dear Md, first thing to do is to choose an extraction method basing on the bibliographic research of the genus ( the corrent secondary metabolites in the genus) then use the extraction to obtain extracts. the separation is basing on your equipment laboratory (if you have HPLC) it(s the best way to separate most constituents of your extracts, but if you don't, you need to use the traditional method by using column chrommatography and preparative TLC to isolate the compounds, each extract need a special protocol or eluents to get the best separation of your natural compounds

It's depend on your plant material and physical properties. you can start with dry plant material or fresh materials. If it is dried metirial, soak in dicholoro methane for 24 h and extractthe crude. then you can purify the compunds using normal phase column chromatography(Silica gel), size exclusion chromatography (sephadex) and HPLC

As Dr. Cheriet mentioned, start with doing some research. Look up your biology, and see what sort of compounds tend to show activity in that biology, and how they were purified. As an example, alkaloids tended to show activity in a muscarinic assay, so we had a short protocol to get those compounds quickly. Also look at the species and genus that is producing your active compound and see what has been purified already, and how those compounds were purified.

Most of us do some sort of screening. I'll describe a method I used for plants. Bacterial/fungal cultures are often done somewhat differently. Most of us will do an initial extraction with a moderate polarity solvent such as methanol or ethanol. Some people will extract their plant starting with hexane, then extract the same material in ethyl acetate, followed by dichloromethane, ethanol, and finally water. Others take the methanol or ethanol extract and dissolve the solid in those same solvents, the solid being mixed with the next higher polarity solvent. One then needs to determine the chromatography. This method gets compounds of different polarity.

I like to do a "column screen". I run several types of columns (silica, alumina, diol, C18, SAX, and SCX) with the ethanol extract. This helps determine which columns might work, the compound polarity, suggest alternative chromatography solvents, and sometimes even suggest the purification procedure.

Here are some links: 

http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/New_Techniques_In_Extraction_Column_Screening.pdf

For the bioguided assay fractionation you need a very simple and reproductible cell based assay to test extract, fraction and pure compounds you will obtained. There is not a standard method for the isolation of pure compound from plant. Depending to the genus and the family of your plant material you can used a lot of method. But the general scheme is: extraction of plant material, using one solvent in the large range of available solvents, next you will performed a fractionation of the obtained extract using a chromatography methods such as flash chromatography, MPLC, CPC and others...furthers, you will purify fractions, using a slight chromatography column, HPLC and/or TLC preparatives. finaly the structure elucidation of compounds will be done using LC-MS, GC-MS, NMR..ect...

Good Luck...

Hi Md Moniruzzaman

First perform qualitative phytochemical testing to find out which type of phytochemicals are present in your extract via chemical tests. Afterwards perform thin layer chromatographic profiling of the extract to see how many compounds might be present in the extract and which one is supposed to be the major one. On the basis of chemical composition and previous findings about the plant, try to figure out the major active compound of your extract or the marker compound. It may be the phytochemical marker or biological marker, not necessary that the activity of the extract will be because of that compound. But it will serve as a identifying parameter for the extract and you can standardize the extract if you have the standard marker with you using HPTLC or HPLC. Now you should try to find out the biological activity of that extract via in-vitro or in-vivo experiments whichever is applicable for particular activity. If activity is evident in the extract, then you can try to isolate the active component via column chromatography or other suitable techniques. Alternatively you can also measure the content of standard compound in the extract and if it has the claimed activity in literature, you can call your extract as bio-active. For characterization of the bioactive fraction or the marker compound, you can use UV, IR, NMR, MS, LCMS, GCMS etc, which will help you elucidate the structure of the marker or standardize the fraction accordingly.

Regards 

Our plant sample is L. Cylindrica.

We have design a tentative procedure. This is-

Will this procedure work properly? If not, What are corrections needed?

Note: We have an HPLC. To the best of my knowledge, our HPLC only allows to detect and quantify compound.

Hi 

Just few modifications required for a proper and flawless study:

2. Use shade drying as far as possible.

10. GC MS is selective. So be prepared if your active compound is not fit for GCMS analysis. May be LCMS would be a better option as a standby. You can use your HPLC to find the Rt of the active compound. If you have it, use it. 

Step no. 1. I am not sure how you are going to develop a mouse model when you don't have any idea of what activity you want to perform. Are you targetting a specific pharmacological activity? If yes, then OK. 

Regards 

Select bioassay and first screen the 70% methanolic extract if it is active then follow polarity based fractionation using petroleum ether(steroid), toluene(terpenoids & alkaloids), chloroform(alkaloid), n butanol(flavonoids& saponins), ethyl acetate(free flavonoids) alcohol (glycosides)and then residuals(water soluble compounds). Now based on bioactivity assay select bioactive fraction and further load to flash column or preparative HPLC to isolate pure compound and check for purity & if required purify by HPLC preparative.

First do a little research on the plant- find out what has been found already. This allows you to determine what sort of compounds from this plant have already been discovered, and if any of them are active in your mouse model.

A Google search quickly yielded:

The structures of arundoin, cylindrin and fernenol: Triterpenoids of fernane and arborane groups of imperata cylindrica var. koenigii from http://www.sciencedirect.com/science/article/pii/0040402068880238;

Anti-bacterial activity was also reported: http://www.ijpsdr.com/pdf/vol4-issue3/8.pdf

Also look here: http://www.ebi.ac.uk/chebi/ and also https://pubchem.ncbi.nlm.nih.gov/ .

Also look in ScienceDirect.com, and the American Chemical Society publications ( http://pubs.acs.org/ ).

Any hits will tell you how these compounds were purified, and alloy you to screen them quickly.

For column chromatography, I like to do a column screen which suggests how to purify compounds. see: http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/New_Techniques_In_Extraction_Column_Screening.pdf and also the attachment in this link.

GC-MS is fine if the compound is volatile enough to run on a GC, many terpenoids will run fine, but many compounds will not. You may need LC-MS as well.

Note that plants produce different compounds depending on their growth cycle- a young plant may produce different compounds compared to an older plant. Different parts of a plant also produce different compounds.

It depends a lot on whether the plant material contains a lot of chlorophyl as in leaves or not as in roots and bark. Extract the dry plant using methanol and dichloromethane (1:1) for extraction of all bioactive compounds. Use column chromatography or HPLC (reverse phase with C18) and (normal phase with silica gel). Choose solvent based on information from literature. If none, search for solvent system that will give excellent separation based on polarity of compounds using Tlc.

If the plant contains the three types of compounds, divide the MeOH extract into two halves. One for flavonoids and steroids by routine chromatographic techniques after fractionation with organic solvents. The second for alkaloids by acid-base procedure after defatting with n-hexane followed by chromatographic methods

To isolate, purify and elucidate the structure of any flavonoid, the method we described in 1994 may best be used. Column chromatography or HPLC with reversed phase C18 material, followed by purification with polyamide, cellulose or silica gel packed columns and elution of column with appropriate solvent system will give fast and excellent results. Reference work in:  Igile, G.O; Oleszek, W; Jurzysta, M;S. Burda; Faunsho M; Fasanmade A (1994). Flavonoids from Vernonia amygdalina and their antioxidant activities. J. Agric Food Chem. 42(11), pp 2445-2448.

plant material need to be extracted on different solvents polar to non polar and the extracts can be subjected to phytochemical screening by TLC. The highly yielded solvents can be used for further extractions and HPLC for charcter elucidation. Bioactivity can be evaluated using different methods like, antimocrobial properties, anti inflammatory properties....

Abridged:

1. Extract using methanol or similar polar solvents (get it concentrated to get the crude extract)

2. Fractionate, using different solvents (like n-hexane, ethyl acetate, methanol; from non-polar to polar). Can use Column Chromatography

3. (if to be used for pharmacological assay, activity-guarded assay should be conducted). This aids to detect the most active fraction.

4. HDLC, or Small column Chromatography, coupled with TLC to isolate the pure compound.

5. Further on structural elucidation, the isolated compound(s) can be characterized using IR, UV, MS, GC-MS, LC-MS, NMR

FReese dry or air dry the plant sample. Mill sample to powder form. Extract with 30 to 80% methanol by reflux over a electro thermal mantle for 3hrs. Filter extract and you may centrifuge at 3000rpm if extract is dirty. Carry out Tlc on cellulose or polyamide plates and run with appropriate solvent mixture. For flavonoids you may use glass column chromatography or HPLC reverse phase, or a combination of both. In both cases with adsorbent material as cellulose or polyamide powder. Elute column with water, then 30% methanol, followed by n-but anoh. Obtain fractions every 1 minute and monitor for flavonoids using Tlc to guide separation and purification. Once pure fractions are obtained evaporate with a rotary evaporator in vacuo, and freeze dry. Flavonoids will be light to deep yellow.. Individual flavonoid compounds will be identified by comparing Tlc patterns, retention times, uv/visit spectrophotometry, mass spectrometry and nmr data.