In vitro and in vivo Mechanistic Studies of the Wound-healing Effects of Astragali Radix and Phytochemical Analysis of Its Active Fractions/Components Isolated Using Bioassay-guided Fractionation

Wound healing process is typically divided into three sequential but overlapping phases, namely inflammation, proliferation, and remodeling. Chronic wounds fail to proceed through an orderly and timely healing process, and hence cannot be recovered due to the underlying pathology. They may lead to ulceration such as diabetic foot ulcers. In our recent study, a simplified diabetic foot ulcer herbal formula NF3 was shown to enhance diabetic wound healing in rats, and Astragali Radix (AR) was demonstrated as the principal herb in NF3. Therefore, the wound healing effects of AR would be further investigated in this study.;We aimed to (1) isolate the AR major active fraction(s) or pure compound(s) which were responsible for the three wound healing related mechanism of actions (antiinflammation, angiogenesis and fibroblast proliferation) by bioassay-guided fractionation; (2) further investigate their underlying mechanisms using different in vitro and in vivo models; (3) identify their chemical constituent(s); (4) investigate their in vivo wound healing effects; and (5) examine their oral bioavailability in in vitro model.;Several AR major active fractions including anti-inflammatory active fractions (P2-3-2-2-2 and P2-3-2-2-3), angiogenic active fraction (P1-1-1) and fibroblast proliferative active fraction (P2-2-3), had been successfully isolated.;Thirteen known AR compounds including five flavonoids and eight triterpenoid saponins were found in P2-3-2-2-2 and P2-3-2-2-3. Formononetin and ononin were the active components which partially contributed to its anti-inflammatory activity due to their mild effects and minute amounts (less than 5 %). MAF (combined fraction of P2-3- 2-2-2 and P2-3-2-2-3) was shown to display anti-inflammatory effects by reducing the release of inflammatory mediators and inactivation of NFeB through MAPK signalling pathway.;P1-1-1 was identified to consist of glycoprotein. It stimulated angiogenesis in zebrafish embryos via up-regulating expression of VEGF and its tyrosine kinase receptors, and proliferation of endothelial cells (HMEC-1) by increasing cell viability, DNA synthesis and the S phase population.;P2-2-3 was found to have trace of adenine, adenosine and guanosine (0.1 %) which were shown to have no proliferative activities. The proliferative property of P2-2-3 was further verified by the increase in cellular DNA synthesis and S phase population in cell cycle analysis.;To investigate their wound healing effects in diabetic foot ulcer rat model, the AR combined fraction which should consist of the three major active fractions (MAF, P1-1- 1 and P2-2-3 in 1:1:1 ratio) was replaced by their corresponding mother active fractions (P2-3, P1-1 and P2-2) due to their low extraction yields. Unfortunately, the AR combined fraction was found to have no significant promoting effect in wound healing when compared with that of the control group.;The oral bioavailability of the three major active fractions were investigated in in vitro Caco-2 model. Among them, only MAF was proved to display in vivo biological activity due to the presence of its absorbable active components (formononetin and ononin).;In conclusion, we had successfully isolated several major AR active fractions which exhibited anti-inflammatory, angiogenic and proliferative properties, by bioassayguided fractionation. The anti-inflammatory active fractions could contribute greatly to the overall wound healing effects when compared with the other two fractions due to their confirmed in vivo biological effects. The wound healing effects of the major active fractions which were combined in different ratios, would be further investigated in the future.