This method is suitable for the determination of proanthocyanidins in various plant tissues, organs and their preparations (such as grape seeds and pine bark extracts).
1. Method summary
Proanthocyanidins (also called condensed tannins) are oligomers and polymers of flavan -3- ol, belonging to polyphenols. Different from other phenolic compounds, flavanols (condensed tannins, monomers, dimers, etc. ) can react with vanillin in acidic medium to generate colored substances with maximum absorption at 500nm, and its content is determined by colorimetry.
2. Tools
Spectrophotometer.
3. Reagent
The water used is deionized water or distilled water with the same purity.
(1) Vanillin, methanol and concentrated hydrochloric acid are all analytically pure grades.
(2) purified proanthocyanidins or catechins.
(3)4% vanillin methanol solution.
(4) Standard use solution: the purified proanthocyanidins are dissolved in distilled water to prepare a stock solution of 1mg/mL, and the stock solution is diluted to a standard use solution with a concentration of 1x 10-2mg/ml to 1mg/mL. The standard solution should be prepared on the day of determination.
If there is no purified proanthocyanidins, catechins can be used instead, and the preparation method is the same as above.
Step 4: Determine the steps
Preparation of proanthocyanidins in (1) sample: plant materials were extracted with 4 times of acetone and water (7+3, volume ratio) or 60% methanol, and the organic solvent was removed by distillation under reduced pressure below 40℃, and the water phase was washed with ether to constant volume.
Freeze-dried solid proanthocyanidins are directly dissolved in water (a small amount of methanol is added to help dissolve) to prepare proanthocyanidins solution.
The proanthocyanidins solution was stored in a dark environment at 5℃ for later use.
(2) Sample determination: Wrap the test tube (14mm×20mm) tightly with tin foil, leaving only the sampling nozzle. Add 0.5mL of sample into the tube, then add 3.0ml of 4% vanillin methanol solution and mix well, then add 1.5mL of concentrated hydrochloric acid, mix well and develop color at room temperature 15min. The above operation can also be carried out in a dark environment. Finally, the color contrast is carried out at 500nm.
The standard curve can be made according to the above steps (that is, the absorption value of 0. 1mg proanthocyanidins at 500nm is 0.55).
5. Performance statistics
Formula for calculating proanthocyanidin content,
Procyanidins (1×10-3 mg) = a 500 nm ÷ 0.55×100× v.
Where v is the dilution volume (multiple) of the sample.
Step 6 take notes
(1) The detection range of this method is (5~500)× 10-3mg/0.5mL sample solution. The precision and accuracy are greater than 1× 10-3mg.
(2) The reaction tube should be soaked in detergent for 24 hours and thoroughly cleaned.
(3) When comparing colors, use water as a blank.
(4) The OD value at 4)500nm should be controlled below 3.
(5) When the content of proanthocyanidins in the sample is high, the value measured in the absence of vanillin should be subtracted from the 500nm value measured in the presence of vanillin.
(6) The chromogenic solution should be placed away from light.
Method 2: Determination of procyanidins in health food.
1, principle
Procyanidins are easily soluble in solvents containing hydroxyl groups, such as methanol. The method is to extract proanthocyanidins from samples with methanol, add hydrochloric acid for hydrolysis, and then convert them into scarlet cyanine (C 15H 10O6 HCl), and then determine them by high pressure liquid chromatography.
2. Reagent:
2. Analytical purity of1dichloromethane
2.2 chromatographic purity of methanol
2.3 Analysis purity of isopropanol
2.4 Analytical purity of formic acid
2.5 Analytical purity of hydrochloric acid
3. Inspection method
3. 1 sample Weigh about 500mg of oil-soluble sample into 100ml conical flask, add 5.0ml of dichloromethane, and shake well to dissolve. Add 45.0ml of methanol, shake for 5 minutes, and then pass through a 20μm filter membrane. Take 5.0 ml of filtrate in a 25 ml volumetric flask, add 5.0 ml of 3 mol/L hydrochloric acid, shake well, and adjust the volume to the scale with isopropanol. Immediately pass through a 5μm filter membrane, take out a part and put it into a test tube, plug it with a cork, put it into an oven at100 5℃ for hydrolysis for 45 minutes, then take it out and let it stand at room temperature for liquid chromatography analysis.
3.2 Standard The steps are the same except that 25.0mg is weighed in the 100ml conical flask.
3.3 Qualitative and quantitative analysis of quantitative standard curve and external standard method.
4, chromatographic analysis conditions
4. 1 mobile phase: water: methanol: isopropanol: formic acid =73: 13:6:8.
4.2 Detection wavelength: 525 nm
4.3 chromatographic column: shimadzu shim-pak CLODS15cm× 6mm.
4.4 Column temperature: 35℃
4.5 Flow: 1 ml/min.