The biosynthetic origin of psychoactive kavalactones in kava.

Kava (Piper methysticum) is an ethnomedicinal shrub native to the Polynesian islands with well-established anxiolytic and analgesic properties. Its main psychoactive principles, kavalactones, form a unique class of polyketides that interact with the human central nervous system through mechanisms distinct from those of conventional psychiatric drugs. However, an unknown biosynthetic machinery and difficulty in chemical synthesis hinder the therapeutic use of kavalactones. In addition, kava also produces flavokavains, which are chalconoids with anticancer properties structurally related to kavalactones. Here, we report de novo elucidation of the key enzymes of the kavalactone and flavokavain biosynthetic network. We present the structural basis for the evolutionary development of a pair of paralogous styrylpyrone synthases that establish the kavalactone scaffold and the catalytic mechanism of a regio- and stereo-specific kavalactone reductase that produces a subset of chiral kavalactones. We further demonstrate the feasibility of engineering styrylpyrone production in heterologous hosts, thus opening a way to develop kavalactone-based non-addictive psychiatric therapeutics through synthetic biology.

Isolation and Purification of Potent Growth Inhibitors from Piper methysticum Root.

Piper methysticum (kava) root is known to possess promising weed suppressing activity. The present study was conducted to search for potent plant growth inhibitors from the root of this medicinal pepper plant. The ethyl acetate (EtOAc) extract exhibited the strongest reduction on growth of Raphanus sativus (radish) (IC50 shoot and root growth = 172.00 and 51.31 µg/mL respectively) among solvent extracts. From this active extract, nine potent growth inhibitors involved in the inhibitory activities of P. methysticum root were isolated, purified and characterized by column chromatography (CC), gas chromatography-mass spectrometry (GC-MS), electrospray ionization-mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR). The six fractions purified by CC included two flavanones: 5-hydroxy-4',7-dimethoxyflavanone (C1) and 5,7-dihydroxy-4'-methoxy-6,8-dimethylflavanone (matteucinol, C2) and six kavalactones: 5,6-dehydro-kavain (C3), a mixture of kavain and yagonin (C4), yagonin (C5) and dihydro-5,6-dehydrokavain, 7,8-dihydrokavain, dihydromethysticin and methysticin (C6). The amounts of 5-hydroxy-4',7-dimethoxyflavanone, matteucinol, 5,6-dehydrokavain and yangonin were 0.76, 2.50, 2.75 and 2.09 mg/g dry weight (DW), respectively. The two flavanones C1 and C2 exhibited the strongest inhibition on shoot elongation (IC50 = 120.22 and 248.03 µg/mL, respectively), whilst the two kavalactone mixtures C4 and C6 showed the highest suppression on root growth of R. sativus (IC50 = 7.70 and 15.67 µg/mL, respectively). This study was the first to report the purification and inhibitory activities of the two flavanones 5-hydroxy-4',7-dimethoxyflavanone and matteucinol in P. methysticum root. The isolated constituents from P. methysticum root including the flavanones C1 and C2 and the mixtures C4 and C6 may possess distinct modes of action on plant growth. Findings of this study highlighted that the combinations of hexane-ethyl acetate by 9:1 and 8:2 ratios successfully purified flavanones and kavalactones in P. methysticum root.

Flavokawains a and B in kava, not dihydromethysticin, potentiate acetaminophen-induced hepatotoxicity in C57BL/6 mice.

Anxiolytic kava products have been associated with rare but severe hepatotoxicity in humans. This adverse potential has never been captured in animal models, and the responsible compound(s) remains to be determined. The lack of such knowledge greatly hinders the preparation of a safer kava product and limits its beneficial applications. In this study we evaluated the toxicity of kava as a single entity or in combination with acetaminophen (APAP) in C57BL/6 mice. Kava alone revealed no adverse effects for long-term usage even at a dose of 500 mg/kg bodyweight. On the contrary a three-day kava pretreatment potentiated APAP-induced hepatotoxicity, resulted in an increase in serum ALT and AST, and increased severity of liver lesions. Chalcone-based flavokawains A (FKA) and B (FKB) in kava recapitulated its hepatotoxic synergism with APAP while dihydromethysticin (DHM, a representative kavalactone and a potential lung cancer chemopreventive agent) had no such effect. These results, for the first time, demonstrate the hepatotoxic risk of kava and its chalcone-based FKA and FKB in vivo and suggest that herb-drug interaction may account for the rare hepatotoxicity associated with anxiolytic kava usage in humans.

Gene expression profiling in male B6C3F1 mouse livers exposed to kava identifies--changes in drug metabolizing genes and potential mechanisms linked to kava toxicity.

The association of kava products with liver-related health risks has prompted regulatory action in many countries. We used a genome-wide gene expression approach to generate global gene expression profiles from the livers of male B6C3F1 mice administered kava extract by gavage for 14 weeks, and identified the differentially expressed drug metabolizing genes in response to kava treatments. Analyses of gene functions and pathways reveal that the levels of significant numbers of genes involving drug metabolism were changed and that the pathways involving xenobiotics metabolism, Nrf2-mediated oxidative stress response, mitochondrial functions and others, were altered. Our results indicate that kava extract can significantly modulate drug metabolizing enzymes, potentially leading to herb-drug interactions and hepatotoxicity.

Role of ethanol in kava hepatotoxicity.

Kava is known for its recreational, ceremonial and medicinal use in the Pacific. The aqueous non-alcoholic drink of kava rhizome produces intoxicating, relaxing and soothing effects. While kava's medicinal effects receive worldwide recognition, kava-containing products came under scrutiny after over 100 reports of spontaneous adverse hepatic effects. Many mechanisms have been postulated to explain the unexpected toxicity, one being pharmacokinetic interactions between kavalactones and co-administered drugs involving cytochrome P450 enzyme system. Alcohol is often co-injested in kava hepatotoxicity cases. This review evaluates the possible hepatotoxicity mechanisms involving alcohol and kava.

Clinical assessment of CYP2D6-mediated herb-drug interactions in humans: effects of milk thistle, black cohosh, goldenseal, kava kava, St. John's wort, and Echinacea.

Cytochrome P450 2D6 (CYP2D6), an important CYP isoform with regard to drug-drug interactions, accounts for the metabolism of approximately 30% of all medications. To date, few studies have assessed the effects of botanical supplementation on human CYP2D6 activity in vivo. Six botanical extracts were evaluated in three separate studies (two extracts per study), each incorporating 16 healthy volunteers (eight females). Subjects were randomized to receive a standardized botanical extract for 14 days on separate occasions. A 30-day washout period was interposed between each supplementation phase. In study 1, subjects received milk thistle (Silybum marianum) and black cohosh (Cimicifuga racemosa). In study 2, kava kava (Piper methysticum) and goldenseal (Hydrastis canadensis) extracts were administered, and in study 3 subjects received St. John's wort (Hypericum perforatum) and Echinacea (Echinacea purpurea). The CYP2D6 substrate, debrisoquine (5 mg), was administered before and at the end of supplementation. Pre- and post-supplementation phenotypic trait measurements were determined for CYP2D6 using 8-h debrisoquine urinary recovery ratios (DURR). Comparisons of pre- and post-supplementation DURR revealed significant inhibition (approximately 50%) of CYP2D6 activity for goldenseal, but not for the other extracts. Accordingly, adverse herb-drug interactions may result with concomitant ingestion of goldenseal supplements and drugs that are CYP2D6 substrates.

Kava consumption and its health effects.

Sakau or Kava is a psychoactive beverage used ceremonially for thousands of years by Pacificans Kava beverage is made from the root of the pepper plant, Piper methysticum. It contains herbal ingredients for reliving anxiety and tension. The Kava's biological effects is due to a mixture of compounds called kava lactones which are reported to include sedative, anxiolytic, anti-stress, analgesic, local anaesthetic, anticonvulsant and neuroprotective properties. A skin disorder or dermopathy, occurs with prolonged use of large amounts of kava and reversible on reduced intake or cessation. Heavy kava drinkers acquire a reversible ichthyosiform eruption, known as kanikani in Fijian. Rare cases of interactions have occurred with pharmaceutical drugs that share one or more mechanisms of action with the kava lactones Kava was banned in the United Kingdom, after concerns that the product can cause liver toxicity. The US Food and drug Administration is still investigating kava's continued use in United States.

In vivo effects of goldenseal, kava kava, black cohosh, and valerian on human cytochrome P450 1A2, 2D6, 2E1, and 3A4/5 phenotypes.

OBJECTIVES: Phytochemical-mediated modulation of cytochrome P450 (CYP) activity may underlie many herb-drug interactions. Single-time point phenotypic metabolic ratios were used to determine whether long-term supplementation of goldenseal ( Hydrastis canadensis ), black cohosh ( Cimicifuga racemosa ), kava kava ( Piper methysticum ), or valerian ( Valeriana officinalis ) extracts affected CYP1A2, CYP2D6, CYP2E1, or CYP3A4/5 activity. METHODS: Twelve healthy volunteers (6 women) were randomly assigned to receive goldenseal, black cohosh, kava kava, or valerian for 28 days. For each subject, a 30-day washout period was interposed between each supplementation phase. Probe drug cocktails of midazolam and caffeine, followed 24 hours later by chlorzoxazone and debrisoquin (INN, debrisoquine), were administered before (baseline) and at the end of supplementation. Presupplementation and postsupplementation phenotypic trait measurements were determined for CYP3A4/5, CYP1A2, CYP2E1, and CYP2D6 by use of 1-hydroxymidazolam/midazolam serum ratios (1-hour sample), paraxanthine/caffeine serum ratios (6-hour sample), 6-hydroxychlorzoxazone/chlorzoxazone serum ratios (2-hour sample), and debrisoquin urinary recovery ratios (8-hour collection), respectively. The content of purported "active" phytochemicals was determined for each supplement. RESULTS: Comparisons of presupplementation and postsupplementation phenotypic ratio means revealed significant inhibition (approximately 40%) of CYP2D6 (difference, -0.228; 95% confidence interval [CI], -0.268 to -0.188) and CYP3A4/5 (difference, -1.501; 95% CI, -1.840 to -1.163) activity for goldenseal. Kava produced significant reductions (approximately 40%) in CYP2E1 only (difference, -0.192; 95% CI, -0.325 to -0.060). Black cohosh also exhibited statistically significant inhibition of CYP2D6 (difference, -0.046; 95% CI, -0.085 to -0.007), but the magnitude of the effect (approximately 7%) did not appear to be clinically relevant. No significant changes in phenotypic ratios were observed for valerian. CONCLUSIONS: Botanical supplements containing goldenseal strongly inhibited CYP2D6 and CYP3A4/5 activity in vivo, whereas kava inhibited CYP2E1 and black cohosh weakly inhibited CYP2D6. Accordingly, serious adverse interactions may result from the concomitant ingestion of goldenseal supplements and drugs that are CYP2D6 and CYP3A4/5 substrates. Kava kava and black cohosh may interact with CYP2E1 and CYP2D6 substrates, respectively. Valerian appears to be less likely to produce CYP-mediated herb-drug interactions.

Determination of six kavalactones in dietary supplements and selected functional foods containing Piper methysticum by isocratic liquid chromatography with internal standard.

Kava (Piper methysticum) dietary products have been sold worldwide for treatment of nervous anxiety, tension, and restlessness. Recent reports showed potential association of kava usage and liver injuries. This study was conducted to develop simple and reliable methodologies for the extraction and determination of 6 major kavalactones: (+)-methysticin, (+)-dihydromethysticin, (+)-kavain, (+)-dihydrokavain, yangonin, and desmethoxyyangonin. Ultrasonic extraction techniques and isocratic reversed-phase liquid chromatography (LC) were optimized for different types of samples, including capsules containing kava root extract or root powder, raw root material, tea bags, and snack bar. A suitable internal standard, 5,7-dihydroxyflavone, was used for LC calibration. Kavalactones were completely separated in 30 min using a Luna C18-2 column at 60 degrees C with an isocratic mobile phase consisting of 2-propanol-acetonitrile-water-acetic acid (16 + 16 + 68 + 0.1, v/v/v/v). Within-laboratory, intraday, and interday method variation (% relative standard deviation) for most samples extracted by methanol or methanol-water mixture were <5%. Lower levels of kavalactone contents and higher variations were observed for tea bags from water extraction or infusion as compared to methanol extraction. Labeling information of tea bags based on methanol extraction could be misleading to consumers. Analytical recoveries of snack bar fortified at 10 and 20 microg/g were >84% with RSD values <8%. Methods developed in this study offer a simple and reproducible means for analysis of kavalactones in various matrixes of dietary products.

Microbial metabolism. Part 5. Dihydrokawain.

Preparative scale fermentation of (6S)-dihydrokawain (1) with Rhizopus arrhizus (ATCC 11145) gave 3'-hydroxydihydrokawain (2) and (8S)-hydroxydihydrokawain (3). Structure elucidation of the metabolites was based on spectroscopic data. The C-8 absolute configuration of (3) was assessed via its Mosher's esters.

Desmethoxyyangonin and dihydromethysticin are two major pharmacological kavalactones with marked activity on the induction of CYP3A23.

Kava kava (Piper methysticum), an herbal remedy, is widely used for the treatment of mild to moderate cases of anxiety. The therapeutic activity is presumably achieved through multiple constituents called kavalactones. Recently, kava extracts were shown to induce CYP3A4 and activate human pregnane X receptor (PXR). This study was undertaken to test the ability of purified kavalactones to induce CYP3A23 and activate PXR. Rat hepatocytes were treated with desmethoxyyangonin, dihydrokawain, dihydromethysticin, kawain, methysticin, or yangonin, and the expression of CYP3A23 was monitored. Among the kavalactones, only desmethoxyyangonin and dihydromethysticin markedly induced the expression of CYP3A23 (approximately 7-fold). A similar magnitude of induction was detected with combined six kavalactones at a noninductive concentration when individually used. The induced expression, however, was markedly reduced or completely abolished if dihydromethysticin, desmethoxyyangonin, or both were excluded from the mixtures. Interestingly, regardless of whether dihydromethysticin or desmethoxyyangonin was used alone or together with other kavalactones, similar amounts of total kavalactones were needed to produce comparable induction, suggesting that the inductive activity of dihydromethysticin and desmethoxyyangonin is additively/synergistically enhanced by other kavalactones. In addition, treatment with dihydromethysticin, desmethoxyyangonin, or pregnenolone 16alpha-carbonitrile (PCN) markedly increased the levels of CYP3A23 mRNA, and inhibition of mRNA synthesis abolished the induction. In contrast to PCN, dihydromethysticin and desmethoxyyangonin only slightly activated rat or human PXR. These findings suggest that the induction of CYP3A23 by dihydromethysticin and desmethoxyyangonin involves transcription activation, probably through a PXR-independent or PXR-involved indirect mechanism.

In vitro toxicity of kava alkaloid, pipermethystine, in HepG2 cells compared to kavalactones.

Kava herbal supplements have been recently associated with acute hepatotoxicity, leading to the ban of kava products in approximately a dozen countries around the world. It is suspected that some alkaloids from aerial kava may have contributed to the problem. Traditionally, Pacific Islanders use primarily the underground parts of the shrub to prepare the kava beverage. However, some kava herbal supplements may contain ingredients from aerial stem peelings. The aim of this study was to test the in vitro effects of a major kava alkaloid, pipermethystine (PM), found mostly in leaves and stem peelings, and kavalactones such as 7,8-dihydromethysticin (DHM) and desmethoxyyangonin (DMY), which are abundant in the roots. Exposure of human hepatoma cells, HepG2, to 100 microM PM caused 90% loss in cell viability within 24 h, while 50 microM caused 65% cell death. Similar concentrations of kavalactones did not affect cell viability for up to 8 days of treatment. Mechanistic studies indicate that, in contrast to kavalactones, PM significantly decreased cellular ATP levels, mitochondrial membrane potential, and induced apoptosis as measured by the release of caspase-3 after 24 h of treatment. These observations suggest that PM, rather than kavalactones, is capable of causing cell death, probably in part by disrupting mitochondrial function. Thus, PM may contribute to rare but severe hepatotoxic reactions to kava.

Identification of novel electrophilic metabolites of piper methysticum Forst (Kava).

Dietary supplements containing Piper methysticum Forst. (kava) have been implicated in multiple cases of liver injury in humans, including 10 recently reviewed cases in which patients required liver transplantation following the usage of kava-containing products (Centers for Disease Control and Prevention, reprinted. (2003) J. Am. Med. Assoc. 289, 36-37). To investigate a possible mechanism(s) of kava-induced hepatotoxicity, an extract of kava was incubated in vitro with hepatic microsomes, NADPH, and GSH. Electrophilic intermediates that were generated via metabolic activation were trapped as GSH conjugates and removed from the protein mixture using ultrafiltration. Positive ion electrospray LC-MS/MS with precursor ion scanning was used for the selective detection of GSH conjugates, and LC-MS(n) product ion scanning was used to elucidate their structures. Using this in vitro MS-based screening assay, two novel electrophilic metabolites of kava, 11,12-dihydroxy-7,8-dihydrokavain-o-quinone and 11,12-dihydroxykavain-o-quinone, were identified. Mercapturic acids of these quinoid species were not detected in the urine of a human volunteer following ingestion of a dietary supplement that contained kava; instead, the corresponding catechols were metabolized extensively to glucuronic acid and sulfate conjugates. These observations indicate that quinoid metabolites, under most circumstances, are probably not formed in substantial quantities following the ingestion of moderate doses of kava. However, the formation of electrophilic quinoid metabolites by hepatic microsomes in vitro suggests that such metabolites might contribute to hepatotoxicity in humans when metabolic pathways are altered (e.g., because of a drug interaction, genetic difference in enzyme expression, etc.) or if conjugation pathways become saturated.

Drug-herb interaction among commonly used conventional medicines: a compendium for health care professionals.

The objective of the review was to consolidate the clinical and pharmacologic aspects of drug-herb interactions to develop a compendium of information to provide prescribers with a measure of the risk of interactions, a description of the clinical consequences, and an assessment of the quality (ie, validity) of evidence. A variety of electronic databases and hand-searched references were used to identify documentation of interactions between herbal products and drugs from the most commonly used therapeutic classes. MEDLINE, Allied and Complementary Medicine Database, CINHAL, HealthSTAR, and EMBASE were searched from 1966 to the present. One hundred sixty-two citations were identified. Only 22 citations met the inclusion criteria. Using a matrix of 165 possible drug-herb interaction pairs (15 therapeutic drug classes by 11 herbal products), we identified 51 (31%) interactions discussed in the literature. Twenty-two of these 51 drug-herb pairs (43%) were supported by randomized clinical trials, case-control studies, cohort studies, case series, or case studies. The remaining interaction pairs reflected theoretic reasoning in the absence of clinical data. Most interactions were pharmacokinetic, with most actually or theoretically affecting the metabolism of the affected product by way of the cytochrome p450 enzymes. In this review, warfarin was the most common drug and St. John's wort was the most common herbal product reported in drug-herb interactions. To create a comprehensive and valid list of herb-drug interactions would require a substantial increase in research activities in this area. Improvements in the quality of methodology used are also necessary.

Herbal medicines and perioperative care.

CONTEXT: Widespread use of herbal medications among the presurgical population may have a negative impact on perioperative patient care. OBJECTIVES: To review the literature on commonly used herbal medications in the context of the perioperative period and provide rational strategies for managing their preoperative use. DATA SOURCES: The MEDLINE and Cochrane Collaboration databases were searched for articles published between January 1966 and December 2000 using the search terms herbal medicine, phytotherapy, and alternative medicine and the names of the 16 most commonly used herbal medications. Additional data sources were obtained from manual searches of recent journal articles and textbooks. STUDY SELECTION: We selected studies, case reports, and reviews addressing the safety and pharmacology of 8 commonly used herbal medications for which safety information pertinent to the perioperative period was available. DATA EXTRACTION: We extracted safety, pharmacodynamic, and pharmacokinetic information from the selected literature and reached consensus about any discrepancies. DATA SYNTHESIS: Echinacea, ephedra, garlic, ginkgo, ginseng, kava, St John's wort, and valerian are commonly used herbal medications that may pose a concern during the perioperative period. Complications can arise from these herbs' direct and pharmacodynamic or pharmacokinetic effects. Direct effects include bleeding from garlic, ginkgo, and ginseng; cardiovascular instability from ephedra; and hypoglycemia from ginseng. Pharmacodynamic herb-drug interactions include potentiation of the sedative effect of anesthetics by kava and valerian. Pharmacokinetic herb-drug interactions include increased metabolism of many drugs used in the perioperative period by St John's wort. CONCLUSIONS: During the preoperative evaluation, physicians should explicitly elicit and document a history of herbal medication use. Physicians should be familiar with the potential perioperative effects of the commonly used herbal medications to prevent, recognize, and treat potentially serious problems associated with their use and discontinuation.