Extraction and detection method of grape seed proanthocyanidins
1 Concept, properties and safety of grape seed proanthocyanidins
1.1 Concept of proanthocyanidins
Research shows that the only proanthocyanidin substance in grape seeds is proanthocyanidin [21. The definition of proanthocyanidins is not uniform. Proanthocyanidins are named because they produce red anthocyanins when heated in acidic media3. However, catechin monomers react without anthocyanins under hot acid conditions, so catechin monomers should not be classified as proanthocyanidins. This concept has also been recognized by the American Grape Seed Method Evaluation Committee and major domestic companies that produce grape seed extract. Grape seed proanthocyanidins are composed of catechin, epicatechin and their gallic acid esters via C4-C or C4-C. It is a polymer composed of valence-linked bonds. The general structural formula is shown in Figure 15J. Dimers to tetramers are usually called oligomers (OPCs), and pentamers and above are called polymers.
R=3p-OH catechin, R=3p-O-gallate catechin O-gallate R-3a-OH epicatechin. R-3a-O epicatechin o-gallate
Figure 1 The structure and constituent units of proanthocyanidins
1.2 The main properties of proanthocyanidins
Proanthocyanidins can produce red anthocyanins under hot and acidic conditions. This property can be used for qualitative and quantitative analysis of proanthocyanidins. There are more hydroxyl groups in the structure and greater polarity, making it well soluble in polar solvents such as water, methanol, acetone, and ethanol, but not soluble in non-polar substances such as benzene, chloroform, and petroleum ether. The more hydroxyl structures also make it a good hydrogen atom donor and has strong antioxidant properties. Studies have shown that among 0:1, ·OH and ·CH, proanthocyanidins have the best scavenging ability for 0:1, and in the range of polymerization degree between 2 and 5, the effect increases with the increase of polymerization degree. Analysis of the structure-activity relationship shows that proanthocyanidins with galloyl groups have stronger antioxidant activity, and the antioxidant activity of dimers is stronger than that of monomeric catechins. The c-c linked dimer has stronger antioxidant activity than the c-c linked dimer. The maximum absorption wavelength of proanthocyanidins is near 280 nm, giving it strong UV absorption ability. The above main properties make proanthocyanidins very suitable for the development of health foods and cosmetics.
13 Safety of Proanthocyanidin Products
The Grape Seed Proanthocyanidin Research Group of Creighton University in the United States and the U.S. Environmental Protection Agency jointly conducted a study on Grape Seed Proanthocyanidin Extract in accordance with the Toxic Substances Control Regulations Health Effects Test Manual (GSPE) a series of toxicity and bioefficacy studies. The results prove that GSPE has high safety and good free radical scavenging and antioxidant capabilities8. Japanese scholar Yamakoshi and others also used a series of toxicology tests to confirm that grape seed extract rich in proanthocyanidins is highly safe. It can be used for the development of functional foods.
2 Extraction methods of proanthocyanidins
Commonly used methods for extracting proanthocyanidins include water extraction, organic solvent-water extraction and instrument-assisted extraction. . The proanthocyanidin substances in grape seeds are usually combined with proteins and cellulose in a bound state fl01, which is generally difficult to extract. It is usually extracted with organic solvents or water, which has the effect of breaking hydrogen bonds. At the same time, due to the poor permeability of organic solvents, they are generally not used alone and often require water as a mass transfer agent.
2.1 Water extraction method
Masquelier~l-Nengzao obtained proanthocyanidins from pine bark by crude extraction with boiling water and purification with ethyl acetate. When water is used as the extraction agent, the extraction takes a long time and the temperature is high, which can easily cause the loss of proanthocyanidins. At the same time, water is more polar and dissolves more impurities.
2.2 Organic solvent-water extraction method
Methanol, acetone, ethanol and ethyl acetate are commonly used organic solvents for extracting grape seed proanthocyanidins. They have good dissolution of proanthocyanidins. properties, their polarity is large II, and the Jt~ order is methanol>ethanol>acetone>ethyl acetate. Ethanol is a commonly used extraction solvent that is cheap and abundant. The proanthocyanidin components extracted with ethyl acetate have good biological activity, but due to low polarity, the extraction of proanthocyanidins is incomplete.
Methanol and acetone aqueous solutions (50% to 75%) have good extraction performance for proanthocyanidins, and are also often used as extraction solvents for determination of proanthocyanidin content. Xiong He-21 compared the extraction effects of methanol, ethanol, and acetone aqueous solutions on polyphenols. The results showed that 70% acetone aqueous solution was the best solvent. The reason for the good extraction effect of acetone aqueous solution: proanthocyanidin molecules contain multiple benzene rings and ether bonds, are highly oil-soluble, and have a large number of hydroxyl groups connected to the molecular skeleton. They have good solubility in water and have both oil and water solubility. When acetone matches each other, the solubility of proanthocyanidins naturally increases, and its extraction rate is improved accordingly.
2-3 Instrument-assisted extraction method
Supercritical extraction and ultrasonic-assisted extraction are increasingly used for the extraction of grape seed proanthocyanidins. Supercritical CO: The extraction rate is high and the proanthocyanidins are not affected by air and light. However, due to the expensive equipment, it is difficult to promote its use. The ultrasonic method is widely used. The strong vibration, high acceleration, strong cavitation effect, stirring and other special effects generated by ultrasonic waves can destroy the cell walls of plants, allowing the solvent to penetrate into the cells, allowing the chemical components to dissolve in the solvent. Thereby improving the extraction efficiency. It shows superior performance in extracting heat-sensitive substances such as proanthocyanidins.
3 Detection of grape seed proanthocyanidins
Since most polyphenols in grape seeds and grape seed extracts are proanthocyanidins ( Generally accounting for 70% to 85%), so many manufacturers use proanthocyanidins to calibrate the content of active ingredients. Proanthocyanidin content is a key indicator that reflects the quality of grape seed extract or grape seeds. There are two main indicators, namely proanthocyanidin value and proanthocyanidin content.
3.1 Determination of proanthocyanidin value
The determination of proanthocyanidin value adopts Bates-smith method and Poaer method. Principle: Procyanidins are heated under acidic conditions and converted into red anthocyanins, while flavan-3-ol monomers such as catechin and epicatechin do not have this reaction (Figure 2). The results they measured are the relative content of proanthocyanidins, expressed by proanthocyanidin index and PVU respectively, which are obtained based on empirical formulas. The proanthocyanidin index in grape seed extract is generally between 80 and 100, and the PVU is generally between 250 and 350.
The proanthocyanidin value is only a relative content, not the actual content of proanthocyanidins. According to the survey, products with a proanthocyanidin value of 95 have a 15% difference in polyphenol content, and the quality is quite different. Many manufacturers use the proanthocyanidin index to express the percentage of proanthocyanidins in grape seed extract which is incorrect.
Figure 2 Anthocyanin production reaction Fig. 2 Reaction of producing cyaniding
3.2 Determination of proanthocyanidin content
There are many methods for determining the content of proanthocyanidins, which are also quite confusing. The following methods are commonly used.
3.2.1 Iron salt catalytic method
The reaction principle of this method is the same as that of proanthocyanidin value determination. Proanthocyanidin standards are used when calculating the proanthocyanidin content. Fe¨ and hydrochloric acid are commonly used catalysts and acidolysis agents. Since the absorbance value is very low when water or ethanol is used as the reaction medium, n-butanol is generally used as the reaction medium15-161. Usual specific operations: Take 1.0 mL sample solution (or proanthocyanidin solution) in a 10 mL graduated test tube, add 6.0 mL n-butanol-concentrated hydrochloric acid (95:5) and 2% ferric ammonium sulfate solution (dissolved in 2 mol/L hydrochloric acid) 0.2mL, mix well, place in a boiling water bath and heat for 40 minutes, immediately take it out and quickly cool to room temperature with ice water, and measure the absorbance value at 550 Nm.
This method is simpler and has better selective response to proanthocyanidins. The iron salt catalytic method has strict requirements on the water content and Fe concentration in the reaction system. Generally, the water content is 6% and the Fe concentration is 4.5x10%. Moreover, an excessively high Fe¨ concentration has no effect on the reaction l51. Fu Wusheng l61 research shows that 3% to 4% is the appropriate water content, and the Fe concentration is selected at 9. About OxlO%.
However, some scholars have concluded that 2% to 6% water content has an inhibitory effect on the formation of anthocyanins, and a slightly higher Fe¨ concentration (>15 g/L) also inhibits the formation of anthocyanins.
On the basis of the iron salt catalytic reaction, Yang Dajin I and others used high-performance liquid chromatography to detect the content of proanthocyanidins. In this method, the deep red anthocyanin ions generated by proanthocyanidins under the above-mentioned iron salt catalytic conditions are analyzed by high performance liquid chromatography to determine the content of proanthocyanidins. This method can eliminate the influence of some impurities and has the advantage of accurate qualitative and quantitative results.
3.2.2 Vanillin method
Measurement principle: The A ring of proanthocyanidins and catechin monomers has high chemical activity. Under acidic conditions, the A ring on it Phloroquinone or phloroglucinol condenses with vanillin, and the product forms a red carbon ion under the action of concentrated acid. The concentration of the sample is positively correlated with the color produced. Its absorption light value is measured at a wavelength of 500 llm l91 (Figure 3)
Figure 3 Phenolic condensation reaction
When measuring vanillin method, catechin is generally used as the standard and methanol is used as the solvent. Both hydrochloric acid and sulfuric acid can be used as catalysts in the reaction process, but when using sulfuric acid, the concentration should not be too high. Too high sulfuric acid can easily cause vanillin to undergo self-condensation reaction and oxidative decomposition. There are many specific operation methods: 1 mL test solution + 2.5 mL 1% vanillin methanol solution + 2.5 mL 25% sulfuric acid or 8% hydrochloric acid (both dissolved in methanol), 30. React twice for 15 min～20 min2l. Ugly; 1 mL test solution + 6 mL 4% vanillin methanol solution + 3 mL concentrated hydrochloric acid, react at room temperature for 15 minL231; some even react at 20°C for 15 h[241. The operation methods are quite different, which is not conducive to the user's choice and needs to be unified.
3.2_3 Ultraviolet spectrophotometry
Proanthocyanidins are colorless substances with no characteristic absorption peak in the visible light region and a unique absorption peak in the ultraviolet region, with the maximum absorption wavelength at 280 Nm. at. Although this method is simple and quick, it is only suitable for products with particularly high purity of proanthocyanidin content and is not suitable for the detection of proanthocyanidins in general raw materials. This is because catechins also have maximum absorption at 280 Nm, V, Vc, Ve. , Ve, rutin, B-carotene and other substances have obvious absorption at this wavelength.
3.2.4 Folin-Ciocaheau and HPLC combination method
This method It is a method recommended by the American Grape Seed Method Evaluation Committee. The Folin-Ciocaheau method determines polyphenol content, generally using gallic acid as a control substance. In alkaline solution, polyphenols can reduce tungstomolybdic acid to form a blue compound with maximum absorption at 760 Nm. The polyphenol content in grape seed extract is generally between 75% and 95%. HPLC was used to measure the contents of four monomers: gallic acid, catechin, epicatechin, and epicatechin gallate to represent the total amount of monomers. This is because the content of these four monomers accounts for more than 90.0% of the monomer content in grape seed extract. The content of proanthocyanidins is the difference between the polyphenol content and the monomer content.
The disadvantage of this method is that easily oxidized substances such as proteins, amino acids, nucleic acids, and ascorbic acid also participate in the Folin-Ciocalteau reaction. At the same time, since the content of gallic acid in grape seed extract is very small (0% ~ 1.2%), it is different from catechin (1.5% ~ 7.3%) and epicatechin (2.0% ~ 5.0%). 1%) content is very different. The content of proanthocyanidins expressed by the amount of gallic acid is not representative.
3.2.5 Ammonium molybdate spectrophotometry
It is based on the formation of yellow molybdate ester between catechol and ammonium molybdate in a weakly acidic medium. The reaction product is It has maximum absorption at the wavelength of 333 nm. Ma Yajun conducted a simple exploration of the inch detection conditions: take 0.08 mol, 1 mL of L ammonium molybdate solution and place it in a 25mL colorimetric tube, add an appropriate amount of test solution, and use 1. OxlO mol/L hydrochloric acid reaches the mark, and the reaction is completed instantly.
According to the reaction principle, anthocyanins, gallic acid, and catechins all have a catechol structure and are also involved in the generation of molybdate esters. The selectivity of measuring proanthocyanidins is not high and is greatly affected by impurities. .
3.2.6 Other determination methods
Ma Yajun studied the determination method of proanthocyanidin content: Ferric salt-potassium ferricyanide spectrophotometry, which is based on the ability of proanthocyanidins to Fe is reduced to Fe, and Fe and potassium ferricyanide form a soluble dark blue coordination compound with maximum absorption at 710 nm; the principle of ceric ammonium sulfate spectrophotometry, which is based on the reaction of proanthocyanidins and Ce in a strong acidic medium Colorless Ce is generated, and Ce "has maximum absorption at a wavelength of 319 nm. By measuring the absorbance of yellow high cerium salts, proanthocyanidins are indirectly determined. There is also a flow injection-inhibited chemiluminescence method [271: Under alkaline conditions, the use of proanthocyanidins to reduce H:O: can inhibit the chemiluminescence of the luminol-H20 system, and the degree of inhibition is linearly related to the concentration of proanthocyanidins. . These three methods are similar to the Folin-Ciocaheau method, which uses the reducing properties of polyphenols to determine the polyphenol content. As a result, the content of proanthocyanidins is expanded.
In summary, the measurement of proanthocyanidin value is only based on empirical formulas, not the true content of proanthocyanidins, and is out of date with modern detection methods. The iron salt catalyzed method for determination of proanthocyanidins is highly specific and has good application prospects, but further research and improvement are still needed. The vanillin method measures the total amount of proanthocyanidins and flavan-3-ol monomers. The content of proanthocyanidins can be calculated by combining the HPLC method to detect the content of flavan-3-ol monomers: catechin and epicatechin. . However, there are many operating methods for the vanillin method, which is not conducive to user choice, and the specific operating methods need to be unified. Some foreign scholars use HPLC/MS technology to analyze and detect proanthocyanidins. The process is complicated and the technical requirements are high, so it cannot be widely used in the determination of proanthocyanidin products.
4 Outlook
Grape seed proanthocyanidins have highly effective anti-aging, anti-cardiovascular disease, and anti-cancer functions. In addition, they also have anti-radiation, anti-fatigue, and improved memory effects, showing that Unparalleled superior biological activity and safety. At present, there are more than 50 companies producing and selling grape seed extract in my country, with an annual production capacity of more than 80 tons. Therefore, in order to adapt to the vigorous development of the grape seed extract industry, there is an urgent need to establish a unified measurement method for the content of proanthocyanidins in grape seeds and their products, so as to facilitate the production and trade of enterprises, product quality control, and customer consumption guidance.