I think some of you might be interested in Dr Lebot's latest papers. Some parts might be a bit overwhelming and confusing to casual readers. I certainly can't claim to understand everything well and I reached out to both Dr Lebot and @verticity for clarification.
Dr Lebot was kind enough not only to clarify some bits, but also provide additional information that might be of interest to those who follow these discussions.
Here's a summary of the key findings and key points made by Dr Lebot both in his paper and in the email replies:
On Flavokavains
Dr Lebot notes that flavokavains (compounds found in much higher concentrations in the non-noble kava than in noble kava) might "present cytoprotective properties and have potential against various diseases. FKA has an anti-inflammation activity and has been found to induce apoptosis of tumour cells and to possess anticancer properties. FKB has cytotoxic and apoptotic effects against different types of cancer cells; FKC is the least studied but has an antiinflammatory
effect."
At the same time: "FKB has also been suspected of being potentially hepatotoxic. FKA and FKB can potentiate acetaminophen-induced hepatotoxicity
and this observation suggested that kava and a drug interaction may account for potential hepatotoxicity. FKA and FKB were probably the major but not the sole compounds responsible for the ethanolic extracts toxicity of commercial kava imported in the USA while aqueous extracts showed no toxicity. The in vitro metabolism of FKA, B and C has also been characterised using human liver microsomes and it was concluded that the absolute amount of conjugated metabolites in vivo maybe even higher than the significant levels observed in vitro." In his other paper he observes that: "FKs have been studied for their potential toxicity, and in vitro studies have shown that they possess increased cytotoxicity comparedto KLs [27]. Consequently, they have been suspected to be the constituents responsible for initiating the skin rash linked to the high consumption of kava [12,28,29]. The mechanism of FKB cytotoxicityhas been described to be mediated through oxidative stress and depletion of glutathione, and this has also been shown for other FKs. When the potential toxicity of the three FKs was compared with six KLs, it was found that all three FKs displayed higher toxicity than the most toxic KL. (...) The occurrence of suspected hepatotoxicity in German acetonic extracts was most likely caused bynon KLs compounds. KLs have been tested in different assays but were never found to be toxic [33,34].FKB and FKA have been found to be potentially hepatotoxic for mice [35]. It has been shown that oralconsumption of FKB leads to the inhibition of hepatic transcriptional activityin vivoand severe liverdamage. FKB has been identified as a potent GSH-sensitive hepatotoxin and it was advised that its levels should be controlled in kava-containing herb products [10]. It has also been shown that FKs are high in the bark of the plant [36] and could contribute to protection against potential predators, due totheir potential cytotoxicity."
He consequently notes that kavas high in flavokavains are more likely to produce "hangovers". In the first paper Dr Lebot admits that the idea of flavokavains themselves being solely responsible for hangovers and nausea (as opposed to it also being caused by the chemotypes) remains a hypothesis: "High levels of FKsin two-day and wichmannii kavas might contribute to the nausea and hangover caused by these varieties, although more research is needed to confirm this hypothesis. " In his other paper, he goes a bit further and more firmly notes that: " HPTLC results are clarifying the potential role of FKs in the different physiological effectsinduced by each varietal group. It appears that the major difference between nobles, two-dayand wichmannii varieties might not be due only to their different KLs chemotypes as previously suggested [7] but rather their very high FKs. Although more research is needed, this observation would suggest that the side effects produced by beverages prepared with these two groups of varieties would result from high FKs and not from DHK and DHM alone [7]."
On the acetone test
In light of the above, Dr Lebot argues there's a strong case to be made for differentiating between noble and non-noble kava and that it "appears appropriate" to exclude high flavokavains cultivars from being exported as food. Dr Lebot notes that the best and most accurate method for identifying cultivars would be "a test for DNA fingerprinting (see VandenBroucke et al 2015 in Genome)" or "morphological assessment in the field". DNA fingerprinting would allow a precise and accurate identification of specific cultivars and could be used by both the exporting nations and importers. In the absence of such testing, the focus is on "the secondary metabolites chemotypes produced by these two different genotypes" (that can be analysed via HPLC, UHPLC, HPTLC). In this context, the acetone test is "just indicating the proportion (content) of non kavalactones compounds". This works because "kavalactones are translucid (or yellow) compounds when diluted in acetone". Flavokavains (but also other pigments etc) would give amber/red/brown colour to the sample. The problem of false positives (i.e. a noble kava testing as non-noble) is, in Dr Lebot's opinion, exclusively due to the samples not being properly peeled/containing parts of stump bark that was exposed to sunlight. Peeled noble rootstock (and roots) "will always look yellow". At the same time, some samples of two-day stumps, when peeled properly, might also "test yellow" (i.e. look noble) due to the fact that most of the flavokavain can be found in the skin/bark. It appears that Dr Lebot doesn't see the problem of false positives as a big issue, in part because in theory (and by law in some places) the stump should always be peeled anyway, so a false positve due to the presence of skin/bark still signals inferior quality.
He argues that his testing revealed that 52% of the variability in the absorbance observed with the colorimeter can be explained by FKs (Lebot's point being the rest of that variability is explained by other colored chemicals that shouldn't be there either). @verticity can definetely explain it better, but if I understood his points correcty, that in reality the % of cases in which it is the FKs that explain the colour is far greater than 52% (about 90%) because most samples are actually peeled (@verticity please correct I misunderstood this point).
Isa
This might come as surprise to many, but Dr Lebot highlighted the fact that it is inaccurate to call Isa kava "two-day" for a number of reasons. First of all, "two-day" is a Vanuatu category, strictly defined by law that lists two-day varieties. Second, it is genetically distinct from Vanuatu two-day. As you know, it originates from PNG, not Vanuatu. Finally, it is also chemically different with lower amounts of flavokavains than average Vanuatu two-day. As a side note, Isa actually became a thing in Hawaii because Lebot himself brought cuttings of Isa with him to Hawaii and plated it for research purposes in the 1980s. After he left some of the isa "escaped" from the university and found its way to farms. Dr Lebot maintains that "traditionally in PNG Isa was very rarely consumed, never regularly, just for funerals .... and consumers are not well the next day, I think that traditionnal knowledge is important". He considers it to be an inferior variety (in terms of it being used as a daily drinking kava/as food), but one that quantitatively (in terms of flavokavain content) lies somewhere between noble and average Vanuatu two-days (not to mention wild kava). In his paper he actually divides kava into: Noble, Isa, Two-Day and Wild.
This is a rather interesting point, because it kind of confirms what we've been seeing on the forums for years. Isa is heavier than nobles, but quite a few people find it acceptable or even suitable for certain applications, especially when consumed in moderation. In this context, it would perhaps make sense to highlight the genetic, chemical and legal difference between Isa and the Vanuatu two-day or wild kava varieties.
Other minor findings
It appears that the previously reported chemotypes might have not been entirely accurate due to lower than actual reported ratios of Y and DHY. As noted by Lebot: "Our findings reveal chemotype groups different than those previously described because wehave used two different detection wavelengths that allow for a better quantification of yangoninand desmethoxyyangonin. It appears that these two compounds were underestimated in previousHPLC studies because they were quantified at 240 nm. "
Dr Lebot was kind enough not only to clarify some bits, but also provide additional information that might be of interest to those who follow these discussions.
Here's a summary of the key findings and key points made by Dr Lebot both in his paper and in the email replies:
On Flavokavains
Dr Lebot notes that flavokavains (compounds found in much higher concentrations in the non-noble kava than in noble kava) might "present cytoprotective properties and have potential against various diseases. FKA has an anti-inflammation activity and has been found to induce apoptosis of tumour cells and to possess anticancer properties. FKB has cytotoxic and apoptotic effects against different types of cancer cells; FKC is the least studied but has an antiinflammatory
effect."
At the same time: "FKB has also been suspected of being potentially hepatotoxic. FKA and FKB can potentiate acetaminophen-induced hepatotoxicity
and this observation suggested that kava and a drug interaction may account for potential hepatotoxicity. FKA and FKB were probably the major but not the sole compounds responsible for the ethanolic extracts toxicity of commercial kava imported in the USA while aqueous extracts showed no toxicity. The in vitro metabolism of FKA, B and C has also been characterised using human liver microsomes and it was concluded that the absolute amount of conjugated metabolites in vivo maybe even higher than the significant levels observed in vitro." In his other paper he observes that: "FKs have been studied for their potential toxicity, and in vitro studies have shown that they possess increased cytotoxicity comparedto KLs [27]. Consequently, they have been suspected to be the constituents responsible for initiating the skin rash linked to the high consumption of kava [12,28,29]. The mechanism of FKB cytotoxicityhas been described to be mediated through oxidative stress and depletion of glutathione, and this has also been shown for other FKs. When the potential toxicity of the three FKs was compared with six KLs, it was found that all three FKs displayed higher toxicity than the most toxic KL. (...) The occurrence of suspected hepatotoxicity in German acetonic extracts was most likely caused bynon KLs compounds. KLs have been tested in different assays but were never found to be toxic [33,34].FKB and FKA have been found to be potentially hepatotoxic for mice [35]. It has been shown that oralconsumption of FKB leads to the inhibition of hepatic transcriptional activityin vivoand severe liverdamage. FKB has been identified as a potent GSH-sensitive hepatotoxin and it was advised that its levels should be controlled in kava-containing herb products [10]. It has also been shown that FKs are high in the bark of the plant [36] and could contribute to protection against potential predators, due totheir potential cytotoxicity."
He consequently notes that kavas high in flavokavains are more likely to produce "hangovers". In the first paper Dr Lebot admits that the idea of flavokavains themselves being solely responsible for hangovers and nausea (as opposed to it also being caused by the chemotypes) remains a hypothesis: "High levels of FKsin two-day and wichmannii kavas might contribute to the nausea and hangover caused by these varieties, although more research is needed to confirm this hypothesis. " In his other paper, he goes a bit further and more firmly notes that: " HPTLC results are clarifying the potential role of FKs in the different physiological effectsinduced by each varietal group. It appears that the major difference between nobles, two-dayand wichmannii varieties might not be due only to their different KLs chemotypes as previously suggested [7] but rather their very high FKs. Although more research is needed, this observation would suggest that the side effects produced by beverages prepared with these two groups of varieties would result from high FKs and not from DHK and DHM alone [7]."
On the acetone test
In light of the above, Dr Lebot argues there's a strong case to be made for differentiating between noble and non-noble kava and that it "appears appropriate" to exclude high flavokavains cultivars from being exported as food. Dr Lebot notes that the best and most accurate method for identifying cultivars would be "a test for DNA fingerprinting (see VandenBroucke et al 2015 in Genome)" or "morphological assessment in the field". DNA fingerprinting would allow a precise and accurate identification of specific cultivars and could be used by both the exporting nations and importers. In the absence of such testing, the focus is on "the secondary metabolites chemotypes produced by these two different genotypes" (that can be analysed via HPLC, UHPLC, HPTLC). In this context, the acetone test is "just indicating the proportion (content) of non kavalactones compounds". This works because "kavalactones are translucid (or yellow) compounds when diluted in acetone". Flavokavains (but also other pigments etc) would give amber/red/brown colour to the sample. The problem of false positives (i.e. a noble kava testing as non-noble) is, in Dr Lebot's opinion, exclusively due to the samples not being properly peeled/containing parts of stump bark that was exposed to sunlight. Peeled noble rootstock (and roots) "will always look yellow". At the same time, some samples of two-day stumps, when peeled properly, might also "test yellow" (i.e. look noble) due to the fact that most of the flavokavain can be found in the skin/bark. It appears that Dr Lebot doesn't see the problem of false positives as a big issue, in part because in theory (and by law in some places) the stump should always be peeled anyway, so a false positve due to the presence of skin/bark still signals inferior quality.
He argues that his testing revealed that 52% of the variability in the absorbance observed with the colorimeter can be explained by FKs (Lebot's point being the rest of that variability is explained by other colored chemicals that shouldn't be there either). @verticity can definetely explain it better, but if I understood his points correcty, that in reality the % of cases in which it is the FKs that explain the colour is far greater than 52% (about 90%) because most samples are actually peeled (@verticity please correct I misunderstood this point).
Isa
This might come as surprise to many, but Dr Lebot highlighted the fact that it is inaccurate to call Isa kava "two-day" for a number of reasons. First of all, "two-day" is a Vanuatu category, strictly defined by law that lists two-day varieties. Second, it is genetically distinct from Vanuatu two-day. As you know, it originates from PNG, not Vanuatu. Finally, it is also chemically different with lower amounts of flavokavains than average Vanuatu two-day. As a side note, Isa actually became a thing in Hawaii because Lebot himself brought cuttings of Isa with him to Hawaii and plated it for research purposes in the 1980s. After he left some of the isa "escaped" from the university and found its way to farms. Dr Lebot maintains that "traditionally in PNG Isa was very rarely consumed, never regularly, just for funerals .... and consumers are not well the next day, I think that traditionnal knowledge is important". He considers it to be an inferior variety (in terms of it being used as a daily drinking kava/as food), but one that quantitatively (in terms of flavokavain content) lies somewhere between noble and average Vanuatu two-days (not to mention wild kava). In his paper he actually divides kava into: Noble, Isa, Two-Day and Wild.
This is a rather interesting point, because it kind of confirms what we've been seeing on the forums for years. Isa is heavier than nobles, but quite a few people find it acceptable or even suitable for certain applications, especially when consumed in moderation. In this context, it would perhaps make sense to highlight the genetic, chemical and legal difference between Isa and the Vanuatu two-day or wild kava varieties.
Other minor findings
It appears that the previously reported chemotypes might have not been entirely accurate due to lower than actual reported ratios of Y and DHY. As noted by Lebot: "Our findings reveal chemotype groups different than those previously described because wehave used two different detection wavelengths that allow for a better quantification of yangoninand desmethoxyyangonin. It appears that these two compounds were underestimated in previousHPLC studies because they were quantified at 240 nm. "
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