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Kava Science Simple Test for Checking if your Kava is Tudei (Please read if you're new to Kava)

Discussion in 'Kava Lounge' started by infraredz, Sep 26, 2013.

  1. infraredz

    infraredz BULA!

    For those who aren't familiar, Dr. Lebot is a leading researcher of Kava who has a PhD in Plant Genetics and authored the book: Kava: The Pacific Elixir. More information can be found here: http://www.kavaforums.com/forum/threads/dr-vincent-lebot-the-kava-expert.2518/#post-25541

    In Dr. Lebot's research, he has created a very simple, cheap and quick way to test a small amount of kava to determine if it is "Noble" or "Tudei" shown in this video:



    Read on to learn how to test your kava!

    For information on why this works [New Information Available], please see the comments below because much of the "mystery" behind this test has been addressed by Dr. Lebot.

    Introduction (Important Notes Regarding the Test):
    [NEW AND IMPORTANT INFORMATION ON THE TEST]

    • The test detects an "non identified molecule" which Dr. Lebot has said is visible using HPTLC at 366nm and hypothesizes that "this molecule is biosynthesed by a peculiar genotype [genetic makeup of an organism]"
    • According to Dr. Lebot, this molecule "is unique to twodays and wichmannii [...] [and] a marker associated to these genotypes"
    • Dr. Lebot has tested 208 samples using this test, from 72 distinct cultivars which were all planted in the same place, in a climate and otherwise controlled environment.
      • 8 clones of both Borogu [a known noble kava] and Palisi [a known tudei kava] were tested to make sure that there was no interclonal variation in the results.
    • From these samples, Dr. Lebot "didn't get false positives nor false [negatives]; All nobles were yellow, all two-days were ambre and all wichmannii were brown"
    The above information is new, and might disprove my hypothesis, though regardless, more testing is required to identify the exact mechanism behind the color change and to identify molecules that are distinct to tudei/isa cultivars.


    How to Get Started Now:
    The beauty of this test is its' simplicity and cheap cost. You only need four, cheap and easily accessible things!
    All you will need is a small glass container, some acetone, and a small amount of kava.
    • Regarding the container, all you need is a small (preferably) glass container of some sort with a lid. I found the vials below for $0.99 as "spice containers" in a big box store.
    • Acetone can be purchased at any home improvement store and is very cheap.
      • Furthermore, all the acetone I came across was quite pure (over 99% purity)
    • The amount of kava doesn't need to be more than a tablespoon (2 flat teaspoons is what I used), so you don't have to worry about wasting your kava!
    The Method (How to):
    The test is usually performed with 10g of powder, and 30ml of acetone. However, you do not need a scale if you do not have one.

    1. Add 30mL of acetone to (10g) or 2 teaspoons (approx. 10mL) of each kava in a small glass container.
    • If you want to keep it simple, just use the measuring glass and keep the ratio of kava/solent at 1/3
    2. Agitate- (shake lightly) for 4 minutes then leave to settle for 24 hours.

    3. Then, visually examine the liquid portion (called supernatant) of the suspension for hue, saturation and darkness as described by Lebot as the method of determination of tudei, wichmanii and noble kavas.​

    As a side note, the amount of kava is approximately 10mL if measured in a measuring device, but mL is a measurement of liquid but this might make the ratio easier to understand [1/3]

    The Results
    Although it is not necessary to use more than one sample, but it can help to test a known "noble" kava as a reference, or "control" to compare your sample to.

    My results: For my purposes, I figured we all know Boroguru is a common noble kava. I'm calling this the control (to show what the color should be).
    "Koniak" is a kava that has been suspected to be tudei. Based on the technique Dr. Lebot outlines, I decided to determine if BKH's Koniak is a tudei (based on the standard in the video [dark color]) and if Solomon's Island by Nakamal at Home is Piper wichmanii (based on the standard in the video [very dark color]) as some have theorized and have been curious about.​

    Kapm also used this technique to test "Hawaiian Kava Center Tudei" (on the Left) and Bula Kava House "Borogu" (on the right) by using one teaspoon of each kava and equal amounts of methanol, which can also be used instead of acetone though I think acetone is more common and probably cheaper though I'm not sure.​

    Violet has also used this technique to test known "ISA" (tudei), Wakacon's "Vanuatu Kava" as well as "Papa Kea". The same method as mine was used and her results are below.

    Chris from Gourmet Hawaiian Kava has also used this test to compare noble Hawaiian varieties and Hawaiian isa. He also has tested varying degrees of noble/isa ratios and the colors they give. More of his testing can be found here

    Roaddog's has also used this to test Bula Kava House's "Melo Melo" and Mood and Mind's "Premium Kava Kava Powder".​

    Below are my results (as well as other members' test results) for reference.
    -My samples were backlit and photographed, though you obviously don't have to do this. This was only meant to help give a "clearer" resulting photograph.

    -The ratios of solvent to sample were kept constant.
    -The reason for seemingly different amounts of kava at the bottom is due to the differing grinds (upon close inspection, the larger [most dense] particles settle lower).

    acetonic_extraction.jpg

    Comparison between Boroguru on the left and Koniak on the right. Both samples were isolated and photographed with black flags on either side. Photos were then merged into Photoshop and 'split' then combined. Any white balance or curve adjustment was done on the combined image as a whole. The reason why the foreground of the Boroguru sample shows yellow is due to the sample being less opaque (therefore allowing more light from backlight through) than the Koniak sample which, as stated above is more opaque.
    acetonic_extraction_comparison.jpg

    Kapm's Results:
    u3e8u5u3.jpg

    Left- Hawaiian Kava Center Tudei
    Right - Bula Kava House Borogu

    Violet's Results:
    violetsolventtest.jpg

    Labels attached
    Chris' Results:
    GHK-noble.jpg GHK-ISA.jpg

    Three varieties of Hawaiian Noble Kava and the results of an Hawaiian ISA

    Roaddog's Results:
    roaddogsolventtest.jpg

    "Melo Melo" on left and "Premium Kava Kava Powder" on right

    Discussion on Results:
    Something that might not be immediately apparent from my photograph is that Boroguru has the clearest supernatant of all the samples.

    I suspect that without a centrifuge, the smaller particles responsible for the cloudiness of the other two samples might not decant completely so cloudiness is not something to worry about.

    The glass quality of my vials is poor as well as fairly thick, giving seemingly different tones from one side to the other.

    Just speaking from photo-geekness, it would be best to evaluate the central part of each sample for the most accurate representation of the actual color (due to refractivity of different wavelengths through the glass)

    Original Thoughts for Color Difference:
    It's interesting to note that flavokavain A, B, and C are all chalconoids and conjugate ring closures result in flavonoids which are responsible for a wide variety of plant pigmentation (eg. red vs yellow, for example). These chalconoids (FKA, FKB, FKC) reportedly possess antibacterial, antifungal, antitumor and anti-inflammatory properties (sound similar to kava?). Some chalconoids have demonstrated the ability to block voltage-dependent potassium channels (which is a proposed hypothesis regarding the MOA of kava, and in my opinion, sounds very likely to play a significant role in kava's effects). [http://www.ncbi.nlm.nih.gov/pubmed/18032041]

    I wonder if these secondary metabolites are in fact more important in the overall biosynthesis of different compounds than I thought. Flavonoids are responsible for a significant amount of color in a plant, and so I'm really interested if the reason we are seeing tudeis (which have FKB, unlike nobles) show a red color vs. the "control" of yellow is because of the conjugation of this phenyl ring which would cause it to to become a flavonoid, which in turn possesses specific color properties. Chalconoids by themselves don't necessarily have color properties, but they serve as the intermediate in the biosynthesis to the flavonoids that do.

    My New Theory (decoded)
    [UPDATED: Currently the theory being used by Dr. Lebot as of 03/21/14]

    FKB, is a chalcone which is a natural biological compound and is an intermediary to flavonoids (which is another type of natural biological compound, common in fruit and vegetables and currently the subject of an abundance of positive health effects).
    When chalcones undergo conversion (conjugate ring closure) in the plant they result in flavonoids.

    I propose that it is not unreasonable to assume that the presence of FKB can cause an increase or difference in the formation of flavonoids (which then have color properties).

    My supposition is that the yellow vs orange tincture could be due to the presence and/or abundance of flavones or flavonoids (in tudei kava). For instance, FKB might result in an orange tint or color whereas noble kava (that has far less FKB, and therefore less substrate [chalcones] being converted to a flavonoid with an orange color) results in a "lighter" color of yellow whereas a higher concentration of FKB corresponds to a higher concentration of a flavonoid that results in an orange color.

    I was, at one time, a photography major and took 2 years of extensive coursework.
    From that, I can say that these colors can be looked at this way:
    1. Red is the highest saturation "amount" of red (Red, Green and Blue being the "primary color")
    2. Orange is the same hue as red, but slightly lower saturation of red.
    3. Yellow is the same hue as red, but even slower saturation than orange.
    Hue is the "color", and "saturation" is the amount of that "hue"

    The essence of this theory is currently the operating hypothesis of Lebot as a possible mechanism behind the differences in supernatant color, though it will take some time before definitive information is available due to having to test hundreds of compounds.
    Last edited: Jul 5, 2014
    Karl, kavadude, HeadHodge and 6 others like this.
  2. Well done Infraredz! Well done indeed.
  3. infraredz

    infraredz BULA!

    Just as a comment, this thread isn't about tudei kava. This thread is solely about the solvent test, how it is done, what it can do, how it works and what you can learn from it.

    I decided to leave the thread open for anyone that might have questions or comments on this test, but I don't want it to become a discussion on tudei.

    Just as a heads up to everyone, that's all.
    violet and HeadHodge like this.
  4. Vekta

    Vekta Notorious Lightweight Review Maestro

    My mind exploded a little about half way through.:woot:

    Good read though.
  5. infraredz

    infraredz BULA!

    I tried to make the last paragraph a little more accessbile and easier to understand. To say that the mechanism behind the color change isn't known, it sure as hell it being tested right now and I'm sort of astounded when I got the reply from Lebot that it was the theory they were using, considering I was expecting some brilliant Botanist to explain how far off I was. I never had any true education in botany, so it made it even the more astounding. The biochemical stuff behind it isn't too hard to see though, and in fact, I can post some images of the chemical pathways, conversions and such if anyone is interested.

    To me, it seems that it's a case of Occam's razor because (what I thought was) a silly and overly-simplistic explanation of the color change is actually the current hypothesis. The problem is they need to spend a lot of time researching hundreds of compounds, and with Lebot being busy as it is, it's certainly not going to be coming out tomorrow.

    I hope though, that understanding that very hypothesis makes this test less "murky" when it comes it its accuracy which seems to be somewhat under fire by some.
    HeadHodge likes this.
  6. Gourmet Hawaiian Kava

    Gourmet Hawaiian Kava Kava Expert Kava Vendor

    Hi Deleted User,

    Concerning the test samples that I did here in this post-
    After I took pictures of all of them, I left them in the sun to evaporate the solvent. When the solvent was gone I noticed the residue in the bottom, (the kavalactones and other stuff) was a darker orange with all the tudei kava, but the noble kava was darker, but still not even close to the tudei kava.

    Let me know if you need some Isa and I will send you some Isa and other plant parts if you want to do some experiments. Let me know if you do need anything. Aloha.

    Chris
  7. Blimpie

    Blimpie Member

    Can nail polish remover be used in this test? Or will the other ingredients (water, gelatin, aloe vera, propylene carbonate, glycerin, etc.) be a problem? Sorry if this is a foolish question; I am lacking in chemistry knowledge and very eager to go ahead and test the kava I have!
  8. Gourmet Hawaiian Kava

    Gourmet Hawaiian Kava Kava Expert Kava Vendor

    I would recommend that you use regular acetone, you can get it at any hardware store and that way you will get the most accurate results, Walmart might have it in there hardware dept, it is easy to get at any local hardware store. Let us know if you have any other questions about this.
    Aloha.

    Chris
  9. violet

    violet Do all things with love Moderator

    My local Walmart also carries "100% Acetone" in the health and beauty section as well with the other nail polish removers. You should get four tests out of a 6 oz bottle.
    Gourmet Hawaiian Kava likes this.
  10. Blimpie

    Blimpie Member

    Would Rust-Oleum acetone be ok to use?
  11. Blimpie

    Blimpie Member

    Thanks for all the quick answers, by the way! :LOL:
  12. Gourmet Hawaiian Kava

    Gourmet Hawaiian Kava Kava Expert Kava Vendor

    What you are looking for is 100% acetone, if the rust oleum is 100% pure acetone then it will work. The 100% acetone is clear and will extract more of what you need to do the test, if there are other things in the acetone then it might throw off the results, it is best to be sure with this test and it is super easy to get the 100% pure acetone. Aloha.

    Chris
  13. infraredz

    infraredz BULA!

    Yes, you need pure acteone without any other ingredients at all. Also, pure ethanol will work if you ever have come across it, as will 100% Methanol, again, these are generally harder to find.

    99% pure acetone (nothing can technically be 100% even sometimes from lab supply stores) is what you want. It's dirt cheap and very easy to find at hardware stores, home improvement stores, and hell, I think even CVS or Rite Aid might carry it.

    Deleted User is definitely correct, all of these above organic solvents are very flammable though I would say that most of the problem is the fumes (which evaporate in a very "rapid" way) so be careful to just take adequate precautions. You don't need a fume hood or even an R/N/P 100 respirator or anything (even a face mask) to do this, but keep in mind that these fumes aren't exactly safe to just use willy nilly all over your kitchen or something.

    Just measure out the amount of solvent, make sure the cap is on when the bottle isn't in use, and keep away from fire, heat or ignition sources and everything is totally safe.