Anti-inflammatory activities of an active fraction isolated from the root of Astragalus membranaceus in RAW 264.7 macrophages.

The root of Astragalus membranaceus (AR), which has been widely used in Traditional Chinese herbal formulae for treating foot ulcer, was found to exhibit anti-inflammatory property, but its molecular mechanism still remains unknown. We previously identified the anti-inflammatory sub-fraction using bioassay-guided fractionation. The objective of the present study was to investigate the anti-inflammatory mechanism of the major active fraction (MAF) (0.039 to 0.156 mg/mL) using lipopolysaccharide (LPS)-stimulated mouse macrophage RAW 264.7 cells. MAF was shown to inhibit LPS-induced mRNA and protein expression of inducible nitric oxide synthase by 54.7% and 65.1%, respectively. Additionally, MAF down-regulated the protein expression of cyclooxygenase-2 and MAPK regulator by 45.0% to 74.6%, as well as the reduction of DNA binding activity of nuclear factor kappa B (NFκB) by 66.5%. It also attenuated the production of prostaglandin E2 , interleukin-1 beta (IL-1ß), IL-6 and tumor necrosis factor alpha by 21.2% to 86.2%. Furthermore, the chemical constituents of MAF were identified. A total of 13 known chemical compounds were found in MAF, including five isoflavonoids and eight saponins. In conclusion, a bioactive fraction of AR was identified which possessed anti-inflammatory property by reducing the release of inflammatory mediators and inactivation of NFκB through MAPK signalling pathway.

Isolation of anti-inflammatory fractions and compounds from the root of Astragalus membranaceus.

Foot ulceration, if not treated properly, will eventually result in amputation. Inflammation may impede the wound healing process if not properly controlled. The root of Astragalus membranaceus (AR) is one of the Chinese herbs commonly found in Chinese herbal formulae used for treating foot ulcer. In this study, we aimed to identify the active fractions and/or compounds from AR aqueous extract, which are responsible for the anti-inflammatory effect using in vitro bioassay-guided fractionation. The anti-inflammatory effect was monitored by the inhibition of nitric oxide (NO) released from lipopolysaccharide-stimulated mouse macrophage RAW 264.7 cells after treated with AR aqueous extract or its fractions and isolated components. Two major active fractions (P2-3-2-2-2 and P2-3-2-2-3) were found to significantly inhibit NO production at 0.156 mg/mL (p < 0.01). In addition, three chemical components (formononetin, calycosin and astragaloside IV) were successfully isolated from P2-3-2-2-3. Only formononetin could significantly inhibit NO production (p < 0.01), whereas the other two components had no significant effects at concentrations ranging from 0.039 to 0.156 mg/mL. In conclusion, two major anti-inflammatory active fractions that may enhance wound healing were identified, and formononetin was one of the active ingredients in the active fractions.

Induction of Angiogenesis in Zebrafish Embryos and Proliferation of Endothelial Cells by an Active Fraction Isolated from the Root of Astragalus membranaceus using Bioassay-guided Fractionation.

The objective of the study was to identify the active fraction(s) from AR aqueous extract responsible for promoting angiogenesis using bioassay-guided fractionation. The angiogenic activity was screened by monitoring the increase of sprout number in sub-intestinal vessel (SIV) of the transgenic zebrafish embryos after they were treated with 0.06-0.25 mg/ml of AR aqueous extract or its fraction(s) for 96 h. Furthermore, the angiogenic effect was evaluated in treated zebrafish embryos by measuring the gene expression of angiogenic markers (VEGFA, KDR, and Flt-1) using real-time polymerase chain reaction (RT-PCR), and in human microvascular endothelial cell (HMEC-1) by measuring cell proliferation using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, (3)H-thymidine uptake assay, and cell cycle analysis. A major active fraction (P1-1-1), which was identified as glycoproteins, was found to significantly stimulate sprout formation (2.03 ± 0.27) at 0.125 mg/ml (P < 0.001) and up-regulate the gene expression of VEGFA, KDR, and Flt-1 by 2.6-fold to 8.2-fold. Additionally, 0.031-0.125 mg/ml of P1-1-1 was demonstrated to significantly stimulate cell proliferation by increasing cell viability (from 180% to 205%), (3)H-thymidine incorporation (from 126% to 133%) during DNA synthesis, and the shift of cell population to S phase of cell cycle. A major AR active fraction consisting of glycoproteins was identified, and shown to promote angiogenesis in zebrafish embryos and proliferation of endothelial cells in vitro.

Stachyose: One of the Active Fibroblast-proliferating Components in the Root of Rehmanniae Radix ( dì huáng).

This study aimed to investigate and compare the fibroblast-proliferating activities of different Rehmanniae Radix (RR) samples and its chemical components using human normal fibroblast cells Hs27. Those active components were quantified in differently treated RR samples using UPLC so as to correlate activity with component content. Our results showed that dried RR aqueous extract exhibited the most potent fibroblast-proliferating activity. Stronger effect was observed when ethanol with heating was applied in the extraction process. Stachyose and verbascoside were demonstrated for their first time to exhibit significant stimulatory effects on fibroblast proliferation. However, the proliferating effect of dried RR extract did not correlate with the stachyose content, and verbascoside was not responsible for the fibroblast proliferative effect of RR since it was undetectable in all samples. In conclusion, stachyose only contributed in part to the activity of RR, suggesting that other active components might be present and yet to be found.