| Melilotus suaveolens Ledeb, with wide resource in China, is a one or two year old plant of Melilotus of Leguminosae, which have been used as a fork medicine on inflammation-related therapy for a long time. However, the research on anti-inflammatory ingredients and mechanisms of the plant is very scant. In this paper, the anti-inflammatory ingredients and mechanisms of M. suaveolens Ledeb were investigated comprehensively. Meanwhile, the quality evaluation and extraction technology of active parts of M. suaveolens Ledeb and the biotransformation of some active ingredients by several medical fungi were explored.Four solution fractions, such as petroleum ether fraction, ethyl acetate fraction, n-butanol fraction and aqueous fraction, were obtained from ethanol extract of M. suaveolens Ledeb, using petroleum ether, ethyl acetate (EtOAc) and n-butanol (BuOH) as solvent. Furthermore, with Dexamethasone (DM) as positive control, the influences of different solution fractions of M. suaveolens Ledeb on TNF-a mRNA of LPS-stimulated RAW 264.7 cell were investigated by the method of real time PCR. The results showed that the aqueous fraction showed no effect on inflammatory. The ethanol extract and organic solution fractions, such as petroleum ether fraction, ethyl acetate (EtOAc) fraction and n-butanol (BuOH) fraction, showed similar effects on inflammation as Dexamethasone.On the base of its anti-inflammation research, 5 compounds were isolated and purified from petroleum ether and ethyl acetate (EtOAc) fraction, they were identified to be coumarin,scopoletin, scoparone, luteolin and 5,7,4'-trihydroxyl-6,3′-dimethoxylflavone, respectively. Among these identified compounds, scopoletin, scoparone, luteolin and 5,7,4'-trihydroxyl -6,3′-dimethoxylflavone are isolated from this plant for the first time. Meanwhile, scoparone and 5,7,4'-trihydroxyl -6,3′-dimethoxylflavone are isolated from the genus for the first time.Furthermore, the anti-inflammatory mechanisms of these active fractions and coumarin were investigated by ELISA, western blot analysis, real-time PCR and immunocytochemistry assay. The results showed that their anti-inflammation mechanisms attributed to down-regulation of iNOS, COX-2, IL-1βand TNF-αgene expression via the suppression of NF-κB activity. At the same time, those active fractions and coumarin could increase the anti-inflammatory cytokines and mediators such as IL-10 and HO-1. HPLC fingerprint of the M. suaveolens Ledeb extracts shows that coumarin is one of the main anti-inflammatory compounds in the plant.TLC and HPLC were used to set up estimate the herb quality with standard substance of coumarin. The results indicated that the third harvest time is best for collection of the herb, and the contents of coumarin are higher in seeds than that in stems. In addition, with coumarin as the standard substance, HPLC fingerprint of M. suaveolens Ledeb at various harvest time and various medical parts of M. suaveolens Ledeb was set up,and 12 same common peaks and 8 same characteristic peaks were verified with a similarity evaluation system for chromatographic fingerprint of TCM.To develop the herb as a drug, the extraction technology of total anti-inflammation active fraction from M. suaveolens Ledeb was studied, The optimum extraction conditions were: adding 50% ethanol to extract for three times, 1.0 h each time, the amounts of added ethanol were 20 times, 15 times and 12 times, respectively. The optimum purifying process was as follows:the best macroporous resin was YWD07D1, the sample concentration was 200 g /L, the ratio of cloumn diameter to cloumn height was 1:7, the ratio of herb weight to macroporous resin weight was 7 :1.The sample was washed with 3 bed volume (BV)of distilled water, then eluted with 3 BV of 80% ethanol,yield of coumarin was 90% and product purity was above 50%. Furthermore, TLC and HPLC are selected as quality evaluation method to estimate the extract.At last, a primary study on the change of herb by fermentation with 9 kinds of medical fungi, such as Schizophyllum sp., Fomitopsis sp.and Myrothecium sp., was taken, using coumarin content as index. The residue of the herb fermented with 9 kinds of fungi was used as control, the results showed that the change of different fungi diverse to each other. Coumarin could be biosynthesized during the herb fermentation by Myrothecium sp.
|
PDF Full Text Request