Development of Certified Reference Material Solutions for Phytochemicals in Ginger and Kava.

BACKGROUND: Dietary supplements derived from botanicals are commonly consumed and investigated in biomedical studies for their potential health benefits. Accurate identification and quantification of key chemical constituents from botanical ingredients is necessary for consistent product preparations and reproducible research results. Manufacturers need quantitative reference materials of the chemical constituents of interest to verify the content of ingredients and products. The rigor and reproducibility of biomedical research is enhanced through thorough characterization of the interventions used in mechanistic, clinical, and safety investigations. Quantitative reference materials enable reliable product quality assessments and reproducible research results. OBJECTIVE: Solution-based certified reference material (CRM) mixes were developed as calibrants for phytochemicals in ginger and kava. The kava CRM contained yangonin, desmethoxyyangonin, dihydrokavain, DL-kavain, methysticin, dihydromethysticin, flavokawain A, flavokawain B, and flavokawain C. The ginger CRM contained 6-gingerol, 8-gingerol, 10-gingerol, 6-shogaol, 8-shogaol, and 10-shogaol. METHODS: Each phytochemical was sourced as an isolated compound and assigned a purity factor by a mass balance approach accounting for residual impurities. The solution standard mixes were formulated by gravimetric addition of each phytochemical incorporating the purity factor and diluting with acetonitrile to the target concentrations of 500 µg/mL for the gingerols and shogaols, 250 µg/mL for the kavalactones, and 25 µg/mL for the flavokawains. RESULTS: The concentration accuracy of each component in the solution mixes was analytically verified by ultra high performance liquid chromatography with ultraviolet detection (UHPLC-UV) assay comparison to an independently prepared calibration solution. Each component in the ginger and kava CRMs were within 5 and 7% of the target concentrations, respectively. CONCLUSION: Homogeneous kava and ginger phytochemical solution mixes were produced with accurate constituent concentrations and demonstrated good stability over 2 years. These solution mixes were launched as commercially available CRMs. HIGHLIGHTS: These mixes can be used as accurate concentration stock solutions to prepare calibrators and controls for botanical dietary supplement product testing and standardization.

Neuroimaging Insights: Kava's (Piper methysticum) Effect on Dorsal Anterior Cingulate Cortex GABA in Generalized Anxiety Disorder.

Generalised Anxiety Disorder (GAD) is a prevalent, chronic mental health disorder. The measurement of regional brain gamma-aminobutyric acid (GABA) offers insight into its role in anxiety and is a potential biomarker for treatment response. Research literature suggests Piper methysticum (Kava) is efficacious as an anxiety treatment, but no study has assessed its effects on central GABA levels. This study investigated dorsal anterior cingulate (dACC) GABA levels in 37 adult participants with GAD. GABA was measured using proton magnetic resonance spectroscopy (1H-MRS) at baseline and following an eight-week administration of Kava (standardised to 120 mg kavalactones twice daily) (n = 20) or placebo (n = 17). This study was part of the Kava for the Treatment of GAD (KGAD; ClinicalTrials.gov: NCT02219880), a 16-week intervention study. Compared with the placebo group, the Kava group had a significant reduction in dACC GABA (p = 0.049) at eight weeks. Baseline anxiety scores on the HAM-A were positively correlated with GABA levels but were not significantly related to treatment. Central GABA reductions following Kava treatment may signal an inhibitory effect, which, if considered efficacious, suggests that GABA levels are modulated by Kava, independent of reported anxiety symptoms. dACC GABA patterns suggest a functional role of higher levels in clinical anxiety but warrants further research for symptom benefit. Findings suggest that dACC GABA levels previously un-examined in GAD could serve as a biomarker for diagnosis and treatment response.

Clinical pharmacokinetics of kavalactones after oral dosing of standardized kava extract in healthy volunteers.

ETHNOPHARMACOLOGICAL RELEVANCE: Piper methysticum G. Forst. (Piperaceae), commonly known as kava, has been used as a traditional beverage for centuries for its relaxing properties. Kavalactones are considered to be the major constituents responsible for kava's beneficial effects. Despite the extensive use of kava, clinical pharmacokinetic data is not available in the literature; therefore, the findings of this study will be critical for the dosage calculations for future clinical evaluation of kava. AIM OF THE STUDY: The aim of the current study is to examine the clinical pharmacokinetics of six major kavalactones following oral dosing of flavokavain A/B-free standardized kava extract capsules in healthy volunteers using two dosage regimens. MATERIALS AND METHODS: A sensitive, reliable, and specific ultra-high pressure liquid chromatography-mass spectrometry (UPLC-MS/MS) method was developed and validated for the simultaneous quantification of six major kavalactones (kavain, dihydrokavain, methysticin, dihydromethysticin, yangonin, and desmethoxyyangonin) and two flavokavains (A and B) in human plasma. Pharmacokinetic profiles were assessed in ten healthy volunteers after oral doses of standardized kava product, and plasma samples were analyzed for six kavalactones and two flavokavains using the validated UPLC-MS/MS method. Concentration-time data was subjected to pharmacokinetic analysis. RESULTS: The systemic exposure of the kavalactones was found to be in the following order: dihydrokavain > dihydromethysticin > kavain > methysticin > yangonin. Desmethoxyyangonin was quantifiable only at a couple of time points, while flavokavain A and flavokavain B were not present in any of the plasma samples. Fast absorption of five kavalactones was observed with time to reach the maximum plasma concentration of 1-3 h. A dose proportionality in pharmacokinetics was established from 75 to 225 mg of kavalactone doses. In the multiple-dose study, a significant reduction in the extent of absorption of kavalactones with food was observed. CONCLUSION: Single and multiple-dose clinical pharmacokinetic studies for kava were performed in healthy volunteers, and higher exposure to the kavalactones was observed after single-dosing (225 mg), while a longer duration of exposure was observed after three times a day (3 x 75 mg) dosing.

Biopharmaceutical evaluation of kavain in Piper methysticum G. Forst dried extract: Equilibrium solubility and intestinal permeability in Caco-2 cell model.

ETHNOPHARMACOLOGICAL RELEVANCE: Piper methysticum G. Forst, popularly known as kava, is a traditional medicinal plant native from South Pacific islands and widely used to treat anxiety, depression and stress. The psychoactive properties are related to the kavalactones, mainly kavain. AIM OF THE STUDY: To evaluate the biopharmaceutical properties of synthetic kavain and when present in kava dried extracts by means of equilibrium solubility and intestinal permeability studies in the Caco-2 cell model. MATERIALS AND METHODS: The equilibrium solubility of kavain was performed using a shake flask incubator at 37 °C in different media at physiological pH range (1.2-6.8). The intestinal permeability of kavain evaluated in Caco-2 cells in the presence and absence of the P-glycoprotein inhibitor verapamil. Kavain concentrations were determined by reversed phase high performance liquid chromatography (HPLC). RESULTS: HPLC methods were developed and fully validated for kavain quantitation. Kavain demonstrated low solubility and the pH of the aqueous media did not affect its solubility. Kavain was found to be highly permeable and efflux of kavain mediated by P-glycoprotein was not significant during intestinal permeation. CONCLUSION: The results of biopharmaceutical studies provided useful information for predicting availability of kavain from the gastrointestinal tract and this compound was ranked as BCS Class II, exhibiting dissolution rate-limited absorption.

Investigating volatiles as the secondary metabolome of Piper methysticum from root powder and water extracts using comprehensive two-dimensional gas chromatography.

ETHNOPHARMACOLOGICAL RELEVANCE: Kava (Piper methysticum G. Forst) is a plant grown in the Pacific that is used in traditional medicines. The roots are macerated and powdered for consumption as a beverage in social settings as well as in ceremonies. Other types of preparations can also be used as traditional medicines. There has been an increase in demand for kava as there is continued traditional use and as it is becoming utilized more both socially and medicinally outside of Oceania. Currently, most research of this plant has focused on bioactive kavalactones and flavokawains, and there are few studies focusing on the other compounds that kava contains, such as volatile and semivolatile components. AIM OF THE STUDY: This study investigated the kava volatile organic compound (VOC) profile from nine different commercially available samples of dried, powdered kava root sourced across the Pacific region. MATERIALS AND METHODS: The headspace above the kava samples was analyzed, both from the root powder as originally purchased and by performing a scaled-down extraction into water mimicking traditional preparation of the beverage. The headspace of each sample was extracted using solid-phase microextraction arrow (SPME Arrow), followed by analysis using comprehensive two-dimensional gas chromatography - quadrupole mass spectrometry/flame ionization detection (GC×GC-qMS/FID). The superior peak capacity of GC×GC was invaluable in effectively separating the complex mixture of compounds found in all samples, which enabled improved monitoring of minor differences between batches. RESULTS: Dry root powder samples contained high levels of ß-caryophyllene while water extracted samples showed high levels of camphene. Many alcohols, aldehydes, ketones, terpenes, terpenoids, and aromatics were also characterized from both types of samples. All water extracted samples from the different brands followed similar trends in terms of compounds being detected or not. Additional major compounds found in water extracts included benzaldehyde, hexanal, methoxyphenyloxime, camphor, limonene, 1-hexanol, endoborneol, and copaene. While some samples could be differentiated based on brand, samples did not group by purported geographic origin. CONCLUSIONS: This study provides foundational data about a different subset of compounds within kava than previous research has studied, and also informs the community of the compounds that transfer into the consumed beverage during the traditional means of preparing kava.

Characterization of Different Forms of Kava (Piper methysticum) Products by UPLC-MS/MS.

There are several forms of kava (Piper methysticum) products available for human consumption, and many factors are known to influence their chemical compositions and therefore their pharmacological properties. Because of the increased popularity of kava intake, a rigorous characterization of their content diversity is prerequisite, particularly due to its known potential to cause hepatotoxicity. To understand the composition diversity of kavalactones and flavokavains in commercial kava products, we developed a UPLC-MS/MS-based analytical method for the quantification of six kavalactones (kavain, dihydrokavain, methysticin, dihydromethysticin, yangonin and desmethoxyyangonin) and two flavokavains (flavokavains A and B) and analyzed their contents in 28 different kava products in the form of capsules, tinctures, traditional aqueous suspensions and dried powders. Our results demonstrated a great variation in terms of the total and relative abundance of the analyzed kavalactones and flavokavains among the analyzed kava preparations. More importantly, the kavalactone abundance in the product label could differ up to 90% from our experimental measurements. Therefore, more rigorous and comprehensive quality control of kava products is required with respect to the content of individual kavalactones and flavokavains. Accurate content information is essential to understand the pharmacological properties and safety of different kava products.

Simultaneous determination of both kavalactone and flavokawain constituents by different single-marker methods in kava.

Kava, the rhizomes and roots of Piper methysticum Forst, is a popular edible medicinal herb traditionally used to prepare beverages for anxiety reduction. Since the German kava ban has been lifted by the court, the quality evaluation is particularly important for its application, especially the flavokawains which were believed to be responsible for hepatotoxicity. Now, by employing two different standard references and four different methods to calculate the relative correction factors, eight different quantitative analyses of multicomponents by single-marker methods have been developed for the simultaneous determination of eight major kavalactones and flavokawains in kava. The low standard method difference on quantitative measurement of the compounds among the external standard method and ours confirmed the reliability of the mentioned methods. A radar plot clearly illustrated that the contents of dihydrokavain and kavain were higher, whereas flavokawains A and B were lower in different kava samples. Only one of eight samples did not detect flavokawains that may be related to hepatotoxicity. In summary, by using different agents as an internal standard reference, the developed methods were believed as a powerful analytical tool not only for the qualitative and quantitative of kava constituents but also for the other multicomponents when authentic standard substances were unavailable.

Flavokawain C exhibits anti-tumor effects on in vivo HCT 116 xenograft and identification of its apoptosis-linked serum biomarkers via proteomic analysis.

Chalcones and their derivatives belong to the flavonoid family. They have been extensively studied for their anticancer properties and some have been approved for clinical use. In this study, the in vivo anti-tumor activity of flavokawain C (FKC), a naturally occurring chalcone found in Kava (Piper methysticum Forst) was evaluated in HCT 116 cells (colon carcinoma). We also attempted to identify potential biomarkers and/or molecular targets in serum with applicability in predicting treatment outcome. The anti-tumor effects and toxicity of FKC were assessed using the xenograft nude mice model. Cisplatin was used as positive control. The anti-proliferative and apoptotic activities were then evaluated in tumor tissues treated with FKC. Furthermore, two-dimensional electrophoresis (2-DE) followed by protein identification using MALDI-TOF/TOF-MS/MS was performed to compare the serum proteome profiles between healthy nude mice and nude mice bearing HCT 116 tumor treated with vehicle solution and FKC, respectively. Our results showed that FKC treatment significantly inhibited HCT 116 tumor growth. In vivo toxicity studies showed that administration of FKC did not cause damage to major organs and had no significant effect on body weight. FKC was found to induce apoptosis in tumor, and this was associated with increased expression of cleaved caspase-3 and decreased expression of Ki67 in tumor tissues. Our proteomic analysis identified five proteins that changed in abundance - Ig mu chain C region (secreted form), GRP78, hemopexin, kininogen-1 and apolipoprotein E. Overall, our findings demonstrated the potential of FKC as an anti-cancer agent for the treatment of colon carcinoma.

Nutraceuticals for Sleep Disorders.

Sleep is considered as one of the most important aspects for maintaining a healthy life. For a person to function normally, at least 6-8 hours of sleep daily is necessary. Sleep not only affects our mood, but also regulates the efficiency of work done. Many complications arise due to inadequacy of sleep. The unhealthy food and lifestyle choices have made us more prone to sleep disorders. The medications used for the treatment of sleep disorders are mainly habit forming and have tendencies of withdrawal symptoms. This inadequacy in medication has lead to search for newer, better options. The field of nutraceuticals fits apt for treating such disorders. The quality of being non-toxic, non-habit forming, and being practically more efficient has had made it an excellent option. Nutraceuticals make use of food or part of food for the treatment or to prevent any disease. Remarkable positive effects of nutraceuticals like Caffeine, Chamomile, Kava kava, Cherries and Cherry juice, L tryptophan, Valerian, Vitamin D, Marijuana, melatonin, Lemon balm had been mentioned in the treatment of sleep disorders. The present review gives a general overview of nutraceuticals and discusses their use in sleep disorders.

Kava decreases the stereotyped behavior induced by amphetamine in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Kava extract (Piper methysticum) is a phytotherapic mainly used for the treatment of anxiety. Although the reported effects of Kava drinking improving psychotic symptoms of patients when it was introduced to relieve anxiety in aboriginal communities, its effects on models of psychosis-like symptoms are not investigated. AIM OF THE STUDY: To investigate the effects of Kava extract on behavioral changes induced by amphetamine (AMPH) and its possible relation with alterations in monoamine oxidase (MAO) activity. MATERIALS AND METHODS: Mice received vehicle or Kava extract by gavage and, 2 h after vehicle or AMPH intraperitoneally. Twenty-five minutes after AMPH administration, behavioral (elevated plus maze, open field, stereotyped behavior, social interaction and Y maze) and biochemical tests (MAO-A and MAO-B activity in cortex, hippocampus and striatum) were sequentially evaluated. RESULTS: Kava extract exhibited anxiolytic effects in plus maze test, increased the locomotor activity of mice in open field test and decreased MAO-A (in cortex) and MAO-B (in hippocampus) activity of mice. Kava extract prevented the effects of AMPH on stereotyped behavior and, the association between Kava/AMPH increased the number of entries into arms in Y maze test as well as MAO-B activity in striatum. However, Kava extract did not prevent hyperlocomotion induced by AMPH in open field test. The social interaction was not modified by Kava extract and/or AMPH. CONCLUSION: The results showed that Kava extract decreased the stereotyped behavior induced by AMPH at the same dose that promotes anxiolytic effects, which could be useful to minimize the psychotic symptoms in patients.

Kava (Piper methysticum) Extract for the Treatment of Nervous Anxiety, Tension and Restlessness.

AIM: Prior to the kava ban of 2002, the indication for kava (Piper methysticum) extracts defined by the German Commission E was "nervous anxiety, tension and restlessness". In 2000, an observational trial was started in Germany with the aim of defining symptoms of these indications best treated with kava extract. The trial was interrupted and archived "unevaluated" in 2001 due to the upcoming safety debate on kava. The data from this study has now been analyzed in order to identify symptoms best treated with kava. METHODS: Documentation was available from 156 patients. Twelve typical symptoms of nervous anxiety, tension and restlessness were assessed on a five-item rating scale, together with the therapeutic context, the perceived time of onset of effects and the safety of application. RESULTS: The median duration of treatment was 28 days. All individual symptoms showed significant and clinically relevant improvements. The most effective results were seen for nervous tension and restlessness, with better effects in patients with acute versus chronic complaints. The safety of the treatment was found to be excellent, which included the assessment of laboratory data. CONCLUSIONS: Overall, the study confirms the effective and safe short-term use of kava in the Commission E-defined indication of "nervous anxiety, tension and restlessness", especially in other than chronic cases. The clinical use of kava might be translated into context-related phobias according to ICD-10 F40, or to nervous tension (ICD10 R45.0) or restlessness and excitation (ICD-10 R45.1).

Kava as a Clinical Nutrient: Promises and Challenges.

Kava beverages are typically prepared from the root of Piper methysticum. They have been consumed among Pacific Islanders for centuries. Kava extract preparations were once used as herbal drugs to treat anxiety in Europe. Kava is also marketed as a dietary supplement in the U.S. and is gaining popularity as a recreational drink in Western countries. Recent studies suggest that kava and its key phytochemicals have anti-inflammatory and anticancer effects, in addition to the well-documented neurological benefits. While its beneficial effects are widely recognized, rare hepatotoxicity had been associated with use of certain kava preparations, but there are no validations nor consistent mechanisms. Major challenges lie in the diversity of kava products and the lack of standardization, which has produced an unmet need for quality initiatives. This review aims to provide the scientific community and consumers, as well as regulatory agencies, with a broad overview on kava use and its related research. We first provide a historical background for its different uses and then discuss the current state of the research, including its chemical composition, possible mechanisms of action, and its therapeutic potential in treating inflammatory and neurological conditions, as well as cancer. We then discuss the challenges associated with kava use and research, focusing on the need for the detailed characterization of kava components and associated risks such as its reported hepatotoxicity. Lastly, given its growing popularity in clinical and recreational use, we emphasize the urgent need for quality control and quality assurance of kava products, pharmacokinetics, absorption, distribution, metabolism, excretion, and foundational pharmacology. These are essential in order to inform research into the molecular targets, cellular mechanisms, and creative use of early stage human clinical trials for designer kava modalities to inform and guide the design and execution of future randomized placebo controlled trials to maximize kava's clinical efficacy and to minimize its risks.

Protective effect of kava constituents in an in vitro model of oral mucositis.

PURPOSE: 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. METHODS: 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 H2O2. The effects were quantified by analysis of ROS production, using a CM-H2DCFDA assay. RESULTS: 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. CONCLUSION: 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.

Synthetic Kavalactone Analogues with Increased Potency and Selective Anthelmintic Activity against Larvae of Haemonchus contortus In Vitro.

Kava extract, an aqueous rhizome emulsion of the plant Piper methysticum, has been used for centuries by Pacific Islanders as a ceremonial beverage, and has been sold as an anxiolytic agent for some decades. Kavalactones are a major constituent of kava extract. In a previous investigation, we had identified three kavalactones that inhibit larval development of Haemonchus contortus in an in vitro-bioassay. In the present study, we synthesized two kavalactones, desmethoxyyangonin and yangonin, as well as 17 analogues thereof, and evaluated their anthelmintic activities using the same bioassay as employed previously. Structure activity relationship (SAR) studies showed that a 4-substituent on the pendant aryl ring was required for activity. In particular, compounds with 4-trifluoromethoxy, 4-difluoromethoxy, 4-phenoxy, and 4-N-morpholine substitutions had anthelmintic activities (IC50 values in the range of 1.9 to 8.9 µM) that were greater than either of the parent natural products-desmethoxyyangonin (IC50 of 37.1 µM) and yangonin (IC50 of 15.0 µM). The synthesized analogues did not exhibit toxicity on HepG2 human hepatoma cells in vitro at concentrations of up to 40 µM. These findings confirm the previously-identified kavalactone scaffold as a promising chemotype for new anthelmintics and provide a basis for a detailed SAR investigation focused on developing a novel anthelmintic agent.

Dihydromethysticin, a natural molecule from Kava, suppresses the growth of colorectal cancer via the NLRC3/PI3K pathway.

Dihydromethysticin (DHM), a natural compound derived from Kava, has been reported to be effective against mental disorders and some malignant tumors. However, little is known about the inhibitory effect of DHM on colorectal cancer (CRC). First, we examined the impact of DHM on human colon cancer cell lines, which demonstrated that DHM inhibits proliferation, migration, and invasion and promotes apoptosis and cell cycle arrest in colon cancer cells in vitro. Using small hairpin RNA, we inhibited nucleotide-oligomerization domain-like receptor subfamily C3 (NLRC3)/phosphoinositide 3-kinase (PI3K) pathway to elucidate the partial signaling of DHM-mediated tumor suppression. Additionally, using an ectopic human CRC model, we verified whether DHM inhibits tumor growth and angiogenesis via the NLRC3/PI3K pathway in vivo. Overall, DHM showed an inhibitory effect on CRC by altering cell proliferation, migration, invasion, apoptosis, cell cycle, and angiogenesis, possibly via the NLRC3/PI3K pathway. Thus, DHM may be a promising candidate for CRC therapy.

The Impact of One-week Dietary Supplementation with Kava on Biomarkers of Tobacco Use and Nitrosamine-based Carcinogenesis Risk among Active Smokers.

Tobacco smoking is the primary risk factor for lung cancer, driven by the addictive nature of nicotine and the indisputable carcinogenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as well as other compounds. The integration of lung cancer chemoprevention with smoking cessation is one potential approach to reduce this risk and mitigate lung cancer mortality. Experimental data from our group suggest that kava, commonly consumed in the South Pacific Islands as a beverage to promote relaxation, may reduce lung cancer risk by enhancing NNK detoxification and reducing NNK-derived DNA damage. Building upon these observations, we conducted a pilot clinical trial to evaluate the effects of a 7-day course of kava on NNK metabolism in active smokers. The primary objective was to compare urinary total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL plus its glucuronides, major metabolites of NNK) before and after kava administration as an indicator of NNK detoxification. Secondary objectives included determining kava's safety, its effects on DNA damage, tobacco use, and cortisol (a biomarker of stress). Kava increased urinary excretion of total NNAL and reduced urinary 3-methyladenine in participants, suggestive of its ability to reduce the carcinogenicity of NNK. Kava also reduced urinary total nicotine equivalents, indicative of its potential to facilitate tobacco cessation. Plasma cortisol and urinary total cortisol equivalents were reduced upon kava use, which may contribute to reductions in tobacco use. These results demonstrate the potential of kava intake to reduce lung cancer risk among smokers.

Kava for generalised anxiety disorder: A 16-week double-blind, randomised, placebo-controlled study.

OBJECTIVE: Previous randomised, double-blind, placebo-controlled studies have shown that Kava (a South Pacific medicinal plant) reduced anxiety during short-term administration. The objective of this randomised, double-blind, placebo-controlled study was to perform a larger, longer-term trial assessing the efficacy and safety of Kava in the treatment of generalised anxiety disorder and to determine whether gamma-aminobutyric acid transporter (SLC6A1) single-nucleotide polymorphisms were moderators of response. METHODS: The trial was a phase III, multi-site, two-arm, 16-week, randomised, double-blind, placebo-controlled study investigating an aqueous extract of dried Kava root administered twice per day in tablet form (standardised to 120 mg of kavalactones twice/day) in 171 currently non-medicated anxious participants with diagnosed generalised anxiety disorder. The trial took place in Australia. RESULTS: An analysis of 171 participants revealed a non-significant difference in anxiety reduction between the Kava and placebo groups (a relative reduction favouring placebo of 1.37 points; p = 0.25). At the conclusion of the controlled phase, 17.4% of the Kava group were classified as remitted (Hamilton Anxiety Rating Scale score < 7) compared to 23.8% of the placebo group (p = 0.46). No SLC6A1 polymorphisms were associated with treatment response, while carriers of the rs2601126 T allele preferentially respond to placebo (p = 0.006). Kava was well tolerated aside from poorer memory (Kava = 36 vs placebo = 23; p = 0.044) and tremor/shakiness (Kava = 36 vs placebo = 23; p = 0.024) occurring more frequently in the Kava group. Liver function test abnormalities were significantly more frequent in the Kava group, although no participant met criteria for herb-induced hepatic injury. CONCLUSION: While research has generally supported Kava in non-clinical populations (potentially for more 'situational' anxiety as a short-term anxiolytic), this particular extract was not effective for diagnosed generalised anxiety disorder.

Kava and its Kavalactones Inhibit Norepinephrine-induced Intracellular Calcium Influx in Lung Cancer Cells.

Kava, the extract of the roots of Piper methysticum, has been traditionally consumed in the South Pacific islands for its natural relaxing property. Epidemiological data suggests that kava consumption may reduce human cancer risk, and in vitro and in vivo models suggest chemopreventive potential against carcinogen-induced tumorigenesis. Therefore, knowledge about its molecular mechanisms and responsible ingredient(s) for these beneficial properties will better guide kava's use for the management of these disorders. Psychological stress typically results in increased production of stress hormones, such as norepinephrine (NE), which activate adrenergic receptors (ARs). Psychological stress has also been associated with increased cancer incidence and poor clinical outcomes in cancer patients. Mechanistically, binding of NE to ARs induces intracellular calcium influx, which activates downstream signaling pathways involved in both stress and cancer development. In this study, we characterized the effect of kava and its components, 3 fractions and 6 major kavalactones, on NE-induced intracellular calcium influx in H1299, a human non-small cell lung carcinoma cell line. Results show that kava extract effectively inhibits NE-mediated intracellular calcium influx in H1299 cells, potentially through antagonizing ß-AR signaling. This inhibitory activity is recapitulated by the major kavalactones in kava. Among the 6 major kavalactones, DHK demonstrated the best potency. Taken together, our study suggests a novel mechanism through which kava and its ingredients potentially offer the anxiolytic and cancer-preventive activity.

Identification of a Kavain Analog with Efficient Anti-inflammatory Effects.

Kavain, a compound derived from Piper methysticum, has demonstrated anti-inflammatory properties. To optimize its drug properties, identification and development of new kavain-derived compounds was undertaken. A focused library of analogs was synthesized and their effects on Porphyromonas gingivalis (P. gingivalis) elicited inflammation were evaluated in vitro and in vivo. The library contained cyclohexenones (5,5-dimethyl substituted cyclohexenones) substituted with a benzoate derivative at the 3-position of the cyclohexanone. The most promising analog identifed was a methylated derivative of kavain, Kava-205Me (5,5-dimethyl-3-oxocyclohex-1-en-1-yl 4-methylbenzoate.) In an in vitro assay of anti-inflammatory effects, murine macrophages (BMM) and THP-1 cells were infected with P. gingivalis (MOI = 20:1) and a panel of cytokines were measured. Both cell types treated with Kava-205Me (10 to 200 µg/ml) showed significantly and dose-dependently reduced TNF-α secretion induced by P. gingivalis. In BMM, Kava-205Me also reduced secretion of other cytokines involved in the early phase of inflammation, including IL-12, eotaxin, RANTES, IL-10 and interferon-γ (p < 0.05). In vivo, in an acute model of P. gingivalis-induced calvarial destruction, administration of Kava-205Me significantly improved the rate of healing associated with reduced soft tissue inflammation and osteoclast activation. In an infective arthritis murine model induced by injection of collagen-antibody (ArthriomAb) + P. gingivalis, administration of Kava-205Me was able to reduce efficiently paw swelling and joint destruction. These results highlight the strong anti-inflammatory properties of Kava-205Me and strengthen the interest of testing such compounds in the management of P. gingivalis elicited inflammation, especially in the management of periodontitis.

Monoamine Oxidase Inhibition by Kavalactones from Kava (Piper Methysticum).

Monoamine oxidases (MAOs) are key metabolic enzymes for neurotransmitter and dietary amines and are targets for the treatment of neuropsychiatric and neurodegenerative disorders. This study examined the MAO inhibition potential of kavain and other kavalactones from the roots of kava (Piper methysticum), a plant that has been used for its anxiolytic properties. (±)-Kavain was found to be a good potency in vitro inhibitor of human MAO-B with an IC50 of 5.34 µM. (±)-Kavain is a weaker MAO-A inhibitor with an IC50 of 19.0 µM. Under the same experimental conditions, the reference MAO inhibitor, curcumin, displays IC50 values of 5.01 µM and 2.55 µM for the inhibition of MAO-A and MAO-B, respectively. It was further established that (±)-kavain interacts reversibly and competitively with MAO-A and MAO-B with enzyme-inhibitor dissociation constants (Ki) of 7.72 and 5.10 µM, respectively. Curcumin in turn, displays a Ki value of 3.08 µM for the inhibition of MAO-A. Based on these findings, other kavalactones (dihydrokavain, methysticin, dihydromethysticin, yangonin, and desmethoxyyangonin) were also evaluated as MAO inhibitors in this study. Yangonin proved to be the most potent MAO inhibitor with IC50 values of 1.29 and 0.085 µM for MAO-A and MAO-B, respectively. It may be concluded that some of the central effects (e.g., anxiolytic) of kava may be mediated by MAO inhibition.