Did Dr. Vincent Lebot ever identify the orange substance in the acetone test?

[Palmetto]

The term tudei technically should be restricted to kavas with strong effects that last more than 24 hours. Noble or (Ig)noble would technically be irrelevant according to that usage. What I mean by ignoble would be a kava that is high in flavokawains (FK) B or C or pipermethysticin (PM) in the roots. Lebot divided tudeis from medicinal kavas, supposedly according to traditional usage, but this distinction was consistent with intermediate flavokawain levels between tudei and noble.
Here is how I would recommend dividing kavas:

Class Keys for classification Other tendencies
1. Noble kavas Low FK B+C, virtually no PM, DHM < 20% K > 20%, DHK < 30%, yellow acetone hue
2. Medicinal kavas Medium FK levels, DHM > 15%, DHK > 30%, low PM orange acetone hue, low kavain
3. Tudei kavas High FK levels, high DHM and DHK orange acetone hue, +/- PM

Okay, so not the most original classification, nor the best defined, but it would certainly solve the argument over whether certain borderline kavas should be called noble or not. If they were "medicinal", they wouldn't get as much stigma as the tudeis, but it would alert the buyer of some potential for hangover or FK levels. A kava would only be eligible for a classification if it met ALL of the minimum requirements, not whether it resembled a noble in some ways. For FK levels, I would use FK B and C, since they are both hepatotoxic alone, whereas FK A has very good antitumor effects without the hepatotoxicity.
www.ncbi.nlm.nih.gov/pubmed/24423570

 

[verticity]

The term tudei technically should be restricted to kavas with strong effects that last more than 24 hours. Noble or (Ig)noble would technically be irrelevant according to that usage. What I mean by ignoble would be a kava that is high in flavokawains (FK) B or C or pipermethysticin (PM) in the roots. Lebot divided tudeis from medicinal kavas, supposedly according to traditional usage, but this distinction was consistent with intermediate flavokawain levels between tudei and noble.
Here is how I would recommend dividing kavas:

Class Keys for classification Other tendencies
1. Noble kavas Low FK B+C, virtually no PM, DHM < 20% K > 20%, DHK < 30%, yellow acetone hue
2. Medicinal kavas Medium FK levels, DHM > 15%, DHK > 30%, low PM orange acetone hue, low kavain
3. Tudei kavas High FK levels, high DHM and DHK orange acetone hue, +/- PM

Okay, so not the most original classification, nor the best defined, but it would certainly solve the argument over whether certain borderline kavas should be called noble or not. If they were "medicinal", they wouldn't get as much stigma as the tudeis, but it would alert the buyer of some potential for hangover or FK levels. A kava would only be eligible for a classification if it met ALL of the minimum requirements, not whether it resembled a noble in some ways. For FK levels, I would use FK B and C, since they are both hepatotoxic alone, whereas FK A has very good antitumor effects without the hepatotoxicity.
www.ncbi.nlm.nih.gov/pubmed/24423570

Yeah, there is the distinction between Tudei and "Medicinal" (as well as "Ceremonial" and "Never Drunk") But in Vanuatu, the medicinal and ceremonial kavas are also banned for export. They have the same legal status as Tudei.. I have never seen "medicinal" kava for sale as such, but it's possible it is used to adulterate noble kava..

 

[dragonaut]

Wow, this thread really took off, sorry for abandoning you guys!
I ended up writing Dr. Lebot and he kindly responded. Basically, what is currently known is already published in his 2016 article with Laurent Legendre, which is also linked to in this thread. Just to sum up what he told me:

- The color is probably coming from flavokawains (makes sense since they are highly conjugated molecules), but unidentified compounds found primarily in the peelings could contribute to the color and hepatoxicity also.
- The nausea from tudei is thought to be from dihydromethysticin and dihydrokawain, but flavokawains may also be contributing (thus, the acetone test may indicate the level of nausea caused by the kava in question).
- Tudeis generally contain low kawain and high flavokawain concentrations.

Identification of the unknown compounds as well as the colored compounds is currently being looked into.

 

[HeadHodge]

@dragonaut, Totally Awesome!
I vote giving you the New Member of the Year Award.

 

[Bula Kava House]

Wow, this thread really took off, sorry for abandoning you guys!
I ended up writing Dr. Lebot and he kindly responded. Basically, what is currently known is already published in his 2016 article with Laurent Legendre, which is also linked to in this thread. Just to sum up what he told me:

- The color is probably coming from flavokawains (makes sense since they are highly conjugated molecules), but unidentified compounds found primarily in the peelings could contribute to the color and hepatoxicity also.
- The nausea from tudei is thought to be from dihydromethysticin and dihydrokawain, but flavokawains may also be contributing (thus, the acetone test may indicate the level of nausea caused by the kava in question).
- Tudeis generally contain low kawain and high flavokawain concentrations.

Identification of the unknown compounds as well as the colored compounds is currently being looked into.

There must be something other than flavokavains. Hate to open up a can of worms that needs to stay shut, but we've seen a kava with very low flavokavains turn acetone orange in the past. I think the correct answer to "What turns the acetone orange?" is still, "We really don't know."

 

[verticity]

There must be something other than flavokavains. Hate to open up a can of worms that needs to stay shut, but we've seen a kava with very low flavokavains turn acetone orange in the past. I think the correct answer to "What turns the acetone orange?" is still, "We really don't know."

In the paper mentioned, Lebot does not actually determine what causes the orange color. That is, he does not fully explain the visible spectrum. What he does, is show that the absorbance at the single wavelengths of 400nm and 440 nm correlate well with FK/KL ratio. If you look at visible spectra you will see that the absorbance is much higher near 400 nm for tudei compared to noble kavas. If you look at the spectra of the flavokavains and kavalactones in the paper, notice that the FKs have significant absorbance in the region of 400-440 nm, whereas KLs do not. Therefore, it is reasonable to conclude that the difference between the portions of the spectrum between 400 and 450 nm for tudei and noble kavas is in fact dominated flavokavains. However the color of the acetone test is influenced by the entire visible spectrum between 400 and 700 nm, and not uniformly so. In fact the color you see, and the color that is measured with a colorimeter, is a more sensitive indicator of what is happening in the region where the sample absorbs green light, which is 500 to 600 nm. This figure represents the sensitivity of the human eye, and thus the spectral regions that contribute most to color, and to the perceived dominant wavlength of a sample:

The paper found that there is a correlation between absorbance points greater than 450 nm and FK/KL, but the standard deviations are greater, so that point absorbances above 500 are not very useful. However it is not clear if those standard deviations are greater simply because the absorbances at those points are numerically smaller--since when you subtract an imprecise number from another imprecise number, the difference is less precise than when you subtract a large number from another large number.

The absorbance between 500 and 600 nm, which accounts for most of the color, and the visible spectrum, can not be accounted for by flavokavains: those chemicals are still unknown.

I'll have more to say on this. But just for the time being, keep in mind that the acetone integrates the entire visible spectrum, and thus uses all the information in the spectrum, instead of correlating absorbances at indiviodual points. Thus one would expect the acetone test as done by @Deleted User to be more accurate that the point correlations described in the paper. I'll talk about this more when I have time.

 

[verticity]

There must be something other than flavokavains. Hate to open up a can of worms that needs to stay shut, but we've seen a kava with very low flavokavains turn acetone orange in the past. I think the correct answer to "What turns the acetone orange?" is still, "We really don't know."

In the case of 11-year waka, there was likely something in it, maybe peelings or something, that contained tudei-type chemicals causing the visible absorbance and dominating the near UV contribution to the dominant wavelength, which as I tried to explain, is small, although it was not tudei according to FK/KL measurement. In other words it was probably adulterated with something. What it was adulterated with is not known.

 

[HeadHodge]

...What it was adulterated with is not known.

Tang?

 

[verticity]

Tang?

Probably.
But seriously it could be anything.
I hate typing on my phone
My fingers are way too big
Friggin lemur finger kbd

 

[HeadHodge]

Probably.
But seriously it could be anything.
I hate typing on my phone
My fingers are way too big
Friggin lemur finger kbd

You have F.F.S.
(fat finger syndrome)

 

[HeadHodge]

I have 'CRTFK'
(can't read the fucking keypad)

 

[verticity]

So let me elaborate on the validity of the acetone test.
Dr. Lebot has said that he has tested hundreds of kava cultivars. They are samples the he has had harvested and prepared in a standard fashion under his own supervision. In all of those cases, the noble ones are yellow, the tudei ones are orange, and the wichmannii are brown in color (hue) when observed by eye. The attached paper actually does not talk about color as such. It does not contain the word "orange" that I can find (nor the words "color" or "hue"). I don't know if Dr. Lebot has published that qualitative observation. If someone knows where he has put that in writing I would be interested to see it. I do know that @Deleted User has also done a lot of research in that area, so I'll ask him: have any of the samples provided to you by Dr. Lebot that are noble had an orange hue, or have any of the tudei samples had a yellow hue? Assuming the answer is no (correct me if I am wrong, @Deleted User) that implies that color is an excellent way to discriminate between noble and tudei kavas for samples that are: (1) Known to be kava. (2) Known not to contain anything other than kava (3) Prepared in a standardized way.

The old 11-year waka tested orange, yet was low in FK, and therefore not tudei. (The new 12-or-whatever-year waka is now yellow.) The conditions under which this kava were grown, harvested and processed are completely unknown. It was made by a company called Wakaya, it was not a sample curated by scientists. What is the logical conclusion?

1) This kava was a lone exception to the general scientific observation that all noble kavas are yellow, due to a freak genetic mutation or something.
- or -
2) This kava was not in fact pure kava prepared in a standardized way, and might have contained some unknown adulterant, or the manner of it's preparation might have been very non-standard (e.g. lots of shavings).

#2 seems much more likely to me. It seems especially likely since it seems that a change in the manner of processing what is claimed to be the same kava has caused to orange color to vanish.

 

[verticity]

I hope that herds the worms back into the can, now that they have gotten out for a bit of exercise.

 

[verticity]

And speaking of Orange, Happy Halloween, everyone.
My daughter did the decorations. We got some glow-in-the dark spider webs, which she attempted to cover the house with. We also got some Styrofoam tombstones, and other things. Unfortunately it is pretty windy here, so the Styrofoam tombstones blew away, and are currently headed due South aloft chilly winds, like a flock of birds with tombstone wings.

 

[verticity]

Every known noble cultivar I have tested was yellow; every known non-noble cultivar I have tested was orange. To go a bit further, noble cultivars intentionally adulterated with non-noble are also easily detectable at concentrations of 20%.

Concerning 11 year, I'm afraid the worms will have to wriggle on. I've tested numerous samples of this kava spanning several years. All failed the acetone test, and all were positively identified as kava with no other substances evident. This applies to numerous BKH samples (including the "ginger" and "splinter" variants), as well as samples from non-USA wholesale sources offered as "11 year old Fiji waka". I've also tested ginger, turmeric, cinnamon and other botanicals individually and mixed with kava. All are evident in HPLC tests, and while some affect coloration in the acetone test, none produced a result that could be mistaken for non-noble kava when mixed with noble kava.

There's even more worms in my can, but we'll see what these stir up...

So, in other words, the only thing you have not tested is flavokavain levels.
If you are interested, I could try to help you tease those out of the UV-VIS spectra using modeling. I believe it might be possible. In particular, by focusing on the absorbance spectra between 400-440nm, which like I was saying above, and as you know, are de-emphasized in the dominant wavelength calculation.

 

[verticity]

When I get time I'm going to try the Meissner HPLC method for FKs, I believe the new column I'm using will make this much easier.

But if you have a theory about FKs involving visible spectrum, I'd love to hear it. Virtually all non-noble kavas have absorption spikes at 430.7 and 440.8nm that are not present in nobles. I can't relate this to any particular component, but it seems a rather reliable indicator.

Well, it's not so much the spikes, as the broader bumps. They are kind of wormlike in shape
Basically, in the paper by Lebot linked here, one of the things he looked at was the absorbance at the single wavelength 400 nm. He found a strong correlation between this absorbance, and FK/KL ratios. Now, ignoring the fine structure, in the noble spectra of yours that I have seen, there is a broad peak between 400 and 450 with a maximum of about 1.25 absorbance units. I think much of that absorbance is caused by the absorbance of flavokavains themselves, plus DMY, and possibly a little M and DHM which have some absorbance there too. Here are the UV-VIS spectra from the paper edited to show only the visible region:
Flavokavains

Yangonin & DMY:

M & DHM (click to enlarge)

K & DHK (click to enlarge)

In these spectra I think the units are arbitrary, not standard "absorbance units". If the actual absorbance units were shown you could compare these directly to your absorbance spectra.

So for noble kava the absorbances at 400 nm for each component are roughly (in "arbitrary units"):
Flavokavains 60 + 65 + 25 = 150
Y & DMY: 5 + 27 = 32
M & DHM: 5 + 5 = 10
K & DHK: basically 0
Total Kavalactone absorbance at 400 nm = 42 arbitrary units.
Total FK + KL absorbance = 192
So, in other words, FKs absorb about 3 or 4 times more strongly than kavalactones at this wavelength.
Since these absorbance units are arbitrary anyway, lets normalize them so that the combined absorbance of FKs KLs is 1, and I'll call these new units Normalize Arbitrary Units (NAU). Then the absorbances are:
- Total FK: 0.78 NAU
- Total KL: 0.22 NAU
In noble kava, the ratio of FK/KL is 0.29. [Lebot, Food Chemistry 151(2014) 554-560 Table 2]. This is the ratio of the masses, so if there are 100 grams of KLs, there are 29 grams of FKs. For spectroscopy, we would like to know the amount in moles instead of the mass in grams, so we need to compare the molecular weights of FKs and KLs. FKB is 284.3065 g/mol and DMY is 228.25 g/mol. Those numbers are close enough that for now I'm going to ignore them and assume the molecular weights of FKs and KLs are the same.

So say we have noble kava with 1 gram of KLs and 0.29 grams of FKs in it.
The absorbance caused by the KLs will be proportional to 1 g * 0.22 NAU = 0.22 g*NAU
And the absorbance caused by the FKs will be proportional to 0.29 g * 0.78 NAU = 0.22 g*NAU
In other words, the absorbances caused by FKs and KLs will be approximately equal at 400 nm for noble kava! (Absorbance ratio: 1)
The total absorbance is 0.44 g*NAU

What about tudei?
For tudei, the FK/KL ratio is 1.02 [Lebot, 2014]
So if we have a similar mass kava sample of tudei, it would contain 1 gram of KLs and 1.02 grams of FKs.
The absorbance caused by the KLs is the same = 0.22 g*NAU
The absorbance caused by the FKs is larger: it is proportional to 1.02 g * 0.78 NAU = 0.80 g*NAU
The total absorbance is 1.0 g*NAU

So, if flavokavains accounted for all the absorbance at 400 nm, the aborbance measured for tudei should be about twice as big as for noble.
The ratio of noble/tudei absorbance would be 0.44.
Here is a typical visible spectrum of a noble and tudei. (from @Deleted User).

For the noble the absorbance at 400 nm is about 1.25, and for the tudei it is about 1.75.
The ratio of the aborbances is noble/tudei = 0.71. So that means there is some stuff absorbing at that wavelength not accounted for in this model. But its actually not bad: the flavokavain level concentrations are pretty clearly evident at that wavelength. A more sophisticated model would be able to give a reasonable estimate of FK/KL ratio, I think.

It would be helpful to have spectra of the pure flavokavains, and of the KLs in your instrument, @Deleted User, to make a better model.

Another thing that might make it easier to actually get an accurate FK/KL measurement would be to look at the entire UV-VIS spectrum from 200-700 nm.

 

[Zaphod]

@Deleted User @verticity great work! Keep it up. This is exactly the kind of science we need to make informed decisions.

 

[dragonaut]

Okay, so I have been looking into TLC analysis of different acetone extracts (I can give you the full protocol if anyone's interested, it is a convenient way of identifying the various compounds in kava, but the equipment needed is a bit expensive and not readily available).

The flavokavains are the major coloured compound in tudei, but they are not present in high enough concentration to really contribute to the colour of noble kavas. The colour in nobles come from one of the kavalactones (I have not isolated the exact one). The kavalactone colour is a pale yellow, whereas flavokavain A and B are more intense yellow, probably turning orange with increasing levels. There is also a brown compound present in stems and also wichmanni root - it is not identified, but the conclusion must be to NOT consume anything that is brown in the acetone test. Lebot already reported most of this, he probably figured it out from looking at his TLC plates before developing (only coloured compunds will show).

The problem with the acetone test is that noble has kavalactones and tudei has kavalactones + flavokavains, so both will be yellow to some degree. To further complicate things, the colour of an acetone extract depends greatly on the grind of the kava used as well as the efficiency of the extraction. I have made a tudei extract that looked very pale and would definitely be identified as noble, simply because it was badly grinded.

So we need another way of differentiating between the colour from kavalactones and the colour from flavokavain. TLC is ideal, but not an option for most people. I am working on a method that can be done at home, but I am not sure how reproducible it is yet. If some of you, who have access to tudei and noble (and ideally stems/wichmanni), have time to test it for me it would be great. You will need less than a gram of each kava and some standard household items.