Robin Y.-Y. Chiou

Neuroprotective Effects of Resveratrol on MPTP-Induced Neuron Loss Mediated by Free Radical Scavenging

Resveratrol is a natural polyphenol and possesses many biological functions such as anti-inflammatory activity and protection against atherosclerosis and myocardial infraction. Parkinsons disease is a common progressive neurodegenerative disease. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is the most useful neurotoxin to induce Parkinsonism. The present study was carried out to elucidate the neuroprotective effect and possible mechanism of resveratrol on MPTP-induced striatal neuron loss. Sixty adult Balb/c mice were divided into four groups: sham operation, MPTP treatment (30 mg/kg, i.p.), MPTP combined with resveratrol administration (20 mg/kg, i.v.), and resveratrol treatment alone. Microdialysis and high-performance liquid chromatography were used to analyze dihydroxybenzoic acid (DHBA) that reflected the hydroxyl radical level. In the present study, we found MPTP chronic administration significantly induced motor coordination impairment in mice. After MPTP administration, the hydroxyl radical levels in substantia nigra were also significantly elevated and animals displayed severe neuronal loss. Resveratrol administration significantly protected mice from MPTP-induced motor coordination impairment, hydroxyl radical overloading, and neuronal loss. Our results demonstrated that resveratrol could elicit neuroprotective effects on MPTP-induced Parkinsonism through free radical scavenging.

Polymerase Chain Reaction-Mediated Characterization of Molds Belonging to the Aspergillus flavus Group and Detection of Aspergillus parasiticus in Peanut Kernels by a Multiplex Polymerase Chain Reaction

The Aspergillus flavus group covers species of A. flavus and Aspergillus parasiticus as aflatoxin producers and Aspergillus oryzae and Aspergillus sojae as koji molds. Genetic similarity among these species is high, and aflatoxin production of a culture may be affected by cultivation conditions and substrate composition. Therefore, a polymerase chain reaction (PCR)-mediated method of detecting the aflatoxin-synthesizing genes to indicate the degree of risk a genotype has of being a phenotypic producer was demonstrated. In this study, 19 strains of the A. flavus group, including A. flavus, A. parasiticus, A. oryzae, A. sojae, and one Aspergillus niger, were subjected to PCR testing in an attempt to detect four genes, encoding for norsolorinic acid reductase (nor-1), versicolorin A dehydrogenase (ver-1), sterigmatocystin O-methyltransferase (omt-1), and a regulatory protein (apa-2), involved in aflatoxin biosynthesis. Concurrently, the strains were cultivated in yeast-malt (YM) broth for aflatoxin detection. Fifteen strains were shown to possess the four target DNA fragments. With regard to aflatoxigenicity, all seven aflatoxigenic strains possessed the four DNA fragments, and five strains bearing less than the four DNA fragments did not produce aflatoxin. When peanut kernels were artificially contaminated with A. parasiticus and A. niger for 7 days, the contaminant DNA was extractable from a piece of cotyledon (ca. 100 mg), and when subjected to multiplex PCR testing using the four pairs of primers coding for the above genes, they were successfully detected. The target DNA fragments were detected in the kernels infected with A. parasiticus, and none was detected in the sound (uninoculated) kernels or in the kernels infected with A. niger.

Arachidin-1, a peanut stilbenoid, induces programmed cell death in human leukemia HL-60 cells.

The stilbenoids, arachidin-1 (Ara-1), arachidin-3, isopentadienylresveratrol, and resveratrol, have been isolated from germinating peanut kernels and characterized as antioxidant and anti-inflammatory agents. Resveratrol possesses anticancer activity, and studies have indicated that it induces programmed cell death (PCD) in human leukemia HL-60 cells. In this study, the anticancer activity of these stilbenoids was determined in HL-60 cells. Ara-1 had the highest efficacy in inducing PCD in HL-60 cells, with an approximately 4-fold lower EC50 than resveratrol. Ara-1 treatment caused mitochondrial membrane damage, activation of caspases, and nuclear translocation of apoptosis-inducing factor, resulting in chromosome degradation and cell death. Therefore, Ara-1 induces PCD in HL-60 cells through caspase-dependent and caspase-independent pathways. Ara-1 demonstrates its efficacy as an anticancer agent by inducing caspase-independent cell death, which is an alternative death pathway of cancer cells with mutations in key apoptotic genes. These findings indicate the merits of screening other peanut stilbenoids for anticancer activity.

Structural identification and bioactivities of red-violet pigments present in Basella alba fruits.

Mature Basella alba L. fruit, with dark blue skin and deep red-violet flesh, is a potential source of natural colorants. Its pigment components and bioactivities deserve particular attention and investigation. In this study, fruit flesh was extracted with 80% methanol (containing 0.2% formic acid) and subjected to solid-phase extraction, semipreparative HPLC isolation, mass spectrophotometric analysis, and structural elucidation. The major red pigment was identified as gomphrenin I. Its quantity increased with the increase of fruit maturity. The gomphrenin I extract yield from ripe fruits was 36.1 mg/100 g of fresh weight. In addition to gomphrenin I, betanidin-dihexose and isobetanidin-dihexose were also detected. The antioxidant activities of gomphrenin I determined by Trolox equivalent antioxidant capacity (TEAC), α,α-diphenyl-β-picrylhydrazyl (DPPH) radical scavenging activity, reducing power, and antioxidative capacity assays were equivalent to 534 μM Trolox, 103 μM butylated hydroxytoluene (BHT), 129 μM ascorbic acid, and 68 μM BHT at 180, 23, 45, and 181 μM, respectively. The anti-inflammatory function was tested at concentrations of 25, 50, and 100 μM in murine macrophages stimulated with lipopolysaccharide (LPS). The results revealed that gomphrenin I suppressed LPS-induced nitric oxide (NO) production in a dose-dependent manner and decreased PGE(2) and IL-1β secretions at the highest concentration tested. The transcriptional inhibitory activities of gomphrenin I on the expression of inflammatory genes encoding iNOS, COX-2, IL-1β, TNF-α, and IL-6 were also observed. It is of merit to identify gomphrenin I as a principal pigment of B. alba fruits and as a potent antioxidant and inflammatory inhibitor. These findings suggest that B. alba fruit is a rich source of betalains and has value-added potential for use in the development of food colorants and nutraceuticals.

Modified Fast Procedure for the Detection and Screening of Antiglycative Phytochemicals

In an attempt to elevate temperature to facilitate glycation, a nonenzymatic reaction by incubation of bovine serum albumin (BSA) and fructose at 50 °C for 24 h has been developed. As conducted and compared to a routine procedure by incubation of BSA and fructose at 37 °C for 168 h, the reactant fluorescence intensities and SDS-PAGE-detected glycated BSA quantities produced by both test temperatures increased with time of incubation. As the Amadori products and α-dicarbonyl compounds during incubation were quantified, both quantities produced at each temperature also increased with an increase of time of incubation, and their trends of changes at both temperatures were similar. In practical application for the detection and screening of the antiglycative phytochemicals, each of 20 peanut root extracts was introduced to a series of BSA-fructose solutions and incubated at 37 and 50 °C for 168 and 24 h, correspondingly. All extracts exhibited notable activities and varied depending on peanut origins. Pair comparison of the resultant antiglycative activities determined at 37 and 50 °C showed that both determined activities for each peanut root extract deviated limitedly. As further analyzed, SDS-PAGE-detected glycated BSA quantities formed at 50 °C were closely proportional to the antiglycative activities determined on the basis of their fluorescence intensities. It is of merit to demonstrate that fluorescence-based determination of BSA-fructose reactant after incubation at 50 °C for 24 h is practical and time-saving in the detection and screening of antiglycative phytochemicals.

Survival of Staphylococcus aureus and Escherichia coli as Affected by Ethanol and NaCl

Growth of three strains of Staphylococcus aureus and two strains of Escherichia coli on nutrient agar (NA) supplemented with ethanol and NaCl was investigated. S. aureus did not grow on NA containing > or =10% ethanol (wt/wt) combined with > or =0% NaCl (wt/wt), or 7.5% ethanol combined with 7.5% NaCl. Neither E. coli nor E. coli O157:H7 grew on NA containing > or =7.5% ethanol combined with > or =0% NaCl, 5% ethanol combined with > or =2.5% NaCl, or > or =5% NaCl combined with > or =0% ethanol. It is apparent that NaCl enhanced the inhibitory effect of ethanol on growth of S. aureus and E. coli When cells were suspended in nutrient broth containing 12.5, 20, or 40% ethanol combined with NaCl, viable cells decreased with an increase of ethanol concentration. Ethanol sensitivity among strains and between genera varied in a limited range. When the cells were exposed to 20% ethanol in combination with 5% NaCl, S. aureus and E. coli lost viability after 30 and 10 min, respectively. When treated with 40% ethanol combined with > or =0% NaCl, all test strains lost viability within 5 min.

Oral Administration of Trapa taiwanensis Nakai Fruit Skin Extracts Conferring Hepatoprotection from CCl4-Caused Injury

As a folk medicine, the hot-water infusion of water caltrop fruits has been used to protect the liver. In this study, the outer skins of mature water caltrop fruits ( Trapa taiwanensis Nakai) were removed, forced-air-dried, pulverized, and subjected to extraction with hot water, and the infusion was lyophilized and pulverized to prepare a hot water extract of T. taiwanensis (HWETT). HWETT was subjected to assays of α,α-diphenyl-β-picrylhydrazyl scavenging activity, reducing power, Trolox equivalent antioxidant capacity, and antioxidative potency, and all determinations showed HWETT to be a potent antioxidant. As further analyzed with LC-MS, two major HPLC-detected components were elucidated as gallic acid and ellagic acid. Hepatoprotective activity of HWETT was assessed with Sprague-Dawley male rats by oral administration. Six groups of rats (n = 8 for each) were respectively treated, namely, control, CCl(4) (20% CCl(4)/olive oil by 2.0 mL/kg bw), CCl(4) and Silymarin (200 mg/kg bw), CCl(4) and low HWETT dose (12.5 mg/kg bw), CCl(4) and medium HWETT dose (25 mg/kg bw), and CCl(4) and high HWETT dose (125 mg/kg bw). After 8 weeks, all animals were fasted for an additional day and sacrificed to collect blood, liver, and kidney for analyses. Histopathological examinations showed that oral administrations with Silymarin and HWETT were effective in protecting the liver from CCl(4)-caused fatty change. Oral administration of HWETT at 125 mg/kg bw was more effective than was Silymarin at 200 mg/kg bw. On biochemical analyses, oral administrations with HWETT at medium and high doses were effective (p < 0.05) in lowering CCl(4)-caused increases of alanine aminotransferase and aspartate aminotransferase activities. It is of merit to demonstrate HWETT as a potent source of antioxidants and hepatoprotective agents.

Identification and mode of action of 5-hydroxymethyl-2-furfural (5-hmf) and 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (MTCA) as potent xanthine oxidase inhibitors in vinegars.

Vinegars have been used as an alternative remedy for treating gout, but the scientific basis remains to be elucidated. In this study, seven commercial vinegars and one laboratory-prepared red-koji vinegar were evaluated for the inhibitory activity of xanthine oxidase (XO), a critical enzyme catalyzing uric acid formation. Red-koji vinegar exhibited potent xanthine oxidase inhibitory (XOI) activity and was used for isolating active compounds. The substances under two peaks with XOI activity from HPLC were identified as 5-hydroxymethyl-2-furfural (5-HMF) and 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (MTCA), by LC-MS-MS and NMR. The XO half-maximal inhibitory concentrations (IC(50)) of 5-HMF and MTCA were 168 and 860 μg/mL, respectively. In further mode-of-action analysis, the inhibitory mechanism of each compound was elucidated at the IC(50) level in the presence of various concentrations of xanthine as the substrate. The following Michaelis-Menten kinetics analysis of XO inhibition revealed uncompetitive and competitive patterns for 5-HMF and MTCA, respectively.

Supplementary health benefits of soy aglycons of isoflavone by improvement of serum biochemical attributes, enhancement of liver antioxidative capacities and protection of vaginal epithelium of ovariectomized rats

BackgroundIn the literature, supplement of soy aglycons of isoflavone as estrogen agonists in improvement of serum biochemical attributes, liver antioxidative capacities and vaginal epithelium protection has been meagerly investigated. In this study, ovariectomized (OVX) rats were used as an animal model to simulate post-menopausal status. Supplementary health benefits of soy aglycons of isoflavone (SAI) on improvement of growth and serum biochemical attributes, enhancement of liver antioxidation-related capacities and protection of vaginal epithelium of the OVX rats were assessed.MethodsAs an in vivo study, 30 OVX Sprague-Dawley rats were distributed into OVX (positive control), OVX/LSAI (low SAI group – supplemented with 0.0135% SAI being equivalent to 80 mg per day for a 60 Kg-human), and OVX/HSAI (high SAI group – supplemented with 0.027% SAI) and 10 rats with sham operation as negative control fed with basal diet.ResultsThe average daily gain (ADG), feed intake and feed/gain ratio were higher for the OVX groups than the sham group (P < 0.05). Serum isoflavone concentrations of the OVX rats were increased by SAI supplementation. In comparison, significantly lower serum cholesterol and LDL (low-density lipoprotein) levels, and higher HDL (high-density lipoprotein) levels were detected for the rats of OVX/HSAI group (P < 0.05). SAI supplementation also increased iron chelating ability and decreased values of TBARS (thiobarbituric acid-reactive substance) (P < 0.05) of liver extracts. Liver catalase activity and total antioxidative activity (trolox equivalency) were enhanced by HSAI supplementation (P < 0.05). Decrease of vagina epithelial cellular linings of the OVX rats were noticeably improved by dietary supplementation with SAI.ConclusionDiets supplemented with soy aglycons of isoflavone have conferred health benefits to the OVX rats, in comparison to the sham rats fed with basal diet, by detection of higher serum isoflavone concentrations, significantly lower contents of serum cholesterol and LDL, and higher contents of serum HDL, increased iron chelating ability, lower contents of TBARS (thiobarbituric acid-reactive substance) and enhanced catalase and total antioxidative (as trolox equivalency) activities of the liver extracts, and protection of the epithelial cellular linings of vagina in the former rather than in the latter. This evidences that estrogen-agonist chemoprevention of menopausal-related cardiovascular diseases, decreased liver antioxidative capacities and epithelial degeneration of vagina could be achieved by dietary supplementation with soy aglycons of isoflavone.

GC-MS determined distribution of urinary equol producers as affected by age, gender, and repeated ingestions of soymilk.

Status of equol excretion is likely a reflection of incidence risk of sex-hormone related diseases and deserves research attention. In this study, urine samples collected from 182 volunteers after soymilk ingestion were subjected to equol quantification using GC-MS. As categorized into male, female, and both genders and each category further classified into 6 age (year) categories, namely, <20, 21 to 30, 31 to 40, 41 to 50, >51 and overall, a trend indicating that the younger age the higher equol producer ratio was observed. For the >51 subjects, the corresponding producer ratios of males and females were 35.3% and 50.0%, respectively. Among the volunteers, 20 nonproducers were further recruited to ingest 1000 mL soymilk weekly for 16 wk and urines were analyzed bi-weekly. As resulted, 8 of 20 nonproducers were induced to become equol producers. The observed change of nonproducers to equol producers induced by repeating ingestions of soymilk is of merit from the viewpoint of healthcare.