Hepa-ToxMOA: a pathway-screening method for evaluating cellular stress and hepatic metabolic-dependent toxicity of natural products.

In the field of drug discovery, natural products have emerged as therapeutic agents for diseases such as cancer. However, their potential toxicity poses significant obstacles in the developing effective drug candidates. To overcome this limitation, we propose a pathway-screening method based on imaging analysis to evaluate cellular stress caused by natural products. We have established a cellular stress sensing system, named Hepa-ToxMOA, which utilizes HepG2 cells expressing green fluorescent protein (GFP) fluorescence under the control of transcription factor response elements (TREs) for transcription factors (AP1, P53, Nrf2, and NF-κB). Additionally, to augment the drug metabolic activity of the HepG2 cell line, we evaluated the cytotoxicity of 40 natural products with and without S9 fraction-based metabolic activity. Our finding revealed different activities of Hepa-ToxMOA depending on metabolic or non-metabolic activity, highlighting the involvement of specific cellular stress pathways. Our results suggest that developing a Hepa-ToxMOA system based on activity of drug metabolizing enzyme provides crucial insights into the molecular mechanisms initiating cellular stress during liver toxicity screening for natural products. The pathway-screening method addresses challenges related to the potential toxicity of natural products, advancing their translation into viable therapeutic agents.

Emodin, an Emerging Mycotoxin, Induces Endoplasmic Reticulum Stress-Related Hepatotoxicity through IRE1α-XBP1 Axis in HepG2 Cells.

Emodin, an emerging mycotoxin, is known to be hepatotoxic, but its mechanism remains unclear. We hypothesized that emodin could induce endoplasmic reticulum (ER) stress through the inositol-requiring enzyme 1 alpha (IRE1α)-X-box-binding protein 1 (XBP1) pathway and apoptosis, which are closely correlated and contribute to hepatotoxicity. To test this hypothesis, a novel IRE1α inhibitor, STF-083010, was used. An MTT assay was used to evaluate metabolic activity, and quantitative PCR and western blotting were used to investigate the gene and protein expression of ER stress or apoptosis-related markers. Apoptosis was evaluated with flow cytometry. Results showed that emodin induced cytotoxicity in a dose-dependent manner in HepG2 cells and upregulated the expression of binding immunoglobulin protein (BiP), C/EBP homologous protein (CHOP), IRE1α, spliced XBP1, the B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 ratio, and cleaved caspase-3. Cotreatment with emodin and STF-083010 led to the downregulation of BiP and upregulation of CHOP, the Bax/Bcl-2 ratio, and cleaved caspase-3 compared with single treatment with emodin. Furthermore, the apoptosis rate was increased in a dose-dependent manner with emodin treatment. Thus, emodin induced ER stress in HepG2 cells by activating the IRE1α-XBP1 axis and induced apoptosis, indicating that emodin can cause hepatotoxicity.

Antibody-free and selective detection of okadaic acid using an affinity peptide-based indirect assay.

Okadaic acid (OA) is a type of marine biotoxin produced by some species of dinoflagellates in marine environments. Consumption of shellfish contaminated with OA can cause diarrhetic shellfish poisoning (DSP) in humans with symptoms that typically include abdominal pain, diarrhea and vomiting. In this study, we developed an affinity peptide-based direct competition enzyme-linked immunosorbent assay (dc-ELISA) for the detection of OA in real samples. The OA-specific peptide was successfully identified via M13 biopanning and a series of peptides were chemically synthesized and characterized their recognition activities. The dc-ELISA system showed good sensitivity and selectivity with a half-maximal inhibitory concentration (IC50) of 148.7 ng/mL and a limit of detection (LOD) of 5.41 ng/mL (equivalent, 21.52 ng/g). Moreover, the effectiveness of the developed dc-ELISA was validated using OA-spiked shellfish samples, and the developed dc-ELISA showed a high recovery rate. These results suggest that the affinity peptide-based dc-ELISA can be a promising tool for detecting OA in shellfish samples.

Plant-based natural flavonoids show strong inhibition of aflatoxin production and related gene expressions correlated with chemical structure.

Aflatoxins are strong carcinogenic and mutagenic fungal metabolites, and aflatoxin contamination is a critical issue in agriculture and food production. Natural flavonoids can suppress aflatoxin biosynthesis; however, the structure-activity relationship remains unclear. In the present study, a total of 36 structurally related natural flavonoids were tested against the aflatoxigenic Aspergillus flavus, both in-vitro and in-situ (on maize kernels), to investigate their structure-activity relationship and biological activity. Aflatoxin production (IC50 values: 10.85-20.09 µg/mL) and the expression of related genes (aflD, aflK, aflQ, and aflR) were found to be strongly inhibited. Structure-activity relationship studies revealed that the [-OH] or [-O-CH3] groups at position 6 of ring A and position 4' of ring B were closely associated with antifungal and antiaflatoxigenic activities. These findings provide valuable information for the development of clean and safe methods to prevent aflatoxin contamination in food.

Antifungal Activity of Essential Oil and Plant-Derived Natural Compounds against Aspergillus flavus.

Aspergillus flavus is a facultative parasite that contaminates several important food crops at both the pre- and post-harvest stages. Moreover, it is an opportunistic animal and human pathogen that causes aspergillosis diseases. A. flavus also produces the polyketide-derived carcinogenic and mutagenic secondary metabolite aflatoxin, which negatively impacts global food security and threatens human and livestock health. Recently, plant-derived natural compounds and essential oils (EOs) have shown great potential in combatting A. flavus spoilage and aflatoxin contamination. In this review, the in situ antifungal and antiaflatoxigenic properties of EOs are discussed. The mechanisms through which EOs affect A. flavus growth and aflatoxin biosynthesis are then reviewed. Indeed, several involve physical, chemical, or biochemical changes to the cell wall, cell membrane, mitochondria, and related metabolic enzymes and genes. Finally, the future perspectives towards the application of plant-derived natural compounds and EOs in food protection and novel antifungal agent development are discussed. The present review highlights the great potential of plant-derived natural compounds and EOs to protect agricultural commodities and food items from A. flavus spoilage and aflatoxin contamination, along with reducing the threat of aspergillosis diseases.

Contamination characteristics and risk assessment of aflatoxins in homemade soybean paste, a traditional fermented soybean food, in South Korea.

Soybean paste is manufactured through microbial fermentation and may become contaminated with aflatoxins. Herein, we conducted nationwide large-scale monitoring (n = 1436) over three years (2018-2020) to investigate aflatoxin levels according to geographic, demographic, manufacturing, quality factors, and risk characteristics of homemade soybean paste produced through fermentation. The mean level of total aflatoxins was 5.88 µg/kg (range, 0.01-281.92), with the most common contaminating type being the B type. Aflatoxin levels significantly differed according to the region, age of the manufacturer, type of starter used, and the amino-type nitrogen content and pH of the homemade soybean paste (p < 0.05). Aflatoxin levels was significantly higher when starters were manufactured using the traditional method (inoculation with a naturally occurring strain in the surrounding environment). The aflatoxin exposure level estimated through the average intake of homemade soybean paste in all age groups was 0.1012 ng/kg body weight/day. The risk assessment for the genotoxic and carcinogenic potential of aflatoxins using the margin of exposure approach revealed values of 3705-3954 for average intake of homemade soybean paste, indicating public health concern. These results suggest that follow-up studies and safety management strategies are needed to reduce aflatoxin levels in homemade soybean paste.

Natural Thiols, but Not Thioethers, Attenuate Patulin-Induced Endoplasmic Reticulum Stress in HepG2 Cells.

Patulin, a mycotoxin, is known to have cytotoxic effects, but few studies have focused on the involvement of the endoplasmic reticulum (ER) stress response in patulin toxicity and the natural compounds that attenuate it in HepG2 cells. This study tested the ability of patulin to induce ER stress, and that of four thiols and three thioethers to attenuate patulin-induced ER stress in HepG2 cells. Patulin dose-dependently inhibited cell proliferation (IC50, 8.43 µM). Additionally, patulin was found to increase the expression levels of ER stress-related genes and/or protein markers, including BiP, CHOP, and spliced XBP1, in HepG2 cells compared to the vehicle control, indicating its potential in ER stress induction. Patulin-induced cytotoxicity in HepG2 cells was reduced by naturally occurring thiol compounds (glutathione, L-acetyl-L-cysteine, cysteine, and captopril), but not by thioether compounds (sulforaphane, sulforaphene, and S-allyl-L-cysteine). Patulin-thiol co-treatment decreased CHOP expression and BiP and CHOP levels in HepG2 cells but did not alter BiP expression. Spliced XBP1 expression was decreased by patulin-thiol co-treatment. Thus, patulin induced ER stress in HepG2 cells and thiols, but not in thioethers, attenuated patulin-induced ER stress.

Effect of plant-based compounds on the antifungal and antiaflatoxigenic efficiency of strobilurins against Aspergillus flavus.

Aflatoxins are a group of carcinogenic and mutagenic fungal secondary metabolites that have threatened human health and global food security. Aflatoxin contamination can be controlled by applying fungicides, such as strobilurins. Although these compounds have been effective, they may be risky to the environment due to their wide usage. In this study, plant-based compounds were tested to promote the performance of strobilurins (azoxystrobin, pyraclostrobin) against aflatoxigenic Aspergillus flavus; six natural compounds, namely baicalein, nobiletin, meso-dihydroguaiaretic acid, pinoresinol, syringaresinol, and celastrol, were found to exhibit synergistic antifungal effects with strobilurins with fractional inhibitory concentration index < 0.5. Among them, baicalein showed no inhibitory effects on A. flavus when applied alone, but strongly enhanced the in vitro and in situ antifungal and antiaflatoxigenic efficacy of strobilurins and transformed them from fungistatic to fungicidal agents. Therefore, baicalein may be used as an effective natural chemosensitizing agent to improve the performance of strobilurins against A. flavus. The findings of this study provide novel insights for the development of safer and more effective strategies for the control of aflatoxin contamination.

Transcriptomic responses of Aspergillus flavus to temperature and oxidative stresses during aflatoxin production.

Aflatoxin is a group of polyketide-derived carcinogenic and mutagenic secondary metabolites produced by Aspergillus flavus that negatively impact global food security and threaten the health of both humans and livestock. Aflatoxin biosynthesis is strongly affected by the fungal developmental stage, cultivation conditions, and environmental stress. In this study, a novel float culture method was used to examine the direct responses of the A. flavus transcriptome to temperature stress, oxidative stress, and their dual effects during the aflatoxin production stage. The transcriptomic response of A. flavus illustrated that the co-regulation of different secondary metabolic pathways likely contributes to maintaining cellular homeostasis and promoting cell survival under stress conditions. In particular, aflatoxin biosynthetic gene expression was downregulated, while genes encoding secondary metabolites with antioxidant properties, such as kojic acid and imizoquins, were upregulated under stress conditions. Multiple mitochondrial function-related genes, including those encoding NADH:ubiquinone oxidoreductase, ubiquinol-cytochrome C reductase, and alternative oxidase, were differentially expressed. These data can provide insights into the important mechanisms through which secondary metabolism in A. flavus is co-regulated and facilitate the deployment of various approaches for the effective control and prevention of aflatoxin contamination in food crops.

TiO2 particles induce ER stress and apoptosis in human hepatoma cells, HepG2, in a particle size-dependent manner.

The cytotoxicity of TiO2 nanoparticles are well-known, but the particle size-dependent induction of ER stress and apoptosis by TiO2 in hepatocytes has not been elucidated clearly. In the present study, we investigated whether a fine TiO2 particle and two types of TiO2 nanoparticles induce ER stress and apoptosis differently in HepG2 cells. A particle size-dependent decrease in cell viability was observed after exposure to the TiO2 particles. The levels of ER stress-related proteins (BiP, CHOP, ATF6α, and p-PERK) were increased with decreasing particle size. TiO2 particles induced ER stress-mediated apoptosis in a particle size-dependent manner as seen by a decrease in the expression of Bcl-2, and increases in the expression of Bax, caspase-12, and cleaved caspase-3. These results indicated that the cytotoxicity produced by TiO2 particles was related to particle size, with smaller TiO2 nanoparticles producing greater toxic effects involving ER stress and apoptosis in the HepG2 cells.

Comparison of the Antifungal and Antiaflatoxigenic Potential of Liquid and Vapor Phase of Thymus vulgaris Essential Oil against Aspergillus flavus.

The antifungal and antiaflatoxigenic activity of Thymus vulgaris essential oil (EO) against Aspergillus flavus was evaluated over a range of concentrations in vapor- and liquid-phase contact tests. Total reduction in mycelial growth in the vapor- and liquid-phase tests was detected at EO concentrations of 20 and 400 µg/mL, respectively. Treatment with 10 µg/mL EO reduced aflatoxin production by 97.0 and 56.4% in the vapor- and liquid-phase tests, respectively. Greater inhibition of the expression of both fungal development-related genes (brlA, abaA, and wetA) and aflatoxin biosynthesis-related genes (aflR, aflD, and aflK) was also observed in the vapor-phase test. A substantial reduction in aflatoxin production was also observed in brown rice (72.7%) and white rice (18.0%). Our results indicate that the way this EO contacts fungal cells significantly affects its antifungal activity and that T. vulgaris EO in vapor phase might be a good strategy to control fungal contamination.

Effects of gamma ray, electron beam, and X-ray on the reduction of Aspergillus flavus on red pepper powder (Capsicum annuum L.) and gochujang (red pepper paste).

Aspergillus flavus is the potential pathogenic mold in red pepper powder (Capsicum annuum L.) and gochujang (red pepper paste), which can produce mycotoxins. This study investigated the effects of gamma ray, e-beam, and X-ray irradiation on the reduction of A. flavus on red pepper powder and gochujang and physicochemical and sensory quality changes. Gamma ray and e-beam at 3.5 kGy reduced A. flavus effectively (>4 log), without deteriorating the physicochemical quality. Same dose of X-ray did not cause any deterioration of the physicochemical quality. However, reduction effect of A. flavus in red pepper powder and gochujang by 3.5 kGy X-ray was under 2 log. Further, sensory quality analysis showed no significant difference in color, appearance, texture, and overall acceptability after three irradiations. However, flavor changes of red pepper powder and gochujang after three irradiations were mentioned by panelists. In this study, gamma ray and e-beam irradiation were effective in eliminating A. flavus present in red pepper powder and gochujang, but X-ray irradiation was not effective. The results indicate gamma ray and e-beam are effective in controlling microorganisms present in powdery or paste foods, but the X-ray was not effective.

A Second Derivative Fourier-Transform Infrared Spectroscopy Method to Discriminate Perilla Oil Authenticity.

The aim of this study was to discriminate the authenticity of perilla oils distributed in Korea using their Fourier-Transform infrared spectroscopy (FT-IR) spectra with attenuated total reflectance accessory. By using orthogonal projections for latent structures discriminant analysis (OPLS-DA) technique, the =C-H cis-double bond, -C-H asymmetric and -C-H symmetric stretching are determined to be the best variables for discriminating the perilla oil authenticity. Comparing the integral and the second derivative methods between authentic and adulterated perilla oil samples, the most obvious and significant differences among the three variables is =C-H cis-double bond stretching. The procedure for applying the second derivative range of variables found in authentic perilla oil samples correctly discriminated between the adulterated samples of perilla oils with soybean oils and/or corn oils added at concentrations of ≥ 5 vol%. These results showed that the second derivative FT-IR analysis can be used as a simple and alternative method for discriminating the authenticity of perilla oil.

Antioxidant and antimicrobial efficacy of a biflavonoid, amentoflavone from Nandina domestica in vitro and in minced chicken meat and apple juice food models.

A biflavonoid, amentoflavone isolated from Nandina domestica and characterized by NMR spectral-data analyses was assessed for its antioxidant, and antibacterial potential in vitro and in food-model systems. Amentoflavone exhibited potent antioxidant ability (19.21-75.52%) on scavenging DPPH, ABTS, superoxide, and hydroxyl radicals. Fluorescent images confirmed bacterial membrane depolarization of both the tested pathogens Staphylococcus aureus and Escherichia coli, with a significant reduction in cell viabilities at their respective MIC of 62.5 and 125 µg/mL. Increasing rates of membrane permeability observed in 260 nm-absorbing material, potassium ion, extracellular ATP, and relative electrical conductivity assays confirmed antibacterial mechanistic role of amentoflavone as also evidenced by microscopic studies of SEM and TEM. There was a marked inhibitory effect of amentoflavone with a significant reduction in cell counts of S. aureus and E. coli in minced chicken and apple juice at 4 °C, thus suggesting its nutritional enhancing efficacy as a natural antioxidant and antimicrobial agent.

A 43 MHz Low-Field Benchtop 1H Nuclear Magnetic Resonance Method to Discriminate Perilla Oil Authenticity.

The aim of this study was to discriminate the authenticity of perilla oils distributed in Korea using their 1H nuclear magnetic resonance (NMR) spectra acquired by a 43 MHz low-field benchtop NMR spectrometer. Significant differences existed in the integration values of all 6 peaks found in the spectrum between authentic and adulterated perilla oil samples. The integration values of 4 peaks that signify the methylene protons present in all fatty acids (FA) and allylic or olefinic protons present in all unsaturated FA were the best variables for establishing perilla oil authenticity. The procedure for applying the range of variables found in authentic perilla oil samples correctly discriminated between the samples of perilla oils with soybean oils added at concentrations of ≥ 6 vol%. The results demonstrated that this NMR procedure is a possible cost-effective alternative to the high-field 1H NMR method for discriminating the authenticity of perilla oils.

Heteroassembled gold nanoparticles with sandwich-immunoassay LSPR chip format for rapid and sensitive detection of hepatitis B virus surface antigen (HBsAg).

This study aimed to develop a more sensitive method for the detection of hepatitis B surface antigen (HBsAg) using heteroassembled gold nanoparticles (AuNPs). A single layered localized surface plasmon resonance (LSPR) chip format was developed with antigen-antibody reaction-based detection symmetry using AuNPs, which detected HBsAg at 10 pg/mL. To further improve the detection limit, a modified detection format was fabricated by fixing a secondary antibody (to form a heteroassembled sandwich format) to the AuNP monolayer, which enhanced the detection sensitivity by about 100 times. The developed heteroassembled AuNPs sandwich-immunoassay LSPR chip format was able to detect as little as 100 fg/mL of HBsAg within 10-15 min. In addition, the heteroassembled AuNPs sandwich-immunoassay LSPR chip format did not show any non-specific binding to other tested antigens, including alpha fetoprotein (AFP), C-reactive protein (CRP), and prostate-specific antigen (PSA). These findings confirm that the proposed detection strategy of heteroassembled AuNPs sandwich-immunoassay LSPR chip format may provide a new platform for early diagnosis of various human diseases.

Effects of microwaves on the reduction of Aspergillus flavus and Aspergillus parasiticus on brown rice (Oryza sativa L.) and barley (Hordeum vulgare L.).

Aspergillus flavus and Aspergillus parasiticus are primary pathogen moulds on brown rice and barley. This study investigated the effects of microwave irradiation (MWI) (2450 MHz, 700 W, 10-50 s) on inactivation of A. flavus and A. parasiticus on brown rice and barley and the quality of these samples. The counts of both strains were significantly (p < 0.05) reduced by the stepwise increase in MWI treatment time. The log reductions of A. flavus on brown rice and barley were 0.05 and 0.04 after 10 s; 1.06 and 1.05 after 20 s; 1.59 and 1.52 after 30 s; and 3.04 and 2.78 after 40 s. The log reductions of A. parasiticus on brown rice and barley were 0.06 and 0.10 after 10 s; 1.20 and 1.00 after 20 s; 2.04 and 1.61 after 30 s; and 2.89 and 2.90 after 40 s. Moreover, neither strain survived after 50 s of MWI. The Hunter colour 'L' gradually increased with increasing MWI treatment time. However, there were no significant differences in the 'L' of brown rice after 10-40 s of MWI treatment and of barley after 10-30 s of MWI treatment. The Hunter colour 'a' and 'b' gradually increased with increasing microwave time. No significant change was observed in the moisture content of either cereal treated with 10-20 s of MWI. The differences in the sensory quality (colour, appearance, flavour, texture and overall acceptability) after 0-30 s of MWI were not significant. However, values for colour, appearance, texture and overall acceptability were significantly reduced when treated with 40-50 s of MWI. Therefore, with 20 s of MWI at 2450 MHz, 700 W could be effective for > 90% reduction of mould without causing deleterious changes to the colour, moisture content and sensory qualities of these cereals.

Development of aflatoxin B1 aptasensor based on wide-range fluorescence detection using graphene oxide quencher.

Aflatoxin B1 (AFB1) is a carcinogenic substance produced by fungi of genus Aspergillus, especially Aspergillus flavus. Few nanograms of AFB1 that permeated through the skin is sufficient to cause liver cancer and stunted growth. In this study, a rapid aptamer-based assay for AFB1 was developed using the fluorescence quenching property of graphene oxide (GO) and a fluorescein amidite (FAM)-modified aptamer specific to AFB1. The aptamer, modified with the fluorescence dye FAM on its 5'-end, was used as a probe. Once bound by AFB1, a conformational change of the aptamer was caused that led to its interaction with the well-known fluorescence quencher GO, resulting in a decrease of the fluorescence intensity of the system. In the absence of AFB1, the fluorescence intensity remained unchanged. The aptamer-based AFB1 assay process was conducted through 3 steps within 40min. The aptamer was incubated with AFB1 before the addition of GO. The amount of AFB1 present was measured by the change in fluorescence intensity. The detection system was evaluated with standard solutions of AFB1 of various concentrations. The results showed that the fluorescence intensity decreased linearly as the concentration of AFB1 gradually increased. Although the assay was specific to AFB1, there was slight interference by other types of aflatoxin. When the assay was applied to a real sample, the limit of detection was 4.5 ppb, which was within the wide detection range of up to 300ppb with good linearity. Thus, this biosensor is considered to be competitive with the conventional detection methods in the field owing to its wide detection range and assay rapidity.

Identification and quantification of adulteration in Garcinia cambogia commercial products by chromatographic and spectrometric methods.

Species of genus Garcinia are rich sources of bioactive constituents with antimicrobial, anticancer, anti-inflammatory, hepatoprotective and anti-HIV activities. Commercial products of Garcinia cambogia are used as anti-obesity drugs with increasing market demand. Because of the high price of its products, it can be adulterated with similar lower-priced species. This study was designed to develop and validate an accurate and efficient method for the detection of any adulteration (G. indica) in G. cambogia products. For this purpose, high performance liquid chromatography (HPLC) was used to analyse the ethanolic fruit rind extracts of G. cambogia and G. indica, their formulations of 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 95% G. indica with G. cambogia, and 11 G. cambogia commercial products. The analytical methods were validated by quality assurance parameters of linearity, sensitivity, precision and accuracy. Two marker peaks were detected in G. indica fruit extract, whereas G. cambogia did not show these peaks. The detected peaks were identified as anthocyanins; cyanidin-3-O-sambubioside and cyanidin-3-O-glucoside. In the study to determine the effect of pH and temperature on the stability of its anthocyanin content, HPLC analysis of G. indica extract showed the highest content at pH 1 and 50°C. Using two different mobile phases, the limits of detection (LOD) for cyanidin-3-O-sambubioside and cyanidin-3-O-glucoside were 0.036 and 0.059, and 0.022 and 0.033 mg kg-1, respectively. Furthermore, the inter-day precision (< 3.2%) confirmed that the applied analytical method fulfils the required criteria of Association of Official Analytical Chemists (AOAC). From this study, it was found that the HPLC method used for the detection of adulteration in G. cambogia products is rapid and accurate.

Combined Analysis of Stable Isotope, (1)H NMR, and Fatty Acid To Verify Sesame Oil Authenticity.

The aim of this study was to verify the authenticity of sesame oils using combined analysis of stable isotope ratio, (1)H NMR spectroscopy, and fatty acid profiles of the oils. Analytical data were obtained from 35 samples of authentic sesame oils and 29 samples of adulterated sesame oils currently distributed in Korea. The orthogonal projection to latent structure discriminant analysis technique was used to select variables that most effectively verify the sesame oil authenticity. The variables include δ(13)C value, integration values of NMR peaks that signify the CH3 of n-3 fatty acids, CH2 between two C═C, protons from sesamin/sesamolin, and 18:1n-9, 18:3n-3, 18:2t, and 18:3t content values. The authenticity of 65 of 70 blind samples was correctly verified by applying the range of the eight variables found in the authentic sesame oil samples, suggesting that triple analysis is a useful approach to verify sesame oil authenticity.