Research On Determination Methods Of Proanthocyanidin From Grape Seed

There is no uniform method for detecting proanthocyandin, although many methods are workable. The determination method for proanthocyanidin of grape seed was studied in this paper and the proanthocyanidin in the ultrafine powder of grape seeds was measured, as well as the development of the extraction method of proanthocyanidin in the ultrafine powder of grape seeds. Main results were as follows:1 The optimum reaction condition of catalytic colorimetry with ferric ions was obtained. The linear region was 10~200μg/mL, RSD was 1.40%(n=9), the recovery was 96.0%~108.4% and the detection limit was 3μg/mL. This method is accurate, sensitive, stable, simple and suitable for the analysis of the proanthocyanidin in the ultrafine powder of grape seed. The content of proanthocyanidin in the ultrafine powder of grape seed was 9.56%.2 Determination of the proanthocyanidin from grape seeds by Folin-Ciocalteu method in combination with HPLC. Folin-Ciocalteu method measured the content of polyphenols and was expressed as catechin equivalents. The improved HPLC method effectively separated the three monomeric phenols including gallic acid, catechin, epicatechin. The results showed that the contents of polyphenols and the three monomeric phenols in the ultrafine powder of grape seed were 10.91%, 1.21%, respectively. The content of proanthocyanidin was 9.70% represented by the difference between amount of polyphenols and the three monomeric phenols.3 Determination of the proanthocyanidin from grape seeds by vanillin method in combination with HPLC. Both flavan-3-ols and proanthocyanidin were determined by vanillin method, flavan-3-ols was determined by HPLC. The proanthocyanidin content can be represented by the difference between the amounts by vanillin method and by HPLC, The reaction condition of vanillin method was obtained. Catechin was applied as standard material in the vanillin method, the linear region was 5~200μg/mL, RSD was 1.69%(n=9), the recovery was 91.0%~118.0% and the detection limit was 1μg/mL. The content of proanthocyanidin in the ultrafine powder of grape seeds was 7.07%.4 Large material-methanol ratio was applied and methanol was used as solvent to the determination of proanthocyanidin. The results of single factor tests and orthogonal test showed that the extraction times was the most important factor, while other factors were not significant. The optimum extraction conditions were: 40KHz of ultrasonic frequency, 1:200 of material-methanol ratio, 40℃of extraction temperature, 45minutes of extract time and 3 times.5 The method of catalytic colorimetry with ferric ions was the best method for determination of the proanthocyanidin in the ultrafine powder of grape seeds by appreciation and comparison of the three detection methods, its manipulation and steps were as follows.(1) Extraction: 0.25~1.0g of the ultrafine powder of grape seeds was quantified accurately. The methanol was extraction solvent with 1:200 of material-methanol ratio, 40KHz of ultrasonic frequency, 40℃of extraction temperature, 45minutes of extract time and 3 times. The extracting solution were blended, centrifuged, and diluted to appropriate concentration for reaction.(2) Determination: 1.0mL of extracting solution with appropriate concentration was drawn into the test tube, 9.0mL of blended reaction solution (the proportion of n-butyl alcohol, HCl, 10% ferric ammonium sulfate is 83:6:1) was also joined in, then plugged cap of the tube, blended, and cooled the tube in the ice water for 4~5min after heated for 40min at 100℃, then reverted the tube to the room temperature, and measured the absorbance value on 550 nm after reagent blank.