The Material Basis Research On Treatment Of Chronic Pelvic Inflammatory Disease By Hongheierwan

“Hongheierwan”, also named Hongbaierwan, Yanwanzi, Yuanyangqi, Shanhaitang, as a folk herb, is originated from dry tuber and whole plant of Genus Begonia and used long time ago. Its tuber is usually harvested in summer and autumn, and the fruit is gathered in early winter, keeping fresh or dried. It grows in dark and wet places near the mountains and ditch beside and humid forest, and distributes in Hebei,Shandong, Jiangsu, Anhui, Zhejiang, Fujian, Jiangxi, Hunan, Taiwan,Guangxi, Shandong and Hainan. Genus Begonia displays its effects such as promoting blood circulation to remove blood stasis, cooling blood to stop bleeding, swelling of the efficacy of detoxification, and is used to treat hematemesis, hemoptysis, spit blood, metrorrhagia, irregular menstruation, traumatic injury, dysentery and leucorrhea. There is no report about Hongheierwan treating on chronic pelvic inflammatory disease. So in this paper, the physiological and pharmacological of Hongheierwan were systematically studied including in vitro study on the antiinflammatory effect, HPLC fingerprint of n-butanol extract, Effective extracts based on rat models of chronic pelvic inflammatory and content determination of flavonoids. The study of preliminary mechanism of Hongheierwan in the treatment of chronic pelvic inflammation will provide a foundation for the follow-up clinical application.The anti-inflammatory effects of different extracts of Hongheierwanwere investigated through inhibition of anti-inflammatory model using LPS stimulating mouse peritoneal macrophages(RAW 264.7) to produce inflammatory mediators. Experimental drug concentration was screened by cytotoxicity assay. The actions of test substances inhibiting the secretion of inflammatory mediators such as TNF- alpha, IL-1 beta and NO were determined after inflammatory stimuli Escherichia coli type cells stimulated by LPS. The results showed that the contents of TNF-α, IL-1β and NO in inflammatory model group were increased significantly, while the generation and release of TNF-α、IL-1 and NO could be markedly inhibited by ethanol extract, ethyl acetate extract, n-butanol extract of Hongheierwan. Thus this indicates that the ethanol extract, ethyl acetate extract and n-butanol extracts of Hongheierwan possess anti-inflammatory effect. The action mechanism is relevant with adjusting macrophage to produce inflammatory factors TNF-α, IL-1β and NO.The efficacy and mechanism of Hongheierwan were investigated through the influence of the two extracts to chronic pelvic inflammatory disease(CPID) rats. Sham operation group only accepted surgery no modeling, while the other rats were treated by mechanical damage and mixed infection and divided into the mode group, butanol high and low groups, ethyl acetate high and low groups, and positive control group after 7 days. CPID rats were separated to 7 groups and 8 rats in each group. After the treatment of administration for 14 days(sham operation group and model group only treated by distilled water as negative control), uterine swelling degree, the uterine specimen and the level of IL-2, IL-6 of all the groups were detected. The results indicated that the two extracts of Hongheierwan could obviously inhibit uterine swelling,depress the degree of inflammation in the uterus, and restore theconcentration of IL-2 and IL-6 to normal level in CPID rats.Hongheierwan could effectively reduce uterine inflammation of rats. The mechanism is relative with the inhibition of inflammatory factors.Tuber samples of Hongheierwan derived from different harvest time and habitats were purified by solvent extraction and polyamide.The solvent of effective parts were recycled, and then extracted using n-butanol as n-butanol extract. The chromatographic fingerprint of n-butanol extract of Hongheierwan was established by LC-MS, and9 flavone glycosides in n-butanol extract were tentatively identified as quercetin-3-O-rhamnose glucoside, quercetin-3-O-glucose-shu Li Tang glycosides,quercetin-3-O-rhamnose glucose wood Arabia glycosides, que rcetin glycoside-3-O-rhamnose glucose-Shu Li, kaempferol glycoside-3-O-rhamnose glucoside, kaempferol-3-O-glucose-Shu Li, kaempferolglycoside-3-O-rhamnose glucose-Shu Li, kaempferol glycoside-3-O-glucose rhamnose-Shu Liglycoside, saponin. This will provide the reference for the quality control of Hongheierwan and wide applications in clinic.Furthermore,7 compounds in ethyl acetate were identified.6 compounds isolated from this plant for the first time including protocatechuic acid,protocatechualdehyde, catechin, epicatechin, rutin, procyanidin B1 and B2.4 compounds, such as protocatechualdehyde, catechin, procyanidin B1 and B2,were obtained and identified from Begonia.A UPLC method was established to detect five flavoniods and phenolic acids including protocatechuic acid, protocatechualdehyde,catechin, epicatechin and rutin in rhizome, stem and leaf of Hongheierwan. The ACQUITY UPLC HSS T3 column(2.1 mm × 100 mm, 1.8 μm) was adopted. Methanol(A)-water containing 0.1% formic acid(B) was used as the mobile phase. The flow rate was 0.2 ml/min. The detection wavelength was set at 280 nm and column temperature was maintained at 30 oC. The five components were separated effectively andgood linear relationships were set up. The recovery rate of protocatechuic acid, protocatechualdehyde, Catechin, epicatechin, and rutin was 99.45%,96.92%, 99.69%, 98.42% and 99.75%, respectively(RSD < 5%). This UPLC method showed high accuracy and it is convenient and fast. This will provide a scientific reference for utilization of Begonia herbs.