Not sure if this is the right place but... How do scientists *know* it's mainly one alkaloid as opposed to 'synergy'?

Engage

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Just a short of meta-question, I guess.

Or, something everyone else knows that I don't. :D

Not sure if synergy is a scientific term. (Though, life evolved like that, right?)

Anyways, do scientists have some "sorter" that they put kratom into and it spits out something akin to a 'nutritional facts' list of molecular ingredients?

How do they know it's one thing causing, for instance, kratom's anxiolytic effects and not a few together or all or something else entirely?

Reality is so friggin complex, the endless interactions, in all dimensions, yikes, it's easy to just give up trying to understand the details and to trust that others have it down, I dunno. Lol. Anyways, I found myself thinking, "meh, nobody knows,", then "someone on bluelight might" and thought I might learn something new.
 

sekio

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1. Acquire a natural product with a known effect, e.g. cinchona bark for treating malaria.
2. Subject the natural product to extraction by some chemical means (vary solvent, pH, temperature as needed), check which extracts contain material that still produces the desired drug-like effect
3. Iteratively separate/purify compounds out of the crude extract, measuring which have the desired effect (via chromatography or other chemical manipulations)
4. Spend time determining the structure of the pure compounds which produce the strongest drug-like effect, e.g. quinine.
 

Engage

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So, it's all theoretical guesswork? In that case, well done everyone! Still, can't wait for the 'molecule sorter' to be invented.
 

Engage

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I don't know. Why would I ask a question I already knew the answer to? If that's like what I asked about above then thanks.
 

Hodor

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I don't know. Why would I ask a question I already knew the answer to? If that's like what I asked about above then thanks.
As sekio said, we already have a "molecule sorter" - it's called a chromatography column.

Basically, a chromatograph is typically a cylinder filled with a special porous material that separates the constituents of a sample based on the time it takes them to transit the column. Depending on the column material , the compounds can be separated (for example) based on their solubility in different solvents, or their size.

Often, these chromatographs are directly connected to a detector - like a mass spectrometer, which ionizes the compounds (allowing you to tell a compound's mass) and partially blasts them to pieces (allowing you to infer details about their structure from fragmentation patterns).

As Medbithead already mentioned, there's also other relevant methods of detection, like NMR ("nuclear magnetic resonance" - it basically tells you which and how many atoms a specific atom is connected to) and IR spectroscopy (infrared radiation makes chemical bonds vibrate, so the frequencies where IR radiation is absorbed lets you draw conclusions on the nature of these chemical bonds).

However, tricky part is actually finding out which of these constituents actually have pharmacological activity - you can attempt to compute whether a compound may fit into a receptor from its structure, or use cloned receptors for an in-vitro assay, or you might have to feed it to an actual lab rat.

If someone had two different species of papaver spp. (i.e. poppies), it would be no problem to infer their psychoactive effects from an analysis of their constituents, because we have a pretty good understanding of what the actual natural opiates (morphine, codeine, thebaine) do.
Kratom, however, has had comparatively little research done on it: We do have some idea on how strongly the major psychoactive alkaloids in kratom bind to opioid receptors (but not necessarily all the other adrenergic, acetylcholinergic, histaminergic, etc. receptors), but still relatively little idea on how they actually behave in living organisms (for example, a compound can be stunningly good at activating opioid receptors in an in-vitro test, but still non-psychoactive if it cannot enter the central nervous system of a living organism - loperamide, for example, is a safe and effective diarrhea medication because its powerful opioid effects remain limited to the bowels).
 

Medbithead

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Hey thanks for the shoutout @Hodor And Very clear
You may want to add in chromatography, at least in simple TLC—is it true— the compounds are separated by solubility in size or in solvents (and thus by solvents, attraction for the column or the solution, where it is understood attraction for the column is what is holding some compounds up)? [time it takes to travel the column, as you said] I believe the compound of interest is then often in solution.
I think gas chromatography may operate by same principle

I forgot about receptors, which is kind of tangential to this discussion.
Not just how they work in an academic sense—which is interesting to talk about—but are pharmaceutical firms still working on synthesis of many new compounds that block the route of compounds of abuse?
Where is the focus lately?

It may be interesting to think about: are the routes of many compounds intentionally blocked by “competitive inhibition” of the receptor (or its enzyme) by another substance in vivo? Or receptor agonist?
Any good examples of these? @Hodor
Does Narcan work by this principle?

Some bell is ringing in my head for meds for myasthenia gravis and competition for the acetylcholinergic receptor—but I think I may have this all confused in memory.
 
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Hodor

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Hey thanks for the shoutout @Hodor And Very clear
You may want to add in chromatography, at least in simple TLC—is it true— the compounds are separated by solubility in size or in solvents (and thus by solvents, attraction for the column or the solution, where it is understood attraction for the column is what is holding some compounds up)? [time it takes to travel the column, as you said] I believe the compound of interest is then often in solution.
I think gas chromatography may operate by same principle
There are different types of chromatography... you can separate compounds based on lipophilicity, size, their ionic nature, interactions with antibodies...

But yes, the bog-standard type of analytical chromatography (the so-called "Reverse-Phase High-Pressure Liquid Chromatography) works like this:
You have a tube, a few inches in size. The material inside is silica gel, which is made non-polar (hence "reverse-phase") by coating the polar silica surface with non-polar organic chains. The time it takes for a compound to traverse the column is mostly dependent on two factors:
For one, the polarity of the compound matters, as a non-polar substance is obviously going to be more likely to temporarily get stuck on the non-polar surface. The other is the polarity of the "eluent"/"mobile phase", i.e. the solvent you use to flush the sample through the column. Making the mobile phase slightly less polar is going to increase the speed at which a non-polar compound "elutes" (i.e. when it exits the column). Sometimes, you'll use one solvent to flush through all the stuff you *don't* want while the target compound is retained in the column, then switch up the solvent mixture to rapidly elute the compound you do want.

Gas chromatography is different in that there is no interaction between the compound and the mobile phase. Since the eluent is an inert gas - typically helium - the speed of the elution is determined solely by how strongly the analyte sticks to the inner coating of the "column". Oh, and the "column" isn't really a "column" in the conventional sense, but rather a glass capillary wound around a spool, with a total length of ~30 meters and an inner diameter that is a fraction of a millimeter. While there are definite drawbacks to GC (for one, the compound needs to be able to be vaporized without burning up), it has the major advantage that the gas can be directly fed into a mass spectrometer, meaning it is ideal for checking a sample for the presence of small molecules (which is why forensic drug testing is often based on GC/MS, i.e. a gas chromatograph for separating the analytes, combined with a mass spectrometer for identifying them).
 

Medbithead

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Just thinking about this @Hodor, (Thanks for playing!) since I had the opportunity to review hydrostatics recently.
What if the specific gravity of the compound is nearly equivalent to the specific gravity of the solvent (you call it “eluent”)—then do you have an adhesion problem with the tube? May be problematic for testing. Is this ever considered
And how likely is that? (But maybe identified by meniscus formed?) Are there any natural metabolites like that?
I may be mixing principles but I’m just naturally curious.
Vaporized without burning up in gas phase, is another problem you presented I’d like to ruminate on. Of course if you have any hints...

WRT gas chromatography discussion: Vaporized without burning up in gas phase. Known compounds have known heats of condensation, yes? How do you determine heat of phase change temperature for unknown compound?
 

Hodor

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Just thinking about this @Hodor, (Thanks for playing!) since I had the opportunity to review hydrostatics recently.
What if the specific gravity of the compound is nearly equivalent to the specific gravity of the solvent (you call it “eluent”)—then do you have an adhesion problem with the tube? May be problematic for testing. Is this ever considered
And how likely is that? (But maybe identified by meniscus formed?) Are there any natural metabolites like that?
I may be mixing principles but I’m just naturally curious.
You're mixing up chromatography with sedimentation here.
Specific gravity generally doesn't play into it. The solvent is called an "eluent" (from the latin word for "washing out") because the tube is open. You keep removing the eluent on one end, and feeding new solvent from the other; also, you will often speed up the process by feeding the eluent under high pressures (we're talking hundreds of atmospheres in analytical HPLC devices).

Also, there are different solvents and column materials used for chromatography. It may very well be that a specific eluent or even the whole principle of separation doesn't work for your sample; but that is why there are so many alternative variants of this technology.
The most commonly used combination in high-pressure liquid chromatography involves a non-polar column material with a polar solvent ("reversed-phase HPLC"), but originally column materials were polar, with the eluents being apolar ("normal phase"). And sometimes, you can vastly improve the separation by gradually altering the composition of the eluent during the elution ("gradient elution").

There are also cases where you basically *want* the tube to be able to retain a specific analyte pretty much indefinitely in a given solvent, so that you can thoroughly wash out everything else, then adjust the the eluent composition to rapidly elute the purified analyte.

Vaporized without burning up in gas phase, is another problem you presented I’d like to ruminate on. Of course if you have any hints...

WRT gas chromatography discussion: Vaporized without burning up in gas phase. Known compounds have known heats of condensation, yes? How do you determine heat of phase change temperature for unknown compound?
Compounds that form strong hydrogen or ionic bonds evaporate at markedly higher temperatures (ex.: the reason H2O is a liquid while H2S is a gas is that H2O forms much stronger hydrogen bonds). This means that acids and amines, for example, may have to be chemically converted to esters and amides in order to be able to efficiently analyze them via GC.
 

Medbithead

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Okay I’m familiar with the high melt temp/boiling temp of hydrogen-bonding (“FON”) so okay acids and amines are ensnared. (I can think of one illicit amine: morphine) But if you make the ester salt or the amide —that’s a lot of interconversion—what about the margin of error ? (Other than say percent yield what’s the metric?) You have to recover the original compound for confirmation or no?

I also understand your points above about chromatography.
I feel like you’ve answered all my points but I haven’t gained anything new lol

Okay I also see what you did now. The chromatograph just isolates the sample which you say is identified in tandem with mass spec. Mass spec really does all the work

You should “quiz” me :p Am I one with the fold lol :p
 
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AlphaMethylPhenyl

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In essence: conduct an experiment. There is always just one variable changed by the researchers, and one variable looked at to see if there was any impact. Basic stats: the independent and dependent variable. Randomization. Double-blind. Minimization of bias. Perhaps even a p-value.
 

Medbithead

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Forgive me if I wasn’t clear @AlphaMethylPhenyl I’m looking for the variable that would tell the researcher amount recovered (percent yield?) since @Hodor alluded to conversion of the compound to avoid vaporization in gas phase for GC
Also I find it interesting to think of potential pitfalls with experimental methods; guess that’s my MO here ..
 
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