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Using tryptophan to increase alkaloids


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#41 Headinthehills

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Posted 13 May 2015 - 06:07 AM

Though I hate to link them in here, I found the study. If I broke any rules by posting this here, I'm sorry, please take it down. If not, enjoy...

 

Attached File  Biotransformation_of_tryptamine_derivatives.pdf   1.35MB   74 downloads

 

[No worries, I downloaded the pdf for you and re-uploaded it here :) -Sidestreet]


Edited by Sidestreet, 13 May 2015 - 06:24 AM.

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#42 Phineas_Carmichael

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Posted 13 May 2015 - 09:19 AM

Sorry to repost and create circular discussion, I smoke a lot of pot and read a lot of threads. . . .

I agree, messing with a different tryptamine could produce some unexpected dose response curves and should be assayed with caution. It would be awesome if someone could locate the German study that Sasha is referencing, I would love to read it. I will leaf thru the Tihkal footnotes and see if I can find some info. Hopefully it's posted online.

Found it, see attached PDF.

 

It occurs to me that Headinthehills is using tryptophan, but should to be using DMT or at the very least, tryptamine.

 

Let's look at our target molecule of this biosynthesis, psilocin:

Psilocin copy.jpg

 

Now let's look at Tryptophan:

tryptophan copy.jpg

 

Tryptophan needs to be decarboxylated (The junk after the Nitrogen on the carbon chain removed) into tryptamine, the primary amine (N at the end of the carbon chain) dimethylated, and then hydroxylated at the 4 position on the indole ring.

 

Let's look at Tryptamine next:

Tryptamine copy.jpg

This is simply decarboxylated tryptophan.

 

Now, DMT:
DMT copy.jpg

This is simply psilocin without the hydroxy at the 4 position.

 

Sorry to contribute to the circular (in my case it is due to a crippling sobriety) discussion, but why is our starting material in this case tryptophan?

 

And I'll end with a quote from The Good Doctor: Shulgin, Alexander. "4-HO-DET Phosphate Ester." TiHKAL. 4th ed. Berkeley: Transform Press, 2012. 465. Print.

 

One Final comment.  When you read a paper or listen to a lecture offered by a researcher of impeccable qualifications, take a moment to look about you to see who is alongside you in the audience that is being addressed.  Who else is reading his paper? Who else is hearing his lecture? How might the presentation be tailored to fit the interests of the recipients? The identification and recognition of your neighbors should play a role in your evaluation and acceptance of his presentation

Attached Files


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#43 TurkeyRanch

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Posted 13 May 2015 - 09:31 AM

Very cool, it was Gartz that published it, who knew. I wonder why Shasha didn't just mention him by name, they were friends. Thanks guys that was a great read.

Very good point Phin, the mushroom would have to do a bit more work than simply adding a hydroxy on the 4 position starting from tryptaphan , it would need to decarboxalate, and add two methyl groups too.

There should be a free public GCMS at every library or university so people like us can use them.

Edited by TurkeyRanch, 13 May 2015 - 09:35 AM.

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#44 Headinthehills

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Posted 13 May 2015 - 01:25 PM

I'm on my phone at the moment but tonight I'll find the tryptophan study and link that too. I want to say it was Goetz that used cubes and his fruits had 3.6%! But the study is unverified by colleagues.
Here's a link that I found right now. Seems they're not sure if it's being produced from tryptophan.
https://www.erowid.o...synthesis-2.pdf

Edited by Headinthehills, 13 May 2015 - 01:31 PM.

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#45 Headinthehills

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Posted 13 May 2015 - 01:33 PM

^^how not if.

#46 Phineas_Carmichael

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Posted 13 May 2015 - 01:39 PM

I'm on my phone at the moment but tonight I'll find the tryptophan study and link that too. I want to say it was Goetz that used cubes and his fruits had 3.6%! But the study is unverified by colleagues.

...

Here's the Gartz paper from 1989 that you're probably thinking of:

 

Planta Medica 55 (1989) page 249 - 250 Jochen Gartz

BIOTRANSFORMATION OF TRYPTAMINE IN FRUITING MYCELIA OF PSILOCYBE CUBENSIS.

Jochen Gartz 

Institute of Biotechnology, Academy of Sciences of the GDR, 

Permoserstrasse 15, GDR-7050 Leipzig, German Democratic Republic

Received: March 13, 1988

 

ABSTRACT

Mycelial cultures of Psilocybe cubensis, with the ability to form psilocybin and psilocin de-novo, also hydroxylated and methylated fed tryptamine to give psilocin in up to 3.3% dry mass of the obtained fruit bodies. By using HPLC and TLC, it was found that these mushrooms contain only a small amount of psilocybin (0.01-0.2% dry mass). The values of psilocin are the highest described in any mushrooms.

 

INTRODUCTION

Psilocybe cubensis (Earle) Sing, is a subs-tropical mushroom and contains the indole alkaloid psilocybin and only small amounts of its dephosphorylated counterpart psilocin (1-4). Variations in these metabolites have been well demonstrated by investigations of fruit bodies cultivated under controlled conditions of a rye-grain medium (2) and rice substratum (3), respectively.

The study of psilocybin biosynthesis in submerged culture of P. cubensis showed that radioactive tryptamine functioned as a better precursor than tryptophan (5-7). It was found that not less than 22.4% of the psilocybin formed was derived from the labeled precursor tryptamine (5). The level of psilocin was generally zero in the mycelial tissue from these experiments (5-7).

In the present paper, the bio-transformation of fed tryptamine in fruiting mycelia of Psilocybe cubensis is described.

 

MATERIALS and METHODS 

 

Cultivation of Psilocybe cubensis

 

A dried cow dung/rice-grain mixture (2:1) with twice the amount of water was used to obtain fast fructifications without casing of a strain (3) of Psilocybe cubensis . A 25 mM concentration of tryptamine (as hydrochloride) was added to this medium. Cultivations without the addition of tryptamine were also tested. The methods of cultivations were described in (3).

The first sporocarps were produced by cultures of Psilocybe cubensis in 3 to 4 weeks. The cultures continued to produce mushrooms in five flushes. Each flush was harvested as soon as the sporocarps were mature. The mushrooms were immediately freeze-dried, sealed in plastic, and stored at -10 degrees C until analysis.

 

EXTRACTION and ANALYSIS The extraction procedure and the analysis of the indole alkaloids by using HPLC and TLC were described in the previous papers (3,8-10). The presence or absence of tryptamine was demonstrated by TLC as described by Stijve et al. (11).

 

RESULTS and DISCUSSION

The cow dung-rice mixture actually produced the first flush of mushrooms earlier than the cultivations on ry (with casing) (2) and rice (3), respectively. They yielded an average of 3 g dry mass per 10 g substratum.

Under the same culture conditions, the fructification times, the yields, and sizes of the mushrooms as well as the bluing feature (3) were equal when the growth media also contained high concentrations of tryptamine. Initial experiments without the addition of tryptamine were performed to determine the content of psilocybin and psilocin in comparison with experiments using other culture conditions and/or media (2,3).

The levels of psilocybin and psilocin varied from one flush to the next, but generally were much the same as those in the other experiments (2,3) (table 1). Consistently low levels of psilocin were found in the mushrooms without the addition of tryptamine to the substratum. Additionally, psilocin generally was absent in the first flush as was also observed in earlier investigations (2,3). Table 1 shows that the fed tryptamine gives high values of psilocin in each flush from the cultures.

 

Table 1 Variation of psilocybin and psilocin levels in Psilocybe cubensis as a function of flush number from the cultivations with (a) and without (b) addition of tryptamine (25 mM concentration).

 

Screen Shot 2015-05-13 at 2.39.55 PM.png

 

These psilocin levels are uncommonly high (from 2.1 to 3.3%) since values reported for psilocin in dried mushrooms are always below 1% (1-4,12,13).

Inocybe Aeruginasens Babos contains only traces of psilocin but high amounts of the incompletely methylated psilocybin (baeocystin) (9). In contrast to the intitial experiments without an addition of tryptamine, the mushrooms generally contained only small amounts of psilocybin. The tryptamine level was always zero in each mushroom. In this case no tryptamine was additionally found in the methanolic extract of the vegetative mycelia from the substratum.

In a previous report, Gartz (3) was unable to detect baeocystin in P. cubensis. But Repke et al. (14) reported traces of baeocystin in other strains of Psilocybe cubensis about 10 years ago. They suggested that many non-specific enzyme systems exist in fungi which have the ability to oxidise exogenously added compounds, as well as normal, obligatory intermediates (14).

The results in Table 1 show that the enzyme systems in Psilocybe cubensis have a high hydroxylation and methalation capacity to convert added Tryptamine to psilocin. It is possible that a reduced amount of phosphate in the culture media decreased the bio-synthesis of psilocybin from psilocin in the media.

P.cubensis also failed to produce detectable amounts of baeocystin under these culture conditions.

Acknowledgments

The author thanks the following persons: G. Drewitz, T. Stijve, G.K.Muller, and M. Gey who generously supplied valuable information.

 

REFERENCES

 

  1. Heim, R., Hoffman, A. (1958) Compt. Rend. 247,557.
  2. Bigwood, J.. Beug, M.W. (1982) J. Ethnopharm. 5, 287.
  3. Gartz, J. (1987) Beitrage zur Kenntnis der Pilze Mitteleuropas 3, 275.
  4. Badham, E. (1984) J. Ethnopharm. 10, 249
  5. Agurell, S., Blomkvist, S., Catalfomo, P. (1966) Acta Pharm. Suecica 3, 37.
  6. Agurell, S., Nilsson, J.L.G. (1968) Acta Chem. Scand. 22, 1210.
  7. Agurell, S., Nilsson, J.L.G. (1968) Tetrahedron Lett. 1063.
  8. Gartz, J. (1985) Pharmazie 40, 134.
  9. Gartz, J. (1987) Planta Med. 53, 539.
  10. Semerdzieva, M., Wurst, M., Koza, T., Gartz, J. (1986) Planta Med. 52, 83.
  11. Stijve, T., Hischenhuber, C., Ashley, D. (1984) Z. Mykol. 50, 361.
  12. Beug, M.W., Bigwood, J. (1982) J. Ethnopharm. 5, 271.
  13. Ohenoja, E., Jokiranata, J., Makinen, T., Kaikkonen, A., Airaksinen, M.M. (1987) J. Nat. Prod. 50, 741
  14. Repke, D.B., Leslie, D.T., Guzman, G. (1977) Lloydia 40, 566.

Edited by Phineas_Carmichael, 13 May 2015 - 01:43 PM.

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#47 TVCasualty

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Posted 13 May 2015 - 03:47 PM

Looks like we'd have to eat mushrooms potentiated with tryptamine while still fresh as I'm under the impression that most if not all of that psilocin would be lost upon drying.


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#48 Headinthehills

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Posted 13 May 2015 - 08:15 PM

I think you're right phin.

#49 BushwickBill

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Posted 21 May 2015 - 07:09 PM

Check out Ksylika & Wurst planta medica 1989 paper for info on optimal solvent systems for extraction from fruit bodies.  Been a while since I looked at it but iirc different ratios of MeOH:EtOH worked better for extracting psilocybin vs. psilocin.  Logically i think we'd expect MeOH to be better for pulling psilocybin.

 

I wonder how promiscuous the enzyme catalyzing indole 4-hydroxylation is - can it tolerate other 6,5 bicyclic heteroaromatics?  Benzimidazole would be a logical extension but things like benzofurans/benzothiophenes and even pyridine derivatives come to mind.  Someone get to work cloning this enzyme and let's find out  :ph34r:


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#50 TurkeyRanch

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Posted 22 May 2015 - 02:10 AM

That would be a trick, but I suspect it is more than a single enzyme, more like a series or combination of enzymes and who knows what other process. Isolating enzymes, your chasing the actual biosynthesis route, which seems exceedingly complex, much more complicated than normal synthesis from an appropriate indole! At that point, I say just toss all the lab glass and grow a cake lol.

A combination of an appropriate enzyme that yielded a consistent result combined with synthetic methods might be easier, but then your making 4-aco in a beaker and not growing mushrooms, and that borders on subjects we don't discuss here (synthesis) so moving on. . .

But certainly an interesting line of thought.

Head, how far along are ya? Get any experiments in yet?

Edited by TurkeyRanch, 22 May 2015 - 02:15 AM.

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#51 Headinthehills

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Posted 22 May 2015 - 08:22 AM

This weekend I'm doing the second blind test. The second batch with double the tryptophan is harvested, waiting to do this second round of tests on the first batch. I'm going to try to do a methanol extraction this weekend, if I have time. Summer gets really busy for me.
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#52 Headinthehills

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Posted 28 May 2015 - 07:03 AM

Something I'm noticing, the enhanced subs are pissing all over themselves. Way more than what I normally start to see after two flushes. The one with double the tryptophan almost smells like piss when I first open the lid.
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#53 BushwickBill

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Posted 28 May 2015 - 08:58 AM

Are you intending to extract 'cin and 'cybin?  If so I'd suggest using maybe 1:1 MeOH:EtOH.  Consider that the O-phosphate is charged and therefore much more polar than the alcohol so a mixed solvent system will definitely to be your benefit if a broader spectrum extraction is your goal.  Maybe even put some IPA in the mix.

 

And I hope it isn't the case for the sake of your experiment, but wouldn't it be funny if the extra myc piss was the fungus essentially saying "wtf is this excess of amino acid doing around, better get rid of that shit"  :tongue:  Thumbing their nose at our silly attempt to supercharge them.


Edited by BushwickBill, 28 May 2015 - 09:00 AM.

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#54 TurkeyRanch

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Posted 28 May 2015 - 10:18 AM

Something I'm noticing, the enhanced subs are pissing all over themselves. Way more than what I normally start to see after two flushes. The one with double the tryptophan almost smells like piss when I first open the lid.


Save the metabolites in a jar, and add it to your water for your herb. Super nutrients. Like Bill mentions, it could be the mycelium getting rid of unwanted ingredients in the sub, or maybe it is the byproduct of overdrive biosynthesis of whatever the mushroom is hydroxylating.

If someone ran for president on the platform that all university labs should be open to the public, I might start voting again. With out analysis this is just a huge tease.
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#55 TurkeyRanch

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Posted 28 May 2015 - 10:28 AM

Regarding Bills post, if you use any IPA, make sure it's anhydrous. 99% is available if you can find it, but getting that last bit of water is a trick. Baked magnesium sulfate might grab some, but it will be pulling h2o out of the air while your messing with it.
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#56 Headinthehills

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Posted 28 May 2015 - 03:34 PM

Bill, thanks for the advice. I was going to do straight methanol but from what I think you're saying, I'll get more of the other alkaloids out of them. I actually have ten grams in methanol as we speak.
Turkey, absolutely! We need more hobbyists to be able to access true scientific labs. I'm poking around to try to get an in at a university for some testing. So far no dice, but you never know.
Saturday night we are doing round two of tests on batch one. The more I read the more I'm thinking tryptophan wasn't quite right, tryptamine seems to give it enough of a head start that they're able to make significant potency increases. I'm guessing this will do something but not a ton.

#57 BushwickBill

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Posted 28 May 2015 - 07:58 PM

Dry solvents!! What a careless chemist I've been giving out this advice - you should always always dry your organics with either magnesium sulfate - commonly available as epsom salts - or sodium sulfate if you're working with something more acid-sensitive.  That's the standard workup procedure - extract product into organic, collect and dry organics, gravity filter, take off solvent under vacuum.  Not sure where you'd pick up the Na2SO4 outside the fischer catalog though.  Some people probably use specific amounts of desiccant but my protocol was usually more along the lines of "dump enough in the flask that it looks right."

 

Turkey, I dunno if I'd want university labs freely open to the public  :biggrin: but from my POV in the academic area the trend right now is money money money.  I'm generalizing obviously but if your research isn't ultimately generating leads for pharmaceutically relevant compounds ($$$$), unfortunately nobody seems to give a shit.  So as interesting as what we're discussing here is, it's not gonna get a grant proposal funded  What we need is more groups like MAPS bringing psychedelics into the realm of 'legitimate' i.e. mainstream acceptable science.

 

Here's one way of quantitating indoles but you'd need a colorimeter, which isn't impossible to obtain.  What did you have in mind for measurements?

 

http://onlinelibrary...e9754e71211f1d9



#58 TurkeyRanch

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Posted 28 May 2015 - 10:02 PM

Na2SO4 is available at almost any drug store like Walgreens or any pharmacy. I just got some at the dollar store actually, 1 lb for $0.99, to soak my feet cause I am sore. :)

You pretty much need a vacuum system to get the epsom salt powder out of solvents, get a cheap vac pump from harbor freight if you don't have one. Sidearm flasks and Buckner funnels are pretty cheap on ebay too. Sometimes there are good deals on packages of all three together, and they often toss in filter paper disks and stuff too.

If your going whole hog, get a vac distillation setup, and strip under vac like bill advises. This will prob cost you about 2-300 in glass, if you only buy the glass and make lab racks and heating apparatus yourself (crockpot with hacked thermo control, or a hot plate and double boiler with a thermometer). You can use an aquarium pump and Home Depot tube for circulating water for the condenser on the cheap.
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#59 Headinthehills

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Posted 29 May 2015 - 05:43 AM

I actually have a complete suction kit. I've used it to remove Epsom salts from acetone a few times. I've done this extraction twice, once anhydrous and once with straight methanol. I really didn't notice. I know crystals of the gods are supposed to be dry, but I'm not entirely sure you can get all of the water out of methanol with out chemicals other than Epsom salt. Ethanol needs to be chemically dried beyond 95%. I might need to open my chemistry book again.
I've seen links online using a soxhlet distiller to do the extraction, Rick Simpson style. That's going to be my next lab purchase.

Edited by Headinthehills, 29 May 2015 - 05:50 AM.


#60 TurkeyRanch

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Posted 29 May 2015 - 11:05 AM

If you don't have a distillation setup I would get that first. Soxhlet units are great for extracting lots of stuff, but I haven't found a use for them with cannabis, it over extracts that particular plant. And in general, you can manually do everything a soxhlet does, often better. I had one for a long time and used it ummm, maybe twice. It is great to dry out solvents with thou, I do admit.

Edited by TurkeyRanch, 29 May 2015 - 11:16 AM.

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