Talekar et al., experimented with extraction with magnetic nanocatalyst of FeSO4 7H2O and FeCl3 6H2O solutions, which yielded 64.2–66.5 mg/g PU . It was lower than their previous research using cellulase . However, the nanocatalysts are recyclable and could reduce the cost of cellulase. Supercritical fluid extraction is another extraction method with increasing popularity. It utilizes supercritical fluid, such as high-pressure water and CO2, instead of chemical solvents that would cause drastic environmental impacts. . Therefore, it is deemed a green and clean method for compound-specific extraction. Bustamante et al. investigated one-passage supercritical CO2 extraction of PP. The optimal extraction condition was 400 bar, 40 °C, and 20% ethanol, obtaining 8.94 mg/g TPC as of ellagic acid equivalent with high antioxidant capacity and 9.5% PU retention . PU content increased with elevated pressure, due to higher fluid density and supercritical fluid solvent power, thus improving the dissolvability of polar compounds. However, the overall percentage of PU was low. Moreover, the scalability of supercritical CO2 extraction is impossible due to the lack of molecular models of solutes diffusion . A summary of the processing methods is shown in Table 1.1, including extraction conditions, total phenolic content, and punicalagin content. Tremendous health benefits of PU have been reported, such as anti-inflammation, anticancer, anti-oxidant, and cardiovascular disease prevention . Processing methods affect the composition of bio-active phytochemicals,gallon nursery pot therefore further are reflected by the functionality. A thorough understanding of the biodegradation of PU will help to improve the processing in return.

To be specific, this section will focus on the effect of PU on gut microbiota. Similar to other ETs with a high molecular weight, such as proanthocyanidins, PU was reported to have low absorption through the gastro-intestine tract . EA that hydrolyzed from PU before gastric digestion will be absorbed in the stomach. Then the remaining part of the PU will be degraded and absorbed in the alkaline intestinal environment . Gut microflora in the colon could also cleave the ester links from the PU to produce EA/GA. Only less than 1% of free EA will be absorbed and metabolized in the stomach and small intestine , while the majority of the EA will arrive in the jejunum and be further transformed to different products in an order of urolithin D , urolithin A , urolithin A and finally urolithin B . This metabolism relies on the sequential removal of hydroxyl groups by gut microbiota in the distal parts of the colon and will form urolithin aglycones . After absorption, UA will undergo an extensive phase II metabolism , which takes place within a large intestine wall and/or within the hepatocytes. The metabolites will further go through enterohepatic recirculation, enter the systemic circulation, and be excreted in urine . In return, ETs also modify the composition of microbiota since they are toxic to certain bacteria. Several mechanisms were proposed. For example, ETs were able to form a complex with proteins, carbohydrates, or metal ions including Fe and Cu. The complex of ETs and cell wall proteins decreases cell wall permeability, which will reduce the substrate transportation among cells. At the same time, Enzyme activity will also be inhibited, since the formation of ETs and enzymes alters the structural conformation . Additionally, the stable complex of ETs and ion decrease metal ion availability to bacteria, which subsequently adversely affects the metalloenzymes activity and selectively inhibits the growth of bacteria .

Besides direct inhibition of bacterial growth, the degradation of ETs can significantly lower the intestinal pH with the production of PA, which will further alter the bacterial population. Puupponen-Pimia et al. investigated the pH lowering ability of berries and their phenolic extracts, which could bring down the liquid media to pH 5 with the organic acids in the extract. Generally, lower pH is suitable for probiotic bacteria rather than pathogenic bacteria. For example, PUs, PCs, GA and EA were proven to significantly improve the growth of Bifidobacterium breve and Bifidobacterium infantis, while they also partially inhibited the growth of Bifidobacterium animalis lactis and Bifidobacterium bifidum. On the contrary, pathogens, such as S. aureus, Clostridium perfringens, Clostridium clostridioforme, Clostridium ramosum, and Bacteroides fragilis, were strongly inhibited with the presence of these polyphenols . Enhanced growth of Bifidobacterium spp. is associated with health benefits, including weight management under a high-fat diet and cardiovascular disease prevention . A summary of the effects of punicalagin on gut microbiota is shown in Table 1.2. Overall, the intact form of PU is desired to avoid significant biodegradation and antioxidant loss during the GI-tract digestion, which could further strengthen the gut microbiota and promote more health benefits. This section discussed the health benefits of punicalagin including modifying the gut microbiota composition and possessing strong antioxidant activity. Figure 1.6 summarizes the effect on punicalagins by processing methods and transformation in GI track and by gut microbiota. Proper novel processing methods, such as ultrasonic-assisted method, microwave-assisted method, enzymatic method, and supercritical fluid CO2, showed promising potential in improving PU yield with high biological activity, but the effects on PU quality need to be further quantified. In the future, research should focus on developing processing methods to recover punicalagins with high yield, purity, and biological activity, while staying low cost, low timeconsuming, and environmental-friendly.

Research has shown that pomegranate fruit might be beneficial for its antioxidant, antimutagenic, and anti-hypertension activities and its ability to reduce liver injury . It has also been studied for therapeutic purposes to alleviate ailments, such as colic, colitis-diarrhea, dysentery, leucorrhea, paralysis, and headache . Pomegranates are considered beneficial for curing chronic stomach ailments and are also known for their anti-inflammatory and anti-atherosclerotic activities against osteoarthritis, prostate cancer, heart disease, and HIV-I . High molecular weight polyphenols are the major high-value phytochemicals in pomegranate peel. They have demonstrated the likelihood of reducing risks of chronic diseases , including type-2 diabetes and cardiovascular diseases . Among all the polyphenols in pomegranate peel, gallic acid, ellagic acid, and punicalagin are most likely responsible for these health benefits . Punicalagin has also demonstrated significant in vitro antioxidant activities with abundant hydroxyl groups, which can trap peroxyl radicals to reduce oxidation. Therefore, it is of great interest to extract punicalagin, ellagic acid, and gallic acid for nutrient fortification and other applications. Extraction is a mass transfer process influenced by the matrix properties of the plant part as well as the solvent, temperature, and time . The particle size and solvent type play an essential role in extraction. Particle size reduction results in higher mass transfer efficiency and product yield,greenhouse ABS snap clamp as smaller particles have a higher surface-to-volume ratio and less internal path . The solvent type impacts extraction through different polarities and affinity to the compounds of interest . However, a better understanding is needed of the effects of extraction conditions on antioxidant yield and antioxidant activity. Most previous research on the extraction of bio-active compounds used organic solvents to improve the extraction rate. However, inappropriate use or recycling of organic solvents in food applications can cause pollution and raise safety concerns. Green extraction of natural products, focusing on modified extraction processes with reduced energy consumption, alternative solvents, and renewable natural products, is in demand . Water is a universal solvent that is generally recognized as safe . Therefore, deionized water was applied for extraction in this study. Nearly all extraction studies of plant-based bio-active compounds have used dried material for extended research time . However, drying increases energy and time consumption, in addition to the excessive preparation requirements. Therefore, a novel extraction approach using fresh wet pomegranate peel was studied in this research. Pomegranate pomace of Wonderful variety was collected from SunnyGem LLC . The wet pomace was stored at -18°C and thawed to room temperature before use. The chemicals used in the experiments, including Folin-Ciocalteu reagent, analytical standards of tannic, gallic, and ellagic acids, and 2,2-diphenyl-1-picrylhydrazyl , were purchased from Sigma-Aldrich . Methanol, HPLC-grade o-phosphoric acid , and analytical-grade sodium hydroxide and sodium carbonate were obtained from Fisher Scientific . A mixture of punicalagin and punicalagin was purchased from ChromaDex Co. . Pomegranate juice had been extracted by carving the peels and separating the peels from the arils. Therefore, the collected pomace contained about 96% peels and 4% seeds. Because the percentage of seeds in the pomace was low, further separation of seeds was not performed. The WPP was sliced into small pieces of less than 5 mm using a continuous-feed food processor . The sliced peel pieces were then ground into two groups of fine particles in a Comitrol processor using 0.024 and 0.012 in. cutting heads. The moisture contents of the two groups were determined by drying about 10 g of particles at 105°C in a hotair oven until a constant weight was obtained . To compare the polyphenol extraction yield and antioxidant activity of polyphenols produced from WPP and DPP, the WPP samples were dried using infrared radiation and hot air .

IR drying was performed by heating a single layer of peels at a surface temperature of 60°C, and HA drying was performed using hot air at 40°C until the moisture content was less than 10% . DPP samples produced by IR and HA drying were milled in a sample mill to less than 0.38 mm size for polyphenol extraction. A demonstration of the extraction process and the corresponding equipment was shown in Figure 2.1.Experiments were conducted using WPP particles of both sizes to determine the effects of single factors, including particle size, solvent ratio, extraction temperature, and extraction time, on the extraction of polyphenols. To study the effect of extraction time, 5 g of WPP was mixed with 40 mL of DI water and extracted at 20°C for 2, 3, 4, 5, and 6 min with a stirring speed of 1200 rpm. The effect of solvent ratio was investigated by extracting 5 g of WPP powder with 5, 10, 20, 30, and 40 mL of DI water for 6 min at 20°C. The effect of temperature was tested by adding 5 g of WPP powder to 40 mL of DI water and extracting for 6 min at temperatures of 20°C, 30°C, 40°C, 50°C, and 60°C. All extractions were conducted in triplicate using a Corning hot plate stirrer properly shielded from light to avoid light induced quality loss. After extraction, the extract mixture was separated by centrifugation at 4400´g at 4°C for 15 min . The liquid extract was collected for the determination of physicochemical qualities.The effects of the ratio of solvent to peel particles on the TEY, TPY, TPC, and DSA of the extract are shown in Figure 2.5. When the solvent ratio increased from 1:1 to 4:1, the TEY, TPY, and TPC significantly increased from 27.53% to 53.20%, from 4.42% to 10.53%, and from 16.06% to 19.53%, respectively. SPP achieved significantly higher TEY, TPY, and phenolic content than LPP, showing that the finer cell structure facilitated mass transfer consistent with Fick’s law . Laroze et al. studied the extraction kinetics of polyphenols from raspberry pomace. They observed that small particles resulted in much higher extraction efficiency compared to large particles with methanol extraction at a 20:1 solvent ratio. The reason could be that smaller particles allowed the solvent to access solutes with less resistance to mass transfer. Smaller particles were also related to more cell breakage, which promoted the release of phytochemicals. Increasing the solvent ratio to greater than 20:1 resulted in a slightly increased TEY, but it was not significantly different, indicating that equilibrium was reached. A possible reason could be that a higher concentration gradient was built up with the increasing solvent ratio, which increased diffusion from the internal structure, thus increasing the extraction rate. Similar findings were observed for the extraction of phenolics from date seeds and dried sage . The results demonstrated that a higher solvent ratio increased the antioxidant yield and content, with a limited effect on the DSA. The DSA was not significantly different regardless of the solvent ratio and peel particle size at this phase. A higher solvent ratio required more water consumption in the extraction and higher energy use for the concentration of the extract. In conclusion, a solvent ratio of 4:1, which resulted in relatively high TEY and TPY, was considered optimal in terms of solvent usage. Therefore, a solvent ratio at 4:1 is recommended for industrial applications.