extraction focusing on denaturing undesirable enzymes

[retack]

hi all. there was a comprehensive report about extractions with pure methanol, acetic acid, and aqueous ethanol/methanol that proved that when you have anything except pure methanol and dried shrooms, phosphatase starts converting psilocybin into psilocin, which is undesired for long-term storage. this showed that previous reports, showing large quantities of psilocin in mushroom strains, were in fact just seeing psilocin that was never in the mushrooms, just created from psilocybin during extraction.

my idea is this - the phosphatase in question is an enzyme, which should be able to be denatured and rendered permanently inactive by exposure to high temperatures, still well below any temperature that psilocybin (what we're after for long term storage) degrades.

so, you boil water for some time to deoxygenate it. you keep heat applied, and add either dried-pulverized OR fresh-finely-minced mushrooms, with the goal of raising the temperature of the shrooms from ~70 degrees to ~200 degrees as fast as possible. you should end up with a pot of water, active chemicals, mushroom gunk, destroyed enzymes (the critical part), and little-to-no dissolved oxygen.

with this done, you should be free to perform a high-temperature (and thus FAST) extraction by keeping the water hot, or adding ethanol when temperature is low enough that it won't boil off. strain the resulting liquid, evaporate, and you should be left with albeit impure, but still very concentrated psilocybin, mushroom gunk and NOTHING that will be able to degrade our psilocybin thereafter.

the same report that mentioned pure methanol extractions stated that at 45 degrees celcius, ALL active chemicals were extracted in 1 hour. room temperature took 12 hours. high-temperature extraction has its advantages!

possible issues i can see:
- from the time the mushrooms are added to the water until the time the enzymes are denatured, they'll be free (and encouraged by the heat) to dephosphorylate and destroy psilocybin. this can be solved by having the temperature change as fast as possible, by using a large quantity of extremely hot water. disadvantage is that you have a lot of water that will later need to be evaporated. i don't know of any way other than trial and error and extremely precise measurements to see 1) exactly how fast psilocybin can be destroyed by the enzymes in question, 2) at what temperature range they are able to function, and 3) at what temperature they're denatured and completely eliminated
- the enzymes somehow 'renature' themselves when temperature is lowered. this seems extremely unlikely, but would completely prevent this method from being feasible
- the psilocybin degrades at high temperatures in water/ethanol. this does NOT seem to have been proven in anything i've read, psilocybin is stable at MUCH higher temperatures than boiling water, and if it's been thoroughly deoxygenated, as it would have been, there should be nothing left to affect it
- the psilocybin is degraded by some other chemical (acid, base, who knows) in the mushroom that is mixed with the water. i know nothing about this possibility. would require fixing ph of water.

any thoughts? yes i know i know, just do standard extractions, just eat the shrooms, just keep the dried shrooms, i know all these options. i'm looking at this from a chemistry perspective, are there any issues i've overlooked here? why wouldn't this work, and be nice and fast at the same time?

i mean, fresh shrooms, thrown in a blender, tossed in a pot of boiling water, ready to strain/evaporate into a stable extract 30 minutes or an hour after being picked. that has some time-saving potential!

[retack]

someone noted that instead of blending fresh mushrooms at room temperature, they could be flash-frozen with some nice cheap dry ice, further reducing the amount of possibly-damaging time around enzymes AND breaking apart cell walls.

could be overkill, considering the time between blending and water immersion is ~10 seconds, but only a lab could do a meaningful test.

[retack]

with even more daydreaming, the final, probably fatally flawed, quick extraction idea:

1) start a pot of distilled water + ascorbic acid rapidly boiling
2) pulverize dried shrooms, or blend up fresh shrooms. add to boiling water asap
3) let simmer with lid on for 20 mins, start a 2nd pot of acidic boiling water
4) filter 1st pot of shroom water (hot!) into desired container
5) add gunk caught in filter to 2nd pot of boiling water for re-extraction
6) simmer 20 mins, filter again into container. discard leftovers, or consume if desired
7a) optional: add some naphtha at this point, shake vigorously, let separate and siphon off to defat. repeat if desired
7b) optional: add a capsule or 2 of bht. this powerful, cheap, safe preservative may even allow some psilocin to stay preserved in the final extraction. must be done after naphtha wash
8) evaporate using preferred method. large flat pan + fan, vacuum, desiccants, etc.

if naphtha wash was used, extract should be 'reasonably' pure. if bht was added, it should be quite stable even if exposed to air. for best storage, put extract in desired sealable container, rest container in a bowl with some vinegar, add baking soda to vinegar to fill bowl with co2, place lid on container. voila.

problem: i can't find any reference anywhere that says conclusively whether psilocybin or psilocin will dissolve in naphtha.
problem: still not sure how hot phosphatase must get before completely destroyed. perhaps edta should be added to water to eliminate enzyme

[limeade]

I haven't met one person that's' tried this with successfully obtaining psilo in a storable salt/crystallin form.

[relic]

Unfortunately the naptha won't work b/c it will absorb some of the psilocybin as I understand it. This has been the bane of all who have wanted to extract psilocybin. The standard a-b extraction doesn't work. It is neither polar nor non-polar. I think lye destroys it? And the only way I have read to purify the extract is through chromatography. It seems to me that the best way is to use methanol. This eliminates the pesky enzymes and gives you a stable extract. If only you could figure out a way to pull the gunk out of this extract without chromatography or massive loss. What I would like afoaf to try is:

1. Extraction with 55C Methanol
2. Filter through fine sieve
3. Filter through fine filter paper w/ vacuum
4. *maybe*Filter extract while still hot through carbon slowly in a column(carbon prewashed with clean methanol to remove any dust or maybe rinsed w/ distilled water and baked would be better)
5. Evaporate methanol with fan
6. Evaporate residue/product under vacuum
7. Attempt recrystalization with minimum nessecary amounts to get the extract to completely dissolve of either: boiling methanol, boiling distilled water, or boiling dried ethanol(probably in that order but with different samples, not progressively on a single sample)
8. Filter through fine paper hot under vacuum
9. Let cool slowly
10. Once cooled to room temps and crystallized, lower temp to 33~F to obtain maximum yeild
11. Filter off crystals, if any, and dry them under vacuum.
12. Repeat from #5

[retack]

i can't find any figures, but apparently acetone makes a TERRIBLE extraction of mushroom actives, this is either because it cannot dissolve them at all, or it rapidly degrades them to uselessness. a 'respectable' older paper mentions acetone as a 3-stage washing agent, and still followed up with impressive yields of actives. it might be the missing ingredient!

i wouldn't expect the result to be crystallized, but the purer it can end up while still retaining stability the better. i've never been a big fan of gunk, and mushrooms inspire that feeling more than anything else. the little gunk that'd be left would probably help longevity more than hinder it.

[relic]

I realize that the recrystallation is probably the weak link here. The problem is that we need a solvent that the psilocybin is barely soluble in. The only info I can find everywhere says the same thing, it is 'soluble in water, partially soluble in methanol and ethanol, and insoluble in most organic solvents'. Of course this is somewhat of a contradiction b/c ethanol, methanol, and acetic acid are all organic solvents. But that does leave many others. We know that hexane and chloroform can be used for this as well as ethyl ether. But getting those is sketchy and they seem even more dangerous than most of this shit already is. Xylene is a possibility but experimentation will have to be done. It seems for now that since psilocybin is only slightly soluble in ethanol that it should be at the top of the list for recrystalization. I would think you would want to do your best to dry it as much as posible with drierite or the like and keep it sealed up or in a vacuum as much as possible as water may prevent some crystalization b/c it will absorb some of the alkaloids.

Yes, I have read some mixed results with acetone as well. What foaf would be trying to do above is purify by filtering and mainly recrystalization. Also using the methanol would yield a better starting point, the initial extract, junk and all would be extremely stable or so it seems. Everything removed after that would not change that. You could throw another carbon run in there after the residue is redisolved in the dry ethanol or other solvent, but it would have to be done hot and fairly quickly. I think we see alot of talk but few go through the trouble of actually experimenting to find the solutions and dead ends.

[retack]

i just read yet another thing i hadn't seen before (funny how that always seems to happen around here )

apparently while researching synthesis of psilocybin and psilocin, it was found that the fumarate salt of psilocin was much more stable than psilocin itself, to the point where psilocybin no longer had any advantage over it. fumaric acid is cheap and widely available, and sounds like another perfect addition!

so boil and strain, evaporate, wash gunk with acetone, re-dissolve in anything, add bht and fumaric acid, evaporate again. success!

edit: yes crystals would be theoretically ideal, but also seem extremely troublesome and even lossy. an amateur tek such as this is going for a balance between speed, convenience, yield and cost, with purity taking an unfortunate back seat. a big part of the reason for skipping methanol is purely that - cost and difficulty of handling. re-capturing the evaporated fumes would be possible but troublesome for the intended audience. i certainly wouldn't want to deal with hot methanol in my kitchen, or even bathroom. another benefit to skipping crystallization and ending up with some level of gunk is ease of dosage - the gunk could easily be cut with a scalpel into small blocks which would retain their shape. how convenient!

and of course this is all talk until some numbers can be actually reported by a foaf. i will have to work to convince one it's worthwhile, we shall see in a month or so.

[relic]

That is an interesting find! What is the foaf going to use to do the extraction, water? I'm sorry for hijacking this thread but afoaf is stuck on methanol. And at no point would you need to boil it if you use something else for the recrystalization. And it is readily available. Of course there is the same risk in boiling ethanol duriong the recrystalization. But crystals are the goal in foaf's mind. You could work with the extract and water. All you need to ensure safety is pure methanol and a vacuum filtering and drying set-up. And as far as that go's you could fan evaporate it outside or under an exhaust fan and then bake the extract at a low temp like 200F to get all of the methanol out. Both readily available and fairly cheap. And acetone isn't exactly o natural and those are some intense fumes but it does evaporate really fast. Existing extractions can give you a fairly stable product but it must be stored properly and consumption of alcohol is generally required.

If the fumeric thing pans out AND the psilocin isn't destroyed in the extraction it could be drawn out after recrystalization in ethanol b/c it is completely soluble in ethanol and will not crystalize out with the psilocybin.

[retack]

well the only way we'll get crystals of desired purity will be with a reliable defatting agent, and acetone is definitely not that one would need to use chloroform, probably with 2 washes, and availability is definitely a problem there. i've not read any method for obtaining pure crystals (apart from unreliable forum posts) that doesn't rely on chloroform.

once you have performed an extraction of the initial shrooms with anything (methanol or water), all you need to do is wash with both acetone and chloroform and you're set. there should be nothing left whatsoever other than pure alkaloids, some inactive but mostly actives, depending on the makeup of the mushroom itself. you can isolate pure psilocin/cybin only with more advanced methods (chromatography, etc.)

[relic]

Good points. One thing to keep in mind though is that all reading that involves getting pure crystals using chloroform and hexane or diethyl ether to clean the extract with is written by chemists who readily have access to these chems. It doesn't mean it cannot be done another way. obviously we will not be able to get a pure isolated alkaloid w/o chromotography. But if we can isolate the alkaloids from the enzymes and all the crap we will be alot closer. If we can find an organic solvent that is readily available that the psilly is completely insoluble in we're almost there.

[retack]

and still more new developments

apparently urea will do a great job of denaturing enzymes, not much is needed, and it'll wash away cleanly with acetone. not to mention 1lb costs $5 or less, and only a few grams are needed per kg of fresh mushrooms. great extra safety step. so that'd make the finalized procedure (with costs):

- get 3 empty pots, some urea (1lb = $5, 5g per 1kg shrooms), bht capsules (100 capsules = $6, 1 capsule per 1kg shrooms), fumaric acid powder (10g = $3, 0.1g per 1kg shrooms), acetone (1 liter = $5, 50ml wash per 1kg shrooms)

- get 2 pots, put 1 liter of water in each, boil, add 5g urea to pot #1
- blend up fresh shrooms, add to pot #1 keeping a boil
- reduce heat, stir, after 30 mins filter into empty pot #3
- re-extract shroom gunk in pot #2 for 30 more mins, filter & combine with pot #3
- discard shroom gunk, or save/eat it if desired, should be fairly inactive now
- evaporate pot #3 with fan, heat, fan + heat, whatever. no color change should be observed now, our actives shouldn't have anything to react with
- once evaporated, wash with acetone, discard acetone. should remove all urea, most of the mushroom taste
- remaining shroom gunk doesn't need to be dried from acetone, just add enough water to re-dissolve all solids, add fumaric acid and bht, evaporate down to solid again

per 1kg of mushrooms we should aim for 1g of actives, about 4g final extracted product, doses are 125g each, so about 30 doses. cost of inputs (water, shrooms and electricity/gas ignored) are $0.39 per 1kg shrooms, or a bit over $0.01 per dose. not bad.

procedure here is just for reference, though afoaf may be trying it in 1 month's time, so a relevant update can be posted.

[Buzztea]

..ok..


someone tried that stuff...

some questions about the washing process..

how does acetone washing work, I imagine something like that

the acetone and water extract would be mixed with the unwanted still inside

mixed, water and acetone (now with the unwanted inside) left alone in fridge until the acetone and water seperate,

then seperate the two liquids..

boil water to evaporte rest of acetone, evaporate down the result..


is that right





or how else should I imagine the acetone wash in your last step

[Psilo-somatic]

Na sorry man; water and acetone are completely miscible. It would have to be dry, then you throw on the acetone, run it through a filter and discard the acetone.

I'm curious as well if anyone has tried the fumaric acid step. SWIM doesnt have alot to play around with and doesnt want to potentially have something screw up and waste half his shit.
Lemme know! im excited

edit: Something that has not been mentioned yet is that acetone, methanol, etc are rarely anhydrous unless you're getting regeant grade chems. This is solved by drying epsom salts in the oven into dry magnesium sulfate and throwing that in your solvent container. Allow to settle and pipette off the top when needed.

[Buzztea]

ok I was afraid..


so we could do the acetone wash prior to the other extractions (apply and other preservative before)

wash the dried shrooms with acetone...dry..and loose a lot of gunk that way

[Soliver]

Did anyone ever try this? Seems to have floated away into the ether...



soliver