What Are the Extraction Methods of Pomegranate Peel Extract Polyphenol?

1 Overview of pomegranate research and polyphenol extraction

Pomegranate (Punica granatum L.) is a plant in the family Punicaceae. It is widely cultivated in tropical and subtropical countries and is not only edible, but also widely used in folk medicine [1-2]. The fruit of the pomegranate is mainly composed of three parts: seeds, flesh and peel. Pomegranate juice is squeezed from the flesh, and its sales are currently increasing rapidly worldwide as it is recommended as a chemical prophylactic, chemotherapeutic, anti-atherosclerotic, anti-inflammatory agent, etc. [3-4]; pomegranate seeds are mainly used for oil extraction, and pomegranate seed oil has antioxidant [5], anti-aging [6], and immunity-enhancing [7] functions, and is now also being sold on the market; Pomegranate peel is a by-product of the processing industry of pomegranate juice and pomegranate wine, accounting for about 40% of the fresh weight of the entire pomegranate fruit [8]. In China, most of the fresh pomegranate peel is discarded, and only a small amount is dried in the sun for medicinal purposes [9].

Pomegranate peel contains a large amount of polyphenols, which are the main active ingredients that exert pharmacological effects. These include various compounds such as punicalagin, gallic acid, procyanidins, chlorogenic acid, epicatechin, caffeic acid, rutin, quercetin, kaempferol, etc. [10], accounting for about 10% to 20% of the dry weight of pomegranate peel. Studies over the past 10 years have shown that it has various effects such as anti-oxidation, anti-atherosclerosis, anti-aging, antibacterial, anti-mutation, lowering blood lipids, lowering blood pressure, and moisturizing and beautifying the skin [11-16]. Therefore, pomegranate peel polyphenols have great application value in food, medicine, and chemical products.

The extraction methods of polyphenols mainly include: solvent extraction, microwave and ultrasonic assisted extraction, enzymatic extraction, supercritical fluid extraction, and ultra-high pressure fluid extraction. Pomegranate peel polyphenols are unstable substances that are easily damaged by oxidation. Prolonged extraction times and high temperatures increase the probability of damage to the structure of the pomegranate peel polyphenols. A low extraction efficiency not only increases production costs, but also reduces the biological activity of the pomegranate peel polyphenols. Therefore, it is particularly urgent to conduct in-depth and meticulous research on the extraction methods of pomegranate peel polyphenols and establish an efficient, fast, economical and practical extraction method. This paper provides a review of the methods used to extract pomegranate polyphenols in recent years.

2 Extraction methods for pomegranate polyphenols

2. 1 Solvent extraction

Solvent extraction is the most traditional method for extracting polyphenols. The method involves separating different components of plants based on their solubility in different solvents. The solvents used are mainly water or organic solvents.

Sun Lanping [17] and others used ethanol to extract the polyphenols from pomegranate peel, and determined that the optimal extraction conditions were: extraction temperature 70 °C, liquid-to-material ratio 25:1 (mL:g), extraction time 1.5 h, ethanol volume fraction 50%, and yield of pomegranate peel polyphenols 16.28%.

Wang Xiaoyu [18] et al. used four solvents, methanol, ethanol, ethyl acetate and water, to extract the total polyphenols from pomegranate peel in Xinjiang. It was found that the extraction results varied greatly with different solvents, and the yield of total polyphenols was: methanol > ethanol > water > ethyl acetate.

Li Guoxiu [19] and others used pomegranate peel powder as the raw material to study the effects of extraction solvent concentration, extraction temperature, extraction time and material-to-liquid ratio on the extraction of polyphenols. The extraction process was optimized by orthogonal experiments under the influence of the above four factors, and it was determined that the optimal process for the ethanol extraction of pomegranate peel polyphenols was: ethanol volume fraction 60%, extraction temperature 60 °C, liquid-to-material ratio of 1:20 (g:mL), extraction temperature of 60 °C, extraction time of 2 h, and extraction for 2 times. Experiments were carried out using this optimized process, and the yield of polyphenols was 23.39%.

Most foreign researchers use water as the extraction solvent, believing that water is cheaper than organic solvents and causes less environmental pollution. In their study on the optimization of the aqueous extraction of polyphenols from pomegranate peel using central composite design, am [20] and others eliminated three factors from the five factors that may affect the yield that had little effect on the yield. The final two factors that had a greater effect, extraction temperature and extraction time, were selected for optimization experiments, The optimal extraction conditions were: extraction temperature 100 °C, extraction time 1 min. The total phenol content of pomegranate peel was determined by HPLC to be 192.0 mg/g dry basis, which was not significantly different from that obtained using the traditional methanol solvent extraction method.

Amyrgialaki [21] used response surface optimization to extract pomegranate peel polyphenols. The experiment was based on a 23-factor central composite design, with ethanol/water/citric acid as the extraction medium. The influencing factors included the pH of the extraction solvent, the concentration of ethanol, and the extraction time. The optimal extraction conditions were obtained: ethanol volume fraction 40%, pH 2, extraction time 1 h, total phenol content of pomegranate peel 324.9 mg/g dry basis.

When using the solvent method to extract pomegranate polyphenols, the main solvents that can be selected are methanol, ethanol, acetone, ethyl acetate and water. From the perspective of energy conservation and environmental protection and food safety, most researchers generally use ethanol or water as solvents, which are low in toxicity and relatively inexpensive. This method is simple to carry out, but it is time-consuming and not very efficient.

2. 2 Enzyme hydrolysis extraction method

The enzymatic extraction method is based on the highly specific nature of enzymatic reactions. The corresponding enzyme is selected to hydrolyze or degrade the components of the cell wall, thereby destroying the cells and making the active ingredients in the cells more easily soluble in the solvent, so as to achieve the purpose of extracting the active ingredients of the plant.

Wang Huabin [22] and others studied the process of enzymatic extraction of polyphenols from pomegranate peel. A single-factor test was used to investigate the effects of different concentrations of cellulase, pectinase, complex enzyme (cellulase and pectinase at different mass ratios), enzymatic hydrolysis time, enzymatic hydrolysis temperature and enzymatic hydrolysis pH value on the polyphenol yield of pomegranate peel, and used a two-stage generalised rotary regression combined design to optimise the optimal process parameters for enzymatic extraction of pomegranate peel polyphenols. The test results showed that the order of influence on the polyphenol yield of pomegranate peel was enzymatic hydrolysis time > enzyme concentration > pH > enzymatic hydrolysis temperature. When the mass concentration of the complex enzyme (cellulase and pectinase in a mass ratio of 2:1) is 0.25 mg/mL, the enzymatic hydrolysis time is 150 min, the enzymatic hydrolysis temperature is 50 °C, and the initial pH of the enzymatic hydrolysis solution is 6.0, the polyphenol yield is 23.87%.

Enzymatic extraction, with its mild conditions and high product yield, is now also being increasingly used in the extraction of active ingredients from food, pharmaceuticals, and animal and plant cells, but the extraction time is longer.

2. 3 Microwave-assisted extraction

Microwave-assisted extraction is a method in which high-frequency electromagnetic waves reach the inside of the material. Due to the absorption of microwave energy, the internal temperature of the material rises rapidly, the cells are broken instantly, and the extractant enters the extraction medium, thereby improving the extraction efficiency [23-24]. Microwave-assisted extraction has been widely used in recent years for the extraction of natural active ingredients. It can increase the mass transfer rate of the target substance from the solid phase to the liquid phase, thereby increasing the extraction yield.

Song Weiwei [25] and others used microwave-assisted extraction to extract polyphenols from pomegranate peel, and determined the optimal process conditions for the extraction of pomegranate polyphenols: 40% (volume fraction) ethanol as a solvent, a liquid-to-solid ratio (g:mL) of 1:35, a microwave power of 242 W, an extraction time of 60 s, and 3 extractions. When extracted under these optimized conditions, the yield of crude polyphenol extract was 26.52%.

Wang Ling [26] and others used microwave-assisted aqueous extraction to extract pomegranate peel polyphenols, and concluded that the optimal extraction process for the crude extract was: 60-80 mesh, extraction power 385 W, material-to-liquid ratio 1:25 (g:mL), extraction time 120 s, and extraction times 3 times. Under these conditions, the yield of pomegranate peel polyphenols can be as high as 21.4%.

Wen Chunquan [27] and others used microwave-assisted ethanol extraction to study the pomegranate peel polyphenols. The results showed that the ethanol volume fraction, microwave power, extraction time and liquid-to-material ratio all had a significant effect on the extraction yield of pomegranate peel polyphenols. The optimal extraction conditions for pomegranate polyphenols are: 50% ethanol volume fraction, 300 W microwave power, extraction time 120 s, and a material-to-liquid ratio of 1:35 (g:mL). Under these conditions, the extraction rate of pomegranate polyphenols can reach 21.41%.

Zhou Ancun [28] and others used an ethanol-ammonium sulfate two-phase system with microwave integration to extract and separate pomegranate peel polyphenols. single-factor experiments and orthogonal experiments showed that the optimal conditions for extracting pomegranate peel polyphenols were m (material) : V (alcohol) : V (water) = 1: 12: 20, (NH4)2SO4 dosage 0.325 g/mL, microwave treatment temperature 55 °C, microwave treatment time 60 s, the crude polyphenol extraction rate was 18.33%, and the polyphenol content in the crude extract was 75.36%.

The microwave extraction method combines microwave with solvent extraction, which is simple and easy to operate, greatly shortening the extraction time. However, special microwave extraction equipment is required, otherwise the extraction temperature cannot be controlled.

2.4 Ultrasonic-assisted extraction method

Ultrasonic extraction and separation is mainly based on the differences in the state of existence, polarity and solubility of the active ingredients and groups of active ingredients in the substance. Ultrasonic vibrations can quickly introduce the solvent into the solid material, dissolving as many of the organic components as possible into the solvent to obtain a mixed extract of multiple components. Ultrasound also causes high-frequency vibration in liquids and produces a “cavitation effect” that can destroy cell tissue and help dissolve and diffuse polyphenolic compounds. It has the advantages of short extraction time and high efficiency [29-30].

In a study on the ultrasonic-assisted extraction of polyphenols from pomegranate peel with ethanol, Tabaraki [31] and others selected three factors that had a significant effect on the yield: extraction time, ethanol concentration, and extraction temperature. The extraction conditions were optimized using response surface methodology. The results showed that when the extraction temperature was 60 °C, the ethanol volume fraction was 70%, and the extraction time was 30 min, the maximum extraction rate was 45.38%, which was three times higher than that obtained using the ultrasonic-assisted water method (Pan and Qu et al. used the ultrasonic-assisted water method to obtain an extraction rate of 11% to 14% [5, 32]).

Fang Yulin [33] and others used an ultrasonic-assisted methanol method to extract polyphenols from pomegranate peel. The results showed that the highest total polyphenol extraction rate was 14.06 mg/g, which was obtained using 80% acidified methanol as the solvent, a solvent-to-material ratio of 1:20 (g:mL), and ultrasonic waves (fixed power of 100 W) at 30 °C for 20 minutes with one extraction.

Wang Huabin [34] and others used pomegranate peel from Xinjiang as the raw material and used a two-stage generalised rotary regression combined design to study the process of ultrasonic-assisted ethanol extraction of pomegranate peel polyphenols. The results showed that the optimal extraction process conditions were: 50% ethanol volume fraction, 1:25 material-to-liquid ratio (g:mL), 30 min ultrasonic time, and 360 W ultrasonic power. Under these optimized conditions, the polyphenol yield from pomegranate peel was (21. 2 ± 0. 06) %.

In the study by RUSHAOGang [35] et al. on the extraction of pomegranate peel polyphenols using an ultrasound-assisted ethanol method, the optimal extraction conditions for pomegranate peel polyphenols were optimized by orthogonal experiments: ethanol volume fraction 40%, pomegranate peel particle size 40 mesh, extraction time 50 min, extraction temperature 30 ℃, material to liquid ratio 1:15 (g:mL), and the average extraction rate of pomegranate peel polyphenols under these conditions was 16.44%.

Guo Qinghe [36] and others used ultrasound-chelating agent assisted extraction of pomegranate peel polyphenols. Through response surface analysis, the optimal process conditions for ultrasound-chelating agent assisted extraction of pomegranate peel polyphenols were determined: ethanol volume fraction 50%, extraction temperature 60 °C, extraction time 50 min, liquid-to-material ratio 1:30 (g:mL), chelating agent addition 0.08%, and the polyphenol extraction rate under these conditions can reach 28.03%. Compared with ordinary hot reflux extraction, ultrasonic-chelating agent assisted extraction has the advantages of less impurities, less time-consuming and higher efficiency. It can be used as a new method for extracting polyphenols from pomegranate peel and has broad application prospects.

Pan [37] and others used two methods, continuous ultrasound-assisted and pulsed ultrasound-assisted, to extract the antioxidant substances (mainly polyphenols) from pomegranate peel, and compared the two methods with the traditional method. The optimal extraction method and process conditions were judged by the extraction rate and the size of the antioxidant activity. The results showed that in continuous ultrasound-assisted extraction (CUAE), the yield was positively correlated with the change of ultrasonic intensity and extraction time, but the extension of extraction time would seriously affect the antioxidant activity of the extract.

For pulsed ultrasound-assisted extraction (PUAE), CUAE), the yield is positively correlated with the change in ultrasonic intensity and extraction time, but the extension of the extraction time will seriously affect the antioxidant activity of the extract. For pulsed ultra-sound-assisted extraction (PUAE), the ultrasonic intensity, pulse repetition rate, pulse duration and pulse interval will affect the product yield, but does not affect the antioxidant activity of the extract. Under the conditions of an ultrasonic intensity of 59.2 W/cm2, an extraction time of 60 min, a temperature of (25 ± 2) °C, a liquid-to-solid ratio of 1:50 (g:mL), and a pulse duration and interval of 5 s for PUAE, the extraction yields of PUAE and CUAE were similar (14.5% and 14.8%, respectively), and compared with the traditional extraction method, the extraction yields of PUAE and CUAE increased by 22% and 24%, respectively, and the extraction time was shortened by 87% and 90%, respectively. In addition, PUAE can save 50% of the electrical energy compared to CUAE. In view of this, the authors recommend PUAE as the best method for extracting antioxidant substances from pomegranate peel.

Although the above scholars all used ultrasound-assisted extraction to extract the polyphenols from pomegranate peel, the solvents and extraction conditions used were different, so the yield of polyphenols also varied greatly. The ultrasound-assisted extraction method also combines ultrasound with solvent extraction, which can greatly save extraction time and improve extraction efficiency.

2. 5 Supercritical fluid extraction

Supercritical fluid extraction is a new modern separation technique that uses a fluid between a gas and a liquid with a temperature and pressure slightly above or close to the critical point as an extractant to extract certain high-boiling-point and heat-sensitive components from solids or liquids for the purpose of separation and purification [38]. At present, there have been few reports on the use of supercritical CO2 extraction technology for the extraction of polyphenols from pomegranate peel.

Feng Wqun [39] and others used supercritical CO2 extraction, ultrasonic extraction, microwave extraction and methanol extraction to extract gallic acid from pomegranate peel and determined its content by HPLC. The results showed that the gallic acid content extracted by the four methods was 0.498%, 0.311%, 0.271% and 0.396%, respectively. Therefore, supercritical CO2 extraction is an efficient method for extracting gallic acid from pomegranate peel polyphenols.

Supercritical extraction is a new and advanced extraction method that has been studied in recent years. It is mainly suitable for extracting non-polar and weakly polar low-molecular components. For the extraction of polar components, the extraction rate can be improved by adding a polar entrainer. Gallic acid (3,4,5-trihydroxybenzoic acid) contains multiple polar groups and is a strongly polar compound. It is suitable for extraction using a polar solvent as an entrainer. Methanol is a commonly used polar solvent. Feng W.Q. [39] and others used methanol as an entrainer, and the gallic acid content obtained was the highest among the four extraction methods.

Supercritical fluid extraction is a highly efficient and environmentally friendly extraction method that has been widely used in laboratory research, but it is relatively expensive during the extraction process [40-42]. Recent studies have found that subcritical water extraction can replace it [43].

2. 6 Subcritical water extraction

Supercritical water, also known as superheated water, high-pressure hot water or hot liquid water, refers to water that is heated to a critical temperature of above 100 °C and below 374 °C under a certain pressure, and the water still remains in a liquid state. In the subcritical state, the hydrogen bonds, ionic hydration, ionic association, and cluster structure of the microscopic structure of the fluid change, so the physical and chemical properties of subcritical water are quite different from those of water at room temperature and normal pressure. Water at room temperature and normal pressure has a strong polarity. In the subcritical state, as the temperature increases, the hydrogen bonds in the subcritical water are opened or weakened, so that the water is extracted from high to low. In this way, by controlling the temperature and pressure of subcritical water, the polarity of water can be varied over a wide range, thereby achieving continuous extraction of the active ingredients in natural products from water-soluble to fat-soluble ingredients, and selective extraction can be achieved. In addition, because subcritical water extraction uses inexpensive, non-polluting water as the extractant, subcritical water extraction technology is considered to be a revolutionary technology with broad prospects for environmental protection [43].

In a study by He [44] et al. on the extraction of pomegranate pomace polyphenols using subcritical water, extraction time, liquid-to-material ratio and temperature were used as single factors. The results showed that the optimal extraction conditions were: extraction time 30 min, liquid-to-material ratio 1:40 (g:mL), and temperature 220 °C. The temperature had the greatest effect on the extraction rate. Within the temperature range of 100–220 °C, the polyphenol content varied within 651.7–4854.7 mg/100 g dry basis.

Supercritical water extraction is an advanced and novel extraction technique. Its advantages over traditional extraction methods include: short extraction time, simple process, low cost, high extraction rate, and environmental protection [43].

2. 7   Ultra-high pressure extraction method

Ultra-high pressure extraction, also known as ultra-high cold isostatic pressure extraction, refers to the application of a hydrostatic pressure of 100 to 1,000 MPa at room temperature to a mixture of an extraction solvent and traditional Chinese medicine, and maintaining the pressure at a predetermined level for a period of time, after which the pressure is quickly released after the pressure inside and outside the plant cells has reached equilibrium. Due to the sudden increase in osmotic pressure inside and outside the cell, the structure of the cell membrane changes, allowing the active ingredients in the cell to pass through various cell membranes and be transferred to the extracellular extraction solution, thereby achieving the purpose of extracting the active ingredients. Recent studies have shown that its application in the extraction of natural products can greatly shorten the extraction time, reduce the dissolution of impurities, increase the yield of active ingredients, avoid changes in active ingredients caused by thermal effects, and will not cause environmental pollution [45].

Yan Longbing [45] and others used the total phenol yield as an index for the purpose of extracting polyphenols from pomegranate peel. Based on single factor experiments, a orthogonal rotation combined test design was used to optimize the process parameters of supercritical fluid extraction. The test results showed that the order of the factors affecting the yield of polyphenols extracted from pomegranate peel by supercritical fluid extraction was: liquid-to-material ratio > pressure > ethanol concentration > holding time. The optimal extraction process conditions are an extraction pressure of 582.7 MPa, a holding time of 2.3 min, a liquid-to-solid ratio of 1:41 (g:mL), and an ethanol volume fraction of 52.8%. Under these conditions, the polyphenol yield exceeds 26%.

In addition to ultra-high pressure, high-pressure solvent extraction is also used. In the study of extracting polyphenols from pomegranate peel by high-pressure water extraction by am [8], the pressure in the extraction vessel was maintained at 102.1 atmospheres (about 10 MPa). It was found that the particle size of the pomegranate peel raw material, the high-pressure treatment temperature and the electrostatic interference time are the main factors affecting the extraction rate of pomegranate peel polyphenols. The research results show that The optimum extraction temperature is 40 ℃, the electrostatic interference time is 5 min, the particle size can be as small as possible but should not be less than 65 μm, and the content of punicalin is (116.6 ± 12.2) mg/g dry basis. At the same time, the author also compared high-pressure water extraction with extraction at atmospheric pressure using different solvents (methanol, ethanol, ethyl acetate, acetone and water) were compared. The results showed that the total phenol content obtained by methanol extraction (252.4 mg/g dry weight) was significantly higher than that obtained by extraction with other solvents (3.9–96.8 mg/g dry weight), while the total phenol content extracted by the high-pressure water method was equivalent to that extracted by methanol (258.2 mg/g dry weight).

In atmospheric pressure solvent extraction, although methanol extraction has a high total phenol extraction rate, methanol reagents are toxic, require large amounts and are costly, and the solvent residue in the product is not easy to completely remove. For atmospheric pressure water extraction, although there is no solvent residue pollution, it is time-consuming, and because the water temperature is high, polyphenols are heat-sensitive ingredients, and their structure is easily destroyed, resulting in a lower total phenol extraction rate. If high-pressure water extraction is used, it can make up for the shortcomings of the above methods. Therefore, the use of high pressure or ultra-high pressure methods to extract pomegranate peel polyphenols has great advantages.

3 Conclusion

Pomegranate peel polyphenols are promising due to their various biological activities. Making full use of pomegranate resources to establish an efficient and inexpensive industrial production process for extracting pomegranate peel polyphenols will not only provide a material basis for developing new uses of pomegranate peel polyphenols, a natural active ingredient, in food, health products and chemical supplies, but also provide a viable way to reuse pomegranate peel resources, increase the added value of the pomegranate processing industry and promote the coordinated development of pomegranate cultivation and processing.

Traditional solvent extraction and enzymatic extraction methods have been well researched, but the high solvent consumption, high cost, long extraction time and low extraction rate have become bottlenecks hindering the development and utilization of pomegranate peel polyphenol resources in China. In recent years, microwave and ultrasonic assisted solvent extraction has been widely used to extract pomegranate peel polyphenols, which can save extraction time and improve the extraction rate. The latest methods, such as supercritical fluid extraction, ultra-high pressure extraction, and subcritical water extraction, have greater advantages than existing methods and have opened up new ways to industrially extract pomegranate polyphenols. However, these techniques are currently limited to laboratory research and are not yet suitable for large-scale industrial production. Therefore, in order to accelerate the comprehensive development and utilization of China's abundant pomegranate resources, it is extremely important to seek new extraction methods that are fast, efficient, and energy-saving, and to improve existing extraction processes that can be adapted to large-scale industrial production. 

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