Detection of trypsin inhibition and antioxidant effects on TLC

Activities of plant extracts in cosmetic preparations include trypsin inhibition and antioxidation. Trypsin inhibition detection was adapted from a HPLC procedure [4]. TLC plates sprayed with 4mg/mL trypsin in TRIS buffer pH 8.2, left at 25° for 10min, then sprayed with 1mg/mL NA-benzoyl-L-arginine 4 nitroanilide (L-BAPA) in TRIS buffer pH 8.2 gave white zones against yellow background for inhibitors after 30min at 25°; minimum detecta\ble amount (MLA) of positive control hexamidine diisethionate 1µg. In situ TLC detection of oxidation of unsaturated lipids through malondialdehyde formation using thiobarbituric acid (TBA) was used [2]. Plates were sprayed with 2.5% v/v linseed oil in dichloromethane, dried for 5min, then sprayed with 15% aq. perchloric acid, heated at 37° for 10min and sprayed with 1% TBA in 50mM aq. NaOH. Antioxidants appear as pale zones against a pink background after 10min. MDA of positive control propyl gallate 1µg. A modification of the Griess reaction was used to detect NO scavengers [3]. The developed plate was sprayed with 20mM aq. sodium nitroprusside, left in full daylight for 10min at 25° then sprayed with 1% w/v sulfanilimide in 2% aq. phosphoric acid, followed immediately by 0.1% N-(1-naphthyl)ethylene diamine in 2% aq. phosphoric acid. NO scavengers appear after 20min as pale zones against a purple background; MLA of positive control carboxy-PTIO potassium salt 100ng.

References: 1. Dickson, R. et al. (2006) Phytother. Res. 20:41–45.

2. Ozgen, U. et al. (2006) Pharm. Biol. 44: 107–112.

3. Fox, J.B (1979) Analyt. Chem. 51:1493–1502.

4. Schlüter, H. et al. (2008) Anal. Bioanal. Chem. 392: 783–789.