Determination of phenolic acid and flavonoids in madimak plant by HPLC-DAD, determination of biological activity

Madımak (Polygonum cognatum Meissn.), belonging to the Polygonaceae family, is a perennial plant that grows spontaneously in our country. This plant is rich in phenolic content. Phenolic compounds constitute the most important secondary metabolite group of plants and are known to have antioxidant, antimicrobial and anticarcinogenic activities. Due to its sensitivity, accuracy and short analysis time, high performance liquid chromatography (HPLC) is one of the most popular analytical methods used for the qualitative and quantitative determination of phenolic compounds. In this thesis study, the antioxidant, antimicrobial and cytotoxic activities of the madimak plant, which was harvested from different regions at different times, were investigated. A new HPLC-DAD method has been developed and validated for the simultaneous qualitative and quantitative determination of 10 phenolic compounds (protocatechuic acid, catechin, gentisic acid, caffeic acid, p-coumaric acid, polydatin, coumarin, resveratrol, ellagic acid, quercetin) in the plant. Antioxidant activity of madimak plants which are collected from Sivas (plant A) and Ordu (plant B) and extracted by soxhlet method was determined by DPPH method. The IC50 values of the plant extracts were determined as 0.781 mg/mL and 0.657 mg/mL for plant A and plant B, respectively. Antimicrobial activity was investigated by diffusion and dilution method. In the Spot-on-Lawn method, the crude extract did not form an inhibition zone against microorganisms. Methanol extract has the strongest antibacterial activity from Gram negative bacteria to Escherichia coli bacteria with a 23 mm inhibition zone; From Gram positive bacteria to Vancomycin resistant Enterococcus faecium (VRE) bacteria with a 20 mm inhibition zone; on the other hand, it has showed the strongest antimicrobial activity against Candida albicans with a 25 mm inhibition zone. An increase was observed in all concentrations of the crude extract, and MIC values in the methanol extract determined as 4.75 mg/mL for all microorganisms. MBC and MFC values were determined as 5.00 mg/mL for Vancomycin resistant Enterococcus faecium (VRE) bacteria and 4.75 mg/mL for all other microorganisms. The determination of cytotoxic activity was performed on L929 and SAOS-2 cell lines by MTT method. Cell viability % of plant A and plant B in L929 cell line was determined as 160.182-121.992% and 156.997-135.546%, respectively. A and B plant extracts showed a proliferative effect compared to the negative control. In SAOS-2 cell line, cell viability % of plant A and B was determined in the range of 111.341-63.492% and 137.141- 99.483%, respectively. While the antiproliferative effect of plant A extract was observed compared to the negative control; the antiproliferative effect of plant B extract was observed only at 10 mg/mL concentration. A new HPLC-DAD method has been developed for the simultaneous determination of phenolic compounds. Chromatographic determination was performed with the gradient program on the SUPELCOSIL LC-18 (25cm x 4.6mm, 5µm) column. The mobile phase, flow rate and column temperature are optimized. In the validation of the developed method, the system suitability was investigated, and it was determined that the developed method was in accordance with the recommended values. LOD and LOQ values were calculated in the range of 0.051- 0.188 µg/mL and 0.169- 0.628 µg/mL, respectively. In intra-day analyzes, recovery % was determined in the range of 99.273-101.032 and RSD% 0.090-0.652; in inter-day analyzes, recovery % was determined in the range of 99.017-101.983 and RSD% 0.059-0.697. The highest amount of resveratrol; the lowest amount of caffeic acid was determined in plants A and B. Gentisic acid compound was not found in both plant extracts.