beverages Article Kavalactones and Flavokavins Profiles Contribute to Quality Assessment of Kava (Piper methysticum G. Forst.), the Traditional Beverage of the Pacific Vincent Lebot 1,* , Serge Michalet 2 and Laurent Legendre 2 1 Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD), Amélioration Génétique et Adaptation des Plantes (AGAP), P.O. Box 946 Port-Vila, Vanuatu 2 CNRS UMR 5557 & INRA UMR 1418, Université de Lyon, F-69622 Villeurbanne, France; [email protected] (S.M.); [email protected] (L.L.) * Correspondence: [email protected]; Tel.: +678-259-47 Received: 18 January 2019; Accepted: 1 April 2019; Published: 2 May 2019 Abstract: Kava (Piper methysticum) is increasingly traded internationally and there is need for a rapid method to analyze kava raw material before export. The objectives of the present study were: (i) to develop a simple and robust protocol for high throughput simultaneous quantification of kavalactones (KLs) and flavokavins (FKs) in kava and (ii) to assess its potential for quality control. Methysticin; dihydromethysticin; kavain; desmethoxyyangonin; dihydrokavain; yangonin; and flavokavin A, B and C were quantified using HPTLC in acetonic extracts of 174 kava varieties. UHPLC analysis was conducted on a subset of six varieties representing the genetic variation of the species. The genetically distinct groups of nobles, two-day and wichmannii varieties were clearly differentiated and multivariate analyses of UHPLC and HPTLC data were congruent. Noble varieties have significantly low FKs/KLs (0.13) and high kavain/flavokavin B (K/FKB = 7.31). Two-day and wichmannii varieties are characterized by high FKs/KLs (0.36, 0.21) and low K/FKB (1.5, 1.7). A high-throughput HPTLC protocol was developed with a total analytical time of 50 min for 20 samples and only 10 mL of mobile phase. The use of acetone, sonication and two different detection wavelengths improves the accuracy compared to previous HPLC studies and confirms that kava varieties exhibit distinct chemotypes clearly differentiated by their FKs/KLs profiles. These results will strengthen the use of Codex Alimentarius regional standards. Keywords: HPTLC; genetic variation; methysticin; dihydromethysticin; kavain; desmethoxyyangonin; dihydrokavain; yangonin; flavokavins A; FKB; FKC 1. Introduction Piper methysticum G. Forst. (Piperaceae) rhizomes and roots are peeled, grinded, macerated in cold water, and pressed through a cloth strainer to prepare kava, a non-alcoholic beverage. In the Pacific Islands, it is consumed on a daily basis for social purposes, at home or in urban kava bars. During the last two decades, it has become more widely used as a recreational beverage by the international community, and it is appreciated for the relaxing and anxiety-relieving properties of its active ingredients called kavalactones [1]. The two major producers are Fiji and Vanuatu and it is estimated that around 4000 ha are harvested every year to satisfy a regional market of approximately 6000 tons (dry weight) per year [2]. In the Pacific, kava is considered to be a safe beverage and no evidence of harm is observed among regular drinkers. In Europe, kava has been used as raw material by the pharmaceutical industry to prepare acetonic or ethanol extracts, and in 2002, kava-based products were suspected of hepatotoxicity and banned by the German health authorities (BfArM). Beverages 2019, 5, 34; doi:10.3390/beverages5020034 www.mdpi.com/journal/beverages Beverages 2019, 5, 34 2 of 14 Beverages 2019, 5, x FOR PEER REVIEW 2 of 14 The German ban has been contested because of the poor evidence of risks associated to kava extracts theand ban two was German inappropriate administrative [3]. However, court decisions a concern concluded regarding in 2015 kava that stems the bannot wasonly inappropriate from Germany, [3]. butHowever, is worldwide a concern (e.g., regarding FDA issued kava stemswarnings). not only Kava from extract Germany, is also but considered is worldwide by the (e.g., International FDA issued warnings).Agency for KavaResearch extract on isCancer also considered (IARC) as by group the International 2B, which is Agencypossibly for carcinogenic Research on to Cancer humans, (IARC) but thisas group classification 2B, which applies is possibly to the carcinogenic extract and tonot humans, the traditional but this beverage classification stricto applies sensuto [4]. the extract and not theThe traditional composition beverage and qualitystricto of sensu kava[4 ].can be highly variable, depending on the age of the plant, the variety,The composition and the part and used quality to prepare of kava the can bevera be highlyge: roots, variable, rhizomes depending or ba onsal thestems. age In of Vanuatu, the plant, the variety,“Kava Act” and the was part passed used toin prepareParliament the beverage:in 2002 to roots, regulate rhizomes the industry. or basal stems. Based Inon Vanuatu, traditional the “Kavaknowledge, Act” was this passedAct stipulates in Parliament that only in 2002 “noble” to regulate varieties the locally industry. known Based for on their traditional pleasant knowledge, effect and beingthis Act harmless stipulates for that daily only “noble”consumption varieties must locally be knownused, while for their “wild pleasant kava” effect (P. and methysticum being harmless var. wichmanniifor daily consumption) and “two-day” must bevarieties, used, while known “wild for kava” their (lastingP. methysticum effect causingvar. wichmannii nausea )and and hangover, “two-day” varieties,are illegal known [5]. DNA for theirmarkers lasting studies effect have causing shown nausea that andthese hangover, varieties arecorrespond illegal [5 ].to DNAthree markersdistinct groupsstudies of have genotypes shown that[6]. these varieties correspond to three distinct groups of genotypes [6]. The six major kavalactones (KLs: (KLs: yangonin yangonin == Y,Y, dihydrokavain dihydrokavain == DHK,DHK, desmethoxyyangonin desmethoxyyangonin = DMY,= DMY, kavain kavain = =K,K, dihydromethysticin dihydromethysticin = =DHMDHM and and methysticin methysticin == M)M) are are responsible responsible for for the physiological eeffectffect and and are are usually usually quantified quantified with with HPLC HPLC [7,8]. There[7,8]. isThere a second is a group second of molecules,group of molecules,flavokavins flavokavins (FKs: A, B, (FKs: C), which A, B, has C), recentlywhich has attracted recently attention attracted (Figure attention1). (Figure 1). Kavalactones R1–R2 C5–C6 C7–C8 Flavokavins R Yangonin (Y) OCH3 = = A HCH3 Dihydrokavain (DHK) B - Desmethoxyyangonin (DMY) = = C HO Kavain (K) = Dihydromethysticin (DHM) OCH2O Methysticin (M) OCH2O = Figure 1. ChemicalChemical structure of kavalactones and flavokavins flavokavins (= : presence of double bond). Some studies suggest that FKA and FKB are cytoto cytotoxicxic to cancer cells [9–11] [9–11] and that FKB is a major hepatotoxin in organic kava extracts [12], [12], althoughalthough others have questionedquestioned whether the levels in these these extracts extracts are are high high enough enough to to produce produce hepato hepatotoxicitytoxicity [13,14]. [13,14 FKC]. FKC has has been been shown shown to have to have the potentialthe potential to prevent to prevent the the inflammation inflammation process process wi withth antioxidant antioxidant properties properties [15]. [15]. However, However, when cytotoxicity againstagainst humanhuman lung lung adenocarcinoma adenocarcinoma A549 A549 cancer cancer cells cells was was tested tested with with kava kava extracts, extracts, FKA FKAand FKB and wereFKB were found found to be to the be major the major but not but sole not compounds sole compounds responsible responsible for the for extract the extract toxicity toxicity while whileKLs were KLs lesswere likely less likely to be involvedto be involved [16]. Codex[16]. Codex Alimentarius Alimentarius regional regional quality quality standards standards have have been beendeveloped developed for kava for kava products products for use for as use a beverage as a beverage [17]. [17]. The chemicalchemical compositioncomposition of of the the kava kava extract extract is stronglyis strongly influenced influenced by theby the extraction extraction solvent solvent [18]. Acetone[18]. Acetone is the is most thee ffmostective effective solvent solvent in terms in of te yieldrms ofof KLs, yield followed of KLs,by followed water whereas by water chloroform, whereas chloroform,hexane, methanol, hexane, and methanol, ethanol are and less ethanol efficient are [19 ].less Sonication efficient has [19]. been Sonication shown to has improve been theshown solvent to improveextraction the effi solventciency [extraction20]. HPLC efficiency protocols [20]. are HP basedLC onprotocols ethanol, are methanol based on or ethanol, chloroform methanol extracts or chloroform extracts to avoid undesirable compounds in the separation columns [8], but their results are biased by the limited efficiency of these solvents. Another major analytical constraint comes from the different absorption wavelengths maxima of the target substances. M, DHM, K, and DHK are better detected at 240 nm while Y, DMY, FKA, FKB, and FKC are well separated at 355 nm [21,22]. Beverages 2019, 5, 34 3 of 14 to avoid undesirable compounds in the separation columns [8], but their results are biased by the limited efficiency of these solvents. Another major analytical constraint comes from the different absorption wavelengths maxima of the target substances. M,