Analysis of Resveratrol in Wine by HPLC

Outline Introduction Resveratrol o o Discovery Biosynthesis HPLC separation Results Conclusion

Introduction Composition of flavoring, coloring and other characteristic ingredients in wine is largely dependent on make and storage White and red wine differ not only in color of the grapes, but also in the way they are produced For white wine, grapes are pressed and the resulting must is fermented into an alcoholic beverage Mostgärung To produce red wine the grapes are squashed to produce a slurry of grape juice, pulp and skin. During fermentation of this mash, the colorants are extracted into solution. Pressing takes place after fermentation Maischegärung.

Introduction Only fermentation from mash can result in extraction of compounds from grape skin and seeds Therefore red wine contains a number of compounds that are not found in white wine. Also after fermentation, the process of aging during the period of storage in a wooden cask results in distinct changes and a unique profile of over 1000 different compounds in each lot.

Introduction Apart from water and ethanol, red wine contains in average: Component Amount Charecteristic Glycerol 1 % Oily, sweetness Organic acids 0.4 % Acidity Anthocyanins Catechins Tannins Flavonols 0.1 % Red colour Bitterness (> 20 mg/l), antioxidants Astringency, bitterness antioxidants Other compounds 0.5 % 5

Resveratrol Another molecule in red wine, however, has been the main focus of health benefits in recent years: Resveratrol It has been shown in studies that resveratrol, as well as having antioxidant properties, can help prevent high blood pressure (hypertension) in mice and also has antiinflammatory effects. It s also suspected to have anticancer, and chemopreventive abilities.

Resveratrol - Discovery Reseveratrol was first discovered byjapanese researcher Michio Takoaka and his group in 1939. They collected veratrum grandiflorum, a stout, exotic and flowering plant from the Hokkaido Island. A phenolic compound was obtained by crystallization and the molecular formula was identified. The new compound was named RES (resorcinol family), VERATR (from veratrum grandiflorum) OL (used to indicate hydroxyl groups)

Resveratrol - Biosynthesis Resveratrol is found in the skin of red grapes, peanuts, in many berries and a few other plants but how does it get there? Resveratrol is derived from p-coumaric acid which is an intermediate in lignin production. The two key enzymes are Coenzyme A (CoA) Ligase (4CL) and Stilbene Synthase (STS) Resveratrol is produced in plants when they are exposed to stress such as UV-light, disease and pests.

HPLC separation Reversed phase HPLC is most appropriate to separate the cis- and trans isomers from each other. Fluorescent detection is used for high sensitivity (LOQ 0.015 mg/l 1 vs. 0.3 mg/l 2 using DAD) Wine sample complexity requires gradient optimization to resolve the trans-resveratrol peak from the rest of fluorescent stilbenes and polyphenols. Fast analysis using Nexera X2 UHPLC system equipped with PDA and RF-20 AXS Fluorescent detector. 1 J. Sep. Sci. 2007, 30, 669 672 2 J. Agric. Food Chem., Vol. 51, No. 18, 200

Method Development Columnn: GIST C18, 100 x 2.1 mm, 2 µm Mobile Phase: A: Water incl. 0.2 % Formic Acid B: Acetonitrile GE Program 0 min BCONC 20% 4 min BCONC 50% 4.01 min BCONC 20% Flow rate: 0.6 ml/min Temperature: 50 C PDA detection: 190 360 nm Detection: RF-20AXS Excitation λ = 300 nm Emission λ = 386 nm SENS = Med Cell Temperature: 20 C Inj. Vol.: 2 µl

Results 17.5 mg /L trans-resveratrol standard PDA detection 291 nm 90 trans-resveratrol/0.889 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 cis-resveratrol/1.241 0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 min

Results 9 mg /L trans-resveratrol standard RF - Ex:300nm Em:386nm mv 210 trans-resveratrol 190 170 150 130 110 90 70 50 30 cis-resveratrol 10 0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 min

Method Development Cell temperature affects sensitivity improve baseline stability and eliminate the effect of temperature uv 350000 Data1:Trans_Res_20ppm_MeOH-Water_40deg_001.lcd Detector A:Ex:300nm,Em:386nm Data2:Trans_Res_20ppm_MeOH-Water_30deg_001.lcd Detector A:Ex:300nm,Em:386nm Data3:Trans_Res_20ppm_MeOH-Water_20deg_001.lcd Detector A:Ex:300nm,Em:386nm 325000 300000 275000 250000 225000 200000 175000 trans-resveratrol 20 ºC 30 ºC 40 ºC 225000 200000 175000 150000 125000 cis-resveratrol uv 250000 Data1:Cis_Res_20ppm_MeOH-Water_40deg_001.lcd Detector A:Ex:300nm,Em:386nm Data2:Cis_Res_20ppm_MeOH-Water_30deg_001.lcd Detector A:Ex:300nm,Em:386nm Data3:Cis_Res_20ppm_MeOH-Water_20deg_001.lcd Detector A:Ex:300nm,Em:386nm 20 ºC 30 ºC 40 ºC 150000 125000 100000 75000 50000 25000 0 1.60 1.65 1.70 1.75 min 100000 75000 50000 25000 0 2.20 2.25 2.30 2.35 2.40 min 13

Sample preparation

Results Wine sample, LL extracted PDA detection 280 nm mau 45.0 trans-resveratrol = 7.7 mg/l 40.0 35.0 30.0 25.0 20.0 15.0 10.0 cis-resveratrol = 1.6 mg/l 5.0 0.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 min

cis-resveratrol trans-resveratrol cis-resveratrol trans-resveratrol Example: Filtered red wine mau 225 291nm,4nm 200 175 150 125 Datafile Name:Shim_Spätburgunder_002.lcd B.Conc Pump A Pressure Pump A Degassing Unit Pressure(x -1000) bar 60.0 55.0 50.0 45.0 100 75 50 25 40.0 35.0 30.0 0 25.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 min Datafile Name:Shim_Spätburgunder_002.lcd 350 mv bar Detector A Ex:300nm,Em:386nm Pump A Pressure 300 250 75.0 200 150 100 50 50.0 25.0 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 min 0.0 17

LL extraction vs filtered (0,2 µm) sample RF Signal uv Data1:Shim-Wine-filtered_GE_.lcd Detector A:Ex:300nm,Em:386nm Data2:Shim-Wine-LL-extract_2ul_IV_GE_002.lcd Detector A:Ex:300nm,Em:386nm 2750000 2500000 2250000 2000000 1750000 1500000 1250000 1000000 750000 500000 250000 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 min 18

LL extraction vs filtered (0,2 µm) sample PDA signal uv Data1:Shim-Wine-filtered_GE_.lcd PDA Ch1 280nm,4nm 225000 Data2:Shim-Wine-LL-extract_2ul_IV_GE_002.lcd PDA Ch1 280nm,4nm 200000 175000 150000 125000 100000 75000 50000 25000 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 min 19

Conclusion The proposed method allows the determination of transresveratrol in wine in a one step procedure Fluorescence detection is highly sensitive and allows for the detection of resveratrol in concentrations present in red wine Animal studies suggest as much as 500 mg daily may be necessary to provide any health benefits, even if a 40 mg daily dose is sufficient, as suggested elsewhere, red wine contains at most 12 mg resveratrol per liter, you'd need to drink a little over 3 liters of wine daily to get that much resveratrol.

Cheers!