I agree with Punit in the selection of the solvent system, which is always depends on the phyto-constituents of your sample, but if you want to extract the whole active ingredients (crude extract), I recommend pure methanol or pure ethanol, through maceration for about 72 hrs with concomitant shaking.

Regarding the selection of animals, usually rats and mice are preferred for the toxicity studies, and the selection of either male or female gender depends on the target of study, in which certain physiological factors sometimes interfere with the results (for examples hormones like female sexual hormones could antagonize the effects of inflammation-induced toxicity).

Hope it is help to clarify your opinion.  

In fact the WHO guidelines for safety studies recommends the use of both sexes of rats or mice so normally two different animal models are used.  For efficacy studies depending on what you are working on rats, mice guinea pigs, rabbits etc can be used, mostly male animals

The  best method of extraction is dependent on the type of solvent you are using which is determined by the way it is taken traditionally. However, cold extraction is preferred because the  heat labile compounds are expected not to be affected.

Extraction must be related to the traditional use of the plant. I do not know the type of constituents that you have in this plant.  But my earlier studies, in vivo and in vitro, on other plants have shown that alcoholic extracts are more toxic than aqueous extracts.

Hi Anthony

I think you should extract the materials taking solvents of increasing polarity. Start with hexane/petroleum ether followed by ethyl acetate, chloroform, ethanol, water. This will enable the separation of the components of the plant material according to their solubility in different solvents with increasing polarity. That will also help to fractionate the extract which will be helpful further in finding the bioactive fraction too. Isolation studies would also become more convenient. Soxhlet extractor can be used for these purpose to attain complete extraction. 

Regarding second part of your question, I would like to quote OECD-423 as below:

Normally females are used (9). This is because literature surveys of conventional LD50 tests show that, although there is little difference in sensitivity between the sexes, in those cases where differences are observed females are generally slightly more sensitive (11). However if knowledge of the toxicological or toxicokinetic properties of structurally related chemicals indicates that males are likely to be more sensitive, then this sex should be used. When the test is conducted in males adequate justification should be provided.

References for above para as listed below:

(9) OECD (2000) Guidance Document on the Recognition, Assessment and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation Environmental Health and Safety Monograph Series on Testing and Assessment No 19.

(11) Lipnick R L, Cotruvo, J A, Hill R N, Bruce R D, Stitzel K A, Walker A P, Chu I; Goddard M, Segal L, Springer J A and Myers R C (1995) Comparison of the Up-and Down, Conventional LD50, and Fixed Dose Acute Toxicity Procedures. Fd. Chem. Toxicol 33, 223-231.

In case of any query, feel free to ask.

Gud Luck and regards 

Thank you guys. Will take your answers into consideration..

Dear Anthony.

A colleague told me that you shouldn't test crude extracts on an in vivo model unless you know which is the active compound (for dosage).

If you are testing an unknown crude extract or medicinal potion you must document in detail exactly how you got the extract, and provide some chromatographic profile. Is very important to take this in account when you do your work.

About the in vivo test. you must establish which via you are going to apply the extract. maybe orally? maybe intraperitoneal? Please be nice with the little guys.

take in account that in order to publish your work you must have a letter of permission of a bioethics comitte of your institution.

you must perform a toxicity test. Please see the attachment. If you do this first, you will be doing an internationally and bioethically correct job. because what is the point of testing an extract in vivo if it will be toxic? Now, If you have passed the toxicity test and your extract is not toxic, then you should make 3 groups of mice. one control and you do to them all that you do to the others using only the vehicle (extract solvent) the positive control group you apply to them the prescription medication used to heal humans in the appropriate dose (mg/weight kilograms). Then your test group. About this group you must answer two questions: What dosage? how often? there are several formulas related to pharmacokinetics that can extrapolate the in vitro activity to the expected in vivo activity intraperitoneally.. but if you are going to test by oral dosage... You will need to search more formulas.

About how often, you must know very well your extract and find support in traditional medicine use.

The parameters you can measure in the mice after your test, are many, always measure the most parameters you can... so you don't have to repeat the experiment ever again... killing mice is not a nice task but still necessary to science.

Collect and measure toxicity in organs such as heart, liver, lungs, brain, intestine and kidneys. (histopathology), blood chemistry levels, if possible, urine presence and quantity of metabolites related to your extract (extract by products).

Now, You must think whether it is important or not to test a second group of mice using a higher or lower dose of extract.

A teacher of the Doctorate (Dr. Manuel Loyola Vargas) said once to me: "good experiments get good results, and make good publications."

Good luck with your research