Proanthocyanidins (PC for short) are a large class of polyphenolic compounds widely present in the plant kingdom. Phytochemists usually refer to all colorless polyphenolic compounds isolated from plants that can produce red anthocyanins (cyanidin) in the presence of inorganic acids and heat treatment as proanthocyanidins. From the early 1960s to the present, a series of chemical reactions such as procyanidin antioxidants and free radical scavenging have been initially revealed. This type of natural products has been increasingly used in the fields of medicine, food, daily chemicals and other fields. The research on proanthocyanidins is getting more and more in-depth all over the world, among which the research on the extraction, separation and purification methods of proanthocyanidins is a major focus. The extraction, separation and purification methods of proanthocyanidins are summarized as follows.
1 Extraction method of proanthocyanidins
The extraction rate of proanthocyanidins in plant materials is closely related to the condition of the material and the extraction conditions. The storage, drying, grinding degree, extraction solvent, temperature, etc. of plant materials may lead to changes in the chemical structure of proanthocyanidins and changes in the extraction rate, thereby changing the physical and chemical properties and biological activity of proanthocyanidins.
When measuring the content of raw materials When the proanthocyanidin content is lower, the longer the storage time, the lower the measurement results may be. At the same time, the moisture content in the sample will also cause the measurement results to decrease. Moreover, different drying conditions will also lead to changes in the extraction rate. It is best to use freeze-drying to avoid high temperatures.
Samples generally need to be crushed before extraction. Usually finer powder is beneficial to extraction, but if it is too fine, the extraction rate will be reduced.
The choice of extractant is also a key factor affecting the extraction rate. Because proanthocyanidins usually form stable molecular complexes with proteins, polysaccharides, etc. in the form of hydrogen bonds and hydrophobic bonds in plants, the same is true between proanthocyanidin molecules. Therefore, the extractant for proanthocyanidins must not only have good solubility, but also must have hydrogen bond breaking effect. Therefore, a composite system of organic solvent and water (organic solvent accounts for 50% to 70% of the total volume) is most suitable for extraction. The order of extraction capacity of organic solvents is propanol lt; ethanol lt; methanol lt; acetone lt; tetrahydrofuran. Among them, acetone-water system and methanol-water system are more commonly used.
When the content of iron and other metal ions in plant samples is large, proanthocyanidins complex and precipitate with metal ions under neutral conditions, and the deposition in the fiber is not conducive to extraction. At this time, acidified solvents must also be used to break the hydrogen and hydrophobic bonds between proanthocyanidins and proteins, polysaccharides and their own ions on the one hand, and on the other hand to break the proanthocyanidin-metal ion complex bonds to increase the extraction rate.
1.1 Traditional organic solvent extraction
In 1991, Ayroles et al. invented a method of extracting proanthocyanidins from Ginkgo leaves using aqueous solutions of ketone compounds as extractants. Use an aqueous solution of ketone compounds as the extractant. After filtering the extract, use alkali to adjust the pH value of the filtrate to about 9 to precipitate proanthocyanidins. Then use acid to adjust the filtrate to a pH value of about 2. In the presence of (NH4) SO, use c ~ c Ketones extract the proanthocyanidins in the filtrate, remove the ketone compounds, and dry.
When Romanczyk et al. invented the method of extracting proanthocyanidins from cocoa, they extracted defatted cocoa beans with a mass fraction of 70% MeOH/deionized water, and then extracted them twice with a mass fraction of 70% acetone/deionized water. Concentrate in a vacuum, remove the organic solvent, then dissolve in water, extract with CHC1, extract the aqueous phase with ethyl acetate, concentrate in a vacuum to remove the ethyl acetate, and freeze-dry the aqueous phase to obtain proanthocyanidins.
1.2 Green solvent - water extraction technology
Since organic solvents such as acetone may cause environmental pollution and toxic organic residues in products, people are vigorously developing environmentally friendly green solvents. Extraction technology. In 1998, Duncan and Gilmour invented a method for extracting proanthocyanidins from plant materials (bark, leaves, grape seeds, skins, soybeans, green tea).
Crush the material (≤15 mm), extract it with deoxygenated hot water (1 min to 20 h) under normal pressure, 60°C to 100°C or high pressure of 100°C to 125°C, and filter using ultrafiltration or reverse osmosis or a combination of both. , concentrate the filtrate, vacuum spray or freeze-dry. This method mainly extracts water-soluble proanthocyanidins with a molecular weight ≤5 000 D. The yield is between 0.5% and 10.0%, usually 6.5% - 9.6 % (depending on the difference in sampling location), proanthocyanidins B, B,, B and c were isolated. The obtained product has a significant inhibitory effect on the oxidation of linoleic acid triggered by AAPH, and an inhibition rate of 70% to 79% can be achieved at 1 g/mL. Toxicological testing showed that no toxicity or side effects occurred within 24 hours in the dose-based dose group and the dose dose group 100 times the human body weight, and there were no obvious toxicities or side effects in the chronic toxicology (5-month) experiment.
In 1999, Karim et al. invented the method of extracting proanthocyanidins from plant materials using deoxygenated deionized water under pressurized conditions. After ultrafiltration of the extract, a column chromatography method using hydrophobic microporous polymer resin as filler is used, and a polar eluent (ethanol and water) is used for elution. The eluent is removed by reverse osmosis to remove ethanol, and dried to obtain proanthocyanidins.
1.3 Supercritical fluid extraction technology
Sun Chuanjing et al. invented a polar modifier composed of supercritical carbon dioxide plus acetone and water, and extracted Ginkgo biloba containing Methods for Proanthocyanidin Extracts. At an extraction temperature of 60°C to 90°C and an extraction pressure of 20 MPa to 35 MPa, add a polar modifier with a volume ratio of acetone to water (50% to 80%): (50% to 20%). 2 h-4 h, perform static and dynamic extraction. The extract is concentrated by traditional resin and dried in a spray dryer to obtain refined Ginkgo leaf extract. The product contains ginkgo flavonoid glycosides gt; 35 g/100 g, terpene lactones gt; 8 g/100 g, proanthocyanidins <7 g/100 g, and phenolic acids <5 mg/kg. The advantage of this method is that the process is short and it can extract the strongest natural antioxidant proanthocyanidins. In 2000, Sun Chuanjing and others invented a supercritical CO method to extract blackcurrant seed oil and proanthocyanidin oligomers from blackcurrant seeds. This method is carried out in two steps: the first step is to use supercritical CO2 to extract blackcurrant seed oil, control the extraction pressure between 25 MPa and 29 MPa, and the temperature is 60°C; the second step is to add supercritical CO2 to the mixture of acetone and water. The volume ratio of the modifier is 70:30, the flow volume ratio of CO to modifier is 4:1, the pressure is 22 MPa~25 MPa, and the temperature is 60°C, and the proanthocyanidin oligomers are extracted. The blackcurrant seed oil yield was 16%, and the proanthocyanidin oligomer yield was 4%. The advantage of this method is that two products are obtained at the same time, the process is simple and reliable, CO and modifiers are recycled, there is no solvent residue in the product, and there is no pollution to the environment.
1.4 Microwave extraction technology
Liu Zhengtao et al. invented a method of using microwaves with a frequency of 2450 MHz or 915 MHz and a power of 500 W to 15 000 W to extract grape seeds. A new method for extracting proanthocyanidins from grape seeds by treating it with water, alcohol with a carbon chain length of C to C, ether, acetone, ethyl acetate, toluene or a mixture thereof. Compared with conventional chemical methods, this method is simple, efficient, fast, low-cost, and has less waste liquid discharge.
1.5 Aqueous two-phase extraction method
From the discovery of the two-phase aqueous system by Albertsson of Lund University in Sweden in 1956 to the two-phase aqueous extraction method by Kula et al. of GBF in Germany in 1979 Although separation technology has only been used for the separation of biological products for more than 20 years, it has been successfully used in the separation and purification of biological products such as proteins, nucleic acids, and viruses due to its mild conditions and easy amplification. In recent years, literature on the extraction of active ingredients from Chinese herbal medicines using aqueous two-phase extraction technology has begun to be reported. Although the number is small, the existing examples fully demonstrate its good application prospects.
Research on enrichment and separation of ginkgo leaf extract using a two-phase aqueous extraction system showed good distribution coefficient and separation effect. Studies have shown that the two-phase aqueous system has fast phase separation, low operating temperature, easy operation and waiting point, and the PEG and salts used are non-toxic to the human body and the environment, and have a high extraction rate, making it an effective method for enrichment and separation of ginkgo flavonoid compounds. method. Although the application of aqueous two-phase extraction to Chinese herbal medicine extraction research is in its infancy, the application of this technology is expected to provide a new idea for extracting active ingredients from natural products.
2 Purification and separation of proanthocyanidins
2.1 Liquid phase extraction method
Ethyl acetate, toluene, dichloromethane, and ether are often used for the purification of proanthocyanidins. Multi-stage organic solvents are used through liquid phase extraction. This method may cause pollution to the environment due to the large amount of organic solvents used, and it is also easy to cause toxic organic residues in the product.
2-2 Column Chromatography
Currently, most of the commonly used purification methods are column chromatography. Wang Jianqing et al. purified the acetone extract of proanthocyanidins from barley using PVPP resin as column chromatography packing and CH CN as the mobile phase.
Ricardo da silva et al. extracted grapes with methanol. After recovering methanol from the extract, preliminary separation was carried out through a polyamide column. The phenolic acid was first washed away with neutral water, and then acetonitrile/acetonitrile in a volume ratio of 30:70 was used. Catechins were eluted with water, and proanthocyanidins were eluted with acetone and water in a volume ratio of 75:25 for purification.
Liu Rui et al. used macroporous resin to purify proanthocyanidins in sorghum using an aqueous ethanol solution, and obtained oligomeric proanthocyanidins with a product purity greater than 95 g/100 g.
2.3 Solid-phase extraction method
Solid-phase extraction (SPE) is one of the most effective methods to selectively extract required components from complex systems. In 1999, Lazarus et al. used SPE method to purify proanthocyanidins in almond skin, grape juice and red wine. Conditions: supelcosil Envi-18 20mL SPE column, mobile phase: acetone: water: acetic acid = 70:29.5:0. 5 (volume ratio). Kennedy and Waterhouse used a c-18 column (Alhech) for proanthocyanidins in red wine. The mobile phase was water and methanol. The extracted proanthocyanidins are purified by elution to remove organic acids, sugars and other compounds insoluble in the organic phase.
2,4 Gel Chromatography
Gel chromatography is also commonly used for the purification of proanthocyanidins. Sephadex LH-20 is a hydroxypropylated dextran gel with high affinity for flavonoids. Sephadex LH-20 gel chromatography is currently used for the purification and separation of proanthocyanidins. However, the physical properties of Sephadex LH-20 gel determine that it cannot separate proanthocyanidins with high efficiency. Therefore, further purification and separation are performed using gel filtration chromatography or HPLC.
In addition, Sepherdex 75HR, as a glucan polymer with an average particle size of 13 m, is also used for the purification and separation of proanthocyanidins. It can withstand a back pressure of more than 1.8 MPa. Although this material has Commercial columns are usually used for protein separation, and their ability to separate proanthocyanidins was found to be better than Sephadex LH-20. McMurrough and Madigan concentrated the barley extract and directly used high-performance gel filtration chromatography (Sepherdex 75HR) to elute with methanol. According to uV detection, the eluates were collected and each component was identified using DMACA. Escribano-Bail 6n et al used Sephadex LH-20 and semi-preparative RP-HPLC to purify proanthocyanidins in grape seeds.
Rigaud et al. used gel permeation chromatography (GPC) TSK G 2500 Hxl and TSK G3000 Hxl to purify cocoa and grape seed extracts by elution with tetrahydrofuran (flow rate 1 mL/min).
2.5 Microbial fermentation method
Ariga et al. invented an active yeast that can ferment and remove the starch in the extract obtained by extraction with water and organic solvents to purify proanthocyanidins. Purpose; It was also found that the metal ions in the purified proanthocyanidins can also be better removed. If the extractant is water and water/ethanol, it can be directly concentrated and then fermented. If the extractant is acetone, the acetone must be removed before fermentation can be carried out. Commonly used yeasts include: Saccharomyces cerevisiae, Saccharomyces genus and Saccharomyces genus strains.
2.6 High-speed countercurrent chromatography
High-speed countercurrent chromatography was pioneered by Dr. Ito Yiochiro of the National Academy of Medicine in the 1960s. It was originally a preparative chromatography technology. This kind of liquid-liquid distribution chromatography does not use solid carriers or supports. It is mainly separated based on the distribution ability of compounds between two immiscible phases. It has high separation efficiency, high product purity, no adsorption and contamination of the sample by the carrier, and low preparation volume. It has the characteristics of large size, low solvent consumption, and simple operating conditions (room temperature, Teflon inert column material). It has been widely used in the preparation and analysis of natural pharmaceutical materials.
At present, high-speed countercurrent chromatography has been successfully developed into two series: analytical type and preparative type. That is, high-speed countercurrent chromatography can be used not only for the preparation and separation of natural pharmaceutical ingredients, but also for quantification. The injection volume ranges from a few milligrams to grams, and the injection volume ranges from a few milliliters to tens of milliliters. It is not only suitable for the separation of non-polar compounds, but also for the separation of polar compounds. It is suitable for coarse separation of functional components of natural products. It can also be further refined and purified.
2-7 Molecular Imprinting Technology
Molecular imprinting technology (MIT) is a highly selective separation technology that emerged at the end of the 20th century. Because MIT imitates the biological The principle of lock-and-key action enables the prepared materials to have extremely high selectivity, so they are quickly used in many related fields such as chiral separation and substrate selective separation, solid phase extraction, chemical or biological sensors, asymmetric catalysis, and simulated enzymes. been applied. In terms of general separation, compared with traditional methods, the MIT method has the advantages of high efficiency, speed, and specificity. The role of the MIT method in chiral separation is even more unparalleled. According to statistics, 60% of existing drugs have one or more chiral centers, and the efficacy and impact on the human body are very different between enantiomers. Therefore, in 1992, the U.S. Food and Drug Administration stipulated that in the future, drugs containing asymmetric The drug in the center must separate the optical isomers. Compared with the futility of traditional methods, the MIT method is very valuable. Pgt;
In 2002, Zhou Li et al. prepared a molecularly imprinted polymer (MIP) using quercetin as a template, and separated and extracted quercetin and isorhamnetin from the crude extract of sea buckthorn. Plant flavonoids and obtain good separation results. Xie Jianchun et al. used a non-ionic method to prepare molecularly imprinted polymers (MIP) in polar solvents using acrylamide as the functional monomer and the highly polar compound quercetin as the template. Liquid chromatography experiments show that MIP has a specific affinity for kaempferin. This MIP is used to directly separate the hydrolyzate of Ginkgo leaf extract to obtain two flavonoids, which mainly contain template quercetin and a compound with a similar structure to quercetin, kaempferol. components. The study confirmed the feasibility of MIP technology for directly separating and extracting specific medicinal compounds from Chinese herbal medicines.