Isolation And Purification Of Phloridzin By High Performance Centrifugal Partition Chromatography And The Preparation Of Yellow Pigment By Oxidation

Phloridzin is glucoside phloretin, it belongs to the dihydrochalcone glycoside in plant flavonoids, present in a variety of plant tissues, especially in apple branches, leaves and the extract of apple fruit, even it has been used to considered as the typical substance in apple fruit.A reversed-phase high performance liquid chromatographic method for determination of phloridzin in apple tree branch and leaf was established in this paper. Chromatographic separation was performed by Waters Symmetry C₁₈ column(150mm×4.6mm,Φ5μm) using the mobile phase isocratic elution of methanol-phosphoric acid(50:50)with a flow rate of 0.6mL·min^-1 at 30℃and the UV detective wavelength was 287nm. Phloridzin in apple tree branch and leaf was separated rapidly within 10min. Results showed that the calibration curve of phloridzin was linear in the range of 0.30 4.50μg with R²=0.9993. The recovery and RSD were 98.95% and 1.22% respectively. This method is accurate and reliable for determination of phloridzin in apple tree branch and leaf.Phloridzin was extracted from apple tree branch by ethanol solvent. The content of phloridzin was determined by high performance liquid chromatography (HPLC). On the basis of the single-factor experiment, the optimum extraction conditions were obtained by orthogonal experiments. The results showed that the order of the main factors influencing the extracting effect from more important to less were as follows: ratio of liquid to material, extraction temperature, ethanol concentration and extraction time. The optimum extraction conditions were that the ratio of liquid to material was 20mL/g, the extraction temperature is 70℃, the ethanol concentration was 60% and the extraction time was 90min. Under the above mentioned conditions, the content of phloridzin in the extracts was 39.2 mg/g.In order to obtain high purity phloridzin,High performance centrifugal partition chromatography was applied to separate and purify phloridzin from the ethanol extract of apple tree branch.Through the system optimization experiments of the two-phase solvent system , rotation speed and flow rate , the satisfactory separation effect were obtained.Results showed that the best two-phase solvent system is composed of n-hexane-ethyl acetate-methanol-water (1:5:1:5,v/v) with the upper organic phase as stationary phase,the lower aqueous phase as the mobile phase; the rotation speed of 1500rpm,the flow rate of 3.0mL/min and the detection wavelength of 287nm.Using this method,phloridzin was successfully purified from the ethanol extract of apple tree branch with the purity of 17.91% by one step in 30min.The purity of isolated compounds were determined to be 91.20% by HPLC analysis,and the yield of phloridzin was 98.10%.Phloridzin oxidation products (POP) was synthesised by polyphenol oxidase (PPO) on phloridzin, oxidation kinetics showed that phloridzin was all oxidized in 40min, and converted to POP within 60min. Its oxidation by polyphenol oxidase followed a very specific pathway: phloridzin was first almost totally converted to a colourless intermediate (POP1), which was then converted to a yellow pigment (POP2) without any significant side reactions.