Antioxidant and antimelanogenic activities of polyamine conjugates from corn bran and related hydroxycinnamic acids

The antioxidant activity of three major polyamine conjugates, N,N'-dicoumaroyl-putrescine (DCP), N-p-coumaroyl-N'-feruloylputrescine (CFP), and N,N'-diferuloyl-putrescine (DFP) isolated from corn bran, and their related hydroxycinnamic acids, p-coumaric acid and ferulic acid, were evaluated by three antioxidant in vitro assay systems, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and superoxide and hydroxyl radicals generated by enzymatic and nonenzymatic reactions. Additionally, five phenolic compounds were evaluated for melanogenesis inhibitory activity using mushroom tyrosinase and B16 melanoma cells. Most of the phenolic compounds significantly scavenged DPPH, superoxide, and hydroxyl radicals in a dose-dependent manner. Particularly, DFP showed potent DPPH (IC50 = 38.46 microM) and superoxide (IC50 = 291.62 microM) radical scavenging activities, while DCP exhibited the strongest hydroxyl radical scavenging activity (IC50 = 120.55 microM). CFP also exerted moderate DPPH, superoxide, and hydroxyl radical scavenging activities. Meanwhile, DCP (IC50 = 181.73 microM) showed potent tyrosinase inhibitory activity toward l-tyrosine as the substrate, whereas DFP (IC50 = 733.64 microM) significantly inhibited melanin synthesis in B16 melanoma cells. These current results indicate that these three polyamine conjugates from corn bran may be useful potential sources of natural antioxidants and skin-whitening agents.     

Purification and characterization of an N-acetylglucosamine-binding lectin from Koelreuteria paniculata seeds and its effect on the larval development of Callosobruchus maculatus (Coleoptera: Bruchidae) and Anagasta kuehniella (Lepidoptera: Pyralidae)

This study describes the purification of an N-acetylglucosamine-binding lectin from Koelreuteria paniculata seeds and its effects on the larval development of Callobruchus maculatus and Anagasta kuehniella. The lectin (KpLec) was characterized and isolated by gel filtration, affinity column, and reverse phase chromatography. SDS-PAGE indicated that this lectin is a dimer composed of subunits of 22 and 44 kDa. The N terminus exhibited 40% similarity with Urtiga dioica agglutinin. KpLec was tested for anti-insect activity against C. maculatus and A. kuehniella. With regard to C. maculatus, an artificial diet containing 0.7 and 1% KpLec produced LD(50) and ED(50) value, respectively. However, for A. kuenhiella, an artificial diet containing 0.65% KpLec produced an LD(50), whereas 0.2% KpLec produced an ED(50). The transformation of genes coding for this lectin could be useful in the development of insect resistance in important agricultural crops.     

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.     

Identification and antioxidant activity of novel chlorogenic acid derivatives from bamboo (Phyllostachys edulis)

One known and two novel antioxidant compounds have been isolated from bamboo (Phyllostachys edulis). The butanol-soluble extract of the bamboo leaves was found to have a significant antioxidant activity, as measured by scavenging the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical and the superoxide anion radical (O(2)(-)) in the xanthine/xanthine oxidase assay system. Antioxidant activity-directed fractionation of the extract led to the isolation and characterization of three structural isomeric chlorogenic acid derivatives: 3-O-(3'-methylcaffeoyl)quinic acid (1), 5-O-caffeoyl-4-methylquinic acid (2), and 3-O-caffeoyl-1-methylquinic acid (3). Compounds 2 and 3 were isolated and characterized for the first time from the natural products. In the DPPH scavenging assay as well as in the iron-induced rat microsomal lipid peroxidation system, compounds 2 (IC(50) = 8.8 and 19.2 microM) and 3 (IC(50) = 6.9 and 14.6 microM) showed approximately 2-4 times higher antioxidant activity than did chlorogenic acid (IC(50) = 12.3 and 28.3 microM) and other related hydroxycinnamates such as caffeic acid (IC(50) =13.7 and 25.5 microM) and ferulic acid (IC(50) = 36.5 and 56.9 microM). Among the three compounds, compound 1 yielded the weakest antioxidant activity, and the DPPH scavenging and lipid peroxidation inhibitory activity (IC(50) = 16.0 and 29.8 microM) was lower than those of chlorogenic and caffeic acids. All three compounds exhibited both superoxide scavenging activities and inhibitory effects on xanthine oxidase. Their superoxide anion (O(2)(-)) scavenging activities (IC(50) = 1, 4.3 microM; 2, 2.8 microM; and 3, 1.2 microM) were markedly stronger than those of ascorbic acid (IC(50) = 56.0 microM), alpha-tocopherol (IC(50) > 100 microM), and other test compounds, although their inhibition effects on xanthine oxidase may contribute to the potent scavenging activity. alpha-Tocopherol exerted a significant inhibitory effect (65.5% of the control) on superoxide generation in 12-O-tetradecanoylphorbol-13-acetate-induced human promyelocytic leukemia HL-60 cells, and compound 3 showed moderate activity (36.0%). On the other hand, other compounds including 1, 2, chlorogenic acid, and other antioxidants were weakly active (24.8-10.1%) in the suppression of superoxide generation.     

New mannose-binding lectin isolated from the rhizome of Sarsaparilla Smilax glabra Roxb. (Liliaceae)

A new mannose-binding lectin, designated SGM2, was isolated from the rhizome of a Chinese medicinal herb Smilax glabra (also known as sarsaparilla in general) by saline extraction, ammonium sulfate precipitation and fractionation, and affinity chromatography on fetuin- and mannose-agarose. SGM2 is shown to have a molecular mass of 37 kDa on gel filtration and 12.5 kDa on SDS-PAGE, indicating that it is a trimeric protein composed of three identical subunits. When the first 30 amino acid residues at the N-terminal were compared, SGM2 had approximately 40% homology with those of some other monocots. SGM2 had the property of hemagglutinating activity toward rabbit erythrocytes, which could be reversed by mannose and mannose polymers. SGM2 exhibited antiviral activities against both herpes simplex virus type 1 (HSV-1) and respiratory syncytial virus (RSV) with the same EC(50) of 8.1 microM.     

Purification of a lectin from the marine red alga Gracilaria cornea and its effects on the cattle tick Boophilus microplus (Acari: Ixodidae)

A lectin was purified from the seaweed Gracilaria cornea by hydrophobic interaction chromatography on phenyl-Sepharose CL-4B followed by affinity chromatography on immobilized mucin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of G. cornea lectin (GCL) revealed a single protein band of approximately 60 kDa, whereas by gel filtration on Sephadex G-100 its native molecular mass was 66 kDa. GCL exhibited a single isoeletric point of 4.3 and a 52.5% content of neutral sugars. Furthermore, the EDTA-treated lectin did not show any significant decrease in its ability to agglutinate trypsin-treated chicken erythrocytes. These data suggest that GCL is an acidic, monomeric glycoprotein that probably does not require divalent metal ions for its hemagglutinating activity. GCL hemagglutinating activity was not inhibited by any of the mono-, di-, and trisaccharides tested but was by the complex glycoproteins fetuin and porcine stomach mucin. Exposure of engorged females of the cattle tick (Boophilus microplus) to 0.1 mg mL(-1) GCL significantly (P < 0.05) reduced the female weight after the oviposition period, the egg mass weight, the hatching period, and the mean larvae survival time.     

Angiotensin I converting enzyme inhibitory peptides purified from bovine skin gelatin hydrolysate.

Bovine skin gelatin was hydrolyzed with sequential protease treatments in the order of Alcalase, Pronase E, and collagenase using a three-step ultrafiltration membrane reactor. The molecular weight distributions of the first, second, and third hydrolysates were 4.8-6.6, 3.4-6.6, and 0.9-1.9 kDa, respectively. The angiotensin I converting enzyme (ACE) inhibitory activity of the third hydrolysate (IC(50) = 0.689 mg/mL) was higher than that of the first and second hydrolysates. Two different peptides showing strong ACE inhibitory activity were isolated from the hydrolysate using consecutive chromatographic methods including gel filtration chromatography, ion-exchange chromatography, and reversed-phase high-performance liquid chromatography. The isolated peptides were composed of Gly-Pro-Leu and Gly-Pro-Val and showed IC(50) values of 2.55 and 4.67 microM, respectively.     

Isolation and characterization of a low molecular weight peptide contained in sourdough

To investigate a sourdough-specific peptide, low molecular weight peptides were extracted from sourdough. The peptide fraction was subjected to two kinds of chromatography to separate the peptides. Reverse-phase chromatography of the peptide fraction in the sourdough showed certain specific peptides. The specific peptide fraction was further separated by gel filtration chromatography. Liquid chromatography tandem mass spectrometry analysis identified one of the peptides as VPFGVG (six-mer). This sequence was estimated to occur at the 287-292 position of a low molecular weight glutenin subunit. The peptide (designed as SDP1) was produced by proteases derived from wheat flour. SDP1 showed angiotensin-converting enzyme (ACE) inhibitory activity, and the 50% inhibitory peptide concentration (IC50) was 336 microM. It is possible that the SDP1 peptide partially confers ACE inhibitory activity in sourdough.     

Novel nitric oxide and superoxide generation inhibitors, persenone A and B, from avocado fruit

One known, (2R)-(12Z,15Z)-2-hydroxy-4-oxoheneicosa-12,15-dien+ ++-1-yl acetate (1), and two novel compounds, persenone A (2) and B (3), have been isolated from avocado fruit (Persea americana P. Mill), as inhibitors of superoxide (O(2)(-)) and nitric oxide (NO) generation in cell culture systems. They showed marked inhibitory activities toward NO generation induced by lipopolysaccharide in combination with interferon-gamma in mouse macrophage RAW 264.7 cells. Their inhibitory potencies of NO generation (1, IC(50) = 3.6; 2, IC(50) = 1.2; and 3, IC(50) = 3.5 microM) were comparable to or higher than that of a natural NO generation inhibitor, docosahexaenoic acid (DHA; IC(50) = 4.3 microM). Furthermore, compounds 1-3 and DHA markedly suppressed tumor promoter 12-O-tetradecanoylphorbol-13-acetate-induced O(2)(-) generation in differentiated human promyelocytic HL-60 cells (1, IC(50) = 33.7; 2, IC(50) = 1.4; 3, IC(50) = 1.8; and DHA, IC(50) = 10.3 microM). It is notable that they were found to be suppressors of both NO- and O(2)(-)-generating biochemical pathways but not to be radical scavengers. The results indicate that these compounds are unique antioxidants, preferentially suppressing radical generation, and thus may be promising as effective chemopreventive agent candidates in inflammation-associated carcinogenesis.     

Isolation of isolectins from Vitis vinifera L. Cv. Chardonnay grape berries

A lectin fraction from Chardonnay grape juice has been isolated by affinity chromatography on a column of p-aminophenyl beta-D-glucoside-derivatized agarose. The lectin fractions agglutinate rabbit and human erythrocytes without serological specificity. None of the usual monosaccharides, glycosides, or glycoproteins inhibit the hemagglutinating activity. Erythroagglutination is only inhibited by nitrophenyl glycosides, p-nitrophenyl beta-D-glucoside being the strongest inhibitor. In SDS-PAGE in the presence of 2-mercaptoethanol and gel filtration HPLC, the lectin fraction gave a single band or peak corresponding to M(r) 13.2-11.9 kDa, thus indicating it to be a monomer. Three bands were observed by isoelectric focusing with pI values of 4.1, 4. 4, and 4.9. The isolectins seem to be glycoproteins since they are bound on a concanavalin A-Sepharose column.     

Alpha-eleostearic acid and its dihydroxy derivative are major apoptosis-inducing components of bitter gourd

Bitter gourd ( Momordica charantia L.) pericarp, placenta, and seed extracts were previously shown to induce apoptosis in HL60 human leukemia cells. To determine the active component that induces apoptosis in cancer cells, bitter gourd ethanol extract was fractionated by liquid-liquid partition and silica gel column chromatography. Several fractions obtained by silica gel column chromatography inhibited growth and induced apoptosis in HL60 cells. Among them, fraction 7 had the strongest activity in inhibiting growth and inducing apoptosis in HL60 cells. A component that induced apoptosis in HL60 cells was then isolated from fraction 7 by another silica gel column chromatography and high-performance liquid chromatography (HPLC) using a C18 column and was identified as (9Z,11E,13E)-15,16-dihydroxy-9,11,13-octadecatrienoic acid (15,16-dihydroxy alpha-eleostearic acid). 15,16-Dihydroxy alpha-eleostearic acid induced apoptosis in HL60 cells within 5 h at a concentration of 160 microM (50 microg/mL). (9Z,11E,13E)-9,11,13-Octadecatrienoic acid (alpha-eleostearic acid) is known to be the major conjugated linolenic acid in bitter gourd seeds. Therefore, the effect of alpha-eleostearic acid on the growth of some cancer and normal cell lines was examined. alpha-Eleostearic acid strongly inhibited the growth of some cancer and fibroblast cell lines, including those of HL60 leukemia and HT29 colon carcinoma. alpha-Eleostearic acid induced apoptosis in HL60 cells after a 24 h incubation at a concentration of 5 microM. Thus, alpha-eleostearic acid and the dihydroxy derivative from bitter gourd were suggested to be the major inducers of apoptosis in HL60 cells.     

Characterization of beta-hydroxy-beta-methylglutaryl coenzyme A reductase inhibitor from Pueraria thunbergiana

This study describes the extraction and characterization of an inhibitor for beta-hydroxy-beta-methylglutaryl (HMG) coenzyme A (CoA) reductase from Pueraria thunbergiana. The maximum HMG-CoA reductase inhibitory activity (IC(50) = 79 microg) was obtained when P. thunbergiana was extracted with 70% ethanol at 30 degrees C for 12 h. After purification of the HMG-CoA reductase inhibitor by means of systematic solvent extraction, silica gel column chromatography, and HPLC, an active fraction with an IC(50) of 0.9 microg (4.25 microM) and a yield of 1.3% was obtained. The purified HMG-CoA reductase inhibitor was identified as daidzein (C(15)H(10)O(4); molecular mass, 254 Da).     

Structure characterization and antioxidant activity of a novel polysaccharide isolated from pulp tissues of Litchi chinensis

A novel polysaccharide (LCP50S-2) with antioxidant activity was isolated from Litchi chinensis Sonn. The structure of LCP50S-2 was elucidated on the basis of physicochemical and instrumental analyses, and its average molecular weight was determined by gel permeation chromatography to be 2.19 × 10(2) kDa. The backbone of LCP50S-2 was composed of (1→3)-linked β-L-rhamnopyranosyl residues, (1→4)-linked α-D-xylopyranosyl residues, (1→4)-linked β-D-glucopyranosyl residues, and (1→4)-linked α-D-glucopyranosyl residues which branched at O-6. The two branches consisted of α-L-arabinopyranosyl residues and (1→6)-linked β-D-galactopyranosyl residues terminated with α-L-arabinopyranosyl residues, respectively. In the in vitro antioxidant assay, LCP50S-2 was found to possess DPPH radical-scavenging activity and hydroxyl radical-scavenging activity with IC(50) values of 220 and 266 μg/mL, respectively.     

Peptide with angiotensin I-converting enzyme inhibitory activity from hydrolyzed corn gluten meal

Corn gluten meal (CGM) was hydrolyzed by Alcalase after starch removal of CGM was applied as a pretreatment. A new inhibitory peptide for angiotensin I-converting enzyme (ACE) was isolated from the hydrolysate of CGM with the use of Bio-Rad P-2 gel filtration and followed by reverse-phase high-performance liquid chromatography (RP-HPLC). The sequence of the active peptide was determined to be Ala-Tyr after the application of amino acid analysis and HPLC/MS. The IC50 of the peptide was 14.2 microM, and it was not affected by preincubation with 30 mU of ACE at 37 degrees C for 3 h. Ala-Tyr also exerted antihypertensive effects after oral administration to spontaneously hypertensive rats. A maximal reduction of systolic blood pressure of 9.5 mmHg was observed 2 h after oral administration of Ala-Tyr at doses of 50 mg/kg.     

Isolation and characterization of a bioactive mannose-binding protein from the Chinese chive Allium tuberosum

A mannose-binding protein was isolated from two different cultivars of the Chinese chive Allium tuberosum by extraction with 0.2 M NaCl, ammonium sulfate precipitation, and affinity chromatography on mannose agarose and fetuin agarose. It exhibited hemagglutinating activity toward rabbit erythrocytes. The lectin (agglutinin) was adsorbed on the mannose-agarose column, but not on the fetuin-agarose column. This A. tuberosum lectin (ATL) is unglycosylated, and not sialic acid binding. Lectins isolated from the two cultivars exhibited the same molecular mass of 25 kDa on gel filtration (Superose 12) and 12.5 kDa on SDS-polyacrylamide gel electrophoresis, indicating that they might be a dimeric protein composed of two identical subunits. The N-terminal amino acid sequence analysis of the lectin of various cultivars of A. tuberosum revealed that they were identical and showed 50%, or more, homology to the lectins from Galanthus nivalis (family Amaryllidaceae), Narcissus tazetta (family Amaryllidaceae), and Aloe arborescenes (family Liliaceae).     

Antioxidant xanthones from the pericarp of Garcinia mangostana (Mangosteen)

As part of ongoing research on cancer chemopreventive agents from botanical dietary supplements, Garcinia mangostana L. (commonly known as mangosteen) was selected for detailed study. Repeated chromatography of a CH2Cl2-soluble extract of the pericarp led to the isolation of two new highly oxygenated prenylated xanthones, 8-hydroxycudraxanthone G (1) and mangostingone [7-methoxy-2-(3-methyl-2-butenyl)-8-(3-methyl-2-oxo-3-butenyl)-1,3,6-trihydroxyxanthone, 2], together with 12 known xanthones, cudraxanthone G (3), 8-deoxygartanin (4), garcimangosone B (5), garcinone D (6), garcinone E (7), gartanin (8), 1-isomangostin (9), alpha-mangostin (10), gamma-mangostin (11), mangostinone (12), smeathxanthone A (13), and tovophyllin A (14). The structures of compounds 1 and 2 were elucidated by spectroscopic data analysis. Except for compound 2, which was isolated as a minor component, the antioxidant activities of all isolates were determined using authentic and morpholinosydnonimine-derived peroxynitrite methods, and compounds 1, 8, 10, 11, and 13 were the most active. Alpha-mangostin (10) inhibited 7,12-dimethylbenz[alpha]anthracene-induced preneoplastic lesions in a mouse mammary organ culture assay with an IC50 of 1.0 microg/mL (2.44 microM).     

pH modulation of zopfiellin antifungal activity to Colletotrichum and Botrytis

Zopfiellin, a novel cyclooctanoid natural product isolated from Zopfiella curvata No. 37-3, was evaluated in a 96-well microtiter assay for fungicidal activity against Botrytis cinerea, Colletotrichum acutatum, Colletotrichum fragariae, Colletotrichum gloeosporioides, and Fusarium oxysporum. Zopfiellin exhibited pH-dependent activity, with the most mycelial growth inhibition demonstrated at pH 5.0. Mass spectrometry and nuclear magnetic resonance spectroscopy studies indicated that zopfiellin undergoes structural changes with changes in pH. At pH 5.0, zopfiellin showed the greatest activity against B. cinerea (IC(80) = 10 microM), C. gloeosporioides (IC(80) = 10 microM), and C. fragariae (IC(80) = 10 microM) and intermediate activity against C. acutatum (IC(80) = 30 microM), and was not active against F. oxysporum (IC(80) > 100 microM).     

LDL-antioxidant pterocarpans from roots of Glycine max (L.) Merr

The methanolic root extract of Glycine max (L.) Merr. was chromatographed, which yielded 10 flavonoids, including three isoflavones 1-3, five pterocarpans 4-8, one flavonol 9, and one anthocyanidin 10. All isolated compounds were examined for LDL-antioxidant activities using four different assay systems on the basis of Cu2+-mediated oxidation. Among them, seven compounds showed potent LDL-antioxidant activities in the thiobarbituric acid reactive substances (TBARS) assay, the lag time of conjugated diene formation, relative electrophoretic mobility (REM), and fragmentation of apoB-100 on copper-mediated LDL oxidation. Three pterocarpans 4, 6, and 7, never reported as LDL-antioxidant, showed potent activities with IC50 values of 19.8, 0.9, 45.0 microM, respectively, in comparison with probucol (IC50 = 5.6 microM) as positive control. Interestingly, coumestrol 6 (IC50 = 0.9 microM) showed 20 times more activity in the TBARS assay than genistein (IC50 = 30.1 microM) and daidzein (IC50 = 21.6 microM), representative antioxidants in soybean. Moreover, coumestrol 6 had an extended lag time of 190 min at 3.0 microM in measuring conjugated diene formation, while both genistein (120 min) and daidzein (93 min) lag times were extended to less than 120 min at the same concentration.     

Inhibition on human liver cytochrome P450 3A4 by constituents of fennel (Foeniculum vulgare): identification and characterization of a mechanism-based inactivator

Fennel, a seed of Foeniculum vulgare, is used as a culinary spice and traditional medicine. The methanolic extract of fennel showed a characteristic of mechanism-based inactivation on erythromycin N-demethylation mediated by human liver microsomal cytochrome P450 3A4 (CYP3A4). The present study was conducted to identify the fennel constituent having the inhibition. Thirteen compounds have been isolated from a methanol extract of fennel and tested for their inhibition on CYP3A4. Among them, 5-methoxypsoralen (5-MOP) showed the strongest inhibition with an IC50 value of 18.3 microM and a mixed type of inhibition. In addition, with the preincubation time of 20 min only 5-MOP showed preincubation time dependency; the IC50 value decreased from 18.3 microM with a preincubation time of 0 min to 4.6 microM with a preincubation time of 20 min. Further investigation on 5-MOP showed the characteristics of time-dependent inhibition, requirement of NADPH, lack of protecting effect of nucleophiles, and recovery of CYP3A4 activity by the competitive inhibitor. This result suggests that the inhibitory activity of CYP3A4 by 5-MOP was a mechanism-based inactivation. The kinetic parameter for mechanism-based inactivation was characterized by a KI value of 15.0 microM and a kinact value of 0.098 min(-1).