Studies On The Chemical Constituents Of Ajuga Ciliata And Their Neuroprotective Activities

The whole plants of Ajuga ciliata Bunge(Labiatae)are used as a folk medicine for the treatment of coughing up blood,injury from fall,tonsillitis,and sphagitis,etc.Athough the genus Ajuga have been reported to be rich sources of bioactive diterpenes,which display insect antifeedant,antibacterial,antimycobacterial,antiplasmodial,cytotoxic,hypotensive activities,the phytochemical and pharmacological studies on A.ciliata are limited.The chemical constituents of the whole plants of A.ciliata have been studied,and partial compounds isolated from A.ciliata have been tested on neuroprotective activities against MPP+-induced neuronal cell death.The purpose of this thesis is to search for lead compound in treatment for serious neurological disorders,such as Parkinson's disease(DP),Alzheimer's disease(AD).Main achievements of the thesis are followings:1.The chemical constituents and pharmacological research of the plants of the genus Ajuga have been reviewed.2.The chemical constituents of the methanol extract of the whole plants of A.ciliata have been investigated.By means of various chromatographic methods such as silica gel,ODS column chromatography,and semi-preparative HPLC,50 compounds(Figure 1)have been isolated from the CH2Cl2 extract of the whole plants of A.ciliata,and their structures were elucidated and identified on the basis of spectroscopic data and physico-chemical methods.They are neo-clerodane diterpenes:ajugaciliatin A?R(1?8,12?16,19?22,and 42),ajugamarin A2 chlorohydrin(9),ajugamarin A1 chlorohydrin(10),ajugacumbin A chlorohydrin(11),ajugacumbin F(17),ajugacumbin A 4,18-diol(18),ajugalide B?D(38,23,and 41),ajuganipponin A?B(36 and 24),ajugacumbin A?B(26 and 25),ajugarin I(27),ajugamarin B2,H1,B1,A2,G1,A1 and C1(28,30,31,32,34,35,and 39),ajugatakasin A?B(33 and 29),ajugapantin A(37),and deacetylajugarin IV(40);fatty acid and fatty acid ester:(10E,15Z)-1-methyl 9,12,13-trihydroxyoctadeca-10,15-dienoate(43),(10E,15Z)-9,12,13-trihydroxyoctadeca-10,15-dienoic acid(44),(2E,6R)-8-hydroxy-2,6-dimethyl-2-octenoic acid(45),and methyl monosuccinate(46);lignanoids:3,3'-bis(3,4-dihydro-4-hydroxy-6-methoxy-2H-1-benzopyran)(47);sterols:stigmasterol(48),?-sitosterol(49),and daucosterol(50).And compounds 1?8,14?16,19?22,and 42 are new compounds,9?11 and 18 are new natural products,and 43?47 are obtained from the genus Ajuga for the first time.3.All the compounds of diterpenes isolated from A.ciliata belong to neo-clerodane diterpenes.Analysis of their constructures,most of the nwo-clerodane diterpenes have a 2-en-?-lactone unit at C-12 position.The neo-clerodane skeleton are divided into three categories of 28-chloro(1?11),18-oxy(12?18),and 4,18-epoxy(22?39),according to different substituents at C-18(except compounds 40?42).In addition,the locations of C-1,C-6,C-12,and C-19 are always substituted by hydroxy,acetoxy,2-methylbutanoyloxy,and tigloyloxy groups.4.The neo-clerodane diterpenes have complicated structures for their chirality carbons.Although all of the known clerodane type diterpenes from the genus Ajuga possess the neo-clerodane absolute configuration,X-ray crystallographic analysis of 1 have be done to give a good evidence to confirm the configurations of the diterpene compounds we have isolated,especially the chiral carbons of 2-methyl butanoate groups and C-12.5.The neo-clerodane diterpenes always have oxygenated substitutes at C-1 and C-12,but the naturally occurring neo-clerodane diterpenes containing a pyran moiety formed by ether linkage spanning C-1 and C-12,are rare,and they all possess 12S-configuration except for synthetic products.This is the first report of compounds 19?21 with 12R-configuration in this type of naturally occurring neo-clerodane diterpenes.6.Ajugaciliatin A?J(1?5,14?15,and 19?21),ajugamarin A2,A1,and A chlorohydrin(9?11),ajugacumbin F and A(17 and 26),ajugalide B?C(38 and 23),ajuganipponin A?B(36 and 24),ajugamarin A1?B1,A2,and G1?H1(35,31?32,34,and 30),ajugatakasin A?B(33 and 29),and ajugapantin A(37)isolated from A.ciliata are evaluated for their neuroprotective activities against MPP+-induced neuronal cell death in dopaminergic neuroblastoma SH-SY5Y cells using an established MTT assay with guanosine as a positive control.Compounds 2,9,10,20,21,29,30,33,35,and 36 exhibited moderate neuroprotective effects,comparing with MPP+-treated group(61.1 ± 4.3%)and positive control(3 ?M,70.0 ± 3.1%;10 ?M,79.7 ± 2.9%;30 ?M,90.1± 3.8%).The results indicate that the cell viabilities of these compounds have significant difference compared with MPP+-treated group(P<0.05);In these compounds,9 and 29 have neuroprotective effects at 3 ?M with the cell viabilities at 71.0 ± 2.5%and 70.2 ± 3.4%,respectively;2 and 9 have neuroprotective effects at 10 ?M with the cell viabilities at 80.4 ± 5.3%and 79.7 ± 3.6%,respectively;2 and 30 have neuroprotective effects at 30 ?M with the cell viabilities at 88.2 ±6.2%and 87.5 ± 2.6%,respectively.The results also indicate that the concentrations and cell viabilities of these compounds have dose-respondes relationship.We will give a further research on these compounds for their neuroprotective activities in future.We hope to search for the leading compounds in treatment of serious neurological disorders on the base of our studies.