Study On Determination Method Of Aconitine Alkaloids In Patent Medicine And Chemical Constituents Of Aconitum Szechenyianum

Ranunculaceae Aconitum of perennial herbs is a kind of broad prospects for the development of medicinal plant resources.Most of the plants of Aconitum are used for medical in Chinese traditional herb.As typical medical plants,Aconitum carmichaeli Debx.,Radix Aconiti Kusnezoffii,Radix Aconitine,have been embodied by Chinese Pharmacopoeia.In recent years,Aconitum szechenyianum Gay.and roots ofAconitum Brachypudum are often as important materials to be used for preparation of medicines.Aconitum szechenyianum Gay.also known as Tieniuqi is growing in Taibai mountain of Shaanxi.As a famous-region Chinese traditional medicine of Shaanxi Provine, Aconitum szechenyianum Gay.is acrid and bitter in taste,poisonous and hot in nature.It performs well in vanishing ecchymosis,dispelling wind and removing dampness,detumescence and detoxification,diminishing inflammation and pain,nerve exciteing and etc.Diester-alkaloids with highly toxicity are the main effective ingredients of Aconitum plants.The therapeutic dose is very close to that of toxicity.In order to ensure the safety and efficience in clinical treatment,a lot of work about the quality evaluation of medicines has been done by domestic and foreign scholars,and some feasible methods for quality control have been developed.But simultaneous determination of four Aconitine alkaloids in patent medicine with aconitum plants with HPLC has not been reported, and a few of research has been made on the chemical constituents ofAconitum szechenyianum Gay.. This paper aims to provide a reference method for the medication safety ofAconitum szechenyianum Gay.and patent medicine with aconitum plants and to investigate the chemical constituents of Aconitum szechenyianum Gay..In this paper,including the introduction and research of two parts:Part 1:IntroductionFirst,the research status about ranunculaceae plant and determination of its patent medicine.Chemical component,clinical application,toxicity and determination of aconitum alkaloids in patent medicine are included.Second,the research survey of Aconitum szechenyianum Gay.and alkaloids.This part relate to chemical constituents,pharmacological effects and toxicity of Aconitum szechenyianum Gay..The general characters and sorts,extraction method,research applications and direction of development of alkoloids are also included.Partâ…¡:Research papersIn this part,three departments are involved:simultaneous determination of four aconitine alkaloids in patent medicine by HPLC;extraction and determination of songorine from Aconitum szechenyianum Gay.;extraction and separation of chemical constituents of Aconitum szechenyianum Gay.by lipophilic organic solvents.First,simultaneous determination of four aconitine alkaloids in patent medicine by HPLC.We chose a few representatives of Chinese medicine,Tibetan and Mongolian medicine to optimize the extraction conditions of alkaloids,and then songorine,mesaconitine,aconitine and hypaconitine was determine by HPLC with a diode array detector,simultaneously.Four alkaloids linear range was 0.1 to 1.5/μg(r=0.9996),0.1 to 2.0μg(r=0.9995),0.1 to 3.0μg(r=0.9996) and 0.1 to 2.5μg(r=0.9997),respectively.A series of parameters were studied and the results showed that this method has high precision,good reproducibility,well stability and higher recoveries.It applies to determine the four aconitine alkaloids from Chinese medicines Wantongjingupian and Fuzilizhongwan,Tibetan medicine Anshenwan,and Mongolian medicine Garidiwuweiwan, simultaneously.Experiment results demonstrated it can be a reference method for the drug quality control of patent medicines.Second,extraction and determination of songorine fromAconitum szechenyianum Gay..The influence of traditional solvent reflux extraction and microwave-assisted extraction on extraction yield of songorine from Aconitum szechenyianum Gay.was investigated.First,we use traditional solvents reflux extraction to optimize the soaking time,extraction solvent and soak menstruum.In that case,we also study on the number of extraction.Finally,we use response surface methodology to investigate the effect of microwave power,irradiation time,solid-liquid ratio and extraction times on extraction yield of songorine in microwave-assisted extraction.The optimized extraction condition was as follows.For traditonal solvent reflux extraction:After soaking haft-hour by 1%ammonia,reflux extracting with 5 times chloroform thrice,each 2 hours,and then reverse extracting with 1% hydrochloric acid three times from chloroform solution.Followed that,10%sodium hydroxide was used to make neutralization,constant volume and determination at last.The content of songorine from Aconitum szechenyianum Gay.was 9.37 mg.g^-1.For microwave-assisted extraction:After soaking haft-hour by 1%ammonia,reflux extracting with 12 times chloroform thrice under 450W power,each 9 minutes,and then extracting with 1% hydrochloric acid three times from chloroform solution.Followed that,10%sodium hydroxide was used to make neutralization,constant volume and determination at last.The content of songorine from Aconitum szechenyianum Gay.was 9.26 mg·g^-1.The two methods of extraction were compared with t test,and results show that the two technics have no significant difference.But the microwave-assisted extraction can greatly reduce the extraction time,and the yield is similar to traditional solvent reflux extraction.Third,study on the extraction and separation of chemical constituents from Aconitum szechenyianum Gay.with lipophilic organic solvents.The fat-soluble crude alkaloids was extracted with chloroform,then extacted with diluted hydrochloric acid from extracting solution,and was divide into chloroform and acid of two layer. Mainly do research on lipophilic components of chloroform layer.It was dissolved by ethanol,and the solution was preliminary separation by a macroporous resin column on Flash.Collecting eluate that eluted by different concentrations of ethanol.Al eluted with water contains a lot of sugar.We use C-18 column and activated carbon column chromatography to separation and purification in succession and collect each part eluted with water,10%and 20%of ethanol in order,respectively. After these series of complex separation and purification,we got the only one pure compound, named Cl.The HR,¹H NMR,¹³C NMR,2D NMR and TOF-MS concluded that the compound actually was unexpected hydrophilic sugar.The reason to be further explored.