Determination of Four Constituents in Rubi Fructus and Optimization of Wine Preparation Process

OBJECTIVE  To develop a HPLC method for determining elleagic acid, kaempferol-3-O-rutinoside, astragalin, and tiliroside in Rubi Fructus, and to optimize the wine preparation process of Rubi Fructus.

METHODS  Waters CORTECS^® C₁₈(4.6 mm×150 mm, 2.7μm) was used as the chromatography column for the analytical methods for elleagic acid and three flavonoids; Mobile phase A and B were 0.1% formic acid-H₂O and 0.1% formic acid-CH₃CN, respectively; Gradient elution, the UV detection wavelength for elleagic acid was 254 nm, the UV detection wavelength for three flavonoids was 266 nm; After determining the level ranges of four factors, including yellow rice dosage, damping time, processing time, and processing temperature by using single factor experiments, definitive screening design was employed to design the experiments. The overall normalized value of ellagic acid, kaempferol-3-O-rutinoside, astragalin, and tiliroside was adopted as the evaluation index, which was used to establish the functional model between the factors and the normalized value of the total evaluation was established. And then the optimum process parameters were predicted.

RESULTS The correlation coefficient r≥0.999 in linearity ranges of four consitutents; The average recoveries were 103.7%, 97.2%, 96.9%, and 97.4%, respectively, with RSD<2%(n=6); The optimized process parameters for the wine preparation process of Rubi Fructus were as follows: the amount of yellow rice wine is 19.4%, the soaking time is 1.8 hours, the processing time is 10.5 min, and the processing temperature is 149 ℃. The RSD values of contents of four constituents were less than 12% for three verification experiments. The contents of ellagic acid were 2.65−3.80 mg·g⁻¹/3.00−4.97 mg·g⁻¹, kaempferol-3-O-rutinoside 0.344−0.476 mg·g⁻¹/0.316−0.460 mg·g⁻¹, astragalin 0.216−0.255 mg·g⁻¹/0.195−0.280 mg·g⁻¹, and tiliroside 0.470−0.568 mg·g⁻¹/0.559−0.787 mg·g⁻¹ in the three batches of Rubi Fructus/wine prepared Rubi Fructus.

CONCLUSION The developed HPLC methods were simple, and applicable, which could be used to determine elleagic acid, kaempferol-3-O-rutinoside, astragalin and tiliroside in (wine) Rubi Fructus. The optimum preparation process of wine prepared Rubi Fructus is stable and reliable, which can obtain high contents of active constituents. The results can provide a scientific basis for the quality standard and clinical application of wine prepared Rubi Fructus.