Studies On Chemical Constituents And Anti - Inflammatory Activity Of Phenols In

Menthae Haplocalycis Herba, as traditional Chinese medicine, has been a medicinal and edible plant more than two thousand years.. Pharmacological study showed that various phenolic compounds from Menthae Haplocalycis Herba are important active components with anti-inflammatory, antibacterial and antiviral actions. However, systematic studies on anti-inflammatory of the phenolic fraction and its chemical constituents of Menthae Haplocalycis Herba have never been reported. In order to fully explore the medicinal value of the phenolic fraction and its chemical constituents of Menthae Haplocalycis Herba, this paper carries out the following research work.HPLC-MS/MS was applied to investigate the chemical composition of the phenolic fraction of Menthae Haplocalycis Herba.15 compounds including protocatechualdehyde, p-coumaric acid, caffeic acid, rosmanic acid ethyl ester, p-CoQA, ethy lithospermate, lithospermic acid, apigenin-7-O-rutinoside, hesperidin, diosmin, rosmarinic acid, salvianolic acid D, salvianolic acid B, linarin and thymonin were isolated from Menthae Haplocalycis Herba. The results of mass spectrometry data provide a reference for the screening of the anti-inflammatory compounds of the phenolic fraction from Menthae Haplocalycis Herba.The HPLC specific chromatogram of the phenolic fraction of Menthae Haplocalycis Herba purchased from different drugstores of Beijing was established and 11 common peaks were chose in the specific chromatograms. This study validated the precision, stability and reproducibility of specific chromatograms of the phenolic fraction of Menthae Haplocalycis Herba, and the results showed that RSD of the relative retention time and relative peak area of every characteristic peak in method validation was less than 3%. The similarity is above 0.9.The content of the phenolic fraction was measured by FeC13-K.3[Fe(CN)6] colorimetry with rosmarinic acid as reference substance. The content of rosmarinic acid and linarin from the phenolic fraction was measured by HPLC.The effects of phenolic fraction, rosmarinic acid and linarin on the activity of RAW264.7 macrophages were investigated by MTT assay. And non-toxic concentration ranges of phenolic fraction, rosmarinic acid and linarin were 0～200 μg/mL,0～160μM and 0～20μM, respectively.Using LPS-induced RAW264.7 macrophages, this paper studied the effects of different concentrations of the phenolic fraction and its chemical constituents of Menthae Haplocalycis Herba on the secretion of NO and inflammatory cytokines including TNF-, IL-1β and IL-6 in LPS-induced RAW264.7 macrophages. The results showed that, compared with the normal control group, LPS increased significantly the secretion levels of NO, TNF-a, IL-1β and IL-6; treating different concentrations of the phenolic fraction (50,100 and 200 1μg/ml), rosmarinic acid (10,40 and 160 μM) and linarin (5,10 and 20 μM) for 1 h inhibited the secretion of NO, TNF-a, IL-1β and IL-6. The above data showed that the phenolic fraction, rosmarinic acid and linarin can exert anti-inflammatory effects by regulating the secretion of cytokines and inflammatory mediators.The effects of phenolic fraction (50,100 and 200 μg/ml) and linarin (5,10 and 20 μM) on the mRNA expression were studied by qRT-PCR. The results showed that, compared with the normal control group, LPS (1μg/ml) increased the expression levels of iNOS, TNF-a, IL-1βp and IL-6 mRNA; treating the different concentrations of the phenolic fraction and linarin for 1 h reduced the expression levels of iNOS, TNF-a, IL-1β and IL-6 mRNA.The effects of phenolic fraction (50,100 and 200μg/ml) and linarin (5,10 and 20μM) on MAPK, NF-kB and PI3K/Akt signaling pathways were studied by Western Bolt. The phenolic fraction and linarin inhibited the phosphorylation levels of IκBa and p65 in LPS-induced RAW264.7 macrophages, and linarin inhibited NF-κB p65 translocation from the cytoplasm to the nucleus in a dose-dependent manner. The phenolic fraction and linarin could significantly inhibit phosphorylation levels of p38, ERK and JNK in LPS-induced RAW264.7 macrophages, and thus blocked the activation of MAPK signaling pathway, and the Akt phosphorylation was not inhibited by pre-treatment with phenolic fraction and linarin. These results exhibited that the phenolic fraction and linarin exerted an anti-inflammatory effect by inhibiting the secretion of NO, TNF-a, IL-1β and IL-6 via MAPK and NF-λB signaling pathways.