Effect of extraction methods on phenolic content and antimicrobial properties of pimpinella affinis leaf

It has been well demonstrated that vegetables provide, in addition to other basic nutrients, bioactive substances with beneficial effects on human health. In fact, the consumption of vegetables has been associated with lower incidence and lower mortality rates of cancer and cardiovascular diseases in humans. Increasing demand for natural additives has shifted the attention from synthetic to natural antimicrobial agents. Leafy vegetables are found to be good source of antimicrobial agents. This study was aimed to examine the antimicrobial activity of leaf extracts of pimpinella affinis. Pimpinella affinis is a member of the family Apiaceae. This biennial herb grows up to 110 cm tall and is native in central and northern parts of Iran. In traditional medicine this herb is being used as carminative agent, appetizer, diuretic, antispasmodic drug, antimicrobial, sedative and lactation medication. It has also been distinguished as an antioxidant and antibacterial agent. There are several methods of obtaining extract from plants including maceration, super critical fluid extraction, subcritical water extraction, microwave and ultrasonic assisted method.
After collection from natural habitats of Pimpinella affinis in Mazandaran Province, it was then approved by the Department of Botany of Faculty of Agriculture of University of Sari. The plant was dried in a dry and dark place away from the sun and then was pulverized in the mill and sieved by a mesh of 80 (800 microns). Pimpinella affinis extract obtained by using maceration extraction (ME), ultrasonic assisted method (UAE) and supercritical fluid extraction (SFE). Ethanol: water in 50:50 ratio used as solvent for extraction. Total phenolic content of different extracts was measured by Folin-ciocalteu method. The phenolic compounds fractions were determined using Liquid chromatography–mass spectrometry system. After preparing the mother culture medium, the bacteria were cultured in MHB medium (37 ° C) for 24-18 hours. Stock solutions were prepared from each of the extracts. Serial dilutions of the extracts at concentrations of 0.01 to 10 mg / ml in 2.5% dimethyl sulfoxide were prepared and then sterilized with 0.22 μm pore size syringe filter. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration assay (MBC) were determined by micro dilution method for listeria monocytogenes (ATCC19112), staphylococcus aureus (ATCC25923), pseudomonas aeruginosa (ATCC9027) and Escherichia coli (ATCC25922). Antimicrobial growth was inhibited by measuring the absorbance and ELISA Reader was used to determine the growth rate of microorganisms, and the first house with the lowest absorption read as MIC (mg /ml) was determined. Statistical analysis of MIC, MBC and phenolic compounds of extracts results was done in a completely randomized design and using SPSS software version 20. The comparison of the means was done using Duncan’s test and one-way ANOVA method. In order to reduce the error, all experiments were performed in triplicate.
Total phenolic content of extracts ranged between 1502.25 to 1836.69 mg GA/100g E. The results showed that the ultrasonic assisted method have highest total phenolic content and the least phenolic content was observed in extract which obtained by supercritical fluid extraction. Chlorogenic Acid, Cafeic acid and Apigenin-6-C-glucoside were the predominant fractions in Pimpinella affinis which detected by Liquid chromatography–mass spectrometry system. The least and highest amount of MIC and MBC were belonged to ultrasonic assisted and supercritical fluid extracts, respectively. Staphylococcus aureus was most sensitive and Escherichia coli and Pseudomonas aeruginosa were most resistance bacteria. Pimpinella extract due to having phenolic compounds such as Gallic acid, Cafeic acid, Chlorogenic Acid, Kaempferol and Apigenin showed antimicrobial activity and can be used as natural antimicrobial agent.