Extraction and Properties of the Polyphenol, Catechin, as an Antioxidant

Transcription

1 Extraction and Properties of the Polyphenol, Catechin, as an Antioxidant Anthony U. Onuzuruike and Jacob J. Woltering Department of Chemistry, University of Missouri-Columbia, Columbia, Missouri

2 Introduction Materials and Methods Grape seeds were collected from a brewery and milled to powder. 1 Then it was defatted with petroleum ether firstly, and then was mixed for in an acetone/water solution for twelve hours. The three macerates were combined and concentrated until no acetone was left. The concentrated solution was extracted four times with 1000 ml of ethyl acetate each time. The ethyl acetate extracts were combined, evaporated to remove ethyl acetate and were lyophilized. 1 After the whole process, the grape seed extract (GSE) was obtained. GSE was dissolved in water and was fractionated on a column (70 8 cm), eluted with methanol in water. Fraction was detected by polyamide TLC and analytic RP- HPLC. The fraction was separated by semi-preparative HPLC, using a Waters ODS semipreparative column (10µm, mm). The HPLC mobile phase contained solvent A (water), solvent B (5% V/V THF in 10% methanol) and solvent C (methanol). The linear gradient system employed, at room temperature, was: 0 60 min 40% solvent A and 60% solvent B to 35% solvent A 55% solvent B and 10% solvent C to 100% solvent C. The solvent flow rate throughout the run was 3 ml/min. The column eluate was monitored at 270 nm UV absorbance in each case and individual polyphenol peaks were collected and the purity was identified by analytic HPLC. 1 2

3 Scheme 1. Extraction of Catechin From Grape Seed Extract. STEP 1 = Gross Extraction STEP 2 = Catechin Extraction Grape Seed Extract Grape Seed Extract Catechin TLC HPLC In the data shown in Table 1, the control is Vitamin C. Catechin is the polyphenol that we are looking at and the ps B 4 is the other polyphenol being compared to it. In the results, we found a correlation between the activity of each enzyme and the concentration of the antioxidants. Overall, the control, Vitamin C, was lower in activity for four out of the five enzymes. Catechin showed similar results to pc B 4. They were both polyphenols and it seemed like they are similar if not the same effect. Scheme 2 shows the structure. 3

4 Scheme 2. Molecular Structures of Sample Antioxidants Scheme 2. Catechin, Pc B 4, and Vitamin C Vitamin C Table 1. The Effects of Different Antioxidants on Various Enzymes Activities Control (Vit. C) Catechin (µm) pc B 4 (µm) SOD (units/mg of protein) 1200 ± ± ± ± ± ± ± 5 Catalase (um/min./mg) 7 ± ± ± 5 25 ± 5 7 ± ± 5 23 ± 5 GP x (um/min./mg) 530 ± ± ± ± ± ± ± 12 4

5 Results and Discussion Cellular SOD Activities (units/mg protein) Catechin Pc B Ct (µm) Cellular Catalase Activity (µm/min/mg protein) Catechin Pc B4 0 Ct (µm) 5

6 600 Cellular GPx Activity (µm/min/mg protein) Catechin Pc B4 100 Ct (µm) Conclusion Supplemental Material Available: A detailed description of the synthesis of Catechin and details its spectroscopic characterization (MS, IR, and NMR). References (1) Fan, P.; Lou, H. Mol. Cell Biochem. 2004, 267,

7 Supporting Information Extraction and Properties of the Polyphenol, Catechin, as an Antioxidant Anthony U. Onuzuruike and Jacob J. Woltering Department of Chemistry, University of Missouri-Columbia, Columbia, Missouri S1

8 Table of Contents Extraction of Catechin S3 IR Spectrum of Catechin... S5 MS Spectrum of Catechin..... S6 Carbon-13 NMR Spectrum of Catechin.... S7 Bibliography.. S8 S2

9 Extraction of the Polyphenol, Catechin, as an Antioxidant Grape seeds were collected from a brewery and milled to powder as seen in Scheme S3. The powder (5 kg) was defatted with petroleum ether firstly, and then was macerated for 12 h at room temperature three times with 2000 ml of water/acetone (30:70, V/V) each time. The three macerates were combined and concentrated until no acetone was left using a rotary evaporator under reduced pressure and a water bath temperature <40. The concentrated solution was extracted four times with 1000 ml of ethyl acetate each time. The ethyl acetate extracts were combined, evaporated to remove ethyl acetate and were lyophilized. GSE was obtained. GSE was dissolved in water and was fractionated on a Toyopearl TSK HW- 40(F) column (70 8 cm), eluted with methanol in water gradiently. Fraction was detected by polyamide TLC [CHCl3-MeOH-H2O as mobile phase, stained by 1% K3Fe(CN)6- FeCl3] and analytic RP-HPLC [using a Phenomenex ODS column (5µm, mm), 0.2% V/V formate in 20% methanol as mobile phase, flow rate 1 ml/min, monitored at 270 nm UV absorbance]. The fraction was separated by semi-preparative HPLC, using a Waters ODS semipreparative column (10µm, mm). The HPLC mobile phase contained solvent A (water), solvent B (5% V/V THF in 10% methanol) and solvent C (methanol). The linear gradient system employed, at room temperature, was: 0 60 min 40% solvent A and 60% solvent B to 35% solvent A 55% solvent B and 10% solvent C to 100% solvent C. The solvent flow rate throughout the run was 3 ml/min. The column eluate was monitored at S3

10 270 nm UV absorbance in each case and individual polyphenol peaks were collected, the purity was identified by analytic HPLC. Scheme S3. Extraction of the Polyphenol, Catechin, as an Antioxidant STEP 1 = Gross Extraction STEP 2 = Catechin Extraction Grape Seed Extract Grape Seed Extract Catechin TLC HPLC S4

11 Figure S1. IR Spectrum of Catechin S5

12 Figure S2. Mass Spectrum of Catechin S6

13 Figure S3. C- NMR Spectrum of Catechin S7

14 Bibliography Azmi, A.; Bhat, S.; Hanif, S.; Hadi, S. FEBS Lett. 2006, 580, Cren-Olive, C.; Wieruszeski, J.; Maes, E.; Rolando, C. Tetrahedron Lett. 2002, 43, Du, Y.; Guo, H.; Lou, H. J. Agric. Food Chem. 2007, 55, Duthie, G.; Duthie, S.; Kyle, J.Nutr Res. 2000, 13, Han, X.; Shen, T.; Lou, H. Int. J. Mol. Sci. 2007, 8, Masella, R.; Benedetto, R.; Vari, R.; Filesi, C.; Giovannini, C. J. Nutr. Biochem. 2005, 16, Spectral Database for Organic Compounds SDBS. (Apr 10, 2014) S8