Characterization of Marker Compounds in Ziziphi Fructus Using TLC

Ziziphi Fructus (the fruit of Ziziphus jujuba Miller var. inermis Rehder; Japanese name "Taiso"), listed in the 18th edition of the Japanese Pharmacopoeia (JP18), is widely used in Kampo (Japanese traditional) medicine. However, a specific identification test for Ziziphi Fructus was not prescribed in JP18. Hence, in this study, we establish a TLC-based identification test for quality assurance of the crude drug Ziziphi Fructus. The HPLC experiments identified 3-O-cis-p-coumaroylalphitolic acid and 3-O-trans-p-coumaroylaliphitolic acid as suitable indicators. After developing the samples to a distance of 10 cm using ethyl acetate (EtOAc)/n-hexane/acetic acid (AcOH)/H2O (60 : 40 : 2 : 1) as the TLC-developing solvent and UV light at 254 nm, two spots with an Rf value of approximately 0.4-0.6 were detected. Additionally, based on the TLC-based experiments, alphitolic and maslinic acids emerged as potential indicator components. Treatment of samples with EtOAc/n-hexane/AcOH (60 : 40 : 1) serving as the developing solvent, followed by exposure to dilute sulfuric acid solution, heat, and UV light at 365 nm, revealed two clear spots with Rf values of approximately 0.2-0.3. These findings potentially serve as a basis for the development of an identification test to identify Ziziphi Fructus.