Tabriz University of Medical Sciences Advanced Pharmaceutical Bulletin 2228-5881 5 4 2015 11 30 Determination of Four Major Saponins in Skin and Endosperm of Seeds of Horse Chestnut (Aesculus Hippocastanum L.) Using High Performance Liquid Chromatography with Positive Confirmation by Thin Layer Chromatography 587 591 10.15171/apb.2015.079 EN Zead Helmi Mahmoud Abudayeh Khaldun Mohammad Al Azzam Ahmad Naddaf Uliana Vladimirovna Karpiuk Viktoria Sergeevna Kislichenko Journal Article 10.15171/apb.2015.079 2015 05 12 2015 08 25 urpose: To separate and quantify four major saponins in the extracts of the skin and the endosperm of seeds of horse chestnut (Aesculus hippocastanum L.) using ultrasonic solvent extraction followed by a high performance liquid chromatography-diode array detector (HPLC-DAD) with positive confirmation by thin layer chromatography (TLC). Methods: The saponins: escin Ia, escin Ib, isoescin Ia and isoescin Ib were extracted using ultrasonic extraction method. The optimized extraction conditions were: 70% methanol as extraction solvent, 80 C as extraction temperature, and the extraction time was achieved in 4 hours. The HPLC conditions used: Zorbax SB-ODS-(150 mm × 2.1 mm, 3 m) column, acetonitrile and 0.10% phosphoric acid solution (39:61 v/v) as mobile phase, flow rate was 0.5 mL min-1 at 210 nm and 230 nm detection. The injection volume was 10 L, and the separation was carried out isothermally at 30 C in a heated chamber. Results: The results indicated that the developed HPLC method is simple, sensitive and reliable. Moreover, the content of escins in seeds decreased by more than 30% in endosperm and by more than 40% in skin upon storage for two years. Conclusion: This assay can be readily utilized as a quality control method for horse chestnut and other related medicinal plants.