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I tested my acidification theory today...


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#1 cosmicaug

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Posted 01 May 2007 - 11:59 PM

I actually tested my theory about dephosphorylation of psilocybin under acidic conditions today. This idea was prompted by a thread here on Mycotopia that I came across well before I was even a member here (I was very skeptical about the spontaneous dephosphorylation mentioned there as a possibility, no matter what the pH, until I realized that the fungal tissue itself is loaded with phosphatases which have activity peaks at different pH's). I used some trace amounts of P. cubensis in the form of a fine dust coating the inside of a small plastic bag as supplied by a FOAF for my assay (I rinsed it with 200-300 microliters of water and centrifuged some of that liquid to remove the mushroom tissue debris). Unfortunately I botched the assay because I did not realize that the end product which is monitored spectrophotometrically, p-nitrophenol, only absorbs heavily at 405 nm when under alkaline conditions (when it's deprotonated --duh!).
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I was doing an assay for alkaline phosphatase for a class project and, for a quick and dirty assay using a microplate reader, the NaOH "stop solution" (which would have brought the acid buffer to a reasonable pH for monitoring the p-nitrophenol) is not required since all it does is stop the reaction --and the pH when set up for assaying for alkaline phosphatase activity is already fine for spectrophotometric tracking of p-nitrophenol (for a more careful assay it is needed to ensure that all the samples have had approximately the same reaction time). However, this does not apply when the reaction solution's pH is low to begin with.
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Needless to say, the signal I got from the acid phosphatase well was small (and, indeed, could have been wholly due to developing psilocin oxidation products which are, as you all know, strongly colored --though I don't know if they absorb significantly at 405 nm since I have never taken a spectrum of such a solution).
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I wish I could say that my official class project turned out better than that but it didn't. I suppose that I could still give the acid phosphatase assay another go if I feel ambitious enough since I did not yet throw away all my samples.

#2 Guest_Mr. Natural #1_*

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Posted 02 May 2007 - 04:17 AM

What is your theory. What was the result.


#3 cosmicaug

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Posted 02 May 2007 - 07:11 AM

What is your theory. What was the result.

Theory was that acid phosphatases in the dried mushroom catalyze a conversion form psilocybin to psilocin when it they are left standing in an acidic solution. There were no meaningful results (beyond phosphatase activity at a high pH which gets greater at a neutral pH) because I screwed up the assay.

#4 cosmicaug

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Posted 02 May 2007 - 08:27 PM

What is your theory. What was the result.

I have done it again. I still didn't do it quite right because the acid phosphatase activity was so strong it went off scale. But as this is what I was trying to test, I consider this a success. I guess it's nothing that Paul Stamets doesn't already know since he has a patent on using this acid phosphatase activity but his information deals with live fungi whereas I just wanted to know if the activity was still there in dry tissue (one would not have to imply the other).
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I wanted to answer two questions:
  • Does dried P. cubensis tissue exhibit phosphatase activity?
  • If the above is true, does said activity display a maximum at a low pH?
The answer to both questions is yes. It's so nice to know for sure that I am not bullshitting about this.
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The way the assay works is that p-nitrophenyl phosphate is used in the assay as a substrate for the enzyme. The phosphatase hydrolyzes the phosphate group and you en up with p-nitrophenol (plus free phosphate).
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Posted Image<br>
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The former is colorless and the latter is quite yellow. As a result, if you start out with a vast excess of p-nitrophenyl phosphate, you can measure the yellow to find out how much has been converted to p-nitrophenol by phosphatase activity there is in a sample.
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Where I went wrong before is that p-nitrophenol is only yellow under alkaline conditions. One needs to stop the reaction by using a strong base when testing for acid phosphatase activity but not when testing for alkaline phosphatase activity.
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The alkaline phosphatase assay was done in a 0.1 M glycine buffer with 1 mM MgCl2 and 1 mM ZnCl2 (0.5 mM concentrations in the final reaction mixture) that was pH adjusted to 10.4. The reason for the ZnCl2 is because the zinc and magnesium cations are required for activity alkaline phosphatases.
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I prepared two additional buffers (both with 1 mM MgCl2 and 1 mM ZnCl2): a 0.1 M Tris buffer prepared from a 1 M stock had been pH adjusted to 7.6 (which should have retained a pH of about 7.6 --though I did not bother to actually measure it) and a 0.1 citrate buffer which was pH adjusted to 3.06. The tris buffer was (to see activity at a near neutral pH) for the hell of it and the citrate buffer was to assay for acid phosphatase activity (which should replicate acidified mushrooms --a pH of around 3 or even lower is not unreasonable).
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I was doing this with a batch of legitimate school work. I was looking at one sample with very weak activity and wating a long time for it to consume the substrate. As a result even with the dilutions I did on the mushroom trace sample for the acidic test I let the reaction go too long (since I had to process everything at once).
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I did get tired of waiting and ran a separate set of assays on only the mushroom traces which used the normal 10 minute incubation period. In this quick attempt I did not bother to do additional dilutions. As a result there was also a huge activity mismatch between acid test and both the alkaline test and the near neutral test.
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In the ten minute test each sample was appropriately blanked with the appropriate buffer (basic, neutral and acidic). With the 110 minute test, I did not bother and I blanked everything with the alkaline buffer. In any case, in the fungal samples, absorbance effects from the buffer are probably minor when compared to interference from blue-green psilocin oxidation products.
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The measurements where all done at 405 nm. Here is my actual data for the 10 minute assay:
<code>
Assay                 Dilution         Absorbance
-----------------     -----------      ---------------
Glycine (pH 10.4)     1:1              0.0687
Tris (pH 7.6)         1:1              0.1511
Citrate (pH 3.06)     1:1              2.8065
</code>
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The last reading is actually out of range and could just as easily indicate 15 times more activity at low pH than at near neutral pH as it could 9000 times (your guess is as good as mine). Nevertheless it is quite clear that the phosphatase activity is much stronger at the lower pH.
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Here is my actual data for the 110 minute assay (I did not bother reading the undiluted fungal samples since they were obviously out of range):
<code>
Assay                 Dilution         Absorbance
-----------------     -----------      ---------------
Glycine (pH 10.4)     1:4              0.0300
Glycine (pH 10.4)     1:16             0.0070
Tris (pH 7.6)         1:4              0.1876
Tris (pH 7.6)         1:16             0.0437
Citrate (pH 3.06)     1:16             2.7827
</code>
Again, the same pattern is seen.
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Actually trying to derive a back of the envelope estimate of the actual conversion rate from psilocybin to psilocin with a similar assay (it would have to involve a carefully weighed sample rather than just traces of mushroom dust) would probably not be that difficult but I'll have to leave that for someone else.
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I am guessing that confirmation of whether soaking in an acidic solution achieves significant psilocybin to psilocin conversion could probably be done with something like thin layer chromatography or with HPLC by comparing he relative sizes of the different alkaloid spots or peaks.
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The next question would be what happens when one consumes a psilocin enriched mixture as compared to the same amount of psilocybin.

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Posted 03 May 2007 - 12:59 AM

Do you know of any way that amateur mycologist could devise a test to accurately measure the psilocybin/psilocin content of a sample. Maybe something better than dropping cocaine into bleach but does not require a lab and an advanced degree.
Keep up the good work.

#6 cosmicaug

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Posted 03 May 2007 - 09:07 PM

Do you know of any way that amateur mycologist could devise a test to accurately measure the psilocybin/psilocin content of a sample. Maybe something better than dropping cocaine into bleach but does not require a lab and an advanced degree.
Keep up the good work.

No, I do not. However, I am no expert.<br>
I will suggest, however, that such a test is probably simply going to indiscriminately look for alkaloids and one who has a known Psilocybian fungus sample would have to simply assume that all the alkaloids in the sample are psilocybin or psilocin. The assumption is going to be close enough to being true if you do know what your sample is (assuming that you are not dealing with identification issues) and that the lack of specificity will not matter.<br>
The real problem that one is going to have is with the quantitation. Even the test I used above requires a decent scale (to weigh the p-nitrophenyl phosphate --though maybe one could just guess since what is important here is that it is in excess), chemicals (specifically the citrate, the p-nitrophenyl and the NaOH used to make the buffer and for the "stop solution" --maybe not that hard to find but the closest to a household chemical in that list is the NaOH but it might not be in a form suitable for use as a reagent), a spectrophotometer (you might be able to get an old clunky one from Ebay for a few hundred dollars that would be more than suitable if you can get it to work but it certainly is not an item that most of us have --hell, I don't even have a toaster, but I digress) plus a means to accurately measure small amounts of liquids such as a micropipetter (though perhaps one might be able to either improvise or procure something like this or change the procedure to not require this).

#7 cosmicaug

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Posted 03 May 2007 - 09:50 PM

Do you know of any way that amateur mycologist could devise a test to accurately measure the psilocybin/psilocin content of a sample. Maybe something better than dropping cocaine into bleach but does not require a lab and an advanced degree.
Keep up the good work.

On the other hand, I remembered running into a possibly relevant thread on this very forum so I looked it up. A fellow who goes by ClearLight posted something which could be of interest. See "How Much You Got? Quantitative TLC Analysis [GC/MS Alternative]"

P.S. I forgot to mention the problem which I see with that. Accuracy might not be great but it might be good enough. The real problem I see with it is that you need a standard and psilocybin and psilocin standards might be a bit hard to come by. Such a description could be relevant to trying to carry out something like what I mentioned in the next to last sentence of post #4.

#8 Hippie3

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Posted 03 May 2007 - 10:16 PM

pretty technical
but if you were to
boil it down into distilled wisdom
is the practical implication
that we should favor teas, brews, etc.
which have an acidic ph ?

#9 cosmicaug

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Posted 16 May 2007 - 10:28 PM

pretty technical
but if you were to
boil it down into distilled wisdom
is the practical implication
that we should favor teas, brews, etc.
which have an acidic ph ?

Distilled wisdom would be that if you want increased psilocin content from conversion of psilocybin, grinding it up and mixing it into black currant juice or any other liquid with a very low pH (lemon juice, orange juice, probably even lemonade) and letting it sit there for a while would probably do the job.

#10 oldiebutnewbie

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Posted 17 May 2007 - 11:11 PM

Distilled wisdom would be that if you want increased psilocin content from conversion of psilocybin, grinding it up and mixing it into black currant juice or any other liquid with a very low pH (lemon juice, orange juice, probably even lemonade) and letting it sit there for a while would probably do the job.

------------------------------

A lot of folks do make smoothies which often incorporate acid fruits like OJ and pineapple. For those of us who prefer the flavor of simply sucking and chewing them whole or cooking with them, we also have acid in our stomachs.

OBN

#11 cosmicaug

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Posted 20 May 2007 - 01:01 AM

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A lot of folks do make smoothies which often incorporate acid fruits like OJ and pineapple. For those of us who prefer the flavor of simply sucking and chewing them whole or cooking with them, we also have acid in our stomachs.
OBN

This is true, but you also have proteases (pepsin, specifically) which may prevent the fungal acid phosphatases from doing their job (by breking them down) which would leave the dephosphorylation to occur sometime post-absorption (absorpton which might be hindered by the big honkin' negatively charged phosphate group of psilocybin).

#12 TVCasualty

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Posted 20 May 2007 - 10:33 AM

Last weekend I witnessed a bioassay of a lemon-juice experiment. I was talking to a friend about how I'd put some powdered cubensis into a shot glass and covered it w/ some fresh squeezed lemon juice, let it soak for 20-30 minutes, and got off great on only a third of a gram.

So he went out and got a bottle of lemon juice, powdered a handful of mushrooms (unmeasured), and soaked them for 20 minutes. He then split it 4 ways with some friends (I was in another room, unaware of all this). About 10 minutes later he comes up to me with his eyes open real wide saying "you said it took an hour before you felt anything, but I'm already fucked up!" And so were the others (for me, it did take an hour, but then I didn't eat a quarter of a handful). It came on very hard and dropped off suddenly after a few hours, bringing the total number of people I know who report a qualitatively different result from a lemon juice soak to 8. It also showed me that having fresh-squeezed juice isn't required, but I suspect doing it at room temperature is.

Very interesting thread, btw! We need as many people out there as possible slipping these experiments into their routine lab work! :reb:

For those of us who prefer the flavor of simply sucking and chewing them whole or cooking with them, we also have acid in our stomachs.


This is something different. Pre-soaking outside your stomach allows the relevant reaction to occur in vitro, which means that by the time it hits your stomach it's already finished the acid reaction. Eating them whole begins the reaction only when they reach your stomach, so it plays out more gradually and the actives enter your bloodstream in more of a time-release manner. One of the startling results of the soaking is how everyone went from baseline to tripping so quickly, with very little sense of the "climb," and no one so far has reported any nausea. I'm not sure if the body's active transport mechanism for vitamin C has a role in the qualitative changes, but it may (which means lowering the pH with no vitamin C present would not produce the same results when we drink it, though the chemistry may be identical).

Also, there's the baeocystin (4-OP-MMT) content to consider, which I have read may be an intermediary molecular step between psilocin and psilocybin, but certainly is psychoactive and could be a factor in these conventional and bio assay results. By itself, baeocystin is present in relatively small amounts, but otherwise not much is known about it.

I highly recommend that everyone bio-assay this recipe... that is, mix it up and take a shot! Ultimately, it's about what happens in our minds and bodies, not what happens in the beaker, that's important (just ask Dr. "Here, try this" Shulgin).

#13 cosmicaug

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Posted 20 May 2007 - 03:11 PM

Last weekend I witnessed a bioassay of a lemon-juice experiment. I was talking to a friend about how I'd put some powdered cubensis into a shot glass and covered it w/ some fresh squeezed lemon juice, let it soak for 20-30 minutes, and got off great on only a third of a gram.
So he went out and got a bottle of lemon juice, powdered a handful of mushrooms (unmeasured), and soaked them for 20 minutes. He then split it 4 ways with some friends (I was in another room, unaware of all this). About 10 minutes later he comes up to me with his eyes open real wide saying "you said it took an hour before you felt anything, but I'm already fucked up!" And so were the others (for me, it did take an hour, but then I didn't eat a quarter of a handful). It came on very hard and dropped off suddenly after a few hours, bringing the total number of people I know who report a qualitatively different result from a lemon juice soak to 8. It also showed me that having fresh-squeezed juice isn't required, but I suspect doing it at room temperature is.

Room temperature (as opposed to refrigerator temperature) should be important if what I think is happening is actually happening (the assays were carried out at 37 degrees Celsius, by the way --though, other than presumably producing a faster reaction rate, the results should still be applicable to room temperature).

FOAF tried the bioassay twice. The first time is probably not meaningful because the dose used was 7 grams (the largest dose which FOAF has ever consumed). The second time involved a 3 gram dose which, though relatively large, is not out of line with what FOAF has consumed on other occasions. Both experiences involved rapid onset (under 20 minutes --maybe less --versus 50 minutes to an hour when simply consuming dried or fresh mushrooms as they are). Both experiences were incredibly intense (perhaps they should be documented here at the appropriate subforum).

Very interesting thread, btw! We need as many people out there as possible slipping these experiments into their routine lab work! :reb:
This is something different. Pre-soaking outside your stomach allows the relevant reaction to occur in vitro, which means that by the time it hits your stomach it's already finished the acid reaction. Eating them whole begins the reaction only when they reach your stomach, so it plays out more gradually and the actives enter your bloodstream in more of a time-release manner.

That, and the fact that acid phosphatases might be degraded in the stomach (not that I know that to be the case). Erowid seems to think that dephosphorylation is catalyzed by alkaline phosphatases (which means in the small intestine). My thinking is that total amount of psilocin that makes the way into circulation might be less important than how hast it gets there (and, if the psilocin is already there to begin with, rather than being produced in the small intestine, I suspect that absorption would even happen as soon as the mixture reaches the stomach --in contrast to psilocybin which probably can only be absobed minimally, if at all, due to the charged phosphate group).

One of the startling results of the soaking is how everyone went from baseline to tripping so quickly, with very little sense of the "climb," and no one so far has reported any nausea.

FOAF reported very intense tripping on 3 grams after under 20 minutes. FOAF expected, maybe, a gradual climb (a feeling of "I think that I might be tripping..." as informed by subtle perceptual details which gradually grows to "I'm certain that I am tripping" when everything starts looking very different) but instead got a full-on trip at onset (that doesn't mean that there wasn't a climb --it may have merely been so brief that it went unnoticed).

I'm not sure if the body's active transport mechanism for vitamin C has a role in the qualitative changes, but it may (which means lowering the pH with no vitamin C present would not produce the same results when we drink it, though the chemistry may be identical).

FOAF's first bioassay involved a lemon concentrate (ReaLemmon brand or equivalent) which should have contained plenty of vitamin C. FOAF's second bioassay involved a fancy vinegar (because that's what FOAF had on hand) which should not have contained significant quantities of vitamin C.

Also, there's the baeocystin (4-OP-MMT) content to consider, which I have read may be an intermediary molecular step between psilocin and psilocybin, but certainly is psychoactive and could be a factor in these conventional and bio assay results. By itself, baeocystin is present in relatively small amounts, but otherwise not much is known about it.

I would not think that baeocystin should matter too much. One reason is simply because, as you point out, the amounts normally found in P. cubensis are fairly insignificant. In any case, if what is going on is pre-consumption dephosphorylation, the mechanism by which it might make a difference would be the same as with the psilocybin to psilocin conversion only in this case the acid phosphatase activity should turn baeocystin into 4-hydroxy-N-methyltryptamine (see the previous Erowid link --or just figure out what happens when you lose the phosphate group).

I highly recommend that everyone bio-assay this recipe... that is, mix it up and take a shot! Ultimately, it's about what happens in our minds and bodies, not what happens in the beaker, that's important (just ask Dr. "Here, try this" Shulgin).

I would think that if what I think is happening is actually what is happening, the experience should more closely resemble consumption of Panaeolus cyanescens, Psilocybe azurescens and Psilocybe cyanescens since these species are known for much having more significant psilocin content than what Psilocybe cubensis has.

#14 cosmicaug

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Posted 20 May 2007 - 04:11 PM

Actually, psilocybin is absorbed according to a paper I have. I'm still probably right regarding relative absorption rates.

P.S. Actually the paper wasn't too clear about that. It cites a couple of papers that claim to find psilocybin in plasma and one which claims complete conversion before entering systemic circulation (in rodent tissue) and makes the point that there is a "lack of reliable analytical methods for the determination of psilocybin in human plasma,"

#15 eatyualive

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Posted 08 July 2008 - 08:44 AM

excellent info. completely missed this thread.




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