Regarding the chromatography of Banisteriopsis caapi alkaloids

[lightwill]

While extracting Banisteriopsis caapi vine in a large soxhlet apparatus using 95% ethanol acididifed with USP HCL, some observations were made. It was first thought that when the elute from the soxhlet chamber was clear or close to clear in color, the extraction would be complete. this was not true. remembering the caapi alkaloids all have a beautiful glow in the UV light, the soxhlet chamber was exposed to UV light and observed periodically. Surprisingly, even when the elute was clear the chamber glowed a brilliant green. this was after about 24 hours of extraction. extraction was continued. over 56 hours later the chamber was still glowing bright green, but it was observed that the top 20% of the chamber no longer glowed, indicating complete extraction of the alkaloids (at least from that region).

the boiling flask which had the extractions from many runs of biomass over the last couple weeks glowed a very bright blue color, with green drips falling into it. before the uv observation came to mind, the previous extractions were stopped early. the biomass set aside of course in case any alkaloids remained.... but they were thought to be mostly spent at the time although smelling strongly of "vanilla bean". (at least in this olfactory system).

anyway, what this boils down to.... the compounds which flouresce blue in the UV light are rinsed out first. at least when using acidic ethanol. this includes all the color and surely many inactive compounds from the biomass. What remains for several days worth of extraction flouresces bright green and in regular light appears as barely tinted clear liquid.

this green fraction is clearly one of several banisteriopsis caapi alkaloids, in quite a pure form one would guess. this compound can be easily seperated with intent to isolate the particular alkaloid the green color is associated with. here is a clip that was read from article regarding caapi and two similar plants which were assayed:

A comparative study of the three extracts was performed using thin-layer chromatography. Prepared aluminum-backed plates coated with silica gel were used. A solvent solution of 100 parts water to 100 parts ethyl acetate to 200 parts methanol to 6 parts ammonia was used in all three TLCs. The separated compounds were then visualized under UV light, and with an ammonia reagent to detect the presence of organic compounds.

TLC analysis detected the presence of alkaloids in all three bark extracts. B. caapi revealed four compounds (pale blue fluorescence at Rf=0.16, medium blue at Rf=0.34, bright green at Rf=0.42, and orange at Rf=0.45). B. muricata revealed four compounds as well (purple fluorescence at Rf=0.07, pale green at Rf=0.20, bright green at Rf=0.42, and blue at Rf=0.50). TLC analysis of B. martiana revealed only one compound (purple fluorescence at Rf=0.7). Comparative analysis found one compound in common between B. muricata and B. caapi (bright green fluorescence at Rf=0.42), as well as a shared compound between B. martiana and B. muricata (purple fluorescence at Rf=0.7).

So it is clear from this study that the different compounds present in Banisteriopsis caapi each flouresce a unique color, green being the most unique and easily identifiable.

Also it is noteworthy that the practice of traditional ayahuasca brewing with water places emphasis on multiple boilings of the biomass to achieve complete extraction. this would make alot of sense seeing as how an extraction using acidic ethanol is not completed after more than 3 days! never would have imagined it requiring so much time as the rinsing is steady and constant. maybe after 4 or 5 days total it will be completed.

My question is this.......... Who knows which compound glows green? The above quoted article made no reference to color id of the compounds.

it would be interesting to seperate this compound, the green glowing compound in the clear liquid, which is the most tenacious of all the ayahuasca alkaloids, remaining after many days, probably left behind in most peoples aqueous brews unless they are *very* thorough... this compound obviously would effect the over tone of the experience so it is of interest to research to identify which compound this is and what possible effect it may have on the experience. to me, metaphysically, there is some significance to the tenacity of this single compound remaining after everything has been rinsed. by the way, after 3 days of constant rinsing the clear liquid glows BRIGHT green as does about 2/3 of the soxhlet chamber... how many people actually extract this compound when they are brewing? I would guess acidic water would have much less of a chance of accessing this compound then acidic ethanol. my point is this compound (at least partially) is probably left out of most brews. it would be interesting and quite easy to seperate and assay.

comments anyone?

i just noticed i put this in the wrong section of the forum maybe... oops. please move if need be.

 

[ismokecrystals]

B. caapi revealed four compounds

pale blue fluorescence at Rf=0.16
medium blue at Rf=0.34
bright green at Rf=0.42
orange at Rf=0.45).

In Caapi there are at least 3 alkaloids, correct? THH, Harmine, Harmaline. I believe THH has the pale blue florescence but I don't know of the others.

 

[Infundibulum]

Fluorescent colours do not really mean much. All these information about colours is poor because it is not rigorous. Many parameters (other than the nature of thee alkaloid can influence fluorescence. For instance, pH; SWIM has observed that P.harmala alkaloids fluoresce bluish in acidic pH and greenish in basic pH.

I would like to see more details, e.g. the wavelength of UV excitation (just "UV" does not mean much either) just as emission wavelength ("blue" or "green" or "maroon blue" also does not mean much, esp to a people like SWIM who has some degree of colour confusion) so as to make a serious discourse.

In addition, the other compounds (that may be extracted at different rates) from the extracted caapi can also influence fluorescence of the very same compound. Lightwill, if you or SWIY has kept those extracted fractions, do an alkaloidal purification, analyse them and report back.

 

[lightwill]

what you say about the ph and color makes good sense to me. thanks. i suppose the cited article which reported the color data was also quite vague with their data. even to the point of excluding the names of the characterized compounds. i imagine they identified each compound during the study, but maybe not.

no way to measure wavelength is at hand so data regarding this would not be possible.

there is one 250gm portion of caapi dust left to extract. this one could be monitored periodically with the uv light and the fractions separated. purified, melting point test, then report back... sound good? i imagine we can get some pretty definitive answers like that.

or maybe chromatograph the complete extraction from the last remaining portion of caapi... that may achieve a cleaner separation than changing the flask on the soxhlet apparatus for each fraction.

will write back soon. thanks.

 

[lightwill]

In Caapi there are at least 3 alkaloids, correct? THH, Harmine, Harmaline. I believe THH has the pale blue florescence but I don't know of the others.

Yeah at least 3. Actually I was digging around the search engines and it appears that recently (although it may not have been so recent who knows) they discovered more alkaloids in caapi than previously though.

Abstract

Three new alkaloids isolated from Banisteriopsis caapi, were identified as harmic amide (1-carbamoyl-7-methoxy β-carboline), acetyl norharmine (1-acetyl-7-methoxy β-carboline) and ketotetrahydronorharmine (7-methoxy-1,2,3,4-tetrahydro-1-oxo-β-carboline)

I thought that was interesting. Caapi is such a mysterious plant. Actually that paper is not new at all it was dated 1976.

Here is a link to the articl3 "Emanations volume 4" about the caapi study I cited earlier.

caapi research data

I thought this was a great article. Of particular interest to me was the "bioactivity study" where they tested the alkaloid extractives against various bacteriums. caapi was shown to be effective against several different strains, validating in a very third dimension ways the reputation caapi holds as a medicine and healing agent. this has been a topic of my recent interest as well although i was not aware of any proven science data regarding caapi and healing. personally i think caapi is an antiparasitide as well as an antibacterial which is why it so effectively solves so many health problems.

 

[Hybridoma]

For someone with the time and access to the proper equipment, suggest the following:

1. Perform the extractions as noted earlier to maximize both yield and range of compounds extracted.
2. Run the thin layer chromatography and see how many spots are generated, and how they individually fluoresce under UV. Measure rf values.
3. Design a silica column with a solvent mix that is most likely able to separate the fractions, monitoring by UV on the fraction collector, and then testing the fractions against the TLC.
4. Once reasonably pure compounds are isolated, use mass spec to make compound identification, which can then be correlated with the TLC and column data.
5. Now you have the data and the samples as controls for optimizing extractions that will preferentially yield the desired alkaloids, and publish the protocols for all of us!

This looks like a good month of a grad student's time, but the results would add a lot of knowledge to our Forum!