Kava (Piper methysticum) Safety Concerns and Studies on

Chapter 17

Kava (Piper methysticum) Safety Concerns and Studies on Pipermethystine, an Alkaloid in Kava Downloaded by UNIV OF TENNESSEE KNOXVILLE on May 24, 2016 | http://pubs.acs.org Publication Date: September 1, 2008 | doi: 10.1021/bk-2008-0987.ch017

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Jin-Woo Jhoo , Catharina Y. W. Ang ,Nan Mei ,Tao Chen , Klaus Dragull , and Chung-Shih Tang 3

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National Center for Toxicological Research, U.S. Food and Drug Administration, 3900 NCTR Road, Jefferson, AR 72079 Department of Food Science and Technology in Animal Resources, Kangwon National University, 192-1 Hyoja-2, Chunchon, Kangwon 200-701, South Korea Department of Molecular Biosciences and Bioengineering, University of Hawaii, Honolulu, HI 96822 2

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Kava (Piper methysticum) extract products have been associated with a number of severe hepatotoxicity cases. Factors influencing the reported kava-linked liver injuries could include the methods of kava preparation, genetic differences between populations, herbal-drug interactions, enzyme inhibition and minor toxic kava constituents in the kava extracts. This article provides highlights of recent findings on kava safety issues, and also presents results of our current studies on pipermethystine, a kava alkaloid, in commercial products and its potential mutagenicity. The level of pipermethystine was found to be negligible in most of the kava dietary supplement products analyzed; however, one product sold as a mixture of kava roots and leaves did contain detectable amounts of pipermethystine. In vitro mutagenicity tests indicated that pipermethystine was negative in Salmonella umu assay at a dose range up to 1000 µM and in the mouse lymphoma assay at concentrations lower than 2.5 µg/mL. However, it showed cytotoxicity in mouse lymphoma cells in a dose-response manner.

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© 2008 American Chemical Society

Ho et al.; Dietary Supplements ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

Downloaded by UNIV OF TENNESSEE KNOXVILLE on May 24, 2016 | http://pubs.acs.org Publication Date: September 1, 2008 | doi: 10.1021/bk-2008-0987.ch017

249 Kava, also has been referred to as kava kava, is Piper methysticum Forst. f. in the family Piperaceae. It is a perennial shrub grown in Pacific islands including Fiji, Tonga, Vanuatu, Samoa, Futuna, New Caledonia, and the Hawaiian islands (7). The rootstocks are commonly used in the preparation of traditional beverages with calm and relaxation effects for social and ceremonial occasions for centuries. Some of the events where kava is commonly used are weddings, funerals, religious activities, welcoming of visitors, and exchanging of gifts. Various kava preparations have also been used as folk medicines for treatments of a range of symptoms, including chills, headaches, gastrointestinal upset and skin disease. Lebot et al (7, 2) described many different aspects about kava as used in the South Pacific region. During the last 20 years, some Western manufacturers have become interested in making kava extracts as an alternative medicine for the treatment of mental disorders, nervous anxiety, tension, and restlessness (5). Upon the passage of the Dietary Supplement Health and Education Act in 1994, more herbal dietary products with perceived beneficial effects became readily available in the U. S. A. The sales of kava dietary supplements or related products were booming worldwide in the late 1990s and early in the year 2000. Consumers were led to believe that kava products were effective, non-addictive, and safe. Kava root extract capsules, kava root powder capsules and kava tea blends became one of the top-selling herbal dietary supplements for a number of years before 2002. Within a few years, however, severe cases of hepatic toxicity, possibly associated with the consumption of herbal products containing kava were reported in several countries. Germany was the first country to ban kava products in 2002, and England, France, and Australia also removed kava products from the market in the following years. The liver injuries included hepatitis, cirrhosis, and liver failure. The U.S. FDA (4) issued an advisory to health care professionals and consumers regarding the potential risks of severe liver injury with use of kava supplements. Up to the present time, there have been 78 cases of hepatotoxicity presumed to be linked to kava ingestion (J), but it is still under debate as to whether the ban of kava products was justified or was an overreaction (6). Many of the liver failure cases were associated with products containing acetone or alcohol extracts of kava (7). Proposed causes of liver injuries associated with kava include the commercial preparation methods (organic solvent extraction), genetic difference in consumers, inhibition of cytochrome P450 (CYP) enzymes by kavalactones, herbal-drug interactions, and the presence of toxic alkaloid, such as pipermethystine, in aerial parts of kava plant. Detailed discussions on the kava controversy and proposed causes of kava hepatotoxicity appeared recently (5, 8-11). The present article provides highlights of some key findings related to the chemical and safety aspects of


250 kava, and also presents results of our current studies on the determination of pipermethystine, a kava alkaloid, in dietary supplement products and testing for cytotoxicity and mutagenicity of pipermethystine.

Factors Related to Kava Safety Issues


Composition and Active Constituents Fresh kava rootstock contains about 80 % water. On a dry weight basis, the composition is approximately 12% water, 43% starch, 10% fiber, 3.2% sugars, 3.6% proteins, 3.2% minerals, and 15% kavalactones which can vary from 3 to 20%. Many factors influence the composition, including the age of plant, cultivars, location and growing conditions (1, 12). Kavalactones, also have been referred to as kavapyrones, are regarded as pharmacologically active constituents responsible for the anti-anxiety and mild sedative action. They are present as a mixture of more than 18 different forms that appear to have synergistic effects. Six kavalactones (Figure 1) are present in highest concentrations and they account for 96% of the lipid extract. These major kavalactones are (+)methysticin, (+)-dihydromethysticin, (+)-kavain, (+)-dihydrokavain, yangonin, and desmethoxyyangonin (13). The total kavalactone content is generally highest in the roots, followed by stumps and basal stems (72), and the lowest content is in the stems and leaves (7). The distribution of various kavalactones is different among different cultivars and kava plant parts. Dihydrokavain and dihydromethysticin are the major constituents in leaves and stems. Kavain and methysticin are the major components of the roots and rhizome (14, 15). The ratios of kavalactones may be important to biological effects. Other constituents from kava rootstock include trace pyrrolidine alkaloids, three chalcones (flavokavains, A, B, C) (73, 14), and a different alkaloid, pipermethystine, in kava leaves, which is specific to P. methysticum (16). There are also alcohols (e.g. dihydrokavain-5-ol), ketones (cinnamalaketone), essential oils, and methylene (e.g dioxy-3,4-cinnamalaketone) (73, 14).

Methods of Kava Preparation for Consumption Traditional Methods In the South Pacific islands, rootstocks are used to prepare kava beverages. The traditional methods involve chewing, masticating, grating, grinding or


251

Kavain Dihydrokavain Methysticin Dihydromethysticin Yangonin Desmethoxyyangonin

*0


R'

R H H H H -O-CHr-O-O-CHr-O-OMe H H

C5-C6

C7-C11

H

)Ac Pipermethystine Figure 1. Structures of six major kavalactones and pipermethystine.

poundingfreshor dried kava rootstocks and stumps, followed by immersing and mixing the processed mass in cold water (7, 77). The solids are strained through a strainer made of certain types of plant barks or leaves. The aqueous infusions of kava rootstock made by these methods are actually emulsions of suspended kava resin in water. Kavalactones are released into the water as a suspension rather than as a true solution (7). The kava beverages made by the traditional methods are generally referred to as aqueous infusions or water extracts.

Industrial Extraction Methods Kava pharmaceuticals or dietary supplements are commonly made using organic solvent, such as acetone and ethanol in a mixture with water. An ethanolwater mixture can be used to extract kava and gives crude extracts containing about 30% kavalactones, while an acetone-water can be used to result in concentrated extracts containing 70% kavalactones (18). The solvents are then dried and the extracts may or may not be standardized to certain concentrations of total kavalactones in the final forms of capsules, tablets, or tinctures. The dietary products made from organic solvent extraction are referred to as herbal products or dietary supplements. However, some capsules may contain only the powdered kava roots, not the concentrated extracts. It is well documented that the compositions of aqueous infusions and organic extracts are very different (79, 20), the former contains more polar components and the latter contains more non-polar constituents. Because the polarity of different kavalactones are different, the ratios of the individual


252 kavalactones are dependent upon the preparation methods (79, 20), and the difference in kavalactone ratios may contribute to different biological effects in consumers.


Aqueous Infusion and Its Effects on Indigenous Populations Some indigenous populations, who are frequent or excessive users of kava beverages (prepared by the traditional methods), have not developed liver problems (27). Approximately 10 % Caucasian populations have a genetic CYP 2D6 deficiency that could contribute to the hepatotoxicity of patients who ingest kava extract products (8, 22), while Polynesians do not have this deficiency (8). A number of studies, however, have also shown various other symptoms possibly caused by the heavy consumption of kava.

General Adverse Effects Some adverse health effects were found in heavy users, such as those reported in Australian aborigines, Arnhem Land (25, 24), and Fiji (25). These adverse effects included general poor health, a "puff face, scaly rash, headache, chest pain, indigestion, (23-25) mild and reversible gastrointestinal disturbance, and kava dermatopathy (scaly skin eruption) (26). A more recent survey by Tavana et al. (77) in Savaii, Samoa also reported that heavy drinkers might experience dizziness, dry pale skin, weight loss, and upset stomach. But the symptoms were reversible. Their data, collected by interviewing with traditional healers and biomedical practitioners, suggest that kava-related hepatotoxicity is lacking in native populations in Savaii, Samoa. No liver problems were reported for patients who had a history of drinking only kava without alcohol.

Effects on Liver Functions Very heavy users of kava can consume 400 g/week of kava root (24) that may contain 40 g total kavalactones (based on 10% kavalactones in roots) and might show weight loss and abnormal liver functions. A survey by Russmann et al. (26) demonstrated that heavy kava drinkers in New Caledonia showed elevated y-glutamyl transferase in 23/27 and minimally elevated transaminases in 8/27. These abnormal enzyme functions were not regarded as a sign of liver injury, but rather as an indication of CYP450 enzyme induction. A study by Clough et al. (27) showed that the liver function changes in users of aqueous kava extracts appear to be reversible. In the case of herbal product


253 hepatotoxicity, the aminotransferase and alkaline phosphatase levels were especially high and not reversible. More recent kava users developed higher levels of liver enzymes y-glutamyl transferase and alkaline phosphatase, but not alanine aminotransferase or bilirubin. The abnormalities in liver function usually return to normal within 1-2 weeks. No evidence for irreversible liver damage was reported.


Correlations to Other Health Effects On the positive side of health effects, populations with more kava consumption may have less cancer incidences. Steiner (28) published a statistical correlation and concluded that there was a close inverse relationship between kava consumption and age-standardized cancer incidence rates for all sites. Countries or cities compared were Vanuatu, Fiji, Western Samoa, Micronesia, New Caledonia, Hawaii/ Hawaiians, New Zealand/Maoris and Los Angeles. Another case-control study by Clough et al. (29) showed that there was no association between kava use and ischaemic heart disease in Aboriginal communities in eastern Arnhem Land Australia.

Organic Solvent Extracts and Kava Toxicity Kava and other herbs including garlic, ginkgo, echinacea, ginseng and St. John's wort have the potential to modulate the activity of drug-metabolizing enzymes (notably cytochromes P450 isozymes) and/or drug transporter Pglycoprotein. All of these products participate in potential pharmacokinetic interactions with anticancer drugs (30).

Enzyme Inhibition and HerbaUdrug Interactions Several studies have indicated that kava extract and kavalactones inhibited human liver microsome enzymes. Mathews, et al (31) reported that whole kava extract caused significant inhibitory effects on the activities of human liver microsomes cytochromes P450 enzymes (CYP1A2, 2C9, 2C19, 2D6, 3A4 and 4A9/11). However, individual kavalactones had varied effects on different P450 enzymes. Some kavalactones also formed 455 nm metabolic intermediate complexes after incubation with human liver microsomes and NADPH, but kavain and desmethoxyyangonin did not. These data indicate that kava has a high potential for causing drug interactions through inhibition of P450 enzymes responsible for the majority of the metabolism of pharmaceutical agents. Unger et al. (32) found that kavaprones exhibited inhibitory effect on CYP3A4 and


254 dihydromethysticin was the main inhibitory component of the ethyl acetate extract of kava.


Organic Solvent Extracts vs. Aqueous Infusions Using 2 cell lines to study the effects of kava, Zou et al. (33) reported that the parent compounds of each of the four test samples (methysticin, yangonin, desmethoxyyangonin and ethanolic extract of root) were primarily responsible for the observed cell toxicity and that CYP 1 A l , 2A6,2E1, and 3A4 or epoxide hydroxylase did not appear to be involved. In in vitro studies, kava was not activated to toxic metabolites by enzymes. Aqueous kava extracts had no effect on liver function tests in rats administered in daily dosages of 200 or 500 mg active kavalactones/kg body weight for 2 or 4 weeks (19). The data showed that alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and lactate dehydrogenase, and malondialdehyde were not elevated, and in some other cases, the enzymes were even significantly reduced, suggesting lack of toxic effect by aqueous kava extracts on the liver. The study also included the analysis of kavalactone content as extracted by different solvents. The ratios of different kavalactones were different for the different extraction methods; kavain and methysticin were predominant in water extracts while the proportions of these two compounds were less in ethanol extracts. Dihydrokavain and dihydromethysticin levels were higher in non-polar solvent extracts. Whether some or all of the kavalactones are hepatotoxic is still unclear; and whether large amounts of these kavalactones or other compounds extracted are responsible for the liver damage remains to be resolved. Cote et al. (20) reported a significant difference in the ratio of the major kavalactones between commercial kava extracts and traditional kava beverage, and found a significant difference in their ability to inhibit of P450 enzymes (CYP3A4, 1A2, 2C9, 2C19). The inhibition was more pronounced for the commercial preparation. Although all of the extracts inhibited human CYP3A4, 1A2,2C9 and 2C19 in a low micromolar range, the aqueous extract was the least potent for inhibiting all these P450s. However, it is not known if the inhibition of P450 is responsible for the hepatotoxicity. Nevertheless, they concluded that if the hepatotoxicity reported for the commercial caplet was the resultfromP450 inhibition, the traditional extract should also be hepatotoxic at high doses.

Toxicological Studies on Pipermethystine The reported kava hepatotoxicity may resultfromminor toxic constituent(s) in kava extracts. Dragull et al. (34) isolated and identified three piperidine alkaloids. They also found that pipermethystine was concentrated in kava stem



255 peelings and leaves; 3a, 4a-epoxy-5p-pipermethystine and awaine were new alkaloids; and the epoxide existed only in cultivar Isa among 11 cultivars examined. None of the compounds was detectable in commercial root powders from Fiji, Tonga or Hawaii. The pipermethystine content was 0.06 to 0.85 % in stem peelings and 0.32 to 2.43% (dry weight basis) in leaves. However, no commercial dietary supplement products were evaluated. Nerurkar et al. (35) reported that pipermethystine significantly decreased cellular ATP levels, mitochondrial membrane potential, and induced apoptosis as measured by the release of caspase-3 after 24 hr of treatment. Their data suggest that pipermethystine is capable of causing cell death, probably in part by disrupting mitochondrial function. As a follow up of the above studies, we collected 12 commercial dietary supplement products from local markets or via internet in the USA and analyzed for their pipermethystine content. We also evaluated in vitro cytotoxicity and mutagenicity of pipermethystine that was purified at the University of Hawaii.

Experimental Sample Preparation Four products of capsule type, five of root powder type and two tincture products of kava-containing dietary supplements were collected in local markets or via Internet. For the preparation of solid samples, 1 g of finely ground sample powders (pre-grind in a coffee grinder as needed) was extracted with 40 mL of ethyl acetate with 30 min sonication. The extract was filtered, and the filtrate was evaporated in vacuo. The resulting residue was dissolved in 2 mL of ethyl acetate for GC-FID analysis. For liquid samples, 5 g of sample solution was dried under reduced pressure. The resulting residue was dissolved in 4 mL of ethyl acetate with sonication. The solution was centrifuged and the clear supernatant was used for GC-FID analysis.

GC-FID Analysis and GC-MS Confirmation The method was modified from Dragull et al. (34). A Hewlett-Packard (HP) 5890 system equipped with FID detector and 6890 auto-injector was employed. Prepared sample solutions were separated on a DB-XLB capillary column (20 m x 0.18 mm, 0.18 pm, J&W Scientific, Folsom, CA). The column temperature initially was held at 150 °C for 1 min, raised to 300 °C at 5 °C/min and held 2 min. The total time was 33 min for each run. Injector and detector temperatures were maintained at 250 °C and 280 °C, respectively. The injection


256 volume was 1 pL in the splitless mode. Quantification of pipermethystine in the sample was conducted by relating peak area to that of external standard curves that were constructed with a range from 10 pg/mL to 1000 pg/mL (10,25, 50, 75, 100,250, 500, 750 and 1000 pg/mL of pipermethystine). The calibration curve was linear over these concentrations, and the correlation coefficient was r > 0.995. The identification of pipermethystine was performed by GC-FID analysis by comparing the retention time with authentic standard mid confirmed by GC-MS analysis. In order to minimize the MS interference, pipermethystine in sample was first separated from major kavalactones by HPLC. The eluate containing the pipermethystine peak was collected and further purified with SPE (Sep-Pak cartridge, C-18) for the GC-MS analysis. The MS spectrum of sample was compared with that of the authentic compound.


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Umu Test This method was based on the report by Oda et al. (36). S. typhimurium TA 1535/pSK1002 was grown overnight in LB-broth supplemented with 50 pg of ampicillin/mL. The culture was then diluted 50-fold with fresh TGA medium (1% trypton, 0.5% NaCl, 0.2% glucose with ampicillin at 50 pg/mL). The culture media were further incubated until a bacterial optical density of 0.25 to 0.30 at 600 nm was reached. A 2.4 mL portion of bacterial culture was divided into test tubes, then 0.1 mL of test compound was added along with either 0.5 mL of 0.1 M phosphate buffer (pH 7.4) or microsome mixture (0.25mM NADP, 5mM glucose-6-phosphate, 0.5 unit glucose-6-phosphatedehydrogenase, 3 mM MgCl , and microsomes in 1 mL) for metabolic activation. Rat liver microsome was prepared from Spargue-Dawley rats which were pre-treated with 3methylcholanthrene (3-MC). After further incubation at 37 °C for 2 hrs, the pgalactosidase activity was measured with procedure reported by Miller (37). The absorbance at 420 nm and 550 nm was measured. 1-Nitropyrene (330 ng/mL) and 2-amino-3-methylimidazol [4,5-/] quinoline (IQ; 330 ng/ml) were used as positive controls in the systems without or with metabolic activation, respectively. DMSO was used as the negative control in both systems. The galactosidase activity was calculated using the following equation: units=[[A o(1.75 x A o)] / (/ x v x A o)] x 1000, where, / is time of the reaction in minutes and v is volume (mL) of culture used in this assay. 2

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Mouse Lymphoma Assay (MLA) +/

The 7fc ~-3.7.2C heterozygote of the L5178Y mouse lymphoma cell line was utilized for this experiment. MLA experiment was performed according to the procedures of Chen and Moore (38). The test compound, pipermethystine,


257 was dissolved in DMSO and added to the suspended cells at concentration range from 0.5 to 3 pg/mL. The cell cultures were incubated for 4 hr, washed twice with fresh medium and then re-suspended in fresh medium. The culture flasks were placed in a humidified incubator at 37 °C in the presence of 5% C 0 and maintained in log phase growth for a 2-day expression period. The cells were counted and the densities were adjusted usingfreshmedium. For the mutant selection, trifluorothymidine (TFT, 3 pg/mL) was added to the cell culture and cells were seeded into four 96-wellflat-bottommicrotiter plates using 200 pL per well and a density of 2000 cells/well. For the determination of plating efficiency, cultures containing 8 cells/mL were prepared with dilution, and 200 pL this culture was aliquoted per well into two 96-well flat-bottom microtiter plates. The colonies were counted after 11 days of incubation at 37 °C in a humidified incubator with 5% C 0 in air. Mutation frequencies (MF) and relative total growth (RTG) were determined according to the method of Chen and Moore (38).


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Results and Discussion Pipermethystine Content in Dietary Supplement Products The results of analysis of pipermethystine and a brief description of the products are shown in Table I. These products varied widely within and between product types as far as labeling for kavalactone content. Analytical results showed that pipermethystine was not detectable in 11 of 12 products tested (Table I). However, one product (No. 5, Table I) composed of kava root chips and leaves was found to contain pipermethystine at 126pg/g. The finding that pipermethystine was present in samples containing kava leaves but not in other samples of root powders agreed with the report of Dragull et al. (34), showing the presence pipermethystine in kava leaves and stems but not roots. Since all kava parts might be used for commercial extracts (34) and human exposure to pipermethystine would then occur, we evaluated the potential cytotoxicity and mutagenicity of pipermethystine in this study.

Umu Test This test system is based on the SOS response using strain Salmonella typhimurium TA1535/pSK 1002 that contains a umuC-lacZ gene. Mutagenic chemicals induce the SOS response that activates umu region of the plasmid, leading to production of lacZ gene product, p-galactosidase whose activity can be measured by colorimetry.


258 Table I. Pipermethystine Content of Dietary Supplement Products in U. S. Market


Product No.

a

Type

1

Capsule

2

Capsule

3

Capsule

4

Capsule

5

Powder and chips

6, 7,8

Powder

9, 10

Powder

11

Tincture

12

Tincture

Description/Comment

Pipermethystine yg/g

Extract of kava rhizome and root, 60 mg kavalactones per capsule Kava, 30% kavalactones, 250 mg per capsule 450 mg per capsule, kava root 500 mg per capsule, kava root extract Kava tea - root chips and leaves

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Kava beverage powder, from three different origins, one supplier Kava root powder of different cultivars, different suppliers Rhizome and root, certified Organic grain alcohol (85-90%) and kava extractives Organic kava root, grain alcohol (50-60%)

Not detectable;

Kava (Piper methysticum) Safety Concerns and Studies on

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