Isolation of Natural Products by Ion-Exchange Methods

[Loveall]

I took 320mg of the powder from the previous post. By weight this is equivalent to 1.9g of dry mushrooms. I felt effects but not where near what one expects from 1.9g dry.

I think this is promising but needs some troubleshooting.

-Where the actives extracted during by the hot vinegar water? I think that yes based on personal multiple bioassays of mushroom tea.
-Did the psilocybin convert to psilocin in hot acidic water? I think yes as this is claimed in the literature
-Did the psilocin move to the Na+ resin? For H+ resin, this seemed to work (older post in this thread). Can repeat that test for Na+ resin.
-Did all the psilocin move out out of the resin during the basified ethanol extraction? Can try more pulls
-Was the psilocin damaged during the basified extraction? Could try less ammonia, colder extraction temp, and faster neutralization
-Did psilocin elute early during the rinses? No UV fluorescence was seen during the rinses, but I'm not sure if psilocin is strongly fluorescent or not.
-Did I have some tolerance at the time (I had bioassayed the golden liquid from the H+ resin the night before with some minor effects). I can wait for a week and try again.

Will need to go through these to try to figure it out. Any other thoughts? What could I be missing.

Thanks.

PS: Below is a a picture of the 320mg bioassayed powder dissolved in a small amount of water for ingestion under UV. It had a beautiful green fluorescence. Not sure if the fluorescence is from psilocin and/or tryptophan proteins. It was very tart at pH of 3, so I added a pinch of baking powder to pH6. The resulting drink was carbonated and delicious.

5/7/2019 Edit: Yep, something was missing. Oxydation from air during the dry step is a possible issue and could explain this result based on subsequent learnings.

 

[Loveall]

-Did the psilocin move to the Na+ resin? For H+ resin, this seemed to work (older post in this thread). Can repeat that test for Na+ resin.

This seems to be a contributing factor. The last extract pulled with Na+ cation resin became blue after a few days (see picture below).

This could make sense since Na+ has more affinity for the resin compared to H+. Therefore, it colluld be more difficult for psilocinH+ to be exchanged by Na+. However, I did not keep track of the mushroom/resin ratio so I can't say for sure.

The resulting powder from the Na+ resin pulls is weak but seemed active. It could simply be that more pulls or simply more resin is needed.

Next, will do tests using more Na+ resin while pulling to see if it can pass this blueing check.

Na+ would be the ideal cation resin to use since pH adjustments are straightforward. I think we need it if this ever becomes a practical mushroom extraction method.

 

[Loveall]

Alright, here is a second attempt using Na+ ion exchange resin.

Started with hot filtered vinegar tea (120g fresh mushrooms, 45m seep). This time I added a lot of Na+ exchange resin (280g) and mixed it all up well for a few minutes by shaking. Samples were taken before/after ion exchange for blueing (will post that later after they have time to oxydize).

Resin was recovered and rinsed with water. First rinses where muddy, last rinses were clear as seen in the two leftmost jars in the image below. These rinses will also be aged and checked for oxydation.

The resin was rinsed with 75% ethanol and the alcohol remained clear.

Next, the resin was pulled with 75% ethanol by adding some drops of ammonia (pH shot up to 10.6, I hope I did not overdo it; no blueing was observed which may be a godd sign) . The alcohol turned tawny under room lighting and fluorescent under UV, indicating that material was being pulled from the resin. The resin was pulled two more times with fresh ethanol (pH remained above 9 on the recovered alcohol). By the third and last pull the twanyness/fluorescence was barely noticeable. The alcohol pulls were combined and made acidic with 1/4 teaspoon of tartaric acid (pH ~ 4). The resulting acidified basic ethanol pull is the rightmost jar of the picture below.

Next the tawny alcohol will be dried (expecting crystalline result) and bioassyed. If potent, the powder will be cleaned up (dry acetone and maybe IPA/naphtha) and sent for analysis.

 

[benzyme]

:thumb_up:

I can appreciate your resilience, especially for an extraction as challenging as this.
I may have to try it sometime.

 

[Loveall]

Thanks benzyme, hopefully we end up with a practical extraction. This is the resin I got for Na+ exchange.

 

[benzyme]

yup, that's the stuff. Looks like Amberlite IR-120

 

[sbc1]

Excellent stuff loveall keep up the good work, look forward to more posts :thumb_up:

 

[Loveall]

Update on the latest test (post #23) using more Na+ resin (also more shaking):

- The water before the Na+ resin pull is changing color to a green-blue, while the water sampled after the ion exchange pull is not. This is a good sign indicating that Na+ resin can also absorb psilocin. If anyone is interested in how it looks I can take a picture later today.
- After the oven dry a flaky residue is obtained (image below for reference). This time I used less tartaric acid and also less fan time and more oven time. Some blueing was observed during the long oven dry (can't be good for final potency). Indeed, a bioassay showed little to no activity.

To summarize, it seems like the Na+ resin is capable of binding to the psilocin (and other stuff). However, the oven dry could be a place where potency is lost.

Will need to troubleshoot this. Mindlusion had some suggestions in the chat last night (thank you).

One thing that will be tried next is acetone crashes. If acetone is added to the alkaline ethanol pull, proteins are expected to crash put. After that, when adding tartaric acid more proteins should crash out in salt for (maybe also the psilocin salt). Will do these kind of tests when possible.

This is all still very much work in progress.

 

[Loveall]

Want to give an update and ask for help.

I'm pretty sure that cation exchange resins can catch and release psiloc(yb)in.

However, every time I evap to recover the drug, it becomes very weak. Seems like it is very sensitive to air and evaporation.

Any ideas on how to overcome this for the kitchen chemist? Looking for something anyone could do, so an inert atmosphere setup may be out.

How about using HPBCD? Would complexing our target molecule avoid damage from the air during final evaporation? If that works, we could also try the technique with Harmalol, another interesting molecule we tend to struggle with.

 

[sbc1]

Want to give an update and ask for help.

I'm pretty sure that cation exchange resins can catch and release psiloc(yb)in.

However, every time I evap to recover the drug, it becomes very weak. Seems like it is very sensitive to air and evaporation.

Any ideas on how to overcome this for the kitchen chemist? Looking for something anyone could do, so an inert atmosphere setup may be out.

How about using HPBCD? Would complexing our target molecule avoid damage from the air during final evaporation? If that works, we could also try the technique with Harmalol, another interesting molecule we tend to struggle with.

Will a vacuum chamber not work

 

[Loveall]

It could, but I really want to make this kitchen-level alchemy.

That is, order some cheap ion exchange resin, maybe some HPBCD and have a simple process we can all follow.

Maybe it's a pipedream, but we can try. I think we've made some good progress since I'm pretty sure the resin catches and releases the actives, just need to get the solution to evaporate safely and/or crash somehow.

 

[downwardsfromzero]

every time I evap to recover the drug, it becomes very weak

Are you sure? Have you tested the potency of the liquid before evaporation?

Anyhow, great work! It's so tantalising... my silly side wonders what would happen if one were to swallow some of the resin with the psilo.xx absorbed in it. Maybe best take that idea with a pinch of salt...

 

[Loveall]

every time I evap to recover the drug, it becomes very weak

Are you sure? Have you tested the potency of the liquid before evaporation?

Anyhow, great work! It's so tantalising... my silly side wonders what would happen if one were to swallow some of the resin with the psilo.xx absorbed in it. Maybe best take that idea with a pinch of salt...

Interesting ideas

Im pretty sure based on where the bluing reaction happens in the different liquids are left out for a while. However to be sure I should test the solution from the extracted resin before evap to confirm as you mention.

It's had a couple drops of ammonia before hence the reluctance, but that can be modified (use baking soda which would remain after evap as part of a a new salt).

 

[Loveall]

Here is an update.

280g of fresh mushrooms were extracted by seeping in plain hot water for 10 minutes with intermittent squeezimg with a potato masher. This tea was filtered.

To the filtered tea, half a cup of Na+ strong cation resin (pictured here) wass added. Tea and resin was shaken multiple times over a 30 minute period. Water was filtered out using a nylon mesh.

The cation resin was repeatedly washed with water until it ran clear (5 times).

75% everclear was added to to the resin. The everclear remained clear at this point. To this, baking soda was added until a pH of 9.1 was reached. At this point, the everclear became tawny under normal light and fluorescent under UV.

The twany everclear was filtered and to this 2g of HPBCD was added and stirred well in the hopes that it would complex with and protect any actives during subsequent steps. This is because in all previous testing, activity was mimal for all dried product with one possibility for this being damage from oxygen while drying.

At this point 250ml of tawny everclear with baking soda and HPBCD were obtained.

100ml were reserved for a later bioassay. This is why baking soda was used and not ammonia, so that a bioassay could be done without evaporating.

50ml were dryed to obtain 900mg of white powder containing HPBCD, baking powder, and mushroom material. This is reserved for later bioassay.

100ml of this were diluted up to 900ml using acetone and put in the freezer. Material began to precipitate out. After two days in the freezer, the solution was filtered and evaporated. The remaining material after evaporation was scraped up relatively easily to form a crystalline fluorescent powder. This has been reserved for bioassay. There are some visible tawny "hot spots" in the powder which will be mixed in before bioassay. Pictures after drying and after scraping in normal and UV light are below.

Next, bioassys will be done. They could all be negative - I'll report results as they come in.

 

[Loveall]

100ml of this were diluted up to 900ml using acetone and put in the freezer. Material began to precipitate out. After two days in the freezer, the solution was filtered and evaporated. The remaining material after evaporation was scraped up relatively easily to form a crystalline fluorescent powder. This has been reserved for bioassay.

561mg were recovered and 125mg were ingested after dilution in a couple fingers of water in a cup. Solution was very heavily fluorescent, but no significant effects noted. I simply fell asleep and got a great nights rest.

Will continue to bioassay the other parts of the cation resin extract and report back.

 

[Loveall]

50ml were dryed to obtain 900mg of white powder containing HPBCD, baking powder, and mushroom material. This is reserved for later bioassay.

350mg of this were consumed and minimal activity was noted. Could have been placebo.

Interestingly, the original liquid that was left over after the first cation pull was acidified with vinegar to pH3.7, freh cation resin added, and left in the closet for several days. This resin was then rinsed and extracted with baking soda in ever clear. A strongly tawny fluorescent liquid resulted. This liquid was bioassaed which had noticeable effect, but it was short lived. This could be from psilocybin being converted to psilocin in the closet which was then extracted with the cation resin and pulled with a weak base.

So were are we after all this. Here are some observations and possible explanations:

- Cation resin is absorbing tawny mushroom material at low pH and releasing it at higher pH
- Fluorescence tends to be greenish (see image below)
- Air evaporation could be damaging psilocin

This is all very interesting to me. I'll be doing more work and testing stuff out. Apparently, the actives fluores blue (?) so the green fluorescence could be due to tryptamine proteins coming along for the ride.

 

[Loveall]

After these tests, here is the next strategy I'm thinking of pursuing.

First a bulk test to confirm that we can use the Na+ cation resin to catch and relese psilocin as follows:
1) Loosely follow Casale's enzymatic psilocin extraction into dilute vinegar. Will likely use fresh mushrooms though (should not matter since this is an aqueous extract).
-Room temp dilute acedic acid extract pH~5.5 (psilocin and psilocybin)
-Heat cycle up to 70C to convert psilocybin to psilocin.
-Pull acidic water with Na+ cation resin
-Rinse resin with neutral water
-Pull resin with bicarbonate water (or alcohol) and neutralize with an organic acid

This should give a potent extract that is relatively clean. A lot of poteins would still be present though. At this point a bioassay would be done to confirm things are as expected.

To create a cleaner extreact:
2) To reduce protein content, add a protein removal step
- Cold acidic water extract (pH ~ 5.5)
- Cation resin protein removal. This will remove any psilocin present at this time also, but it should be a low amount. We are hoping that the dephosphorilating enzymes remain though.
- Heat cycle up to 70 C. If we did not lose the enzymes, psilocybin will convert to psilocin.
- Pull extract with cation resin. Only the new psilocin should be pulled.
- Rinse resin with neutral water
- Pull resin with a slightly basic solution and try to isolate and stabilize psilocin which should be pure. A vacuum evaporation may be needed at this point.

The choice of pH 5.5 is higher than Casale's (which was pH4). Hope is that it still works roughly the same. This pH is chosen as a guess since most proteins and enzymes could be near their isoelectric point (see post #5) and not interact with the resin. This improves the chances that the dephosphorilating enzymes are left in solution with strategy 2).

For reference, attached are a couple papers on the dilute acetic extraction and conversion to psilocin.

 

[sbc1]

Keep up the hard work loveall I appreciate it :thumb_up:

 

[Loveall]

Keep up the hard work loveall I appreciate it :thumb_up:

Thank you sir. I've actually been looking at vacuum chambers to dry the final product per your suggestion. Looks like it is doable to get or build one for a few bucks.

 

[sbc1]

Good can't wait for the results