Today’s fact of the day will focus on one specific piece of data from a very timely and important paper by Lebot, Koah, and Legendre in 2020 [1]. In this paper the authors provide evidence to support the theory that roots, chips and powders can be tested for their combined kavalactone and flavokavain content by way of passing light through their acetonic extracts for absorbance in a colorimeter.
What exactly do they mean by “acetonic extract”?
The way in which this type of test goes about indicating the presence of various chemicals in kava is by using acetone as an extraction medium. This targets the hydrophobic portions of kava such as kavalactones and flavokavains which normally don’t mix well with water. We should keep in mind here that when making kava, we’re not so much looking for kavalactones to be dissolved, as kavalactones themselves are quite difficult to dissolve in water. Kava is more of an emulsion, so while these chemicals may not be able to mix well with water, we’re pushing them out with physical force and they’re still attached to the little particles of kava that settle in our beverages. I say this in order to clarify that although acetone is used here, it doesn’t befuddle the results as what we are looking for would be present in aqueous extracts as well.
What is a flavokavain?
Flavokavains are a constituent of kava known as a “chalcone” or “flavonoid”. They’re pigments. They are found concentrated in the surface cell layers of kava and in the bark of the stump and stem. It’s thought that they play a protective role for kava, as it has been found that other flavonoids in this family often occur for protecting other plant species in the same way. Research suggests that flavokavains contribution to noble kava beverage quality is very limited [1], however it’s turning out to be a good indicator of kava quality when paired with kavalactone quantity. This reveals itself in absorbance through a colorimeter.
Absorbance, what does it mean to us in this situation?
In this instance we’re dealing with a device known as a colorimeter, specifically the colorimeter pictured above. This device is an instrument that compares the amount of light getting through a solution with the amount which can get through a pure solvent [2]. In this instance we’re measuring pigments and kavalactones, and these compounds absorb light in differing amounts that are recorded by the colorimeter. The device was used to ascertain the total quantity of kavalactones and flavokavains together rather than their individual quantities.
The Colorimetric Test
Researchers used 10g of kava powder and 30 mL of acetone in clear test tubes. These were then centrifuged in order to push the sediment to the bottoms of the tubes, leaving a clear, but obviously color filled supernatant such as Figure 1 shows. Approximately 10ml of this was transferred to the colorimeter and tested at 440nm for absorption. The researchers applied this method to over 1,000 samples of commercial kava and compared the results to tests performed on a machine known as HPTLC or “High-performance thin-layer chromatography”. This method is far more sensitive, and can discern between quantities of different kavalactones and flavokavains, whereas the colorimeter cannot.
What they found
Out of over 1,000 kava samples, researchers found an r value of correlation with absorbance being .72 which is quite statistically significant. This comes up to .5211 for an R² value, and here’s where yesterday’s fact of the day comes into play.
This can also be expressed as a percentage, so 52.11%. Essentially there has been much confusion surrounding this number. Some have wondered if this means the test only works ½ of the time seeing as the number = 52%. No. Here is exactly what this means for us: R squared, is the proportion of the variance in the flavokavain amount that is predictable from the absorbance spectra.
This means, verbatim: 52% of the variation in the flavokavain amount can be predicted and explained by the absorbance spectra. This leaves 48% of the variation in these results that may be due to other factors we haven’t seen yet. This in no way invalidates the results of the acetonic test, and in fact strengthens it. Keep in mind the number of samples in this test was 1053.
Via personal communications with Dr. Lebot: " it is just measuring the proportion of non KL compounds in the extracts, especially the amount of pigments, KLs are not pigments, FKs and other pigments are the ones responsible for the colour"
Conclusions:
This test is viable, however it has its limitations. The acetonic test does not represent a quantitative measure, as in it doesn’t give us exact amounts of flavokavains or kavalactones. The test can be used as a fair assessment of the flavokavain content in the acetonic extract, and can be used as an initial indicator of quality.
If you find yourself asking “why this when they have all the other ways of measuring kavalactones and flavokavains”, the answer is simple, cost. HPTLC new costs between $80k-$100k whereas the colorimeter we spoke about costs around $800. This quick and cheap method could easily indicate kavas in which you would want to further test, reducing the amount of time and money required overall. This has been known for a number of years, but this paper [1] adds credibility and evidence towards the fact that this method is repeatable, and usable.
[1] Lebot, Vincent, Juliane Kaoh, and Laurent Legendre. 2020. “High-Throughput Analysis of Flavokawains in Kava (Piper Methysticum Forst. F.) Roots, Chips and Powders and Correlations with Their Acetonic Extracts Absorbance.” Food Analytical Methods 13 (8): 1583–93. https://doi.org/10.1007/s12161-020-01781-9.
https://sci-hub.st/10.1007/s12161-020-01781-9
[2] How does a colorimeter work? (n.d.). Retrieved May 18, 2021, from http://www.saps.org.uk/saps-associates/browse-q-and-a/378-how-does-a-colorimeter-work
[3] Lebot, Vincent, and Laurent Legendre. 2016. “Comparison of Kava (Piper Methysticum Forst.) Varieties by UV Absorbance of Acetonic Extracts and High-Performance Thin-Layer Chromatography.” Journal of Food Composition and Analysis: An Official Publication of the United Nations University, International Network of Food Data Systems 48 (May): 25–33. https://doi.org/10.1016/j.jfca.2016.01.009.
https://sci-hub.st/10.1016/j.jfca.2016.01.009
What exactly do they mean by “acetonic extract”?
The way in which this type of test goes about indicating the presence of various chemicals in kava is by using acetone as an extraction medium. This targets the hydrophobic portions of kava such as kavalactones and flavokavains which normally don’t mix well with water. We should keep in mind here that when making kava, we’re not so much looking for kavalactones to be dissolved, as kavalactones themselves are quite difficult to dissolve in water. Kava is more of an emulsion, so while these chemicals may not be able to mix well with water, we’re pushing them out with physical force and they’re still attached to the little particles of kava that settle in our beverages. I say this in order to clarify that although acetone is used here, it doesn’t befuddle the results as what we are looking for would be present in aqueous extracts as well.
What is a flavokavain?
Flavokavains are a constituent of kava known as a “chalcone” or “flavonoid”. They’re pigments. They are found concentrated in the surface cell layers of kava and in the bark of the stump and stem. It’s thought that they play a protective role for kava, as it has been found that other flavonoids in this family often occur for protecting other plant species in the same way. Research suggests that flavokavains contribution to noble kava beverage quality is very limited [1], however it’s turning out to be a good indicator of kava quality when paired with kavalactone quantity. This reveals itself in absorbance through a colorimeter.
Absorbance, what does it mean to us in this situation?
In this instance we’re dealing with a device known as a colorimeter, specifically the colorimeter pictured above. This device is an instrument that compares the amount of light getting through a solution with the amount which can get through a pure solvent [2]. In this instance we’re measuring pigments and kavalactones, and these compounds absorb light in differing amounts that are recorded by the colorimeter. The device was used to ascertain the total quantity of kavalactones and flavokavains together rather than their individual quantities.
The Colorimetric Test
Researchers used 10g of kava powder and 30 mL of acetone in clear test tubes. These were then centrifuged in order to push the sediment to the bottoms of the tubes, leaving a clear, but obviously color filled supernatant such as Figure 1 shows. Approximately 10ml of this was transferred to the colorimeter and tested at 440nm for absorption. The researchers applied this method to over 1,000 samples of commercial kava and compared the results to tests performed on a machine known as HPTLC or “High-performance thin-layer chromatography”. This method is far more sensitive, and can discern between quantities of different kavalactones and flavokavains, whereas the colorimeter cannot.
What they found
Out of over 1,000 kava samples, researchers found an r value of correlation with absorbance being .72 which is quite statistically significant. This comes up to .5211 for an R² value, and here’s where yesterday’s fact of the day comes into play.
This can also be expressed as a percentage, so 52.11%. Essentially there has been much confusion surrounding this number. Some have wondered if this means the test only works ½ of the time seeing as the number = 52%. No. Here is exactly what this means for us: R squared, is the proportion of the variance in the flavokavain amount that is predictable from the absorbance spectra.
This means, verbatim: 52% of the variation in the flavokavain amount can be predicted and explained by the absorbance spectra. This leaves 48% of the variation in these results that may be due to other factors we haven’t seen yet. This in no way invalidates the results of the acetonic test, and in fact strengthens it. Keep in mind the number of samples in this test was 1053.
Via personal communications with Dr. Lebot: " it is just measuring the proportion of non KL compounds in the extracts, especially the amount of pigments, KLs are not pigments, FKs and other pigments are the ones responsible for the colour"
Conclusions:
This test is viable, however it has its limitations. The acetonic test does not represent a quantitative measure, as in it doesn’t give us exact amounts of flavokavains or kavalactones. The test can be used as a fair assessment of the flavokavain content in the acetonic extract, and can be used as an initial indicator of quality.
If you find yourself asking “why this when they have all the other ways of measuring kavalactones and flavokavains”, the answer is simple, cost. HPTLC new costs between $80k-$100k whereas the colorimeter we spoke about costs around $800. This quick and cheap method could easily indicate kavas in which you would want to further test, reducing the amount of time and money required overall. This has been known for a number of years, but this paper [1] adds credibility and evidence towards the fact that this method is repeatable, and usable.
[1] Lebot, Vincent, Juliane Kaoh, and Laurent Legendre. 2020. “High-Throughput Analysis of Flavokawains in Kava (Piper Methysticum Forst. F.) Roots, Chips and Powders and Correlations with Their Acetonic Extracts Absorbance.” Food Analytical Methods 13 (8): 1583–93. https://doi.org/10.1007/s12161-020-01781-9.
https://sci-hub.st/10.1007/s12161-020-01781-9
[2] How does a colorimeter work? (n.d.). Retrieved May 18, 2021, from http://www.saps.org.uk/saps-associates/browse-q-and-a/378-how-does-a-colorimeter-work
[3] Lebot, Vincent, and Laurent Legendre. 2016. “Comparison of Kava (Piper Methysticum Forst.) Varieties by UV Absorbance of Acetonic Extracts and High-Performance Thin-Layer Chromatography.” Journal of Food Composition and Analysis: An Official Publication of the United Nations University, International Network of Food Data Systems 48 (May): 25–33. https://doi.org/10.1016/j.jfca.2016.01.009.
https://sci-hub.st/10.1016/j.jfca.2016.01.009
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