Optimizing Extraction Process By Orthogonal Test Of The Fruit Of Rhus Typhina L.

Objective: By the detection method of high performance liquid chromatography(HPLC), the evaluation index of the amount of Gallic acid extracted, using orthogonal test to decide the best extraction technology of Phenolic Acids from Rhus typhina; after that, conducting an in vitro antioxidant experiment to scavenge the DPPH radicals of the extracts to test its antioxidant capacity.Method:1) Using Gallic acid as the reference substance, Octadecyl silane bonded silica gel column and Methanol-0.1% aqueous phosphoric acid solution as mobile phase, with the detection wavelength selected to be 273 nm, draw a standard curve of Gallic acid, test the linearity and range, precision, repeatability, and accuracy of the method, and establish a method to measure Gallic acid by High performance liquid chromatography;2) Study the influence of extraction solvent, extraction time, extraction solvent amount and extraction times on efficiency of extracting Gallic acid out of Rhus typhina by refluxing extraction and ultrasonic extraction separately, decide the best extraction method by single factor method and orthogonal experiment.3) Using DPPH method to test the scavenging ability of Rhus typhina extraction against DPPH radicals, assess the in vitro antioxidant ability of the two extractions.Result:1) Using high performance liquid chromatography(HPLC) to measure the amount of Gallic acid extracted, with mobile phase of Methanol-0.1% aqueous phosphoric acid solution 1 mL·min-1 and detection wavelength of 273 nm. This method had good linear relationship in the range of 0-71.0 ?g·ml1, with regression function y=22.615x+6.941 and regression coefficient R=0.9993. This method was good in precision(RSD=1.43%), repeatability(RSD=1.19%), accuracy(RSD=1.88%), and stability in 24 hours(RSD=2.15%). It was applicable.2) The results of orthogonal test showed that the best extraction process by refluxextraction method was A3B2C1D3, which meant reflux time was 4 hours, ethanol concentration was 50%, liquid ratio was 1:5, and extraction times was 3. Repeat the experiment under such conditions for 3 times, resulting in amount of Gallic acid which was 4.509 mg·g-1, 4.547 mg·g-1, 4.540mg·g-1, with an average of 4.532mg·g-1 Amount of Gallic acid extracted from Rhus typhina was approximately the same by the two extraction process, with a higher extraction rate of ultrasonic extraction method over reflux extraction method.3) Conducting in vitro antioxidant experiments to scavenge DPPH radicals of the extracts by the two methods, extract of reflux extraction method had IC50=5.117 mg·ml-1, while extract of ultrasonic extraction method had IC50=5.044 mg·ml-1, a little higher than that from reflux extraction method. The results showed that extract of ultrasonic extraction method had a higher ability to eliminate DPPH radicals than extract of reflux extraction method, which was in line with the difference in Gallic acid extracted. It showed that the antioxidant ingredients in Rhus typhina were mainly Phenolic acids, and reflux extraction method had a capacity as well as ultrasonic extraction method.Conclusion: This experiment employed high performance liquid chromatography(HPLC) to detect the amount of Gallic acid in Rhus typhina, which was easy to operate, accurate and fast to conclude, and simple in practice. In the meantime, this experiment performed orthogonal test on the basis of single factor test, optimized the extracting process to extract Gallic acid from Rhus typhina by reflux extraction method and ultrasonic extraction method; for the first time studied the detection method and extraction process of Gallic acid from Rhus typhina, which laid the foundation for the development and utilization of Gallic acid in Rhus typhina.