Isolation And Purification Of Collagenase Inhibitor From The Sap Of Aloe And Its Inhibition Study

In this paper, we mainly studied the collagenase inhibitory ingredients from the sap of Aloe, including the method of collagenase activity and inhibition test, the extraction and purification of useful ingredients, chemical structure identify, inhibition ratio test, primarily discussed possible inhibitory mechanism and other inhibitory plants extracts in further study.First of all, we studied the effective factors of Hydroxyproline photocolorimetric method, confirm the test condition as: 1.0mL sample add 2.0mL Isopropanol, 1.0mL oxidation reagent and oxidated 5min, then add 2.0mL color reagent, cooled in icy water 5min after heated 20min at 60℃; Test its UV absorption at 561nm, calculate the Hydroxyproline mass by its standard curve.Use the Hydroxyproline photocolorimetric method, we studied the interaction between collagenase and collagen, then designed the method of collagenase activity and inhibition test: 10mg collagen with 0.1mg/mL collagenase solution at 37.0℃in Tris-HCl buffer, collagenase degraded the insoluble collagen to soluble protein into the buffer, Filtrated the undegraded collagen and add HCl heated in a sealed tube. Then test the Hydroxyproline's mass in the tube by Hydroxyproline photocolorimetric method, collaterally calculated the collagen's mass. The difference between blank sample and inhibition sample indicated the effect of inhibitor.Inhibitory test found the Anthraquinones glycoside materials in Aloe sap have obviously collagenase inhibition; Aloe Power was decocted by hot water, extracted by ethyl acetate and got crude extract after dissolvent; This crude material was separated several times by silica column and got an extract mainly contain two ingredients, then isolated to two pure ingredients by Semi- Preparative HPLC. UV, IR, MS & 1H NMR tested their chemical structure, confirmed as Barbaloin & Isobarbaloin respectively. Inhibition ratio test found they have similar and dose dependent inhibitory activity, respective inhibition ratio was 42.4% & 35.0% at 1mg/mL, higher inhibition ratio in low concentration compared with tetracycline.According to the collagenase inhibitory mechanism of Zn2+ chelating theory, we primarily considered Barbaloin & Isobarbaloin also follow this theory, the inhibition maybe due to their Special chemical structure in the molecule. Further studies found Paeonia lactiflora & Ligusticum chuanxing extraction also have collagenase inhibition.