A quantity of 20 mg of A. arguta pectin (m₂) in a beaker was weighed; 50 ml of pure water was added to dissolve; the solution was centrifuged for 30 min; and the supernatant was taken, placed in a beaker with a known constant weight, and dried at 110°C to a constant weight (m₁). The solubility of pectin was calculated according to the formula (2) (38).

A 50 ml centrifuge tube was weighed as M₁; then the dry powder sample of 0.5 g (M₂) was weighed accurately and placed in the 50 ml centrifuge tube; 50 ml ultra-pure water was added, shaken well, centrifuged at a speed of 3,500 r/min for 15 min; the supernatant of the centrifuge liquid was discarded; and the total weight of the centrifuge tube containing sediment M₃ was weighed. Then the sediment weight was calculated as (M₃-M₁). The water-holding capacity of pectin was calculated according to the formula (3) as follows (39).

The pectin solution of A. arguta was prepared with mass concentrations of 0.5, 1, 2, 5, and 10 mg/ml, respectively. Using Vc as a positive control, it was also configured according to the above concentration gradient, and then a certain volume of DPPH ethanol solution was added to the test tube under the conditions of room temperature and dark light, respectively. After the solution was mixed, the absorbance of the sample solution at the wavelength of 517 nm was measured by an ultraviolet spectrophotometer. Three parallel tests were conducted with anhydrous ethanol as the reference. The formula (4) is as follows.

where A₁ is the absorbance of 2 ml of kiwifruit pectin solution and 2 ml of DPPH. A₂ is the absorbance of 2 ml of A. arguta pectin solution and 2 ml of anhydrous ethanol. A₀ is the absorbance of 2 ml of dH₂O+ 2 ml DPPH (40, 41).

The pectin solution of A. arguta was prepared with mass concentrations of 0.5, 1, 2, 5, and 10 mg/ml, respectively. Using Vc as a positive control, it was also configured according to the above concentration gradient, and then the same volume of 6 mmol/L FeSO₄ solution, 6 mmol/L H₂O₂ solution, and different mass concentrations of A. arguta pectin solution or Vc solution were mixed in the test tube. Then, the same volume of 6 mmol/L salicylic acid-ethanol solution was added to the water bath at 37°C for 1 h. After taking it out and cooling it to room temperature, the absorbance of the sample solution at the wavelength of 510 nm was measured by an ultraviolet spectrophotometer. Three parallel tests were carried out with distilled water as the reference. The formula (5) is as follows (40, 41).

where A₁ is the absorbance value of 2 ml FeSO₄ solution + 2 ml H₂O₂ solution + 2 ml salicylic acid solution + 2 ml sample solution; A₂ is the absorbance value of 2 ml FeSO₄ solution + 2 ml dH₂O+2 ml salicylic acid solution + 2 ml sample solution. A₀ is the absorbance value of 2 ml FeSO₄ solution + 2 ml H₂O₂ solution + 2 ml salicylic acid solution + 2 ml dH₂O.

The pectin solution of A. arguta was prepared with mass concentrations of 0.5, 1, 2, 5, and 10 mg/ml, respectively. Using Vc as a positive control, it was also configured according to the above concentration gradient. ABTS+ mother liquor was prepared from 200 mg of accurately weighed ABTS and 34.4 mg of potassium persulfate dissolved in 50 ml of distilled water and placed away from light at room temperature for 24 h. The absorbance was measured to (0.70 ± 0.02) at 734 nm by diluting the mother liquor with 95% ethanol. A volume of 0.4 ml of sample solution was added to 3.6 ml of diluted ABTS solution and allowed to react at 30°C for 5 min. Then, the absorbance A1 is determined at 734 nm, and the absorbance A0 is determined with distilled water instead of the sample solution. The calculation formula (6) is as follows (40, 41).

The pectin solution of A. arguta was prepared with mass concentrations of 0.5, 1, 2, 5, and 10 mg/ml, respectively. Using Vc as a positive control, it was also configured according to the above concentration gradient. A volume of 1.0 ml of pectin solution of different concentrations was taken; 2.5 ml of K₃[Fe (CN)₆] solution (1%, w/v) and 2.5 ml of phosphoric acid buffer (pH = 6.8) were added to the test tube, mixed well, and immersed in water at 50°C for 30 min; and 2.5 ml of TCA solution (10%, w/v) was added after cooling. After 2.5 ml of supernatant was centrifuged, 2.5 ml of phosphoric acid buffer and 0.5 ml of FeCl₃ solution (1%, w/v) were added for 10 min. Abs was determined at 700 nm with Vc as the control (40, 41).

Using galacturonic acid as the standard substance to create a standard curve, the linear regression equation was obtained as y = 0.0131x+0.024, R² = 0.9975, where y is the absorbance and x is the mass concentration (μG/ml). Based on the equation, the yield of the UAP is 23.98 ± 0.21%, and the yield of the SAP is 18.21 ± 0.19%. The result suggested the contribution of ultrasound-assisted treatments to pectin extraction. This is because ultrasonic waves are good at breaking down the cell walls and releasing pectin. So, the ultrasonic-assisted extraction method can improve the extraction yield of the pectin.

The composition of pectin is shown in Table 1. The UAP and the SAP all comply with the National Quality Standards of pectin in the People's Republic of China, GB 25533-2010. Compared with the SAP (71.64 ± 0.33%), the total sugar content of the UAP (78.23 ± 0.98%) is higher, which suggests the cavity, mechanical crushing, and thermolysis effects of ultrasound may destroy the cell walls of plants, causing the intracellular polysaccharides to dissolve out. The essence of pectin is a linear structure of polysaccharides, mainly composed of D-galactonic acid (2). Its galacturonic acid residues are often esterified by some groups such as methoxyl and amide. The degree of esterification is also called methoxylation, which refers to the sum of the proportions of methylesterification, acetylation, and amidation in pectin (3). Although the esterification degree of UAP (66.6%) is lower than the SAP (75%), indicating that the cavity effect of ultrasound may have broken the ester bond, resulting in the esterification degree of pectin decreasing the UAP and the SAP all belong to the high ester pectin (≥50%) (7). The change in esterification degree can affect the application field of pectin. For example, in yogurt products, high esterification degree pectin can stabilize the structure of yogurt, while adding low esterification degree pectin can prevent whey precipitation (24). The ultrasonic-assisted preparation of pectin will not affect its properties or application areas as a food additive.

The monosaccharide composition of the UAP and SAP was determined by HPLC. A. arguta pectin is a heteropolysaccharide composed of multiple monosaccharides. As shown in Table 2, the monosaccharides and their molar ratios in UAP are Man:GlcA:Rha:GalA:Glc:Gal:Xyl:Ara:Fuc = 8.3:7.9:1.6:30:24.8:12.2:1.8:11.9:1.4. The monosaccharides and their molar ratios in SAP are Man: GlcA:Rha:GalA:Glc:Gal:Xyl:Ara:Fuc = 6.4:4.9:1.4:25.2:40.1:11.3:7.6:1.9. The results indicate that UAP will not change the type of monosaccharides, but the compositions of monosaccharides extracted from pectin by the two different extraction methods are different. The contents of GalA in the two pectins were relatively high, proving that they have better antioxidant activity (42). The content of GalA in UAp is higher than that of SAP; therefore, we predict that the antioxidant activity of UAP is stronger than that of SAP, which will be confirmed in subsequent antioxidant experiments. Pectin can be divided into four configurations, namely, homoglycan (HG), rhamnoglucuronic glycan I (RG-I), rhamnoglucuronic glycan II (RG-II), and xyloglucuronic glycan (XG) (43). In the RG-I type structure of pectin, the disaccharide unit is mainly composed of alternating rhamnose and galacturonic acid, which forms the main chain through continuous repetition; HG type and RG-II type pectin are polygalacturonic acid polymers, whose main chains are composed of α-1,4-glycosidic bond binding. The O-3 or O-4 position of the galacturonic acid residue in HG is replaced by xylose, forming XG. Visser et al. proposed a method for determining the structure of pectin molecules, where the structure is mainly RG-I type when the Rha/Gal ratio of pectin molecules is between 0.05 and 1.00; when the ratio is below 0.05, its structure is mainly HG type and RG-II type (44). The results showed that the Rha/GalA ratio of UAP was 0.053 and the one of SAP was 0.055, ranging from 0.05 to 1.00. However, the proportion is close to the lower limit, inferring that the molecular structure of A. arguta pectin belongs to a mixed structure of RG-I and HG-type domain. (Ara+GalA)/Rha reflects the ratio of RG-I branches to Rha, with a higher ratio indicating more RG-I branches. The (Ara+GalA)/Rha ratio of UAP is 15.06, and the one of SAP is 13.5, indicating that the UAP has more branched chains of RG-I than the SAP (45).

The molecular weight of pectin plays an important role in the biological activity and use of pectin (34). The standard curve equation for molecular weight is y = −0.3272+1.7945, R² = 0.9916. Figure 1 shows the molecular weight distribution of UAP and SAP. By analyzing the molecular weight distribution, it was found that the overall distribution of pectin molecular weight in UAP and SAP has changed. The overall molecular weight distribution range of UAP is relatively concentrated, mainly distributed at 422 kDa, as shown in Figure 1A. The molecular weight distribution of SAP is relatively dispersed, with 2,654, 921, and 84 kDa, as shown in Figure 1B. This indicates that the molecular weight distribution of pectin varies with different extraction methods, which may be due to the ultrasonic pretreatment cutting off the sugar or ester chains of long-chain pectin, reducing the distribution of pectin's large molecular weight and lowering the degree of esterification, which is consistent with the results of the previous esterification degree experiment. The change in molecular weight distribution may cause changes in some physical properties of pectin (1), such as solubility and viscosity, which will be verified in the following tests.

FTIR spectrum scanning scans the moisture and CO₂ in the air as blanks and subtracts the atmospheric background from the spectrum of the sample. It can be seen from Figure 2 that pectin has a characteristic absorption peak of sugars within 500–4,000 cm⁻¹. Among them, the broad absorption peak near 3,000–3,600 cm⁻¹ is caused by the O-H stretching vibration on the pectin carbon ring (46). The peak between 2,795 and 2,950 cm⁻¹ is caused by the C-H stretching vibration on methyl galacturonate or the carbon ring. The absorption peaks at 1,635 and 1,746 cm⁻¹ are caused by carboxylate (-COO) asymmetric stretching and the C=O stretching vibration formed by carboxylate (-COOR). The peak value of UAP at the position of the carboxylic acid ester is significantly lower than the peak value of SAP, indicating that some carboxylic acid ester bonds of UAP are destroyed, so the degree of esterification is reflected, and the degree of esterification decreases. The absorption peak near 1,400 cm⁻¹ is caused by the bending vibration absorption of the saturated C-H bond and the absorption peak between 1,300 and 1,000 cm⁻¹ is caused by the C-O stretching vibration (1). These absorption peaks are all characteristic absorption peaks (47) unique to polysaccharide compounds, and the functional groups of pectin were not substantially changed by ultrasonic treatment.

Figure 3A shows the microstructure of UAP at 500 × , 1,000 × , and 2,000 × magnification. As shown in the figure, the surface of pectin is rough, with wrinkles and many pores. The NAP is shown in Figure 3B, which has a relatively flat and smooth surface with a sheet-like structure and small scales stacked on the surface. This is similar to the results determined by Xu et al. (46). The surface of pectin extracted by ultrasonic methods has undergone significant changes after ultrasonic treatment, resulting in porous tissue and the appearance of pores. This may be related to the changes in molecular weight distribution caused by UAP, or it may also be due to the destruction of the crosslinking between pectin molecules and the recombination of the intermolecular network structure of pectin. The surface structure of pectin prepared with ultrasonic assistance has changed, which may lead to changes in some physical properties and biological activities of pectin, which are also reflected in the following test results.

As shown in Table 3, the UAP has better water retention, viscosity, and solubility than SAP. This may be due to changes in the molecular weight distribution of pectin treated with ultrasonic waves and wrinkles in its surface structure and pores, which improve pectin's physical properties such as water-holding capacity and solubility (48). This is consistent with the previous molecular weight distribution and electron microscope observation. The molecular weight and structure of the branching of pectin have important effects on its viscosity. The hydrogen bonds and hydrophobic methyl groups contained in pectin can reduce the intramolecular or intermolecular distance and increase the branching of the pectin structure, thus improving its viscosity (33). As above, ultrasonic-assisted preparation of pectin can improve the physical properties of pectin, which will improve its future application value.

Although ultrasonic treatment can improve the yield of pectin, pectin is a class of bioactive macromolecules. From previous experimental results, it can be seen that ultrasonic treatment can affect the esterification degree, molecular weight distribution, and physical properties of pectin. Whether this change will affect its antioxidant properties is related to its future development and application as an antioxidant food additive. Therefore, we analyzed its antioxidant properties.

DPPH is a relatively stable free radical with a characteristic absorption peak at 517 nm wavelength (33), which is widely used to evaluate the free radical scavenging ability of antioxidant components. DPPH radical scavengers can provide hydrogen and form stable non-free radical molecules (40). Studies have shown that pectin has free radical scavenging ability, possibly due to its hydrogen donor substance, which can react with free radicals to generate more stable products and terminate free radical chain reactions (33). It can be seen from Figure 4A that in the mass concentration range of 0.5–10 mg/ml, as the mass concentration increases, the pectin shows strong DPPH-free radical scavenging ability. At the lowest concentration of 0.5 mg/ml, the DPPH-free radical scavenging ability of SAP is worse than that of UAP, and it basically remains the same as the concentration increases. When the mass concentration is 10 mg/ml, the ability of SAP is worse than that of UAP. The SAP has a stronger DPPH-free radical scavenging ability than the UAP, and the ability of Vc to scavenge hydroxyl-free radicals is always maintained at a very high level. The scavenging rate of the pectin reaches more than 50% of the scavenging rate of Vc, indicating that the A. arguta pectin has a strong ability to scavenge DPPH-free radicals.

The scavenging ability of OH radicals is one of the important indexes of antioxidant activity. Hydroxyl radicals can easily pass through the cell membrane, enter the cell, and react further with biological macromolecules such as carbohydrates, resulting in cell death or damage. Scavenging hydroxyl-free radicals is of great significance to organisms (41). It can be seen from Figure 4B that in the mass concentration range of 0.5–10 mg/ml, as the mass concentration increases, the ability of the pectin to scavenge hydroxyl radicals increases; when the pectin mass concentration increases from 0.5 to 10 mg/ml, the hydroxyl radical scavenging rate of UAP was significantly higher than that of SAP, indicating that ultrasonic treatment will affect the ability of the A. arguta pectin to scavenge hydroxyl-free radicals, which may be related to its high content of polysaccharide and galacturonic acid. The ability of Vc to scavenge hydroxyl-free radicals has always been maintained at a very high level.

ABTS+·scavenging capacity evaluation of antioxidant capacity is based on the fact that antioxidants can provide electrons or hydrogen atoms to inactivate free radicals, resulting in the change of color of ABTS+·solution (33), which is then analyzed and measured by a spectrophotometer (40). It can be seen from Figure 4C that within the mass concentration range of 0.5–10 mg/ml, as the mass concentration increases, the ability of A. arguta pectin to remove ABTS increases, and its ability to remove ABTS increases steadily as the concentration increases. At low concentrations, the scavenging ability of UAP is better than that of NAP, and the concentration exceeds 2 mg/ml. The difference between the UAP and SAP is not significant. The ability of Vc to scavenge hydroxyl radicals is always maintained at a very high level. When the mass concentration is 10 mg/ml, the scavenging rate of A. arguta pectin reaches more than 50% of the scavenging rate of Vc, indicating that A. arguta pectin has strong ABTS-free radical scavenging ability.

The reducing capacity of pectin is closely related to its potential antioxidant activity. Providing hydrogen atoms can break free radical chains and prevent the formation of hydrogen peroxide (33). The higher the absorbance value, the larger the sample's capacity for decrease. The reducing power of both kinds of pectin increased significantly with the increase in concentration. It can be seen from Figure 4D that in the mass concentration range of 0.5–10 mg/ml, as the mass concentration increases, the reducing power of the pectin gradually increases; when the pectin mass concentration increases from 0.5 to 10 mg/ml, the reducing ability of UAP is better than that of SAP, indicating that ultrasonic treatment improves the reducing ability of pectin; when the mass concentration is 10 mg/ml, the UAP reaches 50% of the Vc reducing ability. It shows that the UAP has the strong reducing ability. Generally speaking, the antioxidant activity of pectin is related to its structural characteristics, molecular weight, and monosaccharide composition. It has been reported that the electrophilic groups of pectin contribute to the release of H from O-H bonds, which can improve oxidation resistance. In this study, the high reducing power observed in UAP may be attributed in part to its relatively low molecular weight and high content of unmethylgalacturonic acid (43).