Screening of Prolyl Endopeptidase Inhibitors from Medicinal Plants. Lee Si-young, Lee Jung-han, Paik Young-Sook College of Environment and Applied Chemistry, Kyung Hee University.
Statistical Data of Dementia.
Statistical Data of Dementia.
Extraction procedure. Sample MeOH Extract(3L/8hr/3times). Filteration & Evaporation. CHCl 3 layer O layer 90% MeOH layer n-hexane layer Ethyl Acetate layer O layer layer O layer Each layer Enzyme Assay. Discovered PEP activation layer.
Solvent Concentration. Absorbance(380nm) 2.2 0.1U PEP cont MeOH solvent effect 2.0 1ppm 2.5ppm 1.8 4ppm 1.6 5.5ppm 1.4 7.7ppm 16.6ppm 1.2 1.0 0.8 0.6 Time(Sec)
Enzyme Assay. A. 100mM Phosphate buffer (ph 7.0 / 320/ 37, 30min UV cuvette) 2mM Z-Gly-Pro-pNA 32 Sample 16 0.1U PEP 32(380nm) B. 100mM Phosphate buffer(ph 7.0 / 384) Sample 16
Enzyme Assay. Control 100mM Phosphate buffer (ph 7.0 / 336/ 37, 30min UV cuvette) 2mM Z-Gly-Pro-pNA 32 0.1U PEP 32(380nm) Positive Control 100mM Phosphate buffer (ph 7.0 / 320/ 37, 30min UV cuvette) 2mM Z-Gly-Pro-pNA 32 Z -Pro-Prolinal 16 0.1U PEP 32(380nm) Inhibition(%) = A 380 of Control - (A B) A 380 of Control 100
Positive Control 3.0 2.5 Positive control Absorbance 2.0 1.5 1.0 22ppb Z-Pro-Prolinal 0.5 200 300 400 500 600 700 800 W avelength
0.1 U PEP(pH 7.0 / 37):10 min Interval 2.0 317nm 380nm 1.5 tim e in te rva l 1 0 m in /3 h r Absorbance 1.0 0.5 200 300 400 500 600 700 800 W avelength
PEP control. 1.6 1.4 1.2 Absorbance(380nm) 1.0 0.8 0.6 0.1U PEP Control 10min/380nm 0 20 40 60 80 100 120 140 160 180 200 Min
Absorbance 0.7 0.6 0.5 0.3 O layer 4ppm CHCl 3 EA PEP Absorbance 1.0 0.8 0.6 O layer 4ppm MeOH Hexane EA PEP 0.1
Absorbacne 0.7 0.6 0.5 0.3 CHCl 3 layer 4ppm O EA PEP Absorbance 1.0 0.8 0.6 O layer 4ppm EA Hexane MeOH PEP 0.1 second
Absorbacne 0.6 O layer 4ppm PEP 0.5 MeOH Hexane EA 0.3 0.1 Absorbance 0.35 PEP Hexane layer 4ppm 0.30 MeOH EA 5 O 0 0.15 0.10 5 0
Absorbance 0.6 0.5 0.3 EA layer 4ppm MeOH Hexane O PEP Absorbance 0.30 PEP Hexane layer 4ppm 5 MeOH EA 0 O 0.15 0.10 0.1 5 0
Absorbance 1.0 4ppm PEP CHCL 3 4ppm 0.8 EA 4ppm 0.6 Absorbance 1.2 1.0 0.8 0.6 0.1u pep cont MeOH layer 4ppm EA Hexane O PEP
Absorbacne 0.6 0.5 0.3 O layer 4ppm MeOH Hexane EA PEP Absorbacne 0.6 0.5 0.3 O layer 4ppm EA MeOH Hexane 0.1 0.1
Absorbance 0.6 PEP EA layer 4ppm 0.5 O Hexane MeOH 0.3 Absorbacne 1.4 1.2 1.0 0.8 0.6 Hexane layer 4ppm MeOH O EA PEP 0.1
Absorbance 1.2 1.0 0.8 0.6 PEP O layr 4ppm EA MeOH Hexane Absorbacne 0.30 5 0 0.15 0.10 Hexane Layer 4ppm MeOH EA O PEP 5 0
Absorbance 0.7 PEP Hexane layer 4ppm 0.6 MeOH O 0.5 EA 0.3 Absorbance 0.35 PEP Hexane layer 4ppm 0.30 MeOH EA 5 O 0 0.15 0.10 0.1 5 0
Absorbacne(380nm) 1.6 1.4 1.2 1.0 0.8 0.6 O Layer 4ppm EA MeOH Hexane Ginkgo leaves extracts 4ppm Time(Sec)
Percent inhibition of Compound 1.
PEP percent Inhibition.
Isolation procedure. n-hexane layer Silica gel Column Chromatography. Fr 1 Fr 2 Fr 3 Fr 4 Fr 5 Fr 6 Sephadex LH-20 C.C. Fr 5-a Fr 5-b Fr 5-c Prep TLC. Compound1
Compound 1 from Ginkgo biloba leaves Absorbance(380nm) 0.6 0.5 0.3 2ppm 3ppm 4ppm Compound 1 from Ginkgo L 0.1 Rf = 0.5 Time(Sec)
UV / IR Data. 1.5 1.0 Compound 1 in MeOH/ 1.5 ppm A max 0.79/301nm Absorbacne 0.5 200 300 400 500 600 700 800 Wavelength
NMR Data ( 1 H, 13 C)
NMR Data (DEPT, COSY)
NMR Data (HMQC, HMBC)
EI + / HR-mass.
Determination of Chemical Structure. HO COOH Anacardic acid.