Evaluation of the Feasibility of In Vitro Metabolic Interruption of Trimethylamine with Resveratrol Butyrate Esters and Its Purified Monomers.

Resveratrol (RSV), obtained from dietary sources, has been shown to reduce trimethylamine oxide (TMAO) levels in humans, and much research indicates that TMAO is recognized as a risk factor for cardiovascular disease. Therefore, this study investigated the effects of RSV and RSV-butyrate esters (RBE) on the proliferation of co-cultured bacteria and HepG2 cell lines, respectively, and also investigated the changes in trimethylamine (TMA) and TMOA content in the medium and flavin-containing monooxygenase-3 (FMO3) gene expression. This study revealed that 50 µg/mL of RBE could increase the population percentage of Bifidobacterium longum at a rate of 53%, while the rate was 48% for Clostridium asparagiforme. In contrast, co-cultivation of the two bacterial strains effectively reduced TMA levels from 561 ppm to 449 ppm. In addition, regarding TMA-induced HepG2 cell lines, treatment with 50 µM each of RBE, 3,4'-di-O-butanoylresveratrol (ED2), and 3-O-butanoylresveratrol (ED4) significantly reduced FMO3 gene expression from 2.13 to 0.40-1.40, which would also contribute to the reduction of TMAO content. This study demonstrated the potential of RBE, ED2, and ED4 for regulating TMA metabolism in microbial co-cultures and cell line cultures, which also suggests that the resveratrol derivative might be a daily dietary supplement that will be beneficial for health promotion in the future.

Cardiovascular risk of dietary trimethylamine oxide precursors and the therapeutic potential of resveratrol and its derivatives.

Overall diet, lifestyle choices, genetic predisposition, and other underlying health conditions may contribute to higher trimethylamine N-oxide (TMAO) levels and increased cardiovascular risk. This review explores the potential therapeutic ability of RSV to protect against cardiovascular diseases (CVD) and affect TMAO levels. This review considers recent studies on the association of TMAO with CVD. It also examines the sources, mechanisms, and metabolism of TMAO along with TMAO-induced cardiovascular events. Plant polyphenolic compounds, including resveratrol (RSV), and their cardioprotective mechanism of regulating TMAO levels and modifying gut microbiota are also discussed here. RSV's salient features and bioactive properties in reducing CVD have been evaluated. The close relationship between TMAO and CVD is clearly understood from currently available data, making it a potent biomarker for CVD. Precise investigation, including clinical trials, must be performed to understand RSV's mechanism, dose, effects, and derivatives as a cardioprotectant agent.

Sources, Degradation, Ingestion and Effects of Microplastics on Humans: A Review.

Celluloid, the predecessor to plastic, was synthesized in 1869, and due to technological advancements, plastic products appear to be ubiquitous in daily life. The massive production, rampant usage, and inadequate disposal of plastic products have led to severe environmental pollution. Consequently, reducing the employment of plastic has emerged as a pressing concern for governments globally. This review explores microplastics, including their origins, absorption, and harmful effects on the environment and humans. Several methods exist for breaking down plastics, including thermal, mechanical, light, catalytic, and biological processes. Despite these methods, microplastics (MPs, between 1 and 5 mm in size) continue to be produced during degradation. Acknowledging the significant threat that MPs pose to the environment and human health is imperative. This form of pollution is pervasive in the air and food and infiltrates our bodies through ingestion, inhalation, or skin contact. It is essential to assess the potential hazards that MPs can introduce. There is evidence suggesting that MPs may have negative impacts on different areas of human health. These include the respiratory, gastrointestinal, immune, nervous, and reproductive systems, the liver and organs, the skin, and even the placenta and placental barrier. It is encouraging to see that most of the countries have taken steps to regulate plastic particles. These measures aim to reduce plastic usage, which is essential today. At the same time, this review summarizes the degradation mechanism of plastics, their impact on human health, and plastic reduction policies worldwide. It provides valuable information for future research on MPs and regulatory development.

Investigation of the optimal production conditions for egg white hydrolysates and physicochemical characteristics.

This study aimed to investigate the potential of egg white protein hydrolysate (EWH) as a functional food by identifying the optimum production conditions for EWH with response surface methodology (the results of the sensory evaluation were considered as an essential quality indicator). At the same time, its physicochemical and biological activity was also evaluated. The optimal economic production conditions were selected: substrate concentration of 12.5%, enzyme content of 7.5%, and hydrolysis time at 100 min. The degree of hydrolysis (DH %) was 13.51%. In addition, to the better acceptance of the evaluation, it also helps to reduce the production cost of the protein hydrolysate, which is beneficial to future processing and applications. The antioxidant capacity experiments showed that EWH has good antioxidant activity, which presents a dose-dependent relationship. Hence, this study provides a theoretical basis for future research and application of EWH for processing applications, including dietary supplementation. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05708-0.

The relationship among knowledge, attitude, and behavior of workers on food safety in Taiwan's Company A.

In recent years, consumer awareness of food safety has been increasing, and food workers with professional food safety knowledge, attitude, and practical implementation are essential for maintaining the quality of food as well as preventing foodborne diseases. This study evaluates the knowledge, attitude, and implementation of food safety practices among employees in a food company in Taiwan. The employees were both who had received food safety knowledge and training and had yet to receive it. Data collection was carried out through 68 questionnaires, and analysis methods, including descriptive statistical analysis and multiple regression analysis, have performed. Before the first stage of education and training, 100% of the employees knew that food factories must conduct regular water quality testing. Still, the error rate for personal hygiene and safety was the highest. The second part has the highest average food safety attitude and the concept of food preservation. The third part of personnel behavior was to avoid cross-contamination. After the second stage of education and training of personnel in food safety knowledge, the attitude and behavior have been greatly improved. The findings of this study can be used as a reference for food suppliers to develop employee education and training.

Development of novel green methods for preparation of lead-free preserved pidan (duck egg).

Pidan, a pickled duck egg, is a traditional Chinese cuisine and generally produced by soaking in metal ion containing strong alkaline solution such as NaOH solution. However, nowadays consumers possess negative perception for using strong alkali in food processing. Therefore, the objective of the current study was to determine the potential of incinerated eggshell powder and alkaline electrolyzed oxidized (EO) water for pidan production rather than harmful NaOH use. This study aims to obtain the optimal physicochemical and sensory qualities of pidan. Various dosing (1-5%) of the incinerated eggshell powder solution or alkaline EO water was used as a basic pickling solution. Duck eggs were pickled at 25-27 °C for 15-30 days with 3 days of an observation interval. Actual commercial process commonly undergoes for 14 days of ripening, after 25 days of picking process with incinerated eggshell powder or EO water. Results showed that physicochemical and sensory attributes of pidan obtained by incinerated eggshell powder solution and alkaline EO water were not significantly different (P < 0.05) from the commercial product. This study reports a cost-effective and green alternative method for pidan processing by replacing costly NaOH without compromising their physico-chemical and sensory attributes.

Resveratrol butyrate esters inhibit lipid biosynthesis in 3T3-L1 cells by AMP-activated protein kinase phosphorylation.

Resveratrol butyrate esters (RBEs), which are novel resveratrol-synthesized derivatives, exhibit increased biological activity. This study elucidated the effect of RBEs on fat metabolism and their anti-obesity characteristics. Their molecular mechanism was investigated in the 3T3-L1 murine preadipocyte cells and adipocytes. RBE doses of < 2 µM did not induce a significant change in the viability of 3T3-L1 adipocytes. After RBEs treatment, intracellular lipid droplet accumulation in 3T3-L1 adipocytes was stimulated by methylisobutylxanthine, dexamethasone, and insulin-containing medium. However, a significant dose-dependent reduction in intracellular lipid levels was observed. The mRNA levels of two adipogenic transcription factors (peroxisome proliferator-activated receptor [PPAR] and CCAAT/enhancer-binding proteins [C/EBP]) and lipogenic proteins (fatty acid-binding protein 4 [FABP4] and fatty acid synthase [FAS]) were significantly attenuated by RBE treatment in both MDI-stimulated and differentiated 3T3-L1 adipocytes. Moreover, the phosphorylation level of adenosine monophosphate-activated protein kinase (AMPK) also dramatically increased in the MDI + RBE-treated group compared to that in the MDI + vehicle-treated group. Collectively, our study provides strong evidence that RBEs inhibit adipogenesis by regulating adipogenic protein expression and increasing the p-AMPK/AMPK ratio. Future studies will be conducted on animal models to validate the application of RBEs as a functional food ingredient in improving human health. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05436-x.

Application of egg white hydrolysate (EWH) to improve frothing functionality of pasteurized liquid egg in large quantity production.

Sterilized Liquid Eggs (SLE) are convenient for the baking process by minimizing the food safety risks of fresh eggs. Although these advantages were encouraging, the thermal effects of the pasteurization process had a negative impact on the functionality of the egg whites, thus making them unattractive to the food industry. Therefore, our previous study found that adding 1-5% egg white hydrolysate (EWH) contributed to the foaminess and stability in SLE. This primary purpose of this study was to confirm the feasibility of applying the optimum concentration of EWH for simultaneous evaluation and shelf life for batch production of SLE. The physical characteristics of the foam were analyzed by adding 1 ± 0.2% of EWH to SLE, and it was found that the foam with 1% EWH had better stability (low drainage), better viscosity, and similar distribution of foam bubbles size in the microstructure. No Salmonella infection has been found during the shelf life of 7 days. In addition, the highest overall acceptability has obtained using the large quantity produced SLE with 1% EWH to produce spoon cookies, followed by sensory evaluation. The cross-sectional height of the cookie and the distribution of holes in the structure were in line with those of the non-sterilized liquid egg white (NSLE). Hence, adding 1% EWH was found to the optimum concentration, which provides good foaming performance and stability of SLE. This study conveys a positive assessment to SLE producers and potential users, as it will increase their profitability economically while meeting the market challenges.

Extraction Optimization and Structural Characteristics of Chitosan from Cuttlefish (S. pharaonis sp.) Bone.

In fish processing, reducing the waste rate and increasing the economic value of products is an important issue for global environmental protection and resource sustainability. It has been discovered that cuttlefish bones can be an excellent resource for producing attractive amounts of chitin and chitosan. Therefore, this study optimized chitosan extraction conditions using response surface methodology (RSM) to establish application conditions suitable for industrial production and reducing environmental impact. In addition, Fourier-transform infrared spectroscopy (FTIR), 1H NMR and scanning electron microscope (SEM) characteristics of extracted chitosan were evaluated. The optimum extraction conditions for chitosan from cuttlebone chitin were 12.5M NaOH, 6 h and 80 °C, and the highest average yield was 56.47%. FTIR spectroscopy, 1H NMR, and SEM identification proved that the chitosan prepared from cuttlefish bone has a unique molecular structure, and the degree of deacetylation of chitosan was about 81.3%. In addition, it was also confirmed that chitosan has significant anti-oxidation and oil-absorbing abilities. This research has successfully transformed the by-products of cuttlefish processing into value-added products. The process not only achieved the recycling and utilization of by-products but also enhanced industrial competitiveness and resource sustainability.

Design of an Integrated Microfluidic Paper-Based Chip and Inspection Machine for the Detection of Mercury in Food with Silver Nanoparticles.

For most of the fast screening test papers for detecting Hg2+, the obtained results are qualitative. This study developed an operation for the µPAD and combined it with the chemical colorimetric method. Silver nanoparticle (AgNP) colloids were adopted as the reactive color reagent to combine and react with the Hg standards on the paper-based chip. Then, the RGB values for the color change were used to establish the standard curve (R2 > 0.99). Subsequently, this detection system was employed for the detection tests of actual samples, and the detected RGB values of the samples were substituted back to the formula to calculate the Hg2+ contents in the food. In this study, the Hg2+ content and recovery rate in commercially available packaged water and edible salts were measured. The research results indicate that a swift, economical, and simple detection method for Hg2+ content in food has been successfully developed.

Separation and Identification of Resveratrol Butyrate Ester Complexes and Their Bioactivity in HepG2 Cell Models.

Resveratrol butyrate ester (RBE) complexes have demonstrated higher antioxidant capacity and anti-fat accumulation activity in previous studies. In this study, silica gel, high-performance liquid chromatography, and 1H nuclear magnetic resonance were used for separation and identification of RBE complex components. With the exception of resveratrol, five different structures of ester derivatives were separated from silica gel: 3,4'-di-O-butanoylresveratrol (ED2, 18.8%), 3-O-butanoylresveratrol (ED4, 35.7%), 4'-O-butanoylresveratrol (ED5, 4.4%), 3,5,4'-tri-O-butanoylresveratrol (ED6, 1.5%), and 3,5-di-O-butanoylresveratrol (ED7, 0.7%). Among the ester derivatives obtained, ED2 and ED4 were the main ester derivatives in the RBE complex. Thus, the cellular antioxidant activities of the RBE mixture, ED2, and ED4 were evaluated. Results showed that the antioxidant capacity of ED2 and ED4 was higher than that of the RBE mixture, demonstrating that the number and position of butyrate esterification sites are related to cell survival rate and antioxidant capacity. This study is the first to report the successful isolation, structural identification, and cellular biological antioxidant activity of RBE complex derivatives, which are key characteristics for the potential practical application of RBE complexes.

Process Optimization of Silver Nanoparticle Synthesis and Its Application in Mercury Detection.

Silver nanoparticles (AgNPs) have stable reactivity and excellent optical absorption properties. They can be applied in various industries, such as environmental protection, biochemical engineering, and analyte monitoring. However, synthesizing AgNPs and determining their appropriate dosage as a coloring substance are difficult tasks. In this study, to optimize the process of AgNP synthesis and obtain a simple detection method for trace mercury in the environment, we evaluate several factors-including the reagent addition sequence, reaction temperature, reaction time, the pH of the solution, and reagent concentration-considering the color intensity and purity of AgNPs as the reaction optimization criteria. The optimal process for AgNP synthesis is as follows: Mix 10 mM of silver nitrate with trisodium citrate in a hot water bath for 10 min; then, add 10 mM of sodium borohydride to produce the AgNPs and keep stirring for 2 h; finally, adjust the pH to 12 to obtain the most stable products. For AgNP-based mercury detection, the calibration curve of mercury over the concentration range of 0.1-2 ppb exhibits good linearity (R2 > 0.99). This study provides a stable and excellent AgNP synthesis technique that can improve various applications involving AgNP-mediated reactions and has the potential to be developed as an alternative to using expensive detection equipment and to be applied for the detection of mercury in food.

Polysaccharides Obtained from Cordyceps militaris Alleviate Hyperglycemia by Regulating Gut Microbiota in Mice Fed a High-Fat/Sucrose Diet.

Polysaccharides isolated from fungus Cordyceps militaris display multi-biofunctions, such as immunostimulation, down-regulation of hyperlipidemia, and anti-cancer function. The occurrence of obesity and metabolic syndrome is related to the imbalance of gut microbiota. In this study, the effects of C. militaris and its fractions on modifying metabolic syndrome in mice were evaluated. Mice were fed a high-fat/high-sucrose diet (HFSD) for 14 weeks to induce body weight increase and hyperlipidemia symptoms in mice, and then the mice were simultaneously given a HFSD and C. militaris samples for a further 8 weeks. The results indicated that the fruit body, polysaccharides, and cordycepin obtained from C. militaris had different efficacies on regulating metabolic syndrome and gut microbiota in HFSD-treated mice. Polysaccharides derived from C. militaris decreased the levels of blood sugar and serum lipids in mice fed HFSD. In addition, C. militaris-polysaccharide treatment obviously improved intestinal dysbiosis through promoting the population of next generation probiotic Akkermansia muciniphila in the gut of mice fed HFSD. In conclusion, polysaccharides derived from C. militaris have the potential to act as dietary supplements and health food products for modifying the gut microbiota to improve the metabolic syndrome.

Resveratrol Butyrate Esters Inhibit Obesity Caused by Perinatal Exposure to Bisphenol A in Female Offspring Rats.

Resveratrol butyrate esters (RBE) are derivatives of resveratrol (RSV) and butyric acid and exhibit biological activity similar to that of RSV but with higher bioavailability. The aim of this study was designed as an animal experiment to explore the effects of RBE on the serum biochemistry, and fat deposits in the offspring rats exposed to bisphenol A (BPA), along with the growth and decline of gut microbiota. We constructed an animal model of perinatal Bisphenol A (BPA) exposure to observe the effects of RBE supplementation on obesity, blood lipids, and intestinal microbiota in female offspring rats. Perinatal exposure to BPA led to weight gain, lipid accumulation, high levels of blood lipids, and deterioration of intestinal microbiota in female offspring rats. RBE supplementation reduced the weight gain and lipid accumulation caused by BPA, optimised the levels of blood lipids, significantly reduced the Firmicutes/Bacteroidetes (F/B) ratio, and increased and decreased the abundance of S24-7 and Lactobacillus, respectively. The analysis of faecal short-chain fatty acid (SCFA) levels revealed that BPA exposure increased the faecal concentration of acetate, which could be reduced via RBE supplementation. However, the faecal concentrations of propionate and butyrate were not only significantly lower than that of acetate, but also did not significantly change in response to BPA exposure or RBE supplementation. Hence, RBE can suppress BPA-induced obesity in female offspring rats, and it demonstrates excellent modulatory activity on intestinal microbiota, with potential applications in perinatological research.

Improvement of Foaming Characteristics and Stability of Sterilized Liquid Egg with Egg White Hydrolysate (EWH).

A pasteurized liquid egg leads to protein denaturation and degradation of processing properties, whereas non-pasteurized eggs may have food safety risks. If the negative impact of the pasteurization process on liquid eggs can be reduced, for example, the loss of stability and foamability, companies will be willing to purchase pasteurized eggs, thereby reducing food safety risks. Therefore, in this study, specific hydrolyzation conditions were used to produce egg white hydrolysate (EWH) with a lower molecular mass of amino acid and peptide fragments, and the effects of various concentration of EWH refilling on pasteurized liquid egg properties were investigated. The results showed that up to 30.1% of EWH was hydrolyzed by protease A and papain. Adding 1% (w/w) EWH can improve the negative charge potential value, surface tension, viscosity, and weight loss analysis of the sample. In addition, the cake structure and the appearance was acceptable to consumers. Therefore, to ensure its efficient use in the baking industry and considering the cost and stability, 1% (w/w) EWH was chosen as the best concentration.

Resveratrol Butyrate Esters Inhibit BPA-Induced Liver Damage in Male Offspring Rats by Modulating Antioxidant Capacity and Gut Microbiota.

Resveratrol can affect the physiology or biochemistry of offspring in the maternal-fetal animal model. However, it exhibits low bioavailability in humans and animals. Fifteen-week SD pregnant female rats were orally administered bisphenol A (BPA) and/or resveratrol butyrate ester (RBE), and the male offspring rats (n = 4-8 per group) were evaluated. The results show that RBE treatment (BPA + R30) compared with the BPA group can reduce the damage caused by BPA (p < 0.05). RBE enhanced the expression of selected genes and induced extramedullary hematopoiesis and mononuclear cell infiltration. RBE increased the abundance of S24-7 and Adlercreutzia in the intestines of the male offspring rats, as well as the concentrations of short-chain fatty acids (SCFAs) in the feces. RBE also increased the antioxidant capacity of the liver by inducing Nrf2, promoting the expression of HO-1, SOD, and CAT. It also increased the concentration of intestinal SCFAs, enhancing the barrier formed by intestinal cells, thereby preventing BPA-induced metabolic disruption in the male offspring rats, and reduced liver inflammation. This study identified a potential mechanism underlying the protective effects of RBE against the liver damage caused by BPA exposure during the peri-pregnancy period, and the influence of the gut microbiota on the gut-liver axis in the offspring.

Deep Ocean Water Concentrate Changes Physicochemical Characteristics, the Profile of Volatile Components and Consumer Acceptance for Taiwanese Rice Shochu.

To study the effects of deep-ocean water concentrate (DOWC) on sake quality, Taichung No. 10 indica rice (Oryza sativa subsp. indica) and Tainan No. 11 japonica rice (O. sativa subsp. japonica) were used as raw materials, and basic physicochemical property parameters in shochu were analyzed differentially. Sake fermentation mash analysis results revealed that DOWC addition did not significantly affect the basic physicochemical properties during sake brewing, but it significantly reduced citric acid and malic acid contents in Taichung No. 10 indica rice sake sample by 52-66% and 73-93%, respectively. DOWC addition significantly increased citric acid content in Tainan No. 11 japonica rice sake sample by 32-202%. Rice shochu analysis results revealed that DOWC addition significantly increased isoamyl acetate, ethyl hexanoate, and ethyl octanoate contents in shochu made from japonica rice and indica rice, respectively. The results indicate that rice variety directly affects the types of volatile compounds in rice shochu. Principal component analysis and sensory evaluation results revealed that DOWC addition affected the composition of volatile compounds in the two types of rice shochu and resulted in differences in flavor evaluation. DOWC addition affects yeast metabolites and directly changes the volatile compound composition and flavor of rice shochu.

LC-MS Quantification of Site-Specific Phosphorylation Degree by Stable-Isotope Dimethyl Labeling Coupled with Phosphatase Dephosphorylation.

Protein phosphorylation is a crucial post-translational modification that plays an important role in the regulation of cellular signaling processes. Site-specific quantitation of phosphorylation levels can help decipher the physiological functions of phosphorylation modifications under diverse physiological statuses. However, quantitative analysis of protein phosphorylation degrees is still a challenging task due to its dynamic nature and the lack of an internal standard simultaneously available for the samples differently prepared for various phosphorylation extents. In this study, stable-isotope dimethyl labeling coupled with phosphatase dephosphorylation (DM + deP) was tried to determine the site-specific degrees of phosphorylation in proteins. Firstly, quantitation accuracy of the (DM + deP) approach was confirmed using synthetic peptides of various simulated phosphorylation degrees. Afterwards, it was applied to evaluate the phosphorylation stoichiometry of milk caseins. The phosphorylation degree of Ser130 on α-S1-casein was also validated by absolute quantification with the corresponding synthetic phosphorylated and nonphosphorylated peptides under a selected reaction monitoring (SRM) mode. Moreover, this (DM + deP) method was used to detect the phosphorylation degree change of Ser82 on the Hsp27 protein of HepG2 cells caused by tert-butyl hydroperoxide (t-BHP) treatment. The results showed that the absolute phosphorylation degree obtained from the (DM + deP) approach was comparable with the relative quantitation resulting from stable-isotope dimethyl labeling coupled with TiO2 enrichment. This study suggested that the (DM + deP) approach is promising for absolute quantification of site-specific degrees of phosphorylation in proteins, and it may provide more convincing information than the relative quantification method.

Comparison of Standing Posture Bioelectrical Impedance Analysis with DXA for Body Composition in a Large, Healthy Chinese Population.

Bioelectrical impedance analysis (BIA) is a common method for assessing body composition in research and clinical trials. BIA is convenient but when compared with other reference methods, the results have been inconclusive. The level of obesity degree in subjects is considered to be an important factor affecting the accuracy of the measurements. A total of 711 participants were recruited in Taiwan and were sub-grouped by gender and levels of adiposity. Regression analysis and Bland-Altman analysis were used to evaluate the agreement of the measured body fat percentage (BF%) between BIA and DXA. The BF% measured by the DXA and BIA methods (Tanita BC-418) were expressed as BF%DXA and BF%BIA8, respectively. A one-way ANOVA was used to test the differences in BF% measurements by gender and levels of adiposity. The estimated BF%BIA8 and BF%DXA in the all subjects, male and female groups were all highly correlated (r = 0.934, 0.901, 0.916, all P< 0.001). The average estimated BF%BIA8 (22.54 ± 9.48%) was significantly lower than the average BF%DXA (26.26 ± 11.18%). The BF%BIA8 was overestimated in the male subgroup (BF%DXA< 15%), compared to BF%DXA by 0.45%, respectively. In the other subgroups, the BF%BIA8 values were all underestimated. Standing BIA estimating body fat percentage in Chinese participants have a high correlation, but underestimated on normal and high obesity degree in both male and female subjects.

Isoobtusilactone A-induced apoptosis in human hepatoma Hep G2 cells is mediated via increased NADPH oxidase-derived reactive oxygen species (ROS) production and the mitochondria-associated apoptotic mechanisms.

Chemoprevention by the use of naturally occurring substances is becoming a promising strategy to prevent cancer. In this study, the effects of isoobtusilactone A, a novel constituent isolated from the leaves of Cinnamomum kotoense, on the proliferation of human hepatoma Hep G2 cells were studied. Under our experimental conditions, isoobtusilactone A was found to elicit a concentration-dependent growth impediment (IC(50)=37.5 microM). The demise of these cells induced by isoobtusilactone A was apoptotic in nature, exhibiting a concentration-dependent increase in sub-G(1) fraction and DNA fragmentation. Subcellular fractionation analysis further revealed that Bax translocation to mitochondria resulted in a rapid release of cytochrome c, followed by activation of caspase 3 and PARP cleavage, and finally cell death. Isoobtusilactone A-treated cells also displayed transient increase of ROS during the earlier stage of the experiment, followed by the disruption of mitochondrial transmembrane potential (DeltaPsi(m)). The presence of a ROS scavenger (N-acetyl-L-cysteine) and an inhibitor of NADPH oxidase (diphenyleneiodonium chloride) blocked ROS production and the subsequent apoptotic cell death. In addition, in order to investigate the acute toxicity of isoobtusilactone A, groups of 5-6-week old Sprague-Dawley rats were subjected to oral administration of 350, or 700 mg/kg bw isoobtusilactone A four times each week for two weeks. There was no significant difference between control animals and treated animals with respect to the body weight gain, the body weight ratio of liver, spleen and kidney, haematological and clinical chemistry parameters. Taken together, our data suggest that ROS generated through the activation of NADPH oxidase plays an essential role in apoptosis induced by isoobtusilactone A, and the dosages of isoobtusilactone A tested in this study did not cause animal toxicity.