The Processing Technology And Spectrum-effect Relationship Of Hippophae Rhamnoides Leaves

Hippophae rhamnoides Linn.,is a thorny,nitrogen-fixing deciduous shrub of the genus Sea Buckthorn in the family Hochschiaceae.The active ingredients of Hippophae rhamnoides leaves（HRL）account for about 70%of the whole Hippophae rhamnoides plant,the flavonoid content of which is even higher than that of Hippophae rhamnoides berries,and has a high value for development and application.However,HRL are often treated as waste in the development of Hippophae rhamnoides resources at present,resulting in a serious waste of resources.Most of the research on HRL has focused on the brewing of tea and the analysis of its ingredients,but there is no precedent for the process of processing of HRL.Based on this background,in order to improve the comprehensive utilization of Hippophae rhamnoides by-products,this topic is based on a series of studies to optimize the processing process of HRL as raw material.The purpose of this is to provide reliable data for the further development and utilization of HRL.The main research methods and results are as follows:1.The processing technology and quality standard of HRL:The experiment was carried out using the fried yellow method in the frying method.The total flavonoid content was used as an indicator for the single-factor experiment to screen the frying time,frying temperature,and frying amount.The optimal frying process was then optimised by response surface experimental design,resulting in the following optimal frying process:Frying temperature 3±0.5 min,frying temperature 140±5°C and frying volume 1/3-1/4 of the herb frying machine.All indicators of fried Hippophae rhamnoides leaves（F-HRL）are in accordance with the quality standard of HRL in the"《Sichuan Tibetan Herbal Medicine Standard》（2014 Edition）",and the quality standard of F-HRL was drafted on this basis.2.The effect of frying on the properties and bioactivity of HRL:The changes in the morphological appearance and chemical composition of HRL and F-HRL were compared by scanning electron microscopy（SEM）and high performance liquid chromatography（HPLC）and infrared spectroscopy（FT-IR）,and some of the biological activities of HRL and F-HRL were investigated.The results of SEM demonstrated that frying altered the microscopic morphology of HRL.The results of FT-TR and SD-IR showed that the frying did not change the structure of the active ingredients in HRL,but increased their overall content.The results of HPLC showed that the flavonoids of HRL contained three monomeric compounds which were rutin,ellagic acid and isoquercitrin,with the content of rutin was 10.36μg/m L in flavonoids of F-HRL and 11.79μg/m L in flavonoids of F-HRL.The results of bioactivity tests in vitro showed that both flavonoids of HRL and F-HRL possess strong antioxidant,hypoglycaemic and anticoagulant activities.The IC₅₀of flavonoids of HRL and F-HRL for DPPH radicals were 13.42±0.3μg/m L and 10.92±0.2μg/m L,respectively.The IC₅₀of flavonoids of HRL and F-HRL for hydroxyl radicals were 0.27±0.01 mg/m L and0.23±0.01 mg/m L,respectively.The IC₅₀of flavonoids of HRL and F-HRL forα-glucosidase were 5.25±0.2μg/m L and 3.54±0.3μg/m L,respectively.Both high concentrations of flavonoids of HRL and F-HRL caused a gradual prolongation of PT,TT and APTT values,thus exerting an anticoagulant effect.In summary,it was shown that flavonoids have strong biological activity in vitro,and the activity of F-HRL was higher than that of HRL.3.Spectrum-effect relationship between HPLC fingerprints and antioxidant activity of HRL:The HPLC fingerprints of 10 batches of HRL and F-HRL of different origins were established using HPLC.The spectrum-effect relationship of flavonoids of F-HRL antioxidant was investigated using grey correlation analysis（GCA）and Pearson correlation analysis.The results showed that the fingerprints of the 20 batches of total flavonoids of HRL identified a total of 12common peaks with similarities ranging from 0.813 to 0.993,indicating that they were consistent in composition but with some differences in content.HCA clustered the 10 batches of HRL and their fried products into two main groups.PCA was extracted three principal components,reflecting 85.955%of the information in the original variables and the peak 12 considered to be the component contributing most to the variance.The results of GCA showed that the largest contribution to the antioxidant activity being peak 10（isoquercitrin）with a correlation of 0.771.There were three common peaks with Pearson correlation coefficients greater than 0.400,and the common peak valuable for scavenging DPPH radicals as an antioxidant capacity was peak 10,and the correlation level was significant（P<0.01）.The result that corresponds to the results obtained by GCA.4.Mechanism of antioxidant effects of total flavonoids of F-HRL based on Ultra-high Performance Liquid Chromatography-Triple Quadrupole-Tandem Mass Spectrometry（UPLC-QQQ-MS/MS）and network pharmacology:Analysis of total flavonoids in F-HRL by UPLC-QQQ-MS/MS and network pharmacology was used to explore the mechanism of action of flavonoids of F-HRL exerting antioxidant activity through a multi-component,multi-target and multi-pathway perspective.Eleven monomeric compounds of total flavonoids of F-HRL were obtained by mass spectrometric analysis.The results showed that the flavonoids of F-HRL of exert antioxidant activity mainly through 92 pathways,involving 644 biological processes,64cellular composition and 56 molecular functions,and acting on target proteins such as SRC、EGFR、FYN、LCK and AKT1.The optimized frying process of HRL enhanced the content of active ingredients in HRL and improve its biological activities such as antioxidant,hypoglycemic and anticoagulant,etc.The HPLC fingerprinting antioxidant spectrum-effect relationship study objectively reflect the real differences in the quality of HRL from different origins.The study will provide a theoretical basis for the control and exploitation of ingredients in the production of HRL-related pharmaceuticals and health products,and will be of great significance to the development and industrialisation of HRL resources.