Dose-dependent in vitro inhibition of rabbit corneal matrix metalloproteinases by an extract of Pothomorphe umbellata after alkali injury

Abstract

The in vitro ability of Pothomorphe umbellata ethanolic crude extract to inhibit matrix metalloproteinase (MMP) in normal cornea and in cornea after alkali injury was demonstrated. Corneas of albino rabbits were injured with 1 N NaOH for 20 s. After 48 h the corneas were excised, homogenized and analyzed for MMP-9 (92 kDa), pro-MMP-2 (72 kDa) and MMP-2 (67 kDa) activity by gelatin zymography. The activity was also measured in untreated corneas. After electrophoresis of 20 microg protein, gels were incubated with 50, 100, or 250 microg/mL lyophilized hydroethanolic (1:1) root crude extract of P. umbellata standardized for 4-nerolidylcatechol (7.09%). The activity of the enzymes was compared with that of untreated gel. At 48 h after injury, the activity of all MMPs was increased compared with untreated eyes. When the gels were incubated with P. umbellata extract the activity of MMP-2, pro-MMP-2 and MMP-9 decreased in a dose-dependent manner. MMP-9 activity decreased by approximately 50% after incubation with 50 microg/mL and was completely abolished at 100 and 250 microg/mL of the extract. After incubation with 50 microg/mL the activity of pro-MMP-2 and MMP-2 also decreased by 50%. The activity of pro-MMP-2 was almost completely abolished after incubation with 250 microg/mL of the extract. For MMP-2 the incubation with 100 or 250 microg/mL of the extract of P. umbellata promoted a 10-fold decrease in activity. In conclusion, P. umbellata root crude extract can be useful as an alternative therapy to control MMP activity after corneal injury.