The determination of pycnogenol in some of the pine barks which is found in Turkey

Pine barks have been used since ancient times as medicine, syrup, edema reliever and etc. and due to these reasons they are drunk after boiling or applied for wounds. Old people knew this based on their experiences and this infromation was known and verified by the development of science in our era. Finally, scientist have named this mixture extracted from pine barks as pycnogenol. Pycnogenol is a mixture composed of many constituents like proanthocyanidins, flavonoids and etc. It has been known to have antioxidant properties of 50 times more potent than Vitamin C and 30 times more potent than Vitamin E when compared. Pycnogenol is obtained from Pinus nigra in France near the seaside and is commercially available. In this study, pycnogenol components of different types of pine barks collcted from Manisa, Bilecik and Adana regions were analyzed qualitatively and quantitatively after the optimization of gradient HPLC method. At first, the extraction procedure was performed according to the studies in literature. However, oxidation of pycnogenol components were observed during the removal of ethyl acetae phase. For this reason, removal of ethyl acetate step was realized under vacuum using argon gas. The collected pine bark samples were thoroughly grounded, 20 g of pine bark was weighed and then it was extracted in 150 mL of hot water. The sample was filtrated to remove the barks after cooling to room temperature. After, salt was added until saturation and it was re-filtrated. Then, the filtrate was extracted with ethyl acetate and this phase was collected. Sodium sulfate was added to this ethyl acetate phase in order to remove the remaining water. Later, sodium sulphate was also filtered and the ethyl acetate was removed by argon with vacuum. The solid residue which was off-white was dissolved in 25 mL of 25% methanol-water (v/v) mxture. Afterwards, the solution was injected to the instrument. The samples were analyzed only after the optimization of mobile phase composition. As the mobile phase composition, water, methanol and acetonitrile were used each containing 0.1% ortho-phosphoric acid. The flow rate was adjusetd to 1mL/min. and gradient elution was used. The qualitative and quantitative analyses of pine barks (karaçam, kızılçam, sarıçam, fıstıkçamı, halepçamı) that contain catechin, epicatechin, epicatechingallate, ferulic acid and taxifolin have been performed. According to the types of pine barks analyzed, the pycnogenol components were found to be as: 0.209 mg/g taxifolin, 0.086 mg/g catechin, 0.076 mg/g epicatechin, 0.022 mg/g ferulic acid in karaçam; 23.16 mg/g taxifolin, 1.454 mg/g catechin, 0.027 mg/g epicatechin in kızılçam; 1.923 mg/g catechin, 1.801 mg/g taxifolin in fıstıkçamı; 0.066 mg/g epicatechin, 0.065 mg/g catechin, 0.025 mg/g epicatechingallate, 0.015 mg/g taxifolin, 0.010 ferulic acid in sarıçam; 0.219 mg/g taxifolin, 0.093 mg/g catechin, 0.046 mg/g epicatechin in halepçam.