A convenient spectrophotometric test for screening skin-sensitizing chemicals using reactivity with glutathione in chemico.

To initiate skin sensitization, haptens react with endogenous proteins. During this process, skin sensitizers react with small endogenous molecules containing thiol or amino groups. In this study, a simple spectrophotometric method to identify skin sensitizers in chemico was developed using the reactivity of glutathione (GSH) with test chemicals in a 96-well plate. To quantitate the remaining GSH following the reaction with a skin sensitizer, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) was employed. The optimized experimental conditions included the pH- and time-dependent stability of GSH, stability of derivatized products of GSH with optimal concentration and incubation time of DTNB, incubation time of GSH with the test chemicals, and molar ratios of GSH to the test chemicals. With the optimized conditions with both acetonitrile and DMSO as vehicles and incubation of GSH with test chemicals in 1:10 and 1:15 ratios for 24 h at 4 °C, 23 skin sensitizers and 23 non-sensitizers, based on the local lymph node assay, were tested to determine the predictive capacity of individual conditions. The best result showed a predictive capacity of 95.2% sensitivity, 91.3% specificity, and 93.2% accuracy, with 93.2% consistency in three trials, when 5.8% depletion was used as a cut-off value in 1:10 of GSH:test chemicals in DMSO. It would be an economic and useful screening tool for determining the skin sensitization potential of small molecules, because the present method employs simple endogenous GSH as an electron donor for sensitizers with a spectrophotometric detection system in 96-well plates, and because the method requires neither experimental animals nor cell cultures. Supplementary Information: The online version contains supplementary material available at 10.1007/s43188-023-00218-9.

Effects of faba bean protein isolate on rheological properties of pork myofibrillar protein gels and quality characteristics of pork low-fat model sausages.

BACKGROUND: This study aimed to assess the effect of faba bean (Vicia faba L.) protein isolate (FBPI) on the rheological properties of pork myofibrillar protein gels (MPGs) and the quality characteristics of pork low-fat model sausages (LFMSs). RESULTS: Pork MPGs with 5 or 10 g kg-1 FBPI had higher cooking yield, gel strength, and viscosity than controls. The addition of FBPI to MPGs increased the protein surface hydrophobicity and decreased sulfhydryl groups. Adding FBPI to MPGs changed the protein profile and microstructure. The cooking loss and expressible moisture of LFMSs with 5, 10, or 15 g kg-1 FBPI were lower than those of controls and showed similar results to those with 15 g kg-1 soy protein isolate (SPI). Hardness values of LFMSs with FBPI and SPI were no different, and were higher than those of controls. CONCLUSION: The addition of FBPI potentially improves rheological properties of MPGs and the functional properties of LFMSs, including water-holding capacity and textural properties. © 2024 Society of Chemical Industry.

Evaluation of Gelation Properties of Salt-Soluble Proteins Extracted from Protaetia brevitarsis Larvae and Tenebrio molitor Larvae and Application to Pork Myofibrillar Protein Gel System.

The purpose of this study was to investigate the functional properties of salt-soluble proteins obtained from Protaetia brevitarsis (PB) and Tenebrio molitor (TM) larvae, the interaction between these proteins and pork myofibrillar protein (MP) in a gel system. The gel properties of salt-soluble protein extracts showed that the PB had a higher viscosity than the TM protein. However, the TM protein had higher gel strength compared with the PB protein. The gelation characteristics of the pork MP gel systems added with lyophilized insect salt-soluble protein powder showed to decrease slightly viscosity compared with MP alone. Adding the TM or PB protein powder did not affect the pork MP's hydrophobicity and sulfhydryl group levels. Furthermore, the protein bands of the MP did not change with the type or amount of insect salt-soluble protein. The cooking yields of the pork MP gels containing PB or TM protein powder were higher than those without insect protein. Regardless of the type of insect salt-soluble protein added, the pork MP's gel strength decreased. Furthermore, as the level of insect powder increased, the surface protein structure became rough and porous. The results demonstrated that proteins extracted from PB and TM larvae interfered with the gelation of pork MP in a gel system.

Effect of sea tangle extract on the quality characteristics of reduced-salt, low-fat sausages using pre-rigor muscle during refrigerated storage.

OBJECTIVE: The aim of this study was to investigate quality characteristics of reduced-salt, low-fat pork sausage (PS) using pre-rigor muscle and sea tangle extract (STE) to reduce salt level of sausages during refrigerated storage. METHODS: Pork ham was prepared with pre-rigor and post-rigor muscle from the local market. Sausages using post-rigor muscle were manufactured with the 1.5% of salt content, and samples with pre-rigor muscle were processed by different salt concentrations (0.8%). Accordingly, PSs were prepared in 4 treatments (REF, PS with 1.5% of salt using post-rigor muscle; CTL, PS with 0.8% of salt using pre-rigor muscle; TRT1, PS with 0.8% of salt and 5% of STE using pre-rigor muscle; TRT2, PS with 0.8% of salt and 10% of STE using pre-rigor muscle). For the evaluation of quality characteristics and shelf-life of reduced-salt PS, pH and color values, cooking loss (%), expressible moisture (%), textural properties, lipid oxidation (thiobarbituric reactive substances), protein denaturation (volatile basic nitrogen), and microbiological analysis (total plate counts and Enterobacteriaceae counts) were determined. RESULTS: The pH and temperature of pre-rigor raw pork ham were higher than those of post-rigor pork ham. Hardness of TRT2 was higher than that of REF or CTL. TRT2 had higher gumminess and chewiness than CTL. TRT1 and TRT2 had lower volatile basic nitrogen than CTL. Total plate counts of TRT2 were lower than those of CTL. Expressible moisture values of TRT1 and TRT2 were similar to those of REF. The addition of STE into PS improved functional properties and shelf-life of PS. CONCLUSION: Reduced-salt PS containing pre-rigor muscle and STE had similar functional properties to those of regular-salt ones, while containing approximately 47% less salt compared to regular-salt level.

Evaluation of salt level and rigor status on the physicochemical and textural properties of low-fat pork sausages added with sea tangle extract using rapidly chilled pre-rigor pork ham.

OBJECTIVE: This study was performed to evaluate the quality characteristics of pork sausage (PS) with sea tangle extract (STE) and rapid chilled pre-rigor muscle (RCPM) for the development of reduced-salt low-fat sausage. METHODS: Pre- and post-rigor pork ham muscles were prepared to process PSs. Positive control (reference, REF) using post-rigor muscle were manufactured at a regular-salt level of 1.5%. Fresh and rapid-chilled pre-rigor muscle (FPM and RCPM) were used to manufacture reduced-salt sausages with 0.8% salt. Reduced-salt PSs were prepared with four treatments: FT1 (FPM alone), FT2 (FPM with 5% STE), RT1 (RCPM alone), and RT2 (RCPM with 5% STE). The physicochemical and textural properties of the sausages with reduced-salt levels and RCPM combination were measured to determine if the characteristics of RCPM were similar to those with FPM. RESULTS: The pH values of PS with FPM and RCPM were higher than those of REF with post-rigor muscle. Color values (L*, a*, b*) were not affected by different rigor-states and salt addition level. Textural properties of reduced-salt PSs were similar to those of REF due to the improved functionalities of pre-rigor muscle. RT2 had lower expressible moisture (%) than other treatments with post-rigor muscle and RCPM except for RT1. CONCLUSION: The addition of STE and RCPM to reduced-salt PS increased the waterholding capacity, which was lower than those of PS with STE using RCPM but similar to those of regular-salt sausage.

Ginseng Sprouts Attenuate Mortality and Systemic Inflammation by Modulating TLR4/NF-κB Signaling in an LPS-Induced Mouse Model of Sepsis.

Sepsis leads to multi-organ failure due to aggressive systemic inflammation, which is one of the main causes of death clinically. This study aimed to evaluate whether ginseng sprout extracts (GSE) can rescue sepsis and explore its underlying mechanisms. C57BL/6J male mice (n = 15/group) were pre-administered with GSE (25, 50, and 100 mg/kg, p.o) for 5 days, and a single injection of lipopolysaccharide (LPS, 30 mg/kg, i.p) was administered to construct a sepsis model. Additionally, RAW264.7 cells were treated with LPS with/without GSE/its main components (Rd and Re) to explain the mechanisms corresponding to the animal-derived effects. LPS injection led to the death of all mice within 38 h, while GSE pretreatment delayed the time to death. GSE pretreatment also notably ameliorated LPS-induced systemic inflammation such as histological destruction in both the lung and liver, along with reductions in inflammatory cytokines, such as TNF-α, IL-6, and IL-1ß, in both tissues and serum. Additionally, GSE markedly diminished the drastic secretion of nitric oxide (NO) by suppressing the expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2) in both tissues. Similar changes in TNF-α, IL-1ß, NO, iNOS, and COX2 were observed in LPS-stimulated RAW264.7 cells, and protein expression data and nuclear translocation assays suggested GSE could modulate LPS-binding protein (LBP), Toll-like receptor 4 (TLR4), and NF-κB. Ginsenoside Rd could be a major active component in GSE that produces the anti-sepsis effects. Our data support that ginseng sprouts could be used as an herbal resource to reduce the risk of sepsis. The corresponding mechanisms may involve TLR4/NF-κB signaling and a potentially active component.

Effects of different frozen temperatures of pork sausage batter on quality characteristics of reduced-salt sausages using pre-rigor muscle.

OBJECTIVE: The objective of this study was to evaluate quality characteristics of reduced-salt pork sausage (PS) using pre-rigor muscle compared to those of regular-salt PS. In addition, effects of freezing on sausage batter with different temperatures (-30°C vs -70°C) on quality characteristics of both sausage batter and cooked sausages during frozen storage were observed. METHODS: Pre-rigor and post-rigor pork hams were used to manufacture low-fat sausages. Sausages using post-rigor (Post) muscle were manufactured at a salt level of 1.5%, whereas those with pre-rigor (Pre) muscle were processed at salt level of 1.0%. After these muscles were made at two salt levels (1.5% salt, Post-rigor; 1.0% salt, Pre-rigor), Sausage batters were stored at two frozen temperatures (-30°C vs -70°C). During storage for 12 wks, they were measured for physicochemical and textural properties every 4 wks up to 12 wks. RESULTS: pH values and temperatures of sausage batter of pre-rigor muscle were higher than those of post-rigor muscle regardless of the frozen temperature. The lightness and yellowness values of batter at the initial storage were the highest during storage. For PS, there were no differences in most parameters measured among all treatments. However, expressible moisture values (%) of Pre-30 and Pre-70 were lower than those of Post-30 (p<0.05). CONCLUSION: Regardless of frozen temperature during storage, quality characteristics of pre-rigor PS with salt level of 1.0% salt were similar to those of post-rigor PS with salt level of 1.5%. By using the pre-rigor muscle, salt content could be reduced by one third of the regular-salt level (1.5%) of post-rigor muscle.

Korean Red Ginseng Ameliorates Fatigue via Modulation of 5-HT and Corticosterone in a Sleep-Deprived Mouse Model.

Central fatigue, which is neuromuscular dysfunction associated with neurochemical alterations, is an important clinical issue related to pathologic fatigue. This study aimed to investigate the anti-central fatigue effect of Korean red ginseng (KRG) and its underlying mechanism. Male BALB/c mice (8 weeks old) were subjected to periodic sleep deprivation (SD) for 6 cycles (forced wakefulness for 2 days + 1 normal day per cycle). Simultaneously, the mice were administered KRG (0, 100, 200, or 400 mg/kg) or ascorbic acid (100 mg/kg). After all cycles, the rotarod and grip strength tests were performed, and then the changes regarding stress- and neurotransmitter-related parameters in serum and brain tissue were evaluated. Six cycles of SD notably deteriorated exercise performance in both the rotarod and grip strength tests, while KRG administration significantly ameliorated these alterations. KRG also significantly attenuated the SD-induced depletion of serum corticosterone. The levels of main neurotransmitters related to the sleep/wake cycle were markedly altered (serotonin was overproduced while dopamine levels were decreased) by SD, and KRG significantly attenuated these alterations through relevant molecules including brain-derived neurotropic factor and serotonin transporter. This study demonstrated the anti-fatigue effects of KRG in an SD mouse model, indicating the clinical relevance of KRG.

A convenient fluorometric test method for skin sensitization using glutathione in chemico.

A convenient fluorometrical test method to identify skin sensitizers in chemico was developed using reactivity with glutathione (GSH), a low molecular weight endogenous substance. Following incubation of test chemicals with GSH, the remaining GSH was quantitated fluorometrically by using monobromobimane (mBBr), a thiol-detecting agent, for determining % depletion of this endogenous substance by test chemicals. The experimental conditions optimized were: (1) reactivity of thiol compounds including GSH with mBBr, (2) effects of vehicles on reactivity, (3) molar ratios of GSH to test chemicals, and (4) reactivity of endogenous substance with test substances under different incubation times. When an optimized condition with DMSO as a vehicle for test chemicals and in 1:60 ratio for 24 hr at 4°C was applied to classify 48 well-known skin sensitizers and non-sensitizers, the predictive capacity was as follows: 88.2% sensitivity, 78.6% specificity, and 85.4% accuracy with 95.8% consistency of three trials when 10.3% depletion of GSH was used as a cutoff value. Because the present method employed relatively simple GSH as an acceptor for sensitizers and/or a relatively convenient fluorometric detection system in 96-well plates for a high throughput test, it would be a useful test tool for screening skin sensitization potential of test chemicals.

Characteristics of low-nitrite pork emulsified-sausages with paprika oleoresin solution during refrigerated storage.

The objective of this study was to evaluate quality characteristics of low-nitrite emulsified-sausages (ESs, < 75 ppm) containing paprika oleoresin solution (POS) for replacing sodium nitrite (NaNO2). Pork ESs were prepared with four treatments (reference (REF), 150 ppm NaNO2; TRT1, 0 ppm NaNO2 + 0.1% POS; TRT2, 37.5 ppm NaNO2 + 0.1% POS; and TRT3, 75 ppm NaNO2 + 0.1% POS). The physicochemical and texture properties, microbial counts, residual nitrite and thiobarbituric acid reactant substances (TBARS) were measured during refrigerated storage of 35 days. Although TRT2 and TRT3 had lower levels of NaNO2, they had higher redness and yellowness than REF (p < 0.05). Microbial counts of total bacterial counts and Enterobacteriaceae of TRT2 and TRT3 were similar to those of REF (p > 0.05). Expressible moisture percentages (EM, %) of TRT2 and TRT3 were lower than those of REF (p < 0.05). TBARS values of TRT2 and TRT3 were not different from those of REF (p > 0.05). Among treatments, TRT1 had the highest TBARS values (p < 0.05). In conclusion, 0.1% POS in combination with 37.5 ppm NaNO2 would have quality characteristics similar to those of REF. Therefore, approximately 3/4 of the initial nitrite level could be replaced with 0.1% POS, and eventually developed healthier pork products.

Evaluation of Quality Characteristics of Low-Nitrite Pork Sausages with Paprika Oleoresin Solution during Refrigerated Storage.

The objective of this study was to evaluate quality characteristics of pork emulsified-sausage (ES) containing paprika oleoresin solution (POS) as a replacement for sodium nitrite (NaNO2) during refrigerated storage. ESs were prepared with four treatments: 1) REF, 150 ppm NaNO2; 2) CTL, 75 ppm NaNO2; 3) TRT1, 75 ppm NaNO2+0.1% POS (1% paprika oleoresin+99% sunflower seed oil); and 4) TRT2, 75 ppm NaNO2+0.1% POS (5% paprika oleoresin+95% sunflower seed oil). The addition of POS into ES increased redness and yellowness but decreased lightness (p<0.05). TRT1 and TRT2 had higher redness and yellowness than CTL (p<0.05). TRT1 and TRT2 had lower total plate counts (Log CFU/g) than CTL due to antimicrobial activity of POS, regardless of its levels (p<0.05). Residual nitrite decreased with increasing storage time for all treatments. TRT2 had lower residual nitrite due to nitrite scavenging activity of POS (p<0.05). CTL had the highest thiobarbituric acid reactant substances (TBARS) among all treatments during storage. The addition of POS into ES showed nitrite scavenging activity during refrigerated storage. In conclusion, antimicrobial and antioxidant activities of the ES with a combination of POS and 75 ppm NaNO2 were similar to those of REF (150 ppm NaNO2), and improved color development of redness value. Therefore, the addition of POS could decrease the amount of nitrite in ESs, leading to healthier meat products.

Role of Intestinal Microbiota in Metabolism of Voglibose In Vitro and In Vivo.

BACKGROUND: Voglibose, an α-glucosidase inhibitor, inhibits breakdown of complex carbohydrates into simple sugar units in intestine. Studies showed that voglibose metabolism in the liver might be negligible due to its poor intestinal absorption. Numerous microorganisms live in intestine and have several roles in metabolism and detoxification of various xenobiotics. Due to the limited information, the possible metabolism of voglibose by intestinal microbiota was investigated in vitro and in vivo. METHODS: For the in vitro study, different concentrations of voglibose were incubated with intestinal contents, prepared from both vehicle- and antibiotics-treated mice, to determine the decreased amount of voglibose over time by using liquid chromatography-mass spectrometry. Similarly, in vivo pharmacodynamic effect of voglibose was determined following the administration of voglibose and starch in vehicle- and antibiotic-pretreated non-diabetic and diabetic mice, by measuring the modulatory effects of voglibose on blood glucose levels. RESULTS: The in vitro results indicated that the remaining voglibose could be significantly decreased when incubated with the intestinal contents from normal mice compared to those from antibiotic-treated mice, which had less enzyme activities. The in vivo results showed that the antibiotic pretreatment resulted in reduced metabolism of voglibose. This significantly lowered blood glucose levels in antibiotic-pretreated mice compared to the control animals. CONCLUSION: The present results indicate that voglibose would be metabolized by the intestinal microbiota, and that this metabolism might be pharmacodynamically critical in lowering blood glucose levels in mice.

Assessment of skin sensitizing potential of metals with ß-galactosidase-expressing E. coli culture system.

It has been a challenge to develop in vitro alternative test methods for accurate prediction of metallic products which may exert skin sensitization, as several test methods adopted by OECD were relatively ineffective in assessing the capacity for metallic compounds to exert sensitizing reactions, compared with organic test substances. Based upon these findings, a system that incorporates ß-galactosidase producing E. coli cultures was tested for its predictive capacity to well-known metallic sensitizers. In this system, E. coli cells were incubated with metal salts at various concentrations and ß-galactosidase suppression by each test metal was determined. Fourteen local lymph node assay (LLNA) categorized metal salts were examined. Although color interference from metal salts was minimal, a fluorometric detection system was also employed using 4-methylumbelliferyl galactopyranoside as a substrate for ß-galactosidase to avoid the color interference, concomitantly with the original UV-spectrometric method. Data demonstrated that two detection methods were comparable and complementary. In addition, most of the metallic sensitizers were correctly identified at 0.6 and 0.8 mM concentrations. Despite the lower specificity obtained in the current study and small number of substances tested, the developed method appears to be a relatively simple and effective in vitro method for detecting metallic sensitizers. When 61 chemicals tested in the ß-galactosidase producing E. coli cultures including the present study were collectively analyzed, the prediction capacity was as high as other OECD-adopted tests: 95.6% of sensitivity, 66.7% of specificity, and 88.5% of accuracy. It is important to emphasize that animals or mammalian cell cultures were not required in the current method, which are in accordance with the EU guidelines on restricted or banned animal testing.

Role of Intestinal Microbiota in Metabolism of Gastrodin In Vitro and In Vivo.

Alteration in the number and composition of intestinal microbiota affects the metabolism of several xenobiotics. Gastrodin, isolated from Gastrodia elata, is prone to be hydrolyzed by intestinal microbiota. In the present study, the role of intestinal microbiota in gastrodin metabolism was investigated in vitro and in vivo. Gastrodin was incubated in an anaerobic condition with intestinal contents prepared from vehicle- and antibiotics-treated rats and the disappearance of gastrodin and formation of 4-hydroxybenzyl alcohol (4-HBA) was measured by liquid chromatography coupled to mass spectroscopy (LC-MS/MS). The results showed that almost all gastrodin incubated with control intestinal contents was metabolized to its aglycone in time- and concentration-dependent manners. In contrast, much less formation of 4-HBA was detected in intestinal contents from antibiotics-treated rats. Subsequently, in vivo pharmacokinetic study revealed that the antibiotic pretreatment of rats significantly affected the metabolism of gastrodin to 4-HBA. When administered orally, gastrodin was rapidly absorbed rapidly into plasma, metabolized to 4-HBA, and disappeared from the body within six hours. Interestingly, the pharmacokinetic parameters of 4-HBA were changed remarkably in antibiotics-treated rats, compared to control rats. The results clearly indicated that the antibiotics treatment of rats suppressed the ability of intestinal microbiota to metabolize gastrodin to 4-HBA and that, thereby, the pharmacodynamic action was significantly modulated.

Identification of pre- and pro-haptens with a ß-galactosidase-expressing E. coli culture system for skin sensitization.

Although several in vitro approaches were successful in separating chemicals as skin sensitizers and non-sensitizers, none of the available methods completely mimics the absolute in vivo scenario of skin sensitization. One of the major challenges with currently available systems would be the limited or no metabolic capacity to activate pre- or pro-haptens to reactive metabolites in the system. In the present study, E. coli cells with ß-galactosidase-expressing LacZ gene were combined with either induced rat liver S-9 fractions or microsomal fractions to detect pre- or pro-haptens to cause skin sensitization. Following optimization of some experimental conditions, we examined 20 sensitizers classified as pre- or pro-haptens and 11 non-sensitizers in these E. coli cultures by incubating bacterial cells and test chemicals with and without S-9 or microsomal proteins. After a 6-h incubation in the presence of IPTG, cells were lyzed to determine the suppression of ß-galactosidase enzyme. A cut-off of 17.3% was applied to determine the percent suppression of ß-galactosidase activity by test chemicals to classify skin sensitizers and non-sensitizers. Among chemicals tested, 19 pre- or pro-haptens were categorized as true positives and 8 non-sensitizers were categorized as true negatives. Thereby, the overall sensitivity, specificity and accuracy achieved with microsome-incorporated and S-9 fraction-incorporated group were 95.0%, 72.7% and 87.1% and 80.0%, 81.8% and 80.6%, respectively. The results suggested that the present bacterial system incorporated with the microsomal activation system could be considered as a useful alternative method to classify not only direct-acting sensitizers but also pre- or pro-haptens requiring metabolic activation in vitro.

Atypical femoral fractures after anti-osteoporotic medication: a Korean multicenter study.

PURPOSE: Increasing numbers of atypical femoral fractures have been reported among long-term bisphosphonate users. We evaluated clinical characteristics of atypical femoral fractures throughout Korean multicenter studies. METHODS: We retrospectively analysed the bone mineral density, prodromal symptoms before femoral fracture, and medication history of osteoporosis in 76 cases of atypical femoral fracture. RESULTS: The mean age of cases was 71.4 ± 8.8 (range, 43-89) years old. The mean follow-up period after the fracture operation was 24.5 ± 12.9 (range, 12-79) months. BMI was 23.2 ± 3.0 on average. The mean BMD of femur was -1.9 ± 1.4 (range, -4.8 to 1.3). Prodromal symptoms including thigh pain before femoral fracture appeared in 22 (28.9 %) of 76 patients. All patients included in the study used bisphosphonate. The duration of taking bisphosphonate before fracture was 36.8 ± 50.8 (one-204 months) months. Fifty-seven (75 %) of 76 patients were taking the medication for more than three years. Delayed union occurred in 43 (56.5 %) of 76 patients. Delayed union was defined as a fractured bone that did not completely heal within six months of injury. The group of having taken anti-osteoporotic medication for more than three years showed relatively longer union period compared to that for a shorter period medication group (4.8 ± 2.5 months vs 9.3 ± 3.7 months, p = 0.017). The delayed union developed in 43 (56.5 %) of 76 patients and showed a significantly higher incidence in the group with long-term therapy (five/43 vs 38/43, p = 0.021). The bilateral femoral fractures developed in 23 (30.2 %) of 76 patients and showed a high incidence in the group medicated more than three years (two/23 vs 21/23, p = 0.039). CONCLUSIONS: The longer bisphosphonates are used, the more the cases of delayed union and the more frequent the development of bilateral fractures following unilateral fractures. With regard to the delayed union, the methods of the acceleration of fracture healing may be beneficial in atypical femoral fracture patients who had been receiving long-term bisphosphonates therapy. Careful observation is required for contra-lateral femurs due to a high incidence of bilateral atypical femoral fractures.