Chemical Constituents Of Dracocephalum Moldavica L.and Their Anti-Complementary Activity

The complement system is a component of the innate immune system,which consists of at least 30 proteins.These proteins play major roles in inflammation,immune complex clearance,induction of a normal humoral immune response,and clearance of apoptotic cells.However,excessive activation of this system may result in tissue injure,and may lead to a host of diseases,such as systemic lupus erythematosus,rheumatoid arthritis,atherosclerosis,Alzheimer’s disease and other diseases.According to the traditional efficacy and modern pharmacological action of Dracocephalum moldavica L.as well as immune complement activity.Based on activity orientation,the anti-complementary activities of the aerial parts of D.moldavica were studied.D.moldavica is an annual herb belonging to the genus Dracocephalum and the family Lamiaceae.It is mainly distributed in North China,Northwest China and Northeast China,which has a long and rich history of medicinal in China,and having the functions of benefiting the heart and protecting the brain,dispelling wind clearing heat and opening of the occlud.There are many kinds of chemical constituents and many pharmacological effects such as mycardial protection,central nervous system protection,and anti-tumor activity in D.moldavica.In order to obtain more clearly the active chemical constituents and polysaccharides from this species,seventy nine compounds were isolated from the 75%Et OH extract of D.moldavica using silica gel column,octade cylsilyl（ODS）,Sephadex LH 20,preparative middle pressure liquid chromatography（MPLC）.The chemical structures were elucidated based on extensive spectroscopic experiments,including infrared spectrometry（IR）,ultrviolet（UV）,1D and 2D NMR,HR ESI MS as well as optical rotation,including eight new compounds,including two new monoterpenoid glycosides,（1R,2R,4S）-1,8-epoxy-p-menthan-2-O-β-D-glucopyranosyl-（1→4）-β-D-glucopyranoside（1）,（1R,3S,4S）-1,8-epoxy-p-menthan-3-yl O-β-D-glucopyranoside（2）,one new iridoid glycoside,3’-O-β-D-glucopyranosyl-melampyroside（9）,one new sesquiterpene,1-（2-methylbutanol）-2-pentyl-1,3-cyclohexadiene（11）,two new triterpenoids,28-nor-3β,18β-dihydroxyurs-12-ene（16）,2α,3β,24-trihydroxyolean-12-ene（28）,one new phenolic glycoside,cis-1-（3-hydroxy-4-methoxyphenyl）-2-butene-4-O-α-L-rhamnopyranosyl-（1→3）-O-β-D-glucopyranoside（45）,one new naphthalene derivative,2-octa-2’,4’,6’-atriynenaphthalene（76）,and seventy one known compounds（3-8,10,12-15,17-27,29-44,46-75 and 77-79）.Among them,Compounds 5,8,10,24,26-27,32-33,36,38-40,42,55,58,63,69-72,77 and 79 were isolated from the Lamiaceae family for the first time,compounds 3-4,12-15,19-22,29,31,34,41,44,46,48,50,52,54,59and 60 were identified from the genus Dracocephalum for the first time and compounds6-7,18,23,25,47,53,61 and 62 were reported from the D.moldavica for the first time.Seventy compounds（1-68 and 76-77）were were assessed for their anti-complementary activities using hemolysis and Elisa assay system in vitro,respectively.The CH₅₀,AP₅₀ and LP₅₀ values range from 0.22-1.77 m M,0.47-1.84 m M and 1.56-1.87 m M,respectively.Determined the targets of compounds 4,12,20,38,43,45,48,51,59,65 and 67 in the CP complement activation cascade.Compounds 4 and 59interacted with C3,C5 and C9.Compounds 12 and 48 interacted with C2,C5 and C9.Compound 20 acted on C2,C3,C4,C5,and C9.Compounds 38,65 and 67 interacted with C2 and C5.Compound 43 interacted with C2,C4 and C5.Compound 45 acted on C2 and C4.Compound 51 interacted with C2,C3 and C5.Determined the targets of compounds 20,43 and 65 in the AP complement activation cascade.Compound 20acted on C3,C5,C9 and Factor B.Compounds 43 and 65 interacted with C5 and Factor B.Two homogeneous polysaccharides DMP-1 and DMP-2 were obtained using column chromatography of D101 and DEAE-52.The characteristic groups,molecular weight,monosaccharide composition and glycosidic bond type of two polysaccharides were characterized by UV-vis spectrophotometry,infrared spectroscopy,high performance gel permeation chromatography,high performance liquid chromatography and gas chromatography-mass spectrometry.The results showed that DMP-1 and DMP-2 were composed of mannose,glucose,galactose and arabinose,and their monosaccharide content percentages were 9.20%,31.09%,44.53%,15.18%and 7.22%,44.33%,34.87%,13.57%,respectively.The molecular weights of DMP-1 and DMP-2are 4.1553×10⁵ k Da and 1.9764×10⁵ k Da,respectively.The same six glycosidic bond types were detected in the two polysaccharides,including→5)-Araf-(1→、Manp-(1→、Glcp-（1→、→2）-Manp-（1→、→6）-Glcp-（1→and→3,6）-Galp-(1→.In addition,to assessed for their anti-complementary activities using hemolysis and Elisa assay system in vitro,respectively.The result showed that DMP-1(CH₅₀ value was 0.03 mg/m L)had potent anti-complementary activity on CP,and the anti-complementary activity was stronger than heaprin(CH₅₀ value was 0.11 mg/m L).The anti-complementary activity of DMP-1 and DMP-2 on AP(AP₅₀ value were 0.11,0.07 mg/m L,respectively)was better than that of positive control(AP₅₀ value was 0.13 mg/m L).The inhibitory activity of DMP-1 on LP(LP₅₀ value was 0.25 mg/m L)was stronger than that of positive control(LP₅₀ value was 0.33 mg/m L).Determined the targets of DMP-1 and DMP-2in the CP complement activation cascade.DMP-1 interacted with C2,C3,C5 and C9.DMP-2 acted on C2,C3,C4,C5,and C9.Determined the targets of DMP-1 and DMP-2 in the AP complement activation cascade.DMP-1 acted on C3,C5,C9,Factor B and Factor P.DMP-2 interacted with C3,C5,C9 and Factor B.To summarize,the study not only enriched the chemical constituents of D.moldavica but also found that the polysaccharides of DMP-1,DMP-2 showed better anti-complementary activities.Worth noting is that the anti-complementary activity on lectin pathway of chemical consistent（1-68 and 76-77）were studied for the first time.It is hoped that this study can be able to provide theoretical support for the development of anti-complement drugs from natural products.