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Agricultural waste from the hulls of water caltrop (Trapa taiwanesis Nakai, TT-hull) was extracted by either steeping them in cold 95% ethanol (C95E), refluxing 95E, refluxing 50E, or refluxing hot water (HW) to obtain C95EE, 95EE, 50EE, and HWE, respectively. These four extracts showed acetylcholinesterase (AChE) inhibitory activities and free radical scavenging activities, as well as anti-non-enzymatic protein glycation in vitro. Eight compounds were isolated from TT-hull-50EE and were used to plot the chromatographic fingerprints of the TT-hull extracts, among which tellimagrandin-I, tellimagrandin-II, and 1,2,3,6-tetra-galloylglucose showed the strongest AChE inhibitory activities, and they also exhibited anti-amyloid β peptide aggregations. The scopolamine-induced amnesiac ICR mice that were fed with TT-hull-50EE or TT-hull-HWE (100 and 200 mg/kg) or tellimagrandin-II (100 and 200 mg/kg) showed improved learning behavior when evaluated using passive avoidance or water maze evaluation, and they showed significant differences (p &lt, 0.05) compared to those in the control group. The enriched hydrolysable tannins of the recycled TT-hull may be developed as functional foods for the treatment of degenerative disorders.

Hydrogen peroxide, one of the reactive oxygen species (ROS), can cause intracellular oxidative stress associated with skin aging and/or photoaging. Curcumin, a polyphenol in turmeric, has been reported to exhibit biological activity. In this study, five naturally occurring curcuminoids [curcumin, demethoxycurcumin (DMC), bisdemethoxycurcumin (BDMC), monohydroxy-DMC, and monohydroxy-BDMC] were used to investigate their protective roles against hydrogen peroxide-induced oxidative stress in the immortalized human keratinocyte cell lines (HaCaT cells). These five curcuminoids at 10 μM, but not at 5 μM, were shown to exhibit cytotoxicities toward HaCaT keratinocytes. Therefore, a 5 μM concentration of the five curcuminoids was selected for further investigations. Cells were pretreated with or without curcuminoids for 2.5 h before 24-h hydrogen peroxide (150 μM) treatments. Pretreatments with the minor components monohydroxy-DMC or monohydroxy-BDMC, but not curcumin, DMC, and BDMC, showed protective activity, elevating cell viability compared to cells with direct hydrogen peroxide treatments. Pretreatments with monohydroxy-DMC and monohydroxy-BDMC showed the best protective effects, reducing apoptotic cell populations and intracellular ROS, as demonstrated by flow cytometry, as well as reducing the changes of the mitochondrial membrane potential compared to cells with direct hydrogen peroxide treatments. The pretreatments with monohydroxy-DMC and monohydroxy-BDMC reduced c-jun and c-fos mRNA expression and p53 tumor suppressor protein expression and increased HO-1 protein expression and glutathione peroxidase (GPx) activity, respectively, compared to cells with direct hydrogen peroxide treatments. The five curcuminoids exhibited similar hydrogen peroxide-scavenging activity in vitro. It was proposed that monohydroxy-DMC and monohydroxy-BDMC could induce antioxidant defense systems better than curcumin, DMC, or BDMC could against hydrogen peroxide-induced oxidative stress and apoptosis of HaCaT keratinocytes and that they may have potential as ingredients in antiaging cosmetics for skin care.

Dioscorea japonica Thunb. var. pseudojaponica (DP) is consumed as food and widely used in traditional Chinese medicine in Taiwan. The aims of this study are to investigate the antioxidant and anti-inflammatory effects of ethanol extract of DP (EDP) and its reference compounds. Fingerprint chromatogram from HPLC indicated that EDP contains gallic acid and vanillic acid. EDP was evaluated for its antioxidant effects and LPS-induced nitrite oxide (NO) production in RAW264.7 cells. EDP decreased the LPS-induced NO production and expressions of iNOS and COX-2 in RAW264.7 cells. In-vivo anti-inflammatory activities of EDP were assessed in mouse paw oedema induced by λ-carrageenan (Carr). We investigated the antioxidant mechanism of EDP via studies of the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) and the levels of malondialdehyde (MDA) in the oedematous paw. The results showed that EDP might be a natural antioxidant and anti-inflammatory agent.

Anti-inflammatory effects of tormentic acid ( TA ) were investigated ex vivo and in vivo . TA decreased the paw edema at the 4th and 5th hour after λ-carrageenin (Carr) administration, and increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in the liver tissue. TA also significantly attenuated the thiobarbituric acid reactive substances (TBARS) level in the edematous paw at the 5th hour after Carr injection. TA decreased the nitric oxide (NO) levels on the serum level and diminished the serum tumour necrosis factor (TNF-α) at the 5th hour after Carr injection. Western blotting revealed that the TA decreased Carr-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expressions. As per results, the anti-inflammatory mechanisms of TA might be correlated to the decrease in the level of TBARS, iNOS, and COX-2 in the edema paw via increasing the activities of CAT, SOD, and GPx in the liver.

Aims of the study Cardiospermum halicacabum (CH) has been used in Chinese medicine for a long time. However, its fingerprint chromatogram, antioxidant, anti-inflammatory effects and mechanism are still needed to be explored. Therefore, the aims of this study investigated the antioxidant and anti-inflammatory effects of CH extracts and its reference compounds ex vivo and in vivo. Materials and methods In HPLC analysis, the fingerprint chromatogram of ethanolic extract of CH (ECH) was established. The effects of ACH (aqueous extract of CH) and ECH extracts were assessed for the antioxidant and LPS-induced NO production in RAW264.7 cells. In vivo anti-inflammatory activities of ECH were evaluated in mouse paw edema induced by λ-carrageenan (Carr). We investigate the anti-inflammatory mechanism of ECH via studies of the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in the liver and the levels of malondialdehyde (MDA) and nitrite oxide (NO) in the edema paw. Serum NO and TNF-α were also measured. Results ECH had better antioxidant activity than that of ACH. In the anti-inflammatory test, ECH inhibited the development of paw edema induced by Carr and increased the activities of CAT, SOD and GPx in the liver tissue. ECH also decreased the level of NO in edematous paw tissue and in serum level, and diminished the level of serum TNF-α at the fifth hour after Carr injection. Conclusions ECH exerts anti-inflammatory effects by suppressing TNF-α and NO. The anti-inflammatory mechanism of ECH might be related to the decrement of the level of MDA in the edema paw via increasing the activities of CAT, SOD and GPx in the liver. The results showed that ECH might serve as a natural antioxidant and anti-inflammatory agent.

A protein isolate was produced from cellulase-treated defatted flaxseed meal followed by hydrolysis with seven proteases and evaluation of the hydrolysates for antioxidant and anti-inflammatory properties. The flaxseed protein hydrolysates (FPH) were processed by ultrafiltration and ion-exchange chromatography to isolate low molecular weight (LMW) and cationic peptide fractions, respectively. The peptides showed antioxidant properties in scavenging 2,2-diphenyl-1-picrylhydrazyl radical, superoxide anion radical, electron-spin resonance-detected hydroxyl radical and nitric oxide. In addition, all peptide fractions inhibited semicarbazide-sensitive amine oxidase activity. Antioxidant activities of these peptides were dependent on the specificity of proteases and size of the resulting peptides. The LMW fractions from pepsin, ficin and papain FPH also inhibited lipopolysaccharide-induced nitric oxide productions in RAW 264.7 macrophages without apparent cytotoxicity; thus, these peptides may act as anti-inflammatory agents. Thus, flaxseed protein hydrolysates may serve as potential ingredients for the formulation of therapeutic products.

Enzymatic hydrolysates from flaxseed protein were investigated for in vitro inhibition of angiotensin I-converting enzyme (ACE) and renin activities. Pepsin, ficin, trypsin, papain, thermolysin, pancreatin and Alcalase were used to hydrolyze flaxseed proteins followed by fractionation using ultrafiltration to isolate low-molecular-weight peptides, and separation of the Alcalase hydrolysate into cationic peptide fractions. Using N -(3-[2-furyl]acryloyl)-phenylalanylglycylglycine as substrate, the protein hydrolysates showed a concentration-dependent ACE inhibition (IC 50 , 0.0275–0.151 mg/ml) with thermolysin hydrolysate and Alcalase cationic peptide fraction I (FI) showing the most potent activity. Flaxseed peptide fractions also showed no or moderate inhibitory activities against human recombinant renin (IC 50 , 1.22–2.81 mg/ml). Kinetics studies showed that the thermolysin hydrolysate and FI exhibited mixed-type pattern of ACE inhibition whereas cationic peptide fraction II inhibited renin in uncompetitive fashion. These results show that the protein components of flaxseed meal possess peptide amino acid sequences that can be exploited as potential food sources of anti-hypertensive agents.

Taiwanofungus camphoratus (T. camphoratus), a fungus and a Taiwan-specific, well-known traditional Chinese medicine, has long been used to treat diarrhea, hypertension, itchy skin, and liver cancer. To gain a large amount of T. camphoratus, several culture techniques have been developed, including solid-state culture and liquid-state fermentation. Peroxisome proliferator-activated receptor gamma (PPARgamma) has been described as a hypoglycemic agent that increases insulin sensitivity in peripheral tissues and results in reduced blood glucose, insulin, and triglyceride levels in insulin-resistant animals and in type-2 (non-insulin-dependent) diabetic patients. In this study, we investigate the possibility that T. camphoratus might activate PPARgamma in vitro and hypolipidemic activity in vivo. The results show that an aqueous extract of the wild fruiting bodies of T. camphoratus was able to increase the PPARgamma activity in cells transfected with the PPARgamma expression plasmid and the AOx-TK reporter plasmid. Based on the cell experiment, we examined the hypolipidemic effect of wild fruiting bodies (WFT) and a solid-state culture (SST) of T. camphoratus on SD rats fed on a high-cholesterol (HC) diet. The results show that WFT significantly decreased the serum triglyceride level, but could not affect the cholesterol level. SST only slightly decreased the serum triglyceride level. In addition, both WFT and SST significantly decreased the serum alanine transaminase (ALT) level and protected against the liver damage induced by the HC diet from the results of a histological examination. These results suggest that T. camphoratus might contain PPARgamma ligands and result in a hypotriglyceridemic effect, and that it also exhibits a liver protective activity.

The increasing prevalence of obesity continues to gain more attention worldwide. In this study, diet-induced obese mice were used to evaluate the antiobesity effects of extracts, fractions, and purified compounds from Vitis thunbergii var. taiwaniana (VTT). The C57BL/6J mice were fed a 5-week high-fat diet (HF) concurrently with ethanol extracts (Et-ext, 80 mg/kg) from roots (R), stems (S), and leaves (L) by oral gavage daily. Only R-Et-ext interventions showed significant weight reduction in mice compared with those in the HF group; however, mouse plasma contents of total cholesterols (TC), total triglycerides (TG) and low-density lipoproteins (LDL) of all three Et-ext intervened groups showed significant reductions compared with those in the HF group. Furthermore, intervention with the ethyl acetate-partitioned fraction (EA-fra, 60 mg/kg) from R-Et-ext but not the n-butanol-partitioned fraction or water fraction from R-Et-ext showed significant weight reduction in mice compared with those in the HF group. The same molecular weights of three resveratrol tetramers, (+)-hopeaphenol, (+)-vitisin A, and (-)-vitisin B, were isolated from the EA-fra of VTT-R. The (+)-vitisin A and fenofibrate (25 mg/kg) but not the (+)-hopeaphenol and (-)-vitisin B interventions showed significant weight reduction in mice compared with those in the HF group. The total feed intake among the HF groups with or without interventions showed no significant differences. The mouse plasma contents of TC, TG, LDL, free fatty acid, and plasma lipase activity of the three resveratrol tetramer-intervened groups showed reductions in the mice compared with those in the HF group. It was proposed that the lipase inhibitory activities of VTT extracts and purified resveratrol tetramers might contribute in part to the antiobesity effect, and these results suggested that VTT may be developed as functional food for achieving antiobesity objectives and requires further investigation.

Background Large amounts of Ganoderma lucidum (GL) commercial products are provided in the worldwide market such as powders, tea bags, or capsules as dietary supplements which contained triterpenoids and/or polysaccharides. Therefore, it was estimated that several thousand tons of GL residues (GLR) are produced and discarded. For recycling uses, the aim of this study was to evaluate the benefits of two hot-water extracts from GLR (HWP_GLR) and solid-state fermentation GLR inoculated with GL mycelia (HWP_GLRF) on the growths of Lactobacillus rhamnosus and Bifidobacterium longum. The RAW264.7 cells were used to investigate the effects of HWP_GLR and HWP_GLRF on nitric oxide productions, phagocytic activities against FITC-labeled E. coli, and to lower lipopolysaccharide (LPS)-binding capacities. The powders of GLR and GLRF were used as additives in the commercial feeds for feeding broiler chicks in vivo to evaluate the immune-stimulatory and prebiotic activities. Results HWP_GLR and HWP_GLRF with molecular size 5 to 8 kDa were showed to stimulate growths of L. rhamnosus and B. longum. It was found that in the presence of polymyxin B HWP_GLR and HWP_GLRF could stimulate nitric oxide productions, elevate phagocytic activities against FITC-labeled E. coli, and to lower lipopolysaccharide-binding capacities in RAW264.7 cells. The broiler chicks were selected for feedings in vivo. The 1-day-old chicks were fed commercial feeds for 1 week, and then were fed without or with 4 or 8 % of GLR and GLRF additives for 3 weeks. There was no significant weight difference among feeding groups. However, the phagocytosis and natural killer cytotoxicity in the peripheral bloods, and prebiotic activities of bifidobacteria in feces of GLR and/or GLRF groups were significantly different compared to the control (P Conclusions The GLR, GLRF, and their hot-water extracts with beneficial activities could be processed as feed additives which could increase the waste-recycling.

The dried rhizome of Bai Zhu (Atractylodes ovata) is widely used as a Chinese herbal medicine. Two sesquiterpenolides of similar structures (atractylenolide I, AT-I; atractylenolide III, AT-III) were isolated from dried rhizome of Atractylodes ovata. Incubation of AT-I with recombinant human Cu,Zn-superoxide dismutase (rhCu,Zn-SOD) resulted in rhCu,Zn-SOD fragmentations and Zn releases. However, these were not observed in the AT-III reaction. The AT-1 showed dose-dependent cytotoxic activities (7.5, 15, and 30 lg/ml) on the human promyeloleukemic HL-60 cells while AT-III did not, and the IC50 of the former being 10.6 lg/ml (corresponding to 46 lM) on 12 h-treated cells. The results of DNA ladder and DNA contents in sub-G1 type revealed that AT-I induced apoptosis in human promyeloleukemic HL-60 cells. The cytotoxic and pharmacological mechanisms of AT-I against human promyeloleukemic HL-60 cells was investigated. The AT-I appeared to exhibit both pro-oxidant and antioxidant properties after an ESR spectrometer was used to detect hydroxyl radical productions in vitro and flow cytometry to detect intracellular ROS productions in AT-I treated cells. The AT-1 also showed dose-dependent Cu,Zn-SOD inhibitory activity in HL-60 cells treated for 12 h, confirmed by activity and immune stainings. However, catalase, Mn-SOD, and glutathione peroxidase did not apparently change activities under the same treatments. The addition of commercial rhCu,Zn-SOD (25–100 U/mL) to the AT-I-treated HL-60 cells (15 lg/ml) resulted in significant differences (p < 0.01) and could reduce the AT-I cytotoxicity from 78% to 28% on HL-60 cells. It was proposed that the AT-I might work via Cu,Zn-SOD inhibition in HL-60 cells to induce apoptosis and bring about cytotoxicity. � 2006 Elsevier Ltd. All rights reserved.

Granulins are a family of evolutionarily ancient proteins that are involved in regulating cell growth and division in animals. In this report a full-length cDNA, SPCP3, was isolated from senescent leaves of sweet potato (Ipomoea batatas). SPCP3 contains 1389 nucleotides (462 amino acids) in its open reading frame, and exhibits high amino acid sequence homologies (ca. 64-73.6%) with several plant granulin-containing cysteine proteases, including potato, tomato, soybean, kidney bean, pea, maize, rice, cabbage, and Arabidopsis. Gene structural analysis shows that SPCP3 encodes a putative precursor protein. Via cleavage of the N-terminal propeptide, it generates a protein with 324 amino acids (from the 139th to the 462nd amino acid residues), which contains two main domains: the conserved catalytic domain with the putative catalytic residues (the 163rd Cys, 299th His and 319th Asn) and the C-terminal granulin domain (from the 375th to the 462nd amino acid residues). Semi-quantitative RT-PCR and protein gel blot hybridization showed that SPCP3 gene expression was enhanced significantly in natural senescent leaves and in dark- and ethephon-induced senescent leaves, but was almost undetectable in mature green leaves, veins, and roots. Phylogenic analysis showed that SPCP3 displayed close association with a group of plant granulin-containing cysteine proteases which have been implied to be involved in programmed cell death. In conclusion, sweet potato SPCP3 is a functional, senescence-associated gene. Its mRNA and protein levels were significantly enhanced in natural and induced senescing leaves. The physiological role and/or function of SPCP3 associated with programmed cell death during leaf senescence were also discussed.

Yam storage protein (YSP) was purified from tubers of Dioscorea alata L. Tainong No. 1 (TN1) to homogeneity by DE-52 ion-exchange chromatography. The short-term (24 h) and long-term (25 days) antihypertensive effects of YSP-TN1 and its peptic hydrolyzates (PH-TN1) were measured in spontaneously hypertensive rats (SHRs). For 24-h antihypertensive measurements, SHRs (age 10 weeks, body weight from 240 to 250 g) were administered orally once (YSP-TN1 and PH-TN1, 40 mg kg−1 SHR) to measure the mean blood pressure (MBP), systolic blood pressure (SBP) and diastolic blood pressure (DBP). For a long-term antihypertensive measurement, SHRs (age 12 weeks, body weight from 250 to 270 g) were administered orally once a day for 25 days (YSP-TN1, 40 mg kg−1 SHR) to measure SBP, DBP and MBP. Captopril (10 or 15 mg kg−1 SHR) was used as a positive control. It was found that short-term administration of 40 mg kg−1 SHR of YSP-TN1 and PH-TN1 effectively lowered SHRs' MBP, SBP and DBP (For YSP-TN1, the lowest blood pressure was reached in the fourth hour and for PH-TN1 in the eighth hour). The lasting effects of PH-TN1 on reduced SHRs' BP were better than those of YSP-TN1 for one oral administration. For oral administration of 40 mg YSP-TN1 kg−1 SHR, the reduced MBP was 21.5 mmHg, which was comparable to 25.2 mmHg (the fourth hour) of 10 mg captopril kg−1 SHR oral administration. For oral administration of 40 mg PH-TN1 kg−1 SHR, the reduced MBP was 33.7 mmHg, comparable to 38.4 mmHg (the fourth hour) of 15 mg captopril kg−1 SHR. For long-term 25-day oral administration of 40 mg YSP-TN1 kg−1 SHR once a day, it was found that a feeding trial of YSP-TN1 effectively lowered SHRs' SBP, DBP and MBP. The greatest reduction in SHRs' blood pressure was reached on the ninth day, for the reduced SBP, 27.7 mmHg; for the reduced DBP, 28.3 mmHg; and for the reduced MBP, 27.5 mmHg. Copyright © 2006 Society of Chemical Industry

Commercial pectin (with a 94% degree of esterification, DE94) suspended in methanol was reacted with methanolic alkaline hydroxylamine at room temperature for 20 h to prepare pectin hydroxamic acids (PHAs). The prepared PHA was coupled to the epoxy-activated Sepharose 6B gel to get immobilized PHA resins. The immobilized PHA resin was then balanced in column with 2 mM ZnCl2 in 50 mM Tris-HCl buffer (pH 7.9) to test the immobilized Zn-PHA gel as solid phase for immobilized metal affinity chromatography for the purification of trypsin inhibitors (TIs) from soybean and sweet potato. Using TI activity staining, it was found that purified TIs from the commercial soybean and sweet potato after trypsin affinity column purification could be adsorbed onto an immobilized Zn-PHA affinity column and eluted by 100 mM EDTA in 10 mM Tris-HCl buffer (pH 7.9). The immobilized Zn-PHA affinity column was used for TI purifications from crude extracts of sweet potato. The recovery of TI activity for one step was 90%, with 19.74-fold purification increase.

Cysteine proteinase (CPR) cDNA clone (SPCPRPP) of sweet potato ( Ipomoea batatas [L.] Lam ‘Tainong 57’) storage roots were isolated by differential display. The open reading frame in this cDNA encodes a pre-proprotein of 371 amino acids with conserved catalytic amino acids of papain. Examination of the expression patterns in sweet potato by Northern blot analyses revealed that the transcripts of SPCPRPP were specifically induced in the storage roots. Recombinant SPCPRPP protein overproduced in Escherichia coli (M15) was purified by Ni 2+ -chelated affinity chromatography. Active recombinant SPCPRPP protein was able to digest the 22 kDa sweet potato trypsin inhibitor (TI) protein when the latter was reduced by DTT (dithiothreitol) or NTS (NADPH/thioredoxin system). A smaller peptide (14 kDa) was obtained as a digestion product. These results suggest that CPR is responsible for initiation of degradation and re-mobilization of stored 22 kDa TI during sprouting of SP storage roots after the reduction of 22 kDa TI by the NTS.

The major root storage proteins of sweet potato (Ipomoea batatas Lam.), trypsin inhibitors (TIs), were purified from tuberous roots of two cultivars, Tainong 57 (T57) and T65, by trypsin-Sepharose 4B affinity column. The major protein was 28 kDa TI from two sweet potato cultivars. The purified TIs from T57 and T65 showed dose-dependent DPPH and hydroxyl radical-scavenging activities with glutathione as a control, and casein or bovine serum albumin were used for comparisons. In average, scavenging capacity for superoxide radical of 100 μg TN57 TIs and TN65 TIs were equivalent to 10.49 and 6.13 units of recombinant human superoxide dismutase, respectively. TN65 TIs had activity in the protection of Cu2+-induced human LDL peroxidation, about three-fold that of TN57 TIs. The capacity of TN65 TIs to protect calf thymus from hydroxyl radical-induced DNA damage was about 10-fold that of TN57 TIs. The TN57 TIs showed dose-dependent (1–8 mg/ml) protection effect on dihydrorhodamine 123 (DHR) against peroxynitrite-mediated oxidation. Modifications of denatured TIs by iodoacetamide, or N-bromosuccinimide showed that Trp (tryptophan) residues mainly contributed scavenging activity against hydroxyl radical, while Cys (cysteine) residues mainly contributed scavenging activity against DPPH radical. Putting the above data together we propose that the 3D conformations of native molecular forms of both TN65 TIs and TN57 TIs are important factors that determine the relative capacities of the samples when different antioxidant test systems with different pH, ionic strength, chemical composition and temperature are used. Pepsin treatment or sequential treatments of pepsin and chymotrypsin for various times showed that the DPPH radical-scavenging activity of TI hydrolysates increased during hydrolysis.

Commercial pectins with different degrees of esterification (DE) were reacted with equal volumes of 2 M alkaline hydroxylamine (pH 12.0) at room temperature for 4 h to prepare pectin hydroxamic acids (PHAs; DE94T4, DE65T4, and DE25T4) according to a previously reported method (Hou et al., J. Agric. Food Chem. 2003, 51, 6362-6366) and were used to test the antioxidant and antiradical activities in comparison with those of DE94, DE65, and DE25 pectins. The half-inhibition concentrations, IC(50), of scavenging activity against DPPH were 1.51, 5.43, and 5.63 mg/mL for DE94T4, DE65T4, and DE25T4, respectively, and were much lower than those of corresponding DE pectins under the same concentrations. The scavenging activities of PHAs for DPPH radicals were positively correlated with original DE values of pectin. The optimal pH of DE94T4 for scavenging DPPH radicals was 7.9 or 8.0. Using electron spin resonance (ESR) for scavenging hydroxyl radicals, under the same concentrations of 125 microg/mL, DE94T4, DE65T4, and DE25T4, respectively, exhibited 73.53, 69.01, and 55.17% antiradical activities. PHAs also exhibited protection against hydroxyl radical-mediated DNA damage and anti-human low-density lipoprotein peroxidation tests.

Asparaginyl endopeptidase is a cysteine endopeptidase that has strict substrate specificity toward the carboxyl side of asparagine residues, and is possibly involved in the post-translational processing of proproteins. In this report one full-length cDNA, SPAE, was isolated from senescent leaves of sweet potato (Ipomoea batatas (L.) Lam). SPAE contained 1479 nucleotides (492 amino acids) in the open reading frame, and exhibited high amino acid sequence homologies (c. 61-68%) with asparaginyl endopeptidases of Vicia sativa, Phaseolus vulgaris, Canavalia ensiformis, and Vigna mungo. SPAE probably encoded a putative precursor protein. Via cleavage of the N- and C-termini, it produced a mature protein containing 325 amino acids (from the 51st to the 375th amino acid residues), the conserved catalytic residues (the 173rd His and 215th Cys amino acid residues), and the putative N-glycosylation site (the 332nd Asn amino acid residue). Semi-quantitative RT-PCR and western blot hybridization showed that SPAE gene expression was enhanced significantly in natural senescent leaves and in dark- and ethephon-induced senescent leaves, but was much less in mature green leaves, stems, and roots. Phylogenic analysis showed that SPAE displayed close association with vacuolar processing enzymes (legumains/asparaginyl endopeptidases), which function via cleavage for proprotein maturation in the protein bodies during seed maturation and germination. In conclusion, sweet potato SPAE is probably a functional, senescence-associated gene and its mRNA and protein levels were significantly enhanced in natural and induced senescent leaves. The possible role and function of SPAE associated with bulk protein degradation and mobilization during leaf senescence were also discussed.

Three full-length cDNA clones, designated TRX1, TRX2, and TRX3 encoding different but similar thioredoxin h polypeptides, were isolated from sweet potato (Ipomoea batatas[L.] Lam ‘Tainong 57’) storage roots. These three thioredoxin h clones were similar to each other and contained the canonical WCGPC active site and the important structural and functional amino acids that were conserved in thioredoxin sequences. Recombinant thioredoxin h (TRX2) overproduced in Escherichia coli (M15) was purified by Ni 2+ -chelated affinity chromatography. The molecular mass of TRX2 is ca. 1.4 kDa determined by SDS–PAGE. TRX2 carries an N-terminal 17-amino acid extension enriched in hydrophobic residues. In Northern blot analysis, mRNAs corresponding to all three thioredoxin genes were found to have the highest level in the storage roots; those corresponding to TRX2 and TRX3 were detected at the next higher level in flowers. All three transcripts were detected at very low levels in sprouts of storage roots, roots, veins, and leaves. In Western blot analysis, the thioredoxins were found to have the highest level in the storage roots and veins; higher level in leaves; and very low levels in sprouts of storage root and roots. Active recombinant TRX2 protein is able to reduce the TI proteins in NTR system. Thioredoxin TRX2 was not reduced efficiently when NTR was removed from the NTR system. Three thioredoxin h genes of sweet potato storage roots display differential gene expression patterns, which may be associated with the diverse roles and functions they play in plant physiology in order to cope with particular developmental and environmental cues. © 2003 Elsevier Ireland Ltd. All rights reserved.

Sweet potato ( Ipomoea batatas (L.) Lam) storage roots were sprouted in the dark for two months, and the sprouts were used as source materials for purification of thioredoxin reductase successively via the DE-52, Sephadex G-75, and 2′,5′-ADP Sepharose 4B column chromatography. Thioredoxin reductases with a molecular mass of ca 33 kDa were obtained. The purification was 813-fold and the yield was 2.5%. The effect of thioredoxin reductase on trypsin inhibitor activity (TIA) was analyzed with a TIA assay method. Native, oxidized form of trypsin inhibitors (TIs) inhibited trypsin activity to 24% of its original value. Trypsin activity recovered to 38 and 46% of its original value, respectively, when TI was reduced in advance by NTR system and DTT. The data indicate that NTR-reduced TIs lost the capacity to inhibit trypsin activity in vitro. mBBr/SDS-PAGE was used to analyze the reduction of TIs on gels. The reduced TIs were more sensitive to heat denaturation, and exhibited increased digestibility by partially purified proteases from sweet potato sprouts. Based on these results, we conclude that the NADPH/thioredoxin system is likely associated with the reduction of TIs and initiation of degradation and re-mobilization of stored TIs during sprouting of sweet potato storage roots. Meanwhile we may expect sprouts to be a better protein source for consumption than SP storage root itself.

Summary Metallothionein (MT) is a group of proteins with low molecular masses and high cysteine contents, and is classified into different types, which in general contains two domains (domain 1 and domain 2) with typical amino acid sequences (Rauser 1999). In this report two full-length cDNAs (Y459 and G14) encoding MT-like proteins were isolated from leaves of sweet potato (Ipomoea batatas). Their open reading frames contained 249 and 195 nucleotides (82 and 64 amino acids) for Y459 and G14, respectively, and exhibited a relatively low amino acid sequence similarity (ca. 25.8 percnt;). Gene structure studies showed that Y459 had the conserved domain 1 region of type 2 MT; however, the domain 2 region was not conserved and contained additional amino acids between the CxC and CxC spacing. G14 had conserved domains 1 and 2 of type 4 MT except that the last CxC of domain 2 was changed to RxC. Semi-quantitative RT-PCR showed that Y459 was expressed in significant quantity in roots and stems, but was much less in green leaves. During natural and induced (with dark and ethephon, an ethylene-releasing compound, treatments) leaf senescence, Y459 gene expression was significantly enhanced. In contrast, relatively constant gene expression levels were found for G14 in all tissues or treatments analyzed. In conclusion, the two MT-like protein genes of sweet potato display differential gene structures and gene expression patterns, which may be associated with the diverse roles and functions they play in plant physiology in order to cope with particular developmental and environmental cues.

Summary Salicylic acid (SA) plays a central role in plant acquired resistance and induces gene expression of a group of pathogenesis-related (PR) proteins. We have previously reported that tobacco cells display Ca2+-dependent and Ca2+-independent excretion modes of SA in suspension culture depending on the applied SA concentrations (Chen et al. 2001). In this study, same concentrations of SA (200 μmol/L and 20 μmol/L) were used to compare their effects on PR-N, an acidic β-1,3-glucanase, gene induction and mRNA accumulation in tobacco cell suspension culture. SA at both concentrations induced PR-N gene expression. The induction by 200 μmol/L SA was significantly inhibited by EGTA (a Ca2+ ion chelator) or reduced glutathione (an active oxygen species scavenger). However, these two compounds have little or no effect on PR-N gene induction by 20 μmol/L SA. Either calcium ionophore (A23187) or catalase inhibitor (3-amino-1,2,4-triazole; 3AT) activated PR-N gene expression. As observed for 200 μmol/L SA, the induction by A23187 and 3AT was also blocked by EGTA and reduced glutathione, respectively. A SA functional analog, 2,6-dichloroisonicotinic acid (INA), induced PR-N gene expression. However, EGTA and reduced glutathione had little or no effect on the induction by 20 μmol/L INA, same as observed for the induction by 20 μmol/L SA. Based on these data, we conclude that SA could mediate alternative signal transduction pathways leading to PR-N gene induction in tobacco cell suspension culture. SA concentration seems to play a key role in the activation of different components of signal transduction pathways. Active oxygen species elevation and external Ca2+ influx are components likely associated with 200 μmol/L SA activation mechanism. Another signal transduction pathway not associated with these two components, however, may be responsible for the effect of SA at 20 μmol/L.

Among the eight amino acid hydroxamates tested, Glycine hydroxamate (GH) was the best inhibitor of mushroom tyrosinase (TYR). With l-tyrosine as substrate, the GH inhibition of the monophenolase activity of the mushroom TYR was noncompetitive. GH decreased not only TYR protein expression, but also melanin content, tyrosinase-related protein (TRP)-1, TRP-2, and microphthalmia-associated transcription factor (MITF) expression in B16F10 melanoma cells while in the presence of α-melanocyte-stimulating hormone (α-MSH). GH also significantly decreased the isobutylmethylxanthine (IBMX)-induced increase in melanin content, which was not prevented by the ERK inhibitor PD98059. These results suggest GH has the potential for use in cosmetic hypopigmentation.

Yam tubers, a common tuber crop and an important traditional Chinese medicine in Taiwan, have many bioactive substances, including phenolic compounds, mucilage polysaccharides, steroidal saponins and proteins. Among the total soluble proteins, 80% of them are dioscorins. In the past two decades, many studies showed that dioscorins exhibited biological activities both in vitro and in vivo, including the enzymatic, antioxidant, antihypertensive, immunomodulatory, lectin activities and the protecting role on airway epithelial cells against allergens in vitro. Some of these activities are survived after chemical, heating process or enzymatic digestion. Despite of lacking the intact structural information and the detail action mechanisms in the cells, yam dioscorins are potential resources for developing as functional foods and interesting targets for food protein researchers.

Trypsin inhibitors (TIs), root storage proteins, were purified from sweet potato (Ipomoea batatas[L.] Lam cv. Tainong 57) roots by trypsin affinity column according to the methods of Hou and Lin (Plant Sci. 1997, 126, 11-19 and Plant Sci. 1997, 128, 151-158). A single band of 33 kDa TI was obtained by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. This purified 33 kDa TI had scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. There was positive correlation between scavenging effects against DPPH (2 to 22%) and amounts of 33 kDa TI (1.92 to 46 pmol). The scavenging activities of 33 kDa TI against DPPH were calculated from linear regression to be about one-third of those of glutathione between 5 and 80 pmol. Using electron paramagnetic resonance (EPR) spectrometry for hydroxyl radical detection, it was found that 33 kDa TI could capture hydroxyl radical, and the intensities of EPR signal were significantly decreased from 1.5 to 6 pmol of 33 kDa TI compared to those of the controls. It is suggested that 33 kDa TI, one of the sweet potato root storage proteins, may play a role as an antioxidant in roots and may be beneficial to health when it is consumed.

A complex containing trypsin inhibitor (TI) activity was extracted with 0.1 M TRIS buffer (pH 7.9) from trypsin-treated mitochondria of etiolated mung bean seedlings, and further purified with a Superdex 200 FPLC column. This partially purified complex with an M(r) about 820 kDa exhibited additional dehydroascorbate (DHA) reductase activity with specific activities of 0.21, 1.53 and 1.54 mumol ascorbate formed min-1 mg-1 protein at pH 6.0, 6.5 and 7.0, respectively, when glutathione was added. Much lower DHA reductase activity (0.013 and 0.026 mumol ascorbate formed min-1 mg-1 protein at pH 6.5 and 7.0, respectively) was found when glutathione was omitted. The isolated complex gave positive results when it was tested by TI activity staining after SDS-PAGE, and could be recognized by a polyclonal antibody which was raised against 38 kDa sweet potato Kunitz-type TI, one of the root storage proteins of sweet potato. The possible physiological functions of this complex with both TI and DHA reductase activities were discussed.

Dioscorins, the major storage proteins of yam (Dioscorea batatas Decne), were purified from the tubers according to the methods of Hou et al. (W.C. Hou, J.S. Liu, H.J. Chen, T.E. Chen, C.F. Chang, Y.H. Lin, Dioscorin, the major tuber storage protein of yam (Dioscorea batatas Decne), with carbonic anhydrase and trypsin inhibitor activities, J. Agric. Food Chem. 47 (1999) 2168‐2172.). Dioscorins reduced dehydroascorbate (DHA) in the presence of glutathione to regenerate ascorbate (AsA). However, without glutathione, dioscorins has very low DHA reductase activity. Intermolecular thiol‐disulfide interchanges of dioscorins on SDS‐PAGE gels were found during DHA reduction in the absence of glutathione. AsA was oxidized by AsA oxidase to generate monodehydroascorbate (MDA) free radicals. MDA was also reduced by dioscorins to AsA in the presence of NADH mimicking the MDA reductase catalyzed reaction. These data suggest that dioscorins have both DHA reductase and MDA reductase activities. © 1999 Elsevier Science Ireland Ltd. All rights reserved.

Dioscorin, the tuber storage protein of yam (Dioscorea batatas Decne), was purified successively by ammonium sulfate fractionation, DE-52 ion exchange chromatography, and Sephadex G-75 column. Two protein bands (82 and 28 kDa) were found under nonreducing conditions after SDS-PAGE; but only one band (32 kDa) was detected under reducing conditions. The first 21 amino acids in the N-terminal region of the 28 kDa form were VEDEFSYIEGNPNGPENWGNL, which was highly homologous to deductive sequence of dioscorin from cDNA of another yam species (Dioscoreacayenensis Lam) reported by Conlan et al. (Plant Mol. Biol. 1995, 28, 369-380). Hewett-Emmett and Tashian (Mol. Phylogenet. Evol. 1996, 5, 50 -77) mentioned that, according to DNA alignments, dioscorin from yam (D. cayenensis) was alpha-carbonic anhydrase (alpha-CA) related. In this report, we found that the purified dioscorin showed both CA dehydration activity using sodium bicarbonate as a substrate and CA activity staining after SDS-PAGE. A polyclonal antibody, which was raised against trypsin inhibitor (TI), a storage protein of sweet potato (Ipomoea batatas [L.] Lam var. Tainong 57), cross-reacted with dioscorin, which also showed TI activity determined by both activity staining after SDS-PAGE and trypsin inhibition determination.

Trypsin inhibitors (TIs) were purified from the storage roots of sweet potato ( Ipomoea batatas [L.] Lam var. Tainong 57) with ammonium sulfate precipitation, Sephadex G-75 gel filtration chromatography, and trypsin-Sepharose 4B affinity chromatography as previously reported [1] . Dehydroascorbate (DHA) was reduced by TIs independent of glutathione to regenerate ascorbate (AsA) contrasting with the function of DHA reductase with glutathione as a cofactor. Intermolecular thiol-disulfide interchanges of TIs were found during DHA reduction. AsA was oxidized by AsA oxidase to generate monodehydroascorbate (MDA) free radicals. MDA was reduced also by TIs to AsA in the presence of NADH, which was the function of MDA reductase. The physiological significance of TIs with both DHA reductase and MDA reductase activities are discussed.

Background Advanced glycation end products (AGE) are substances that can induce insulin resistance in adipocyte, hepatocyte and muscle cells. This resistance correlates highly with cardiovascular disease and diabetic complications. Acteoside (A), a phenylethanoid glycoside, is an active compound in several plants and traditional herbal medicines. Acteoside, its structural isomer, isoacteoside (I), and their constituents, caffeic acid (C) and 3,4-dihydroxyphenylethanol (D), were used in the study to investigate the inhibitory activity against AGE formations in vitro. Results AGE formations were detected by anti-(Nϵ-(carboxymethyl)lysine (anti-CML), using bovine serum albumin (BSA)/glucose (glc) and BSA/galactose (gal) as models, or by anti-argpyrimidine (anti-AP), using BSA/methylglyoxal (MGO) as models. It was found that A, I, C, or D, each at 5 mM, could attenuate the CML formations detected by ELISA in the BSA/gal model of a 3-day or 5-day reaction, and showed significant differences (P < 0.01 or P < 0.001) compared to the control. However, these compounds showed a minor effect after a 7-day incubation. It was also found that C or D could lower the CML formations in the BSA/glc model and showed significant differences (P < 0.05 or P < 0.01) compared to the control after a 3-day, 5-day and 7-day reaction. It was found that A, I, C, or D, each at 0.5 mM or 5 mM, could attenuate the AP formations in the BSA/MGO model of a 3-day reaction and showed significant differences (P < 0.001) compared to the control. Conclusions The results suggest the potential anti-glycation activities of A and I in vitro may apply to cell models at higher glucose concentrations or to diabetic animal models, and need further investigation.

Eburicoic acid (TR1) and dehydroeburicoic acid (TR2), an active ingredient from Antrodia camphorata (AC) solid-state culture, were evaluated for analgesic and anti-inflammatory effects. Treatment with TR1 and TR2 significantly inhibited a number of acetic acid-induced writhing responses and formalin-induced pain in the late phase. In the anti-inflammatory test, TR1 and TR2 decreased paw edema at the fourth and fifth hour after λ-carrageenan (Carr) administration and increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in the paw edema tissue. We also demonstrated that TR1 and TR2 significantly attenuated the malondialdehyde (MDA), nitric oxide (NO), tumor necrosis factor (TNF-α), and interleukin-1β (IL-1β) levels in either edema paw or serum at the fifth hour after Carr injection. Western blotting revealed that TR1 and TR2 decreased Carr-induced inducible nitric oxide synthase (iNOS) and cycloxyclase (COX-2) expressions at the fifth hour in paw edema. Treatment with TR1 and TR2 also diminished neutrophil infiltration into the paw edema at the fifth hour. The present study suggests that the anti-inflammatory mechanisms of TR1 and TR2 might be related to the decrease of inflammatory cytokines and an increase of antioxidant enzyme activity.

Many vegetables exhibit a firming effect after precooking at a temperature between 50 and 70C. This effect has generally been attributed to the action of endogenous pectinesterase which hydrolyzes the methyl ester linkages in pectin molecules. The resulting free carboxyl groups then form Ca-bridges between pectin molecules. We have shown, by using the pectinesterases of pea sprouts, that the enzymes catalyzed not only the hydrolysis of the methoxyl groups of pectin molecules, but also a transacylation reaction of the galacturonic acyl groups from methanol to other hydroxyl groups of pectin. The latter reaction results in the formation of new ester linkages between pectin molecules, which also contributes to the firming of the tissue. The pectinesterases have been separated into four isozymes, PE1, PE2, PE3, and PE5. The isozymes exhibited similar transacylation activity, with the exception of PE5 that did not catalyze transacylation.

Oxidative stress and inflammation are related to several chronic diseases including cancer. Actinidia callosa var. ephippioides (ACE) is a special folk medicinal plant from Taiwan. The aim of this study is to evaluate the antioxidant, anti-inflammatory, and antiproliferative activities of the methanol extract and fractions from the stem of ACE. Trolox Equivalent Antioxidant Capacity (TEAC), 1,1-Diphenyl-2-picrylhydrazyl (DPPH) scavenging activity, total phenolic content, flavonoid content, inhibition on nitric oxide (NO) productions by LPS-induced RAW264.7 cell, and on lung cancer cell proliferation were employed. Among all fractions, ethyl-acetate fraction (EA-ACE) showed higher TEAC, DPPH radical scavenging activities, polyphenol and flavonoid contents, respectively. EA-ACE also decreased the LPS-induced NO production and expressions of inducible nitric-oxide synthase (iNOS) in RAW264.7 cells. EA-ACE had the highest antiproliferative activity with an IC50 (The concentrations required for inhibition of 50% of cell viability) of 469.17 ± 3.59 μg/mL. Catechin also had good effects in the antioxidant and anti-inflammatory activities. Catechin might be an important bioactive compound in the stem of ACE. The above experimental data indicated that the stem of ACE is a potent antioxidant medicinal plant, and such efficacy may be mainly attributed to its polyphenolic compounds.

We have investigated the anti-inflammatory effects of Cinnamomum cassia constituents (cinnamic aldehyde, cinnamic alcohol, cinnamic acid, and coumarin) using lipopolysaccharide (LPS)-stimulated mouse macrophage (RAW264.7) and carrageenan (Carr)-induced mouse paw edema model. When RAW264.7 macrophages were treated with cinnamic aldehyde together with LPS, a significant concentration-dependent inhibition of nitric oxide (NO), tumor necrosis factor (TNF-α), and prostaglandin E2 (PGE2) levels productions were detected. Western blotting revealed that cinnamic aldehyde blocked protein expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear transcription factor kappa B (NF-κB), and IκBα, significantly. In the anti-inflammatory test, cinnamic aldehyde decreased the paw edema after Carr administration, and increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in the paw tissue. We also demonstrated cinnamic aldehyde attenuated the malondialdehyde (MDA) level and myeloperoxidase (MPO) activity in the edema paw after Carr injection. Cinnamic aldehyde decreased the NO, TNF-α, and PGE2levels on the serum level after Carr injection. Western blotting revealed that cinnamic aldehyde decreased Carr-induced iNOS, COX-2, and NF-κB expressions in the edema paw. These findings demonstrated that cinnamic aldehyde has excellent anti-inflammatory activities and thus has great potential to be used as a source for natural health products.

In this study, we have investigated the anti-inflammatory effects of imperatorin, a compound isolated from the roots of Glehnia littoralis, using a lipopolysaccharide (LPS)-stimulated mouse macrophage (RAW264.7) in vitro and a carrageenan (Carr)-induced mouse paw edema model in vivo. When RAW264.7 macrophages were treated with imperatorin together with LPS, a significant concentration-dependent inhibition of NO production was detected. Western blotting revealed that imperatorin blocked the protein expression of iNOS and cyclooxygenase-2 (COX-2) in LPS-stimulated RAW264.7 macrophages significantly. In the anti-inflammatory test, imperatorin decreased the paw edema at 4 and 5 h after Carr administration and increased the activities of catalase, superoxide dismutase, and glutathione peroxidase in paw edema. We also demonstrated that imperatorin significantly attenuated the malondialdehyde level in the edema paw at the fifth hour after Carr injection. Imperatorin decreased the NO and tumor necrosis factor and prostaglandin E2 levels on serum at 5 h after Carr injection. Western blotting revealed that imperatorin decreased Carr-induced iNOS and COX-2 expressions at 5 h in edema paw. An intraperitoneal injection treatment with imperatorin also diminished neutrophil infiltration into sites of inflammation as did indomethacin. The results suggested that imperatorin had anti-inflammatory effects in LPS-stimulated RAW 264.7 cells and Carr-injected mice, respectively. In addition, inhibition of elevated iNOS and COX-2 protein expression as well as neutrophil infiltration of Carr-injected paws may be involved in the beneficial effects of imperatorin.

This study investigated the protective effect of the aqueous extract of Flemingia macrophylla (AFM) against hepatic injury induced by CCl4. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected as biomarkers in the blood to indicate hepatic injury. Product of lipid peroxidation (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and reduced glutathione (GSH) contents were evaluated for oxidative stress in hepatic injury. Moreover, histopathological observation was assayed for the degree of hepatic injury. After oral administration of AFM, 0.5 g/kg and 1.0 g/kg doses significantly decreased ALT and AST, attenuated the histopathology of hepatic injury, ameliorated oxidative stress in hepatic tissue, and increased the activities of CAT, SOD and GSH-Px. The hepatoprotective effect of daidzein and genistein were consistent to that of AFM. This study demonstrated for the first time that AFM has hepatoprotective effect on acute liver injuries induced by CCl4, and the results suggested that the effect of AFM against CCl4-induced liver damage was related to antioxidant properties.

Asiatic acid (AA), a pentacyclic triterpene compound in the medicinal plantCentella asiatica, was evaluated for antinociceptive and anti-inflammatory effects. Treatment of male ICR mice with AA significantly inhibited the numbers of acetic acid-induced writhing responses and the formalin-induced pain in the late phase. In the anti-inflammatory test, AA decreased the paw edema at the 4th and 5th h afterλ-carrageenan (Carr) administration and increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in the liver tissue. AA decreased the nitric oxide (NO), tumor necrosis factor-α(TNF-α), and interleukin-1β(IL-1β) levels on serum level at the 5th h after Carr injection. Western blotting revealed that AA decreased Carr-induced inducible nitric oxide synthase (iNOS), cyclooxygenase (COX-2), and nuclear factor-κB (NF-κB) expressions at the 5th h in the edema paw. An intraperitoneal (i.p.) injection treatment with AA also diminished neutrophil infiltration into sites of inflammation as did indomethacin (Indo). The anti-inflammatory mechanisms of AA might be related to the decrease in the level of MDA, iNOS, COX-2, and NF-κB in the edema paw via increasing the activities of CAT, SOD, and GPx in the liver.

Isocitrate was cleaved into succinate and glyoxylate by isocitrate lyase (ICL) in the glyoxylate cycle. We used lactate dehydrogenase as an ancillary enzyme to further metabolize the glyoxylate to glycolate in the presence of NADH. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 2,6-dichlorophenol-indolphenol (DCPIP) were used in the coupling reactions for detecting ICL activity after electrophoresis on either native or sodium dodecyl sulfate (SDS) polyacrylamide gels. This fast and sensitive method can be used in the process of ICL enzyme purification and characterization.

Ergostatrien-3beta-ol (ST1), an active and major ingredient from Antrodia camphorata (AC) submerged whole broth was evaluated for the analgesic and anti-inflammatory effects. Treatment of male imprinting control region (ICR) mice with ST1 (1, 5, and 10 mg/kg) significantly inhibited the numbers of acetic-acid-induced writhing response in 10 min. Also, our result showed that ST1 (10 mg/kg) significantly inhibited the formalin-induced pain in the late phase (p0.001). In the anti-inflammatory test, ST1 (10 mg/kg) decreased the paw edema at 4 and 5 h after lambda-carrageenin (Carr) administration and increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in the liver tissue. We also demonstrated that ST1 significantly attenuated the malondialdehyde (MDA) level in the edema paw at 5 h after Carr injection. ST1 (1, 5, and 10 mg/kg) decreased the nitric oxide (NO) levels on both the edema paw and serum level at 5 h after Carr injection. Also, ST1 (5 and 10 mg/kg) diminished the serum tumor necrosis factor (TNF-alpha) at 5 h after Carr injection. Western blotting revealed that ST1 (10 mg/kg) decreased Carr-induced inducible nitric oxide synthase (iNOS), and cycloxyclase (COX-2) expressions at 5 h in the edema paw. An intraperitoneal (ip) injection treatment with ST1 also diminished neutrophil infiltration into sites of inflammation, as did indomethacin (Indo). The anti-inflammatory mechanisms of ST1 might be related to the decrease in the level of MDA, iNOS, and COX-2 in the edema paw via increasing the activities of CAT, SOD, and GPx in the liver through the suppression of TNF-alpha and NO.

Anredera cordifolia (Ten.) Steenis, or the synonymous name of Boussingaultia baselloides or Boussingaultia gracilis var. pseudobaselloides, is a South American species of ornamental succulent vine, commonly known as the madeira-vine. The fresh leaves of madeira-vine are frequently used as vegetables. A. cordifolia is an evergreen climber that grows from fleshy rhizomes. The rhizome contained one major (23kDa) protein band under non-reducing condition in the SDS-PAGE. The first 15 amino acids in the N-terminal region of the major protein band (23kDa), named tentatively ancordin, were KDDLLVLDIGGNPVV which were highly homologous to sequences of winged bean seed protein ws-1, Medicago truncatula proteinase inhibitor, soybean trypsin inhibitor, and sporamin. By using activity stains, the ancordin showed trypsin inhibitory activity in the SDS-PAGE gel which was found not only in rhizomes but also in aerial tubers, but few in fresh leaves. The crude extracts from rhizomes of madeira-vine were directly loaded onto trypsin-Sepharose 4B affinity column. After washing with 100mM Tris-HCl buffer (pH 7.9) containing 100mM NaCl, the ancordin was eluted directly by 0.2M KC1-HC1 buffer (pH 2.0). In calculation, the purified protein exhibited 0.0428mug trypsin inhibition/mug ancordin (corresponding to 0.53 unit of TPCK-treated trypsin inhibited/mug ancordin). The purified ancordin was used to evaluate the nitric oxide productions in RAW264.7 cells in the presence of polymyxin B (poly B, 50microg/ml) to eliminate the lipopolysaccharide (LPS) contaminations. It was found that ancordin (1.25-5microg/ml) could dose-dependently (R=0.954) stimulate the nitric oxide (NO) productions (expressed as nitrite concentrations) in RAW264.7 cells without significant cytotoxicity, and kept the similar effects in NO production in 6.25microg/ml ancordin.

Pectinesterase (PE), from commercial orange peels or ammonium sulfate fractionation (50-80% saturation) of pea pods, was detected on polyacrylamide gels after native acidic polyacrylamide gel electrophoresis (PAGE) or sodium dodecyl sulfate (SDS)-PAGE by using the synthetic substrate beta-naphthyl acetate (beta-NA). The release of beta-naphthol (at 322 nm) from beta-NA was proportional to PE activity. The PE activity bands on polyacrylamide gels after native acidic PAGE or SDS-PAGE were stained with a combination of tetrazotized o-dianisidine and beta-NA. This fast and sensitive method can be used for enzyme purification and characterization.

A new C19 gamma-lactone, cinnakotolactone (1), along with a known analogue, isolinderanolide B (2), were isolated from the n-hexane layer of the leaf extracts of Cinnamomum kotoense. Their structures were elucidated on the basis of spectroscopic analysis. The anti-proliferation activities of 1 and 2 were evaluated against human HT29 and MCF-7 cancer cell lines, and their IC50 values ranged from 3.3 +/- 0.3 to 25.8 +/- 5.3 microM.

The purified dioscorin from yam (Dioscorea alata L. cv. Tainong 1) tuber was previously reported (Hsu et al., 2002. J. Agric. Food Chem., 50, 6109-6113). In this report, we evaluated its immunomodulatory ability in vitro in the presence of polymyxin B (50 microg/ml) to eliminate lipopolysaccharide (LPS) contamination. Dioscorin (5-100 microg/ml) was able to stimulate nitric oxide production (expressed as nitrite concentrations) in RAW264.7 cells. The stimulation index on the phagocytosis of RAW264.7 cells against E. coli and the oxidative burst (determined by the intensity of rhodamine fluorescence) of RAW264.7 cells were both enhanced by different concentrations of dioscorin (5-100 microg/ml). The cytokine production, including IL-6, TNF-alpha, and IL-1beta in dioscorin-treated RAW264.7 cells or human monocytes, was measured in the cultured medium. Dioscorin (5-100 microg/ml) was found able to induce IL-6, TNF-alpha, and IL-1beta production in RAW264.7 cells and human monocytes. To evaluate the effects of dioscorin on the proliferation of spleen cells from BALB/c mice, phytohemagglutinin (PHA, 2 microg/ml) alone or PHA mixed with different concentrations of dioscorin (10, 25, and 50 microg/ml) was used to treat spleen cells for 24h. The stimulated proliferation index of splenic cells ranged from 1.38- to 1.48-fold of PHA alone for PHA mixed with different concentrations of dioscorin (10, 25, and 50 microg/ml). We suggest that the tuber storage protein of yam dioscorin functions as an immunomodulatory substance.

Attenuation of monoamine oxidase B (MAO-B) activity may provide protection against oxidative neurodegeneration. For this reason, inhibition of MAO-B activity is used as part of the treatment of Parkinson's and Alzheimer's patients. The hook of Uncaria rhynchophylla (Miq.) Jacks. (Rubiaceae) is a traditional Chinese herbal drug that is generally used to treat convulsive disorders. In this study, the fractionation and purification of Uncaria rhynchophylla extracts using a bioguided assay isolated two known compounds, (+)-catechin and (−)-epicatechin. The compounds inhibited MAO-B, as measured by an assay of rat brain MAO-B separated by electrophoresis on a 7.5% native polyacrylamide gel. The IC 50 values of (+)-catechin and (−)-epicatechin were 88.6 and 58.9 μM, respectively, and inhibition occurred in a dose-dependent manner, as measured by the fluorescence method. The Lineweaver–Burk plot revealed K i values for (+)-catechin and (−)-epicatechin of 74 and 21 μM, respectively. This suggests that these two compounds, isolated here for the first time from Uncaria rhynchophylla , might be able to protect against neurodegeneration in vitro, and, therefore, the molecular mechanism deserves further study. This finding may also increase interest in the health benefits of Uncaria rhynchophylla .

To determine the effects of chromium (Cr) supplementations on oxidative stress of type 2 diabetes and euglycemic (EU) subjects, adult having HbA(1C) values of6.0% (EU), 6.8-8.5% (mildly hyperglycemic, MH), and8.5% (severely hyperglycemic, SH) were supplemented for 6 months with 1000 microg/day of Cr (as Cr yeast) or with a placebo. In the beginning, the levels of the plasma Cr in the MH and SH groups were 25-30% lower than those of the EU subjects. The values of thiobarbituric acid reactive substances (TBARS) and total antioxidative status (TAS) of the MH and SH groups were significantly higher than those of the EU ones. Following supplementations, the levels of plasma TBARS in the Cr groups of MH and SH groups were significantly decreased (the inverse was found in the EU) and showed no significant changes in the placebo group. The levels of plasma TAS in the Cr groups of EU and MH were significantly decreased (the inverse was found in the SH) and showed no significant changes in the placebo group. No significant difference was found in the antioxidant enzyme (superoxide dismutase, glutathione peroxidase, catalase) activities during supplementations. These data suggest that Cr supplementation was an effective treatment strategy to minimize increased oxidative stress in type 2 diabetes mellitus patients whose HbA(1C) level was8.5%, and the Cr in EU groups might act as a prooxidant.

Solutions of 100 mL of 1% commercial pectin each with a different degree of esterification (DE), DE94, DE65, and DE25, were reacted with 100 mL of 2 M alkaline hydroxylamine (pH 12.0) at room temperature for 4 or 18 h. These pectin hydroxamic acids (PHAs; DE94T4, DE94T18, DE65T4, and DE25T4) were used to test the inhibitory activities against semicarbazide-sensitive amine oxidase (SSAO) and angiotensin-converting enzyme (ACE). Compared to different DE pectins (DE94, DE65, and DE25), the PHAs of DE94T4, DE94T18, DE65T4, and DE25T4 showed different inhibition activities against SSAO or ACE. Commercial pectins with different DE values showed negligible SSAO or ACE inhibitions. The order of SSAO inhibition was DE65T4 > DE94T18 approximately DE25T4 >> DE94T4. However, the order of ACE inhibition was DE94T4 > DE94T18 >> DE65T4 > DE25T4. The SSAO activity staining or ACE-hydrolyzed products on TLC chromatogram also confirmed the inhibitory activities of PHAs against SSAO or ACE.

The potato (Solanum tuberosum L.) tuber storage protein, patatin, was purified to homogeneity with a molecular mass of 45 kDa. The purified patatin showed antioxidant or antiradical activity by a series of in vitro tests, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical (half-inhibition concentration, IC(50), was 0.582 mg/mL) scavenging activity assays, anti-human low-density lipoprotein peroxidation tests, and protections against hydroxyl radical-mediated DNA damages and peroxynitrite-mediated dihydrorhodamine 123 oxidations. Using electron paramagnetic resonance (EPR) spectrometry for hydroxyl radical detections, it was found that the intensities of the EPR signal were decreased by the increased amounts of patatin added (IC(50) was 0.775 mg/mL). Through modifications of patatin by iodoacetamide or N-bromosuccinimide, it was found that the antiradical activities of modified patatin against DPPH or hydroxyl radicals were decreased. It was suggested that cysteine and tryptophan residues in patatin might contribute to its antioxidant activities against radicals.

Male adult Wistar rats were randomly divided into six groups in a 2 x 3 factorial design and fed diets containing different levels of Tremella fuciformis Berk (TFB) dietary fiber (0, 5, or 10%) and 1 g of cholesterol/100 g of diet with or without 0.7% Nebacitin for 4 weeks. TFB contained 6.2% soluble dietary fiber and 57.3% insoluble dietary fiber. The results showed that the serum LDL-cholesterol, hepatic total cholesterol, and triglyceride levels were significantly decreased (P < 0.05) in the rats fed diets with TFB content with or without Nebacitin. However, the serum total cholesterol, VLDL-cholesterol, and triglyceride levels were significantly decreased (P < 0.05) by Nebacitin. In feces, the presence of TFB (T5, T10, AT5, and AT10) in the diet significantly increased the total neutral steroids and bile acid excretions and undigested fiber concentrations as compared to T0 or AT0. In the small intestine, the Nebacitin diets increased the weights of both cecum and colon-rectum contents and lowered short-chain fatty acid (SCFA) concentrations of serum and cecal contents more than no Nebacitin diets did. It was suggested that the hypocholesterolemic effect of TFB dietary fiber may be mediated by the increase in fecal neutral steroids and total bile acids excretion and the increase in SCFA productions. The TFB edible mushroom dietary supplement altered the intestinal physiology of the rats.

Monoamine oxidase type B (MAO-B) activity and free radicals are elevated in certain neurological diseases. Four natural flavonoids, quercitrin, isoquercitrin, rutin, and quercetin, were isolated for the first time from the leaves of Melastoma candidum D. Don. They exhibited an inhibitory effect on MAO-B. These potent flavonoids were purified using bioassay-guided fractionation and were separated by Diaion, Sephadex LH-20, and MCI CHP20P columns. The IC(50) values of the four potent flavonoids, quercitrin, isoquercitrin, rutin, and quercetin on monoamine oxidase were 19.06, 11.64, 3.89, and 10.89 microM and enzyme kinetics analysis revealed apparent inhibition constants (K(i)) of 21.01, 2.72, 1.83, and 7.95 microM, respectively, on the substrate, benzylamine. The four potent compounds also exhibited hydroxyl radical scavenging activity as determined using a spin trapping electron spin resonance method. This suggests that the four flavonoids from M. candidum possess both MAO-B inhibitory and free radical scavenging activities. These important properties may be used for preventing some neurodegenerative diseases in the future.

Dioscorin, the storage protein of yam (Dioscorea batatas Decne) tuber (which is different from dioscorine found in tubers of Dioscorea hirsuta), was purified to homogeneity after DE-52 ion exchange column according to the methods of Hou et al. (J. Agric. Food Chem. 1999, 47, 2168-2172). A single band of 32 kDa dioscorin was obtained on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel with 2-mercaptoethanol treatment. This purified dioscorin was shown by spectrophotometric method to have scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical in a pH-dependent manner. There is a positive correlation between scavenging effects against DPPH (8-46%) and amounts of 32 kDa dioscorin (5.97-47.80 nmol) added in Tris-HCl buffer (pH 7.9), which are comparable to those of glutathione at the same concentrations. Using electron paramagnetic resonance (EPR) spectrometry for DPPH radical detection, it was found that the intensities of the EPR signal were decreased by 28.6 and 57 nmol of 32 kDa dioscorin in Tris-HCl buffer (pH 7.9) more than in distilled water compared to controls. EPR spectrometry was also used for hydroxyl radical detection. It was found that 32 kDa dioscorin could capture hydroxyl radical, and the intensities of the EPR signal were significantly decreased dose-dependently by 1.79-14.32 nmol of 32 kDa dioscorin (r = 0.975) compared to the control. It is suggested that 32 kDa dioscorin, the storage protein of yam tuber, may play a role as antioxidant in tubers and may be beneficial for health when people take it as a food additive or consume yam tubers.