Study On Anti-inflammatory Material Base And Total Flavonoids Of Kummerowia Striata(Thunb)Schindl

The traditional Chinese medicine Jiyancao, with wide resource in China, also known as Kummerowia striata(Thunb)Schindl. It is a one year old plant of Kummerowia striata of Leguminosae, which have been used as a folk medicine on inflammation-related therapy for a long time. However, the research on anti-inflammatory ingredients and quality standards of the plant is almost empty. In this paper, the anti-inflammatory Material base and quality standard of Renzicao were investigated comprehensively. Meanwhile, the preparation technique and preliminary pharmacodynamics of anti-inflammatory active fractions of Jiyancao were explored.Writing the literature review of Renzicao by consulting domestic and international literature, including Pharmacognosy, Chemical composition, pharmacology and Clinical application.RAW264.7macrophage line in mice was induced by LPS to set up the inflammatory model. ELISA assay was used to measure the secretion of TNF-α, IL-1β and IL-6in the supernatant of MSL treated and untreated macrophages. The mRNA expression of TNF-α, iNOS, COX-2and the protein expression of COX-2, HO-1were detected by real-time PCR and Western blot respectively. The results showed that the ethanol extracts from Renzicao had similar effects on inflammation as Dexamethasone, it could inhibit inflammation by down-regulating the expression and secretion of pro-inflammatory factors, and enhancing the expression of HO-1. Four solution fractions, such as cyclohexane fraction, ethyl acetate fraction, n-butanol fraction and aqueous fraction, were obtained from ethanol extract of Renzicao, using cyclohexane, ethyl acetate and n-butanol as solvent. Furthermore, with Aspirin as positive control, the influences of different solution fractions of Renzicao were investigated by the method of mice acute ear swelling model. The experimental results showed that only the ethyl acetate fraction had a significant anti-inflammatory effect. On the base of the anti-inflammation research,10compounds were isolated and purified from the ethyl acetate fraction, they were identified to be β-sitosterol, apigenin, kaempferol, genistein, quercetin, isoquercitrin, rutin, luteoloside, isovitexin and isoorientin. Among these identified compounds, P-sitosterol, kaempferol, genistein, isoquercitrin, rutin, luteoloside, isovitexin and isoorientin are isolated from this plant for the first time.Luteoloside are chosen as the standard control, Renzicao are identified by TLC without interruption. With rutin as the control, the total flavones in Renzicao are3.32%respectively determined by the method of UV-VIS spectrophotometric at the wavelength of510nm. Meanwhile, RP-HPLC was used to determine the content of quercetin and luteoside in Renzicao hydrolyzate, the chromatographie conditions included Zorbax SB-C18column (4.6mm×250mm,5μm) and the mobile phase consisting of methanol-0.2%phosphorie acid (50:50), the flow rate was0.1mL/min, the detection wave-length was360nm, the colum temperature was30℃. The linear range of quercitin and luteolin were5.28-26.4μg/mL(r=0.9995, n=5),4.12-20.6μg/mL(r=0.9991, n=5). The isoorientin was also determined by RP-HPLC in Renzicao, the chromatographie conditions included Welchrom-C18column (4.6mm×250mm,5μm) and the mobile phase consisting of Acetonitrile-0.2%phosphorie acid (15:85), the flow rate was0.1mL/min, the detection wave-length was360nm, the colum temperature was30℃. The linear range of isoorientin was8.08-40.4μg/mL(r=0.9999, n=5). The contents of quercetin and luteoloside in Renzicao hydrolyzate is0.233%and0.032%respectively; the content of isoorientin in Renzicao is about0.305%. Furthermore, the best collecting time for the plant is from August to September by comparing the contents of total flavones. Such results supply a better basis for its harvesting and the quality control.On the basis, the optimum total flavonoids preparation process of Renzicao was carried out by orthogonal test and single factor compared, practicable extraction technology, Clarification technology and purification technology are selected as follows:the optimization of the extraction technology is:70%ethanol15times capacity of Renzicao, extracted2times,60min per time. And then made the filter solution at the concentration rate of1:6(weight of herb to the volume), added1.0%chitosan acetate acid solution to0.60%(g/100g) at60℃, deposited24h, filtrated. Then the concentrated sample was purified by Macroporous resin D-101, the ratio of cloumn diameter to cloumn height was1:8,4BV of the Renzicao solution (6.0mg/mL), filling rate of2min/mL, flushing with2BV of water, followed by4BV of80%ethanol, the desorption flow rate was3mL/min, then the flushed solution is treated by decompressed-concentration, desiccated, shattered.Learn from the methods of Renzicao, The quality of Renzicao total flavonoids extraction is examined. The contents of the total flavonoids, luteoloside are measured, and are about60%and2.4%.To study the the anti-inflammation effects of the total flavonoids from Renzicao. Through two pharmacological models:xylene-induced ear swelling in mice, eggwhite-induced toe swelling in mice. The experimental results showed that when the total flavonoids from Renzicao was125-500mg/kg, it had a significant anti-inflammation effect, showing that the anti-inflammation effect increased with the dose of the total flavonoids. These studies provided a preliminary pharmacodynamic data for the development and application of Renzicao and its total flavonoids.In general, what we have done in the paper could provide some scientific basis for the further research on Renzicao.