Quince (Cydonia oblonga Miller) fruit (pulp, peel, and seed) and Jam: antioxidant activity

To study the antioxidant activity of quince fruit (pulp, peel, and seed) and jam, methanolic extracts were prepared. Each extract was fractionated into a phenolic fraction and an organic acid fraction and was analyzed by high-performance liquid chromatography (HPLC)/diode array detection and HPLC/UV, respectively. Antiradical activities of the extracts and fractions were evaluated by a microassay using 1,1'-diphenyl-2-picrylhydrazyl. The phenolic fraction always exhibited a stronger antioxidant activity than the whole methanolic extract. Organic acid extracts were always the weakest in terms of antiradical activity, which seems to indicate that the phenolic fraction gives a higher contribution for the antioxidant potential of quince fruit and jam. The evaluation of the antioxidant activity of methanolic extracts showed that peel extract was the one presenting the highest antioxidant capacity. The IC50 values of quince pulp, peel, and jam extracts were correlated with the caffeoylquinic acids total content. Among the phenolic fractions, the seed extract was the one that exhibited the strongest antioxidant activity. The IC50 values of quince pulp, peel, and jam phenolic extracts were strongly correlated with caffeoylquinic acids and phenolics total contents. For organic acid fractions, the peel extract was the one that had the strongest antiradical activity. The IC50 values of quince pulp, peel, and jam organic acid fractions were correlated with the ascorbic acid and citric acid contents.     

Catalytic inhibition of human DNA topoisomerase II by interactions of grape cell culture polyphenols

Previously, we isolated mixed polyphenolic fractions on a toyopearl matrix (TP-2 to TP-6) from grape cell cultures that were highly potent catalytic inhibitors in a human DNA topoisomerase II assay for cancer chemoprevention. The objectives of this study were to evaluate the potency of, and potential interactions between, individual fractions and some of the purified bioactive polyphenols that comprise these fractions on human DNA topoisomerase II catalytic activity. Treatments that combined anthocyanin-rich fractions (TP-2; 0.5 or 2.0 microg of dried material/mL), fractions containing catechins, procyanidin dimers, and flavanones (TP-4; 0.25 microg of dried material/mL), and/or fractions enriched with procyanidin oligomers and polymers (TP-6; 0.15 or 0.5 microg of dried material/mL) showed additive effects toward catalytic inhibition of the enzyme. Epicatechin gallate (IC50 = 0.029 microM), myricetin (0.39 microM), procyanidin B2 (PB2, 4.5 microM), and resveratrol (65.7 microM), constituents of the most bioactive mixed fraction from grape cell culture (TP-4), each individually provided potent catalytic inhibition of topoisomerase II. In addition, potentiating interactions between the PB2 and the other polyphenolic constituents mentioned above and between myricetin and resveratrol were clearly demonstrated. A synergistic interaction between myricetin and resveratrol was also confirmed with isobolographic analysis at a molar ratio of 1:70.     

Grape-seed procyanidins act as antiinflammatory agents in endotoxin-stimulated RAW 264.7 macrophages by inhibiting NFkB signaling pathway

Procyanindin extract (PE) is a mixture of polyphenols, mainly procyanidins, obtained from grape seed with putative antiinflammatory activity. We evaluated the PE effect on RAW 264.7 macrophages stimulated with lipopolysaccharide plus interferon-gamma that show a rapid enhanced production of prostaglandin E2 (PGE2) and nitric oxide (NO). Our results demonstrated that PE significantly inhibited the overproduction of NO, dose and time dependently. PE caused a marked inhibition of PGE2 synthesis when administered during activation. Moreover, PE pretreatment diminished iNOS mRNA and protein amount dose dependently (10-65 microg/mL). PE (65 microg/mL) pretreatment inhibited NFkappaB (p65) translocation to nucleus by nearly 40%. Trimeric and longer oligomeric-rich procyanidin fractions from PE (5-30 microg/mL) inhibited iNOS expression but not the monomeric forms catechin and epicatechin. Thus, we show that the degree of polymerization is important in determining procyanidin effects. PE was considerably a more effective inhibitor of NO biosynthesis (IC50 = 50 microg/mL) in comparison to other antiinflammatories, such as aspirin (3 mM), indomethacin (20 microM), and dexamethasone (9 nM). In conclusion, PE modulates inflammatory response in activated macrophages by the inhibition of NO and PGE2 production, suppression of iNOS expression, and NFkB translocation. These results demonstrate an immunomodulatory role of grape seed procyanidins and thus a potential health-benefit in inflammatory conditions that exert an overproduction of NO and PGE2.     

Effects of grape cell culture extracts on human topoisomerase II catalytic activity and characterization of active fractions

Grape and its cell culture extracts are rich in flavonoids and stilbenes that are biologically active. The objective of this study was to evaluate possible inhibitory effects of grape (a Vitis hybrid Bailey Alicant A) cell culture extract and subfractions on human DNA topoisomerase II catalytic activity and to characterize constituents in the most potent fractions. At 5 microg/mL, grape cell crude extract and Toyopearl (TP) fractions 2-6 provided significantly greater inhibition of topoisomerase II catalytic activity than quercetin, a chemopreventive agent previously known as a topoisomerase catalytic inhibitor. The most potent topoisomerase II catalytic inhibitors from grape cell culture extracts in descending order of potency were TP fractions 4 and 6 (IC(50) = 0.28-0.29 microg/mL), TP-3 (IC(50) = 0.74 microg/mL), and crude extract (IC(50) = 1.02 microg/mL); each was significantly more potent than resveratrol (IC(50) = 18.0 microg/mL), another well-known chemopreventive topoisomerase II catalytic inhibitor. Using both high-performance liquid chromatography and liquid chromatography electrospray ionization mass spectrometry, constituents in TP-4 and TP-6 were characterized. These constituents included cyanidin-3,5-diglucoside, malvidin-3-acetylglucoside, peonidin-3-coumaryl-5-diglucoside, procyanidin B(1), procyanidin B(2), procyanidin B(5), procyanidin dimer digallate, procyanidin C(1), myricetin, and rutin, none of which have been previously characterized from grape cell cultures. The significant potency especially of TP-4 and TP-6 from grape cell cultures suggests that these fractions may have potential as chemopreventive agents.     

Inhibitory effects of oolong tea polyphenols on pancreatic lipase in vitro

Fifty-four polyphenols isolated from tea leaves were evaluated for their inhibitory activities against pancreatic lipase, the key enzyme of lipid absorption in the gut. (-)-Epigallocatechin 3-O-gallate (EGCG), which is one of major polyphenols in green tea, showed lipase inhibition with an IC50 of 0.349 microM. Moreover, flavan-3-ol digallate esters, such as (-)-epigallocatechin-3,5-digallate, showed higher activities of inhibition on lipase with an IC50 of 0.098 microM. On the other hand, nonesterified flavan-3-ols, such as (+)-catechin, (-)-epicatechin, (+)-gallocatechin, and (-)-epigallocatechin, showed zero and/or the lowest activities against pancreatic lipase (IC50 > 20 microM). These data suggested that the presence of galloyl moieties within the structure was required for enhancement of pancreatic lipase inhibition. It is well-known that flavan-3-ols are polymerized by polyphenol oxidase and/or heating in a manufacturing process of oolong tea. Oolonghomobisflavans A and B and oolongtheanin 3'-O-gallate, which are typical in oolong tea leaves, showed strong inhibitory activities with IC50 values of 0.048, 0.108, and 0.068 microM, respectively, even higher than that of EGCG. The oolong tea polymerized polyphenols (OTPP) were prepared for the assay from oolong tea extract, from which the preparation effectively subtracted the zero and/or less-active monomeric flavan-3-ols by preparative high-performance liquid chromatography. The weight-average molecular weight (Mw) and number-average molecular-weight (Mn) values of OTPP were 2017 and 903, respectively, by using gel permeation choromatography. OTPP showed a 5-fold stronger inhibition against pancreatic lipase (IC50 = 0.28 microg/mL) by comparison with that of the tannase-treated OTPP (IC50 = 1.38 microg/mL). These data suggested that the presence of galloyl moieties within their chemical structures and/or the polymerization of flavan-3-ols were required for enhancement of pancreatic lipase inhibition.     

Radical scavenging and anti-lipoperoxidative activities of Smallanthus sonchifolius leaf extracts

Radical scavenging and anti-lipoperoxidative effects of two organic fractions and two aqueous extracts from the leaves of a neglected Andean crop-yacon (Smallanthus sonchifolius Poepp. & Endl., Asteraceae) were determined using various in vitro models. The extracts' total phenolic content was 10.7-24.6%. They exhibited DPPH (IC50 16.14-33.39 microg/mL) and HO* scavenging activities (4.49-6.51 mg/mL). The extracts did not scavenge phenylglyoxylic ketyl radicals, but they retarded their formation. In the xanthine/xanthine oxidase superoxide radical generating system, the extracts' activities were 26.10-37.67 superoxide dismutase equivalents/mg. As one of the extracts displayed xanthine oxidase inhibitory activity, the effect of the extracts on a nonenzymatically generated superoxide was determined (IC50 7.36-21.01 microg/mL). The extracts inhibited t-butyl hydroperoxide-induced lipoperoxidation of microsomal and mitochondrial membranes (IC50 22.15-465.3 microg/mL). These results make yacon leaves a good candidate for use as a food supplement in the prevention of chronic diseases involving oxidative stress.     

Assay-guided fractionation study of alpha-amylase inhibitors from Garcinia mangostana pericarp

Alpha-amylase inhibitor (alpha-AI) activity of Garcinia mangostana, commonly known as mangosteen, pericarp extracts was studied by assay guided fractionations from lipophilic to hydrophilic using combined solvent extraction and Amberlite XAD2 adsorption chromatography. Neither the lipophilic, xanthone containing fraction, nor the highly polar fraction, which has no affinity on Amberlite XAD2, showed any alpha-AI. The fraction that shows very high inhibitory activity contains primarily polyphenols and can be adsorbed on Amberlite XAD2. The IC50 of 5.4 microg/mL of this fraction is comparable to that of acarbose, a prescribed alpha-AI used in the control of type II diabetes, at 5.2 microg/mL. Total phenolic content (TPC) of each fraction was measured and the TPC has no correlation with the alpha-AI activity. The lipophilic fraction contains mainly xanthones as revealed by HPLC-MS analysis. Colorimetric analysis coupled with UV-vis and IR spectroscopic analysis demonstrated that the fractions with high alpha-AI activity are primarily oligomeric proanthocyanidins (OPCs) with little gallate moiety. There is also evidence to show that the alpha-AI by these OPCs is not purely by nonspecific protein complexation. Both tannic acid and G. mangostana OPCs precipitate BSA equally well but G. mangostana OPCs are 56 times more effective in inhibiting alpha-amylase.     

大孔树脂纯化黄精多酚及其抗氧化性与组成分析

考察4种树脂对黄精多酚粗提物的吸附-解吸特性,筛选出最佳的纯化树脂,并研究其纯化工艺,分析黄精多酚的体外抗氧化活性、红外光谱特性和单酚组成。结果表明,AB-8型大孔树脂最适合于对黄精多酚粗提液的纯化,静态吸附5 h达到饱和,静态解吸3 h达到平衡;在室温下,用质量浓度为0.80 mg/mL的黄精多酚粗提液以0.8 mL/min的流速上样;吸附平衡后以70%的乙醇为洗脱剂,在洗脱流速为1 mL/min时,大孔树脂纯化效果最好,经纯化后黄精多酚的纯度提高了3.37倍。黄精多酚具有较好的抗氧化能力,且具有良好的量效关系,纯化前后黄精多酚总还原能力的IC50值分别为(27.48±1.93)、(19.01±1.48)μg/mL,清除DPPH·的IC50值依次为(5.21±0.48)、(4.00±0.26)μg/mL,清除ABTS+·的IC50值依次为(4.89±0.82)、(4.21±0.53)μg/mL,经纯化后黄精多酚的抗氧化活性明显增强。红外光谱分析表明其具有多酚和黄酮的特征峰;HPLC-DAD分析表明,黄精多酚主要含绿源酸,阿魏酸,芦丁和熊果酸。     

Chemical characterization of red wine grape (Vitis vinifera and Vitis interspecific hybrids) and pomace phenolic extracts and their biological activity against Streptococcus mutans

Grapes are rich sources of potentially bioactive polyphenols. However, the phenolic content is variable depending on grape variety, and may be modified during vinification. In this study, we examined the chemical composition and biological activity of phenolic extracts prepared from several red wine grape varieties and their fermented byproduct of winemaking (pomace) on some of the virulence properties of Streptococcus mutans a well-known dental pathogen. Grape phenolic extracts were obtained from Vitis vinifera varieties Cabernet Franc and Pinot Noir and Vitis interspecific hybrid varieties Baco Noir and Noiret. The anthocyanins and flavan-3-ols content were highly variable depending on grape variety and type of extract (whole fruit vs fermented pomace). Nevertheless, all grape phenolic extracts remarkably inhibited glucosyltransferases B and C (70-85% inhibition) at concentrations as low as 62.5 microg/mL (P < 0.01). Furthermore, the glycolytic pH-drop by S. mutans cells was inhibited by the grape extracts without affecting the bacterial viability; an effect that can be attributed to partial inhibition of F-ATPase activity (30-65% inhibition at 125 microg/mL; P < 0.01). The biological activity of fermented pomace was either as effective as or significantly better than whole fruit grape extracts. The results showed that grape phenolic extracts, especially from pomace, are highly effective against specific virulence traits of S. mutans despite major differences in their phenolic content.     

Chemical composition of volatile extract and biological activities of volatile and less-volatile extracts of juniper berry (Juniperus drupacea L.) fruit

Volatile chemicals in a dichloromethane extract from a steam distillate of juniper berry fruit (Juniperus drupacea L.) and its two column chromatographic fractions (eluted with hexane and ethyl ether) were analyzed by gas chromatography/mass spectrometry. The major compounds in the dichloromethane extract were alpha-pinene (23.73%), thymol methyl ether (17.32%), and camphor (10.12%). A fraction eluted with hexane contained alpha-pinene (44.24%) as the major constituent. A fraction eluted with ethyl ether had thymol methyl ether (22.27%) and camphor (19.65%) as the main components. Three samples prepared from the distillate and two additional samples prepared by petroleum ether and ethanol extraction directly from juniper berry fruits exhibited clear antioxidant activities with dose response in both 1,2-diphenyl picrylhydrazyl and beta-carotene assays. All samples except the hexane fraction showed comparable activities to that of the synthetic antioxidant t-butyl hydroquinone at a level of 200 microg/mL in the two testing systems. The extracts of dichloromethane, petroleum ether, and ethanol exhibited appreciable antimicrobial activities against six microorganisms with minimum inhibitory concentrations ranging from 0.5 mg/mL (volatile extract against Candida albicans ) to 1.2 mg/mL (ethanol extract against Aspergillus niger ). The results of the present study suggest that this fruit could be a natural antioxidant supplement for foods and beverages.     

Biologically active carbazole alkaloids from Murraya koenigii

The bioassay guided fractionation of the acetone extract of the fresh leaves of Murraya koenigii resulted in the isolation of three bioactive carbazole alkaloids, mahanimbine (1), murrayanol (2), and mahanine (3), as confirmed from their (1)H and (13)C NMR spectral data. Compound 2 showed an IC(50) of 109 microg/mL against hPGHS-1 and an IC(50) of 218 microg/mL against hPGHS-2 in antiinflammatory assays, while compound 1 displayed antioxidant activity at 33.1 microg/mL. All three compounds were mosquitocidal and antimicrobial and exhibited topoisomerase I and II inhibition activities.     

Chemical composition and antifungal activity of essential oils from different tissues of Japanese Cedar (Cryptomeria japonica)

In this study antifungal activities of essential oils from different tissues of Japanese cedar (Cryptomeria japonica D. Don) against four wood decay fungi and six tree pathogenic fungi were investigated. In addition, the yields of essential oils obtained by water distillation were compared and their constituents determined by GC-MS analyses. The yield of essential oils from four tissues of Japanese cedar is in the decreasing order of leaf (27.38 mL/kg) > bark (6.31 mL/kg) > heartwood (3.80 mL/kg) > sapwood (1.27 mL/kg). Results obtained from the antifungal tests demonstrate that the essential oil of Japanese cedar heartwood used against Laetiporus sulphureus and Trametes versicolor and sapwood essential oil used against L. sulphureus had strong antifungal activities at 500 mug/mL, with IC(50) values of 39, 91, and 94 microg/mL, respectively. Besides, the essential oils of Japanese cedar heartwood used against Rhizoctonia solani, Collectotrichum gloeosporioides, Fusarium solani, and Ganoderma australe had strong antifungal activities at 500 microg/mL, with IC(50) values of 65, 80, 80, and 110 microg/mL, respectively. GC-MS analyses showed that the sesquiterpene hydrocarbon compounds dominate in the essential oil from Japanese cedar heartwood, amounting to a total percentage of 82.56%, with the major compounds of delta-cadinene (18.60%), isoledene (12.41%), and gamma-muurolene (11.82%). It is proposed that the excellent antifungal activities of Japanese cedar heartwood essential oils might correlate with the presence of these compounds.     

Inhibitory effects of muscadine anthocyanins on α-glucosidase and pancreatic lipase activities

Inhibitory effects of the Noble muscadine grape extracts and the representative phytochemicals for anthocyanins (i.e., cyanidin and cyanidin-3,5-diglucoside) on two enzymes, that is, α-glucosidase and pancreatic lipase, were investigated regarding their antidiabetic activities. The study demonstrated that the anthocyanin extracts and the selected chemicals obeyed the competitive mode against the enzymes. The methanolic extracts of whole fruit and skin of the muscadine showed inhibitory activities against the α-glucosidase with their IC(50) values at 1.50 and 2.73 mg/mL, and those against the lipase at 16.90 and 11.15 mg/mL, respectively, which indicated that the muscadine extracts possessed strong antidiabetic activities. Particularly, the ethyl acetate (EtoAc) extract and the butanol (BuOH) extract exhibited much higher inhibitory activities against both enzymes than the CHCl(3) and water extracts, while the majority of anthocyanins existed in the BuOH fractions. Moreover, cyanidin exhibited a much stronger antidiabetic activity than cyanidin-3,5-diglucoside, suggesting that anthocyanins may have higher inhibitory activities after being digested. Further chromatographic analysis by high-performance liquid chromatography-mass spectrometry identified five individual anthocyanins, including cyanidin, delphinidin, petunidin, peonidin, and malvidin glycosides.     

Potential food additives from Carex distachya roots: identification and in vitro antioxidant properties

In the present study, the chemical composition and antioxidant properties of root methanol extract of Carex distachya Desf. (Cyperaceae) were assessed to use this plant as sources of food additives and nutraceuticals. The IC50 of the extract (4.2 microg/mL), derived from the DPPH radical scavenging capacity assay, was similar to those of ascorbic acid, alpha-tocopherol, and BHT. These results revealed a strong antioxidant activity because of the presence of an extraordinary quantity of bioactive phytochemicals. The phytochemical study of the root extract led to the isolation and identification of new and known polyphenols, most of them common constituents of plant foods. A total of 16 polyphenols, identified on the basis of spectroscopic data as 7 lignans, 4 phenylethanoids, 3 resveratrol derivatives, a monolignol, and a secoiridoid glucoside, were isolated. The tentative structural elucidation of the new metabolites 5'-O-beta-D-glucopyranosyloxy-3,3'-dimethoxy-7,9'-epoxylignan-4,8',9-triol and 3,5-bis-O-beta-D-glucopyranosyloxy-3'-methoxy-trans-stilben-4'-ol have been performed by a combined approach using ESI/TQ/MS techniques and 1D and 2D NMR experiments. All of the compounds have been tested for their antioxidant activity using six different antioxidant and radical scavenging tests. Interestingly, the extract contained high quantities of polyphenols, most of them reported as constituents of edible plants, such as grape and olive, suggesting that the methanol root extract of this plant could be used as a source of natural antioxidants useful as potential food additives.     

Total cranberry extract versus its phytochemical constituents: antiproliferative and synergistic effects against human tumor cell lines

Cranberries (Vaccinium macrocarpon Ait.) are an excellent dietary source of phytochemicals that include flavonol glycosides, anthocyanins, proanthocyanidins (condensed tannins), and organic and phenolic acids. Using C-18 and Sephadex Lipophilic LH-20 column chromatography, HPLC, and tandem LC-ES/MS, the total cranberry extract (TCE) has been analyzed, quantified, and separated into fractions enriched in sugars, organic acids, total polyphenols, proanthocyanidins, and anthocyanins (39.4, 30.0, 10.6, 5.5, and 1.2% composition, respectively). Using a luminescent ATP cell viability assay, the antiproliferative effects of TCE (200 microg/mL) versus all fractions were evaluated against human oral (KB, CAL27), colon (HT-29, HCT116, SW480, SW620), and prostate (RWPE-1, RWPE-2, 22Rv1) cancer cell lines. The total polyphenol fraction was the most active fraction against all cell lines with 96.1 and 95% inhibition of KB and CAL27 oral cancer cells, respectively. For the colon cancer cells, the antiproliferative activity of this fraction was greater against HCT116 (92.1%) than against HT-29 (61.1%), SW480 (60%), and SW620 (63%). TCE and all fractions showed >/=50% antiproliferative activity against prostate cancer cells with total polyphenols being the most active fraction (RWPE-1, 95%; RWPE-2, 95%; 22Rv1, 99.6%). Cranberry sugars (78.8 microg/mL) did not inhibit the proliferation of any cancer cell lines. The enhanced antiproliferative activity of total polyphenols compared to TCE and its individual phytochemicals suggests synergistic or additive antiproliferative interactions of the anthocyanins, proanthocyanidins, and flavonol glycosides within the cranberry extract.     

Evaluation of antioxidant activity and preventing DNA damage effect of pomegranate extracts by chemiluminescence method

The antioxidant activities of three parts (peel, juice, and seed) and extracts of three pomegranate varieties in China were investigated by using a chemiluminescence (CL) method in vitro. The scavenging ability of pomegranate extracts (PEs) on superoxide anion, hydroxide radical, and hydrogen peroxide was determined by the pyrogallol-luminol system, the CuSO4-Phen-Vc-H2O2 system, and the luminol-H2O2 system, respectively. DNA damage preventing the effect of PE was determined by the CuSO4-Phen-Vc-H2O2-DNA CL system. The results showed that the peel extract of red pomegranate had the best effect on the scavenging ability of superoxide anion because its IC50 value (4.01 +/- 0.09 microg/mL) was the lowest in all PEs. The seed extract of white pomegranate could scavenge hydroxide radical most effectively of the nine extracts (the IC50 value was 1.69 +/- 0.03 microg/mL). The peel extract of white pomegranate had the best scavenging ability on hydrogen peroxide, which had the lowest IC50 value (0.032 +/- 0.003 microg/mL) in the nine extracts. The seed extract of white pomegranate (the IC50 value was 3.67 +/- 0.03 microg/mL) was the most powerful on the DNA damage-preventing effect in all of the PEs. Also, the statistical analysis indicated that there were significant differences (at P < 0.05) among the extracts of the different varieties and parts in each system.     

Inhibition of gastric H(+),K(+)-ATPase and Helicobacter pylori growth by phenolic antioxidants of Curcuma amada

Gastric ulcer is the most prevalent gastrointestinal disorder, resulting from oxidative stress, Helicobacter pylori infection, up-regulation of proton potassium ATPase (PPA) activity, down-regulation of gastric mucosal defense, etc. In this paper it is reported that phenolic fractions of Curcuma amada, commonly known as mango ginger, acted as potent inhibitors of PPA and H. pylori growth. Mango ginger free phenolics (MGFP) and mango ginger bound phenolics (MGBP) inhibited PPA at IC50 values of 2.2 +/- 0.21 and 0.7 +/- 0.08 microg/mL, respectively, exhibiting 9-27-fold better potency over lansoprazole (IC(50) of 19.3 +/- 2.2 microg/mL). MGFP is constituted by caffeic (26%), gentisic (24%), ferulic (20%), gallic (10%), cinnamic (7%), and protocatechuic acids (7%) and MGBP by ferulic (47%), cinnamic (29%), p-coumaric acid (11%), and syringic (5%) acids as major phenolic acids. MGFP and MGBP further exhibited free radical scavenging (IC(50) of 2.2 +/- 0.17 and 4.2 +/- 0.36 microg/mL), reducing power abilities (193-104 units/g), inhibition of lipid peroxidation (IC(50) of 10.3 +/- 0.91 and 15.6 +/- 1.6 microg/mL), and DNA protection (80% at 4 microg), indicating strong antioxidative properties. MGFP and MGBP thus may be potential and inexpensive multistep blockers against ulcers.     

In vitro inhibition of the activation of Pro-matrix Metalloproteinase 1 (Pro-MMP-1) and Pro-matrix metalloproteinase 9 (Pro-MMP-9) by rice and soybean Bowman-Birk inhibitors

The in vitro inhibitory activity of the rice Bowman-Birk inhibitor (rBBI) or soybean Bowman-Birk inhibitor (sBBI) against trypsin-catalyzed activation of pro-matrix metalloproteinase 1 or 9 (pro-MMP-1 or pro-MMP-9), respectively, was investigated using electrophoresis with silver staining, heparin-enhanced zymography, biotinylated gelatin, Biotrak assay, and fluorescence quenched substrate hydrolysis. rBBI at concentrations of 0.08-0.352 mg/mL dose-dependently inhibited the in vitro activation of 45 microg/mL pro-MMP-1 by trypsin. Heparin-enhanced zymography analysis of pro-MMP-1, trypsin-activated MMP-1, and a mixture of pro-MMP-1-trypsin-rBBI showed clear zones associated with trypsin-activated MMP-1 and the absence of clear zones in lanes containing pro-MMP-1 or a mixture of pro-MMP-1, trypsin, and rBBI. The results of the Biotrak assay also indicated that rBBI dose-dependently suppressed the activation of pro-MMP-1 by trypsin. sBBI dose-dependently inhibited the activation of 100 microg/mL of pro-MMP-9 by trypsin. Biotinylated gelatin assays demonstrated that pro-MMP-9 or pro-MMP-9 in the presence of trypsin and BBI did not hydrolyze gelatin, whereas p-aminophenylmercury acetate (APMA)-activated MMP-9 and trypsin-activated MMP-9 caused significant hydrolysis of gelatin. Quenched fluorescence substrate hydrolysis for total MMP activity showed that pro-MMP-1 or pro-MMP-9 did not hydrolyze the substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2; active MMP-1 or MMP-9 hydrolyzed the substrate, but lower substrate hydrolysis was obtained when pro-MMP-1 or pro-MMP-9 was incubated with trypsin in the presence of increasing concentrations of rBBI. The results are discussed in light of the role of MMP-1 and MMP-9 in the process of angiogenesis and the potential of rBBI or sBBI as a functional food ingredient.     

Phenolic constituents and antioxidant properties of Xanthosoma violaceum leaves

An extract of Xanthosoma violaceum leaves was subjected to a polyphenol profile determination, including total polyphenols, and antioxidant activity evaluation. Analysis of the extract resulted in the isolation of a new flavone C-glycoside, apigenin 6-C-beta-D-glucopyranosyl-8-C-beta-D-apiofuranoside (1), as well as known flavone C-glycosides, including vitexin (2), isovitexin (3), isovitexin 4'-O-rhamnopyranoside (4), apigenin 6-C-[beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside] (5), and apigenin 6,8-diC-beta-D-glucopyranoside (6). The antioxidant activity of the extract was assessed by means of two different in vitro tests: bleaching of the stable 1,1-diphenyl-2-picrylhydrazyl radical (DPPH test) and peroxidation induced by the water-soluble radical initiator 2,2'-azobis(2-amidinopropane) hydrochloride, on mixed dipalmitoylphosphatidylcholine/linoleic acid unilamellar vesicles (LP-LUV test). In both tests used, the extract and a fraction II showed a significant antioxidant/free-radical scavenging effect (fraction II, EC(50) = 11.6 microg/mL) in comparison to alpha-tocopherol (EC(50) = 10.1 microg/mL).