Abstract
Introduction: Collagenase plays an important role in the degradation of dermal matrix proteins leading to wrinkle formation. The objectives of this study were to evaluate the inhibitory effect of water-pepper (Polygonum hydropiper L.) sprout extract on the activity of collagenase and to identify the inhibitory compounds.Methods: Collagenase inhibitory activity was measured by spectrophotometric assay. Activity-guided fractionation was performed using liquid-liquid extraction of water and n-butanol and Diaion HP-20 column chromatography, followed by high-performance liquid chromatography (HPLC) fraction collection.Results: A methanolic extract of water-pepper sprout inhibited collagenase activity in a concentration-dependent manner with an IC50 value of 156.7 μg/mL. Collagenase inhibitory activity (IC50 = 23.5 μg/mL) was found in 50% methanol eluate from the HP-20 column chromatography of the n-butanol soluble fraction. The active compound (IC50 = 1.9 μg/mL) in the eluate was isolated by HPLC and identified as quercetin-3-O-galactoside (hyperoside) from comparing retention time, UV-Vis absorption, and mass spectra with those of the standard. Lineweaver-Burk plots revealed that hyperoside was an uncompetitive inhibitor against collagenase. Hyperoside was also the most abundant flavonoid present in the methanolic extract.Conclusion: These results suggest that water-pepper sprouts could be beneficial as a natural source of collagenase inhibitor which might be used for the treatment of skin aging.
Highlights
Collagenase plays an important role in the degradation of dermal matrix proteins leading to wrinkle formation
Ethylenediamine tetraacetate (EDTA), a known collagenase inhibitor that removes metal ions required for enzyme stability and activity, was used as a positive control
Activity-guided fractionation was used to determine the active fraction in the methanolic extract
Summary
Collagenase plays an important role in the degradation of dermal matrix proteins leading to wrinkle formation. The objectives of this study were to evaluate the inhibitory effect of water-pepper (Polygonum hydropiper L.) sprout extract on the activity of collagenase and to identify the inhibitory compounds. Results: A methanolic extract of water-pepper sprout inhibited collagenase activity in a concentration-dependent manner with an IC50 value of 156.7 μg/mL. Conclusion: These results suggest that water-pepper sprouts could be beneficial as a natural source of collagenase inhibitor which might be used for the treatment of skin aging. Water-pepper (Polygonum hydropiper L.) contains bioactive compounds such as polygodial, hydropiperoside, and several flavonoid compounds, such as quercetin, Materials and Methods Materials Collagenase from Clostridium histolyticum type IV and quercetin were obtained from Sigma-Aldrich (St. Louis, MO, USA), Pz-Pro-Leu-Gly-Pro-D-Arg-OH (Pz-peptide) from Bachem (Bubendorf, Switzerland), hyperoside from Extrasynthese (Genay, France), isoquercitrin, quercitrin, and quercetin 4-glucoside from Tokiwa Phytochemical (Chiba, Japan), and rutin from Tokyo Chemical Industry (Tokyo, Japan). Water-pepper sprouts were obtained from JA Chikuzen Asakura (Fukuoka, Japan)
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