methysticin and yangonin

Oral mucositis is a debilitating inflammatory disorder observed in patients undergoing active cancer treatment, particularly cancer of the head and neck region. A key pathway believed to be involved in the pathogenesis of oral mucositis is the formation of reactive oxygen species (ROS). The identification of compounds that can inhibit this pathway may therefore be of benefit in treating this disorder. The kava plant (Piper methysticum) contains various constituents, including flavokawain A (FKA), flavokawain B (FKB), yangonin, methysticin and kavain. These constituents are known to be biologically active and possess anti-oxidative properties. This study therefore focused on examining these constituents for their effect on ROS formation in an in vitro oral mucositis model.. Cell proliferation was assessed in normal oral keratinocytes (OKF6) treated with and without kava constituents, namely FKA, FKB, yangonin, methysticin and kavain using an MTS in vitro assay. Oxidative stress was assessed by co-treating and pre-treating OKF6 cells with H. Pre-treatment of cells for 24 h with 2.5 μg/ml kavain and 5 μg/ml FKA demonstrated a significant protective anti-oxidative effect. Similarly, FKB at a concentration of 2.5 μg/ml, demonstrated a trend of ROS reduction but was observed to be cytotoxic at concentrations greater than 5 μg/ml. Reduction in ROS production by methysticin and yangonin was compromised by their cell cytotoxicity.. This was the first study to identify the anti-oxidative effects and safety of FKA and kavain with regard to oral keratinocytes, highlighting their potential use in the development of a preventative treatment for oral mucositis.

Topics: Antioxidants; Cell Line, Tumor; Cells, Cultured; Humans; Hydrogen Peroxide; Kava; Keratinocytes; Oxidative Stress; Plant Extracts; Protective Agents; Pyrans; Pyrones; Reactive Oxygen Species; Stomatitis

Kava is a beverage made from the ground roots of the plant Piper Methysticum. Active compounds of Kava have previously been demonstrated to exert an antiproliferative effect through cell cycle arrest and promotion of apoptosis. Our aim was to investigate the in vitro effects of the main constituents derived from Kava on oral squamous cell carcinoma (OSCC) activity. Gas chromatography mass spectrometry (GCMS) was used to characterise the main constituents of two Kava preparations. Cell proliferation was assessed in two human OSCC cell lines (H400 and BICR56) and in normal oral keratinocytes (OKF6) treated with the identified Kava constituents, namely Flavokawain A (FKA), Flavokawain B (FKB), yangonin, kavain and methysticin using an MTS in vitro assay. Cell migration at 16 h was assessed using a Transwell migration assay. Cell invasion was measured at 22 h using a Matrigel assay. Cell adhesion was assessed at 90 min with a Cytoselect Adhesion assay. The two Kava preparations contained substantially different concentrations of the main chemical constituents. Treatment of malignant and normal oral keratinocyte cell lines with three of the identified constituents, 10 μg/ml FKA, 2.5 μg/ml FKB and 10 μg/ml yangonin, showed a significant reduction in cell proliferation in both H400 and BICR56 cancer cell lines but not in normal OKF6 cells. Remarkably, the same Kava constituents induced a significant reduction of OSCC cell migration and invasion. We have demonstrated, for the first time, that Kava constituents, FKA, FKB and yangonin have potential anticancer effects on OSCC. This highlights an avenue for further research of Kava constituents in the development of future cancer therapies to prevent and treat OSCC.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Chalcone; Flavonoids; Gas Chromatography-Mass Spectrometry; Humans; Kava; Keratinocytes; Mouth Neoplasms; Plant Extracts; Pyrans; Pyrones; Squamous Cell Carcinoma of Head and Neck

Kava is an Oceanic plant in which the root is consumed as a beverage and is becoming increasingly popular. The effects of kava consumption may include sedation, euphoria, and impairment of motor coordination. This article demonstrates kava impairment through four cases of self-reported kava use supported with Drug Recognition Expert (DRE) evaluations of each subject. Subject's urines screened negative for common drugs of abuse by immunoassay analysis. Urine from cases 3 and 4 were analyzed by liquid chromatography-tandem mass spectrometry, and gas chromatography-mass spectrometry, which yielded the presence of kavalactones. Subjects exhibited poor driving behavior and signs of intoxication. Indicators of impairment from multiple drug categories, central nervous system (CNS) depressants, CNS stimulants, and cannabis were observed, which may be consistent with the presence of multiple kavalactones and their diverse array of mechanisms of action. The consumption of kava can hinder one's ability to operate a vehicle safely.

Topics: Adult; Chromatography, Liquid; Driving Under the Influence; Gas Chromatography-Mass Spectrometry; Humans; Kava; Male; Plant Roots; Pyrans; Pyrones; Substance Abuse Detection; Substance-Related Disorders; Tandem Mass Spectrometry; Young Adult

Topics: Calibration; Chromatography, High Pressure Liquid; Dietary Supplements; Kava; Lactones; Limit of Detection; Plant Roots; Pyrans; Pyrones

Topics: Chromatography, Supercritical Fluid; Kava; Lactones; Molecular Structure; Plant Roots; Pyrans; Pyrones

While cases of severe kava hepatotoxicity have been reported, studies examining the toxicity of individual kavalactones are limited. The present study examined the in vitro hepatotoxicity of kavain, methysticin and yangonin on human hepatocytes (HepG2) and the possible mechanism(s) involved. Cytotoxicity was assessed using lactate dehydrogenase (LDH) and ethidium bromide (EB) assays. The mode of cell death was analysed with acridine orange/ethidium bromide dual staining with fluorescence microscopy. Glutathione oxidation was measured using the ortho-phthalaldehyde (OPT) fluorescence assay. Kavain had minimal cytotoxicity, methysticin showed moderate concentration-dependent toxicity and yangonin displayed marked toxicity with ~ 40% reduction in viability in the EB assay. Acridine orange/ethidium bromide staining showed the predominant mode of cell death was apoptosis rather than necrosis. No significant changes were observed in glutathione levels, excluding this as the primary mechanism of cell death in this model. Further studies may elucidate the precise apoptotic pathways responsible and whether toxic kavalactone metabolites are involved.

Topics: Apoptosis; Cell Survival; Glutathione; Hep G2 Cells; Hepatocytes; Humans; L-Lactate Dehydrogenase; Lactones; Pyrans; Pyrones

To examine the bioavailability of kavalactones in vitro and the possible differences in their bioavailability because of variations in either chemical structure or the method of extraction used.. Caco-2 cell monolayers were used to determine the potential bioavailability of kavalactones. Kavalactones were added to the apical layer and basolateral samples were taken over 150 min to examine the concentration diffusing across the cell monolayer. Kavalactone concentrations in these samples were determined by high pressure liquid chromatography.. Kavalactones were found to be potentially bioavailable as they all readily crossed the Caco-2 monolayers with apparent permeabilities (P(app)) increasing from 42 x 10(-6) cm/s and most exhibiting more than 70% crossing within 90 min. Not all differences in their bioavailability can be related to kavalactone structural differences as it appears that bioavailability may also be affected by co-extracted compounds. For example, the P(app) for kawain from ethanol extracts was higher than the values obtained for the same compound from water extracts or for the kavalactone alone.. While the extraction method used (ethanol or water) influences the total (but not the relative) concentrations of kavalactones, it does not markedly affect their bioavailability. Hence, any differences between an ethanolic or an aqueous extract in terms of the propensity of kava to cause liver damage is not because of differing kavalactone bioavailabilities.

Topics: Biological Availability; Biological Transport; Caco-2 Cells; Cell Membrane Permeability; Chromatography, High Pressure Liquid; Humans; Kava; Kinetics; Lactones; Models, Biological; Molecular Structure; Plant Extracts; Pyrans; Pyrones; Rhizome

In recent years, Kava kava (Piper methysticum, Forst. f., Piperaceae), a folkloric beverage and popular herbal remedy, has been implicated in a number of liver failure cases. Many hypotheses as to the mechanism of its hepatotoxicity, for example interactions with other co-ingested medication, have been postulated. This present study investigated whether pharmacokinetic interactions between kava constituents and alcohol via alcohol dehydrogenase (ADH) inhibition by individual kavalactones might explain its claimed hepatotoxic effects. Four kavalactones, (+/-)-kavain, methysticin, yangonin and desmethoxyyangonin, fail to inhibit ADH in vitro at 1, 10 or 100 microM concentrations.

Topics: Alcohol Dehydrogenase; Alcohol Drinking; Chemical and Drug Induced Liver Injury; Herb-Drug Interactions; Kava; Lactones; Plant Extracts; Pyrans; Pyrazoles; Pyrones; Spectrophotometry

To determine whether kava (Kava kava, 'Awa, Yaqona, Piper methysticum Forst.), the popular herbal product associated recently with possible human hepatotoxicity, is bioactivated by cytochrome P450 enzymes to cytotoxic metabolites, three kava lactones (methysticin, yangonin, and desmethoxyyangonin) and an ethanolic extract of dried kava root were incubated over time in culture with MCL-5 cells, a human lymphoblastoid cell line that has been stably transfected with five human P450's (CYP 1A1, 1A2, 2A6, 2E1, and 3A4) and human epoxide hydrolase. Incubations were performed concurrently with a control cell line (cH2) that is derived from the same parental line as MCL-5, but transfected with two empty vectors. The kava compounds displayed varying degrees of toxicity (IC (50) values ranged from 50 to > 100 microM) to the MCL-5 and cH2 cell lines; however, both cell lines were equally sensitive to the test compounds. These results suggest that the parent compound for each of the four test compounds was primarily responsible for the observed cell toxicity and that CYP 1A1, 1A2, 2A6, 2E1, and 3A4 or epoxide hydroxylase did not appear to be involved. Thus, in vitro kava does not appear to be activated to toxic metabolites by enzymes known to be important in metabolic toxicity.

Topics: Cell Line; Cytochrome P-450 Enzyme System; Humans; Inhibitory Concentration 50; Kava; Lactones; Phytotherapy; Plant Extracts; Plant Roots; Pyrans; Pyrones

The effects of the herbal product kava (Kava kava, 'Awa, Yaqona, Piper methysticum) on human P450 isoforms were studied in vitro using both cDNA-expressed human enzymes and cryopreserved human hepatocytes. Increasing concentrations of an ethanolic extract of dried kava root and three purified kava lactones (methysticin, desmethoxyyangonin, and yangonin) were tested for their ability to inhibit the catalytic activity of a panel of P450 isoforms (1A2, 2A6, 2C9, C2C19, 2D6, 2E1, and 3A4) present as c-DNA expressed-enzymes and in previously cryopreserved human hepatocytes. In addition, the test compounds' effect on hepatocyte viability was evaluated by measuring cellular ATP content. In both models, the kava extract and the three kava lactones were found to be potent inhibitors of CYPs 1A2, 2C9, 2C19, 2E1, and 3A4 with IC50 values of approximately 10 microM. The test compounds were also moderately cytotoxic to human hepatocytes (EC50 values of approximately 50 microM). Methysticin was the most potent enzyme inhibitor as well as the most cytotoxic, followed by (in order of potency:) the kava root extract, desmethoxyyangonin, and yangonin. Our results suggest that the drug interaction and hepatotoxic potential of kava should be further investigated.

Topics: Cryopreservation; Cytochrome P-450 Enzyme System; DNA, Complementary; Enzyme Inhibitors; Hepatocytes; Humans; Inhibitory Concentration 50; Kava; Phytotherapy; Plant Extracts; Plant Roots; Pyrans; Pyrones

Methanolic leaf and root extracts of the Hawaiian kava (Piper methysticum Forst.) cultivars, Mahakea, Nene, Purple Moi and PNG, were tested on binding affinities to CNS receptors including GABAA (GABA and benzodiazepine binding site), dopamine D2, opioid (mu and delta), serotonin (5-HT6 and 5-HT7) and histamine (H1 and H2). HPLC analysis was carried out in order to determine the amount of the main kavalactones kavain, 7,8-dihydrokavain, methysticin, 7,8-dihydromethysticin, yangonin and 5,6-demethoxyyangonin. The most potent binding inhibition was observed for leaf extracts to GABAA receptors (GABA binding site) with IC50 values of approximately 3 micrograms/ml, whereas root extracts were less active with IC50 values ranging from 5 micrograms/ml (Nene) to 87 micrograms/ml (Mahakea). Since the leaf extracts generally contained lower amounts of the kavalactones than the root extracts, there might exist additional substances responsible for these activities. Leaf extracts also inhibited binding to dopamine D2, opioid (mu and delta) and histamine (H1 and H2) receptors more potently than the corresponding root extracts with IC50 values ranging from 1 to 100 micrograms/ml vs. > or = 100 micrograms/l, respectively. Significant differences in the potential of binding inhibition were also observed between cultivars. Binding to serotonin (5-HT6 and 5-HT7) and benzodiazepine receptors was only weakly inhibited by both root and leaf extracts of all four cultivars. In conclusion, our investigation indicates that the GABAA, dopamine D2, opioid (mu and delta) and histamine (H1 and H2) receptors might be involved in the pharmacological action of kava extracts. Since the cultivars contained similar amounts of kavalactones, while their pharmacological activities differed markedly, other constituents may play a role in the observed activities. Additionally, leaves generally exhibited more potent binding inhibition than roots, therefore leaf of P. methysticum might be an interesting subject for further pharmacological studies.

Topics: Animals; Brain; Cattle; Cells, Cultured; Chromatography, High Pressure Liquid; Cricetinae; Dose-Response Relationship, Drug; Humans; In Vitro Techniques; Kava; Plant Extracts; Plant Leaves; Plant Roots; Plants, Medicinal; Pyrans; Pyrones; Rats; Rats, Inbred Strains; Rats, Wistar; Receptors, Dopamine D2; Receptors, GABA-A; Receptors, Histamine; Receptors, Opioid; Receptors, Serotonin; Semliki forest virus

A simultaneous HPLC separation of the enantiomers of kavain, dihydrokavain, methysticin and dihydromethysticin, as well as the achiral dienolides yangonin and desmethoxyyangonin was carried out on a ChiraSpher NT column. For quantitative determinations, calibration curves with correlation coefficients between 0.9982 and 0.9996 were established for the genuine kavapyrones. Detection limits between 0.25 microg and 0.5 microg per injection were measured at 240 nm. The defined scopes of work corresponded with the different kavapyrone amounts, depending on growth factors of distinct plant locations. The precision of the method was verified by analysing a phytopharmacon with a nominal value of 40 mg kavapyrones per tablet. The evaluation revealed 39.62 mg per tablet by the sum of single calculated kavapyrones. Relative standard deviations between 1.06% and 2.39% were found for the compounds under investigation. The accuracy of the method was proved by a recovery of 99.7%. To simplify the determination of the total kavapyrone amount, response factors and correlation factors for (+)-dihydrokavain, (+)-methysticin, (+)-dihydromethysticin, yangonin and desmethoxyyangonin were calculated relative to (+)-kavain.

Topics: Anti-Anxiety Agents; Chromatography, High Pressure Liquid; Pyrans; Pyrones; Reproducibility of Results; Sensitivity and Specificity; Stereoisomerism