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Washing the Psilocybin Extract

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blue.magic

blue.magic

Esteemed member
I am planning a methanol extraction of P. Cubensis.

The solvent used is very pure and will be completely evaporated using a rotavap.

My concern is that the initial extract might become a sticky mess so the washing might not be effective. Even scraping the sludge off the rotavap flask might be difficult.

Maybe using warm/hot water to dissolve the extract and than washing it in a liquid-liquid fashing might work better?

Unfortunately, there is also little information about psilocybin solubilities. It is insoluble in benzene, chloroform and hexane. It is difficultly soluble in anhydrous ethanol and anh. acetone.

Could DCM be used instead of chloroform?

I think final washing with cold anhydrous acetone and/or cold anhydrous ethanol might be a good idea as these solvents evaporate easily and thus will also dry the extract.

The whole purpose of washing is to obtain somewhat powdery product that can be weighed.

I plan to dry the product in a vacuum desiccator.

Have you any experience with psilocybin extracts and whether they can be turned into a powdery form by washing?
 
KloudQ7 said:
There has been some amazing progress with mushroom extraction by loveall and others in another thread.

Click to expand...

Thanks, I will go through the thread.

I observed Loveall's impressive work, but it is still a work in progress so I just wanted some working approach.

But it seems I will have to experiment on microscale too, before extracting a larger batch.
 
Yep, still very much experimental.

I would recommend you try this in one of your experiments:

1) Add 5x volume of acetone to methanol extract, incubate for a day filter out proteins. Using UV it looks like the actives are still in solution.
2) Add fumaric acid at a rate of 5% of mushroom weight and evap, you should get a fluffy powder at this point :). This could already be a powerful product.
3) To clean moar, dissolve in water, filter, wash with naptha (Xylene may wash better). During the first solvent wash you may see a protein pellet form at the water/solvent interface. This is from proteins unfolding at that interface and exposing their hydrophobic/philic sides to each liquid phase and getting trapped at the interphase. Kind of cool. The pro's use this to recover protein pellets in some situations.
4) Add methanol with some fumaric acid to the cleaned up water. Maybe like 1% of the mushroom weight. Dry this. If you use a little heat while drying you may get some blueing (maybe a cool factor for some but you pay with some oxydation).
5) This second fluffy/crystaline stuff may be very concentrated and powerful. Or maybe not, time will tell.

There are probably other/better ways to clean stuff up, but I think the protein precipitation may allow for workable powdery products without the need for column separation. For example this may be interesting based on ongoing work:

A) After step 1. above simply dry. Then wash gunky residue with acetone (which unfortunately removes any psilocin) and naptha. String-like structures remain. Add fumaric acid and water and dissolve everything together. Filter any excess fumaric that does not go into solution. Dry.
B) After step 1 add 10% by volume of tartaric acid. Let sit and see if tartarates precipitate due to common ion effect. This can also be tried before protein precipitation straight on the methanol extraction (but protein salts may crash out first ?).


Cheers and good luck. Looking forward to any results.
 
Could DCM be used instead of chloroform?
Click to expand...
I can't really answer this except to say sometimes DCM unexpectedly dissolves salts, so be sure to follow the adage of "don't throw anything away until you're sure you know where the goods are" should you decide to use it.


Does anyone here have experience with working under inert atmosphere? It would be my choice for minimising oxidation during the process and it isn't that difficult to rig up if you already have a half-decent lab set-up.
 
Thanks. I've just spent 2 hours reading through most of the current psilocybin extraction threads. Wow that was exhausting...

So many thanks for a summary of your current "state of the art".

Yes I plan using methanol as the primary extraction solvent.

Okay I will synthesize some chloroform for the washing (finally have a use for that 10L canister of strong hypochlorite solution 😁 ).

Apart of being rigorous on drying solvents and fine filtration, I think there won't be any modifications.

I am thinking about making a Keller's reagent to indentify psilocybin (unfortunately, I have not yet found proper ratio of chemicals to prepare it). This has been used by A. Gottlieb (Psilocybin Producer's Guide, 1976). His extraction technique is trivial, using just methanol, then evaporating the solvent to dryness. According to Gottlieb, the purity of this simple extract is 25-50% psilocin/psilocybin (we now know psilocin should be out from the game at this point) - which sounds too good to be true - he however worked with mycelium, not fruits.
 
How's this work going?

I've learned a couple new things while working on this on my side (items 2 and 3):

1) Acetone can crash proteins when added to an extract.
2) Heat can denature and agregate proteins, this can make them easier to crash out. Extreme example is a poached egg. Usually getting above 75F is needed from what I gather, which can be done by 75% ethanol in a water bath.
3) When washing mushroom methanol extract with naphta, a removable protein pellet forms between the inmisible layers (bonus: the methanol may be defatted). I think this is from a mechanism similar to the better known methanol/chlorophorm protein pellet separation.

Hope this helps during your mushrooms extraction adventures. Good luck!
 
Thanks, Loveall, for the tips. I will certainly do the protein precipitation.

I am going very very slowly because having several other projects going. I spent last two weeks just recycling and purifiyng my solvents as well as doing lab cleanup.
 
Loveall said:
How's this work going?

I've learned a couple new things while working on this on my side (items 2 and 3):

1) Acetone can crash proteins when added to an extract.
2) Heat can denature and agregate proteins, this can make them easier to crash out. Extreme example is a poached egg. Usually getting above 75F is needed from what I gather, which can be done by 75% ethanol in a water bath.
3) When washing mushroom methanol extract with naphta, a removable protein pellet forms between the inmisible layers (bonus: the methanol may be defatted). I think this is from a mechanism similar to the better known methanol/chlorophorm protein pellet separation.

Hope this helps during your mushrooms extraction adventures. Good luck!
Click to expand...

Here is more info on 1) and 3) and they also have other info on managing proteins (which I think may be important in mushroom extraction). They mention TCA, but I think that can be overcome by using more acteone volume.
 
Interesting, TCA is used for tattoo and genital wart removal and available OTC in general. I was not aware of this at first.

However, care should be taken with it, as it can form salts and endup in a hypothetical product. There are protocols out there where acetone is used to wash TCA from the protein
pellet. The thing to do here is to simply stick to acetone for protein precipitation experiments, unless you really know what you are doing.
 

Attachments

  • %5BJon_Lorsch_(Eds)%5D_Laboratory_Methods_in_Enzymolo(b-okcc).pdf
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if anyone sends me samples for analysis, please don’t use PEG. It sticks out like a sore thumb, and takes a while to eliminate from the system.

or clean the sample thoroughly.
thanks 😁

looks like this

PEG_mass_spectra.png
 
blue.magic,

A kind soul in the Nexus chat pointed me to this patent, has some really good info - including protein precipitation (they added ethanol to water insetad of acetone).

One thing that I have not seen seen mentioned yet and I'm getting preliminary promising results with is non anhydrous acetone. It seems to pick up actives and very little else. I'm starting to think that acetone/water and IPA/water may behave in similar ways, which could open up a path for selective washes/pulls by simply going from anhydrous to slightly wet solvents.

This may all be similar to the what happens with ethanol. Form some reason, Psilocybin does not like pure ethanol, but add a little water and voila! sudently it goes into solution as the many folks with alcohol tinctures will tell you. Maybe this has something to do with the dielectric constant of the liquid mix. This parameter is very important for protein solubility, and by extension maybe zwitterions in general too. Perhaps psylocybin is happy to go to into solution above a certain dielectric constant as long as some water is available and is not too picky about the solvent mixed with the water (?).

So perhaps after the methanol evap and naptha wash (plus a possible quick anhydrous acetone wash which I did not do), a very concentrated acetone pull (minimal but some water) is worth trying. I think I got some promising results this way after drying the wet acetone pull. If it works and repeats it is pure dumb luck on my side, I was trying to do a lazy acetone wash straight out of the can (without drying it) and evaporated the acetone out of curiosity because it was fluorescent under UV.

Essentially this is the water pull with acetone precipitation done up-front, starting with the final acetone/water mix. Playing with the temperature may also be interesting also.

Anyway, just another thing to try. Usual disclaimer is that this is work in progress and may not pan out. Good luck with your experiments. If all else fails we can disaolve the gunk in alcohol, add fumaric acid (about 1 to 5% of the dry mushroom weight depending on how good your cleans are), and dry that to a white fluffy powder :thumb_up:
 

Attachments

  • dielectric_chart%5B1%5D.pdf
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Thanks, I have downloaded the files.

I have enough materials to experiment.

I was actually once thinking about the other way around: precipitating psilocybin from aqueous extract by adding ethanol.

Finally I have recycled my methanol so I will proceed to extraction soon.
 
3A molecular sieves are widely available now available OTC. Something we can try with them is:

After acetone protein precipitation, instead of evaporating everything, add 3A molecular sieves. They can be in a mesh bag hanging from the top of the container. They will absorb the water, and Psilocybin may begin to crash out and/or form crystals (but maybe something else crashes out too depending on how clean the water extract is at this point). The resulting dry acetone could be reused by dumping the next cleaned up water extract.

I'll try this out eventually and report back.
 
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