Anti-complementary Constituents Of Four Medicinal Plants Including Arnebia Euchroma And Fagopyrum Dibotrys

The complement system, a key component of the innate immune response, composed of more than30species protein widespread in the serum, tissue fluid and the cell membrane, was found to complement the action of antibody directed against bacteria and erythrocytes. When activated inappropriately, it may evoke pathologic reactions in a variety of inflammatory and degenerative diseases such as Alzheimer’s disease(AD), systemic lupus erythematosus(SLE), rheumatoid arthritis(RA), multiple sclerosis(MS), myasthenia gravis(MG), myocardial infarction(MI), cardiopulmonary bypass(CPB), as well as acute respiratory distress syndrome(ARDS) which is complication of intense inflammatory response of the host lung to infectious or noninfectious insult, especially when induced by avian influenza A(H5N1) virus or severe acute respiratory syndrome(SARS) coronavirus. Therefore, inhibition of individual complement is a promising approach for the prevention and treatment of these diseases. Numerous natural products have been reported to possess anti-complementary effect, which provides a broad prospect of inexpensive and non-toxic strategies for the inhibition of complement. As part of our continuous effort to search for anti-complementary agents from TCMs, bioactivity-directed fractionation and isolation of four medicinal plants were carried out, and the main findings are as follows:1. Isolation and Identification of Anti-complementary Constituents from Arnebia euchroma63compounds were identified from the EtOAc extract of A. euchroma including11new compounds as arnebial D, isoalkannin A-B, neoshikonin A-G and tetradecyl ferulate. Isoalkannin A-B, neoshikonin A-G were compounds with novel skeleton of derivatives of alkannin and shikonin, respectively. The known compounds included steroids such as stigmast-6β-hydroxy-4-en-3-one and (3β,4β)-ergosta-5-ene-3,4-diol; derivatives of caffeic acid and ferulic acid such as propyl caffeate, isooctyl ferulate, octadecyl caffeate, eicosyl caffeate, et al; terpenoids as hopenone-I and2β-hydroxybryonolic acid, et al; four derivatives of arnebinol such as arnebinol D, leucocordiachrome H, arnebinol C and isoarnebinol and one alkaloid as echimidine. Among these compounds,26compounds including arnebinols, derivatives of phenol and derivatives of alkannin, showed anti-complementary activity. Mechanism studies demonstrated that arnebial D acted on C1q, C3, C4, C5and C9components,1,2,4-benzenetriol interacted with C1q, C2, C3, C5and C9components, isoalkannin A and Neoshikonin A acted on C1q, C2, C3, C4and C5components.Neoshikonin B, isoalkannin A and neoshikonin E possessed in vitro cytotoxicity against A549(lung carcinoma) with IC50values of16.6, less than0.2and5.43μg/ml, respectively. In addition, neoshikonin E showed cytotoxic acvitivity against DU145(Prostatic carcinoma, IC505.89fig/ml), KB (epidermoid carcinoma of the nasopharynx, IC504.13μg/ml) and its drug-resistant variant (KBvin, IC504.96μg/ml).The method of the hemolytic assay on the classical pathway was used as biological-activity guide in the fractionation of the anti-complementary polysaccharides from the roots of A. euchroma. Fractionation on SepharoseTM and SephacrylTM CC led to the isolation of two anti-complementary polysaccharides, APS-1and APS-2. The HPGPC and HPCE profiles showed single and symmetrically sharp peaks, indicating that they were homogeneous polysaccharides. Through GC, GC-MS, NMR, IR and methylation analysis, the chemical characterization of APS-1and APS-2were detailed described. APS-1was pale yellow powder, a branched polysaccharide with average molecular weight about above1106Da and less than2106Da, composed of Rha, Ara, Xyl, Man, Glc and Gal in the ratio of1.3:6.2:1.0:1.4:3.0:3.5. APS-1was found to contain80.3%of total carbohydrate,12%of uronic acid, only2.79%of protein and3.01%of sulfate group. Furthermore, terminal,1,5-linked and1,3,5-linked Ara; terminal,1,4-linked,1,6-linked,1,4,6-linked Gal and terminal,1,3-linked,1,4-linked Glc were identified in APS-1, among which, terminal,1,5-linked Ara and terminal,1,3-linked Gal amounted to61%. APS-2was brown powder, a branched polysaccharide with average molecular weight more than1106Da and less than2106Da, composed of Rha, Ara, Xy1, Man, Glc and Gal in the ratio of1.0:24.2:1.2:1.1:1.0:2.2. In addition, APS-2was found to contain81.1%of total carbohydrate,10.8%of uronic acid,4.38%of protein and3.70%of sulfate group. In addition,3,5-Me3-linked,2,3-Me2-linked and3,5-Me2-linked Ara and2,3,4,6-Me4-linked^2,3,6-Me3-linked and3,4-Me2-linked Glc and2,4,6-Me3-linked^2,3,6-Me3-linked Gal were identified in APS-2.APS-1and APS-2showed anti-complementary activity on both the classical and alternative pathways with CH50values of203±20μg/ml and282±11μg/ml, AP50 values of45±8μg/mland144±17μg/ml, respectively, weaker than heparin against the CP and AP. Preliminary mechanism studies by using complement component depleted-sera indicated that APS-1interacted with Clq, C2, C5and C9while APS-2acted on Clq, C5and C9components of complement system, and APS-1and APS-2had no effect on recalcification time and thrombin time prolongation.2. Isolation and Identification of Anti-complementary Constituents from Fagopyrum dibotrys41compounds were identified of the n-BuOH extract of F, dibotrys, including3new compounds named as bis-(4-hydroxyl-2,3-di-t-Bu-phenyl-ethylalcoholglycol) ethers, seco-docetaxel A and fagopyritol A4. It needs to be pointed out that seco-docetaxel A was a new compound with a4,5open-looped skelton of docetaxel. This is the first discovery of the derivative of docetaxel from natural resource, which might be a good explanation to the effect of anti-tumor activity of F. dibotrys as a famous TCM. The known compounds included steroids such as stigmast-4-en-3-one and stigmast-3-O-β-D-glucopyranoside; two triterpenes as glutinone and neohecogenin; four flavonoids as (2S,3(S,4R)3’,5’-di-r-Bu-3,4,5,7,4‘-fivedroxyflavan and catechin-3-(O-β-D-gluco(2-cinnamoyl)pyranoside; ten derivatives of phenol as β-D-fructo furanosyl-a-D-glucopyranoside, hydroquinone, et al; and three tannins as procyanidin B-2, procyanidin C-1and3-O-galloyl-procyanidin B-2. Among these compounds,9compounds showed anti-complementary activity, including flavonoids and tannins. Tannins, as the main components of F. dibotrys, were the first time to be found with anti-complementary activity, which might be ascribed to its good activity against the complement system. In addition, seco-docetaxel A was found to act on the C1q, C2, C3, C4, C5and C9components of the complement system.3. Isolation and Identification of Anti-complementary Constituents from Amomum tsao-ko59compounds were identified from the n-BuOH extract of A. tsao-ko, among which (2R,3R,4R)-3,4,7,4’-tetrahydroxy-3’,5’-dimethoxyflavan and2-(4-hydroxy-3-methoxybenzoyl)-4-methoxybenzaldehyde were new compounds. The known compounds included flavonoids such as4’-methoxy-4-hydroxychalcone,4’-hydroxy-2’-methoxychalcone,4,4’-dimethoxychalcone, lhfiwne B, et al; derivatives of phenol such as3-hydroxy-4-methoxybenzaldehyde,3-hydroxy-4-methoxybenzaldehyde, 2-methoxy-1,3-benzenediol,3,5-dihydroxybenzoic acid,4-(2-hydroxypropyl)phenol,3-hydroxybenzoic acid, et al; derivatives of indene such as indene-4-aldehyde, indene-6,7-dihydroxy-4-aldehyde, et al; diarylheptane compounds as l-(4-methoxy henyl)-7-(4-methoxyphenyl)-4(E)-heptene-3-one and1-(4-hydroxyphenyl)-7-3-methoxy-4-hydroxyphenyl)-4(E),6(E)-heptadiene-3-one and five terpenoids such as3-hydroxyl-p-methyl-l-en-7-al, et al. In vitro anti-complementary tests showed that14compounds possessed inhibitory activity. Hydroquinone showed the strongest inhibitory effect on the CP and AP with CH50values of61μg/ml and AP50values of58μg/ml. Among these active compounds, benzenediols such as hydroquinone,2-methoxy-1,3-benzenediol, and4-methoxy-l,2-bnzenediol demonstrated the most potent activities with the CH50and AP50values of less than100μg/ml. Preliminary mechanism studies showed that hydroquinone interacted with C1q, C2, C3, C5and C9components of the complement system and1,7-bis(4-hydroxyphenyl)-4(E)-hepten-3-one acted on C1q, C2, C3, C4, C5and C9components.4. Isolation and Identification of Anti-complementary Constituents from Commelina communis24compounds were obtained and identified from the PE and n-BuOH extracts of C. communis including six steroids such as (222?,24R)-ergosta-6,8,8(14),22-tetraen-3-one, cholest-5-en-3β-o1, lanostan-3β-ol,(22Z)-ergosta-5,7,22-trien-3-ol and cholest-5-en-3-acete, three triterpenoids as quinovic acid and a-amyrin, two flavonoids as catechin-3-O-β-D-gluco(2-cinnamoyl)pyranoside and apigenin-4’-O-glucoside and derivatives of phenol such as6-methoxy-3-methylbenzene-1,2,4-triol, hydroquinone,4-methylbenzaldehyde and1,2-dimethylbenzene. All isolates were tested in vitro for their complement-inhibiting properties on the CP and the AP. Furthermore, targets on the complement activation cascade were identified. The results showed that six of the isolates possessed anti-complementary activities on CP and AP with the CH50values of49-127μg/ml and AP50values of55-761μg/ml, respectively. Mechanism studies using complement-depleted sera demonstrated that quinovic acid acted on C1q, C2, C3, C4, C5and C9, while three derivatives of phenols and flavonoid glycoside interacted with C1q, C2, C3, C5and C9components.