Study On Extraction,Separation And Antiasthmatic Activity Of Total Flavonoids From Elaeagnus Pungens Leaf

In recent years,environmental problem has become a worldwide issue.With the air pollution,respiratory diseases such as chronic bronchitis,bronchial asthma,chronic cough have been becoming ordinary,which are threatening human health.The morbidity and mortality of chronic bronchitis and bronchial asthma,two typical diseases of respiratory system,are increasing as well.They have serious effect on the health and the quality of daily life in patients because they are difficult to be cured as chronic diseases.Therefore,it's very important to develop effective and therapeutic drugs.As a traditional medicine,Elaeagnus pungens leaf has some effects on restraining lung,relieving cough and drawing asthma for the treatment of asthma,chronic asthmatic bronchitis in clinic.In order to explore the medicinal resource of Elaeagnus pungens leaf and clarify the relationship between the components and the pharmacological activities,our project is purposed to extract,purify and investigate the effects of flavonoids from Elaeagnus pungens leaf.Firstly,the content of flavonoids was determined.It is known that every flavonol compound of Elaeagnus pungens leaf is difficult to be measured.Thus,the content of total flavonoids was determined by UV-vis spectrometry.Also,the contents of quercetrin,kaempferol and isorhamnetin were determined by HPLC after acid hydrolysis.The two methods were simple,reliable and repeatable,which can be used together for the determination of flavonoids in Elaeagnus pungens.Secondly,the extraction of flavonoids in Elaeagnus pungens was optimized.On the basis of single factor experiment,orthogonal experiment was used to optimize the extraction process of flavonoids.The optimal conditions were as follows: 30-folds of 70% ethanol,extracting for three times.Under the conditions,the average content of flavonoids in the validation experiments was 0.243 %.The method is reliable and repeatable.Thirdly,the flavonoids was isolated and purified by column chromatography.Five types of resin including polyamide,HP-20,HPD600,NKA-2 and D-101 were used to purify flavonoids by evaluating static adsorption rate and desorption rate.Polyamide and HP-20 macroporous resins were selected.After purification,the content of total flavonoids increased less than 85 %.Continuously,the content of total flavonoids increased to 96.68 % by column chromatography of ODS.Fourthly,the effect of purified flavonoids on airway smooth muscle was studied by investigating the relaxation of the airway smooth muscle.The result indicated that the total flavonoids of Elaeagnus pungens leaf completely inhibited the precontraction induced by high K+ in a concentration-dependent way.Furthermore,patch clamp technique was used to detect voltage-dependent L-type Ca²⁺ channel?VDCC?-mediated currents.The results indicate that the total flavonoids of Elaeagnus pungens leaf blocked voltage-dependent L-type Ca²⁺ channel?VDCC?-mediated currents.Taken together,it is concluded that the total flavonoids of Elaeagnus pungens leaf blocked VDCC to relax airway smooth muscle.