# When an aqueous extract of a substance is said to be administered at a certain quantity, is the quoted mass analogous to one as original substance?

I'm trying to read studies about the possible toxicity of the leaves of some plant often said to possess positive medicinal properties.

In the studies, performed on rats, it is usually said that an aqueous and/or ethanol extract of the plant was administered at $$x \ \pu{mg/kg}$$ of mice mass, and from that data possible lethal or detrimental dose is extrapolated.

What I don't understand and is not explained in the texts (therefore I assume there is standard practice I'm not aware of) is how concentrated is $$\pu{1 g}$$ of the aqueous extract compared to $$\pu{1 g}$$ of the original fresh leaves. For example, one study simply states, referring to the administered aqueous extract: "The $$\mathrm{LD_{50}}$$ was estimated to be $$\pu{1585 mg/kg}$$." Should one infer that the amount of chemical substances administered in this scenario is equal to that found in $$\pu{1,585 mg}$$ of fresh leaves? Or is it possibly less/more concentrated?

• The denominator in LD is the unlucky animal mass, nothing about the original concentration in the leaves or the extract. – Alchimista Apr 27 at 8:59
• I agree with @Alchimista about animal mass. However, numerator of $\mathrm{LD_{50}}$ is 50% of the lethal dose of plant extract or fresh plant material. We cannot say exactly what it is until we look at the procedure. Hence, It'd be beneficial if you can put the reference. – Mathew Mahindaratne Apr 27 at 10:19
• @MathewMahindaratne yes but for precision: From the data LD we only know the concentration as amount of X per mass of the animals causing the death of half (this is the meaning of 50% ) of them (in absolute or within a frame time, this I don't know). For acquatic organism it can also be a normal concentration (of X in the water where the organisations live), though. – Alchimista Apr 27 at 10:43
• The concentration in the leaves is sort of irrelevant if you are administering an extract, no? I would not infer anything about the amount of fresh leaves that roughly corresponds to the amount of active substance in extract. One important reason is that the amount may vary between leaves and with season, plant, growing conditions...... Even if explicitly stated that an extract was obtained from a certain amount of plant material, there is no guarantee that the yield will be the same next time. It is similar to the yield of a chemical reaction, which may vary batch to batch. – Buck Thorn Apr 27 at 10:50
• *above organisation reads as organisms. Keyboard :( – Alchimista Apr 27 at 10:51

You probably get the $$\mathrm{LD_{50}} = \pu{1585 mg/kg}$$ value by reading the abstract of the paper. That's why you got confused by that value. However, it is not $$\pu{1585 mg}$$ of fresh leaves. It is actually $$\pu{1585 mg}$$ of dry leave extract. I mean dry residue obtained by removal of water from an aqueous extract of dried leaves. The authors have made a stock solution of that dry residue in $$\pu{200 mg/mL}$$ concentration by dissolving in enough distilled water ($$\mathrm{pH} = 6.7$$). Thus, $$\pu{7.9 mL}$$ of that solution is lethal to a rodent weighing $$\pu{1.00 kg}$$. On the other hand, since they have used rodents averaging $$\pu{100 g}$$ of body weight, each of them need only $$\pu{0.79 mL}$$ of that solution to be lethal.

Hope this explain what you are looking for.

Reference:

Olufunsho Awodele, Ibrahim Adekunle Oreagba, Saidi Odoma, Jaime A. Teixeira da Silva, Vincent Oluseye Osunkalu, "Toxicological evaluation of the aqueous leaf extract of Moringa oleifera Lam.(Moringaceae)," Journal of Ethnopharmacology 2012, 139(2), 330–336 (DOI: https://doi.org/10.1016/j.jep.2011.10.008).

• You accessed the full article in order to have that information, I assume, because I can't see any mention of this method in the redacted version. So to be certain, they basically completely (or close enough to) removed the water component from the fresh leaves (possibly then turning them into powder), and then when administering them to the rats they dissolved the dry leaves in distilled water at a 20mg per 1ml concentration? Did they also mention by what factor were the fresh leaves concentrated through dehydration? Thanks. – TLSO Apr 27 at 15:42
• They air-dried leaves and then added certain amount of water. After $\pu{24 h}$ or so, they remove solid leaves particles (say filtration). The water from liquid filtrate was removed in an oven. What remain was a solid residue. That residue they dissolve in water to get $\pu{200 mg/mL}$ concentration. That is the solution they administered to animals. – Mathew Mahindaratne Apr 27 at 16:24
• So they air-dried the leaves in order to have new water more easily filter the chemical components through the dried leaves, and after that re-dried the solution in order to be left only with the dry components they managed to extract from the leaves (and then dissolved them once again in water in order to have a standardized solution to give the rats)? So from that I understand two things: first, it's not explained what is the exact chemical makeup of the dry extract and at what ratios, so even that processed substance might be different from one leaf batch to another. – TLSO Apr 27 at 16:50
• Second, the 1,585 mg/kg result involves 1,585 milligrams of a certain concentrate of chemicals from fresh leaves, and not 1,585 milligrams of fresh leaves, and thus even though we don't know by how much were the leaves concentrated during the dehydration->aqueous extract->dehydration process (quite important), we at least know the approximate LD50 in fresh leaves would probably be greater than 1,585 mg/kg, perhaps several times as much. Yes? – TLSO Apr 27 at 16:54
• You understand it correctly. Actually this is very crude determination. And also, I think its some what sloppy work and the value might be way off. For example, according to their research, $\pu{158.5 g}$ of leave extract is lethal for a man of $\pu{100 kg}$. I know Moringa is eaten in tropical countries as a vegetable (both leaves and fruit). The leave extract is $\approx 20\%$ of leave weight. Hence, if a certain person eats a one pound of leaves for lunch he may endup in a hospital! :-) – Mathew Mahindaratne Apr 27 at 21:35

At the core of your question there is misunderstanding of what the papers you refer to are discussing. The LD per se in not related to the extract concentration nor to the naturally occurring principle(s) in the plants. You seem interested in a relation between the original concentration and the extracts concentration. This will depend on several factors from the molecular properties to the the extraction technique used. Furthermore, note that the extracts can be further concentrated or diluted according to uses and convenience.

In short, the information you are looking for would be typically found in the experimental part.

LD50 is the concentration (whatever expressed depending on the case)* leading to the death of half of the test animals**. These are the data listed in the papers as per your resume. There is not relation to the concentration of the extract except for trivial reason, ie a more concentrated extract will require a smaller amount to reach the same LD.

*it can be the amount of X per kg of the body and thus a practical concentration rather than a chemical-physical one; the amount of X per volume of water (as in the case of fishes); the amount of X per amount of gas, etc.

**unfortunately used even in routine test, when chemical analysis could be adopted instead.

• I understand what LD50 itself refers to, but it must be correlated with a specific form of the substance tested in order for it to be meaningful, no? As you said, if extract B is more concentrated than extract A, the lethal dose of extract B should be smaller than that of extract A. I simply can't find any sentence within the several studies I've read (although not all are entirely available for free) and these basically discuss a substance (plant matter) that is commonly taken as a traditional medicine in its leaf-form. Providing an average extract-to-fresh leaves ratio seems important. – TLSO Apr 27 at 12:35
• That is the point. It is LD of X, not LD of a specific extract. If, say, the daily alcohol intake should not exceed Y, it does not matter (for this discussion) if you drink whisky or beer. Approaching Y alcohol you must stop. Of course one could define a limit to beer wine etc. And the same for the extracts. If you know what LD is, than it is meaningful for sure. Let me add that speed of administration etc can influence toxicity, but this ceases to be matter for our format/scope. – Alchimista Apr 27 at 12:39
• @TLSO of course many things are or can be important. It would sound like "the LD50 for X is Y, this corresponds roughly to three cups of an infusion of Z grams of fresh leaves...". All those aspects cannot be deduced from LD data. In a way I don't see what is your question. One can prepare differently concentrated cyanide solutions. The LD of CN- won't change. And still can be compared to that of another poison. From this comparison, how the solution were prepared is irrelevant. – Alchimista Apr 27 at 12:50
• The way the leaf solutions were made in the study, or perhaps what are the concentration levels compared to the source matter (fresh leaves in this situation) does seem relevant, if it could mean "1,585 mg containing 50% fresh leaves 50% water", or perhaps "1,585 mg containing 70% dry leaf powder 30% water" etc. I didn't understand what were they referring to in the redacted paper, but Mathew Mahindaratne apparently has access to the full paper and he explained what was the mentioned process for creating the doses and what were they composed of. – TLSO Apr 27 at 17:12
• And thus I now understand that the milligrams given refer to a completely dry matter extracted from the leaves through air-drying, extracting in water and then dehydrating the aqueous extract. Perhaps that was the standard practice one might be familiar with, but I didn't see anyone mention this up until Mathew cited it from the full paper. I still would have been interested in knowing how concentrated was the dry mass versus the original mass of fresh leaves, even though such a processed concentrated extract could likely still differ from one batch of leaves to another. – TLSO Apr 27 at 17:16

They have to analyse extracts for the plant drug concentration. They have idea about typical drug content in plant and use accordingly the ratio between solvent volume and plant leaves mass, together with various concentration techniques like vaporization or solvent/solvent extraction.

The drug concentration in final solution is then multiplied by the used extract volume to get the contained mass of the drug. Finally is calculated the drug/mice mass ratio.

Alternative is calculation leaves/mice mass ratio, if the direct drug content analysis is not available. If you do not know what the drug concentration is, you can relate only to the plant mass. Like plant mass / solvent volume, or plant mass / mice mass.

• Perhaps I'm misunderstanding, but you're saying that unless an analysis of the chemicals within the extract + a comparison to the chemicals' concentration within the plant was explicitly said to take place in the study, one cannot say what is the correlation between the chemical concentration of the aqueous extract and that of the original plant matter? edit: I can see you've updated your answer, so I need to read it anew. – TLSO Apr 27 at 7:25
• I guess you got it. If you do not know what the drug concentration is, you can relate only to the plant mass. Like plant mass / solvent volume, or plant mass / mice mass. – Poutnik Apr 27 at 7:30
• OK, so to my understanding they are supposed to standardize the chemical concentration to some manner, but you suggested what's quoted in the results is a drug (as in mass of actual studied chemical rather than the entirety of the aqueous solution)/mice ratio, while at least when I'm reading the studies it seems to refer to the water containing the leaf extract, which as I said isn't clarified in regards to its relative concentration. – TLSO Apr 27 at 7:37
• It would have been fine if I could infer it parallels plant mass/mice mass (even if regarding plant matter chemical concentration could vary widely), but as I said I'm really not certain from the wording this is the case. Unless it has to be the case due to standard practice. – TLSO Apr 27 at 7:39
• Well, particular interpretation obviously depends on the particular wording. It may be they calculate mg of extract per g of mice, with well defining the extract elsewhere. – Poutnik Apr 27 at 7:40