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Are Harmine and Harmaline the only alkaloids extracted by NaCl tek?

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Rising Star
Hi,
I have done the Harmine&Harmaline extraction from Syrian rue seeds.
Thanks for the guide "Harmalas Extraction and Separation Guide".
I know the tek will produce at least Harmine and Harmaline in the final product.
The Syrian rue seeds contains various alkaloids like the followings.
My question is will Harmane/Harmalol/THH also in final HCl as Harmalol/THH have similar chemical formula like Harmine&Harmaline?
Thanks.

Harmane: 0.16%
Harmine: 0.44% (The coatings of the seeds are said to contain large amounts of harmine.)
Harmaline: 0.25%
Harmalol: 0.6%
Tetrahydroharmine: 0.1%
Vasicine (peganine): 0.25%<
Vasicinone: 0.0007%
 
Oh, did you say Harmalol... :lol: :d 😁

Harmalol has an -OH group which makes it soluble during the basing step. Unfortunately, in our extractions we typically thrown it away. This is also what makes psiloc(yb)in difficult to extract.

PS: Take a look at this thread, it talks about other molecules.
 
And what about harmane? Anyone knows how it would behave and does it get picked up in the common rue extraction? Since it is a neurotoxin we better not have it in the end product, i did search about this for some time and found nothing conclusive
 
Sakkadelic said:
And what about harmane? Anyone knows how it would behave and does it get picked up in the common rue extraction? Since it is a neurotoxin we better not have it in the end product, i did search about this for some time and found nothing conclusive

Harmane is soluble in methanol. Is a study [1] they added methanol to a chromatographic column increase resolution of harmane.

The referenced study used ethyl acetate as a solvent and a common base (sodium bicarbonate or sodium hydroxide) to pick up broad spectrum of harmala alkaloids, but unfortunately they measured just harmine and harmaline contents.

I found pKas for harmane:

pKa (Strongest Acidic) 13.72 ChemAxon
pKa (Strongest Basic) 5.97 ChemAxon

I am not a chemist, don't know why there are 2 numbers. Does it mean we need pH over 13.7 to precipitate harmane?

Sometimes I add excess base after pH separation of harmine/harmaline and get bunch of other chemicals (alkaloids?) precipitating. Could these be the companion, possibly desirable harmala alkaoids?

[1] Brobst, Alyssa, et al. "The free base extraction of harmaline from Peganum harmala." Am J Undergrad Res 8 (2009): 2-3.
 
Regarding the pKa, see first image below.
Below the first pKa a hydrogen is added (+1 charge) at the ternary amine
Between the two pKas molecule is neutral (least water soluble).
At the high pKa a hydrogen is stripped (-1 charge) from the secondary amine.

I'm not sure what happens during extraction. I assume it precipitates during the basing step (see second plot), so any trace content in rue seed could make it through the extraction (?). In the plot you can also see why harmalol does not precipitate like the others.

Note: the plots are theoretical (a calculation by the chemaxon program), not real data.
 

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I have analyzed several rue extractions, my own and from other people around the world, and never saw any harmane (it pretty much only contains harmine and harmaline in variable proportions)..

Is it possible harmane was never there in the first place and thats why it didnt show up? Yes... Is it possible you would be the unlucky minority that would get a rue with harmane and that would end up in your final product? Very unlikely but I cant say absolutely not because Im unaware of the solubility of harmane in a basic solution.. My hypothesis is that you have nothing to worry about since Ive never seen it in many different tests.

Also, if you drink coffee, eat barbeque or smoke cigarretes, you'll be ingesting harmane on a regular basis.
 
Loveall said:
Hello Endlessness. Have we checked by marking where harmene not be in the MS? Is that area completely empty or do we see small peaks there from time to time? For example, in this plot from burnt there is a peak to the left of harmalol, considered and unknown.
yeah that's what i thought too after seeing the plots. bcz all the focus is on harmine and harmaline in the studies. i tried to find how much harmane there is in coffee to see if rue would be better or worse but all i can find is blood concentrations
i'm not worried about it but it's good to know
thanks everyone for the replies
 
Yeah harmane is in the NIST database so it should be automatically detected using the software we were using. I can try to find some of the mass spectra files and post here if people want to double check!

As for amounts in food, this one has some numbers: https://www.tandfonline.com/doi/abs/10.1080/02652030400019844?src=recsys&journalCode=tfac19

They seem low in general but yeah, you prob dont want to get that daily in you, specially from multiple sources adding up.
 
thank you endlessness
these numbers are so low, making the 0.16% seem dangerous, the numbers on that wiki article do not seem right though bcz we get 6% alkaloids from rue
 
Sakkadelic said:
these numbers are so low, making the 0.16% seem dangerous, the numbers on that wiki article do not seem right though bcz we get 6% alkaloids from rue
Sakkadelic, do you have any good references for your earlier statement about harmane toxicity? I have seen similar warnings claimed wrt. other harmala alkaloids and I did not find those very convincing everytime I looked into it.
 
Well there's the wiki article and the references in it saying that it is neurotoxic and related to essential tremor, other articles say it's potentially neurotoxic.. looking more into it now i became less convinced haha
In some of these references they say it's a co-mutagenic compound and that it's related tremors and parkinson and some other things and to be honest it's beyond my knowledge, i'm still wikipedia level person :lol:
 
Sakkadelic said:
thank you endlessness
these numbers are so low, making the 0.16% seem dangerous, the numbers on that wiki article do not seem right though bcz we get 6% alkaloids from rue

I attached the article wikipedia references. They macerated the seeds in methanol 4x at 50 C. After that they did their own cleanup. It looks like they did not squeeze the seeds, so they probably did not get all the alkaloids out 😉

attached paper said:
Four grams of dried and powdered seeds of P. harmala seeds were macerated four times with 50 mL methanol at 50◦C in a water bath for 1 h. After com-bination of the extracts, they were evaporated to dryness

They checked their spectral measurement vs. some standards to validate their spectrometer readings. However, the key phrase in the paper is:

attached paper said:
the method could predict the concentration of the alkaloids in the seed’s extract correctly

So they are reporting on the alkaloids in the extract, not in the seeds (per their own words). In general, this is the typical way of reporting results, because how can you tell how well the extraction and cleanup went? That's very difficult, I think. That is why sometimes (I think), depending on the extraction process, some papers find no harmalol while others see some. Then at the end, when comparing their results to previous papers, they give the usual disclaimer that seeds are variable. While that is true, why not also say that the extraction efficiency is another variable? :?: IDK, maybe I'm missing something.

But there is more. Wikipedia seems to have the numbers wrong. From the paper wikipedia references:

attached paper said:
1.84%, 0.16%,
0.25% and 3.90% for harmine, harmane, harmalol and harma-
line, respectively.

Yet wikipedia got two of these wrong when referencing the paper: harmalol and harmaline are flipped in wikipedia! The attached paper is reference [32]


wikipedia said:
Harmane, 0.16%[32]
Harmine, 0.44%[33]–1.84%[32]–4.3%[34]

The coatings of the seeds are said to contain large amounts of harmine.[7]

Harmaline, 0.25%[32]–0.79%[33]–5.6%[34]
Harmalol, 0.6%[34]–3.90%[32]
Tetrahydroharmine, 0.1%[34]

Total harmala alkaloids were at least 5.9% of dried weight, in one study.[32]

Vasicine (peganine),[16] 0.25%[33]
Vasicinone,[16] 0.0007%[33]

Oh, and sorry but there is more. The paper contradicts itself and in the conclusion gives 0.18% for Harmane instead of 0.16% like it does in the main body :shock: Am I missing something???

attached paper said:
the seed extracts of P. harmala showed that harmaline was the major alkaloid (3.90%) and its content was higher than that of the others (harmine, 1.84%; harmalol, 0.25% and harmane, 0.18%).

Moral of the story is that these extract papers seem to report on the measured plant extract, not the plant itself. There is an important factor between the two since alkaloids are lost during the extraction process.

Then, when papers disagree they typically bring up the "plants are variable" comment, and rarely say something like "we are really reporting on the measured extract, we don't know how efficient the extract itself was, so this is another source of variability".

Thing is, to shed more light on extract efficiency, the scientist could keep the extracts separate and trend the amount of alkaloids in each one (e.g. if the macerate a 5th time do they get 0?). But no, they don't typically do this. They have all this fancy extract analysis stuff, but why not focus on the plant material moar? Seems like they are more interested in the reducible analysis techniques once they get to a cleaned up extract rather than the amazing complex and rich plant they started with.

Credit to VDS for grinding the seeds after several extracts and keeping each one separate in his published work (but he was not doing this reductionist/math/spectral stuff). Why can't we get both?

Sorry for all the complaining. It's just that a lot of papers suffer from this issue. They get fancy analyzing the extract and forget about the plant. Then, when they have different results from other papers out comes the "plants are variable" verbiage. Well, what about doing more work on the efficiency of the extraction and cleanup steps??? Or at least mention that explicitly as a source of variation when trying to compare results.
 

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endlessness said:
Yeah harmane is in the NIST database so it should be automatically detected using the software we were using. I can try to find some of the mass spectra files and post here if people want to double check!

As for amounts in food, this one has some numbers: https://www.tandfonline.com/doi/abs/10.1080/02652030400019844?src=recsys&journalCode=tfac19

They seem low in general but yeah, you prob dont want to get that daily in you, specially from multiple sources adding up.

That would be a great cross-check if you are willing to do it. Maybe if the peak is small the auto-detect is not triggered? However, if you know where to look, you may see a consistent little blip in the data. I don't really know though.
 
Here some of the spectras I found, in .MS format. I havent had time to check the history behind each of those samples but seems they are all different rue extracts I or friends did that I tested in the past.

You can open in openchrom, just have to install the agilent plugin to open the .ms files, it should be free, needed an email to get the key to register it after the try phase.

If anyone needs help opening the files let me know.

Edit: For the record, thats what you should see if harmane is any of the smaller peaks:
bXjSPvA.png
 

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Thanks for the data endlessness :d

I checked and cannot see anything showing up in the 182 au range where harmene would be (images below). However, in the Harmalene control (second image below), the peak is at 207 (shouldn't it be 214?). Anyway, even if the calibration is off by 7au, looks like there is nothing in the 182-189 au range where Harmane would be.

Note: This is the first time I do this so I may have done something wrong.
 

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