| The complement system plays an important role in host immune defense against infection. However, when activated inappropriately, the complement system may cause a variety of diseases such as system lupus erythematosus (SLE), rheumatoid arthritis (RA), and acute respiratory distress syndrome (ARDS). The over-activation of the complement system may induce the increased vascular permeability, angiectasis and topical or systemic edema. The agents of clearing heat and eliminating toxins exert their inhibitory effects against the exudation, the hyper-functional vascular permeability and edema during the early phase of inflammation. Clinical examples proved the traditional Chinese medicine of clearing heat and eliminating toxins to be effective in the treatment of severe acute respiratory syndrome (SARS). It has been reported that the over-activation of the complement system takes part in the pathogenesis of SARS. Thus, we may infer that the traditional Chinese medicine of clearing heat and eliminating toxins attenuate the inflammation through its inhibition of the complement system.Houttuynia cordata Thunb., a traditional Chinese Medicine with the main function of heat-removing and detoxification, showed inhibitory activity on complement in vitro and was chosen as the object of this study. The isolation and characterization of anti-complementary ingredients from H. cordata were presented. The in vivo effects of active agents of H. cordata and another heat-removing herb, Arnebia euchroma, on complement-related diseases were also described. Furthermore, the exploratory research was carried out for the application of anti-complementary assay in the quality evaluation of traditional Chinese Medicine.1. Anti-complementary effect of different extracts from H. cordataThe optimal dilutions of guinea-pig sera (resources of complements) that gave submaximal lysis in the absence of complement inhibitors were determined to perfect the hemolysis assay. The results showed crude polysaccharide (CHCP, CH50=0.092±0.020 mg/ml, AP50=0.221±0.017 mg/ml) and EtOAc extract (CH50=0.378±0.029 mg/ml, AP50= 0.571±0.061 mg/ml) of H. cordata were more active than petroleum extract (hemolysis at high concentrations) and n-BuOH extract (CH5o=1.324±0.102 mg/ml, AP50=2.084±0.147 mg/ml).2. Isolation and characterization of active compounds of EtOAc extract from H. cordataBioactivity-guided fractionation led to the isolation of 23 compounds, including seven flavonoids [quercitrin (1), quercetin (2), afzelin (3), isoquercitrin (4), apigenin (5), hyperoside (6), scutellarein (7)], six alkaloids [aristolactam B (8), cepharadione B (9), piperolactam A (10), norcepharadione (11), arisolactam A II (12), 5-dioxodehyroasmilobine (13)], one monoterpene [vomifoliol (14)], one lignan [epishicine methyl ether (15)], one coumarin [esculetin (16)], and seven phenols [3,4-dihyroxybenzoic acid (17), vanillin (18),1,4-benzendiol (19), p-hydroxybenzoic acid (20),3,4-dihyroxybenzoic folmadehyde (21),p-dimethoxy benzene (22), and o-dihydroxy dimethoxy benzene (23)]. These compounds were assayed in vitro for their anti-complementary activity. Flavonoids have been found to be the main anti-complementary principles of H. cordata. Afzelin (3) showed the strongest anti-complementary activity (CH50=0.125±0.012 mg/ml, AP50=0.242±0.004 mg/ml). Other flavonoids, like quercitrin, quercetin and isoquercitrin, also had great activity.Results of primary mechanism study indicated that quercitrin, afzelin and isoquercitrin acted on all the tested complement components (Clq, C2, C3, C4, C5 and C9); quercetin selectively blocks Clq, C2, C5 and C9. As two representative flavonoids, quercetin and quercitrin were tested for their influence on the coagulation system. There were no adverse effects of these two flavonoids on the coagulation system.We also studied the anti-viral, anti-bacterial and anti-tumor activity of the compounds.3. Anti-complementary pharmacological effects of crude polysaccharides from H. cordata (CHCP)CHCP was brown powder composed of Glu:Gal:Ara:Rha in the ratio of 3.40:2.14:1.17:1.00, along with trace amount of Xyl and Man. CHCP contained 77.21%of total carbohydrate,37.36%of uronic acid and 6.17%of protein. CHCP mainly blocked C3 and C4 components.The in vivo anti-complementary effect of CHCP was studied in acute lung injury (ALI) rat model. CHCP significantly attenuated pulmonary injury in ALI rats and inhibited in vivo over-consumption of complement system. In addition, it improved the oxidant-antioxidant imbalance, reduced pulmonary edema and protein exudation.Febrile response was induced in Wistar rats by intraperitoneal injection of LPS. The oral administration of CHCP led to the significant anti-pyretic effect and the inhibition on the over-activation of the complement system. The reduction of the leukocyte number also indicated that CHCP could attenuate the inflammation in vivo. The data suggested that the anti-inflammation and anti-pyretic effects of H. cordata are associated with its inhibition of the complement system.Experimental results suggested that CHCP had no effect on recalcification time, thrombin time, activated partial thrombin time and prothromin time prolongation. Thus, CHCP may have bright prospect in complement-inhibiting therapy.4. Anti-complementary pharmacological effects of crude polysaccharide from Arnebia euchroma (CAEP)CAEP was brown powder composed of Ara:Glc:Gal:Man:Rha in the ratio of 3.69:2.57:1.47:1.20:1.00, along with trace amount of Fuc and Xyl. CAEP contained 76.36%of total carbohydrate,24.37%of uronic acid and 9.37% of protein. CAEP showed great anti-complementary activity in vitro (CHso= 0.12 mg/ml, AP50= 0.52 mg/ml).In vivo study suggested significant therapeutic effects of CAEP on acute lung injury and LPS-induced febrile response. Hemolytic assay of rat sera indicated that the in vivo activity of CAEP was associated with its inhibitory effect on the over-activation of complement system. The experiment also proved that the heat-removing and detoxification herb may exert its function with the inhibition on the complement system.5. Exploratory research of anti-complementary assay in the quality evaluation of traditional Chinese medicineAccording to the requirements of "Chinese Medicine Bioassay Guidelines" in Appendix XVIIIC of Chinese Pharmacopoeia 2010 Volume I, the methodology study of anti-complementary assay was carried out with the water extract of H. cordata as the test substance and heparin sodium as the positive control. The anti-complementary assay was reliable and accurate with good repeatability and feasibility.The correlations between components and the anti-complementary activity of H. cordata herb were also studied. Volatile oils from H. cordata showed no anti-complementary activity; total flavonoids and crude polysaccharides with good activity. Further research indicated that the activity of the total flavonoid fraction and its total flavonoid content were quite related (Rcp=-0.736, and Rap=-0.844). And there was good correlation between the uronic acid content and anti-complementary activity of crude polysaccharide (Rep=-0.922 and Rap=-0.686). However, the relevance between the activity of the herb and components contents is not significant; which means, the content of one component is not able to reflect the activity of the herb.The applicability of anti-complementary assay was conducted in 11 batches of H. cordata. For classical pathway, CH50 value should be lower than 1.618 mg/ml; and AP50 lower than 4.198 mg/ml for alternative pathway.The results of in vivo study indicated that herbs may exert the "heat-clearing" activity through the inhibition of the complement system. CHCP and some flavonoid components, as well as CAEP, have shown great potential in the development of anti-complementary agents due to their great anti-complementary activity and little adverse effects. CHCP and CAEP could be the candidates for the treatment of ALI and fever. The anti-complementary activity may stand for the therapeutic activity and quality of H. cordata; therefore, the anti-complementary activity could be one of the indexes for the quality evaluation of heat-removing herbs, such as H. cordata. |
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