Extraction and purification of polyphenols and determination of antioxidant activity

Abstract: Cerasus humilis is naturally rich in a variety of vital vitamins and minerals, such as calcium and polyphenols, providing great benefits for health care functions, particularly on improving human immunity. Polyphenols are significant natural antioxidant substances for various functions, including softening blood vessels, promoting digestion, lowering blood lipids, diuresis, enhancing immunity, preventing arteriosclerosis and thrombus formation, even to inhibit the growth of bacteria and cancer. Specifically, an emphasis is put effect on inhibiting the proliferation of human liver cancer (HepG2), colon cancer (HCT116), and gastric cancer (BGC823). However, only a few reports focused on the natural active substances that contained in plum fruit. In this study, a novel extraction and purification process of plum polyphenols was proposed to determine the types and contents of polyphenols, and thereby to explore the in vitro effects of polyphenols. The processing parameters were optimized in the extraction and purification of polyphenols, together with the antioxidant activity of plum cerasus humilis. A single factor and response surface optimization tests were used to investigate the ultrasonic assisted enzymatic extraction process of polyphenol of Cerasus humilis. Macroporous resin was also selected to purify the crude extract of polyphenols. Moreover, high solution liquid chromatography was used to identify the types of phenolic substances, and the spectrophotometry was used to determine the total reducing power of polyphenol in cerasus humilises, and the scavenging effect of free radicals ·OH, DPPH·, ABTS·. The results showed that the optimum extraction parameters of polyphenol of cerasus humilis were as follows: The ultrasonic power was 105 W, temperature of enzymatic hydrolysis was 50 ℃, enzymatic hydrolysis time was 80 min, the ratio of liquid to material was 30:1 (mL/g), and the extraction amount of polyphenols was 42.63 mg/g. After the purification with macroporous resin, the total polyphenol content of cerasus humilises was 73.42 mg/g. All the 6 polyphenols were detected in the plum fruit of cerasus humilises, where their contents were ranked in order, chlorogenic acid > epigallocatechin gallate> caffeine > p-hydroxybenzoic acid >protocatechuic acid gallic > gallic acid. The total reducing power of polyphenol in cerasus humilises was not different from that of Vitamin C, indicating a significant scavenging effect on free radicals, such as ·OH, DPPH·, ABTS·. In the case of the same mass concentration, the reduction ability of purified product, crude extract, and Vc of the cerasus humilis were as follows: Vitamin C (IC50=35.05μg/mL) > the cerasus humilis polyphenols (IC50 =32.42 μg/mL) ＞ the crude extraction of Oleum Phenol (IC50=30.13 μg/mL). The reduction ability of purified cerasus humilis was not significantly different from that of vitamin C (P>0.05), but there was totally difference from the crude extract (P<0.05). The purification process can be used to remove some impurities without affecting the activity of the main components, indicating that it can improve the total reduction capacity. Cerasus humilis showed the good scavenging ability on DPPH·, ABTS·, and ·OH radicals. The purified product of cerasus humilis demonstrated an IC50 value of 72.65 μg/mL for the removal of ·OH, and an IC50 value of 110.12 μg/mL for the removal of DPPH·. The IC50 value for the removal of ABTS· was 55.45 μg/mL, and the total reducing power EC50 value was 32.42 μg/mL, indicating equivalent to the effect of Vc. The IC50 value for the crude extract of Prunella vulgaris polyphenols was 66.92 μg/mL for removing ·OH, the IC50 value for removing DPPH· was 98.42 μg/mL, the IC50 value for removing ABTS· was 49.87 μg/mL, and the total reducing power EC50 value was 30.13 μg/mL. The finding can provide a sound theoretical basis and technical support for the development and utilization of cerasus humilis in the production of plum fruits.