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Talk to me about potency!


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#41 PistolPete13

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Posted 15 January 2019 - 04:58 PM

This is potentially important work, PP13! I'm really impressed with your determination and focus.

 

I have good access to literature, so if there are any papers you need, I might be able to help.

 

Thank you! After reading some of your posts, I am impressed that you are here and decided to become a part of it! I was initially going to take this on by myself but I honestly underestimated the work load I took on in a very short time frame. Installing the hood alone was more work than I was planning on, I think maybe I should start a thread in mad scientists dedicated to this project. So I dont think you understand how much support from people like yourself and Deleena (everyone in this thread really) actually means (its massive!). Thanks guys!

 

 

Well, sounds pretty cool. Maybe do a simple run of just a couple of fruits. Perhaps mature vs aborts or just a couple different ones plus a control of some sort. Don't get held up with the 1 part in 1000 precision unless you already have the equipment laying around but just go through the process - dry the fruit, pulverize, MeOH, apply to the plate, run TLC, scan and let us know how it goes. Looking forward to hearing more.

 

Hey Ray! I guess it is only fair if you are now contributing that you know where we stand! So a quick recap, then expect a new thread within a week (or two, busy).

20190116_075115xx.jpg

I still have not finished the lab and the TLC station might end up being where the sterilizer is at the moment, so I have a side bench for the hood. I still have not brought in a lot of stuff I need for it to be fully functional. But hopefully that will give you a good idea that I am not pulling your leg, those scales weigh down to a milligram, that pipettor down to one microliter, and the microcaps are spot on! You cant see the developing chamber, reagents and a few other items for TLC. And I still dont even have anything to dry the specimens with though, and a few other things I still need to get relating to growing. And am still trying to dial in the fruiting chamber. I know what you mean about not being too anal about quantities ect, but for what I want to do, I need it. For other things like extracts it is not needed at all, and I would probably even go with a good quality lab filter paper it is cheaper than the glass plates you can see in the pic and the resolution will be acceptable. But I am going to need to quantify the metabolites of small populations, with possibly very small differences between them so not worrying too much just nullifies the results.

 

Now where we stand on the mushroom front, I have two 0.66 gallon mason jars filled with WBS that have been inoculated with a PE clone(all I could get at the time) about 15% colonized and is obviously not a MS (which I really wanted). But as you said sample runs are going to have to be made, those figures I gave above are what I am expecting would be optimal (also had some literature to back it up). But no doubt there are going to be issues (however small) that will have to be sorted, and those numbers will change a little I am sure. Maybe the spot wont be big enough, maybe trailing ect, Regarding the first run I was going to run them on two different substrates, coffee/coir I thought was a good substrate that could be used as a benchmark. And the other was going to be in a substrate I came up with (i think you guys are going to like it!) I call it Jurassic Poo! And the potency of shrooms growing on different substrates can be tested and like you said things aborts vs the best fruit of the batch are going to be interesting. I also have a PDA plate with a creeper MS that has germinated, so its not going to be long before we see some creeper MS grows(which is exciting!).

 

I will take all the help I can get regarding the best growing strategies ect, as I know there are many people here that leave me in dust when it comes to growing experience....


Edited by PistolPete13, 15 January 2019 - 05:02 PM.

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#42 MysticalMyco

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Posted 16 January 2019 - 12:58 AM

For me it might be placebo but I've noticed personal differences cube variance to variance.

So when I eat golden teacher for whatever reason I have the wierdest trips. They always smack me in the face and start bad/dark/evilish to the point I almost start freaking out then they level out to a moderately visual very philosophical trip. This happens every single time I've eaten personally grown GT or any time someone has sold me mushrooms calling them GT. I have a similar trips on Penis Envy.

Now the reverse happens when I eat Psilcybe Cubensis var. mexican. Same Exact dosing equals light hearted, fun, slightly visual, laugh till I cry type of trip. I get similar trips when I eat Thai variances such as Ban Hua Thanon.

Both instances have been replicated multiple times. I also notice that when I start tripping all crazy on GT or PE Its not necessarily dose dependent. I can go off the deepend on doses as small as 1.75g. The only thing that changes is the visual intensity when I increase dose. I don't take heroic doses. The most I've taken off any strain is 5g but in graduated steps.

When I eat Psilocybe Cubensis var Mexican or Thai. I can start the light laugh my ass off trip with doses as small as 1.75g and can eat up to 5g+ with the only thing changing is me laughing harder with visuals bumping up to moderate. But always a light happy trip.

I don't know what causes this. I know set and setting are super important so I try to trip the same way every single time for the most part. I try to trip outside in nature during early evening maybe 2-3 hours before sunset then I either start a fire or move inside.

I never trip in bad moods and always trip with my fiance.

I kind of look at it like weed. For the longest time everyone said weed is weed and weed gets you high. There were different variances, strains, strengths. Then the mexicans learned how to grow more potent seedless females to increase profits. Then in the last 15-20 years there has been a Renaissance with marijuana research.

I feel like we're missing something here and that yes a cube is a cube just like marijuana is marijuana. Someday we may learn that this variance of cube helps this and that variance of cube helps for that, when more research dollars are dumped into it and laws become less strict regarding research the veil may be lifted.

I eat the mexicans/Thai when I want to relax outside aimlessly , laugh my ass off and look at cool shit. I take golden teachers and PE when I want to blast off into the outter realms and contemplate Philosophy.

I've taken enough strains repeatedly to notice "trends" in the different cube variances. And for me these trends are absolutely repeatable. As I grow more variances and test them I'm going to try to maintain a good handle of the types of trips I have per variance.

Edited by MysticalMyco, 16 January 2019 - 01:12 AM.


#43 jkdeth

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Posted 16 January 2019 - 09:17 AM

I've never heard anybody say weed is weed. Not the same at all.

I have often wondered if there were possible noticeable geographic differences.

The thing is the longer we grow them indoors, the more chance there is they lose any geographic distinctiveness.

A cube is a cube is true in the genetic sense. In other words, no DNA difference from one strain to another. Even with recognizable differences, its a matter of expression.

If one strain gives a repeatable difference in trip than another, there has to be a different chemical make up. That would be the thing to identify.

#44 Deleena24

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Posted 16 January 2019 - 11:51 AM

Man, it's so awful we can bring samples to our local college or pay for maybe gas chromatography or whatever to see exactly what's in them, and the exact concentrations.

This isnt possible, of course, with today's laws. Scientists cant study illegal specimens, which is why no figures of potency have been available since the late 60s

#45 PistolPete13

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Posted 16 January 2019 - 02:29 PM

"I've never heard anybody say weed is weed. Not the same at all."
 

"A cube is a cube is true in the genetic sense. In other words, no DNA difference from one strain to another. Even with recognizable differences, its a matter of expression."

 

This is the type of logic I was hoping would prevail, before one test has even been conducted. There are a few papers documenting the potency of different cube varieties from different locations and the potency all varied. No two tests were identical. Gartz noted in his work on substrates that different strains of cubensis produced different amounts of alkaloids on the same medium. This is just one example from one of his patents;

 

Example 1

 

A primary culture of Psilocybe cubensis (Earle)Singer was used for inoculation which was obtained by germination of the spores on 100ml of 6% malt agar, 14 days after innoculation and subsequent cultivation (20 days) on 100ml of the same agar.

A piece of mycelium about 2 x 2cm was removed from it with a sterile spatula and by shaking with 30ml of sterile water and 15g saddle packing, generated a suspension of fine mycelium flakes.

10ml of the suspension was used to inoculate an autoclaved mixture of 100g rice grains and 200ml of water. After 3 weeks the formation of the fruiting bodies began, which in 5 fructification waves appeared.

 

Yield: 21g dry fruiting bodies

HPLC analysis: Psilocybin content: 0.7%

Psilocin content: 0.1%

 

Workup is carried out in a known manner by extraction of the powdered material with methanol and subsequent column chromatography on cellulose.

 

Example 2

 

A second strain of Psilocybe cubensis (Earle)Singer was used. Under analogous conditions, the first fruiting bodies were reached after 4 weeks.

 

Yield: 25g dry fruiting bodies

HPLC: Psilocybin-content: 0.95%

Psilocin-content: 0.2%

 

 

source;

DD254395(A1) -1988- VERFAHREN ZUR GEWINNUNG VON TRYPTAMINDERIVATEN DURCH KULTIVIERUNG HOEHERER PILZE

 

 

 

 

 

If one strain gives a repeatable difference in trip than another, there has to be a different chemical make up. That would be the thing to identify.

 

This is where HookWorms comments about subjectivity start becoming extremely interesting, what if for example the mutation in PE is in the biosynthetic pathway to its actives? That for example hinders the expression of the gene that produces N-methyl transferase, or just hinders the reaction. In such a case it would then produce a ton more Baeocystin or Norbaeocystin(or both), in such a case it would usually produce say for example half to no psilocybin at all, and what would have been psilocybin is now baeocystine and norbaeocystin. So the overall potency would technically not change but just as you point out only the chemical makeup would change and without any doubt at all the perceived potency as well. Baeocystin and norbaeocystin are usually produced in some amount in cubes even if its only traces. The same would be true if there was some sort of mutation which silenced the kinase enzyme or some facilitator it uses to make psilocybin resulting in 100% psilocin and that would definitely be perceived as being much stronger because of the extremely quick onset all at once compared to psilocybin, but once again the potency does not have to be different for this to happen. It could even produce a novel compound that is a close analog of psilocybin and active???? This is going to be interesting, bring on the strain fingerprinting! I will try as hard as I can to document the provenance of the strains tested, where it originally came from, what vendor, anything I can get really. As I think also that Hawk and Workmans PE are going to test differently than some of the other very weird looking ones you see at other vendors and on forums. Testing the same strain from different sources, will be also interesting!

 

Oh BTW, Mr. Deth. If you could stick around your 2cents would be greatly appreciated in the coming weeks!

 

 

Man, it's so awful we can bring samples to our local college or pay for maybe gas chromatography or whatever to see exactly what's in them, and the exact concentrations.

This isnt possible, of course, with today's laws. Scientists cant study illegal specimens, which is why no figures of potency have been available since the late 60s

 

Where I live they will not even let teens test the pills they are about to take at festivals, and warn on the news people are dying from taking bad ones????



#46 Deleena24

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Posted 16 January 2019 - 03:48 PM

I noticed the literature you quoted is from 1988? I can see it wasn't in the US, is that Germany or maybe Dutch?

The comments about how the different actives may be affect by different gene expressions, and so on...

If we could pinpoint WHY these substances are produced in the first place, it would make finding the Gene's that regulate alkaloid production that much easier.

Are they a defensive mechanism? Do they benefit the organism in any way? If the Gene's for production of alkaloids are somehow shut off, would it affect how the fruits look and grow?

So many questions...

Edited by Deleena24, 16 January 2019 - 03:55 PM.


#47 PistolPete13

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Posted 16 January 2019 - 05:29 PM

German, he patented a whole heap of stuff around that time frame, here are the ones he patented only in Germany I put an English version of the title underneath them;

 

 

DD290916(A5) -1991- VERFAHREN ZUR MIKROBIELLEN GEWINNUNG VON INDOLYLGLYOXYLSAEUREN  
PROCESS FOR MICROBIAL RECOVERY OF INDOLYLGLYOXYLIC ACIDS

DD287053(A5) -1991- VERFAHREN ZUR GEWINNUNG VON BAEOCYSTIN DURCH BIOTRANSFORMATION VON TRYPTAMIN
PROCESS FOR OBTAINING BAEOCYSTIN BY BIOTRANSFORMATION OF TRYPTAMINE

DD278600(A1) -1988- VERFAHREN ZUR GEWINNUNG VON TRYPTOPHANDERIVATEN DURCH KULTIVIERUNG HOEHERE PILZE
PROCESS FOR OBTAINING TRYPTOPHAN DERIVATIVES BY CULTURING HIGHER MUSHROOMS

DD276420(A1) -1990- VERFAHREN ZUR HERSTELLUNG PILZLICHER BIOMASSEN
METHOD FOR PRODUCING FUNGAL BIOMASSES

DD273449(A1) -1989- VERFAHREN ZUR GEWINNUNG VON 4-SUBSTITUIERTEN INDOLVERBINDUNGEN
METHOD FOR OBTAINING 4-SUBSTITUTED INDOLE COMPOUNDS

DD266811(A1) -1989- VERFAHREN ZUR GEWINNUNG VON INDOLEN DURCH KULTIVIERUNG VON AGARICALES UND NAEHRBODEN HIERFUER
PROCESS FOR OBTAINING INDOLES BY CULTURING AGARICALS AND NUTRIENT MEDIA FOR THIS PURPOSE

DD265636(A1) -1989- VERFAHREN ZUR GEWINNUNG VON INDOLDERIVATEN AUS PILZMYCELIEN UND NAEHRMEDIUM HIERFUER
METHOD FOR OBTAINING INDOLE DERIVATIVES FROM MYCELIUM OF FUNGI AND NUTRIENT MEDIA FOR THIS PURPOSE

DD264023(A1) -1998- VERFAHREN ZUR EXTRAKTION VON INDOLALKALOIDEN AUS PILZMATERIAL
PROCESS FOR EXTRACTION OF INDOLE ALKALOIDS FROM MUSHROOM MATERIAL

DD255169(A1) -1988- VERFAHREN ZUR ABTRENNUNG VON BAKTERIENBIOMASSE
METHOD OF SEPARATING BACTERIA FROM BIOMASS

DD254395(A1) -1988- VERFAHREN ZUR GEWINNUNG VON TRYPTAMINDERIVATEN DURCH KULTIVIERUNG HOEHERER PILZE
PROCESS FOR OBTAINING tryptamine derivatives by cultivating higher fungi

DD255749(A1) -1988- VERFAHREN ZUR GEWINNUNG VON INDOLALKALOIDEN AUS PILZMYCELIEN UND NAEHRMEDIUM HIERFUER
METHOD FOR OBTAINING INDOLE ALKALOIDS FROM MUSHROOM MYCELIUM AND NUTRIENT MEDIA FOR THIS PURPOSE

DD252613(A1) -1987- ABTRENNUNG VON BAKTERIEN AUS SUSPENSIONEN
SEPARATION OF BACTERIA FROM SUSPENSIONS

DD249281(A1) -1987- VERFAHREN ZUR RUECKGEWINNUNG VON BAKTERIEN
METHOD OF RECOVERING BACTERIA

DD245901(A1) -1987- VERFAHREN ZUR FAELLUNG VON BAKTERIEN AUS SUSPENSIONEN
METHOD OF PRECIPITATING BACTERIA FROM SUSPENSIONS

DD245443(A1) -1987- VERFAHREN ZUR KOAGULATION VON BAKTERIEN AUS SUSPENSIONEN
PROCESS FOR COAGULATING BACTERIA FROM SUSPENSIONS

DD240030(A1) -1986- VERFAHREN ZUR KOHLENWASSERSTOFFBESTIMMUNG IN MIKROORGANISMENHALTIGEN DISPERSIONEN
METHOD FOR THE HYDROCARBON DETERMINATION IN MICRO-ORGANISM-CONTAINING DISPERSIONS

DD240027(A1) -1986- VERFAHREN ZUR GEWINNUNG VON TANNIN-PROTEIN-ADDUKTEN AUS BAKTERIELLEN SUSPENSIONEN
METHOD OF OBTAINING TANNIN PROTEIN ADDUCTS FROM BACTERIAL SUSPENSIONS

DD201026(A1) -1983- VERFAHREN ZUR HERSTELLUNG VON ALKYLPEROXYCARBONSAEUREN
PROCESS FOR THE PREPARATION OF ALKYLPEROXYCARBONIC ACIDS

DD201302(A1) -1983- VERFAHREN ZUR HERSTELLUNG VON ALKYLPEROXYALKYLHYPOCHLORITEN
METHOD OF PREPARING ALKYLPEROXY ALKYLHYPOCHLORITES

DD148218(A1) -1981- VERFAHREN ZUR HERSTELLUNG VON BIFUNKTIONELLEN ORGANISCHEN PEROXIDEN
PROCESS FOR PREPARING BI-FUNCTIONAL ORGANIC PEROXIDES

DD148217(A1) -1981- VERFAHREN ZUR HERSTELLUNG VON BIFUNKTIONELLEN OXALSAEUREPERSTERN
PROCESS FOR THE PREPARATION OF BIFUNCTIONAL OXALIC ACID STARTERS

DD147235(A1) -1981- VERFAHREN ZUR HERSTELLUNG VON BIFUNKTIONELLEN AZOPERESTERN
METHOD OF PREPARING BI-FUNCTIONAL AZO STARTERS

 

If you are interested in any let me know, I translated most of the cubensis related ones.

 

It is also possible that it may turn out there is no grand scheme to creating these substances. It has been speculated in papers and it would not be the first case in nature where an organism has to deal with an excess of a certain compound (say tryptophan for example).

 

"Alkaloid formation is suggested to reflect a regulatory device to keep endogenous tryptophan levels in balance."

 

And the  mushrooms that come up with a way to make some beneficial compound from it (anti-oxidant ect) had the evolutionary advantage?

 

It also could be the actual biosynthesis is beneficial, removing compounds that are potentially harmful at the cellular level like oxygen that causes oxidative damage and methyl groups that can damage DNA ect????


Edited by PistolPete13, 16 January 2019 - 05:29 PM.

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#48 WalkingCatfish

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Posted 17 January 2019 - 08:06 AM

The gene-cluster associated with psilocybin seems to have been transferred laterally between distantly-related mushroom clades. The trait seems to be strongly adaptive, and not just preserved neutrally in a particular lineage.  There's some interesting speculation about possible "ecological drivers" here: https://onlinelibrar...10.1002/evl3.42  I like the notion that it influences arthropod behaviour in ways that help the fungus.

 

The discovery of psilocybin in the cicada pathogen Massospora adds some support to that idea:  https://www.biorxiv....18/07/24/375105


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#49 Deleena24

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Posted 17 January 2019 - 12:01 PM

Very very interesting stuff. Psilocybin as a pathogen for changing insect behavior?

So many questions.

#50 raymycoto

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Posted 17 January 2019 - 01:02 PM

Ha, so cool. Mushies were like crack for ancient cicadas for mutual benefit of both species.



#51 PistolPete13

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Posted 17 January 2019 - 04:33 PM

The gene-cluster associated with psilocybin seems to have been transferred laterally between distantly-related mushroom clades. The trait seems to be strongly adaptive, and not just preserved neutrally in a particular lineage.  There's some interesting speculation about possible "ecological drivers" here: https://onlinelibrar...10.1002/evl3.42  I like the notion that it influences arthropod behaviour in ways that help the fungus.

 

The discovery of psilocybin in the cicada pathogen Massospora adds some support to that idea:  https://www.biorxiv....18/07/24/375105

 

Yes I am really loving some of the research thats starting to come out, now the political climate around shrooms is starting to swing as it is being considered as the next medical marijuana style drug. About 3 days ago they have just starting prescribing psilocybin to the elderly in my country to help them deal with the anxiety and fear of dying.

 

You seem very knowledgeable in these areas, are you familiar with the biosynthesis of psilocybin (i.e. The shikimate pathway and the gene-cluster responsible for psilocybin production)?? As I have some rather interesting information, but it seems it is lost on most people I have shown. I need someone with some knowledge of the biosynthetic pathway to review it (and you may just be the catfish that could do it?). If you get time please peruse the article at your leisure, any one else that wants to have a crack please do. I will be around to answer any questions.....

 

Proteome and Transcriptome Reveal Involvement of Heat Shock Proteins and Indoleacetic Acid Metabolism Process in Lentinula Edodes Thermotolerance

 

https://www.karger.c...FullText/494784


Edited by PistolPete13, 17 January 2019 - 04:34 PM.


#52 WalkingCatfish

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Posted 18 January 2019 - 09:44 AM


You seem very knowledgeable in these areas, are you familiar with the biosynthesis of psilocybin (i.e. The shikimate pathway and the gene-cluster responsible for psilocybin production)?? As I have some rather interesting information, but it seems it is lost on most people I have shown. I need someone with some knowledge of the biosynthetic pathway to review it (and you may just be the catfish that could do it?). If you get time please peruse the article at your leisure, any one else that wants to have a crack please do. I will be around to answer any questions.....

 

I wish I were that catfish.  :biggrin: My understanding of biochemistry is pretty sketchy, I'm afraid. I'm out of my comfort zone in fungal biology, too, though I've been trying to get up to speed on the basics. I can admire biosynthesis of alkaloids in these beautiful organisms from afar, but I just don't have the background to discuss it knowledgeably! 



#53 PistolPete13

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Posted 20 January 2019 - 01:37 AM

Did anyone notice the second patent on that list of Jochen Gartz' German patents?

 

 

DD287053(A5) -1991- VERFAHREN ZUR GEWINNUNG VON BAEOCYSTIN DURCH BIOTRANSFORMATION VON TRYPTAMIN
PROCESS FOR OBTAINING BAEOCYSTIN BY BIOTRANSFORMATION OF TRYPTAMINE

 

Claims:

1. A process for the production of beocystin by biotransformation of tryptamine, characterized in that the aqueous solution of tryptamine with the addition of methionine, phosphates and yeast extract under sterile conditions with basidiospores of higher fungi, such as those of the genera Psilocybe, Conocybe, Panaeolus and Gymnopilus is inoculated and after the enzymatic biotransformation, the Baeocystin is separated in known manner by evaporation, extraction or chromatography.

2. The method according to claim 1, is characterized in that the biotransformation at 1 to 30*C depending on the type of fungus and with or without the addition of antibiotics to the medium within 14 days.

Field of application of the invention

The invention relates to a process for the production of Baeocystin by biotransformation of precursors by basidiospores. The substance Baeocystin is a pharmaceutically active substance and can be used as a model substance in basic neurochemical research.

Characterization of the known technical solution

The tryptamine derivative Baeocystin(4-phosphoryloxy-N-methyltryptamine) is found as an accompanying alkaloid of psilocybin and psilocin in naturally occuring higher mushrooms of various genera in small to very small quantities. It is well known that the alkaloid can be isolated from the extracts of natural fungi by using chromatographic methods (Pharmazie 1985, 274).
The method described has several disadvantages. The yield of alkaloid is only small because simple operating methods do not allow for separation of psilocybin which has very similar physiochemical properties, chemically differing only by an additional methyl group. In addition the naturally occurring mushrooms are not economically accessible. The biotransformation of tryptamine is known in cultured mycelia of higher fungi(DD-PS 265636) 0.25% calculated for the separation can then take place only under complex separation methods with loss of yield. Other mycelial ingredients(e.g.lipids) require additional work up. Finally, the cultivation of the mycelia takes at least 30 days in the biotransformation of tryptamine.

Object of the invention

The aim of the invention is to develop a simple and less expensive process for the production of baeocystin by biotransformation of tryptamine, which is in aqueous solution with a short reaction time, achieves a high yield of Baeocystin without disturbing the accompanying alkaloids and organic substances.

Explanation of the nature of the invention

The inventions object is to select such media for biotransformation of tryptamine, which in aqueous solution ensures rapid formation of Baeocystin in high yield without additional alkaloids.
According to the invention the object is achieved in that the aqueous solution of tryptamine with the addition of methionine, phosphates and yeast extract under sterile conditions with basidiospores and higher fungi, such as the genera Psilocybe, Baeocystin in a conventional manner by evaporation, extraction or chromatography.
In the biotransformation, a selective hydroxylation and phosphorylation takes place in the 4-position on the indole ring of the tryptamine, at the same time the amino nitrogen is mono-methylated on the side chain.
For the culture media solutions of 1.5 to 3% methionine, 1 to 4% tryptamine or its water-soluble salts, 1.5 to 3% soluble phosphate, 0.1 to 0.3% yeast extract and water to 100%, also the addition of antibiotics if appropriate, basidiospores are transferred in an amount of 0.1 to 0.2%.
Fungal spores of the genera Psilocybe, Panaeolus, Gymnopilus and Conocybe are used for the biotransformation reaction. After a reaction time of 5 to 14 days at 1 to 30*C, the Baeocystin is isolated in a known manner by filtration, evaporation, preferably in vacuo and subsequent extraction and/or chromatography in a yield of 70 to 90%.
The invention will be explained in more detail by the following example.

Embodiment

Basidiospores of Psilcybe cubensis(Earle) Sing. were used for inoculation, which were obtained from the lamellae of mature fruiting bodies by release under sterile conditions in a humidity chamber.
0.1g of spore dust was added under sterile conditions to a mixture of 1.5g methionine, 2g tryptamine hydrochloride, 1.5g KH2PO4 and 0.1g yeast extract in 95ml water in a 300ml Erlemeyer flask with a cotton plug. After stirring for 10 days(magnetic stirrer) at 20*C, the bluish solution was filtered after 150ml of water had been added. It was then extracted by shaking with 50ml of chloroform, the aqueous phase evaporated(vacuum), 20ml of methanol at 0*C added, filtered, the residue obtained dissolved with 50ml of acetone and then with 50ml of acetone/diethyl ether and the solid residue dissolved with 500ml of methanol. After filtration to remove the inorganic salt is the methanolic solution of Baeocystin i.Vak. eingedunstet. Yield of Baeocystine after recrystallization in n-butanol: 2.1g.

 

https://worldwide.es...287053A5&KC=A5#

 

That is only a final draft so some of language still gets a bit curly, it gets boring after awhile. But I made sure all the important things are correct....


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#54 raymycoto

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Posted 20 January 2019 - 09:04 AM

Am I correct on my take on that patent:

 

1) It uses enzymes present in the spores to cause the transformation of the reagents into Baeocystin (4-HO-NMT).

 

Shulgin does not address 4-HO-NMT's clinical effect.

 

My other thoughts and questions, if statement #1 is correct:

 

Questions:

  • What would be the reaction rate with fewer spores? 0.1 gm of spores is a LOT of spores. OTOH, the yield is impressive.
  • Could the same results occur with a fruit tissue extract? That would be much easier to obtain (and in sterile fashion) than 0.1 gm of spores.
  • Does 4-HO-NMT yield a clinical effect similar to 4-HO-DMT?
  • Is conversion of 4-HO-NMT to the 4-HO-DMT feasible?

Edited by raymycoto, 20 January 2019 - 09:06 AM.


#55 Deleena24

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Posted 20 January 2019 - 02:32 PM

Yup. There is some awesome research in there.

#56 PistolPete13

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Posted 20 January 2019 - 04:01 PM

I assumed the spores would have germinated within that 5 to 14 day window they specify, and thats why the large excess of spores to make sure you quickly get an abundance of mycelium to transform your substrates. And as soon as they consume the tryptamine you harvest before they make anything else. Also they refer to it as a 'culture media' and you generally culture things to get enzymes, and they also add a little yeast extract so the mycelium has enough nutrition to produce the enzymes needed. If the enzymes were already present they would just add precursors and enzymes adjust pH and keep at a certain temp for (generally a much shorter time) and the time frame would be known down to the hour at least whereas 5 to 14 days is a big window, when it goes 14 days they are waiting for something!

 

In one of the last comments in Gartz' famous papers from 89 where he added tryptamine to the substrate and saw record levels of psilocin he speculated;

 

 

It is possible that a reduced amount of phosphate in the culture media decreased the biosynthesis of psiocybin from psilocin in the mycelia.

 

And that was the last thing published on the matter, this patent proves that the work continued but the results were never published. As you can see from the patent phosphate is now added and the phosphorylated compound is indeed formed.  But in his famous papers why does adding tryptamine alone to the substrate yield the N,N-dimethylated compound psilocin. And this one he adds tryptamine and a carefully measured amount of methionine (which provides the methyl group), if you added twice the amount of methionine you would get psilocybin, leave out the phosphate get psilocin ect.. In his initial paper they use cowdung/rice grains as the substrate and rice is relatively high in methionine.

 

Regarding just using fruit tissue; I know from the literature they generally have a little bit of trouble getting a culture to produce actives in submerged culture. They usually have to find the right strain and culture it on the right media, so it may just work? Or may take take a bit of experimentation to find the right strain for the job if tissue culture is used???? If you found the right strain it would be pretty obvious because the culture media turns blue!

 

According to wiki;

 

Little information exists with regard to human pharmacology, but in the book Magic Mushrooms Around the World, author Jochen Gartz reports being aware of a study in which "10 mg of baeocystin were found to be about as psychoactive as a similar amount of psilocybin."[5] Gartz also reported in a research paper that a self-administered assay of 4 mg of baeocystin caused "a gentle hallucinogenic experience".[6]

https://en.wikipedia...wiki/Baeocystin

 

 

  • Is conversion of 4-HO-NMT to the 4-HO-DMT feasible?

 

As stated above you would just double the amount of the amino acid methionine added to the culture media.


Edited by PistolPete13, 20 January 2019 - 04:07 PM.


#57 raymycoto

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Posted 20 January 2019 - 05:57 PM

 

I assumed the spores would have germinated within that 5 to 14 day window they specify

 

Oops, I missed, perhaps, the obvious. Why else add yeast extract?

 

BTW, I was mistaken above when I either stated or implied that Baeocystin was 4-HO-NMT. It is actually the (guessing this is the proper chemical term in bold - please correct me) 4 position phosphate ester.

 

And, my question of adding an emulsion of the fruit was simply looking at it as a source of the enzymatic process, not thinking of a metabolic process.

 

I was not even thinking in terms of thermodynamics of the process. That is, do the products have more chemical enthalpy (probably not the right term) than the reagents.

 

With this in mind but in any case, it would seem interesting or puzzling but quite delightful that the little buggers would care to convert environmental tryptamine to either either of the phosphorylated pro-drugs to psilocin or it's mono methylated relative 4-HO-NMT.  That is, the gravimetric assay of the baeocystin would seem to be orders of magnitude beyond what one would hope to get from the fungus in the mixture.

 

Let's say the spores double in mycelial mass every 4 days and I bet that is an overstatement. That would put their mass at 0.8 grams, let's say. Then suppose they contain 0.1% actives. That would yield 0.8 mg of actives. And . . . the author states that he comes up with 2.1 grams of baeocystin.

 

That would be (drum roll . . .) around 2600 times the possible actives that the mycelium might otherwise contain. That myc has been pretty darn busy!

 

Almost as if they are doing a cleanup of their surroundings to get rid of that nasty environmental tryptamine.

 

Is that really true? Would have to put it to the test. That is, simply take a solid and stable LC, add the aforementioned reagents, incubate, extract. For this, we might need your TLC assay. And, ahem, take a look at my thoughts about LC

https://mycotopia.ne...liquid-culture/

which I would love to be super solid at for this very reason - it could be a foundation for this sort of research.

 

Also, it should be duly noted that, for all our discussions of "Hey, whats the best way to extract . . . " This guy outlines the (or at least one) extraction process in his patent. Someone just do that and report back to us.

 

Is tryptamine a common  reagent?

 

Tryptophan is.

 

How about tryptophan decarboxylase - does it exist?

 

Silly question. Amazon says if I order within the next 72 minutes I'll have it tomorrow afternoon.


Edited by raymycoto, 20 January 2019 - 06:26 PM.


#58 PistolPete13

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Posted 20 January 2019 - 07:55 PM

 

BTW, I was mistaken above when I either stated or implied that Baeocystin was 4-HO-NMT. It is actually the (guessing this is the proper chemical term in bold - please correct me) 4 position phosphate ester

 

Exactly and they refer to that as a 'Phosphoryloxy' group.

 

 

That would be (drum roll . . .) around 2600 times the possible actives that the mycelium might otherwise contain. That myc has been pretty darn busy!

 

lol, also dont forget enzymes keep going for awhile before they end up denaturing. They suck in a molecule that fits and do their job on it, and spit it out, just to suck in the next molecule that fits that is floating by and constantly repeating that process.

 

 

Almost as if they are doing a cleanup of their surroundings to get rid of that nasty environmental tryptamine.

 

It is well documented when mycelium exhausts a substrate and there is nothing more to eat, it starts releasing protease enzymes which break down the enzymes it was using to break down the substrate. And gets some of the nutrition and energy back from them when they are no longer needed.

 

Tryptamine is hard AF to get, but tryptophan is easy decarboxylated with peppermint oil and a high boiling solvent. PS: I had no idea tryptophan decarboxylase was so easy to get, this could be exiciting!!!(note to self, look into this asap!).

 

 

Is that really true? Would have to put it to the test. That is, simply take a solid and stable LC, add the aforementioned reagents, incubate, extract. For this, we might need your TLC assay. And, ahem, take a look at my thoughts about LC

https://mycotopia.ne...liquid-culture/

which I would love to be super solid at for this very reason - it could be a foundation for this sort of research.

 

If you are serious, I would be interested. I would also highly recommend starting here;

 

https://open.library...items/1.0094541

 

That is a thesis where he does pretty much exactly what we are talking about, and the trials and tribulations he had trying to get actives in submerged culture(it was not that hard). And all the relevant papers he has referenced all in there, it is a worthwhile read!



#59 raymycoto

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Posted 21 January 2019 - 11:24 AM

Nice bit of research. I read about 1/3 of the way then was losing concentration so I'll look at further. Looks like he was able to create some of the active alkaloids but I will probably have to really study it to figure out what the yield is.

 

Some interesting points I picked up:

 

  • Sucrose is indeed a suitable nutrient for LC, at least he does use this successfully. Sucrose is common table sugar, a disaccharide of glucose and fructose and the component in almost all common 'sugars' or sweeteners we have access to. These also include maple syrup, honey, molasses and many others. I have been thinking that the best LC really needed glucose, a simpler sugar. This not the case - see comment below re. corn syrup.
  • Page 6 - "Baeocystin and nor-baeocystin are found to be toxins (74)."
  • 74. Bandoni, R. J. and Szczawinski, A. F. (1975). "Guide to Common Mushrooms of British Columbia".

Don't know what he means referring to baeocystin as a toxin.

 

Corn Syrup - Sorry folks, I have been telling you that Karo was sucrose or HFCS. It generally is not the case. "Corn syrup" on an ingredient label, as it turns out, means pure glucose from enzymatic action on corn starch. And "regular Karo" is pure corn syrup and thus glucose. Now, note that there are some 'Karo - like' sweetners that also add either fructose or HFCS to the corn syrup to enhance sweetness. But they will have that on the label.

https://www.thekitch...fference-196819

 

So, as noted above, the fructose variant of 'Karo' would not be bad for an LC. It is, however, a nutritionally unhealthy product as it is the fructose component of all dietary sugars that we overdose on in our sugar laden diet. Fructose in excess is the cause of all aspects of metabolic syndrome and has the same hepatotoxicity as alcohol (17gm max per day for a 70 kg individual).  Too much dietary glucose will make you fat but is not generally the cause of the other aspect of metabolic syndrome (dyslipidemia, hypertension, vascular disease, diabetes).


Edited by raymycoto, 21 January 2019 - 11:30 AM.


#60 PistolPete13

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Posted 21 January 2019 - 03:27 PM

That paper is full of gems like that, I should have warned you to start at page 54 read the discussion and work backwards from there skipping the really analytical stuff first and then going back and just reading the detailed procedures and analytical diagrams of the things that interest you. As you found out if you try and read it cover to cover it gets a bit full-on going through all the techniques and procedures and diagrams that dont even make all that much sense until you have read the whole thing and have an idea of what he was doing and why.

 

 

 

It was noticed that only when the fungus was maintained on Sabouraud agar plates (neopeptone-dextrose, 10 gm-40 gm). would, it produce psilocybin on subsequent transfer to liquid medium. If it was kept on malt extract-yeast-soytone agar plate (MYP, 7 gm-0.5 gm-1 gm) , it did not produce psilocybin even after a 15-day-period of incubation and the cell extracts gave an UV spectrum of ergosterol (Figure 17) instead of that.of an indole.

 

However, this does not preclude the possibility that the fungus when grown on MYP agar will eventually produce psilocybin if a longer incubation is allowed.

Catalfom6 and.Tyler (1) as well as Leung (12) maintained their cultures on potato-dextrose agar plates (PDA, 200. gm—15 gm) and succeeded in obtaining psilocybin.

 

For example that is a great little tip for anyone interested in submerged culture, and that was the first couple of sentences! Also when trying to read the paper you are flicking past NMR and UV spectrum analysis of these compounds without knowing what it is and what it is for, after reading this part you can see that the ergosterol UV spectrum is there for example as proof of his claim that the cubensis cultures they maintained on MEA did not produce psilocybin in submerged culture(not with his strain and methods anyway).

 

Regarding the toxin stuff, I have seen people referring to coffee as a toxin!

https://www.healthst...s-put-bodies-2/


Edited by PistolPete13, 21 January 2019 - 03:30 PM.





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