One more thing, about the gramine phenotypes Dozuki commented, this was proposed by Marum et Al 1979, (with some similar evidence in Gander et al 1976) and reviewed in Festi & Samorini 1994. It does seem to be true in general but there are exceptions. For example IIRC benzyme personally tested one phalaris (brachystachys?) with LC-MS that had significant amount both of gramine and DMT, and als in the Gander article the talk about paper chromatography of Phalaris that contained both gramine and other tryptamines.

Also another thing about what you commented, Dozuki, is that beta carbolines arent always trace. The same Gander et al 1976 talks about Phalaris arundinacea clone R16 containing large amounts of DMT and trace amounts of 2MTHBC, while clone R38 has the inverse, large amounts of of 2MTHBC and low of DMT. Also clone R504 had large amounts of DMT with intermediate amounts of hordenine.

I think safest bet to get DMT out of phalaris is to grow AQ1, and follow the conditions given by the samorini article to increase alkaloid content.

I was going off of memory, and believe this to be an accepted genetic model, however a simple one.



In my original post I was referring specifically to P. arundinacea. This is the species that I have focused most of my attention on. I'm sure that the other species of Phalaris will differ. The articles done with PC were shown to not adequately separate the compounds and Ehlrich's reagent (the viz. used) showed the same colors for T,D and 2 of the beta-carbolines. This is stated clearly in the above referenced Gander et al 1976, The occurrence of... paper:





Thanks for pointing that out. I corrected the above post. Also, all 3 genotypes have shown hordenine according to table #2 in Gander et al 1976. One of the things about the older papers on P. arundinacea is that you have to look at the procedural section to determine if they were actually getting good separations with the compounds, especially with paper chromo. The Gander et al paper is a good one because they did GC-MS and clear a lot of that up. The same mistake can be made with TLC if not careful. This error in the earlier papers led many to believe that G, M, and T could all occur with one another, while in fact they don't when carefully going back through the older articles with this knowledge.

I also ran across a paper that showed a clone with beta-carbolines only in one example, so there *might* be that possibility, unless they were mistaking one of the beta-carbs for a main compounds I will have to go back and look into that further.



While this may be true, I'm personally still more interested in P. arundinacea as it is pretty much established as wild stands of grass pan-globally. It is also more tolerant of extremes in weather conditions. But that doesn't mean the others don't need looked into as well. Probably also helps that there is a ton of it around here

-D.

ginkgo wrote:

..hey ginkgo, i hope i didn't create the impression gramine was without any toxicity, i said it probably wasn't "acutely toxic", meaning that the amounts present in a typical vaporized (30-50mg) extract should not cause serious problems (even if all gramine), in single administrations (i should have added) ..no i don't know for certain, but am basing this on what i've been able to read..of course i would advise extreme caution in bioassaying it..most substances display some toxicity at some dosage..

now, i don't have the full Bourke 1988 paper (a friend may), nor most of the animal study papers, but as far as i can ascertain from abstracts, the toxic effects, particularly serious ones, were the result of either huge doses (400-600mg/kg! repeat: per kg) , or long-term extended feeding [Goelz et al 1980] (but this could also have been due to it's MAOI activity [Ho et. al 1970;])..lower doses (e.g. 10-30mg/kg I.V.) were found to be 'psychotropic' but not otherwise harmful to sheep..here's a rat-feeding gramine paper abstract Response of animals to dietary gramine.

.."may have ephidrine-like effects" is taken from Voogelbreinder (2009), sorry no reference there..
that book also notes that gramine appaered a few years ago as a purported human health supplement..from Designed Nutritional Products:
"Suggested use is as a sedative and nerve tonic"...suggested doses are 100-400mg (see p.403)
i can't verify it, but, interesting..they don't seem to stock it anymore, but some human data must have been aquired

and finally, it appears in a lot of plants, including phalaris spp which have some history of ingestion. so i would think if gramine were 'acutely toxic' in humans we would have some data on this by now..
the death of animals due to Phalaris aquatica 'staggers syndrome' may be due to MAO inhibition interaction (ß-carbolines), or unknown compounds, and the grazing of massive amounts..from Bourke et al 1988 (Aust Vet J. 1988, Jul; 65(7):218-20.)

gramine (3-(dimethylaminomethyl)-indole) was also apparently "behaivourally-active in rats" [Gessner et al. 1961]

i'll see, when i get time, what more i can find out..
.
ps. i think Phalaris may be 'the way of the future' so let's all research together..

we need some method dev in the area
of prep LC, i don't want to be the only one working on it.

was thinking along the lines of silica treatment with tween, or adogen 464 (which I have).
the latter may be able to separate the acidic variants, as an anion exchange resin. you can treat with whatever reagent you want to visualize it, or run tlc plates all day, but only a bed of stationary phase will suffice for separating any usable yields

Another possibly interesting detail for Phalaris extraction:

Justin Case (in Trout's Notes) claims that when extracting DMT from Phalaris, it is important to, immediately after harvesting, soaking the leaves in alcohol. If this is not done and grass is just dried, enzymatic processes will break down the alkaloids, reducing yields significantly. But even in alcohol apparently after a while there will be loss of alkaloids, so processing should begin as soon as possible.

Im not sure what would be the ideal in this case, soak in alcohol and then as soon as possible evaporate it and then extract, or maybe extract straight away. Maybe mixing the fresh leaf with a base, pulling with acetone and adding FASA to precipitate fumarates would work, to help fast separating the alkaloids from possible enzymatic activity?

Benz, can you clarify if indeed you have tested a Phalaris that had significant amounts of both gramine and DMT? Do you remember the relative ratio of alkaloids? Do you still have the mass spectra/TIC somewhere? What species of Phalaris was it?

Also, why prep LC? I mean, sure its interesting but isnt that very limiting for the chemists or very chem-inclined only? I guess ideally we should develop a simpler extraction method for all (which may be already there, I would guess limo or xylene and FASI/FASA, re-convertion, and heptane/hexane re-x, should be able to separate alkaloids from plant fats, as well as from gramine, but this is still untested). But then of cours another issue is to fist of all make sure that the specific Phalaris strain is high yielding.

relative signal intensities were 90 and 10 for 188 m/z and 174, respectively (p. brachystachys)

I mention prep LC as a broad term to describe any methodology using a column of silica or alumina to separate the analytes of interest; this is accessible, and more useful than TLC, for collecting solute fractions. if one can do TLC plating, the next logical step is column chrom.

..benzyme, i recall detailed separation of indoles papers using LC with cation exchange resins,
have you covered these elsewhere or could you explain a bit about them?
..endlessness..maybe what benz is talking about could be expanded on in Separation Techniques threads..?

as a matter of fact, I recently did some method development of SCX (strong cation exchange, resin with alkylsulfonyl groups) of monoclonal antibodies. it involves using a mildly acidic binary buffer system (pH 5-6) with two different concentrations of NaCl. this method may also be applied to free base alkaloids

I agree that LC would be the next logical step for one who has been doing TLC. It should be an easier way to separate the alkaloids/compounds than with a liquid/liquid extraction procedure. I don't think that it is necessary per se, but might shed some light on research. TLC, OTOH, seems almost essential for coming up with a liquid/liquid extraction procedure for verifying that wanted compounds are actually being extracted.

Some standardization for TLC might be helpful if multiple people are researching this way and comparing notes. Using similar/same procedures should produce very similar rf values as well as make things repeatable. Looking at the literature (that I have) there are some suggestions for standardization. Also it seems that the compounds at least in P. arundinacea can be tricky to separate and identify correctly using TLC. I feel this needs addressed and worked on.

What would the goals of such research be? A liquid/liquid extraction technique for a certain compound? A way to identify a high alkaloid producing plant? Finding a plant that displays a particular chemical genotype? A way of determining the alkaloid content of a new/newer spp. of Phalaris, or other grass for that matter?

I think the toxicity factor needs looked at as well. Some of the early reports of Phalaris use in The Enthogen Review report a 'Toxic effect'. I also remember seeing a post here. Johnny Appleseed addresses this in The Entheogen Review Vol. X! No. 1, 2002: (My paraphrasing) Talking of doing an A/B extraction, one of the two reasons for doing it is to eliminate 'a somewhat toxic substance' that is otherwise left in an extraction of the grass when it is only simmered in slightly acidic conditions for a more prolonged period of time. Later he states: 'Thus, when a research colleague reported to me that the 15 minute quick-simmer seemed to extract the tryptamine alkaloids, but leave behind the toxic elements in Phalaris, I realized that this was the breakthrough we had been looking for with Phalaris. I have spent the past eight years in breeding work trying to isolate the toxicity, but with no success.' From the article it seems that he is more interested in Phalaris as an Ayahuasca Analog for a more traditional type of 'Brew' and this (I'm presuming) is the reason for the breeding effort.

When comparing this to the research of Gander et al from above, it would seem that Hordenine could be the culprit, as it was found in all three genotypes of Phalaris a. Presuming that this is the species he was working with. While this would possibly counter Gramine in this instance, I think its potential toxicity is still in the equation.

Good post Dozuki. I do think its important we indeed question what is would be the goal. I think the ideal outcome of this would be to find one (or more) phalaris seed vendor, that the phalaris grown under easily-reproducible conditions X (many of which are already expressed in the Festi & Samorini paper) would result in a good yielding and alkaloid profile using a tek with easy-to-find materials and proceedures (or a few different proceedures).

In the case of grasses with co-occuring DMT (or 5-MeO) and hordenine and/or gramine, it would be interesting to find a good way to separate the alkaloids. Both gramine and hordenine seem poorly soluble in pet ether/heptane/hexane/naphtha according to Merck, so a simple recrystallization (or two or three) in those solvents could help out.

If one would have some hordenine and gramine standards, what would be good things to test? I was wondering if FASA or FASI would form precipitates with these compounds. What else?

Regarding which seeds, I think AQ1 seems like a good bet. Some people would love to explore wild grasses found near them for obvious reasons, but I would guess this is a bit like finding needle in a haystack.

Why do you think its hordenine? I mean, if hordenine is found in cactus, and from info we can gather on safety of cactus ingestion, it doesnt seem toxic, right?

My initial Hypothesis that hordenine may be a contributing factor in the reported 'toxic' effects are based on the above quotes from Johnny Appleseed. My reasoning is thus: It would seem from his writings that he has mostly worked with Phalaris a. which was shown to have hordenine in all 3 genotypes by the Gander et al 1976 reference. Also, in the Appleseed article he is talking about Phalaris 'big medicine' which he bred for a DMT specific strain (tho he never directly states that it is Phalaris a, just a cross from his breeding program). This would then seem to factor out gramine, if it is indeed Phalaris a. Looking at the properties of hordenine from the Merck Index, 7gm dissolves in 1000ml (1l) of H2O (.7 gm in 100ml and .07gm in 10ml) which I believe would put it into the slight soluble category and it also states 'needles from H2O'. Appleseed's statement that a short (15) simmer eliminates the 'toxic' effect would point to a substance that is only slightly soluble in lightly acidic conditions, and this would increase with heat and time. By simmering for a shorter time he *might be* pulling out less hordenine, though it could also be something else.

One factor of this that possibly points away from hordenine is that he is using a slightly acidic simmer using lemons. The Merck Index 1983states that the hydrochlorides of hordenine are very soluble in H2O, but no mention of the citrate, if that is indeed being formed with his simmering. The solubility of the citrate is an unknown.

So, there are my reasonings for and against that hypothesis, but is something that could be sussed out with a little bit of work.



I think that testing them with FASA and or FASI would be good. It would also be good to run these in a standard solvent system on TLC plates to try and get some reference Rf values for them. This was also my thinking of trying to get a standard set for testing with TLC in my above post (the Nexus standard?). It would also be good to see how these react to different visualization reagents on those plates. Xanthydrol, Ehrlich's, Marquis and possibly Sillicotungstic acid. I think in the case of hordenine I would also be interested in the solubility of the citrate. And if you really wanted to push things you could start filling in the holes in the solubility of both compounds. However, that is a lot to ask, and those would be my suggestions of things to look at.

Whichever Phalaris species is studied (the more the merrier I believe) it looks like the seperation of T,M, and G would still be a good thing to come up with. Its been quite a while since I've looked into P. aquatica, but I do have a reference from Mulvena and Slaytor 1983 that shows all three of these compounds in 7 day old seedlings and another by Mulvena et al 1983 titled: 'Methoxylated Gramine Derviatives from Phalaris Aquatica', and yet another reference for beta-carbolines from P. aquatica. I don't know if this follows any proposed genetic model like P. arundinacea or not. But it does show that experiments will be running into same/similar compounds when studying either of these two species. I do not have any info on P. brachystachys other than the Festi and Samorini 1994 article and the scattered info in some of the Entheogen Review back catalog that I have. That being said, I personally feel that those three are very likely candidates for further investigation.

no way.

liquid-liquid is a binary solvent system, LC uses at least a binary solvent system, and a stationary phase. the dynamics are a lot more complex than LLE and even TLC, since it involves more surface area; therefore, it requires some skill to do it properly, technique like the old-school "slurry" method. it's definitely an art which requires some practice, especially in a method dev context. IEX is even more complex.

simple LLE probably won't separate gramine from DMT, but a charge-based assay (IEX) will;
but if you believe the contrary, then develop an LLE method, and I'll come up with an IEX method, and compare notes.

reagents for visualizing (i.e Van Urk's, etc.) may also be used in column chrom.
here's a simple one I did in a glass pipette using a single solvent (EtOAc), with a sample (ergolines) treated with van urk's. Better selectivity may be obtained using a binary, tertiary, or quaternary mobile phase system, of course

..Dozuki, i recall those E.R. articles, but don't remember the kind of claimed toxicity being described..G. Samorini reported some Phalaris extracts as being much more potent than the amount of tryptamines would indicate..
..the Bourke et al. 1988 paper, mentioned earlier, from it's POV established that 5meoDMT, gramine and hordenine were NOT responsible for acute phalaris aquatica toxicity..they led to CNS 'nervous' 'toxicity'..only in extreme doses did they display physiological toxicity..in fact, this paper could be said to have found 5methoxyDMT to be more 'toxic' than gramine or hordenine..
..ß-carboline MAOIs have been suggested as the cause of acute toxicity as they interact with potentially toxic dietarily acquired amines..
..i have heard of human ingestion of hordenine, a friend told me it was slightly sedative..i don't know the dosages..
sure, it's possible there could be unknown strong toxic effects of hordenine or gramine in humans, but there isn't strong evidence to date..like endlessness said, we do not know..

ps. benzyme, thanks for the picture!

I just had an idea. DMT has a melting point of 44-68°C, 5-MeO-DMT of 66-70°C. Gramine doesn't melt before 138-139°C, and other impurities have melting points such as 88°C for NMT and 117-118°C for hordenine. If you were to warm an impure extract to no more than 70°C, the DMT and 5-MeO-DMT should become liquids, while the impurities should stay as crystals. If you were to warm this extract from below in a coffee filter, cheesecloth or similar, the liquid should pass through, while the impurities should stay as crystals. Couldn't that be an efficient way of removing gramine from an extract? That is, if the DMT and 5-MeO-DMT would really pass through.

On the subject of gramine, I've found some more literature, and it points to what nen888 said, that gramine isn't toxic in small doses. Still I think we should be cautious. It is a medium potent 5HT-2A antagonist, which means it will counteract effects of DMT and similar psychedelics, as they do most of their magic on that very receptor type, so gramine should nevertheless be removed from an extract. We also need a way to separate DMT and 5-MeO-DMT. Maybe one of their salt forms have different solubility properties, enabling one to separate them.

Hordenine is a stimulant, by the way, it is well documented as a stimulator of the release of norepinephrine, and is used by body-builders.

I thought of the same the other day, ginkgo, but did you ever try this in a mixture of crystals with different melting points? I thought that possibly when its a mix, the whole thing will change the melting point, so that you couldnt really separate it (sort of like in the movie Blow, when they are testing for the purity of cocaine by the melting point). Maybe im wrong though ...

What makes you think it isnt toxic in small doses?

As for 5-MeO-DMT and DMT, indeed separating through solubilities might be interesting, but it might be that solubilities are very similar with all typical salt forms. Another possiblity: How available is silica gel for most people ? A column with silica gel and dissolving the 5-MeO-DMT and DMT in acetic acid (maybe even crude vinegar) should work well... I think it was rf 0.5 and 0.4 with 2N acetic acid as eluent, I can check later. We could even try to get some standardized column method to work, using 5% white vinegar, and as a column use a glass pipette of a certain size (maybe with a good supplier link for it that others can buy the same) filled with a certain amount of silica gel. Then measure the volume of eluent it takes to elute one and the other. Given a certain column width and amount of silica and how tight its packed (which could be standardized by volume it occupies in column), and the temperature, and eluent, it should be reproducible.

Any other ideas?

Gramine has been used as a 'health supplement', and have high LD50s of 44.6 mg/kg IV in mice and 62.9 mg/kg IV in rats. In sheep it shows toxicity symptoms first at 500 mg/kg orally, and the sheep survives. This is supposedly the alkaloid that kills so many sheeps. I think that a combination of the alkaloids, with the beta-carbolines in particular, are responsible for the sheep deaths. Beta-carbolines are known to be toxic to animals. I think we should still be careful with gramine, it's not something we want to smoke, but I'm thinking that is mostly because it counteracts the effects of DMT. That's enough reason to stay away from it anyway.

Silica gel is commonly available as drought absorbent bags that come with many products, see this picture. I've read it is a pain in the ass to crush them to a fine powder though. I'm attaching a guide on how to make your own TLC plated with silica gel. I've not read through it yet, so I'm pretty green at this subject. It would be awesome if you could work on some ideas concerning TLC and separation of compounds. Do ask me if there's anything on your mind, but as I've said I know practically nothing about testing and separating compounds, however I know practically everything else there is to know concerning Phalaris grass now. Think I've read over a thousand pages the last month!

there are a few threads on the matter, it's been kicked around quite a bit.
how to do TLC has been thoroughly discussed, we need more Rf values from repeated experiments, and positive ID.

..thanks for putting that together Ginkgo , the TLC paper looks handy
the original main reason i pointed to gramine not being highly toxic in small amounts was that, if it was present at say 5% or less, removal may not be crucial. While it may have as one if it's actions the 'opposite effect of dmt' synaptically, at what relative concentrations to dmt does it achieve a relative counter effect? ..possibly at much higher doses..that said, I dig TLC and separation techniques, so thanks for all the info., benzyme, Dozuki & endlessness too!
..a quick thought, if gramine and hordenine have higher melting points, could an extract be carefully vaporized below say these temperatures so that only the dmt vapour would be inhaled..?
.

we've also discusssed that in threads regarding sublimation; we concluded that this method would be the most difficult to achieve, given the necessary control of
ramping up the temp under reduced pressure.

some references list the boiling point of DMT at 160 C at 0.8 hPa, that's approx 375 C @ atmospheric pressure. having attempted sublimation in vacuo before, I find this value more accurate than the ones previously listed

again, the 'simple' route would be to separate over a bed of stationary phase.

..i'm a fan of the simple way..worked previously for experiments...