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OfflineMcJosh13
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Preserving psilocybin in extractions by deactivating degradation enzymes
    #5925358 - 08/02/06 12:56 PM (17 years, 9 months ago)

The extraction of psilocybin/psilocin with methanol is considered superior to using aqueous solutions of ethanol (which is really the only type of ethanol readily available ,everclear, 151.) The main reason for this seems to be because the enzyme responsible for the breakdown of psilocybin to psilocin is soluble in aqueous ethanol and not methanol. This is obviously undesirable since psilocybin is long-lasting and resilient where as psilocin is easily broken down.

Instead of concentrating on using toxic methanol why not take a look at ways to deactivate this undesirable enzyme? Apparently the enzyme responsible for this, alkaline phosphatase, is pretty heat tolerant and is not denatured easily by mild heating, say like in boiling ethanol. In my on-line studies I have found that alkaline phosphatase is a dimeric metalloenzyme, which means that it has an active site containing a tight cluster of two metal zinc ions and one magnesium ion. It cannont function with out these.

It seems that this enzyme can be deactivated, at least to a significant degree, by chelaters such as edta or cysteine which are both readily available as dietary supplements and are water soluble. These chelaters steal the zinc and magnesium ions from the enzyme thus deactivating it. With this enzyme deactivated psilocybin will be preserved and the extract would presumably be alot longer lasting since it won't be continually broken down into psilocin and then oxidized. Thoughts on this and other possible methods of deactivation are welcome.

Edited by McJosh13 (08/02/06 01:11 PM)

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Invisiblefastfred
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Re: Preserving psilocybin in extractions by deactivating degradation enzymes [Re: McJosh13]
    #5925495 - 08/02/06 01:54 PM (17 years, 9 months ago)

Good post, interesting topic and ideas. I'll start you off with a 5 shroom rating for it.

I've not considered this in any detail. Many enzymes are affected by pH, so that might be an option. Most chelating agents only work on free ions, I think. I don't think they would work that effectively on ions bound to enzymes, but maybe I'm not too sure about that.

Alkaline solutions destroy psilocin fairly easily, so lowering the pH might work. Theoretically, in my view, acidic solutions would dephosphorylate psilocybin though, so it might be tough to work that out.

I actually recently attended a lecture on the genetics of zinc metabolism, so I'll try to ask an expert on this what he thinks.

Some sort of protease might work well, but I have no idea if there is any easy source.

"Proteases (proteinases, peptidases or proteolytic enzymes) are enzymes that break peptide bonds between amino acids of proteins. The process is called proteolytic cleavage, a common mechanism of activation or inactivation of enzymes especially involved in blood coagulation or digestion. They use a molecule of water for this and are thus classified as hydrolases."


-FF

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OfflineMcJosh13
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Re: Preserving psilocybin in extractions by deactivating degradation enzymes [Re: fastfred]
    #5925565 - 08/02/06 02:14 PM (17 years, 9 months ago)

I will have to do some research on the action of proteases. There actually are studies on pubmed and other places around the web demonstrating the ability of both EDTA and L Cysteine to deactivate alkaline phosphatase to a significant degree. When you get some time do a search on alkaline phosphatase + EDTA, or Cysteine and I think you will see what I mean. It definatley looks promising to me, and as a plus the ingredients are cheap, I just found some food grade EDTA disodium salt on EBAY for $8.00. The disodium salt is perferable to the calcium salt they sell in many supplements I believe.

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Invisiblefastfred
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Re: Preserving psilocybin in extractions by deactivating degradation enzymes [Re: McJosh13]
    #5925817 - 08/02/06 03:33 PM (17 years, 9 months ago)

Good info. I'll have to look into that.

That might be able to really increase the potency of extracts!


-FF

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OfflineMcJosh13
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Re: Preserving psilocybin in extractions by deactivating degradation enzymes [Re: fastfred]
    #5925991 - 08/02/06 04:34 PM (17 years, 9 months ago)

Here are a few abstracts I stumbled on...they involve alkaline phosphatase obtained from diffrent animals but I am confident that the same theory applies to alkaline phosphatase obtained from from mushrooms as it is the same enzyme with minimal diffrences. There are other studies similar to these using alkaline phosphatase from all kinds of diffrent sources.

http://www.ncbi.nlm.nih.gov/entrez/query...t_uids=12945337

[Kinetics of inactivation of calf intestine alkaline phosphatase by EDTA with absorption spectrum method]

Calf intestinal alkaline phosphatase (EC.3.1.3.1) is a dimeric metalloenzyme composed of two identical subunits, the each active site of which contains a tight cluster of two zinc ions and one magnesium ion. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou has been applied for a study on the kinetics of the course of inactivation of the enzyme by EDTA. The kinetics of the substrate reaction with different concentrations of the substrate p-nitrophenylphosphate (PNPP) and inactivator EDTA suggested a competitive complexing mechanism for inactivation by EDTA, and the process of inactivation composed of the rapid initial formation of an enzyme-EDTA complex, in which the conformation of enzyme has been changed, and then zinc ions are finally removed from the enzyme.

http://www.ncbi.nlm.nih.gov/entrez/query...l=pubmed_docsum

Metal ion-complexing agents, like KCN, EDTA etc., inactivate alkaline phosphatase of pig kidney. This inactivation is reversible at low concentrations of the complexing agents and irreversible at high concentrations. The reversible inhibition is probably due to removal of Zn2+ ions from the active site, where they are necessary for catalytic action, whereas the irreversible inhibition results from the removal of Zn2+ ions necessary for preservation of the structure. The inactivation is pseudo-first order. It depends on the concentration, size and charge of the complexing agents. Beta-Glycerophosphate and Mg2+ ions protect the enzyme from inactivation by complexing agents. Quantitative examination of the effect of substrate leads to a model that is similar to the "sequential model" proposed by D.E. Koshland, G. Nemethy & D. Filmer (1966) (Biochemistry 5, 365-385) to explain allosteric behavior of enzymes. It describes the sequential addition of two substrate molecules at two active centres of the dimer enzyme. The binding of the substrate molecules is accompanied by changes in the conformation, which lead to stabilization of the enzyme against attack by complexing agents.

Edited by McJosh13 (08/02/06 05:02 PM)

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Invisiblefastfred
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Re: Preserving psilocybin in extractions by deactivating degradation enzymes [Re: McJosh13]
    #5926122 - 08/02/06 05:17 PM (17 years, 9 months ago)

Awesome! See if you can come up with a paper detailing the concentrations of EDTA required. I can get most journal articles for you if needed, I just don't have a ton of time at the moment to go searching them out.

I guess it just remains to be tested. The theory seems sound. As long as there are no problems with it reacting with psilocybin it should work!


-FF

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Offlineudok
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Re: Preserving psilocybin in extractions by deactivating degradation enzymes [Re: McJosh13]
    #6495092 - 01/25/07 06:25 AM (17 years, 3 months ago)

The dephosphorylation take place in an acid environment.(Gartz, (Casale) et al.)
I think in this case (acid+heat)it is an acid phosphatase encyme, like that what is found in Agaricus bisporus. (EC-Number 3.1.3.2 )
Note: The link may take some time to load.
http://www.brenda.uni-koeln.de/php/result_flat.php4?ecno=3.1.3.2&Suchword=&organism%5B%5D=Agaricus+bisporus&show_tm=0


--------------------
And on the 7. day the creator designed the psychedelic drugs. Holy shit. Thats intelligent design far beyond my scope. :wink:
Namaste

Edited by udok (01/25/07 12:01 PM)

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Offlinepscyanescens
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Re: Preserving psilocybin in extractions by deactivating degradation enzymes [Re: udok]
    #6510598 - 01/29/07 11:17 PM (17 years, 3 months ago)

OK I am no scientist, but i might want to add something that a friend pointed out to me.

What about honey? It contains enzymes that are used as a natural preservative. This is the reason honey will not mold. Could it be possible that there could be certain enzymes with in the honey, that could possibly neutralize the alkaline phosphatase? Would neutralizing the enzyme deactivate it?

I think of this because most people have talked to me about using honey to preserve the mushrooms. However the honey usually turns blue, indicating that some amount of psilocin was oxidized. I believe this is because most people powder the mushrooms, before they add it to the honey. What if you crush the mushrooms inside the honey??? It would make sense if it were in an oxygen free environment, right?

Well back to the real point. How would the addition of honey help with a finished extraction? Would it absorb the extraction? Or would they seperate from each other?


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"With an abundance of Cyanescens... i would never touch another Cubensis again."

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Invisibledeeptraveller
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Re: Preserving psilocybin in extractions by deactivating degradation enzymes [Re: pscyanescens]
    #6544180 - 02/08/07 01:43 PM (17 years, 3 months ago)




whether there is a way to transform pscn in pscb?


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we makes only that we makes...... this is very amusing idea if you under mushrooms_try this if you are not afraid
I have told it or have thought?

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Invisiblefastfred
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Re: Preserving psilocybin in extractions by deactivating degradation enzymes [Re: deeptraveller]
    #6549829 - 02/10/07 05:52 AM (17 years, 3 months ago)

Looks like you just posted it.

All you need is some N-Butyllithium, BnO2PO2 (whatever that is), tetrahydrofuran, hydrogen, paladium on carbon, and methanol!

Let us know how well it works...


-FF

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Re: Preserving psilocybin in extractions by deactivating degradation enzymes [Re: fastfred]
    #6551009 - 02/10/07 03:27 PM (17 years, 3 months ago)

fastfred your sense of humour is excellent....I am not the chemist.... I am the engineer which were interested in a narrow direction of chemistry

probably there is a transition to any salt of pscn, that will make its more stable....why nobody writes about it/and why nobody carries out the experiments with the PF-crystals, these experiments can give many answers. We receive not pure substances, but group of substances therefore a problem becomes complicated, but I shall try for interest (one crystal more, one crystal is less.... losses are not great)

if I will be occurred with an idea which is pleasant to me and will not seem silly then I necessarily shall write about ...to discuss it

p\s\I read some books about extraction of various substances..... probably I shall come to any conclusion (I hope for it)


--------------------
we makes only that we makes...... this is very amusing idea if you under mushrooms_try this if you are not afraid
I have told it or have thought?

Edited by deeptraveller (02/11/07 02:12 PM)

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