Determination of effective components in grape seed extract

Grape seed extract is a kind of polyphenols extracted from grape seeds, mainly composed of procyanidins, catechins, epicatechins, gallic acid, epicatechin gallate and other polyphenols.

Determination of effective components in grape seed extract

Bate Smith method

This method determines the Proanthocyanidins in grape seed extract, and its content is expressed by proanthocyanidins index. Since there is no standard of proanthocyanidins, this method only determines the relative value of proanthocyanidins in grape seed extract.

Principle

Procyanidins are converted into red anthocyanins by heating under acidic conditions.

Operating procedures

Weigh an appropriate amount of grape seed extract (about 15 ~ 40mg) and dissolve it in methanol, and finally dissolve it in 100ml. Take 1ml of sample solution from it and add it to 10ml colorimetric tube, then add 6ml of hydrochloric acid n-butanol solution (5/95, v/v), react at 97 ℃ for 40min, take it out and cool it quickly, measure its absorbance at 550nm, and use methanol instead of the extract solution as blank.

Proanthocyanidin index = a ×  7.0/W。 A: Absorbance; W: Sample mass (g).

The proanthocyanidin index of grape seed extract measured by bate Smith method is generally between 80 and 100, and sometimes it may be greater than 100.

Porter method

Because the reproducibility of bate Smith method is very poor, and the reaction of procyanidins under this condition is not very thorough. Therefore, Porter et al. Improved the bate Smith method. The improvement of Porter method is mainly to add fe3+ to the reagent to improve the degree of reaction and the stability of color. This method also determines the relative content of procyanidins, and the results are expressed in Pvu (portal value unit). The principle of Porter method is the same as that of bate Smith method.

Operating procedures

Weigh an appropriate amount of grape seed extract (about 10 ~ 30mg) and dissolve it in methanol, and finally dissolve it in 100ml. Take 1ml of sample solution from it and add it to 10ml colorimetric tube, then add 6ml of hydrochloric acid - n-butanol (5/95, v/v), 0.2ml of 2.0% ammonium ferric sulfate solution (dissolved with 2mol.l-1 hydrochloric acid), react at 97 ℃ for 40min, take it out and cool it quickly, measure its absorbance at 550nm, and replace the extract solution with methyl alcohol as blank.

PVU = A  ×  7.2/W。 A: Absorbance; W: Sample mass (g).

The American grape seed method evaluation committee believes that the Pvu of grape seed extract is between 250 and 350. Pvu is too low, which may indicate that the content of proanthocyanidins in this product is low; Pvu is too high, indicating that the content of high polymer procyanidins in the product is high. However, the porter method cannot distinguish which proanthocyanidins are detected, so if the Pvu of a grape seed extract is 300, the product may have more oligomeric proanthocyanidins, or more monomer and high polymer proanthocyanidins.

The American grape seed method evaluation committee believes that the above two methods cannot be used for the quantitative analysis of total polyphenols or proanthocyanidins in grape seed extracts.

Folin ciocalteau method

The content of total polyphenols in grape seed extract is determined by this method, and gallic acid is generally used as the reference substance. The advantage of this method is that it can quantitatively analyze the content of total polyphenols in the extract. The disadvantage is that it cannot distinguish the types of polyphenols (for example, whether the determination is monomer or polymer); In addition, proteins, nucleic acids, ascorbic acid and other easily oxidized substances also participate in this reaction, and it is impossible to distinguish whether grape seed extract is adulterated. Because the factors of monomer and proanthocyanidins reaction are different, there is a certain deviation between the measured content and the real content in grape seed extract.

Principle

In alkaline solution, phenolic compounds can reduce wolfram molybdic acid (w6+ into w5+) to produce blue compounds. The depth of color is positively correlated with the content of polyphenols. Blue compounds have the maximum absorption peak at 760nm.

Operating procedures

Weigh an appropriate amount of grape seed extract, dissolve it with water, and the concentration is about 0.1mg/ml. Take 1ml of sample solution and add it to a 10ml colorimetric tube, then add 1ml of deionized water, 0.5ml of Folin ciocalteau reagent and 1.5ml of 26.7% Na2CO3 solution in sequence, finally fix the volume with water to 10ml, react at room temperature for 2 hours, and measure its absorbance at 760nm.

Take gallic acid as the reference substance, prepare solutions with different concentrations, react according to the above method, and measure the absorbance. Draw a standard curve with absorbance versus concentration. The content of total polyphenols in the extract can be obtained from the standard curve (expressed as the content equivalent to gallic acid). The American grape seed method evaluation committee agreed to use this method to determine the total polyphenols in grape seed extract, but suggested that this method should be further improved to confirm the difference between the reaction of monomer and proanthocyanidins with Folin ciocalteau reagent.

Vanillin detection method

This method is used to determine the content of flavane-3-ol in grape seed extract, but it cannot distinguish between monomers and polymers. Catechin is generally used as a standard for quantitative determination.

Principle

Under acidic conditions, phloroglucinol, Resorcinol, flavanol of a ring and poly (procyanidins a ring) have high chemical activity. Phloroglucinol or resorcinol on it can condense with vanillin, and the product forms colored positive carbon ions under the action of acid. The concentration of the sample is positively correlated with the color produced.

Operating procedures

Weigh an appropriate amount of grape seed extract and dissolve it with a certain volume of methanol. Finally, the concentration of grape seed extract solution is about 0.1mg/ml. Add 1ml of sample solution to 10ml colorimetric tube, and then successively add 2.5ml of vanillin methanol solution (1.0% w/v) and 2.5ml of 25% sulfuric acid methanol solution. React at room temperature for 15min, and measure its absorbance at 510nm.

Take catechin as the reference substance, prepare solutions with different concentrations, react according to the above method, measure the absorbance, and draw the standard curve with the absorbance to the concentration. The content of flavane-3-alcohol in the extract can be obtained from the standard curve.

Vanillin detection method for the determination of flavane-3-ol in grape seed extract is generally to dissolve the extract with methanol and prepare reagents. In addition, glacial acetic acid can also be used to dissolve the extract and prepare reagents, but the measured data of the two are very different. When methanol is used as solvent, the vanillin method measures the amount of total flavane-3-alcohol, which can occur for the amount of all flavane-3-alcohol in polymerized procyanidins. When glacial acetic acid is used as solvent, the vanillin method measures the amount of monomer flavane-3-alcohol and polymerized proanthocyanidins terminal flavane-3-alcohol, while the flavane-3-alcohol in the middle of polymerized proanthocyanidins cannot be measured. Methanol is mostly used as a solvent to determine the amount of flavane-3-ol in grape seed extract. The average polymerization degree of proanthocyanidins in the extract can be roughly measured by using two solvents at the same time.  

The American grape seed method evaluation committee has agreed not to use this method to detect the active ingredients in grape seed extract.

HPLC method

This method determines the content of each monomer, oligomer and homopolymer in the extract. However, due to the limitations of reference substances and analytical methods, only the contents of monomers and some oligomers in grape seed extract can be determined. In practical application, it is only necessary to determine the monomer content. The main monomers in grape seed extract are catechin, epicatechin, gallic acid, epicatechin gallate.

The content of monomers in grape seed extract is generally about 10.0%, up to 30.0%. Due to the complexity of the components of grape seed extract, if the sample is not pretreated, it is generally difficult to completely separate a variety of monomers by HPLC. Therefore, before injection, the extract needs to be pretreated to remove the high polymer and improve the content of monomer. Common pretreatment methods include silica gel column chromatography, sephadexlh-20, Sephadex G25, toyopeah TSK hw40 and other column chromatography methods. The sample can also be pretreated by thin-layer chromatography, generally using toluene: acetone: acetic acid = 3:3:1 (v/v/v) as the developing agent.