which were isolated from EtOAc-soluble fractions of EPM. The above structures were elucidated by their spectra and comparison with the literatures. Among them, hispidin, hispolon, and inotilone exhibited potent against α-glucosidase inhibitor activity with IC(50) values of 297.06 ± 2.06, 12.38 ± 0.13, and 18.62 ± 0.23 μg/mL, respectively, and aldose reductase inhibitor activity with IC(50) values of 48.26 ± 2.48, 9.47 ± 0.52, and 15.37 ± 0.32 μg/mL, respectively. These findings demonstrated that PM may be a good source for lead compounds as alternatives for antidiabetic agents currently used. The importance of finding effective antidiabetic therapeutics led us to further investigate natural compounds.     

Improved synthesis of lysine- and arginine-derived Amadori and Heyns products and in vitro measurement of their angiotensin I-converting enzyme inhibitory activity

The L-lysine- and L-arginine-derived Amadori and Heyns products consisting of N-(1-deoxy-d-fructos-1-yl)amino acid and N-(2-deoxy-d-glucos-2-yl)amino acid were prepared by reaction of d-fructose and d-glucose with l-lysine hydrochloride and l-arginine hydrochloride using commercial zinc powder as deprotonating reagent and also as catalyst precursor in a simple synthetic route in high yield. These compounds were screened for angiotensin I-converting enzyme (ACE) inhibitory activity using a high-throughput colorimetric assay (utilizing porcine kidney ACE). The IC(50) values fall in the range of 1030-1175 μM, with N(α)-(1-deoxy-d-fructos-1-yl)arginine showing the best IC(50) value (1030 ± 38 μM). This study demonstrates an improved synthetic method for simple Amadori and Heyns products and their moderate ACE inhibitor activity.     

Inhibitory effects of (S)- and (R)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acids on tyrosinase activity

The inhibition of (R)-, (S)-, and (+/-)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acids (HTCCA) on mushroom tyrosinase was evaluated. All HTCCAs inhibited the tyrosinase activity. The ID(50) values were 1.88, 1.84, and 1.88 for the (R)-, (S)-, and (+/-)-HTCCAs, respectively. The inhibition kinetics analyzed by Hanes-Woolf plots indicated that both (R)- and (S)-HTCCAs are competitive inhibitors of the tyrosinase, with K(i) values of 0.83 and 0.61 mM, respectively. Dimethyl sulfoxide (DMSO) was also tested for its direct inhibitory activity against the tyrosinase and its potential influence on the tyrosinase inhibitory effects of (R)- and (S)-HTCCAs. DMSO, a widely used solvent for tyrosinase inhibitors, was found to dose-dependently inhibit the tyrosinase activity. Addition of DMSO in a tyrosinase digest containing either (R)- or (S)-HTCCA further dose-dependently reduced the tyrosinase activity. These data indicated a potential to use a HTCCA as a tyrosinase inhibitor in food, cosmetic, and medicinal products and a need to improve the solvent system for the studies of tyrosinase inhibitions.     

Inhibition of gastrointestinal lipolysis by green tea, coffee, and gomchui (Ligularia fischeri) tea polyphenols during simulated digestion

Green tea, coffee, and gomchui (Ligularia fischeri) tea, which are rich in polyphenols, may exhibit antiobesity effects by inhibiting pancreatic lipase. However, the bioavailability of some polyphenols is poor due to either degradation or absorption difficulties in the gastrointestinal tract, thus making their beneficial effects doubtful. This study was conducted to evaluate the inhibitory effect of three beverages on lipolysis and the contribution of their major polyphenols during simulated digestion. During simulated digestion, gomchui tea was the most potent at inhibiting gastrointestinal lipolysis, whereas green tea was the least potent. The strongest lipase inhibitor among purified major polyphenols was a green tea polyphenol, (-)-epigallocatechin gallate (EGCG, IC(50) = 1.8 ± 0.57 μM), followed by di-O-caffeoylquinic acid isomers (DCQA, IC(50) from 12.7 ± 4.5 to 40.4 ± 2.3 μM), which are gomchui tea polyphenols. However, the stability of DCQA was greater than that of EGCG when subjected to simulated digestion. Taken together, gomchui tea, which has DCQA as the major polyphenol, showed stronger lipolysis inhibitory activity during simulated digestion compared to both green tea and coffee.     

Fungistatic activity of bicyclo[4.3.0]-γ-lactones

Five optically active and sixteen racemic lactones (nine of them new) of bicyclo[4.3.0]nonane structure were synthesized. IC(50) values for the following phytopathogens were determined: Aspergillus ochraceus AM 456, Fusarium culmorum AM 282, Fusarium oxysporum AM 13, Fusarium tricinctum AM 16. Effect of compound structures, especially stereogenic centers, on fungistatic activity has been discussed. The highest fungistatic activity was observed for trans-7,8-dibromo-cis-3-oxabicyclo[4.3.0]nonan-2-one (3c), IC(50) = 30.1 μg/mL (0.10 μM/mL), and cis-7,8-epoxy-cis-3-oxabicyclo[4.3.0]nonan-2-one (3b), IC(50) = 72.2 μg/mL (0.47 μM/mL), toward F. oxysporum AM 13.     

Profiles and α-amylase inhibition activity of proanthocyanidins in unripe Manilkara zapota (chiku)

Proanthocyanidins in unripe Manilkara zapota (chiku) were isolated using solvent extraction followed by Sephadex LH-20 fractionation with a yield of 0.9%. HPLC analysis using a diol column revealed well-resolved oligomers ranging from dimer to hexamer. The majority of the proanthocyanidins are composed of higher-degree oligomers appearing as one large peak in the chromatogram. Analysis of the proanthocyanidins using LC/MS showed that (epi)gallocatechins were the dominant extension unit in the proanthocyanidins. In agreement with this result, thiolysis treatment of the proanthocyanidins using mercaptoacetic acid produced thioether derivatives of (epi)gallocatechins as the major product and (epi)gallocatechin gallate derivatives as the minor product. The mean of the degree of polymerization was estimated to be 9.0. From MALDI-TOF MS, B-type gallocatechin oligomers up to decamer could be detected. The unripe chiku proanthocyanidins are thus good starting material for preparation of (epi)gallocatechin derivatives. The proanthocyanidins was shown to inhibit α-amylase with an IC(50) value of 4.2 ± 0.2 μg/mL and inhibit α-glucosidase with an IC(50) of 16.6 ± 0.3 μg/mL.     

Isolation and identification of flavonoids in licorice and a study of their inhibitory effects on tyrosinase

Five different flavonoids were isolated from licorice after multistep chromatographic fractionation. The aim was to identify and characterize active components in licorice responsible for antibrowning activities and to seek new tyrosinase inhibitors for applications as antibrowning and depigmenting agents in the food and cosmetic industries. The isolated flavonoids were identified as liquiritin, licuraside, isoliquiritin, liquiritigenin (from Glycyrrhiza uralensis Fisch.), and licochalcone A (from Glycyrrhiza inflate Bat.) by UV, MS, (1)H NMR, and (13)C NMR analyses. The inhibitory potencies and capacities of these flavonoids toward monophenolase activity of mushroom tyrosinase were investigated. The IC(50) values of licuraside, isoliquiritin, and licochalcone A for monophenolase activity were 0.072, 0.038, and 0.0258 mM, respectively. A study of the mechanisms of monophenolase inhibition by these flavonoids indicated that they are all competitive inhibitors. Different from the above flavonoids, no inhibitory activity was observed for liquiritin, whereas liquiritigenin activated the monophenolase activity as a cofactor. The inhibitory effect of licuraside, isoliquiritin, and licochalcone A on diphenolase activity with l-DOPA as the substrate was much lower than those with l-tyrosine. Results suggest that licuraside, isoliquiritin, and licochalcone A have the high potential to be further developed into effective antibrowning and depigmenting agents.     

Protein from red cabbage (Brassica oleracea) seeds with antifungal, antibacterial, and anticancer activities

A 30 kDa antifungal protein was purified from red cabbage ( Brassica oleracea ) seeds. It exhibited a molecular mass and N-terminal amino acid sequence disinct from those of previously isolated Brassica antifungal proteins. The protocol used entailed ion exchange chromatography on Q-Sepharose and SP-Sepharose followed by fast protein liquid chromatography on Mono S. The protein hindered mycelial growth in Mycosphaerella arachidicola (with an IC50=5 μM), Setospaeria turcica, and Bipolaris maydis. It also inhibited the yeast Candida albicans with an IC50=96 μM. It exerted its antifungal action by permeabilizing the fungal membrane as evidenced by staining with Sytox green. The antifungal activity was stable from pH 3 to 11 and from 0 to 65 °C. It manifested antibacterial activity against Pseudomonas aeruginosa (IC50=53 μM). Furthermore, after 48 h of culture, it suppressed proliferation of nasopharyngeal cancer and hepatoma cells with IC50=50 and 90 μM, respectively.     

Development of a sensitive Indirect Competitive Enzyme-Linked Immunosorbent Assay (ic-ELISA) based on the monoclonal antibody for the detection of the imidaclothiz residue

An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on monoclonal antibodies (MoAbs) for imidaclothiz was developed. The hapten of imidaclothiz was synthesized and conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) to form the artificial antigens. MoAbs were obtained by immunizing BALB/c mice. Under the optimized conditions (10% methanol, 0.14 M Na(+), and pH 7.4), the half-maximal inhibition concentration (IC(50)) was 0.0875 ± 0.0034 mg/L and the limit of detection (IC(20)) was 0.0178 ± 0.0018 mg/L for imidaclothiz. There were no obvious cross-reactivities with most of the structural analogues of neonicotinoid insecticides, except imidacloprid. The recoveries of imidaclothiz in environmental and agricultural samples, including tap water, paddy water, soil, and cabbage, ranged from 80.43 to 113.83%, well within the requirements of residue detection. These results showed that this immunoassay could be used for the determination of imidaclothiz in environmental and agricultural samples.     

Theaflavins depolymerize microtubule network through tubulin binding and cause apoptosis of cervical carcinoma HeLa cells

Here we studied the antiproliferative activity of theaflavins in cervical carcinoma HeLa cells by investigating their effects on cellular microtubules and purified goat brain tubulin. Theaflavins inhibited proliferation of HeLa cells with IC(50) value of 110 ± 2.1 μg/mL (p = < 0.01), caused cell cycle arrest at G(2)/M phase and induced apoptosis with alteration of expression of pro- and antiapoptotic proteins. Along with these antiproliferative activities, theaflavins act as microtubule depolymerizers. Theaflavins disrupted the microtubule network accompanied by alteration of cellular morphology and also decreased the polymeric tubulin mass of the cells. The polymerization of cold treated depolymerized microtubules in HeLa cells was prevented in the presence of theaflavins. In vitro polymerization of purified tubulin into microtubules was also inhibited by theaflavins with an IC(50) value of 78 ± 2.43 μg/mL (P < 0.01). Thus, disruption of cellular microtubule network of HeLa cells through microtubule depolymerization may be one of the possible mechanisms of antiproliferative activity of theaflavins.     

Isolation of a new phlorotannin, a potent inhibitor of carbohydrate-hydrolyzing enzymes, from the brown alga Sargassum patens

Ethanol extracts from 15 kinds of marine algae collected from the coast of the Noto Peninsula in Japan were examined for their inhibitory effects on human salivary α-amylase. Four extracts significantly suppressed the enzyme activity. An inhibitor was purified from the extract of Sargassum patens . The compound was a new phloroglucinol derivative, 2-(4-(3,5-dihydroxyphenoxy)-3,5-dihydroxyphenoxy) benzene-1,3,5-triol (DDBT), which strongly suppressed the hydrolysis of amylopectin by human salivary and pancreatic α-amylases. The 50% inhibitory activity (IC(50)) for α-amylase inhibition of DDBT (3.2 μg/mL) was much lower than that of commercially available α-amylase inhibitors, acarbose (26.3 μg/mL), quercetagetin (764 μg/mL), and α-amylase inhibitor from Triticum aestivum (88.3 μg/mL). A kinetic study indicated that DDBT was a competitive α-amylase inhibitor with a K(i) of 1.8 μg/mL. DDBT also inhibited rat intestinal α-glucosidase with an IC(50) value of 25.4 μg/mL for sucrase activity and 114 μg/mL for maltase activity. These results suggest that DDBT, a potent inhibitor of carbohydrate-hydrolyzing enzymes, may be useful as a natural nutraceutical to prevent diabetes.     

Identification and kinetic study of tyrosinase inhibitors found in sake lees

The present study found that the n-hexane extract of freeze-dried sake lees inhibits tyrosinase activity and showed that the constituents isolated from the n-hexane extract are the mixture of triacylglycerols. The inhibitory effects of triolein and trilinolein found as the triacylglycerols were examined using tyrosinases from mushroom and Streptomyces castaneoglobisporus. The IC50 values of the triacylglycerol mixture for the oxidase activity on mushroom and Streptomyces tyrosinases were 20 and 0.14 microg/mL, respectively. The IC50 values of trilinolein for the oxidase activity on mushroom and Streptomyces tyrosinases were 8.4 and 0.1 microM, respectively. However, the inhibitory effect of triolein (IC50=30 microM) was lower than that of trilinolein, even when the Streptomyces tyrosinase was used for the assay. Kinetic analyses indicate that both trilinolein and triolein inhibit the tyrosinase activity noncompetitively. When transformed with a plasmid carrying the Streptomyces tyrosinase gene, the melanin-synthesizing ability of the transformed Escherichia coli host was dose-dependently interfered with by trilinolein.