Hi Camille

Soxhlet extraction will be good for fractionation of your crude sample. You can move from lower to higher polarity solvents and can get different fractions for which antimicrobial activity can be easily tested.

Initial screenings of plants for possible antimicrobial activities typically begin by using crude aqueous or alcohol extractions and can be followed by various organic extraction methods. Since nearly all of the identified components from plants active against microorganisms are aromatic or saturated organic compounds, they are most often obtained through initial ethanol or methanol extraction. In fact, many studies avoid the use of aqueous fractionation altogether.The exceptional water-soluble compounds, such as polysaccharides (e.g., starch) and polypeptides, including fabatin and various lectins, are commonly more effective as inhibitors of pathogen (usually virus) adsorption and would not be identified in the screening techniques commonly used. Occasionally tannins and terpenoids will be found in the aqueous phase, but they are more often obtained by treatment with less polar solvents. See attached paper for more details and relevant references to follow.

Good luck & Regards

@Yes, first you check the antimicrobial activity of the plant extract. If it is antimicrobial, then study the plant for various phytochemicals present in it. The phytochemicals can be studied by using different solvents so as to get the information about the maximum possible chemical compounds. After this, each of the phytochemicals needs to be tested individually for its antimicrobial activity. I think only then one can say that this compound of a particular plant is having antimicrobial activity. Different phytochemicals can be separated using standard procedure.

When you want to isolate the antimicrobial compounds present in plants, first you have to check the crude extracts of a particular plant prepared in different solvents for antimicrobial activities. Starting from non polar solvent to a polar one e.g. start from n-hexane, ether, chloroform,ethyl acetate, acetone, ethanol, methanol and water etc. Prepare extracts of your selected plant in all these solvents and check their antimicrobial activities, then select the extract of that solvent which has the maximum antimicrobial activities e.g. among your plant extracts ethyl acetate extract has the maximum activities , go further for the isolation of compounds from the ethyl acetate extract .

For this you have to follow a specified method of isolation like PTLC and Column Chromatography(CC).

Most antimicrobial natural compounds in plants are alkaloids, flavonoids, and terpenoids and to extract natural products one has to consider reducing the chance of the degradation of the compounds of interest. In looking for procedures you need to be extra careful not to degrade them. If to do distillation, it must NOT be done directly but instead look for other medium like steam to distill off these compounds so that their functionality is preserved. The same case holds true in using solvents, selection of what solvent to use must be based on this as well - stable solvent and NOT than one which is so reactive that would initiate chemical change in these compounds. I attached two files that I believe will be of great help to you.

Here are the file attachments

Here is the other one.

We can use the cleevenger for the extraction of essential oils called hydrodistillation

Soxhlet is ok but there are also many terpenes you can extract through steam or hydrodistillation.

SPE might be an a affordable method to have a rough isolation. Using various stationary phases and solvent you can narrow down the potential classes of chemicals you have in your extract a further move to more precise chromatography.

Isolation of plant metabolites can be a very long process... I am working since 3.5 years on the same root exudate, highly bioactive as crude extract, without having been able to have a clue on what could be the involved compounds! So good luck Camille with this exiting project!

Hi!

Preliminar selection of bioactive extract is important and then cromatography and HPLC bio-guided method,

best regards

use a bioasay-guided fractionation from an active antimicrobial extract. Liquid/Liquid or Solid/Liquid extraction under hot or not cnodition) depend of the preliminar active extract. Chromatography methods are required.for the final isolation of active compounds

Dear friends,

The medicinal plant parts used by the ethnic people are generally at it's fresh and crude form,generally just after collection from the living plants.The green leaf extracts or wet root extracts deffinitely contain many arometoc substances of very negligible amounts.It also contain many phytochemicals at a very minute amounts which can not be evaluated after extraction by polar or non polar solvents.We can not evaluate the cumilative effects of such phytochemicals by present western evaluation system.We should think the subject from another angle.We should think to evaluate plants from its instant fresh extracts.

With regards,

Dr.S.Pattanayak

In addition to the classical extraction methods, low-pressure solvent extraction (Soxhlet, percolation, agitation, centrifugation), hydrodistillation, and steam distillation, you can use more modern extraction techniques, such as ultrasound-assisted extraction, microwave-assisted extraction, and supercritical fluid extraction. I have recently editted the book "Natural Product Extraction: Principles and Applications", by RSC (http://pubs.rsc.org/en/Content/eBook/978-1-84973-606-0#!divbookcontent). Hope it will help you.

you can perform an extraction at room temperature using 1:1 dichloromethane/methanol mixture and you make sure that you will obtain your extract at 40°C maximum on a rotavapor connected to a good vacuum and a water cooler and recycling system. During the fractionation, make sure that the temperature of the rotavapor bath will never exceed 50°C. The obvious one is the selection of a good solvent system for seperation and the good stationary phase.

The extraction procedure is very much individualized ,depending on the compound that you are going to extract. If the compound is thermo-labile (sensitive to temperature) you can go for cold extraction or extraction under reduced pressure. If not soxhelet is the best option as the consumption of solvent is less. Regarding the solvents that you use. , Ethanol or hydro ethanolic mixutre (50:50) is better because that will yield maximum number of compounds. But if the compound that you suspect is non polar (like flavonoids or essential oils) then you may have to go for hexane or petroleum ether extract. If the compound is volatile (like aromatic or essential oils go in for Clevenger extraction. Before proceeding with antibacterial activity, find out the chemical composition by chemical methods (Harborne or any published paper will give the procedure) to asseess the chemical composition (ie, whether it contains alkaloids, glycosides, tannins etc)

Following paper may be useful to you:

Vijay Kothari, Ankit Gupta, Madhu naraniwal (2012). Comparative study of various methods for extraction of antioxidant and antibacterial compounds from plant seeds. Journal of Natural Remedies, 12(2):162-173.

Following article may be consulted.

If you feel any answer is helpful to you, recommend such answer/s.