Thiolysis-HPLC Analysis Of Proanthocyanidins In Fruit And Their Products

Proanthocyanidins (PC) is a complex polyphenolic plant secondary metabolite, which is polymerized in different ways taking flavanols as the main structural unit. Fruits are the main source of dietary PC. The results show that, PC has a series of important physiological effects, such as strong antioxidant, eliminating free radicals, anti-hypertensive, and anti-tumor. Its physiological effects and absorption rate are closely related to the average degree of polymerization. Currently, the analytical methods of PC content in fruits are spectrophotometry and HPLC method. The spectrophotometry method has a larger system errors and the HPLC method fails in providing the total content and the average degree of polymerization. In recent years, a number of thiolysis-high performance liquid chromatography methods have been used for the analysis of PC content and average degree of polymerization of some fruit samples. However, the results of those methods all have such flaws as little variety analyzed and standard product incomplete. According to these deficiencies, this study develops and evaluates a new method based on a large number of actual samples analysis.This study conducts research on the PC thiolysis reaction conditions, and investigated the effects of the Benzyl-thiol reagent concentration, ratio of acid concentration, reaction time and reaction temperature on the derivative reaction. It is finally determined that when the PC concentration level is less than 0.4%, the benzyl mercaptan concentration in the reaction solution is 1%; Acetic acid:1 M HCl = 5:1 (v/v), with the reaction temperature is 90℃and the reaction time is 60 minutes. This condition can guarantee that the PC polymer can be completely degraded and generate the corresponding flavanols and the benzyl mercaptan derivatives.Taking the industrial Grape Seed Extract as the raw materials and through the following steps including thiolysis, normal-phase HPLC, and RP-HPLC, we purified such two derivatives as epicatechin-benzyl sulfide (EC-S) and gallic acid epicatechin-benzyl sulfide (ECG-S). The Normal phase chromatography conditions are: column SinoChrom SiO2 (250 mm×20 mm i.d, 10μm); mobile phase: Ethyl acetate: petroleum ether(60:40,v/v); flow: 10 mL/min; detection: UV at 280 nm. The RP chromatographic conditions are: column SinoChrom ODS-BP (250 mm×20 mm id, 10μm); mobile phase: 70% (v/v) methanol (0.05% formic acid); flow: 10 mL/min; detection: UV at 280 nm. Using the LC-MS method and the ¹H and ¹³C high-resolution two-dimensional NMR profiles method to analyze and confirm their structure and purity. The results show that the two prepared components are indeed the target compounds.The HPLC analysis method was established to analyze the PC content and average degree of polymerization of the fruit. The chromatographic conditions are: column: Hypersil BDS C18(250 mm×4.6 mm i.d., 5μm); mobile phase A:500μL/L aqueous Formic acid; mobile B: methanol: acetonitrile = 1:2 v/v ; 500μL / L of formic acid. The gradient procedure was: 0 min～8 min, 13% B (isocratic); 8 min～16 min, 13%～40% B (linear gradient); 16 min～24 min, 40%～50% B (linear gradient); 24 min～27 min, 50%～100% B (linear gradient); 27 min～34 min, 100% B (isocratic), 34 min～37 min, 100%～13% B (linear gradient); 37 min～45 min, 13% B (isocratic). Detection UV: 280 nm; flow: 10 mL/min; temperature: 35℃; injection quantity: 10μL. The external standard calibration curves were used in quantification. Count respectively the peak area of gallic acid (GA), catechin (CT), epicatechin (EC), (ECG), EC-S, and ECG-S. Obtain the content of various monomer and the total content of PC through the Standard curve. And then, calculate the PC average degree of polymerization of the samples by using the total concentration/flavanols monomer concentration of the flavanols monomer and the derivatives, or (CCT+CEC+CECG+ CEC-S +CECG-S)/(CCT+CEC+CECG). By this method, the actual samples of hawthorn, grape et al. were measured. The main components were separated and determined well. RSD of these components were 0.4%～4.2%. The detection limit was 0.4 ng～4 ng, The linearity range was 0.8 ng～4000 ng. The correlation coeffeicent (r) was 0.9992～0.9999. The analytic time of one sample was 45 min.Using the new method, analyze the PC content and average degree of polymerization of the grape, apple, hawthorn and its processing products. The results showed that, the total PC content is 0.14 mg/g～0.26 mg/g in such five kinds of apples as Hongxing, Jinaixing, Qiaonajin, Wanglin and Fushi, and the average degree of polymerization is 2.0～2.78. The total content of the proanthocyanidins in the seed of such seven table grapes as Bantian, Jingya, Tiankang, Jixuan, Qianfeng, Jufeng, and Mowang is 5.2 mg/g～26 mg/g. The average degree of polymerization is below 2. Analyze the PC content of the seed of 12 kinds of wine grape such as Zengfangde, Chixianzhu, Meizhenge, Xila, Heipinle, Qiongyaojiang, Changxiangsi, Mile, Baoshi, Baisinan, Leisiling, and Meirong. The total content of PC is 8.4 mg/g～32 mg/g, the average degree of polymerization is 1.8～4.0. Analyze a kind of grape seed extract industrial product. The total PC content is 370 mg/g, and the average degree of polymerization is 6.08. In the six kinds of commercial health products, the total PC content is 6 mg/g～51 mg/g, and the average degree of polymerization is between the 2.70～4.30. The total content of proanthocyanidins in the hawthorn fruit is 4.8 mg/g, and the average degree of polymerization is of 1.9. The PC content of Huiyuan Hawthorn Juice and Huaqi Hawthorn Juice are respectively 0.065 and 0.11 mg/L. While in the four kinds of processed hawthorn products including the hawthorn cake, hawthorn soup, hawthorn slice, and sweetend roll, the PC content is 1 mg/g～2.4 mg/g, and the average degree of polymerization of the PC of the six kinds of products is between 1.77～2.23.This research also studied the stability of hawthorn PC during the drying and storage process. The results show that among the three drying conditions including vacuum freeze-drying, 80℃drying, and natural drying, the Vacuum freeze-drying has the minimum loss of PC components, and the retention rate of the whole amount is 88%; the retention rate of the PC components of hot air drying and natural drying are 76% and 25%. The results of the experiment on the frozen fruits show that after stored for 110 days in 0℃, the total amount of proanthocyanidins in the hawthorn fruits is 80% of that in the initial storage, the rate of good fruit is high, and the color and luster of the fruit meet the normal standard. Through the comparison with the result of dried and dehydrated hawthorn fruit, we can see that under refrigeration, the fruit has a lower loss rate of its PC components and is suitable for short-term storage of the hawthorn raw materials, while the dried method can be used for long-term storage.The thiolysis-HPLC method, developed in this study to analyze PC in the fruit, takes the prepared EC-S, ECG-S derivatives as the standard reference substance, and has the advantages of simple, accurate, and quick operation. It can not only measure the total content of the PC components, but also can provide important information of the degree of polymerization of PC. It can be used the analysis and quality evaluation of apples, grapes, hawthorn and its extracts, related industrial products, and health food. From the analysis results of the actual sample, we can see that there is a large variety in the PC content and the average degree of polymerization between different species. The hawthorn fruit has a higher PC content, even close to that in the the grape seed extract health products, while its average degree of polymerization is much lower than the analyzed health care products. Therefore, it is a good fruit PC resource.