Studies On Hemostatic And Anti-inflammatory Effective Substances Of Sedum Aizoon L. And Its Quality Evaluation

Sedum aizoon L. (SA), alternately named Yang xin cao and Tu san qi, is widely distributed in Fujian, Jiangsu, Zhejiang, and Henan provinces of China. This herb is useful in stopping bleeding without stasis, tranquilization, and detoxification. It has been used to treat insomnia, pain or trauma, especially various hemorrhages in folk medicine. However, the reports regarding the overall effect, active ingredients, and its mechanism are scarcely found in the literature so far, especially for its anti-inflammatory effect. And it would be serious constraints of its clinical application and quality evaluation of this herb or its preparations. Therefore, this study was aimed to investigate the chemical constituents which were responsible for their hemostatic and anti-inflammatory activities as well as its quality evaluation in order to provide a basis for the application of this herb in folk and clinical use.Firstly, we have provided a brief introduction on pharmacognosy, chemical constituents, quality control, and pharmacological and clinical application of Sedum aizoon L. based on its related literatures.In our previous study, the EtOAc fraction of aqueous extract of S A exhibited the hemostatic activity, but its hemostatic constituents are not yet clear. Therefore, the chemical constituents of EtOAc fraction from SA and their hemostatic activity were investigated to clarify its hemostatic material. Eleven isolated compounds were identified as p-hydroxybenzoic acid (IⅠ), gallic acid (Ⅱ), protocatechuic acid (Ⅲ), vallinic acid (Ⅳ), thymine (Ⅴ), caffeic acid (Ⅵ),5,7-dihydroxy chromone (Ⅶ), pyrogallol (Ⅵ), quercetin (Ⅸ), kaempferol (Ⅹ) and luteolin (Ⅺ). Compounds Ⅲ-Ⅵ are the first report of isolation from this plant. Then 8 compounds were tested for hemostatic activity using capillary method and coagulation assays including blood clotting time and PT, APTT, TT in vivo. According to results, gallic acid, vallinic acid, and luteolin could significantly reduce the clotting time and values of PT, APTT, TT. Moreover, caffeic acid also shortened the clotting time, PT and APTT. Thus, it was suggested that SA produced the hemostatic activity possibly related to the presence of gallic acid, vallinic acid, caffeic acid, and luteolin.Next, the anti-inflammatory effect of the fractions and pure compounds isolated from SA by modern separation technology was assessed in LPS-stimulated RAW 264.7 macrophages (inflammatory cell model) in vitro. The priliminary results showed that the EtOAc fraction of alcohol extract of SA displayed the anti-inflammatory activity. Then, the chemical constituents were isolated from this effective fraction by column chromatography and identified as:(1) P-sitosterol, (2) β-daucosterol, (3) oleanolic acid, (4)p-hydroxybenzoic acid, (5) gallic acid, (6) protocatechuic acid, (7) vallinic acid, (8) caffeic acid, (9) 5,7-dihydroxy chromone, (10) methyl gallate, (11) ethyl gallate, (12) quercitrin, (13) myricetin, (14) quercetin, (15) kaempferol, (16) luteolin, (17) isorhamnetin, (18) 3’,4’,5,7-tetrahydroxy flavanone, (19) galuteolin, (20) iriflophene, and (21) iriflophene-2-O-β-D-glucoside. Compounds 18,19,20, and 21 are also the first report of isolation from this plant. Among them, vallinic acid, quercetin, and isorhamnetin inhibited LPS-induced NO and IL-6 production. Protocatechuic acid, caffeic acid, and luteolin also inhibited the pro-inflammatory cytokines TNF-a and IL-6 generation. Moreover, myricetin, 3’,4’,5,7-tetrahydroxy flavanone, and galuteolin could reduce NO, TNF-a, and IL-6. Thus, These results indicated that SA and its constituents such as protocatechuic acid, vallinic acid, caffeic acid, quercetin, luteolin, isorhamnetin, myricetin,3’,4’,5,7-tetrahydroxy flavanone, and galuteolin exhibited anti-inflammatory activity which might attribute to inhibition of NO or TNF-a, and IL-6 generation.Finally, the assessment of the quality of SA besed on the HPLC fingerprint combined with multicomponents quantitative analysis. A HPLC fingerprint of SA from 16 different origins was established. According to results, each batch of the herb was integrally stable with certain differences via similarity evaluation, cluster analysis and principal component analysis. Compared with the other origins, the herbs from Fujian Nanping, Henan, Zhejiang, and Fujian Liancheng indicated a bigger difference and were classified as Ⅱ、Ⅲ、Ⅳ categories based on cluster analysis and principal component analysis. Hence, the overall quality of SA from Shanxi, Gansu, Guangxi, Hubei, and Yunnan provinces showed more stable and homogeneous by comprehensive analysis. Then, a quantitative analysis of quercetin, kaempferol, luteolin, isorhamnetin, gallic acid,p-hydroxybenzoic acid, and methyl gallate was established by HPLC. The results revealed that above compounds of each batch showed obvious differences in quantity or quality.