Enhanced inhibitory efficiency against toxic bloom forming Raphidiopsis raciborskii by Streptomyces sp. HY through triple algicidal modes: Direct and indirect attacks combined with bioflocculation.

Raphidiopsis raciborskii (R. raciborskii) forms harmful cyanobacterial blooms globally, and poses a great threat to the safety of drinking water and public health. There is a great need to develop eco-friendly biological alternative measures to mitigate mass blooms of R. raciborskii. However, previous rare studies on algicidal microorganisms against R. raciborskii restricted this aim. Recently, an algicidal bacterium Streptomyces sp. HY (designated HY) was identified with flavones producing ability, and could remove up to 98.73 % of R. raciborskii biomass within 48 h by directly attacking the cyanobacterium and release of algicidal substances (i.e., flavonoids) with a inoculum ratio of 5 %. Algicidal rate of HY was enhanced by 88.05 %, 89.33 % under dark and light, and full-light conditions respectively, when compared with the dark condition. Its algicidal substances were stable in a broad range of temperature (-80-55 °C) and pH (3-11) conditions, and all treated groups exhibited ≈ 100 % algicidal rate at day 3. HY treatment disrupted the photosynthesis system and triggered serious oxidative stress resulting in severe morphological injury. Thereby, HY treatment significantly affected expression levels of several essential genes (i.e., psbA, psaB, rbcL, ftsZ, recA, grpE), and simultaneously inhibited the biosynthesis and release of cylindrospermopsin. Yet, HY treatment didn't show any toxicity to zebrafish test embryos. Such results indicate that HY is a promising algicidal candidate strain to control global R. raciborskii blooms, and holds great promises for an effective biological measure to sustain water safety.

Phlomis crinita Cav. From Algeria: A source of bioactive compounds possessing antioxidant and wound healing activities.

ETHNOPHARMACOLOGICAL RELEVANCE: Phlomis crinita Cav. (Lamiaceae), locally known as "El Khayata" or "Kayat El Adjarah", is traditionally used in Algeria for its wound-healing properties. AIM OF THE STUDY: Investigate, for the first time, the phytochemical profile, safety, antioxidant and wound-healing activities of the flowering tops methanolic extract of P. crinita (PCME) collected from Bouira Province in the North of Algeria. MATERIALS AND METHODS: Preliminary phytochemical assays were carried out on PCME to quantify the main classes of bioactive compounds, such as total phenols, flavonoids, and tannins. An in-depth LC-DAD-ESI-MS analysis was carried out to elucidate the phytochemical profile of this plant species. Antioxidant activity was investigated by several colorimetric and fluorimetric assays (DPPH, TEAC, FRAP, ORAC, ß-carotene bleaching and ferrozine assay). The acute oral toxicity of PCME (2000 mg/kg b.w.) was tested in vivo on Swiss albino mice, whereas the acute dermal toxicity and wound-healing properties of the PCME ointment (1-5% PCMO) were tested in vivo on Wistar albino rats. Biochemical and histological analyses were carried out on biological samples. RESULTS: The phytochemical screening highlighted a high content of phenolic compounds (175.49 ± 0.8 mg of gallic acid equivalents/g of dry extract), mainly flavonoids (82.28 ± 0.44 mg of quercetin equivalents/g of dry extract). Fifty-seven compounds were identified by LC-DAD-ESI-MS analysis, belonging mainly to the class of flavones (32.27%), with luteolin 7-(6″-acetylglucoside) as the most abundant compound and phenolic acids (32.54%), with salvianolic acid C as the most abundant compound. A conspicuous presence of phenylethanoids (15.26%) was also found, of which the major constituent is forsythoside B. PCME showed a strong antioxidant activity with half-inhibitory activity (IC50) ranging from 1.88 to 37.88 µg/mL and a moderate iron chelating activity (IC50 327.44 µg/mL). PCME appears to be safe with Lethal Dose 50 (LD50) ≥ 2000 mg/kg b.w. No mortality or toxicity signs, including any statistically significant changes in body weight gain and relative organs' weight with respect to the control group, were recorded. A significant (p < 0.001) wound contraction was observed in the 5% PCMO-treated group with respect to the untreated and petroleum jelly groups between 8 and 20 days, whereas no statistically significant results were observed at the two lower doses (1 and 2% PCMO). In addition, the 5% PCMO-treated group showed a statistically significant (p < 0.05) wound healing activity with respect to the reference drug-treated group, showing, at the end of the study, the highest wound contraction percentage (88.00 ± 0.16%). CONCLUSION: PCME was safe and showed strong antioxidant and wound-healing properties, suggesting new interesting pharmaceutical applications for P. crinita based on its traditional use.

"GC-MS profiling, sub-acute toxicity study and total phenolic and flavonoid content analysis of methanolic leaf extract of Schima wallichii (D.C.) Korth-a traditional antidiabetic medicinal plant".

ETHNOPHARMACOLOGICAL RELEVANCE: Schima wallichii (D.C.) Korth is traditionally used in Manipur, India for treatment of diabetes and hypertension. However, there is no data reported regarding safety profile of this medicinal plant upon repeated per oral administration over a period of time. AIM OF THE STUDY: In the current study phytochemical profile, toxicological profile and total phenolic and flavonoid compound content of Schima wallichii leaves extract were evaluated. MATERIALS AND METHODS: Gas chromatography coupled to mass spectrometry was performed for chemical profiling by using Gas Chromatography-Mass Spectrometry/Mass Spectrometry (GC-MS/MS), Shimadzu, TQ8040 system. A 28 days sub-acute toxicity study was carried out using albino Wistar rats by administering 3 different doses (200, 400 and 800 mg/kg body weight per oral) of methanol leaves extract. Changes in body weights were recorded weekly. Serum biochemical parameters were estimated as well as blood-cell count was done to check the effect of extract on haematopoietic system. Histopathology of vital organs viz. kidney, heart, brain, liver was performed to find any pathological indications. Since, liver is main the site for xenobiotic metabolism, estimation of the level of glutathione, catalase and lipid peroxidation were done. Further, total phenolic and flavonoid compound content estimation was performed for the leaves extract. RESULTS: GC-MS revealed 14 major compounds with area percentage >1% of which quinic acid, n-Hexadecanoic acid, 9,12,15-Octadecatrienoic acid, (Z,Z,Z)-, Octatriacontyl trifluoroacetate, are three major compounds. No mortality was observed after the treatment with extract. Blood-cell count and biochemical parameters didn't show significant deviation as compared to control group. Histopathology study of vital organs viz. (liver, kidney, heart and brain) showed normal cellular construction comparing to control group. There was no sign of membrane lipid peroxidation, depletion of catalase level and glutathione level in liver. The result demonstrates that NOAEL (no-observed-adverse-effect levels) in the sub-acute toxicity was above 800 mg/kg. The leaves extract showed significant total phenol and flavonoid content. CONCLUSION: The present study revealed that Schima wallichii possessed important bioactive compounds with therapeutic values. The plant was safe for consumption after repeated high doses administration in rats and possesses significant amount of total phenol and flavonoid content.

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.

Bavachin induces liver injury and cell apoptosis by targeting Wnt/ß-catenin/DRP1 signaling pathway mediated mitochondrial dysfunction.

Psraleae Fructus (PF) is a well-known traditional Chinese medicine in China. While numerous liver injury reports caused by PF limits its clinical application. Bavachin, a flavonoid compound isolated from the fruits of Psoralea corylifolia L., has been validated to induce direct apoptosis in hepatocytes and liver tissues in our previous studies. However, the subcellular mechanisms of bavachin induced liver injury is still elusive. Here, utilizing 6-week-old C57BL/6 J mice and human embryonic hepatocytes (L02 cells), we report that bavachin activates dynamic-related protein 1 (DRP1) mediated excess mitochondrial fission and endoplasmic reticulum (ER) stress related apoptosis via Wnt/ß-catenin signaling pathway. Notably, DRP1 knockdown or XAV-939 induced Wnt/ß-catenin inhibition decreased bavachin-induced ER stress and cell apoptosis in L02 cells. In addition, bavachin impaired mitochondrial structural and function in the mice liver tissues. Mdivi-1, a mitochondrial fission inhibitor targeting DRP1, prevented bavachin-induced mitochondrial and ER structural damage, ER stress, and liver injury. Our results demonstrated that bavachin induced mitochondrial fission plays a crucial role in bavachin induced ER stress related liver injury, via the mechanism that involved activation of Wnt/ß-catenin signaling pathway.

The natural flavonoid compound deguelin suppresses sperm (Sus Scrofa) functions through abnormal activation of the PI3K/AKT pathway.

Deguelin is a natural flavonoid extracted from plants belonging to the Lonchocarpus, Derris, or Tephrosia genera. It inhibits AKT activity in tumors and has the potential to be used as a treatment for malignant tumors. However, the risks associated with the use of deguelin on male fertility have not yet been explained in detail. Therefore, this study was conducted to investigate the effects of deguelin on sperm functions during capacitation. First, boar spermatozoa were exposed to different concentrations of deguelin (0.1, 1, 10, 50, and 100 µM). Next, sperm functional assessments, such as sperm motility, capacitation status, intracellular ATP level, and cell viability, were performed. The expression levels of PI3K/AKT-related proteins and the phosphorylation of their tyrosine residues were also evaluated by western blotting. No significant difference was observed in cell viability; however, deguelin considerably decreased sperm motility and motion kinematics in a dose-dependent manner. Although no significant difference was observed in the capacitation status, acrosome reaction decreased at high concentrations of deguelin (50 and 100 µM). Furthermore, intracellular ATP levels were significantly decreased in all deguelin treatment groups compared with those in the control group. Results of western blotting revealed that deguelin substantially diminished tyrosine phosphorylation. Interestingly, in contrast to previous studies showing that deguelin inhibits AKT activity, our results showed that it increased the expression of PI3K/AKT pathway-related proteins. Collectively, these findings indicate that deguelin exerts negative effects on sperm functions due to abnormal PI3K/AKT signaling activation. We believe that this is the first study to provide evidence that deguelin can regulate sperm functions independent of PI3K/AKT pathway inhibition. Furthermore, its detrimental effects on male fertility should be considered while developing or using deguelin as a therapeutic agent.

An integrated strategy combining network toxicology and feature-based molecular networking for exploring hepatotoxic constituents and mechanism of Epimedii Folium-induced hepatotoxicity in vitro.

Epimedii Folium (EF), a commonly used herbal medicine to treat osteoporosis, has caused serious concern due to potential hepatotoxicity. Until now, its intrinsic hepatotoxic mechanism and hepatotoxic ingredients remain unclear. Here, a novel high-throughput approach was designed to investigate the intrinsic hepatotoxic of EF. High-content screen imaging (HCS) and biochemical tests were first performed to obtain the cytotoxicity parameter matrix of 17 batch EF samples. EF-treated alpha mouse liver 12 (AML12) cells showed increased reactive oxygen species (ROS), reduced glutathione (GSH) and mitochondrial membrane potential (MMP), and apoptosis and cholestasis were further observed. Network toxicology predicted that EF-triggered hepatotoxiciy was involved in transcription factor (TF) activity. The FXR expression, screened by a TF PCR array, exhibited down-regulation following EF extract administration. Moreover, EF inhibited bile acid (BA) metabolism pathway in an FXR-dependent manner. Pearson correlation between the cytotoxicity parameter matrix and quantification feature table obtained from UHPLC-QTOF data of EF suggested 7 prenylated flavonoids possessed potent hepatotoxicities and their cytotoxicity order was further summarized. The transcriptional repression effects of them on FXR were also verified. Collectively, our findings indicate that FXR is probably responsible for EF-induced hepatotoxicity and prenylated flavonoids may be a major class of hepatotoxic constituents in EF.

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.

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.

Analysis of the toxicological and pharmacokinetic profile of Kaempferol-3-O-β-D-(6"-E-p-coumaryl) glucopyranoside - Tiliroside: in silico, in vitro and ex vivo assay / Análise do perfil toxicológico e farmacocinético de Kaempferol-3-O-β D-(6"-E-p-cumaril) glucopiranosídeo - Tilirosídeo: ensaio in silico, in vitro e ex vivo

Tiliroside is a glycosidic flavonoid present in many plants species including Helicteres velutina K. Schum (Malvaceae sensu lato), commonly known in Brazil as "pitó". This molecule has been shown to have many biological activities, however no study has been carried out to investigate the toxicity of this substance. The present work aimed to evaluate the possible cellular toxicity in silico, in vitro and ex-vivo of the kaempferol-3-O-β-D-(6”-E-p-coumaroyl) glucopyranoside (tiliroside), through chemical structure analysis, toxicity assessment and predictive bioactive properties, using human samples for in vitro and ex-vivo tests. The in silico analysis suggests that tiliroside exhibited great absorption index when penetrating biological membranes. In addition, it also displayed considerable potential for cellular protection against free radicals, and anticarcinogenic, antioxidant, antineoplastic, anti-inflammatory, anti-hemorrhagic and antithrombotic activities. The assessment of the hemolytic and genotoxic effects of tiliroside showed low hemolysis rates in red blood cells and absence of cellular toxicity in the oral mucosa cells. The data obtained indicate that this molecule could be a promising therapeutic approach as a possible new drug with biotechnological potential.

O tilirosídeo é um flavonóide glicosídico presente em muitas espécies de plantas, incluindo Helicteres velutina K. Schum (Malvaceae sensu lato), conhecida no Brasil como “pitó”. Esta molécula mostrou ter muitas atividades biológicas, porém nenhum estudo foi realizado para investigar a toxicidade dessa substância. O presente trabalho teve como objetivo avaliar a possível toxicidade celular in silico, in vitro e ex-vivo do kaempferol-3-O-β-D- (6 ”-Ep-coumaroil) glucopiranosídeo (tilirosídeo), por meio de análises de estrutura química, toxicidade avaliação e propriedades bioativas preditivas, utilizando amostras humanas para testes in vitro e ex-vivo. A análise in silico sugere que o tilirosídeo exibe bom índice de absorção para penetrar nas membranas biológicas. Além disso, apresentou considerável potencial de proteção celular contra os radicais livres e com atividades anticarcinogênica, antioxidante, antineoplásica, antiinflamatória, anti-hemorrágica e antitrombótica. A avaliação dos efeitos hemolíticos e genotóxicos do tilirosídeo mostrou baixas taxas de hemólise nas hemácias e ausência de toxicidade em células da mucosa oral. Os dados obtidos indicam que esta molécula pode possuir uma abordagem terapêutica promissora como uma possível nova droga com potencial biotecnológico.

Safety assessment of marigold flavonoids from marigold inflorescence residue.

ETHNOPHARMACOLOGICAL RELEVANCE: Marigold flavonoids, extracted from marigold (Tagetes erecta L.) inflorescence residues, have attracted significant attention with respect to antioxidant, anti-inflammatory and chelating properties. However, the toxicity of marigold flavonoids have not yet been fully investigated. AIM OF THE STUDY: The main purpose of this study was to assess the safety of marigold flavonoids extracted from Marigold (Tagetes erecta L.) in order to provide information on its nonclinical safety. Thus, the acute oral toxicity, in vitro Ames test, sperm aberration study, bone marrow micronucleus test, subchronic oral toxicity test, and teratogenic potential were carried out in rats or mice. MATERIALS AND METHODS: For an acute oral toxicity test, SD rats and ICR mice (male and female, n = 5) orally received a single dose of 5000 mg/kg marigold flavonoids. Evaluation of marigold flavonoids genotoxic potential with a battery of tests, including an in vitro bacterial reverse mutation test using four mutant strains of Salmonella typhimurium (TA97、TA98、TA100、TA102), an sperm aberration test and an in vivo micronucleus test using bone marrow cells ICR mice that were orally administered marigold flavonoids, an subchronic oral toxicity study and teratogenic test employing male and female SD rats that were orally administered marigold flavonoids. All animals tests were completed in accordance with GB 15193 for toxicity tests. RESULTS: In the acute oral toxicity test, marigold flavonoids given at the dose of 5000 mg/kg body weight for 14 days didn't produce any abnormal clinical symptoms or mortality in SD rats and ICR mice (both sex, n = 5). There was no evidence of genotoxicity of marigold flavonoids based on the results of the in vitro bacterial reverse mutation test (up to 1250 µg/plate), the sperm aberration test (up to 5000 mg/kg body weight), the in vivo micronucleus test (up to 5000 mg/kg body weight), the subchronic oral toxicity study (up to 10 g/kg feed dose) and the teratogenic test (up to 1250 mg/kg body weight). CONCLUSIONS: We found that marigold flavonoids are safe with regard to acute toxicity in rats or mice as well as genotoxicity such as mutagenesis or clastogenesis under the present experimental conditions. These results might support the safety of marigold flavonoids as a potential therapeutic material for the traditional use of herbal medicines and for the further development of novel antioxidant.

A flavonoid-rich fraction of Monolluma quadrangula inhibits xanthine oxidase and ameliorates potassium oxonate-induced hyperuricemia in rats.

Hyperuricemia represents a risk factor for the progression of chronic kidney disease. Oxidative stress and inflammation are implicated in the mechanisms underlying hyperuricemia-mediated kidney injury. Monolluma quadrangula possesses several beneficial effects; however, its effect on hyperuricemia has not been investigated. This study evaluated the renoprotective and xanthine oxidase (XO) inhibitory activity of M. quadrangula in hyperuricemic rats. Phytochemical investigation revealed the presence of six known flavonoid isolated for the first time from this species. The rats received M. quadrangula extract (MQE) and potassium oxonate (PO) for 7 days. In vitro assays showed the radical scavenging and XO inhibitory activities of MQE, and in silico molecular docking revealed the inhibitory activity of the isolated flavonoids towards XO. Hyperuricemic rats showed elevated serum uric acid, creatinine, urea, and XO activity, and renal pro-inflammatory cytokines, MDA and NO, and decreased GSH, SOD, and catalase. MQE ameliorated serum uric acid, urea, creatinine, and XO activity, and renal pro-inflammatory cytokines. In addition, MQE attenuated renal oxidative stress, enhanced antioxidants, downregulated URAT-1, and GLUT-9 and upregulated OAT-1 in PO-induced rats. In conclusion, M. quadrangula attenuated hyperuricemia and kidney impairment by suppressing XO activity, oxidative stress and inflammation, and modulating urate transporters.

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.

The chemical composition of different leaf extracts of Lantana fucata Lindl. influences its cytotoxic potential: A study using the Allium cepa model.

ETHNOPHARMACOLOGICAL RELEVANCE: One of the most popular plants used to treat diseases in Brazil is Lantana fucata. Like most herbal medicines, its consumption is based on popular knowledge, which, despite being considered effective, may cause side effects. AIM OF THE STUDY: Since the scientific data on the pharmacological properties of L. fucata are still incipient, this research aimed to evaluate the cytotoxic and genotoxic potential of different types of extracts (infusion, aqueous and hydroalcoholic), characterizing them chemically. MATERIALS AND METHODS: The cytotoxicity assay was performed by the A. cepa model. The cytotoxicity parameters studied were number of dividing cells and percentage mitotic index (%MI). RESULTS: The result of the A. cepa assay showed that there was a decrease in the number of dividing cells and the percentage mitotic index as concentrations increased, for all extracts, indicating cytotoxicity. However, the hydroalcoholic extract was the most cytotoxic. Chromatography analysis allowed the characterization of secondary metabolites in the extracts, which were very similar. However, a greater abundance of flavonoids and triterpenoids was observed in the hydroalcoholic extract, suggesting that these compounds are responsible for its greater toxicity. CONCLUSIONS: Since the highest doses of extracts showed to have a cytotoxic effect, it is suggested that the ingestion of this species occurs in a moderate way.

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.

Glabrol impurity exacerbates glabridin toxicity in zebrafish embryos by increasing myofibril disorganization.

ETHNOPHARMACOLOGICAL RELEVANCE: Glabridin, extracted from Glycyrrhiza glabra L., is widely used for the treatment of hyperpigmentation because of its anti-inflammatory and antioxidant activities and its ability to inhibit melanin synthesis. This led to the strict regulation of its quality and safety. However, traditional quality control methods used for plant extracts cannot reflect the product quality owing to multiple unknown impurities, which necessitates the further analysis of impurities. AIM OF THE STUDY: The study identified the toxic impurities of glabridin and their toxicological mechanism. MATERIALS AND METHODS: In total, 10 glabridin samples from different sources were quantified using high-performance liquid chromatography. Sample toxicities were evaluated using zebrafish and cell models. To identify impurities, samples with different toxicity were analyzed by ultra-high-performance liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry. The toxicity of related impurities was verified in the zebrafish model. Phalloidin stain was used to evaluate subtle changes in myofibril alignment. RESULTS: Although glabridin content in the samples was similar, there were significant differences in toxicity. The results were verified using four different mammalian cell lines. Higher contents of glabrone and glabrol were identified in the sample with the highest toxicity. In the zebrafish model, the addition of glabrol reduced the LC50 of glabridin to 9.224, 6.229, and 5.370 µM at 48, 72, and 96 h post-fertilization, respectively, whereas glabrone did not have any toxic effect. Phalloidin staining indicated that a glabrol impurity exacerbates the myotoxicity of glabridin in zebrafish embryos. CONCLUSION: Glabrol, but not glabrone, was identified as a key impurity that increased glabridin toxicity. This finding indicates that controlling glabrol content is necessary during glabridin product production.

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.

Ação antimicrobiana e antibiofilme de flavonóis isolados ou incorporados em hidrogéis termossensíveis e sua influência sobre marcadores de mineralização em células odontoblastóides para aplicação em endodontia / Antimicrobial/antibiofilm action of flavonols alone or incorporated in thermosensitive hydrogels and their influence on mineralization markers in odontoblast-like cells for endodontic applications

Ainda não foi encontrado um medicamento capaz de desinfetar os canais radiculares e permitir a recuperação celular e a regeneração tecidual em dentes permanentes jovens com comprometimento endodôntico. Dois importantes flavonóis detectados no vinho tinto, morina (MO) e miricetina (MY), são atualmente estudados por suas amplas propriedades biológicas, incluindo atividade antimicrobiana. No entanto, o desenvolvimento de sistemas de liberação controlada pode ser útil para a liberação desses flavonóis para fins de terapia endodôntica. Este estudo avaliou a citocompatibilidade e os efeitos antimicrobianos/antibiofilme de MO e MY, isolados ou incorporados em hidrogéis termorreversíveis de quitosana-poloxamer-ß-glicerofosfato de sódio (CPG), além dos efeitos de MO e MY, isolados e combinados sobre a viabilidade, atividade de ALP e produção de nódulos de mineralização em células MDPC-23. A atividade antimicrobiana dos compostos foi avaliada em Streptococcus mutans, Enterococcus faecalis, Actinomyces israelii e Fusobacterium nucleatum em condições planctônicas, em biofilmes dual-espécies e multiespécies e analisadas por contagem bacteriana e microscopia de varredura. Os hidrogéis CPG foram caracterizados por reometria de fluxo e oscilatória, temperatura de gelificação, perfil de textura e análise de bioadesão em espécimes de dentina. MO, MY e controles (hidróxido de cálcio ­ CH e clorexidina ­ CHX) foram incorporados em hidrogéis de CPG e o efeito do antibiofilme sobre biofilmes multiespécies formados em amostras de dentina radicular foi avaliado por microscopia confocal. O efeito de toxicidade dos compostos isolados ou incorporados em hidrogéis de CGP foi determinado em cultura de fibroblastos por ensaios de resazurina. Os dados foram analisados estatisticamente pelos testes ANOVA e Tukey considerando p < 0,05. A combinação de MO e MY foi sinérgica ou aditiva contra bactérias endodônticas testadas a partir de concentrações de 0,03 mg/mL MO + 0,06 mg/mL MY e não foram tóxicas para fibroblastos até 0,125mg/mL. MO + MY apresentou melhor efeito sobre biofilmes dual-espécies e multiespécies considerando suas menores concentrações quando comparados com os flavonóis isolados. Os hidrogéis CPG foram caracterizados como termorreversíveis e com propriedades mecânicas e bioadesivas adequadas. Hidrogéis de CPG carregados com MO+MY, CH e CHX apresentaram efeitos inibitórios semelhantes quando aplicados em biofilmes multiespécies formados no interior dos túbulos dentinários radiculares por 48h e seus extratos apresentaram citotoxicidade acima de 50% de diluição. As células semelhantes a odontoblastos (MDPC-23) foram expostas a diferentes concentrações de MO, MY, isoladamente ou em combinação e CH como controle positivo por 24h e 48h, e troca contínua de meio osteogênico por 8 dias e 14 dias. As combinações de MO+MY ou CH também foram incorporadas em hidrogéis de quitosana-poloxamer-ß-glicerofosfato e seus extratos em meio de cultura celular foram coletados após 48h e 7 dias. Viabilidade celular, atividade de fosfatase alcalina (ALP) e ensaios de deposição de nódulos mineralizados (MN) foram realizados pelo método de resazurina, ensaios de monofosfato de timolftaleína e coloração com vermelho de alizarina, respectivamente. Todos os compostos não causaram citotoxicidade nas concentrações testadas em 24h e 8 dias e 0,5 mg/mL MO e MY isolados reduziram a viabilidade celular em 48h. A atividade de ALP e a deposição de MN foram aumentadas para a combinação MO+MY e CH em células MDPC-23. Extratos de hidrogel de 7 dias contendo ou não MO+MY não foram citotóxicos até diluição de 25% em 48h e em baixas concentrações estimularam a atividade de ALP e deposição de MN aos 8 e 14 dias de avaliação. Em conclusão, a combinação de morina e miricetina incorporada ou não em hidrogéis de CPG apresentou efeito antibiofilme sobre patógenos orais e baixa toxicidade sobre fibroblastos. Morina e miricetina em baixas concentrações, isoladas, em combinação ou em hidrogéis CPG não foram citotóxicas e foram eficazes na indução de marcadores de mineralização em células semelhantes a odontoblastos(AU)

A drug capable of disinfecting the root canals and allow cell recovery and tissue regeneration in permanent young teeth with endodontic problems has not been found yet. Two important flavonols detected in red wine, morin (MO) and myricetin (MY), are currently studied for their wide biological properties including antimicrobial activity. However, the development of controlled release systems could be useful for the delivery of these flavonols for endodontic therapies. This study evaluated the cytocompatibility and antimicrobial/antibiofilm effects of MO and MY, alone or incorporated in thermoreversible chitosanpoloxamer hydrogels containing sodium ß-glycerophosphate (CPG), in addition to the effects of isolated and combined morin and myricetin flavonols on viability, ALP activity and production of mineralization nodules in MDPC-23 cells. Antimicrobial activity of the compounds was evaluated on Streptococcus mutans, Enterococcus faecalis, Actinomyces israelii, and Fusobacterium nucleatum under planktonic conditions, on dual-species and multispecies biofilms and analyzed by bacterial counts and scanning microscopy. CPG hydrogels were characterized by flow and oscillatory rheometry, gelation temperature, texture profile and bioadhesion analysis in dentin specimens. MO, MY and controls (calcium hydroxide ­ CH and chlorhexidine ­ CHX) were incorporated in CPG hydrogels and antibiofilm effect on multispecies biofilms formed in radicular dentin samples were evaluated by confocal microscopy. Cytotoxicity of the compounds alone or incorporated in CGP hydrogels was determined on fibroblasts culture by resazurin assays. Data were statistically analyzed by ANOVA and Tukey considering p < 0.05. The combination of MO and MY had synergistic or additive against oral bacteria tested starting at concentrations of 0.03 mg/mL MO + 0.06 mg/mL MY and they were not toxic to fibroblasts up to 0.125mg/mL. MO + MY had better effect on dual-species and multispecies biofilms considering their lower concentrations when compared with the flavonols alone. CPG hydrogels were characterized as thermoreversible and with adequate mechanical and bioadhesive properties. CPG hydrogels loaded with MO+MY, CH and CHX have similar inhibitory effects when applied on multispecies biofilms formed inside root dentin tubules for 48h and their extracts were cytotoxicity above 50% dilution. Furthermore, the effects of morin, myricetin, alone or in combination or incorporated in chitosan-based hydrogels on cytotoxicity and expression of mineralization markers in odontoblast-like cells. The MDPC-23 cells were exposed to different concentrations of morin (MO), myricetin (MY), alone or in combination and calcium hydroxide (CH) as a positive control for 24h and 48h, and continuous osteogenic medium changing for 8 days and 14 days. The combinations of MO+MY or CH were also incorporated in chitosan-poloxamer-ß- glycerophosphate hydrogels and their extracts in cell culture media were collected after 48h and 7 days. Cell viability, alkaline phosphatase (ALP) activity and assays mineralized nodules (MN) deposition were performed using resazurin method, thymolphthalein monophosphate assays and alizarin red staining, respectively. Data were statistically analyzed considering p< 0.05. All compounds were non-toxic at the concentrations tested at 24h and 8 days and 0.5 mg/mL MO and MY alone reduced cell viability at 48h. ALP activity and deposition of MN were increased for MO+MY combination and CH in MDPC-23 cells. 7 days hydrogel extracts containing or not MO+MY were not cytotoxic up to 25% dilution at 48h and at low concentrations stimulated ALP activity and MN deposition at 8 and 14 days of evaluation. In conclusion, the combination of morin and myricetin incorporated or not in CPG hydrogels presented antibiofilm effect on oral pathogens and low cytotoxicity on fibroblasts. Morin myricetin at low concentrations, alone, in combination or in CPG hydrogels were not cytotoxic and were effective in inducing mineralization markers in odontoblast-like cells(AU)