Try extracting directly in methanol with ultrasonication and then separate the extract.

Try extracting with ethanol or with hydro-alcohol (50:50).

Try simple method by extracting air dried and powdered plant material with 80% (v/v) aqueous methanol at 38 0C (for Methanol Extract) and Triple distilled water (pH-7) at 96 0C (for Water Extract) using thermo-water fixed to 180 rpm. Filter and distil the extract on rotary evaporator to remove solvent under reduced pressure. Keep/store the crude extract in refrigerator in air tight glass bottles for further bio assay/experiments.

I've prepared the extract following the similar procedure but the assay didn't work well.

Dear Abhin

You have not mentioned the species of Origanum which a genus of Mint (Lamiaceae) family. The most common plant Origanum vulgare (Oregano) is a perennial herb, sometimes called wild marjoram, and its close relative Origanum majorana known as sweet marjoram. Oregano reported to be antiseptic as well as a cure for stomach and respiratory ailments. Due to a high content of phenolic acids and flavonoids, Oregano exhibit antioxidant activity. It has also shown antimicrobial activity against strains of the food-borne pathogen. Since your extracts in polar solvents (methanol & Water) are not showing any significant antibacterial activity, there is possibility of finding antimicrobial activity in the extracts derived from less polar or non-polar solvents. Being a plant of mint family, chance of the presence of terpenoid constituents in the plant are there. Terpenoids, which are normally extracted in less polar solvents are also reported to be antimicrobial. Hence, you may also attempt bioassay of pet. ether/benzene extracts of the plant.

Good Luck

Thank you Yogesh for your valuable suggestion. I've been working on Origanum vulgare L. that was carried from mukteshwar and was propagated in Bhimtal. I'll definitely follow your suggestions and hope for better result.

You may try Microwave Assisted Extraction (MAE). We have found it good for extraction of antimicrobials from plant seeds.

Thank you so much Vijay. I will try following your procedure.