As the name is self-suggestive, the isolation of compounds are followed for the plant extract and the subsequent fractions' bioactivity testing results. The extract showing desired activity is tested and a defined threshold of expected activity is taken as good to be pursued further for investigation only to be followed for the active fraction from multiple major fraction/sub-extracts which is then pursued further to sub-fractions till the activity is demonstrated at the threshold levels. The most active sub-fractions or the FINAL active fraction beyond the described sub-fraction is obtained to analyze and isolate the active constituents for the bioactivity for which it was pursued. The sub-fractions or later fractions can also be combined (enriched) to give the defined/desired (threshold) level of activity to be pursued for isolation and activity confirmation in the isolated compound(s).

Plant material

The plant materials were collected from Artvin, east part of Turkey during the flowering period in July 2014 and identified by Prof. Dr Ozgur Eminagaoglu, specialists of Plant Taxonomy at Department of Forestry Engineering, Faculty of Forestry, Artvin Coruh University where a voucher specimen was deposited (No: ARTH 5247). The O. rotundifolium was dried in shade, finely powdered for extraction process.

Extraction and isolation

Origanum rotundifolium aerial part was dried and powdered (1.0 kg ±2.0 g) then extracted with hexane (3 × 1 L) for a week at room temperature, filtered and solvent was removed to obtain hexane extract. Solid material of hexane extract was re-extracted with ethyl acetate (3 × 1 L) for a week at room temperature, filtered and after removal of the solvent, ethyl acetate extract was achieved. Methanol extract was acquired by extracting (3 × 1 L) the solid part of ethyl acetate extract for a week at room temperature. The solid part of methanol extract was heated with distillated water (500 mL) at 80 °C for 2 h, filtered then 100 mL of which was lyophilized to get the water extract. The other part of the water extract (400 mL) was partitioned with ethyl acetate, and ethyl acetate phase was separated by separator funnel, dried with (MgSO4), filtered then solvent was removed to obtain the water/ethyl acetate extract revealing the most antioxidant activity and including the highest phenolic contents. Therefore, chromatographic techniques were applied for water/EtOAc extract to isolate the bioactive compounds. The water/EtOAc extract (14 g) was subjected to silica gel column chromatography (100 × 2.5 cm), eluted with a solvent system with increasing polarity from hexane to EtOAc and EtOAc-MeOH. 400 fractions, each 100 ml were collected. After checking the thin-layer chromatography, the compounds having same Rf values were combined. Apigenin (1) was isolated from the fractions of 154–182 with further purification of silica gel column chromatography. The fractions 183–188 included the ferulic acid (2). 190–220 fractions were combined and chromatographed on silica gel to isolate vitexin (3). Caprolactam (4) was isolated as pure form from fractions 230–260. Rosmarinic acid (5) was isolated from the fractions 300–330. The fractions 340–400 were combined and subjected to column chromatography to isolate the globoidnan A (6).

more on

Article Bioassay-guided isolation, identification of compounds from ...

Check https://link.springer.com/content/pdf/10.1007/978-1-4899-1778-2_6.pdf

Check:

https://doi.org/10.1016/j.sajb.2010.09.011

https://doi.org/10.1155/2016/9729275.

These journals may help out.

Thanks you Nnyeneime Bassey

check

https://link.springer.com/content/pdf/10.1007/978-1-4899-1778-2_6.pdf

It is a common practice in isolation of these bioactive compounds that a number of different separation techniques such as TLC, column chromatography, flash chromatography, Sephadex chromatography and HPLC, should be used to obtain pure compounds.

Check https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3218439/

Hello Prof. Rauf,

Check on this paper.

https://www.hindawi.com/journals/bmri/2016/9729275/