Flavonoids commonly induce hormetic responses.

The present paper provides a new perspective of previously published findings by Siwak (Food Chem 141:1227-1241, 2013) which showed that 15 structurally diverse flavonoids reduced toxicity (i.e., enhanced cell viability) from hypochlorite using the MTT assay within a pre-conditioning experimental protocol, with each agent showing a similar biphasic concentration response relationship. We use this Commentary to point out that each of the concentration response relationships are consistent with the hormetic dose response. The paper of Siwak (Food Chem 141:1227-1241, 2013) is unique in that it provides a comparison of a relatively large number of agents using the identical experimental protocol.

A toxicological evaluation for safety assessment of ruthenium-based diosmetin complex in rats.

The flavonoid-based organometallic complexes have been identified as novel bioactive compounds with enhanced pharmacological and therapeutic activity. In this study, the ruthenium-p-cymene diosmetin complex was synthesized, characterized, and investigated for toxicological profiling through different toxicological and genotoxicological studies which include acute and sub-acute toxicity, chromosomal aberration, and bone marrow micronucleus study. The acute oral toxicity study demonstrated the LD50 dose of the complex at 500 mg/kg body weight which further instigated the sub-acute doses i.e. 50, 100, and 200 mg/kg. The histopathological analysis demonstrated that the 400 mg/kg dose was associated with severe toxicological incidences of the vital organs (liver, kidney, pancreas, testis, and stomach) except the ovary with increased levels of ALP, AST, ALT, and WBC count. However, 50, 100, and 200 mg/kg doses did not show any toxicological alteration and maintained the normal levels of hematological and serum biochemical parameters. The genotoxicological assessment of the complex depicted no such genetic damage or mutagenicity in any complex treated groups. In conclusion, the 50, 100, and 200 mg/kg doses were determined as therapeutic dose of the novel ruthenium-p-cymene diosmetin complex without any genotoxic and mutagenic potential which can be further implemented in the investigation of various pharmacological and therapeutic interventions.

Acute and 13 weeks subchronic toxicological evaluation of the flavonoid-rich extract of Sophora flavescens.

The roots of Sophora flavescens have a long history of use in Chinese medicine for the treatment of various medical conditions. Flavonoids from the ethyl acetate extract of S. flavescens have shown anti-inflammatory, anticancer, and antidiabetic properties. The objective of this study was to evaluate the toxicological profile of a flavonoid-rich extract of S. flavescens (SFEA). We conducted acute and sub-chronic oral toxicity studies of SFEA in Kunming (KM) mice and Sprague-Dawley (SD) rats. Acute oral administration of 9.0 g/kg SFEA did not result in mortality, clinical signs of toxicity, or abnormal changes in the body weight or food consumption patterns. No significant changes in hematological, blood biochemical, or histopathological parameters were observed. A 13-week sub-chronic toxicity study was conducted in SD rats; the rats were orally administrated with various doses of SFEA (in mg/kg): 0 (control), 40, 80, 400, 800, and 1200. Mortality, clinical signs, or treatment-related changes in body weight, food consumption, hematological parameters, blood biochemical parameters, organ weights, or histopathological parameters were not observed. We found that SFEA is practically nontoxic to KM mice at a dose of 9.0 g/kg and that the no-observed-adverse-effect-level (NOAEL) of SFEA in SD rats is greater than 1200 mg/kg.

Ninety-day oral toxicological profiling of Kolaviron (an extract of Garcinia kola) in male and female rats.

There remains an insufficiency of data on the long-term toxicological profile of Garcinia kola Heckel and its extract, Kolaviron (KV), despite several studies on its pharmacological effects. This research was designed to investigate the long-term histopathological, hematological, biochemical, hormonal, reproductive, and oxidative effects of 90 days administration of KV to male and female rats, as well as additional 30 days reversibility study to assess the potential for reversal of induced effects. Fifty-six male and female Wistar rats divided into four groups were treated orally with distilled water/propylene glycol, 20 mg/kg KV, 100 mg/kg KV, and 500 mg/kg KV for 90 days. At the end of 90 days and 30 additional days of reversibility study, 5 ml blood was collected from animals for relevant analyses. Vital organs were harvested for histopathological assessments. In this study, KV did not elicit any adverse effect on histopathological presentations of vital organs which were generally non-abnormal. There was significant increase (p < 0.05) in LEU, MON, EOS%, BAS%, HCT (male animals) and LYM%, EOS%, BAS%, RBC, hemoglobin and MCH (female animals). There was significant diminution (p < 0.05) in cholesterol, triglycerides, LDL, and VLDL levels, with significant increase (p < 0.05) in HDL level in both male and female animals. KV elicited a non-significant increase in sperm count accompanied by a significant increase (p < 0.05) in levels of Follicle stimulating hormone (FSH) and testosterone in male rats. Furthermore, KV elicited significant (p < 0.001-0.05) elevation in the levels of GSH, SOD and CAT, and diminution in the level of MDA. The findings in this study suggest that long-term administration of KV is considerably safe with some variations in response between male and female animals. The possible sustenance of observed effects after cessation of KV administration, lipid lowering, erythropoiesis inducing, and immune system boosting activities of KV were confirmed in this study.

Glycoalkaloid Composition and Flavonoid Content as Driving Forces of Phytotoxicity in Diploid Potato.

Despite their advantages, biotechnological and omic techniques have not been applied often to characterize phytotoxicity in depth. Here, we show the distribution of phytotoxicity and glycoalkaloid content in a diploid potato population and try to clarify the source of variability of phytotoxicity among plants whose leaf extracts have a high glycoalkaloid content against the test plant species, mustard. Six glycoalkaloids were recognized in the potato leaf extracts: solasonine, solamargine, α-solanine, α-chaconine, leptinine I, and leptine II. The glycoalkaloid profiles of the progeny of the group with high phytotoxicity differed from those of the progeny of the group with low phytotoxicity, which stimulated mustard growth. RNA sequencing analysis revealed that the upregulated flavonol synthase/flavonone 3-hydroxylase-like gene was expressed in the progeny of the low phytotoxicity group, stimulating plant growth. We concluded that the metabolic shift among potato progeny may be a source of different physiological responses in mustard. The composition of glycoalkaloids, rather than the total glycoalkaloid content itself, in potato leaf extracts, may be a driving force of phytotoxicity. We suggest that, in addition to glycoalkaloids, other metabolites may shape phytotoxicity, and we assume that these metabolites may be flavonoids.

Standardization, in-silico and in-vivo safety assessment of methanol extract of Ziziphus mauritiana Lam leaves.

Ziziphus mauritana Lam leaves were used to treat asthma, diabetes, pain, and inflammation in the Indian traditional system of medicine. The leaves of the Ziziphus mauritiana Lam were consumed as a vegetable in Indonesia and India. The present study aims to predict the pharmacokinetic properties of flavonoids identified & quantified through U(H)PLC and to evaluate the safety of methanol extract of Ziziphus mauritana Lam leaves (MEZ) in rats. A U(H)PLC-ESI-QTOF-MS/MS was performed to identify flavonoids present in MEZ and quantified using U(H)PLC method. The in-silico ADME properties of the flavonoids were analyzed using Schrodinger Maestro software. The acute oral toxicity study was performed by administering a single dose of MEZ (5000 mg/kg) in female rats and observed for 14 days. The sub-chronic studies were carried out by oral administration of MEZ at 500, 750, and 1000 mg/kg daily for 90 days. The changes in hematological parameters, clinical biochemistry, and histopathology were observed after the treatment period. Eight flavonoids rutin, kaempferol, luteolin, myricetin, catechin, and apigenin were identified from were identified in UPLC-QTOF-MS/MS analysis. These results showed the highest amount of luteolin (5.41 µg/ml) and kaempferol (4.02 µg/ml) present in MEZ. No signs of toxicity or mortality were observed in acute toxicity studies. In the sub-chronic studies, data showed that MEZ does not produce any changes in hematological and clinical biochemical parameters compared to control rats. MEZ (1000 mg/kg) significantly (p < 0.05) reduced total cholesterol, triglycerides, in male rats, which was more prominent on day 90. The histopathological analysis also revealed no changes in the vital organs. These results conclude that MEZ was considered safe and well-tolerated in rats.

Comparative study of the genotoxic activity of Artemisia vulgaris L. and Artemisia alba Turra extracts in vitro.

In this study, the genotoxic activity of acetone and aqueous extracts of two species of genus Artemisia (Artemisia vulgaris L. and Artemisia alba Turra), and possible role of their polyphenolic composition in the observed activities were investigated. Polyphenolic contents were evaluated by high-performance liquid chromatography (HPLC-PDA), while the genotoxic activity was tested using cytokinesis block micronucleus (CBMN) assay on human peripheral blood lymphocytes (PBLs) in vitro. HPLC-PDA showed that both A. alba extracts were richer in polyphenolic contents than A. vulgaris extracts. The acetone A. alba extract was the richest of polyphenolic content where we detected six phenolic acids and two flavonoids. CBMN assay showed that aqueous extract of A. vulgaris significantly increased micronucleus (MN) frequency in the PBLs treated with all tested concentrations (10, 50, 100, and 250 µg/mL), while A. alba did not significantly affect the mean MN frequency. Further, both acetone extracts were genotoxic in all tested concentrations, except the lowest tested (10 µg/mL) of A. alba. All tested extracts affected the nuclear division index (NDI) except the aqueous A. alba extract (p < 0.05). Based on our results, we can conclude that both acetone and aqueous A. vulgaris extracts and A. alba acetone extract were genotoxic in PBLs in vitro. A. alba aqueous extract was not genotoxic and cytotoxic in tested concentrations. We suggest that the aqueous extract of A. alba can be used in treatment, which has been confirmed by traditional medicine, but with a high dose of caution and not in high concentrations.

Assessment of the risks of copper- and zinc oxide-based nanoparticles used in Vigna radiata L. culture on food quality, human nutrition and health.

The present article aims to assess the phytotoxic effects of copper and zinc oxide nanoparticles (Cu NPs, ZnO NPs) on mung bean (Vigna radiata L.) and their possible risk on food quality and safety. We also study the molecular mechanisms underlying the toxicity of nanosized Cu and ZnO. Seeds of mung bean were germinated under increasing concentrations of Cu NPs and ZnO NPs (10, 100, 1000, 2000 mg/L). We analyzed levels of free amino acids, total soluble sugars, minerals, polyphenols and antioxidant capacity. Our results showed that depending on the concentrations used of Cu NPs and ZnO NPs, the physiology of seed germination and embryo growth were modified. Both free metal ions and nanoparticles themselves may impact plant cellular and physiological processes. At 10 mg/L, an improvement of the nutritive properties, in terms of content in free amino acids, total soluble sugars, essential minerals, antioxidant polyphenols and flavonoids, was shown. However, higher concentrations (100-2000 mg/L) caused an alteration in the nutritional balance, which was revealed by the decrease in contents and quality of phenolic compounds, macronutrients (Na, Mg, Ca) and micronutrients (Cu, Fe, Mn, Zn, K). The overall effects of Cu and ZnO nanoparticles seem to interfere with the bioavailability of mineral and organic nutrients and alter the beneficial properties of the antioxidant phytochemicals, mineral compounds, phenolic acids and flavonoids. This may result in a potential hazard to human food and health, at some critical doses of nanofertilizers. This study may contribute in the guidelines to the safe use of nanofertilizers or nanosafety, for more health benefit and less potential risks.

In vitro anti-Acanthamoeba activity of flavonoid glycosides isolated from Delphinium gracile, D. staphisagria, Consolida oliveriana and Aconitum napellus.

Acanthamoeba spp. are widely distributed in the environment and cause serious infections in humans. Treatment of Acanthamoeba infections is very challenging and not always effective which requires the development of more efficient drugs against Acanthamoeba spp. The purpose of the present study was to test medicinal plants that may be useful in the treatment of Acanthamoeba spp. Here we evaluated the trophozoital and cysticidal activity of 13 flavonoid glycosides isolated from Delphinium gracile, D. staphisagria, Consolida oliveriana and from Aconitum napellus subsp. Lusitanicum against the amoeba Acanthamoeba castellanii. AlamarBlue Assay Reagent® was used to determine the activity against trophozoites of A. castellanii, and cytotoxic using Vero cells. Cysticidal activity was assessed on treated cysts by light microscopy using a Neubauer chamber to quantify cysts and trophozoites. Flavonoids 1, 2, 3 and 4 showed higher trophozoital activity and selectivity indexes than the reference drug chlorhexidine digluconate. In addition, flavonoid 2 showed 100% cysticidal activity at a concentration of 50 µm, lower than those of the reference drug and flavonoid 3 (100 µm). These results suggest that flavonoids 2 and 3 might be used for the development of novel therapeutic approaches against Acanthamoeba infections after satisfactory in vivo evaluations.

AMPK/PGC-1α/GLUT4-Mediated Effect of Icariin on Hyperlipidemia-Induced Non-Alcoholic Fatty Liver Disease and Lipid Metabolism Disorder in Mice.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the world. Therapeutic activity of icariin, a major bioactive component of Epimedii Herba, in NAFLD is still unknown. Herein, the C57BL/6J mice were fed with a high-fat diet for 16 weeks to establish a NAFLD model. Mice were assigned to five groups: control group, NAFLD group, and icariin treatment groups. Effects of icariin on blood indices, glucose tolerance, insulin sensitivity, histopathological morphology, cell apoptosis, lipid accumulation, and AMPK signaling were analyzed. In addition, another cohort of mice were assigned to five groups: control group, NAFLD group, dorsomorphin treatment group, icariin treatment group, and dorsomorphin + icariin treatment group. Expression of proteins in liver tissues associated with AMPK signaling, and levels of ALT and AST were evaluated. Icariin attenuated the NAFLD-induced increase of the TG, TC, LDL-C, ALT, AST levels. HDL-C levels were affected neither by NAFLD nor by icariin. Furthermore, icariin treatment (100-200 mg/kg) counteracted the NAFLD-reduced glucose tolerance and insulin sensitivity and modulated histopathological changes, cell apoptosis, and lipid accumulation in liver tissues. Additionally, icariin mitigated the NAFLD-induced up-regulation of the cleaved caspase 3/9, SREBP-1c, and DGAT-2 levels, and enhanced the expression level of CPT-1, p-ACC/ACC, AMPKα1, PGC-1α, and GLUT4. Effects of icariin on the AMPK signaling and levels of AST and ALT could be reversed by AMPK inhibitor, dorsomorphin. This paper investigates the glucose-reducing and lipid-lowering effects of icariin in NAFLD. Moreover, icariin might function through activating the AMPKα1/PGC-1α/GLTU4 pathway.

Phytochemical screening, phenolic and flavonoid contents, antioxidant and cytogenotoxicity activities of Combretum leprosum Mart. (Combretaceae).

Combretum leprosum Mart. (Combretaceae), a shrub popularly known as mofumbo, is used in folk medicine for treatment of uterine bleeding, pertussis, gastric pain, and as a sedative. The aim of this study was to (1) determine the phytochemical profile,(2) identify chemical constituents and (3) examine antioxidant and cytogenotoxic activity of ethanolic extracts and fractions of stem bark and leaves. The plant material (leaf and stem bark) was submitted to extraction with ethanol, followed by partition using hexane, chloroform, and ethyl acetate. It was possible to identify and quantify the epicatechin in the ethanolic stem bark extract (0.065 mg/g extract) and rutin in the leaf extract (3.33 mg/g extract). Based upon in vitro tests a significant relationship was noted between findings from antioxidant tests and levels of total phenolic and flavonoid. Comparing all samples (extracts and fractions), the ethyl acetate fractions of stem bark (411.40 ± 15.38 GAE/g) and leaves (225.49 ± 9.47 GAE/g) exhibited higher phenolic content, whereas hexanic fraction of stem bark (124.28 ± 56 mg/g sample) and ethyl acetate fraction of leaves (238.91 ± 1.73 mg/g sample) demonstrated a higher content of flavonoids. Among the antioxidant tests, the intermediate fraction of stem bark (28.5 ± 0.60 µg/ml) and ethyl acetate fraction of leaves (40 ± 0.56 µg/ml) displayed a higher % inhibition of free radical DPPH activity, whereas intermediate fraction of stem bark (27.5 ± 0.9 µg/ml) and hydromethanol fraction of leaves (81 ± 1.4 µg/ml) demonstrated inhibition of the free radical ABTS. In biological tests (Allium cepa and micronucleus in peripheral blood), data showed that none of the tested concentrations of ethanolic extracts of leaves and stem bark produced significant cytotoxicity, genotoxicity, and mutagenic activity.Abbreviations AA%: percentage of antioxidant activity; ABTS: 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid); CEUA: Ethics Committee in the Use of Animals; TLC: Thin Layer Chromatography; DNA: deoxyribonucleic acid; DPPH: 1,1-diphenyl-2-picrylhydrazyl; ROS: Reactive oxygen species; EEB: ethanol extract of the stem bark; HFB: Hexanic fraction of stem bark; IFB: Intermediate fraction of stem bark; CFB: Chloroform fraction of stem bark; EAFB: Ethyl acetate fraction of stem bark; HMFB: Hydromethanol fraction of the stem bark; EEL: Ethanol extract from leaves; HFL: Hexane fraction of leaves; CFL: Chloroform fraction of leaves; EAFL: Ethyl acetate fraction of leaves; HMFL: Hydromethanol fraction of leaves; GAE: Gallic Acid Equivalent; IC50: 50% inhibition concentration; HCOOH: Formic acid; HCl: hydrochloric acid; HPLC: High-performance liquid chromatography; MN: micronucleus; WHO: World Health Organization; UFLC: Ultra-Fast Liquid Chromatography; UESPI: State University of Piauí.

Evaluation of in vitro cytotoxicity and in vivo potential toxicity of the extract from in vitro cultivated Anoectochilus roxburghii Lindl.

Anoectochilus roxburghii Lind. (A. roxburghii) has promising anti-oxidant, hyperglycemic, hepatoprotective, and immunomodulatory activities as well as anti-tumor effects. However, the pharmacological actions of in vitro cultured plants remain to be determined. Therefore, the objective of the study was to assess in vitro cytotoxicity and in vivo potential toxicity of an extract derived from in vitro cultivated A. roxburghii, termed as iARE. The total flavonoid content and predominant flavonoid compounds of extract were identified and quantitatively analyzed. The in vitro cytotoxicity of iARE was examined using several cancer and normal cell lines. The apoptotic activity and expression of apoptosis-associated genes were also examined in MCF7 cells to determine the underlying mechanisms related to anti-proliferative effects. In vivo potential toxicity of iARE was assessed following acute and subchronic oral administration in Sprague Dawley rats. Quercetin, kaempferol, and isorhamnetin were three flavonoid components identified in iARE. The extract exerted cytotoxic effects on various cancer cells but not normal fibroblasts. Apoptosis in MCF7 cells was induced by iARE in a concentration-dependent manner associated with increased Bax/Bcl-2 ratio and reduced mitochondrial membrane potential ΔΨm, leading to release of cytochrome c, activation of caspase-3/7 and caspase-9, and cleavage of PARP. In the acute oral toxicity study, no mortality or toxicological signs were observed in rats at 1000 or 5000 mg/kg. In a subchronic oral toxicity study, iARE at a dosage of up to 1000 mg/kg produced no mortality or treatment-related adverse effects on general behavior, food intake, body weight, relative organ weights. No apparent marked changes in the histopathology of the liver and kidney were detected. Data demonstrated that iARE induced in vitro cytotoxic effects in cancer cells are associated with lackof invivo toxicity. Thus, iARE was suggested to be considered as apotential therapeutic candidate for cancer treatment.

Screening of hepatotoxic compounds in Psoralea corylifolia L., a traditional Chinese herbal and dietary supplement, using high-resolution mass spectrometry and high-content imaging.

Owing to the complexity of the composition of herbal and dietary supplements, it is a challenging problem to efficiently screen and identify active or toxic compounds. Psoralea corylifolia L. (PCL) was selected as the subbject to establish a methodology for rapid screening and identification of hepatotoxic compounds. High-content imaging, ultra-performance liquid chromatography and high-resolution mass spectrometry were used in this study to detect the hepatotoxicity and identify unknown compounds in PCL samples. Then, putative toxic compounds which are highly related to hepatotoxicity were screened by spectrum-toxicity correlation analysis, and the toxicity intensity verified by high-content imaging. The maximum nontoxic dose of processed samples with good detoxification effect reduced more than 9 times compared with unprocessed raw medicinal materials. Spectrum-toxicity correlation analysis showed that bavachinin A, bavachin, isobavachalcone and neobavaisoflavone had high correlation with the hepatotoxicity of PCL, and psoralen and isopsoralen had low correlation with hepatotoxicity. This study verified the hepatotoxicity of these six putative compound monomers, proving the results of spectrum-toxicity correlation analysis. Based on the correlation analysis of high-resolution mass spectrometry of detection compounds and high-content imaging of hepatocyte toxicity data, the potential toxic compound of herbal and dietary supplement products can be quickly and accurately screened.

Polyphenols as anticancer agents: Toxicological concern to healthy cells.

Polyphenols are a group of diverse chemical compounds present in a wide range of plants. Various biological properties such as antiallergic, antiviral, antibacterial, anticarcinogenic, antiinflammatory, antithrombotic, vasodilatory, and hepatoprotective effect of different polyphenols have been reported in the scientific literature. The major classes of polyphenols are flavonoids, stilbenoids, lignans, and polyphenolic acids. Flavonoids are a large class of food constituents comprising flavones, isoflavanones, flavanones, flavonols, catechins, and anthocyanins sub-classes. Even with seemingly broad biological activities, their use is minimal clinically. Among the other concurrent problems such as limited bioavailability, rapid metabolism, untargeted delivery, the toxicity associated with these polyphenols has been a topic of concern lately. These polyphenols have been reported to result in different forms of toxicity that include organ toxicity, genotoxicity, mutagenicity, cytotoxicity, etc. In the present article, we have tried to unravel the toxicological aspect of these polyphenols to healthy cells. Further high-quality studies are needed to establish the clinical efficacy and toxicology concern leading to further exploration of these polyphenols.

Toxicological evaluation of a flavonoid, chrysin: morphological, behavioral, biochemical and histopathological assessments in rats.

Nowadays, medicines from plant sources play a vital role in healthcare management. Chrysin, a plant flavonoid, possesses a wide range of pharmacological activities. The aim of present investigation was to evaluate the safety of chrysin by determining toxicity after acute and sub-chronic oral administration in rats. Acute oral toxicity (AOT) and sub-chronic oral toxicity studies of chrysin were carried out according to OECD 425 and OCED 408 in Sprague Dawley rats. In AOT, oral administration of chrysin (5000 mg/kg) showed 40% mortality. In the sub-chronic toxicity study, daily oral administration of chrysin (1000 mg/kg) showed significantly decreased body weight whereas liver weight was increased significantly in male rats. A significant alteration in the hematology (RBC, MCH, MCHC, TLC, lymphocytes, and neutrophil) and blood chemistry (albumin, bilirubin, ALT, AST, creatinine, and GGT) were found in chrysin (1000 mg/kg) treated rats which were either limited to one sex or lacked dose-response or were within the normal laboratory ranges. There was a significant increase in hepatic and renal oxido-nitrosative stress in chrysin (1000 mg/kg) treated rats. There was no significant change in electrocardiographic (except heart rate), hemodynamic, the left ventricular function, and lung function test. Renal and hepatic histological aberrations were induced in chrysin (1000 mg/kg) treated rats. In conclusion results of the present investigation determined the LD50 value of chrysin to be 4350 mg/kg whereas NOAEL and LOAEL of chrysin was found to be 500 and 1000 mg/kg, respectively for both the sexes.

Synthesis and Evaluation of the Acetylcholinesterase Inhibitory Activities of Some Flavonoids Derived from Naringenin.

Alzheimer's disease (AD) is an irreversible neurodegenerative disease that affects many older people adversely. AD has been putting a huge socioeconomic burden on the healthcare systems of many developed countries with aging populations. The need for new therapies that can halt or reverse the progression of the disease is now extremely great. A research approach in the finding new treatment for AD that has attracted much interest from scientists for a long time is the reestablishment of cholinergic transmission through inhibition of acetylcholinesterase (AChE). Naringenin is a flavonoid with the potential inhibitory activity against AChE. From naringenin, many other flavonoid derivatives, such as flavanones and chalcones, can be synthesized. In this study, by applying the Williamson method, nine flavonoid derivatives were synthesized, including four flavanones and five chalcones. The evaluation of AChE inhibitory activity by the Ellman method showed that there were four substances (2, 4, 5, and 7) with relatively good biological activities (IC50 < 100 µM), and these biological activities were better than that of naringenin. The molecular docking revealed that strong interactions with amino acid residue Ser200 of the catalytic triad and those of the peripheral region of the enzyme were crucial for strong effects against AChE. Compound 7 had the strongest AChE inhibitory activity (IC50 13.0 ± 1.9 µM). This substance could be used for further studies.

Microbial Conjugation Studies of Licochalcones and Xanthohumol.

Microbial conjugation studies of licochalcones (1-4) and xanthohumol (5) were performed by using the fungi Mucor hiemalis and Absidia coerulea. As a result, one new glucosylated metabolite was produced by M. hiemalis whereas four new and three known sulfated metabolites were obtained by transformation with A. coerulea. Chemical structures of all the metabolites were elucidated on the basis of 1D-, 2D-NMR and mass spectroscopic data analyses. These results could contribute to a better understanding of the metabolic fates of licochalcones and xanthohumol in mammalian systems. Although licochalcone A 4'-sulfate (7) showed less cytotoxic activity against human cancer cell lines compared to its substrate licochalcone A, its activity was fairly retained with the IC50 values in the range of 27.35-43.07 µM.

Neurotoxic Effect of Flavonol Myricetin in the Presence of Excess Copper.

Oxidative stress (OS) induced by the disturbed homeostasis of metal ions is one of the pivotal factors contributing to neurodegeneration. The aim of the present study was to investigate the effects of flavonoid myricetin on copper-induced toxicity in neuroblastoma SH-SY5Y cells. As determined by the MTT method, trypan blue exclusion assay and measurement of ATP production, myricetin heightened the toxic effects of copper and exacerbated cell death. It also increased copper-induced generation of reactive oxygen species, indicating the prooxidative nature of its action. Furthermore, myricetin provoked chromatin condensation and loss of membrane integrity without caspase-3 activation, suggesting the activation of both caspase-independent programmed cell death and necrosis. At the protein level, myricetin-induced upregulation of PARP-1 and decreased expression of Bcl-2, whereas copper-induced changes in the expression of p53, p73, Bax and NME1 were not further affected by myricetin. Inhibitors of ERK1/2 and JNK kinases, protein kinase A and L-type calcium channels exacerbated the toxic effects of myricetin, indicating the involvement of intracellular signaling pathways in cell death. We also employed atomic force microscopy (AFM) to evaluate the morphological and mechanical properties of SH-SY5Y cells at the nanoscale. Consistent with the cellular and molecular methods, this biophysical approach also revealed a myricetin-induced increase in cell surface roughness and reduced elasticity. Taken together, we demonstrated the adverse effects of myricetin, pointing out that caution is required when considering powerful antioxidants for adjuvant therapy in copper-related neurodegeneration.

Applicability of the OECD 455 in-vitro assay for determination of hERa agonistic activity of isoflavonoids.

The Organisation for Economic Co-operation and Development (OECD)-validated transactivation assay using the human estrogen receptor alpha (hERα) Hela9903 cell line is used for activity evaluation of hERα agonists and antagonists. Due to many advantages, this assay is broadly used as an initial screening process. However, response significantly higher from that of 17-ß estradiol (E2) was observed with phytoestrogens for concentrations commonly above 1 µM in previous studies. The main aim of this study was thus to ascertain the applicability of OECD protocol 455 for evaluation of estrogenic activity of natural flavonoids, including known phytoestrogens. The estrogenic activities of aglycones as well as of O-methylated and glycosylated flavonoids were evaluated. Supra-maximal luciferase activity was seen for most of the flavonoids tested at concentrations even below 1 µM. hERα-mediated luciferase expression was confirmed with the competition assay specified in OECD protocol 455. However, at concentrations above 1 µM, non-specific interactions were also observed. Instead of EC50 values, which could not be determined for most of the isoflavonoids tested, the concentrations corresponding to 10% (PC10) and 50% (PC50) of the maximum activity of the positive control, E2, were used for quantitative determination of estrogenic activities. Appropriate evaluation of the data obtained with the current OECD protocol 455 validated assay represents a valuable tool for initial screening of natural flavonoids for estrogenic activity.

Skullcapflavone II inhibits osteoclastogenesis by regulating reactive oxygen species and attenuates the survival and resorption function of osteoclasts by modulating integrin signaling.

Many bone diseases, such as osteoporosis and rheumatoid arthritis, are attributed to an increase in osteoclast number or activity; therefore, control of osteoclasts has significant clinical implications. This study shows how skullcapflavone II (SFII), a flavonoid with anti-inflammatory activity, regulates osteoclast differentiation, survival, and function. SFII inhibited osteoclastogenesis with decreased activation of MAPKs, Src, and cAMP response element-binding protein (CREB), which have been known to be redox sensitive. SFII decreased reactive oxygen species by scavenging them or activating nuclear factor-erythroid 2-related factor 2 (Nrf2), and its effects were partially reversed by hydrogen peroxide cotreatment or Nrf2 deficiency. In addition, SFII attenuated survival, migration, and bone resorption, with a decrease in the expression of integrin ß3, Src, and p130 Crk-associated substrate, and the activation of RhoA and Rac1 in differentiated osteoclasts. Furthermore, SFII inhibited osteoclast formation and bone loss in an inflammation- or ovariectomy-induced osteolytic mouse model. These findings suggest that SFII inhibits osteoclastogenesis through redox regulation of MAPKs, Src, and CREB and attenuates the survival and resorption function by modulating the integrin pathway in osteoclasts. SFII has therapeutic potential in the treatment and prevention of bone diseases caused by excessive osteoclast activity.-Lee, J., Son, H. S., Lee, H. I., Lee, G.-R., Jo, Y.-J., Hong, S.-E., Kim, N., Kwon, M., Kim, N. Y., Kim, H. J., Lee, Y. J., Seo, E. K., Jeong, W. Skullcapflavone II inhibits osteoclastogenesis by regulating reactive oxygen species and attenuates the survival and resorption function of osteoclasts by modulating integrin signaling.