Study On Extraction And Purification Of Hypericin From Hypericum Perforatum And Its Antioxidant Mechanism

Hypericin,one of the most biologically active of allantoin,is of great interest for its wide range of biological activities and has broad promise in antidepressant and antioxidant applications,making it one of the hot herbs for research and development worldwide.The increased market demand for Hypericum perforatum,in which the content of hypericin is also less than 0.3%,the high cost of extraction and isolation of high-purity hypericin,and the harsh preservation conditions,resulting in the industrial production of hypericin APIs and drug research is hampered.In addition,as the standard of living continues to improve,people pay more and more attention to health,and the nutrients contained in health-related foods are receiving more and more attention,and antioxidants are also attracting more attention from researchers because of their antioxidant effects.Hypericin,as a new type of antioxidant,is able to scavenge free radicals and can effectively ameliorate the harm caused by free radicals in living organisms and foods.At the same time,there are several active sites on the structure of Hypericin,which play different ways to scavenge free radicals.In the study of the mechanism of free radical scavenging action,the nature of free radical scavenging by the active ingredients in Hypericum perforatum was studied in depth by quantum chemical calculation method,which provides an important theoretical basis for the study of the antioxidant effect of new antioxidants in human body.In this paper,we used the raw material of Hypericum perforatum as the raw material,screened the best extraction process by UV detection method of hypericin and purified the alcohol extract to obtain the high-purity hypericin;In addition,the in vitro antioxidant experiments were conducted to evaluate the antioxidant activity of the active substances of Hypericin,Quercitrin and Quercetin in Hypericum perforatum,while the physical parameters of Hypericin,Quercitrin and Quercetin were calculated by Gaussian,and the free radicals were selected as hydroxyl radicals（·OH）to study the antioxidant mechanism and theoretically analyze the antioxidant activity between the three.Regarding the study on the extraction,isolation and purification process of hypericin,the absorbance of hypericin at 589 nm was firstly obtained by UV detection,and the standard curve of hypericin was calculated by external standard method as:y=0.00787+75.25714x to facilitate the calculation of its content.The best extraction process of Hypericum perforatum was screened by orthogonal experiment with the following conditions:the solid-liquid ratio of extraction was 1:8,the extraction temperature was 80℃,the concentration of ethanol was 70%,the extraction time was2h,and the number of times of extraction was 2.The crude product was obtained with the content of 0.3226%,which was in accordance with the international export standard of hypericin content greater than 0.3%.The alcoholic extracts of Hypericum perforatum were separated by column chromatography with petroleum ether-ethyl acetate-methanol as eluent,and high purity of hypericin was isolated with 97.96%purity.Nine compounds were also isolated and identified as bis（2-ethylhexyl）adipate（1）,9,12-Octadecyl linoleic acid-monoglycerides（2）,n-Hexacosanol（3）,Quercetin（4）,Rutin（5）,Micaceol（6）,Quercitrin（7）,Hypericin（8）and Hyperoside（9）.of which 1,2,3,and 6 were newly identified compounds from Hypericum perforatum.Concerning the study of antioxidants of active ingredients of Hypericum perforatum,the scavenging ability of hydroxyl radicals（·OH）,superoxide anion radicals（·O₂^-）,DPPH radicals（·DPPH）and lipid peroxyl radicals（·RO₂）was evaluated by calculating the scavenging rate of free radicals by UV detection of three active molecules for the study subjects of hypericin,quercitrin and quercetin.Hypericin,quercitrin and quercetin have some scavenging effects on all four free radicals.Among the three active ingredients scavenging hydroxyl radicals,when the scavenging rate reached 50%,the concentrations of hypericin,quercitrin and quercetin were:0.0206,0.0101 and 0.0061 mg/m L,respectively,then IC_{50 hypericin}>IC_50quercitrin>IC_{50 quercetin};Among the three active ingredients scavenging superoxide anion radicals,when the scavenging rate reached 50%,the concentrations of hypericin,quercitrin and quercetin were:0.0039,0.0035 and 0.0027 mg/m L,respectively,then IC_{50 hypericin}>IC_{50 quercitrin}>IC_{50 quercetin};Among the three active ingredients scavenging DPPH radicals,when the scavenging rate reached 50%,the concentrations of hypericin,quercitrin and quercetin were:0.0160,0.0013 and 0.0008 mg/m L,respectively,then IC_{50 hypericin}>IC_{50 quercitrin}>IC_{50 quercetin};Among the three active ingredients scavenging lipid peroxyl radicals,when the scavenging rate reached 50%,the concentrations of active molecules of hypericin,quercitrin and quercetin were:0.0124,0.0056 and0.0050 mg/m L,respectively,then IC_{50 hypericin}>IC_{50 quercitrin}>IC_{50 quercetin}.In summary:the order of scavenging activity of free radicals were:quercetin>quercitrin>hypericin.Regarding the study on the mechanism of hydroxyl radical scavenging by hypericin,quercitrin and quercetin,hypericin,quercitrin and quercetin were used as antioxidants for the study of hydroxyl radicals.By HPLC,hypericin,quercitrin and quercetin were found to be able to scavenge hydroxyl radicals,and the product was only one compound.Structural analysis and electrostatic potential analysis of hypericin,quercitrin and quercetin,the active site of the dehydrogenation reaction is on their hydroxyl or phenolic hydroxyl groups.All six active sites on hypericin are very active;quercitrin has the potential to react with hydroxyl groups as hydrogen on O8’,O11 and O7’;quercetin has the potential to react with hydroxyl groups as hydrogen on O8’,O11,O12 and O7’.The dissociation energy analysis,post-dissociation spin density analysis and dissociation frontline orbital analysis of hypericin,quercetin and quercetin showed that the hydrogen on the C7-OH of hypericin was the largest active site for the occurrence of hydroxyl radical scavenging reaction,the hydrogen on the C4’-OH of quercitrin was the largest active site for the occurrence of hydroxyl radical scavenging reaction,and the hydrogen on the C4’-OH of quercetin was the largest active site for the occurrence of hydroxyl radical scavenging reaction.The analysis of the transition states of hypericin,quercitrin and quercetin with hydroxyl radicals,the energy barrier analysis of the reaction and the calculation of the reaction rate,RHY19in hypericin reacts with·OH radicals as the optimal reaction path,RQci8’in quercitrin reacts with·OH radicals as the optimal reaction path,and RQce8’in quercetin reacted with·OH radicals as the optimal reaction paths.The reaction rates of the three reaction paths were K_RHY19=1.562×10⁴s^-1M^-1、K_RQci8’=5.126×10⁴ s^-1M^-1,K_RQce8’=9.013×10⁹s^-1M^-1 and It is obvious that K_RQce8’>>K_RQci8’>K_RHY19,quercetin is more readily associated with·OH radicals,followed by quercitrin,and the weakest is hypericin.