Safety evaluation of kaempferol glycosides-rich standardized roasted goji berry leaf extract.

Goji berry leaf (GL) has been used for medicinal foods for its pharmacological effects, including anti-oxidative and anti-obesity activities. Nevertheless, toxicological information on GL is limited for developing health functional ingredient. The aim of the research was to evaluate the single dose acute, 14-day repeated oral toxicity, and genotoxicity of standardized roasted GL extract (rGL) rich in kaempferol-3-O-sophoroside-7-O-glucoside. Tested rGL was found to be stable as kaempferol-3-O-sophoroside-7-O-glucoside, showing 0.7-2.1% of analytical standard variance. According to the single dose toxicity for 14 days, the lethal dose of rGL was determined to be ≥ 2000 mg/kg. Repeated doses of 0-1000 mg/kg of rGL per day for 14 days did not show any toxicity signs or gross pathological abnormalities. No genotoxic signs for the rGL treatment appeared via bacterial reverse mutation up to 5000 µg/plate. There was no significant increase in chromosomal aberration of rGL irrespective of metabolic activation by using CHO-K1 cells (p > 0.05). Regarding carcinogenic toxicity, chromosomal aberrations were not induced at 2000 mg of rGL/kg by using the in vivo bone marrow micronucleus test (p > 0.05). Results from the current study suggest that rGL could be used as a functional ingredient to provide various effects with safety assurance.

Kaempferol inhibits airway inflammation induced by allergic asthma through NOX4-Mediated autophagy.

BACKGROUND: Kaempferol has important medicinal value in the treatment of asthma. However, its mechanism of action has not been fully understood and needs to be explored and studied. METHODS: A binding activity of kaempferol with nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) was analyzed by molecular docking. Human bronchial epithelial cells (BEAS-2B) were treated with different concentrations (0, 1, 5, 10, 20, 40 µg/mL) of kaempferol to select its suitable concentration. In the transforming growth factor (TGF)-ß1-induced BEAS-2B, cells were treated with 20 µg/mL kaempferol or 20 µM GLX35132 (a NOX4 inhibitor) to analyze its effects on NOX4-mediated autophagy. In the ovalbumin (OVA)-induced mice, 20 mg/kg kaempferol or 3.8 mg/kg GLX351322 administration was performed to analyze the therapeutic effects of kaempferol on NOX4-mediated autophagy. An autophagy activator, rapamycin, was used to confirm the mechanism of kaempferol in treatment of allergic asthma. RESULTS: A good binding of kaempferol to NOX4 (score = -9.2 kcal/mol) was found. In the TGF-ß1-induced BEAS-2B, the NOX4 expression was decreased with kaempferol dose increase. The secretions of IL-25 and IL-33, and the NOX4-mediated autophagy were significantly decreased by kaempferol treatment in the TGF-ß1-induced BEAS-2B. In the OVA-challenged mice, kaempferol treatment improved airway inflammation and remodeling through suppressing NOX4-mediated autophagy. The rapamycin treatment clearly hampered the therapeutic effects of kaempferol in the TGF-ß1-induced cells and OVA-induced mice. CONCLUSIONS: This study identifies kaempferol binds NOX4 to perform its functions in the treatment of allergic asthma, providing an effective therapeutic strategy in the further treatment of asthma.

Analysis of the toxicological and pharmacokinetic profile of Kaempferol-3-O-ß-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-ß-D- (6 "-E-p-cumaril) glucopiranosídeo - Tilirosídeo: ensaio in silico, in vitro e ex vivo

Abstract 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.

Resumo 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.

Subacute 28 days oral toxicity study of kaempferol and biochanin-A in the mouse model.

The present study was undertaken to investigate the safety of kaempferol (KEM) and biochanin-A (BCA) following subacute exposure in mice. KEM and BCA were administered in three different doses by oral administration for 28 days. Evaluation of general toxicity parameters by examining the clinical signs, body weight, organ weights, haematological, biochemical, oxidative stress parameters, and histopathology was done. Administration of KEM and BCA for 28 days did not show any clinical signs of toxicity, nor any treatment-related changes in body weight and organ weights in comparison to control. The haematological parameters such as red blood cell, white blood cell, platelets count, haemoglobin (Hb) level, haematocrit, mean corpuscular haemoglobin concentration, red cell distribution width, and platelet distribution width did not show any change in the treated groups and control. Furthermore, different biochemical parameters like markers of the liver (alanine aminotransferase and aspartate aminotransferase), kidney (creatinine and urea), and heart (creatinine kinase-myocardial band and lactate dehydrogenase) injury along with other biochemical parameters showed nonsignificant differences between treated groups and control. Results of oxidative stress parameters in treated groups showed insignificant variations with control. The level of antioxidant enzymes such as superoxide dismutase and catalase were markedly increased in the treated groups; however, these were nonsignificant in comparison to control. In histopathology, evaluation of all vital organs, such as liver, kidney, heart, and lungs, did not show any morphological abnormalities and lesions in treated groups and control. The present study suggests that KEM and BCA have no adverse effects on the general physiology in mice.

Analysis of the toxicological and pharmacokinetic profile of Kaempferol-3-O-ß-D-(6"-E-p-coumaryl) glucopyranoside - Tiliroside: in silico, in vitro and ex vivo assay.

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-ß-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.

Bioflavonoids cause DNA double-strand breaks and chromosomal translocations through topoisomerase II-dependent and -independent mechanisms.

Bioflavonoids have a similar chemical structure to etoposide, the well-characterized topoisomerase II (Top2) poison, and evidence shows that they also induce DNA double-strand breaks (DSBs) and promote genome rearrangements. The purpose of this study was to determine the kinetics of bioflavonoid-induced DSB appearance and repair, and their dependence on Top2. Cells were exposed to bioflavonoids individually or in combination in the presence or absence of the Top2 catalytic inhibitor dexrazoxane. The kinetics of appearance and repair of γH2AX foci were measured. In addition, the frequency of resultant MLL-AF9 breakpoint cluster region translocations was determined. Bioflavonoids readily induced the appearance of γH2AX foci, but bioflavonoid combinations did not act additively or synergistically to promote DSBs. Myricetin-induced DSBs were mostly reduced by dexrazoxane, while genistein and quercetin-induced DSBs were only partially, but significantly, reduced. By contrast, luteolin and kaempferol-induced DSBs increased with dexrazoxane pre-treatment. Sensitivity to Top2 inhibition correlated with a significant reduction of bioflavonoid-induced MLL-AF9 translocations. These data demonstrate that myricetin, genistein, and quercetin act most similar to etoposide although with varying Top2-dependence. By contrast, luteolin and kaempferol have distinct kinetics that are mostly Top2-independent. These findings have implications for understanding the mechanisms of bioflavonoid activity and the potential of individual bioflavonoids to promote chromosomal translocations. Further, they provide direct evidence that specific Top2 inhibitors or targeted drugs could be developed that possess less leukemic potential or suppress chromosomal translocations associated with therapy-related and infant leukemias.

Effect of Kaempferol Pretreatment on Myocardial Injury in Rats.

The present study was undertaken to evaluate the effect of kaempferol in isoprenaline (ISP)-induced myocardial injury in rats. ISP was administered subcutaneously for two subsequent days to induce myocardial injury. Assessment of myocardial injury was done by estimation of hemodynamic functions, myocardial infarcted area, cardiac injury markers, lipid profile, oxidative stress, pro-inflammatory cytokines and histopathology of heart and liver. Rats pretreated with kaempferol showed reduction in the myocardial infarcted area and heart rate. However, no improvement was observed in change in body weight, mean arterial, systolic and diastolic blood pressure. Kaempferol showed significant decrease in serum LDH, CK-MB, troponin-I and lipid profile. However, highest dose of kaempferol did not reduce the serum triglyceride level. Further, antioxidant enzymes, SOD and catalase, were also higher. However, reduced glutathione, serum SGOT and creatinine did not show any improvement. Kaempferol showed reduction in MDA level. Kaempferol at highest dose showed reduction in pro-MMP-2 expression and MMP-9 level. mRNA expression level of TNF-α was not different in kaempferol-pretreated myocardial injured rats with ISP-alone group. Pretreatment with kaempferol at highest dose showed mild mononuclear infiltration and degenerative changes in heart tissue section of myocardial injured rats. Rats pretreated with kaempferol at higher concentration showed normal cordlike arrangement of hepatocytes with moderate swelling of hepatocytes (vacuolar degeneration) around the central vein. Study suggests that kaempferol attenuated lipid profile, infarcted area and oxidative stress in ISP-induced myocardial injury in rats.

Correlation of binding efficacies of DNA to flavonoids and their induced cellular damage.

Flavonoids are dietary intakes which are bestowed with several health benefits. The most studied property of flavonoids is their antioxidant efficacy. Among the chosen flavonoids Quercetin, Kaempferol and Myricetin is catagorized as flavonols whereas Apigenin and Luteolin belong to the flavone group. In the present study anti-cancer properties of flavonoids are investigated on the basis of their binding efficacy to ct-DNA and their ability to induce cytotoxicity in K562 leukaemic cells. The binding affinities of the flavonoids with calf thymus DNA (ct-DNA) are in the order Quercetin>Myricetin>Luteolin>Kaempferol>Apigenin. Quercetin with fewer OH than myricetin has higher affinity towards DNA suggesting that the number and position of OH influence the binding efficacies of flavonoids to ct-DNA. CD spectra and EtBr displacement studies evidence myricetin and apigenin to be stronger intercalators of DNA compared to quercetin. From comet assay results it is observed that quercetin and myricetin when used in combination induce higher DNA damage in K562 leukemic cells than when tested individually. Higher binding efficacy has been recorded for quercetin to DNA at lower pH, which is the micro environment of cancerous cells, and hence quercetin can act as a potential anti-cancer agent. Presence of Cu also increases cellular damage as recorded by comet assay.

Natural Compounds as Occult Ototoxins? Ginkgo biloba Flavonoids Moderately Damage Lateral Line Hair Cells.

Several drugs, including aminoglycosides and platinum-based chemotherapy agents, are well known for their ototoxic properties. However, FDA-approved drugs are not routinely tested for ototoxicity, so their potential to affect hearing often goes unrecognized. This issue is further compounded for natural products, where there is a lack of FDA oversight and the manufacturer is solely responsible for ensuring the safety of their products. Natural products such as herbal supplements are easily accessible and commonly used in the practice of traditional eastern and alternative medicine. Using the zebrafish lateral line, we screened a natural products library to identify potential ototoxins. We found that the flavonoids quercetin and kaempferol, both from the Gingko biloba plant, demonstrated significant ototoxicity, killing up to 30 % of lateral line hair cells. We then examined a third Ginkgo flavonoid, isorhamnetin, and found similar levels of ototoxicity. After flavonoid treatment, surviving hair cells demonstrated reduced uptake of the vital dye FM 1-43FX, suggesting that the health of the remaining hair cells was compromised. We then asked if these flavonoids enter hair cells through the mechanotransduction channel, which is the site of entry for many known ototoxins. High extracellular calcium or the quinoline derivative E6 berbamine significantly protected hair cells from flavonoid damage, implicating the transduction channel as a site of flavonoid uptake. Since known ototoxins activate cellular stress responses, we asked if reactive oxygen species were necessary for flavonoid ototoxicity. Co-treatment with the antioxidant D-methionine significantly protected hair cells from each flavonoid, suggesting that antioxidant therapy could prevent hair cell loss. How these products affect mammalian hair cells is still an open question and will be the target of future experiments. However, this research demonstrates the potential for ototoxic damage caused by unregulated herbal supplements and suggests that further supplement characterization is warranted.

A New Iridoid Dimer and Other Constituents from the Traditional Kurdish Plant Pterocephalus nestorianus Nábelek.

Accompanied by other rare compounds, a new iridoid dimer, named kurdnestorianoside (1), showing an unprecedented secologanol configuration, has been isolated for the first time from the Kurdish medicinal plant Pterocephalus nestorianus, which is used in Kurdistan for treating oral diseases and inflammation. The structure of 1 was established from 1D- and 2D-NMR spectroscopic data. Kaempferol 3-O-[3,6-di-O-(E)-p-coumaroyl]-ß-d-glucopyranoside (7) showed a remarkable antiproliferative activity against several human tumor cell lines.

Preliminary in vitro and ex vivo evaluation of afzelin, kaempferitrin and pterogynoside action over free radicals and reactive oxygen species.

Biological activities of flavonoids have been extensively reviewed in literature. The biochemical profile of afzelin, kaempferitrin, and pterogynoside acting on reactive oxygen species was investigated in this paper. The flavonoids were able to act as scavengers of the superoxide anion, hypochlorous acid and taurine chloramine. Although flavonoids are naturally occurring substances in plants which antioxidant activities have been widely advertised as beneficial, afzelin, kaempferitrin, and pterogynoside were able to promote cytotoxic effect. In red blood cells this toxicity was enhanced, depending on flavonoids concentration, in the presence of hypochlorous acid, but reduced in the presence of 2,2'-azo-bis(2-amidinopropane) free radical. These flavonoids had also promoted the death of neutrophils, which was exacerbated when the oxidative burst was initiated by phorbol miristate acetate. Therefore, despite their well-known scavenging action toward free radicals and oxidants, these compounds could be very harmful to living organisms through their action over erythrocytes and neutrophils.

Mechanistic evaluation of Ginkgo biloba leaf extract-induced genotoxicity in L5178Y cells.

Ginkgo biloba has been used for many thousand years as a traditional herbal remedy and its extract has been consumed for many decades as a dietary supplement. Ginkgo biloba leaf extract is a complex mixture with many constituents, including flavonol glycosides and terpene lactones. The National Toxicology Program 2-year cancer bioassay found that G. biloba leaf extract targets the liver, thyroid gland, and nose of rodents; however, the mechanism of G. biloba leaf extract-associated carcinogenicity remains unclear. In the current study, the in vitro genotoxicity of G. biloba leaf extract and its eight constituents was evaluated using the mouse lymphoma assay (MLA) and Comet assay. The underlying mechanisms of G. biloba leaf extract-associated genotoxicity were explored. Ginkgo biloba leaf extract, quercetin, and kaempferol resulted in a dose-dependent increase in the mutant frequency and DNA double-strand breaks (DSBs). Western blot analysis confirmed that G. biloba leaf extract, quercetin, and kaempferol activated the DNA damage signaling pathway with increased expression of γ-H2AX and phosphorylated Chk2 and Chk1. In addition, G. biloba leaf extract produced reactive oxygen species and decreased glutathione levels in L5178Y cells. Loss of heterozygosity analysis of mutants indicated that G. biloba leaf extract, quercetin, and kaempferol treatments resulted in extensive chromosomal damage. These results indicate that G. biloba leaf extract and its two constituents, quercetin and kaempferol, are mutagenic to the mouse L5178Y cells and induce DSBs. Quercetin and kaempferol likely are major contributors to G. biloba leaf extract-induced genotoxicity.

Mechanisms underlying apoptosis-inducing effects of Kaempferol in HT-29 human colon cancer cells.

We previously noted that kaempferol, a flavonol present in vegetables and fruits, reduced cell cycle progression of HT-29 cells. To examine whether kaempferol induces apoptosis of HT-29 cells and to explore the underlying molecular mechanisms, cells were treated with various concentrations (0-60 µmol/L) of kaempferol and analyzed by Hoechst staining, Annexin V staining, JC-1 labeling of the mitochondria, immunoprecipitation, in vitro kinase assays, Western blot analyses, and caspase-8 assays. Kaempferol increased chromatin condensation, DNA fragmentation and the number of early apoptotic cells in HT-29 cells in a dose-dependent manner. In addition, kaempferol increased the levels of cleaved caspase-9, caspase-3 and caspase-7 as well as those of cleaved poly (ADP-ribose) polymerase. Moreover, it increased mitochondrial membrane permeability and cytosolic cytochrome c concentrations. Further, kaempferol decreased the levels of Bcl-xL proteins, but increased those of Bik. It also induced a reduction in Akt activation and Akt activity and an increase in mitochondrial Bad. Additionally, kaempferol increased the levels of membrane-bound FAS ligand, decreased those of uncleaved caspase-8 and intact Bid and increased caspase-8 activity. These results indicate that kaempferol induces the apoptosis of HT-29 cells via events associated with the activation of cell surface death receptors and the mitochondrial pathway.

Kaempferol from Semen cuscutae attenuates the immune function of dendritic cells.

Dendritic cells (DCs) are the critical leukocytes in regulating immune responses. Accordingly, DCs are the major target in the development of immunomodulators. In this study, we examined the effect of Semen cuscutae (SC), an important traditional Chinese medicine, on mouse bone marrow-derived DCs. We found that the n-butanol and methanol extracts of SC significantly suppressed LPS-stimulated DC activation. Several flavonoids were verified in the extracts using HPLC, and then kaempferol was identified as the major flavonoid in the methanol fraction of SC. Kaempferol was able to reduce cytokines and chemokines produced by LPS-stimulated DCs, and this reduction was not due to its cytotoxicity on DCs. In addition, DC maturation was impaired by kaempferol. Furthermore, kaempferol abrogated the ability of LPS-stimulated DCs to promote Ag-specific T cell activation, both in vitro and in vivo. Thus, we show for the first time that SC exhibits an immunosuppressive effect on DCs and that the active ingredient kaempferol attenuates DC function, which suggests that kaempferol has potential in the treatment of chronic inflammatory and autoimmune diseases.

Podophyllin, but not the constituents quercetin or kaempferol, induced genotoxicity in vitro and in vivo through ROS production.

The genotoxic potential of podophyllin (PD) was investigated in this study. PD increased bacterial revertants and abnormal chromosomal structures in a concentration-dependent manner, both with and without metabolic activating enzymes, and increased the incidence of micronuclei in imprinted control region mouse reticulocytes. Results from three studied constituents of PD, such as podophyllotoxin, kampferol, and quercetin, suggested that the mutagenic effect of PD was not due to the presence of podophyllotoxin, kampferol, and quercetin and might be related to other components and the formation of reactive oxygen species. The detailed mutagenic mechanisms need further investigation, and the medicinal use of PD needs to be cautioned against.

Kaempferol, a mutagenic flavonol from Helichrysum simillimum.

Helichrysum simillimum is native to South Africa. It is used for the treatment of coughs, colds, fever, infections, headache, and menstrual pain. Extracts of this species showed mutagenic effects in the Salmonella/microsome assay. The aim of this study was to isolate and determine the mutagenic constituents of H. simillimum. Bioassay-guided fractionation of 90% aqueous methanol extracts, using Salmonella typhimurium TA98, led to the isolation of the flavonol kaempferol.

Bioassay-guided isolation of kaempferol-3-O-beta-D-galactoside with anti-inflammatory and antinociceptive activity from the aerial part of Calluna vulgaris L.

Calluna vulgaris L. (Ericaceae) is used for the treatment of various inflammatory ailments in traditional medicines. In order to evaluate this ethnobotanical information, its anti-inflammatory and antinociceptive activities were studied using in vivo experimental models in mice. The ethanolic extract of the plant was first fractionated into five extracts; namely, n-hexane, chloroform, ethyl acetate (EtOAc), n-butanol, and water fractions. Among them, the EtOAc Fr. was found to be the most effective and was further subjected to bioassay-guided fractionation and isolation procedures. After successive column chromatography applications, on Sephadex LH-20 and silica gel, a component, which is responsible for the above-mentioned activities of this species of Turkish origin, was isolated and its structure was elucidated as kaempferol-3-O-beta-D-galactoside, a common flavonol derivative by means of spectral techniques.

Kaempferol inhibits IL-4-induced STAT6 activation by specifically targeting JAK3.

IL-4 is involved in several human diseases including allergies, autoimmunity, and cancer. Its effects are mainly mediated through the transcription factor STAT6. Therefore, investigation of compounds that regulate STAT6 activation is of great interest for these diseases. Natural polyphenols are compounds reported to have therapeutic properties in diseases involving IL-4 and STAT6. The aim of this study was to investigate the effect of these compounds in the activation of this transcription factor. We found that in hemopoietic cells from human and mouse origin, some flavonoids were able to inhibit the activation of STAT6 by IL-4. To identify molecular mechanisms, we focused on kaempferol, the compound that showed the greatest inhibitory effect with the lowest cell toxicity. Treatment of cells with kaempferol did not affect activation of Src kinase by IL-4 but did prevent the phosphorylation of JAK1 and JAK3. Further enzymatic analysis demonstrated that kaempferol blocked the in vitro phosphorylation activity of JAK3 without affecting JAK1, suggesting that it specifically targeted JAK3 activity. Accordingly, kaempferol had no effect on STAT6 activation in nonhemopoietic cell lines lacking JAK3, supporting its selective inhibition of IL-4 responses through type I receptors expressing JAK3 but not type II lacking this kinase. The inhibitory effect of kaempferol was also observed in IL-2 but not IL-3-mediated responses and correlated with the inhibition of MLC proliferation. These findings reveal the potential use of kaempferol as a tool for selectively controlling cell responses to IL-4 and, in general, JAK3-dependent responses.

In vitro basal and metabolism-mediated cytotoxicity of flavonoids.

The purpose of this study was to compare the basal cytotoxicity and metabolism-mediated cytotoxicity of kaempferol, quercetin and rutin. McCoy cells were exposed to various concentrations of the flavonols with and without the S9 system. The neutral red uptake assay was used to determine viability after 24 h at 35-37 degrees C. Dose-response curves were established for each flavonol in the presence and absence of external metabolizing systems. Kaempferol and quercetin were cytotoxic and provoked a dose-dependent decrease in cell viability, without the S9 system. The hepatic S9 microsomal fraction metabolized these compounds to less cytotoxic metabolites. In contrast, rutin at 500 microg/ml failed to produce any overt signs of toxicity in either assay.

Protective and detrimental effects of kaempferol in rat H4IIE cells: implication of oxidative stress and apoptosis.

Flavonoids are ubiquitous substances in fruits and vegetables. Among them, the flavonol kaempferol contributes up to 30% of total dietary flavonoid intake. Flavonoids are assumed to exert beneficial effects on human health, e.g., anticancer properties. For this reason, they are used in food supplements at high doses. The aim of this project was to determine the effects of kaempferol on oxidative stress and apoptosis in H4IIE rat hepatoma cells over a broad concentration range. Kaempferol is rapidly taken up and glucuronidated by H4IIE cells. The results demonstrate that kaempferol protects against H2O2-induced cellular damage at concentrations which lead to cell death and DNA strand breaks in the absence of H2O2-mediated oxidative stress. Preincubation with 50 microM kaempferol exerts protection against the loss of cell viability induced by 500 microM H2O2 (2 h) while the same concentration of kaempferol reduces cell viability by 50% in the absence of H2O2 (24 h). Preincubation with 50 microM kaempferol ameliorates the strong DNA damage induced by 500 microM H2O2 while 50 microM kaempferol leads to a significant increase of DNA breakage in the absence of H2O2. Preincubation with 50 microM kaempferol reduces H2O2-mediated caspase-3 activity by 40% (4 h) while the same concentration of kaempferol leads to the formation of a DNA ladder in the absence of H2O2 (24 h). It is concluded that the intake of high dose kaempferol in food supplements may not be advisable because in our cellular model protective kaempferol concentrations can also induce DNA damage and apoptosis by themselves.