The issue of Gramine

[dithyramb]

Dear Nexians,

As a pursuer of the Phalaris grass I am feeling the need for more clarity on this alkaloid. There is a general caution about it, which is a good thing when it comes to an unfamiliar alkaloid, however I could not find conclusive evidence that gramine is detrimental to human health in the amounts found in grass.

İt is obvious that cattle eat way more grass than in human psychoactive consumption.

According to the research I have uncovered so far: the proposal that cattle sudden death syndrome is caused by gramine has been disproven. That it is part of the neurological poisoning symptoms in the staggers is possible but not well studied.

Experimental evidence that tryptamine alkaloids do not cause Phalaris aquatica sudden death syndrome in sheep - PubMed

The acute toxicity for sheep of 3 alkaloids that occur in Phalaris acquatica was examined by intravenous and oral administration. The lowest tested dose rates that produced clinically observed signs were, for 5-methoxy dimethyltryptamine, 0.1 mg/kg body weight intravenously and 40 mg/kg orally...

pubmed.ncbi.nlm.nih.gov

https://www.researchgate.net/publication/268078891_Phalaris_toxicoses_in_Australian_livestock_production_systems_prevalence_aetiology_and_toxicology

Gramine (a tryptamine) and
hordenine (a tyramine) and 5-MeO-DMT have also been implicated
in cases of phalaris ‘sudden death’, although symptoms of ‘staggers’
could only be produced by experimental administration of 5-MeO-
DMT

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

On the general safety and properties of gramine, here is what I have found so far:


Safety evaluation of an oat grain alkaloid gramine by genotoxicity assays

These results indicate significant antioxidant, non-mutagenic as well as non-genotoxic activity of gramine in vitro and in vivo in the given doses.

Gramine derivatives as neuroprotective Alzheimer medicines

Gramine (Donaxine) | Reverse Transcriptase Inhibitor | MedChemExpress

Gramine (Donaxine) is a natural alkaloid isolated from giant reed, acts as an active adiponectin receptor (AdipoR) agonist, with IC50s of 3.2 and 4.2 µM for AdipoR2 and AdipoR1, respectively. Gramine is also a human and mouse β2-Adrenergic receptor (β2-AR) agonist. Gramine (Donaxine) has...

www.medchemexpress.com

Gramine (Donaxine) is a natural alkaloid isolated from giant reed[2], acts as an active adiponectin receptor (AdipoR) agonist, with IC50s of 3.2 and 4.2 µM for AdipoR2 and AdipoR1, respectively[1]. Gramine is also a human and mouse β2-Adrenergic receptor (β2-AR) agonist[2]. Gramine (Donaxine) has anti-tumor, anti-viral and anti-inflammatory properties[1].

Potential Neuroprotective Effects of Adiponectin in Alzheimer’s Disease

Gramine: A Vasorelaxing Alkaloid Acting on 5-HT2A Receptors
Article in Planta Medica 70(4):373

These results suggest that gramine is a vasorelaxing agent acting mainly by antagonism at 5-HT (2A) receptors.

GRAMINE - Latoxan Valence France

In vitro, it inhibits acetylcholinesterase and butyrilcholinesterase. At 1/5,700 concentration, it lowers the brain and blood serum cholinesterase activity by 61 %.

Chemistry of N-(1H-indol-3-ylmethyl)-N,N-dimethylamine (Gramine): A Review - Pharmaceutical Chemistry Journal

link.springer.com

Gramine was reported to exhibit radioprotector activity
[333]. Experiments on sea urchins showed the absence of
embryotoxicity [334]. Gramine hydrochloride introduced to
experimental animals induced tremor, moderate lacrimation,
and cyanosis with characteristic coloration of the skin (re-
lated to insufficient oxygen supply developed in cases of car-
diac insufficiency), salivation, clonic convulsions (deliberate
contractility of a muscle or a group of muscles), and loss
within an hour. The LD50 upon peroral administration is
542 mgkg for mice and 575 mgkg for rats. The intravenous
introduction of gramine hydrochloride in a dose of 50 mgkg
led to the loss of experimental rabbits. Gramine belongs to
the group of moderately toxic substances and produces no
skin-resorptive, irritant, cumulative, and allergic effects

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

Experientially, orally in combination with rue, I have felt significant toxicity from crude extracts of certain wild Phalaris aquatica strains and Phalaris paradoxa. I cannot know if gramine played a role in it. I could classify the toxicity into two kinds. First, the cardiovascular complications, which felt very dangerous and bordering on lethality. I still assume that this is caused by tyramines. Second is the neurological symptoms - tremors, a disconnection, and unconsciousness. This is also highly undesirable. I am not sure which components might be the cause, and gramine might play a role in it.

In my brachystachys experiences so far I have not experienced any of these toxicities, and I don't know if it contains gramine or not as I don't know how to recognize gramine.

I hope that the issue of Gramine gets fully elucidated for the whole community.

 

[dithyramb]

That's a lot of properties; gramine has a complex pharmacology in humans.

A lot of it is positive: antioxidant, acetylcholinesterase inhibitor, antiviral, antitumor, anti-inflammatory, neuroprotective.

Some of the rest I don't know how to interpret.

The LD50 seems to be high.

The last source indicates it is "moderately toxic" and has no cumulative toxicity. We don't know the doses used in that study, and the symptoms include tremors and convulsions.

I have two specific questions.

1) Are the neurological toxicity symptoms (tremors, unconsciousness) that I have experienced with P. aquatica due to gramine? The "disconnection/unconsciousness" effects were like an inhibition of the effects of DMT. If not gramine, what else could it be?

2) If so, is the toxicity dose dependent, and is gramine safe or even beneficial in lower doses? I am thinking of beta carboline's, especially harmaline. These molecules are also said to be toxic and bring on serious symptoms with excessive doses and unusual administration methods, but are highly medicinal in appropriate doses.

 

[endlessness]

Thanks for the topic and adding sources

Interesting that gramine is 5-HT2A antagonist, I hadn't seen that paper before.. This suggests it can diminish psychedelic effects of tryptamines. So even if it's "safe", it's probably unwanted in combination with psychedelics.

How did you prepare your 'crude' extracts?

Regarding the effects you felt, it's very hard to say because phalaris is a mixed back of alkaloids. People normally worry about gramine itself but there are other gramine analogues as well as other alkaloids of unknown safety profile. According to festi and samorini (1994), these are the alkaloids sometimes found in phalaris:

NMT (n-methyl-tryptamine)
5-MeO-NMT
DMT
5-MeO-DMT
Bufotenine
5-Me-Tryptamine
5-MeO-Tryptamine

2MTHBC
2-Me-6-MeO-THBC
2,9-DMe-6-MeO-THBC

Gramine
7-MeO-Gramine
5,7-DMeO-Gramine

Hordenine

According to Trout, phalaris also occasionaly has 3-methylindole (known to produce Bovine Pulmonary Emphysema in cattle)


IMO considering the variability in genetics and unknown safety profile of many of those compounds, I would not recommend people to consume crude extracts of Phalaris.

I'd advise people to extract and test with TLC or at least colorimetric reagents. And specifically about gramine, it is possible to detect it (and differentiate from just DMT) in an extract even in small amounts with mandelin (as shown here)

 

[dithyramb]

Hey Endlessness. Thanks for your reply

I had been suspecting that gramine might be blocking DMT, also taking into account the 5ht2a antagonism.

To prepare my crude extracts, I simply boil in water with vinegar, multiple washes and combine and reduce.

Wild Phalaris aquatica and arundinacea are often a whole cocktail of alkaloids, and the aquaticas which I have come across have been super potent. Unfortunately I could not find any study revealing the alkaloid content of brachystachys. It supposedly has a cleaner alkaloid profile.

 

[dithyramb]

I am very happy to have found a strain of P. brachystachys which (experientially) lacks all the incompatible alkaloids for an ayahuasca analogue - Hordenine, tyramines, 5 MeO DMT. All but gramine. After a time of study I believe I can now recognize gramine and it's undesirability despite it's relative harmlessness. İt is sedative and blocks/buffers the effects of DMT as a 5HT2A antagonist.

Gramine is said to be practically insoluble in water. Could I be feeling it in my crude water extract because of the vinegar I add in the boiling process? Could it be that if I boiled without vinegar, gramine would not be present in the crude water extract?

 

[endlessness]

Gramine as a freebase is insoluble in water (merck index). Most salt forms, including the one naturally in the plant material, will likely be soluble in water, so it doesn't matter if you added vinegar.

I personally don't believe us humans are equipped with such sensitivity as to be able to subjectively tell apart different ratios of psychoactive + potentially toxic compounds (specially if you don't have a baseline to compare to, having access to pure amounts of all possible compounds involved and testing the different combinations) . It is not possible to discount self-suggestion and other psychological factors at play, as well as the natural variability of subjective experiences even with the same substance.

I highly suggest you acquire some sort of analytical method, whether its TLC or just reagents (like, say, marquis, mecke, mandelin and ehrlich).

I'm attaching Festi and Samorini's paper. Indeed I couldn't find much info on brachystachys phytochemistry from a quick search.

Good luck either way and keep us informed!

 

[dithyramb]

Thank you very much for your help, Endlessness.

I personally don't believe us humans are equipped with such sensitivity as to be able to subjectively tell apart different ratios of psychoactive + potentially toxic compounds (specially if you don't have a baseline to compare to, having access to pure amounts of all possible compounds involved and testing the different combinations) .

İ know that the dominant paradigm here is purely scientific and subjective experience is not considered reliable. İt's probably the way it should be. I personally believe we have the capacity to be as sensitive as you describe, even if not tapped most of the time in the modern lifestyle. Traditional ayahuasca practice is based entirely on study through subjective experience, and it requires highly strict dietary conditions including periods of isolation. I have been doing such diets, with this paradigm, to the best that I can in my life circumstances.

My baseline gold standard to compare my Phalaris experiences is chacruna, with which İ have hundreds of experiences. İ am not saying this to claim authority, but simply trying to explain where I am coming from.

All my Phalaris experiences with the exception of P. truncata have involved sleepiness and a "distancing" of the DMT effects. This was extreme with a strain of aquatica, leading to unconsciousness. I have had two strong experiences with a strain of brachystachys and on the second experience İ recognized this effect, less pronounced than in aquatica,

Thanks for the festi and Samorini paper. According to this study P. brachystachys has a single alkaloid - DMT and has no gramine. However I remember reading an analysis on this forum which found gramine in brachystachys. İs it true that there are strains with only DMT (no gramine)? Will I have to find them or is there a way to purify the tea from gramine.. This is one important question for me at the moment. I think finding such a technique would revolutionize our relationship with the grass, opening the door to the possibility of a true local ayahuasca analogue using P. brachystachys.

Thanks for your recommendation for analytical methods. I might catch up with that some day.

 

[endlessness]

I don't discount the possibility we can tell some things apart. But which things, in which doses? And how can we know if we're mistaken in some particular case or not? The only way is to test it, right? I'm of the opinion that we should always question our own assumptions, and experiment to prove/disprove impartially (if it's something that is possible to test).

I definitely don't doubt that you have extensive experience with chacruna or phalaris, but the question is whether the perceived difference you have really does relate with a specific alkaloid content. There's always the component of self-suggestion, different dosaging with different batches, and also it can be other alkaloids but not the ones you are supposing. So to eliminate some of these different unknown factors it would be ideal to test if possible.

But yeah take your time in informing yourself about the analytical methods. Here are a couple of links:

TLC Megathread - How to test plants, extracts and other drugs - Plant Analysis and Substance Testing - Welcome to the DMT-Nexus

Colorimetric test results (Marquis, Mecke, Ehrlich, etc) for different alkaloids - Plant Analysis and Substance Testing - Welcome to the DMT-Nexus

As for brachystachys, I had forgotten but as I mention here, benzyme has tested one sample containing 9:1 DMT to gramine. One more data point to consider.

 

[dithyramb]

Alongside valuing the traditional approach, I agree with you that testing/analysis is a very important tool for us modern day explorers. Thanks for the helpful links, I will note them down for if I decide to learn this work. İt definitely seems there is a void here, with very little analysis done on the grass. I am surprised to see this. Just one analysis on P. brachystachys throughout the history of this forum?

Are there any others out there who can offer their analysis results for P. brachystachys?

Also, is there anybody out there equipped with the skill to do analyses, willing to receive my seeds for testing?

I also have a special strain of Syrian rue I have been harvesting from the wild for a decade, exceptionally feminine and compassionate which needs to be tested to understand the chemical aspect of it's uniqueness.

 

[dithyramb]

Endlessness, here is a post from you from the link you shared:

Im not sure if this was noticed for those looking at this thread but hot limonene pulls on that wild arundinacea (sample 123) yielded equal amounts of DMT and Gramine, while the room temperature limonene pulls in the Yugo Red (sample 2661) greatly favored the ratio to DMT and only had small amounts of gramine. This is a good indication that, hot limo is much less selective and will pull more of the potentiall toxic alkaloids, while room temperature limonene will nicely pull DMT but only very little gramine.

It appears you compared results from two different strains of arundinacea, so are you sure this proposal holds?

If so, do you think the same would apply to water, i.e. that a cold water extraction would leave out most of the gramine?

...

Also, I have no idea if this would make any sense, but is there a possibility that juicing the grass could leave out gramine..?

 

[Jagube]

If so, do you think the same would apply to water, i.e. that a cold water extraction would leave out most of the gramine?

I can't answer with certainty, but this case is very different for the following reason:

Water is a polar solvent and (optionally acidified) water extractions aim to pull alkaloid salts. The comment amount limonene pulls, on the other hand, refers to non-polar pulls from an alkaline soup, which aim to pull freebase alkaloids.

There is no general rule that would say that if an alkaloid (such as gramine) in its freebase form is considerably less soluble in cold (vs warm) non-polar solvents, then its salts are considerably less soluble in cold (vs warm) water.

Alkaloid salts are ionic compounds. Dissolution of non-polar compounds (such as alkaloid freebases) in non-polar solvents is a very different concept to the dissolution of ionic compounds in water. The former is driven by entropy, the latter is driven by polarity (opposite electric charges) and happens through dissociation into cations and anions, a.k.a. ionization. The intra- and intermolecular forces at play are very different in these two cases. So I wouldn't expect any correlation between the solubilities of a compound in these two different 'worlds'.

That doesn't mean the answer to your question is no, but if it's yes, the fact in the limonene quote is unrelated to it.

 

[dithyramb]

Thank you, Jagube.

 

[dithyramb]

I just came upon a quote by Appleseed that drying reduces the "unwanted alkaloids."

The last factor to consider is whether to dry the grass
or extract the alkaloids from the fresh material. Drying tends to reduce the alkaloid harvest, but in some cases, especially that of the Turkish variety of P. arundinacea, drying may reduce the presence of unwanted alkaloids. — Johnny Appleseed

I wonder if that means / includes gramine?
If so would drying really destroy more gramine than DMT?
What about oven drying which is said to preserve the DMT content?

Pleasantly surprised today from a GC-MS analysis of a methanol extraction of some oven-dried phalaris brachystachys. I heard talk of "up to 3% DMT", but I was sure it was only hype. I don't have a "calibration standard" for DMT but compared to some MHRB crystals, it could very well be 3% (of dried weight). The other good news was that the gramine peak was only about 1/20th or less of the DMT peak.

Does that translate into a 20:1 ratio of DMT to Gramine?

 

[bezevo]

seems i read .........somewere that when extracting grasses that gramine would not pass from an Aquious solution tnto Naptha ?
but of coures DMT Does ..
does anyone have info or an opinion on this ?

thanks

Bez

 

[braindrops]

HERE, in the section "3. Presence of other potentially undesirable compounds", it says

Gramine and hordenine no longer seem to be a problem, since they appear to be barely soluble in naphtha and d-limonene at room temperature. See: HERE, HERE & HERE

There are still some uncertainties with other compounds, however, and though some of them may only have been detected in negligible quantities so far, the high variability of relative alkaloid levels warrants further investigation. See: HERE

Some prevalent compounds are 2-MTHBC, 5-MeO-NMT, and 6-MeO-2-MeTHBC(?).

Anecdotal reports suggest some pronounced differences in the effects of vapourised phalaris extracts (vs. MHRB extracts, etc), and it's unclear how much of this is attributable to 5-MeO-DMT, or to other compounds that weren't able to be separated during the extraction. Significant quantities of NMT may also be a problem, as well as bufotenin.

These various compounds need to be ruled out as benign, separable during extraction, or only ever occurring in negligible quantities.

And then this is said in the "How Can I Extract DMT From Phalaris?" section of the FAQ

Regarding what solvents to use, hexane/heptane or equivalent solvents such as naphtha should work well as gramine is very poorly soluble in it, but if using naphtha make sure that it doesn't contain xylene or aromatics mixed in because they would pull gramine and other unwanted alkaloids.

 

[brokedownpalace10]

Following this thread with interest.
Desmanthus illinoensis appears to have high Gramine, without the 5-Meo, etc..
However, it also appears to have low yields (usually).

I have had a member report to me a good Ayahuasca experience with Desmanthus at 20 Grams.
That would be too low a percentage to extract, I think. But perfectly fine for Aya brews.

It's an easy plant to grow, a weed.

 

[Jahvisions]

Also following with interest.

Also agree that subjectivity can play a mayor part, and a lot depends on dose and set and setting. Also indeed without testing you can't make a definite conclusion which alkaloids give negative side effects.

Maybe I am contradicting myself but I crude extracts give more negative side effects which I felt of which I was sure this was not caused by dose or set and setting. Take for example: eating whole cactus(different story and maybe wrong example), but although (maybe) there is no stuff that gives toxic side effects or to less to notice. Digesting a cactus means you body has to process that and work and all of us know your stomach and intestinnes won't thank you for that.

So generally speaking although some other alkaloids can have a positive synergy. Until that is proven and there is a way to keep only the positive and get rid of the negatives you want the highest purity (leaving more accurate dosing out of the equation). Since you should assume that any other alkaloid can be potentially harmfull or cause negative side effects and should therefore be seen as unwanted.

Again, some give postive synergy but it's my personal believe or hypothesis/assumption that all other stuff in general often will or can be of more harm then good.

So the most interesting question for me is, not if it contains gramine, if it's harmfull or not, or other alkaloids, can be the cause side effects. Why take any risks while you can also dose more accurate?

The main question is: which methods, extraction steps, solvents and purification steps are needed to be left with only 99% of pure product of the active substance(s) you are looking for. But I guess there is no definite answer for that since if don't all the alkaloids which it contains?

TLC probably then is the best option. But is everything visible then? All alkaloids I think are also visible with ninhydrin? Whatabout terpenes, isoquinolines?

Endlessness suggests: (like, say, marquis, mecke, mandelin and ehrlich). Which ones exclude what?

I hope I don't have to buy all this and doing multiple extractions and recrystallizations with all kind of solvent combinations? Sure to be sure it's pure. Of course a melting point test is a good indicator but don't this is in someone kitchen without having the right and expensive materials.

Probabably it writin already somewhere eIse, but for everyone who reads this thread.

Is there an easy extraction method that can everyone can do? (and maybe also inexpensive, with or preferrable without special equipment needed and a relatively easy and safe procedure) So everyone can be pretty sure to have a save, pure product without worrying about gramine).
(I don't mean for kids, of course you should not do anything if you have no basic understanding of the principles of what you are doing. You don't have to be an expert chemist but if you are seriously interrested and make a effort and do some reading you at least avoid stupid accidents)

And if not, what are the methods that have proven succesfull an provide certainty? And If there is no proven method that will give certainty, which following tests are needed then so one can be sure? (I don't no all the Rf of all alkaloids in all solvents, so what am I seeing then on my TLC? I only know if there is one or more alkaloids but not what it is, do I? Or is there a reagent that specifically reacts only on DMT and not Gramine, or vice versa?)

 

[Sidisheikh.mehriz]

Personally i think at least gramine is safe in low doses even with rue. I strongly believe that tyramine + MAOI is the real culprit for the most pronounced reported toxicity (cardiovascular).
My second bet is the betcarboline content of some phalaris strains. They are poorly understood family of betacarbolines and some of them are highly potent MAOI'S.

Betacarbolines according to literature are rare in aquatica but gramine is prevalent at different concentrations ranging from very low to very high. Methy-tyramine is even more potent stimulant than tyramine. If your strain of aquatica contains tyramines (majority of strains do) then your only option is to extract to get rid of the tyramines. There is evidence in literature that suggests tyramines does not get through in an acid base extraction.

more recent TLC analysis of the aquatica strain am working with shows that gramine is there in acid base extracts from young seedlings at fair amounts but was absent or in trace amounts in older clones. But i have smoked this strain of aquatica in all it's growth stages from very young seedlings to fully mature flowering specimens both as clones and from seeds without experiencing any apparent toxicity at least of there's any it's not symptomatic in the course of three years.

On the other hand i experienced some cardiovascular toxicity from the same strain in an Ayahuasca from even 1g dry worth of tea with rue. A subsequent Ayahuasca from the same strain at the flowering stage proven very beautiful and balanced without any toxicity noticed so am suspecting the tyramine levels have dropped during that late spring harvest.

I find these observations are correlating with the limited available literature on gramine. But as Endlessness said we have to also think about other analogues of gramine as they might prove toxic.

 

[dithyramb]

Personally i think at least gramine is safe in low doses even with rue. I strongly believe that tyramine + MAOI is the real culprit for the most pronounced reported toxicity (cardiovascular).
My second bet is the betcarboline content of some phalaris strains. They are poorly understood family of betacarbolines and some of them are highly potent MAOI'S.

Betacarbolines according to literature are rare in aquatica but gramine is prevalent at different concentrations ranging from very low to very high. Methy-tyramine is even more potent stimulant than tyramine. If your strain of aquatica contains tyramines (majority of strains do) then your only option is to extract to get rid of the tyramines. There is evidence in literature that suggests tyramines does not get through in an acid base extraction.

more recent TLC analysis of the aquatica strain am working with shows that gramine is there in acid base extracts from young seedlings at fair amounts but was absent or in trace amounts in older clones. But i have smoked this strain of aquatica in all it's growth stages from very young seedlings to fully mature flowering specimens both as clones and from seeds without experiencing any apparent toxicity at least of there's any it's not symptomatic in the course of three years.

On the other hand i experienced some cardiovascular toxicity from the same strain in an Ayahuasca from even 1g dry worth of tea with rue. A subsequent Ayahuasca from the same strain at the flowering stage proven very beautiful and balanced without any toxicity noticed so am suspecting the tyramine levels have dropped during that late spring harvest.

I find these observations are correlating with the limited available literature on gramine. But as Endlessness said we have to also think about other analogues of gramine as they might prove toxic.

As I have also been saying. The major life threatening (cardiovascular) components in Phalaris are likely the tyramines. Gramine is possibly completely safe, perhaps even desirable in the right dose range for some. And the stuff that poisons the nervous system could as well be some beta carbolines like you guys are suggesting. Keep on the controlled studies

 

[Jahvisions]

Don't know if I understand the comment before last one completly?

So a normal A/B extraction does remove all the tyramine for sure (or at least most of it to be relevant)

But it doesn't remove any gramine at all or only just a small part, leaving most behind in the final end-product right?

Or just partially, removing most of it or at least a large part so it "might be" of no or of a lesser relevance?


I think the almost all the effects tyramine, are well researched and are known quite well already and we know it's dangerous with MAOI's.
That is why all food that contains (high) amount of tyramine is on every diet-list if you want to take/do Ayahuasca
(because tyramine isn't broken down anymore by MAO, the tyramine levels in your blood can raise your bloodpressure to such high levels it can be fatal or in the best scenario will leave you behind with a big headache)

I may hope that this common knowledge is known by everyone who takes ayahuasca? So we can at least conclude already then that Phalaris is not suitable in ayahuasca brews? (at least if it is not tested beforehand on tyramine?)

But I am still curious about a convenient or reliable method on how to remove gramine, if a normal A/B extraction doesn't do the job?