What Are Cranberry Proanthocyanidins Extraction Methods?

Cranberries (Vaccinium macrocarponn), also known as lingonberries, are herbaceous plants in the family Ericaceae, widely cultivated in parts of North America. There are many wild cranberries in China's Greater Khingan Mountains region, and about 3,000 to 4,000 hectares are cultivated in Fuyuan, Heilongjiang Province. Cranberries are known for their high vitamin C content and medicinal value [1].

Research has found that proanthocyanidins in cranberries have functions such as anti-oxidation and elimination of free radicals, and are widely used in food, cosmetics, pharmaceuticals and other fields [1-2]. Proanthocyanidins (PC) are polyphenolic compounds formed by the combination of different amounts of catechins or epicatechins. They are common in plants and are natural antioxidants found in plant stems[2]. They are also known as condensed tannins. In the 21st century, with the development of biomedicine and nutrition, the research and application of proanthocyanidins have received wider attention. On December 7, 2021, a Chinese research team extracted proanthocyanidin C1 from specific grape seeds, which can effectively clear senescent cells[3].

In the study of domestic and foreign researchers exploring the purification process and function of cranberry proanthocyanidins, it was found that the anthocyanins in cranberries have the effects of preventing urinary tract infections, inhibiting oral bacteria, anti-tumor and anti-oxidation, and have broad application prospects [4]. This paper reviews the extraction and purification process and function of cranberry proanthocyanidins, providing a theoretical basis for the further development and utilization of cranberry proanthocyanidins.

1 Extraction technology of cranberry proanthocyanidins

With the deepening of research on cranberry proanthocyanidins, there are more and more extraction methods for cranberry proanthocyanidins. At present, the main extraction methods used at home and abroad are ethanol extraction, countercurrent extraction, and ultrasonic-assisted extraction.

1.1 Ethanol extraction

The ethanol extraction method is a common method for extracting active plant ingredients. Ethanol is used as an organic solvent to dissolve the active ingredients of the plant in ethanol, and the desired ingredients are obtained by evaporating the ethanol. Ke Chunlin et al. [5] used the ethanol extraction method to extract cranberry proanthocyanidins and measured their antioxidant activity in an in vitro antioxidant experiment. The experiment found that the optimal extraction conditions for cranberry proanthocyanidins were an ethanol concentration of 65%, a material-to-liquid ratio of 1:15 (g/mL), an extraction time of 30 min, and an extraction temperature of 70 °C. It was also found that the cranberry proanthocyanidin extract had good superoxide anion scavenging and reducing abilities. The ethanol extraction method is simple to operate and low-cost, but it also has some limitations.

For example, ethanol has limited solubility for some active ingredients, which can affect the extraction effect and may cause some active ingredients to be inactivated or degraded.

1.2 Countercurrent extraction

Countercurrent extraction is a common extraction and separation technique that uses the principle of simultaneous but opposite movement of the material and solvent to separate the desired substance. Compared with existing methods, countercurrent extraction has the advantages of simple material processing, solvent saving, high extraction efficiency, and low cost, making it suitable for industrial production. Ji Jinjun [6] disclosed a method for preparing a proanthocyanidin extract from cranberries, in which cranberry fruits are taken, crushed, and an ethanol solution with a pH of 1.0 to 3.0 and a mass fraction of 65% to 85% is added. The mixture is extracted countercurrent for 4 to 6 h at 30 to 60 °C, filtered, and the filtrate is collected and concentrated to obtain a proanthocyanidin extract from cranberries. This patented countercurrent extraction method can more fully extract the proanthocyanidin components of cranberries.

1.3 Organic solvent degreasing extraction method

Organic solvents can dissolve lipids in plants, and the lipids in the plant extract can be removed by evaporation. During the degreasing process, the organic solvent contacts the oil on the surface of the material by diffusion, dissolving the oil in the solvent. The solvent and dissolved oil are then removed from the surface of the material by washing or volatilization to achieve the purpose of degreasing. Organic solvent degreasing and extraction has the advantages of fast degreasing speed and little or no corrosion of the substrate, but also has some disadvantages, such as incomplete degreasing, flammable, explosive and toxic solvents, which require special attention to safety during use; solvents are volatile and may cause environmental pollution. Shi Tong et al. [7] placed cranberries in an extraction solution containing 70% acetone, 30% methanol, and 0.1 g/100 mL citric acid, and added a fat-soluble organic solvent, petroleum ether, for degreasing to obtain cranberry extract. The volume ratio of cranberry extract to fat-soluble organic solvent is 1:1–1:3. This extraction process can significantly improve the extraction rate of proanthocyanidins.

1.4 Ultrasonic-assisted extraction method

The ultrasonic-assisted extraction method has the characteristics of high extraction rate and simple operation. With the cooperation of organic solvents, the extraction rate is even higher. During the extraction of proanthocyanidins, ultrasound produces strong mechanical vibrations and heat energy. These vibrations can act on the cranberry tissue, causing particle displacement, collision and shearing, thereby increasing the surface area of the cranberry tissue; the generation of heat energy increases the temperature, which helps to change the solubility and diffusion rate of proanthocyanidins and promotes their dissolution and extraction in the solvent. Increasing the contact area between the solvent and cranberry proanthocyanidins is beneficial to the dissolution and release of proanthocyanidins [8]. Qian Yuling et al. [9] used an ultrasonic-assisted extraction method to extract cranberry proanthocyanidins. The highest yield of proanthocyanidins, 11.65%, was obtained when the extraction was carried out for 40 minutes at 70°C with a material-to-liquid ratio of 1:20 and an ethanol volume fraction of 50%. Wang Kaiyin [10] used a combination of ultrasound and solvent extraction to extract proanthocyanidins. After optimization and verification, the optimal process conditions were ethanol concentration 70%, material to liquid ratio 1:20 (g/mL), ultrasound power 180 W, ultrasound temperature 75 °C, and ultrasound time 50 min, with a proanthocyanidin extraction rate of 2.0278%.

2. Purification and separation technology for cranberry proanthocyanidins

Proanthocyanidins extracted from cranberries contain a variety of impurities and need to be purified. The main purification methods for cranberry proanthocyanidins include the macroporous resin method, membrane separation and filtration method, and gel chromatography method.

2.1 Macroporous resin method

Macroporous resin is a synthetic adsorbent made from organic polymer copolymers with a macroporous structure. It has selectivity and adsorption properties due to its porous three-dimensional structure. The macroporous resin method has the characteristics of large adsorption capacity and high elution rate, and is therefore widely used in industrial wastewater treatment, vitamin purification, and the decolorization of chemical products. He Xinhua et al. [11] used an HPD600 macroporous resin column to purify and separate proanthocyanidins from cranberry residue. The optimal process was to load 500 mL of resin, with a sample solution of 10 mg/mL, an elution flow rate of 2.0 BV/h, and 600 mL of 75% ethanol for resolution. The eluate was collected, concentrated, and dried. The process can be used to purify and separate cranberry pomace proanthocyanidins, and can purify cranberry proanthocyanidins with a purity of ≥95%. The yield of cranberry proanthocyanidins is 1.5%.

2.2 Membrane separation and filtration

Membrane separation and filtration can be carried out at room temperature and low pressure, and is characterized by high efficiency and energy conservation. Membrane separation is a dynamic cross-flow process in which macromolecules are blocked by the membrane and thus purified and separated. At a pH of 2.54 in cranberry juice, proanthocyanidins and anthocyanins are positively charged, as they always migrate in the direction of the cathode. When using configuration 2 of the membrane separation system, the migration yield of proanthocyanidins and anthocyanins is higher. Compared to untreated juice, the concentrated cranberry juice has a more intense flavor. The total mass fractions of proanthocyanidins and anthocyanins in the cranberry juice treated with the membrane filtration system increased by 34.8% and 52.9%, respectively. In addition, the antioxidant capacity of the concentrated cranberry juice increased by 18% after treatment with the membrane filtration method. Membrane filtration can be used to enrich phenolic substances in cranberry juice [12].

2.3 Gel chromatography

Gel chromatography is a purification and separation method developed in the early 1960s. It is based on the exclusion effect of porous gel materials on molecules of different sizes to achieve separation. Elizra [13] used a Sephadex LH-20 gel chromatography column to separate and purify proanthocyanidins from grape seeds by gradient elution with methanol. Grape seed extracts were extracted with organic solvents of different polarities. Among the extracted components, ethyl acetate had the strongest antioxidant and non-enzymatic glycosylation inhibitory effects. Among the components obtained after separation with a macroporous resin, the antioxidant activity and non-enzymatic glycosylation inhibitory effect of the component eluted with 30% ethanol were higher than those of the other separated components. Gel chromatography can also be used to extract proanthocyanidins from cranberries, but there have been few reports on this so far.

3 Functions of cranberry proanthocyanidins

Cranberries have many beneficial effects on the human body. Phenolic compounds have been isolated from cranberry extracts and studied, and it is inferred that cranberry proanthocyanidins have antibacterial, anti-adhesion, antioxidant and anti-inflammatory properties [14-15].

3.1 Antibacterial

Studies have shown that cranberry proanthocyanidins can inhibit the adhesion of P. aeruginosa to urinary epithelium, thereby preventing urinary tract infections [16]. Wang et al. [17] conducted a systematic search of MEDLINE, EMBASE and the Cochrane Central Register of Controlled Trials and found that that cranberries have been used traditionally to prevent urinary tract infections. Babar et al. [18] recruited 145 women with a history of recurrent urinary tract infections and randomly assigned the subjects to consume a high dose of standardized cranberry proanthocyanidins or a low dose of a control for 24 weeks. The results showed that a high dose of proanthocyanidins may have a preventive effect on the recurrence of symptomatic urinary tract infections in women who have fewer than 5 infections per year. Current clinical evidence suggests that consuming cranberries can prevent bacteria from adhering to urinary epithelial cells, thereby reducing urinary tract infection-related symptoms; it can also reduce urinary tract infection-related symptoms by inhibiting the inflammatory cascade response, which is an immune response to bacterial invasion [19].

Non-toxic and highly effective natural ingredients found in plants, such as proanthocyanidins and flavan-3-ols, can have an anti-inflammatory effect on periodontitis [20-21]. The colonization of subgingival tissue by Porphyromonas gingivalis is a key first step in the development of periodontitis. Studies have shown that cranberry proanthocyanidins, although they cannot kill Porphyromonas gingivalis or inhibit its growth or reproduction, can strongly inhibit the formation of gingival biofilms, thereby reducing the ability of bacteria to colonize the gums. Proanthocyanidins were isolated from geranium root extract and compared with the whole extract. The results showed that cranberry proanthocyanidins have stronger antioxidant capacity than the root extract and exhibit unique antibacterial effects [22]. Researchers extracted proanthocyanidins from grape seeds and demonstrated that proanthocyanidins have a significant therapeutic effect on experimental periodontitis induced by endotoxin (Escherichia coli) in rats [23].

3.2 Anti-cancer

Studies have shown that cranberries can be used as chemopreventive agents to reduce the risk of cancer by inhibiting oxidative and inflammatory processes in cells. They can also inhibit cell proliferation and angiogenesis, induce apoptosis, and reduce the ability of tumor cells to invade and metastasize, further demonstrating their potential anti-cancer activity [ 24]. Zulfiqar et al. [25] showed that proanthocyanidins can be used as a therapeutic agent. Cranberry proanthocyanidins have obvious anticancer properties, and further extraction of proanthocyanidins from cranberries can treat ovarian cancer through combination or monotherapy.

3.3 Anti-aging

Nutrition plays an important role in aging. Therefore, nutrient absorption and metabolism, as well as diet, are important factors influencing aging-related diseases. The nutritional needs of the elderly are different from those of young people due to factors such as drug-nutrient interactions, age-related diseases, and socioeconomic factors [26]. Researchers have investigated whether cranberry concentrate (CBC), which is rich in proanthocyanidins (PACs) of type A, has anti-aging and redox-state-regulating effects, and compared the effects with those of grape seed extract (GSE), which is rich in PACs of type B. These findings provide groundbreaking in vivo evidence that, compared to the well-known B-type PAC-rich GSE, A-type PAC-rich CBC has significant potential for anti-aging and redox status regulation [27].

3.4 Prevention of cardiovascular disease

Vascular health is crucial in modern society, and the use of drugs to treat vascular problems is a routine approach to the treatment of blood pressure problems. Researchers conducted experiments on obese middle-aged people and demonstrated that emerging cardiovascular disease risk factors can be modified through a food-based intervention with cranberry juice [28]. Thim6teo et al. [29] found that cranberry products can significantly improve several indicators of cardiovascular risk and symptoms such as adhesion molecules, homocysteine and arterial stiffness, based on primary interventions that take into account other cardiovascular risk markers. A growing number of studies have shown that polyphenols may reduce the risk of cardiovascular disease by increasing the resistance of low-density lipoproteins to oxidation, inhibiting platelet aggregation, lowering blood pressure, and through other anti-thrombotic and anti-inflammatory mechanisms [30-31]. Although most of the studies are derived from in vitro studies and animal models, research has shown that these phytochemicals are bioavailable and biologically active. More research is needed on the bioavailability and metabolism of cranberry polyphenols and the relationship between cranberry dose and duration of use to better understand their effects on cardiovascular disease risk factors, especially clinically meaningful parameters such as inflammation, insulin resistance, vascular reactivity and vascular remodeling [32].

3.5 Antioxidant

Studies have shown that proanthocyanidins have 20 times the antioxidant power of vitamin E and 50 times that of vitamin C. Proanthocyanidins help protect the body from sun damage, improve eyesight, increase the flexibility of body tissues such as joints, arteries and the heart, and improve blood circulation by strengthening capillaries, arteries and veins [33]. In a cell model, ARPE-19 cells were irradiated with blue light at a wavelength of 450 nm for 10 h, and cell viability or proliferation rate was assessed using the MTT assay (reparative effect). The study showed that cranberry juice extract containing proanthocyanidins may exhibit better radical scavenging activity and thus effectively protect ARPE -19 cells, hindering the progression of AMD. The effect of cranberry juice on human retinal pigment epithelial cells exposed to blue light, ARPE-19 cells mimic age-related macular degeneration [34].

4 Outlook

Researchers have improved the extraction rate of cranberry proanthocyanidins by analysing the advantages and disadvantages of the extraction and purification methods of cranberry proanthocyanidins and the optimal extraction ratio. The next step is to strengthen research on the extraction and purification process, and to carry out functional research on cranberry proanthocyanidins in terms of their bacteriostatic and anti-functional properties, and continue to develop their functions and apply them to cranberry juice and various non-staple foods, so as to promote the development of the industry.

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