Studies On Diterpenoid Alkaloids From Delphinium Grandiflorum,Aconitum Bulbilliferum,aconitum Hemsleyanum Var. Circinatum And Aconitum Coreanum

"Caowu",consisting of the Aconitum L.and Delphinium L.,has been used in traditional Chinese medicine to treat rheumatic arthralgia and articular pain for thousands of years.Diterpenoid alkaloids are a class of structurally complex natural products that are the characteristic active constituents of Aconitum and Delphinium species,and exhibit a wide range of pharmacological activities,including analgesic,anti-inflammatory and cardiotonic activities.The in-depth research of active constituents from Caowu is of great significance to the development of new drugs.In this thesis,phytochemical studies were carried out on four kinds of"Caowu"including Delphinium grandiflorum L.,Aconitum bulbilliferum Hand.-Mazz.,Aconitum hemsleyanum var.circinatum W.T.Wang and Aconitum coreanum Levl.Ninety-six alkaloids were obtained by multiple chromatographic methods from these four plants,and all isolated compounds were structurally identified based on spectroscopic and chemical methods.Twenty-six new compounds,including two C₂₀-diterpenoid alkaloids with same novel skeleton,were identified.In order to find the components with excellent biological activities and to lay the foundation for the quality evaluation of"Caowu",the analgesic,anti-inflammatory,anti-tumor,and acetylcholinesterase inhibitory activities of some isolates were evaluated by the acetic acid twist method,Griess method,MTT method and Ellman method according to the relationship between different structural characterization units and biological activities.The details are as follows:（1）The phytochemistry and biological research of Delphinium grandiflorum L.Delphinium grandiflorum L.（Ranunculaceae）is a perennial herbaceous plant belonging to Subsect.Grandiflora of Delphinium.It is mainly distributed in northwestern Yunnan,Sichuan,Shanxi,Hebei provinces and the northeastern of China.The study on the whole herb of this plant resulted in the isolation and structural determination of four new C₂₀-diterpenoid alkaloids,grandiflonines A-D,seven new lycoctonine type C₁₉-diterpenoid alkaloids,grandiflonines E,grandiflonine F,grandiflonine G,N-（2′′-methylsuccinamide）-delectine,N-（2′′-methylsuccinyl）-delectine,N-（3′′-methylsuccinyl）-delectine and N-（2′′-methylsuccin-amide）-andersonidine,together with seventeen known compounds,umbrosumine C,umbrosumine A,trifoliolasine B,delsemine A,14-O-acetyldelectinine,lycoctonine,delphatine,browniine,14-acetylbrowniine,delectinine,14-dehydrobrowniine,guan fu base A,yunnanensine C,delgrandine,lappaconitine,deoxylappaconitine and predicentrine.Grandiflonines A and B represent a new skeleton type with C（18）-Me inα-configuration,which derived from the hetisine type C₂₀-diterpenoid alkaloids through the E ring opening reaction.The stereostructure of grandiflonine A was clarified by the Mosher method and X-ray diffraction analysis.Most of the isolates from this plant were evaluated for their inhibitory activities against LPS-induced NO production in the RAW 264.7 cell line,except for grandiflonines D-F,trifoliolasine B and delsemine A.Unfortunately,none of the tested compounds showed significant inhibitory activity at a concentration of 10μM.Acetic acid-induced writhing experiments were used to test the analgesic activity of grandiflonine G,lycoctonine,browniine,deoxylappaconitine,8-acetyl-deoxylappaconitine and 8-acetyl-lappaconitine.As a result,deoxylappaconitine showed notable analgesic activity,with ED₅₀of 0.35 mg/kg and LD₅₀ of 16.18 mg/kg（TI=46.22）,stronger than the reference drug lappaconitine.（2）The phytochemistry and biological research of Aconitum bulbilliferum Hand.-Mazz.Aconitum bulbilliferum Hand.-Mazz.belongs to the Ser.Volubilia of the genus Aconitum and its bioactive components have not yet been reported.In order to search for more bioactive substances,phytochemical studies on the roots of A.bulbilliferum were conducted to afford five new diterpenoid alkaloids,named bulbilliferines A-B and bulbillifedines A-C,along with seven known diterpenoid alkaloids,such as 8-O-methyl-14-O-anisoylchasmanine,geniculatine B,austroconitine B,14-acetylkaracoline,aconitramine C,vilmorrianine A,genicunine B,14-O-acetylsachaconitine,14-acetoxy-8-O-methylsachaconitine,geniculatine C,acoforesticine,ezochasmanine,chasmanine,condelphine,columbianine,15-α-hydroxy-neoline,anisoylyunaconine,aconitramine A,aconosine and panicutine.Bulbilliferine A is a new denudatine-type C₂₀-diterpenoid alkaloid,bulbilliferine B is a new hetisine-type C₂₀-diterpenoid alkaloid,and bulbillifedines A-C are three new aconitine–type C₁₉-diterpenoid alkaloids.Furthermore,the analgesic effects of some isolated compounds were evaluated.8-O-methyl-14-O-anisoylchasmanine and austroconitine B displayed strong pain relief effects,reducing writhing by 83.6%and 83.1%at 10 mg/kg,respectively.At a 0.1 mg/kg dose,aconitramine C and vilmorrianine A showed moderate analgesic activities with 41.7%and58.8%inhibition,respectively.（3）The phytochemistry and biological research of Aconitum hemsleyanum var.circinatum W.T.WangAconitum hemsleyanum var.circinatum W.T.Wang is a perennial herbaceous plant belonging to the Ser.Volubilia of Aconitum.Chemical investigation on the roots of this plant led to the isolation of twenty-one alkaloids,including six new compounds,namely circinatines A-F.Among them,circinatine A is a 7,17-seco type C₁₉-diterpenoid alkaloid.Circinatine E is a C₁₉-diterpenoid alkaloid with N-CHO,and its stereochemistry was elucidated through X-ray analysis.Circinatine F is a new diterpenoid alkaloid with a nitrone functionality.The known compounds were identified as vilmorrianine D,nagadine,14-acetylnagadine,talatisamine,hemsleyanine C,circinasine D,hemsleyanine E,hemsleyanine D,hemsleyanisine,hemsleyadine,hemsleyasine,indaconitine,crassicauline A,yunaconitine,anisoylyunaconine and kongboendine.In order to evaluate the biological activities of these compounds isolated from A.hemsleyanum var.circinatum,all compounds were tested for their in vitro cytotoxicity against human cancer cell lines,including HCT116,786-0,MCF-7and A549 cells by the MTT method.Unfortunately,all compounds were inactive(IC₅₀>60μM,n=6).（4）The phytochemistry and biological research of Aconitum coreanum Levl.Aconitum coreanum Levl.is a member of Ser.Brachypoda of the genus Aconitum.In the search for structurally unique and biogenetically interesting alkaloids,the chemical investigation of this plant led to the isolation of four new hetisine diterpenoid alkaloids,named coreanines A-D,as well as seventeen known compounds.The known compounds were identified as guan fu base A,guan fu base Y,guan fu base F,guan fu base G,guan fu base O,guan fu base P,guan fu base Z,acoridine,trichodelphinine D,triacetylhetisine,hetisine,guan fu base R,anthoroisine F,talatisamine,14-acetyltalatizamine,condelphine and columbidine.Some of the isolated compounds were evaluated for the acetylcholinesterase inhibitory effects.Unfortunately,the results showed that these compounds did not exhibit obvious inhibitory activity at concentrations up to 50μM.