Comparative study of estrogenic activities of phytoestrogens using OECD in vitro and in vivo testing methods.

With growing scientific interest in phytoestrogens, a number of studies have investigated the estrogenic potential of phytoestrogens in a wide variety of assay systems. However, evaluations of individual phytoestrogens with different assay systems make it difficult for predicting their relative estrogenic potency. The objective of this study was to compare estrogenic properties of fifteen known phytoestrogens using an estrogen receptor-α (ER-α) dimerization assay and Organization for Economic Cooperation and Development (OECD) standardized methods including in vitro estrogen receptor (ER) transactivation assay using VM7Luc4E2 cells and in vivo uterotrophic assay using an immature rat model. Human ER-α dimerization assay showed positive responses of eight test compounds and negative responses of seven compounds. These results were consistently found in luciferase reporter assay results for evaluating ER transactivation ability. Seven test compounds exhibiting relatively higher in vitro estrogenic activities were subjected to uterotrophic bioassays. Significant increases in uterine weights were only found after treatments with biochanin A, 8-prenylnaringenin, and coumestrol. Importantly, their uterotrophic effects were lost when animals were co-treated with antagonist of ER, indicating their ER-dependent effects in the uterus. In addition, analysis of estrogen responsive genes revealed that these phytoestrogens regulated uterine gene expressions differently compared to estrogens. Test methods used in this study provided a high consistency between in vitro and in vivo results. Thus, they could be used as effective screening tools for phytoestrogens, particularly focusing on their interactions with ER-α.

Development and characterization of a human cell line-based transactivation assay to assess thyroid EDCs.

Thyroid hormones (THs) are one of the most important hormones, playing key roles in the regulation of various physiological functions. Although THs have important function in human, in vitro test methods based on human cells are currently insufficient to effectively screen and test TH-related endocrine disrupting chemicals (EDCs). We established a TH agonist TA assay using the adenocarcinomic human alveolar basal epithelial cell line A549 to test and screen potential TH agonists. To establish the TH agonist TA assay, a TRE-secNluc-IRES-EGFP reporter cassette was constructed and transfected into the A549 cell line using a retrovirus. We evaluated the TH agonistic properties of several chemicals which were tested by existing thyroid agonists testing method (OECD GD 207). Comparing the results of the TH agonist TA assay with the OECD GD 207, T3, T4, tiratricol, and tetrac (natural TH and 3,3',5,5'-tetraiodothyroacetic acid derivatives), which are TH agonists according to the OECD GD 207, also tested positive in the TH agonist TA assay using the A549 cell line. These results suggested that the TH agonist TA assay developed in this study using a human cell line can provide the information, such as accuracy and specificity to TH agonistic properties of chemicals.

Identification of metabolic pathways related to the bisphenol A-induced adipogenesis in differentiated murine adipocytes by using RNA-sequencing.

We evaluated the effect of bisphenol A and its metabolites on the 3T3-L1 cells, in terms of glucose and lipid metabolism. We also aimed to obtain the information on the genome-wide expression changes in the 3T3-L1 cells treated with Bisphenol A by using RNA-seq, which involves whole-transcriptome sequencing. Differentially Expressed Genes (DEGs) collected from RNA-seq can be used to produce a complete picture of related metabolism pathways. The KEGG pathway was extracted based on the DEGs. Bisphenol A significantly increased the mRNA level of Sterol regulatory element binding transcription factor 1 (Srebf1) and CCAAT/enhancer binding protein alpha (Cebpa). Lipoprotein lipase (Lpl) was also significantly influenced by bisphenol A and its metabolites. Acetyl-Coenzyme A carboxylase beta (Acacb) and Fatty acid synthase (Fasn) mRNA levels were elevated by bisphenol A and its metabolites. The insulin signaling pathway, neurotrophin signaling pathway, and endometrial cancer-related pathway were focused by the functional enrichment analyses, and the pathways were well coincided with recent previous reports. DEGs collected from RNA-seq were confirmed as a reliable evidence in the exposure to the chemicals such as bisphenol A. Collecting pieces of the puzzles obtained from the RNA-seq will help us to produce a complete picture of the metabolic pathway for such chemicals.

Enhancement of androgen transcriptional activation assay based on genome edited glucocorticoid knock out human prostate cancer cell line.

Endocrine-disrupting chemicals (EDCs) interfere with the biological activity of hormones. Among EDC's, (anti-)androgenic compounds potentially cause several androgen-related diseases. To improve the accuracy of an in vitro transactivation assay (TA) for detection of (anti-)androgenic compounds, We established the glucocorticoid receptor (GR) knockout 22Rv1/MMTV cell line by using an RNA-guided engineered nuclease (RGEN)-derived CRISPR/Cas system. The 22Rv1/MMTV GRKO cell line was characterized and validated by androgen receptor (AR)-mediated TA assay compared with the AR-TA assay using 22Rv1/MMTV. In conclusion, the AR-TA assay with the 22Rv1/MMTV GRKO cell line was more accurate, excluding the misleading signals derived from glucocorticoids or equivalent chemicals, and might be an effective method for screening potential (anti-)androgenic compounds.

Effects of probiotic supplementation on post-infectious irritable bowel syndrome in rodent model.

BACKGROUND: Probiotics have been reported to be the active component used in the treatment of many functional gastrointestinal symptoms and syndromes. Lactobacillus and yeast culture are extensively used in probiotic supplements and traditional treatments for irritable bowel syndrome (IBS). The aim of this study was to investigate the effects of probiotic treatments (Lactobacillus acidophilus LA5, Bifidobacterium animalis subsp. lactis BB12 and Saccharomyces cerevisiae var. boulardii) on the behavioral response, targeted gene expression and pro-inflammatory cytokine levels of Pi (Post infectious)-IBS -induced mice. METHODS: Pathogen-free male C57L/B6 mice and the Trichinella-infected mice were used to measure the score of abdominal withdrawal reflex (AWR). To compare molecular, biological and biochemical evidences of given probiotics with normal and positive control groups in mice, we conducted quantitative reverse transcription polymerase chain reaction (RT-qPCR), western blotting, and cytokine analysis. RESULTS: Pi-IBS-induced immune response was confirmed that PAR-2 mRNA level was significantly increased by Trichinella infection (P < 0.05). The reduction of Pi-IBS symptoms through Trichinella infection and the effects of given probiotics were confirmed by a change in the protein levels of cytokines (P < 0.05). In addition, the administration of DW (Daewon) probiotics significantly decreased serum levels of IL-1 and IL-6 (P < 0.05). CONCLUSIONS: We have demonstrated that the given probiotics decreased pro-inflammatory cytokine levels in both the control and Pi-IBS induced mice. Taken all the results together, the results support that DW probiotics has a potential as a probiotic medication for patient with IBS via regulating TNF-α and IL-6 protein levels and serum IL-1 and IL-6 levels.

Romaine Lettuce/Skullcap Mixture Improves Sleep Behavior in Vertebrate Models.

The aim of this study is to investigate the effects of romaine lettuce leaves extract (RE), skullcap root extract (SE) and their mixture on sleep behaviors in vertebrate models. HPLC analysis showed that RE contains lactucopicrin (0.02±0.01 mg/g extract), chlorogenic acid (4.05±0.03 mg/g extract), caffeic acid (2.38±0.03 mg/g extract), and chicoric acid (7.02±0.32 mg/g extract) as main phenolic compounds, while SE includes baicalin (99.4±0.5 mg/g extract), baicalein (8.28±0.21 mg/g extract), and wogonin (3.09±0.32 mg/g extract). The mixture of RE (100 mg/g extract) and SE (40 mg/g extract) increased total sleep time by 50.9% compared with the control in pentobarbital-induced sleep model. In electroencephalography (EEG) analysis, RE/SE mixture significantly increased Non-Rapid Eye Movement (NREM), in which delta wave was enhanced by around 40% compared with normal control, leading to the increase of sleep time. In caffeine-induced wake model, RE/SE mixture greatly decreased (53%) caffeine-induced wake time, showing a similar level to normal control. In addition, caffeine-induced decreased of NREM and delta wave effectively increased with RE/SE mixture; NREM and delta wave increased by 85% and 108%, respectively. Furthermore, RE/SE mixture was shown to bind to a gamma-aminobutyric acid type A (GABAA)-benzodiazepine (BZD) receptor stronger than RE or SE single extract. Taken together, RE/SE mixture effectively improved sleep behavior with the increase of NREM via GABAA-BZD receptor binding. RE/SE mixture can be used as an herbal agent for sleep disorders.

Topical application of spent coffee ground extracts protects skin from ultraviolet B-induced photoaging in hairless mice.

The aim of this study was to evaluate the protective effect of spent coffee ground (SCG) on ultraviolet (UV) B-induced photoaging in hairless mice. The oil fraction (OSCG) and ethanol extract (ESCG) of SCG were prepared from SCG. OSCG contained a much higher level of caffeine (547.32 ± 1.68 µg mg(-1)) when compared to the sum of its chlorogenic acid derivatives (∼119 µg mg(-1)), and pyrazines were the major aromatic compounds in OSCG. OSCG effectively inhibited the UVB-induced increase in intracellular reactive oxygen species in HaCaT cells. Topical application of OSCG or ESCG significantly reduced the UVB-induced wrinkle formation in mice dorsal skin. The combined application of OSCG and ESCG (OEH) led to a decrease in the wrinkle area by over 35% when compared with the UVB-treated control (UVBC). Epidermal thickness was also reduced by 40%. This result was connected to the significant reduction in transdermal water loss (27%) and erythema formation (48%) that result from UVB irradiation. Polarization-sensitive optical coherence tomography (PS-OCT) and antibody-based histological analyses showed that OSCG and ESCG effectively suppressed the UVB-induced decrease in collagen content. The level of type 1 collagen (COL1) in the OEH group was enhanced by around 40% compared with the UVB control group (UVBC). This was attributed to the down-regulation of matrix metalloproteinases (MMP2, 9, and 13), which are known to be responsible for collagen destruction. Our results indicate that topical treatment with OSCG/ESCG protects mouse skin from UVB-induced photoaging by down-regulating MMPs; therefore, suggesting the potential of SCG extracts as a topical anti-photoaging agent.

Deer Bone Oil Extract Suppresses Lipopolysaccharide-Induced Inflammatory Responses in RAW264.7 Cells.

The aim of this study was to investigate the effect of deer bone oil extract (DBOE) on lipopolysaccharide (LPS)-induced inflammatory responses in RAW264.7 cells. DBOE was fractionated by liquid-liquid extraction to obtain two fractions: methanol fraction (DBO-M) and hexane fraction (DBO-H). TLC showed that DBO-M had relatively more hydrophilic lipid complexes, including unsaturated fatty acids, than DBOE and DBO-H. The relative compositions of tetradecenoyl carnitine, α-linoleic acid, and palmitoleic acid increased in the DBO-M fraction by 61, 38, and 32%, respectively, compared with DBOE. The concentration of sugar moieties was 3-fold higher in the DBO-M fraction than DBOE and DBO-H. DBO-M significantly decreased LPS-induced nitric oxide (NO) production in RAW264.7 cells in a dose-dependent manner. This DBO-M-mediated decrease in NO production was due to downregulation of mRNA and protein levels of inducible nitric oxide synthase (iNOS). In addition, mRNA expression of pro-inflammatory mediators, such as cyclooxygenase (COX-2), interleukin (IL)-1ß, and IL-12ß, was suppressed by DBO-M. Our data showed that DBO-M, which has relatively higher sugar content than DBOE and DBO-H, could play an important role in suppressing inflammatory responses by controlling pro-inflammatory cytokines and mediators.

Evaluation of Prebiotic Effects of High-Purity Galactooligosaccharides in vitro and in vivo.

Galactooligosaccharides (GOS) are an important class of dietary prebiotics that exert beneficial effects on intestinal microbiota and gut barrier function. In this study, high-purity GOS (HP-GOS) were investigated in vitro and in vivo and confirmed as prebiotic ingredients in rat diet. HP-GOS were successfully produced using a two-step process, enzymatic hydrolysis and fermentation by yeast. They were found to serve as a good substrate and carbon source for supporting the growth of probiotic bacteria more effectively than other commercial GOS. Following administration of 1% (by mass) of HP-GOS to rats, the growth of Bifidobacterium bifidum and B. longum in the gut increased most rapidly up to 12 h, and thereafter the increase was slow. Therefore, 1% HP-GOS was found to be acceptable for the growth of probiotic bacteria. Groups of animals that were orally administered HP-GOS and bifidobacteria during the study, and the group administered HP-GOS during the 2nd (days 13-15) and 4th (days 28-30) period of the study had significantly (p<0.05) higher numbers of bifidobacteria in faeces than groups receiving a single dose of bifidobacteria. HP-GOS affected the expression of genes encoding glucagon-like peptide-1 (GLP-1) and peptide YY (PYY). There was a significant upregulation of GLP-1 and PYY mRNA with HP-GOS and bifidobacteria intake. We propose that the prebiotic properties of HP-GOS are potentially valuable for the production of functional foods for human consumption.

Aspergillus oryzae fermented germinated soybean extract alleviates perimenopausal symptoms in ovariectomised rats.

BACKGROUND: Soybeans have been widely used to alleviate climacteric symptoms. In this study, we investigated the oestrogenic activities of isoflavones extracted from Aspergillus oryzae-challenged germinated soybeans (AO-GS). Eight-week-old virgin Sprague-Dawley female rats were ovariectomised (OVX). The rats were orally administered 0.1 mg kg(-1) 17α-ethinyl oestradiol or three different doses of AO-GS (0.5, 1.0 2.0 g kg(-1) day(-1)) in distilled water for 6 weeks, while control rats were administered vehicle alone. Uterine weights and levels of oestradiol and testosterone in serum were measured. In addition to serum parameters, bone parameters were also acquired by using micro-computed tomography scanning. RESULTS: Treatments of OVX rats with AO-GS changed the secretory profile of serum oestradiol and testosterone. Serum oestradiol levels were significantly increased in OVX rats treated with and AO-GS (0.5, 1.0, 2.0 g kg(-1) day(-1)), while serum testosterone levels were not significantly increased in OVX rats treated with 1.0 g kg(-1) day(-1) of AO-GS. Furthermore, AO-GS (2.0 g kg(-1) day(-1)) significantly attenuated bone loss, increased trabecular bone volume fraction and trabecular thickness, and significantly decreased trabecular pattern factor. CONCLUSION: AO-GS treatments caused moderate oestrogenic activity in OVX rats compared to those treated with oestradiol, suggesting the potential for the use of AO-GS in the treatment of menopausal symptoms and in osteoporosis caused by oestrogen deficiency.

Hepatoprotective effects of soluble rice protein in primary hepatocytes and in mice.

BACKGROUND: The production of rice-derived by-products has increased owing to the growing use of processed rice products. The objective of this study was to isolate highly purified proteins from a rice by-product, rice syrup meal, and to examine their hepatoprotective effects in vitro and in vivo. RESULTS: Soluble rice protein (SRP70) was obtained via enzymatic processing of rice syrup meal using Termamyl SC and Alcalase. Mass spectrometry analysis showed that SRP70 contained low-molecular-weight (<600 Da) peptides. SRP70 did not affect the viability of rat primary hepatocytes and ameliorated tert-butyl hydroperoxide (t-BHP)-induced cytotoxicity. t-BHP-induced elevations in hepatocyte alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase activities were reduced by SRP70 in a dose-dependent manner. In addition, t-BHP exposure increased the level of malondialdehyde, a toxic reactive aldehyde, which was dose-dependently decreased by SRP70 treatment. These SRP70-induced decreases in biochemical parameters were also observed in vivo in mice. In particular, SRP70 increased the activities of liver antioxidant enzymes in t-BHP-treated mice, including catalase and glutathione peroxidase, as well as increasing the level of glutathione, an antioxidant peptide. SRP70-mediated activation of antioxidant enzymes was shown to be due to the up-regulation in their gene expressions, while nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase 4 (NOX4), a pro-oxidant enzyme, was down-regulated by SRP70. Hematoxylin and eosin staining also showed that SRP70 protected the liver from histopathological changes induced by t-BHP. CONCLUSION: Taken together, these data showed that SRP70, which is derived from a rice-processing by-product, had hepatoprotective effects in vitro and in vivo.

Hypoglycaemic effects of functional tri-peptides from silk in differentiated adipocytes and streptozotocin-induced diabetic mice.

BACKGROUND: In this study, the tri-peptides Gly-Glu-Tyr (GEY) and Gly-Tyr-Gly (GYG), identified previously as active compounds from the silk peptide E5K6, significantly stimulated basal and insulin-mediated glucose uptake by 3T3-L1 fibroblasts in a dose-dependent manner. RESULTS: Synthetic GEY and GYG peptides at a concentration of 500 µmol L(-1) significantly increased glucose transporter type 4 expression by 157% and 239%, respectively. Differentiation of 3T3-L1 cells into adipocytes leads to accumulation of intracellular fat droplets, and GEY and GYG at a concentration of 250 µmol L(-1) suppressed this effect by 72% and 75%, respectively. GYG improved glucose tolerance in steptozotocin (STZ)-induced diabetic mice in a dose-dependent manner. CONCLUSION: These results suggest that GYG isolated from E5K6 has anti-diabetic potential and silk waste products containing bioactive peptides could be used to the developments of treatments to lower blood glucose.

Photoprotective effects of galacto-oligosaccharide and/or Bifidobacterium longum supplementation against skin damage induced by ultraviolet irradiation in hairless mice.

This study aimed at examining whether oral administration of galacto-oligosaccharide (GOS) and Bifidobacterium longum, individually or in combination, could exert photoprotective effects on the skin of hairless mice. GOS and/or Bifidobacterium were administered orally to hairless mice for 12 weeks. Mice were irradiated with UV light daily for four consecutive days. GOS administration increased the water-holding capacity of the skin and prevented transepidermal water loss compared with the control. A reduction in the erythema formation of 16.8% was also observed in the GOS-treated group compared with the control, and CD44 gene expression was significantly increased. Oral administration of GOS or Bifidobacterium significantly increased TIMP-1 and Col1 mRNA expression compared with the control. Our findings support that prebiotics, including GOS, are beneficial not only to the intestine, but also to the skin, and present the possibility of new nutritional strategies for the prevention of UV-induced skin damage.

Lipase-mediated lipid removal from propolis extract and its antiradical and antimicrobial activity.

BACKGROUND: Propolis contains many antioxidants such as polyphenols and flavonoids. However, propolis-derived lipid components interrupt an efficient isolation of antioxidants from propolis extract. We examined the effectiveness of various lipase treatments for the removal of lipids from propolis extract and evaluated the biological features of the extract. RESULTS: Lipase OF and Novozyme 435 treatments did not reduce fatty acid level in propolis extract. However, Lipozyme TL IM-treated propolis extract showed a significant decrease in fatty acid level, suggesting the removal of lipids. Lipozyme RM IM also significantly decreased the fatty acid level of the extract, but was accompanied by the reduction of polyphenols and flavonoids, which are antioxidants. In Lipozyme TL IM treatment, an increase in active flavonoids, such as Artepillin C and kaempferide, was observed, with a slight increase of ferric reducing/antioxidant power (FRAP) radical-scavenging activity. In addition, antimicrobial activity towards skin health-related bacteria such as Staphylococcus epidermidis and Propionibacterium acnes was enhanced by Lipozyme TL IM treatment. CONCLUSION: Lipozyme TL IM treatment effectively removes lipids from propolis extract and enhances antibacterial activity. Therefore, we suggest that Lipozyme TL IM is a useful lipase for lipid removal of propolis extract.

Biotransformation of catechin and extraction of active polysaccharide from green tea leaves via simultaneous treatment with tannase and pectinase.

BACKGROUND: Green tea is a dietary source of bioactive compounds for human health. Enzymatic treatments induce the bioconversion of bioactive components, which can improve biological activities. In this study, we investigated the effect of simultaneous treatment with tannase and Rapidase on biotransformation of catechins and extraction of polysaccharide from green tea extract (GTE). RESULTS: Tannase and pectinase treatments induced the biotransformation of catechins and altered tea polysaccharide () content. The addition of GTE to the enzyme reaction resulted in a significant increase in degallated catechins, including gallic acid, a product of the tannase reaction (314.5-4076.0 µg mL(-1)) and a reduction in epigallocatechin gallate (EGCG). Biotransformation of catechins improved the radical scavenging activity of GTE. Pectinase treatment led to change of TPS composition in GTE by hydrolyzing polysaccharides. In addition, pectinase-driven hydrolysis in polysaccharides significantly increased TPS-induced Interleukin 6 (IL-6) production in macrophages. In particular, treatment of Rapidase (TPS-Ra) led to the highest IL-6 production among TPS samples, similar to treatment of highly purified pectinase (TPS-GTE), a positive control. CONCLUSION: Simultaneous processing with tannase and Rapidase can be an efficient method for the extraction of bioactive polysaccharides and biotransformation of catechins with enhanced radical scavenging activity from green tea.

Permethrin alters adipogenesis in 3T3-L1 adipocytes and causes insulin resistance in C2C12 myotubes.

Pyrethroids are a class of insecticides structurally derived from the naturally occurring insecticides called pyrethrins. Along with emerging evidence that exposure to insecticides is linked to altered weight gain and glucose homeostasis, exposure to pyrethroids has been linked to altered blood glucose levels in humans. Thus, the purpose of this study was to determine the role of permethrin on lipid and glucose metabolisms. Permethrin was treated to 3T3-L1 adipocytes and C2C12 myoblasts to determine its role in lipid and glucose metabolisms, respectively. Permethrin treatment resulted in increased expression of key markers of adipogenesis and lipogenesis in adipocytes. Permethrin significantly reduced insulin-stimulated glucose uptake in myotubes. This is the first report on the role of permethrin in altered lipid metabolism in adipocytes and impaired glucose homeostasis in myotubes. These results may help elucidate fundamental underlying mechanisms between insecticide exposure, particularly permethrin, and potential risk of developing obesity and its comorbidities.

Enhancement of exercise endurance capacity by fermented deer antler in BALB/c mice.

To investigate the activity of fermented deer antler on exercise endurance capacity, we evaluated endurance capacity in five-week-old male BALB/c mice by administering the fermented deer antler extract (FA) or the non-fermented deer antler extract (NFA) and then subjected the mice to exercise in the form of swimming. The mice administered 500 mg/kg/day of FA showed a significant increase in swimming time compared with mice administered placebo (16.55 min vs. 21.64 min, P<0.05). Serum lactate dehydrogenase (LDH), the marker of the liver and muscle damage, was significantly lower in FA groups. However, NFA groups did not show significantly different swimming time or serum LDH from that of the control group. Moreover, the FA-500 group had significantly higher hepatic superoxide dismutase (SOD) activity after forced swimming than the control and NFA groups (P<0.05). These findings suggest that fermentation may increase the exercise endurance capacity of the deer antler.

Effects of deer bone extract on the expression of pro-inflammatory cytokine and cartilage-related genes in monosodium iodoacetate-induced osteoarthritic rats.

Deer bone extract has the potential to relieve the discomfort or the articular cartilaginous damage associated with osteoarthritic (OA) and may be useful as a natural supplement for OA treatment without serious side effects. We analyzed the expression of pro-inflammatory cytokine and cartilage-related genes in monosodium iodoacetate-induced OA rats. Increases in the levels of serum pro-inflammatory cytokines, such as interleukin-1ß, interleukin-6, and tumor necrosis factor-α were significantly inhibited by the administration of deer bone extract (p<0.05). Decreases in the expression of collagen type II (COL2) and tissue inhibitors of metalloproteinases (TIMPs) mRNAs in the cartilage were significantly inhibited by deer bone extract treatment (p<0.05). The deer bone extract significantly suppressed the expression of matrix metalloproteinases (MMPs) mRNAs in the cartilage. The deer bone extract induced the up-regulation of COL2 and TIMP mRNAs and the down-regulation of MMP mRNAs by suppressing the expression of pro-inflammatory cytokine mRNAs.

Enzyme-assisted extraction of cactus bioactive molecules under high hydrostatic pressure.

BACKGROUND: To improve the extraction and recovery of bioactive materials from cactus, the present study investigated the effect of polysaccharide-degrading enzymes [Rapidase-Viscozyme mixture, 1/3 (v/v)] treatment under high hydrostatic pressure (HHP). RESULTS: The dry weight of the extract increased with the use of increasing pressure regardless of enzyme treatment. However, the polyphenol content showed a tendency to decrease with the increase in pressure in the cactus extract with or without enzyme treatment. The enzyme-assisted extraction resulted in an increase of dry weight and polyphenol content in the cactus extract. The total sugar and reducing sugar contents of the cactus extract increased with increasing pressure in enzyme-assisted extraction. The uronic acid content of the cactus extract showed a pattern similar to that of the reducing sugars. The enzyme-assisted extraction also increased the contents of taxifolin, quercetin and isorhametin. The cactus extract obtained through enzyme-assisted extraction showed intense scavenging activity of both DPPH and ABTS radicals. The crude polysaccharides isolated from the extract (51.2% at 1000 µg mL⁻¹ for HHP extraction at 300 MPa) had higher anti-complementary activity than the others except for lipopolysaccharide (60.00% at 1000 µg mL⁻¹). HHP extraction and enzyme-assisted extraction using HHP showed an increase of anti-complementary activity compared with the heat and enzyme controls, respectively. CONCLUSION: Overall, the use of HHP in enzyme-assisted extraction resulted in more efficient extraction than the use of enzyme treatment alone.

Comparative study on estrogen receptor alpha dimerization and transcriptional activity of parabens.

Parabens are used as preservatives in various household products, including oral products, cosmetics, and hair/body washes. In recent years, the widespread use of parabens has raised concerns due to the potential health risks associated with their estrogenic effects. In the present study, we evaluated and compared the estrogenic activity of parabens using two cell-based in vitro tests: (1) bioluminescence resonance energy transfer (BRET)-based estrogen receptor alpha (ERα) dimerization using HEK293 cells that were stably transfected with ERα-fused NanoLuc luciferase (Nluc) and HaloTag (HT) expression vector, and (2) stably transfected transcriptional activation (STTA) assays using ERα-HeLa9903 cells. The following parabens were tested using the BRET-based ERα dimerization assay and showed estrogenic activity (PC20 values): methyl paraben (MP, 5.98 × 10-5 M), ethyl paraben (EP, 3.29 × 10-5 M), propylparaben (PP, 3.09 × 10-5 M), butyl paraben (BP, 2.58 × 10-5 M), isopropyl paraben (IsoPP, 1.37 × 10-5 M), and isobutyl paraben (IsoBP, 1.43 × 10-5 M). Except MP, all other parabens tested using the STTA assay also showed estrogenic activity: EP, 7.57 × 10-6 M; PP, 1.18 × 10-6 M; BP, 3.02 × 10-7 M; IsoPP, 3.58 × 10-7 M; and IsoBP, 1.80 × 10-7 M. Overall, EP, PP, BP, IsoPP, and IsoBP tested positive for estrogenic activity using both assays. These findings demonstrate that most parabens, albeit not all, induce ERα dimerization and possess estrogenic activity.