Analysis of Phenolic Acids and Flavonoids in Rabbiteye Blueberry Leaves by UPLC-MS/MS and Preparation of Nanoemulsions and Extracts for Improving Antiaging Effects in Mice

Rabbiteye blueberry leaves, a waste produced after harvest of blueberry, are rich in polyphenols. This study aims to analyze phenolic acids and flavonoids in blueberry leaves by UPLC-MS/MS and prepare nanoemulsions for determining anti-aging activity in mice. Overall, 30% ethanol was the most suitable extraction solvent for total phenolic acids and total flavonoids. A total of four phenolic acids and four flavonoids were separated within seven minutes for further identification and quantitation by UPLC-MS/MS in selective reaction monitoring (SRM) mode, with 3-O-caffeoylquinic acid being present in the highest amount (6474.2 μg/g), followed by quercetin-3-O-galactoside (1943.9 μg/g), quercetin-3-O-rutinoside (1036.6 μg/g), quercetin-3-O-glucoside (867.2 μg/g), 5-O-caffeoylquinic acid (815.8 μg/g), kaempferol-3-O-glucoside (309.7 μg/g), 3,5-dicaffeoylquinic acid (195.3 μg/g), and 4,5-dicaffeoylquinic acid (60.8 μg/g). The blueberry nanoemulsion was prepared by using an appropriate ratio of soybean oil, Tween 80, glycerol, ethanol, and water at 1.2%, 8%, 2%, 2%, and 86.8%, respectively, and mixing with dried blueberry extract, with the mean particle size and zeta potential being 16 nm and −54 mV, respectively. A high stability was observed during storage of nanoemulsion for 90 days at 4 °C and heated at 100 °C for 2 h. An animal study revealed that this nanoemulsion could elevate dopamine content in mice brain as well as superoxide dismutase, glutathione peroxidase, and catalase activities in mice liver while reducing the contents of malondialdehyde and protein carbonyl in mice brains. Collectively, the high-dose nanoemulsion possessed the highest efficiency in improving mice aging with a promising potential for development into a health food.


Introduction
Rabbiteye blueberry (Vaccinium virgatum), an evergreen shrub of the Ericaceae plant group rich in phenolic compounds, is known to possess a delicate flavor and high nutritive value. In addition, many reports have shown that blueberry has several important biological functions such as anti-inflammation [1], anti-obesity [2], anti-diabetes [3], anti-tumor [4], and protection of cardiovascular disease [5], all of which can be due to the presence of bioactive compounds, including anthocyanins, flavonoids, and phenolic acids. Compared to blueberry fruits, blueberry leaves were reported to contain higher amounts of phenolic compounds. For instance, Li et al. [6] showed that the total phenolic content (TPC) and flavonoid content (TFC) were higher in the ethanolic extracts of blueberry leaves (339.10 mg gallic acid equivalent (GAE)/g and 198.10 mg quercetin equivalent (QE)/g dry weight) than in blueberry fruits (216.43 mg GAE/g and 115.55 mg QE/g dry weight). Likewise, more recently, Debnath-Canning et al. [7] reported that phenolic compounds were present
Solvents such as acetonitrile and ethanol were from Merck Co. (Darmstadt, Germany) and Sigma-Aldrich Co., respectively. Yu Pa Co. (Taipei, Taiwan) provided both Tween 80 and glycerol used for the nanoemulsion preparation, while Sigma-Aldrich supplied both D-galactose and potassium dihydrogen phosphate. Milli-Q water purification system supplied the deionized water for all dilution purposes (Millipore Co., Bedford, MA, USA).
The assay kits, including superoxide dismutase, TBARs, glutathione peroxidase, protein carbonyl, and catalase, were from Cayman Chemicals Co. (Ann Arbor, MI, USA). The ELISA kit for determining mouse dopamine was from CUSABIO Co. (Houston, TX, USA). A total of 36 6-week-old C57BL/6 male mice was obtained from National Experimental Animal Center (Taipei, Taiwan). After pre-feeding for two weeks, a 12-week animal experiment was conducted.

Determination of Total Phenolic Content (TPC) and Total Flavonoid Content (TFC)
Initially, for comparison of extraction efficiency, 0.5 g of rabbiteye blueberry leaf powder was mixed with 30 mL of 30%, 50%, or 70% ethanol (n = 3), followed by shaking each mixture in a 60 • C water bath for 3 h, centrifuging at 1789× g for 30 min (25 • C), collecting the supernatant, filtering with a 0.6-µm filter paper, evaporating to dryness in a N-1200A model Eyela rotary evaporator (Sunway Scientific Corporation, Taipei, Taiwan) at 40 • C, dissolving in 10 mL of deionized water, and filtering with a 0.22-µm polyvinylidene fluoride (PVDF) membrane filter for the analysis of TPC and TFC.
A method based on Kao et al. [25] was used to determine TPC in rabbiteye blueberry leaves. In brief, 200 µL of Folin-Ciocalteu reagent was added separately to five different concentrations of gallic acid standard (50-440 µg/mL). After mixing for homogeneity, the mixture was left in dark for 5 min, and 1 mL of sodium carbonate (15%) was added and allowed to react at 25 • C for 1 h for measuring the absorbance at 750 nm using a VersaMax ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA). Next, a 50 mL of sample extract was collected, and the absorbance was measured at 750 nm for determination of TPC based on the gallic acid standard curve.
Similarly, a method based on Kao et al. [25] was used to determine TFC in rabbiteye blueberry leaves. Initially, 30 µL of 5% sodium nitrite solution was mixed separately with 200 µL of 6 concentrations of quercetin standard (25-400 µg/mL) and was allowed to stand for 5 min. Following that, 60 µL of aluminum chloride (10%) was added and, after 5 min, 300 µL of sodium hydroxide (1 M) and 200 µL of chloroform were added, followed by centrifuging and collecting the supernatant for measuring absorbance at 510 nm in a VersaMax ELISA microplate reader. By using the linear regression equation of quercetin Foods 2023, 12,1942 4 of 21 standard curve, the TFC in rabbiteye blueberry leaves was determined by measuring the absorbance of sample extract (200 µL) at 510 nm.

Analysis of Individual Phenolic Acid and Flavonoid by UPLC-MS/MS
Following the comparison of extraction efficiency of various ethanol proportions, 30% ethanol was chosen for extraction and for subsequent UPLC-MS/MS analysis. In brief, 0.5 g sample was mixed with 30 mL of 30% ethanol and shaken in a 60 • C water bath for 3 h, after which the mixture was centrifuged at 1789× g (25 • C) for 30 min and the supernatant was collected for filtering through a filter paper, followed by evaporating to dryness in an Eyela rotary evaporator at 40 • C and dissolving in 10 mL deionized water. One mL was collected, evaporated to dryness, dissolved in 1 mL of 30% ethanol, and the internal standard EGCG was added (10 µg/mL). After filtration through a 0.22-µm PVDF membrane filter, a 5 µL sample was injected for UPLC-MS/MS analysis.
A method based on Chen et al. [26] was modified and used for the determination of individual phenolic acid and flavonoid compounds in rabbiteye blueberry leaf powder. By using a Waters Acquity UPLC BEH C18 column (100 × 2.1 mm ID, particle size 1.7 µm) coupled with a triple quadrupole tandem mass spectrometer (QqQ) (Dionex Ultimate 3000 Open Sampler XRS System (Thermo Fisher Scientific Co., San Jose, CA, USA) in electrospray ionization mode, a total of four phenolic acids and four flavonoids were separated within 7 min with flow rate at 0.3 mL/min, column temperature at 30 • C, and the following gradient mobile phase: 0.1% formic acid solution (A) and 100% acetonitrile (B) with 100% A and 0% B in the beginning, changed to 20% B at 4 min, 30% B at 9 min, and 100% B at 14 min.
For identification by UPLC-MS/MS, the selected reaction monitoring (SRM) with negative ion mode was used with ion transfer tube temperature, vaporizer temperature, sweep gas flow rate, sheath gas flow rate, auxiliary gas flow rate, and spray voltage at 329 • C, 279 • C, 0 arbitrary units (a.u.), 38 a.u., 12 a.u., and 3000 V, respectively. Following that, the various phenolic acids and flavonoids in rabbiteye blueberry leaves were identified by comparison of retention time, precursor ion, and product ion of unknown peaks with that of reference standards.
For the determination of limit of detection (LOD) and limit of quantitation (LOQ), a total of 13 concentrations of individual phenolic acid and flavonoid standards (0.0001-0.1 µg/mL) were prepared separately. After the injection of each concentration into UPLC-MS/MS three times, the standard curve was obtained by plotting concentration against peak area. Both slope (S) and the standard deviation (δ) were obtained to calculate LOD and LOQ by using the following formula: The intra-day variability (repeatability) was obtained by analyzing individual phenolic acid and flavonoid in rabbiteye blueberry leaves with the internal standard EGCG (10 µg/mL) three times a day with each time analyzed in triplicate for a total of nine analyses. Likewise, the inter-day variability (intermediate precision) was determined using the same approach as the intra-day variability, with the exception that blueberry leaves were analyzed on three consecutive days with triplicate analyses each day for a total of nine analyses.   (17.5 and 175 µg), were added to blueberry leaves separately for extraction and quantitation by UPLC-MS/MS. The recovery was calculated by using the following formula: Recovery (%) = amount found after UPLC − original amount before UPLC amount spiked before UPLC × 100

Quantitation of Individual Phenolic Acid and Flavonoid
A method based on Huang and Chen [27] was modified and used for the quantitation of individual phenolic acid and flavonoid compounds in rabbiteye blueberry leaf powder. A total of six concentrations (0.01, 0.1, 0.5, 1, 5, and 10 µg/mL) were each prepared for each phenolic acid and flavonoid standard and were then mixed with EGCG at 10 µg/mL. After injection into UPLC-MS/MS, the standard curve of individual phenolic acid and flavonoid was prepared by plotting concentration ratio (standard versus internal standard) against area ratio (standard versus internal standard) to obtain both linear regression equations and R 2 . The individual phenolic acid and flavonoid content in rabbiteye blueberry leaves was determined using the formula shown below: where A s : peak area of phenolic acid or flavonoid; A i : peak area of internal standard (EGCG); a: slope of the standard curve; b: intercept of the standard curve; C i : concentration of internal standard (µg/mL); V: final volume of sample extract (mL); DF: dilution factor; W s : weight of the sample (g).

Preparation of Rabbiteye Blueberry Leaf Nanoemulsion
Initially, 0.5 g of rabbiteye blueberry leaf powder from NTU 104 (fresh leaves) with the highest phenolic acid and flavonoid contents was extracted with 30% ethanol by adopting the same procedure described under Section 2.3. A method based on Chen et al. [26] was used for the preparation of nanoemulsion with slight modification. 10 mL of rabbiteye blueberry leaf sample extract containing four phenolic acids at 5 mg/mL was prepared and evaporated to dryness under nitrogen, followed by adding 0.12 g (1.2%) of soybean oil, 0.2 g (2%) of 30% ethanol, 0.8 g (8%) of Tween 80, and 0.2 g (2%) of glycerol with intermittent stirring after each ingredient addition, and finally 8.68 g (86.8%) of deionized water was added, stirred thoroughly, and was sonicated for 30 min for successful preparation of a transparent nanoemulsion.
2.6. Determination of Nanoemulsion Characteristics 2.6.1. Particle Size and Polydispersity Index A 100 µL sample of nanoemulsion was diluted 50-fold with potassium dihydrogen phosphate (pH 5.3-5.5), followed by placing in a styrene tube for measurement of mean particle size and polydispersity index (PDI) by a dynamic light scattering instrument (90 plus particle size analyzer) from Brookhaven Instruments (Holtsville, NY, USA) [28].

Zeta Potential
The zeta potential was determined by 200-fold dilution of 100-µL nanoemulsion sample with deionized water and measurement of zeta potential by a SZ-100 Horiba analyzer (Kyoto, Japan) [27].

Transmission Electron Microscopy
After diluting 100 µL of nanoemulsion sample with deionized water, 20 µL was placed on a copper grid, allowed to stand for 90 s, and the surplus sample was removed by a filter paper. For negative staining, uranyl acetate was added, allowed to stand for 2 min, and the surplus uranyl acetae was removed using a filter paper and was then dried in a desiccator

Encapsulation Efficiency
For determination of encapsulation efficiency, 100 µL of nanoemulsion sample was diluted 10 times with deionized water. The sample was then poured into a centrifuge tube equipped with a 3 kDa dialysis membrane, followed by centrifuging at 13,800× g (25 • C) for 20 min, collecting the bottom layer for determination of four phenolic acids by UPLC-MS/MS. The encapsulation efficiency of four phenolic acids was calculated using the following formula [26]: Encapsulation efficiency = total amount of 4 phenolic acids − amount of 4 free phenolic acids total amount of 4 phenolic acids × 100

Stability of Nanoemulsion
The stability of the nanoemulsion was determined by storing 10 mL of sample at 4 • C for 90 days and a portion was collected at regular intervals for determination of mean particle size, PDI and zeta potential. For heating stability, 200 µL of nanoemulsion sample was placed in a water bath with temperature controlled at 40, 60, 80, or 100 • C, as well as heating time length at 0.5, 1.0, 1.5, or 2 h. At the end of each time interval, the sample was collected for the determination of mean particle size, PDI, and zeta potential [27].

Animal Experiment
A total of 36 6-week-old C57BL/6 male mice were raised in Fu Jen University Experimental Animal Center with temperature at 21 ± 2 • C, relative humidity at 55 ± 10%, and light circle for 12 h. Both animal handling and experimental procedures were based on the guidelines set by Fu Jen Catholic University Experimental Animal Care and Use Committee (approval number A 10901).
Both diet and water were provided ad libitum. After a two-week adaptation period, all the 8-week-old mice with a body weight of about 27 g each were subjected to a 12-week animal experiment by dividing into six groups with six mice each:

1.
Control group (C): mice were provided with sterilized water by subcutaneous injection once a day for the first 6 weeks and then tube feeding with sterilized water, starting at the 7th week once a day for another 6 weeks.

2.
Induction group (D): mice were provided with D-galactose at a dose of 300 mg/kg bw by subcutaneous injection once a day for the first 6 weeks and then tube feeding with sterilized water starting at the 7th week once a day for another 6 weeks.

3.
Low-dose extract (LE): mice were provided with D-galactose at a dose of 300 mg/kg bw by subcutaneous injection once a day for the first 6 weeks and then tube feeding with blueberry extract (dissolved in 0.2 mL of deionized water) at a dose of 8 mg/kg bw containing 0.2 mg of four phenolic acids starting at the 7th week once a day for another 6 weeks.

4.
High-dose extract (HE): mice were provided with D-galactose at a dose of 300 mg/kg bw by subcutaneous injection once a day for the first 6 weeks and then tube feeding with blueberry extract (dissolved in 0.2 mL of deionized water) at a dose of 40 mg/kg bw containing 1 mg of four phenolic acids starting at the 7th week once a day for another 6 weeks.

5.
Low-dose nanoemulsion (LN): mice were provided with D-galactose at a dose of 300 mg/kg bw by subcutaneous injection once a day for the first 6 weeks and then tube feeding with 0.2 mL of nanoemulsion at a dose of 8 mg/kg bw containing 0.2 mg of four phenolic acids starting at the 7th week once a day for another 6 weeks. 6.
High-dose nanoemulsion (HN): mice were provided with D-galactose at a dose of 300 mg/kg bw by subcutaneous injection once a day for the first 6 weeks and then tube feeding with 0.2 mL of nanoemulsion at a dose of 40 mg/kg bw containing 1 mg of four phenolic acids starting at the 7th week once a day for another 6 weeks.
After feeding for 12 weeks, all the mice were euthanized with carbon dioxide, and both brain and liver were collected for weight measurement and then stored at −80 • C for subsequent analyses. By adopting the instructions provided by the assay kits from Cayman Chemical Co. (Ann Arbor, MI, USA), the activities of SOD, GSH-Px, and CAT antioxidant enzymes were determined in mice liver through measuring absorbance using VersaMax ELISA microplate reader.
In brief, a total of six concentrations, including 0.005, 0.010, 0.020, 0.030, 0.040, and 0.050 U/mL, were prepared for the bovine erythrocyte SOD (Cu/Zn) standard, after which a 10 µL standard from each concentration and sample was collected separately and added to a 96-well plate, followed by adding 200 µL of radical detector and mixing homogeneously. Following that, 20 µL of xanthine oxidase was added to initiate the reaction, and the absorbance at 450 nm was measured following shaking at room temperature for 30 min, while the SOD activity of each sample was calculated based on the standard curve [26].
For GSH-Px activity, a 20 µL of GSH-Px standard and sample were added to a 96well plate separately and then 50 µL of assay buffer, 50 µL of co-substrate mixture, and 50 µL of NADPH were added and mixed homogeneously, followed by adding 20 µL of cumene hydroperoxide to initiate the reaction. Following shaking for a few seconds at room temperature, the absorbance was measured at 340 nm once every minute for five times in which the values at two time points were selected to calculate the GSH-Px activity of each sample [26].
For CAT activity, a total of six concentrations, including 5, 15, 30, 45, 60, and 75 µM, were prepared for the formaldehyde standard, after which a 20 µL standard from each concentration and sample were added to a 96-well plate separately and then 100 µL of the assay buffer and 30 µL of methanol were added. After mixing homogeneously, 20 µL of hydrogen peroxide was added to initiate the reaction, followed by shaking for 20 min at room temperature, adding 30 µL of potassium hydroxide to terminate the reaction, adding 30 µL of catalase purpald, shaking at room temperature for 10 min, and then adding 10 µL of potassium periodate. Following standing for 5 min, the absorbance was measured at 540 nm for calculation of the CAT activity of each sample based on the standard curve [26].

Determination of Dopamine, Malondialdehyde (MDA), and Protein Carbonyl (PC) Contents in Mice Brain
The contents of dopamine, MDA, and PC in mice brains were determined by following the instructions in the assay kits obtained from Cayman Chemical Co (Ann Arbor, MI, USA), while that of dopamine from CUSABIO Co. (Houston, TX, USA) measured the absorbance in a VersaMax ELISA microplate reader.
Briefly, a total of seven concentrations, including 1.562, 3.125, 6.25, 12.5, 25, 50, and 100 ng/mL, were prepared for the dopamine standard, after which a 50 µL standard from each concentration and sample was collected separately, followed by adding 50 µL of biotin-labeled antibody and 50 µL of horseradish peroxidase conjugate. After mixing homogeneously at 37 • C for 1 h, this mixture was washed with wash buffer three times, followed by adding 50 µL of substrate A and 50 µL of substrate B, mixing thoroughly, reacting at 37 • C for 15 min, and adding 50 µL of stop solution to terminate the reaction. The absorbance at 450 nm was measured for calculation of the dopamine content in each sample based on the standard curve [29].
For MDA content, a total of seven concentrations, including 0.625, 1.25, 2.5, 5, 10, 25, and 50 µM, were prepared for the MDA standard, after which a 100 µL standard from each concentration and sample was collected separately, followed by adding 100 µL of sodium dodecyl sulfate (SDS) and 4 mL of coloring agent, placing in a water bath (100 • C) for reacting for 1 h, cooling on ice for 10 min, centrifuging at 1600× g for 10 min (4 • C), collecting the supernatant, and adding to a 96-well plate. The absorbance was measured at 535 nm for the calculation of the MDA content in samples based on the standard curve [26].
For PC content, a 200 µL of sample was poured into a sample centrifuge tube and control centrifuge tube separately, followed by adding 800 µL of DNPH to the former and 800 µL of hydrochloric acid (2.5 M) to the latter. Both tubes were stood in the dark for 1 h and then 1 mL of 20% trichloroacetic acid (TCA) was added. After mixing homogeneously, this mixture was placed on ice for 5 min and then centrifuged at 10,000× g for 10 min (4 • C). The supernatant was removed and 1 mL of 10% TCA was added to the protein pellet, homogenized again, placed on ice for 5 min, and centrifuged at 10,000× g for 10 min (4 • C). The supernatant was removed again, and 1 mL of ethanol/ethyl acetate was added to the protein pellet, homogenized, centrifuged at 10,000× g for 10 min (4 • C), and the supernatant removed. One mL of ethanol/ethyl acetate was added twice, followed by adding 500 µL of guanidine hydrochloride to the protein pellet, centrifuging at 10,000× g for 10 min (4 • C), collecting the supernatant, and measuring absorbance at 370 nm in a 96-well plate for calculation of PC contents in samples [30].

Statistical Analysis
Statistical analysis of all the experimental data was done by using statistical analysis system (SAS) software [31] for analysis of variance by ANOVA and Duncan's multiple range test for significant difference of mean values (p < 0.05).

Effect of Solvent Variety on Total Phenolic Content (TPC) and Total Flavonoid Content (TFC) in Rabbiteye Blueberry Leaves
Owing to the more hydrophilic nature of polyphenols, they are commonly extracted from plant products by solvents such as methanol, ethanol, acetonitrile, and acetone alone or in combination with water [27,28]. However, in this study, an aqueous solution of ethanol was used as the extraction solvent, as it is relatively safer (less toxic) and used as a universal solvent with a polarity index of 5.2 and dielectric constant of 24.55 capable of solubilizing both polar and less polar polyphenols [32]. Table 1 shows the effect of different ethanol proportions on TPC and TFC in rabbiteye blueberry leaves. The highest TPC, expressed as gallic acid equivalent (mg/g), was shown for 50% EtOH, followed by 30% EtOH and 70% EtOH. However, the difference in TPC between 30% EtOH and 50% EtOH was not significant (p > 0.05). For the TFC expressed as quercetin equivalent (mg/g), 30% EtOH produced the highest yield, followed by 50% EtOH and 70% EtOH. Furthermore, a lesser volume of ethanol used in the preparation of blueberry leaf extract when compared to 50% ethanol favors the green sustainable strategy of reducing solvent usage. Thus, 30% EtOH was selected as the extraction solvent for subsequent experiments. In several previous studies, Routray and Orsat [33] reported that a higher TPC from Bluetta blueberry leaves was obtained by 30% EtOH than that by 15% EtOH. Likewise, compared to 95% EtOH, 50% EtOH was shown to result in a higher yield of TPC in blueberry leaves [34]. Additionally, an average of TPC (137.61 mg/g) and TFC (249.88 mg/g) from 73 different cultivars of rabbiteye blueberry leaves in southeastern China was reported by Wu et al. [35]. Apparently, the difference in TPC and TFC in blueberry leaves can be affected by species, harvest time, and the extraction method. Table 2 shows TPC and TFC in different cultivars of rabbiteye blueberry leaves used in this study. A total of 20 cultivars were collected in this study, with the cultivar NTU 104 showing the highest TPC (136.09 mg/g) and a medium TFC (36.25 mg/g). Therefore, the cultivar NTU 104 (fresh leaves) was selected for subsequent experiments.   Table 3). Figure S1 shows UPLC-MS/MS chromatograms of phenolic acid and flavonoid standards and rabbiteye blueberry leaf extract, including one internal standard (EGCG), as detected by the SRM mode.

Method Validation and Quantitation
The LOD and LOQ of all eight phenolic acid and flavonoid compounds, as determined by UPLC-MS/MS, ranged from 0.372-0.839 ng/mL and 1.128-2.542 ng/mL, respectively (Table S1). The linear regression equations, along with the coefficient of determinations (R 2 ) obtained from the calibration curves of eight phenolic acid and flavonoid compounds, are also shown in Table S1. Table 3 also shows the contents of eight phenolic acid and flavonoid compounds, with their amounts ranging from 60.8-6474.2 µg/g. Obviously, 3-Ocaffeoylquinic acid, quercetin-3-O-galactoside, and quercetin-3-O-rutinoside dominated in the NTU 104 cultivar of the rabbiteye blueberry leaves used in our study. Similar outcomes were reported by Cezarotto et al. [36] and Wu et al. [35].  a Peaks were positively identified based on comparison of retention time and mass spectra of unknown peaks in rabbiteye blueberry leaf extract with that of standards. b The MS/MS data (precursor ion and product ion) obtained for eight phenolic acid and flavonoid compounds in both standard solution and rabbiteye blueberry leaf extract were found to be the same. c Data are presented as mean ± standard deviation (n = 3).  a Peaks were positively identified based on comparison of retention time and mass spectra of unknown peaks in rabbiteye blueberry leaf extract with that of standards. b The MS/MS data (precursor ion and product ion) obtained for eight phenolic acid and flavonoid compounds in both standard solution and rabbiteye blueberry leaf extract were found to be the same. c Data are presented as mean ± standard deviation (n = 3).
Both the repeatability (intra-day variability) and intermediate precision (inter-day variability) data are shown in Table S2, with the relative standard deviation (RSD) being from 1.14-4.83% for the former and from 1.61-6.33% for the latter. All the precision data are in agreement with a report issued by TFDA [37], stating that the RSD should be <10% for the analyte concentration ≥1 ppm for both repeatability and intermediate precision data. Similarly, the recovery data of phenolic acid and flavonoid compounds in rabbiteye blueberry leaves ranged from 90.37-102.65% (Table S3), which also meets the requirement set by TFDA [37], stating that the recovery should be from 85-110% for the analyte concentration ≥100 ppm. Thus, all the method validation data demonstrated that a high precision and accuracy was achieved for the method employed in this study. Figure 2 shows the appearance of blueberry nanoemulsion (2A), particle size distribution (2B), and TEM images (2C). As mentioned in the Methods section, a 10 mL transparent nanoemulsion containing four phenolic acids (5 mg/mL), including 3-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid, was successfully prepared, with the mean particle size being 16.0 nm as determined by DLS and 19 nm by TEM, as well as PDI being 0.214 and zeta-potential −53.8 mV. Both DLS and TEM analyses showed a similar mean particle size of the blueberry leaf nanoemulsion. Additionally, the encapsulation efficiency based on the content of four phenolic acids was calculated to be 84.13%, which should be responsible for the nanoemulsion's high stability during storage at 4°C for 3 months (Table 4) and during heating at 40-100 • C for 2 h (Table 5), as only a slight change in mean particle size, PDI, and zeta potential was observed. However, we must point out here that the total amount of four phenolic acids was used as the indicator components instead of flavonoids for calculation of encapsulation efficiency, as the former was shown to be present at a much higher level in rabbiteye blueberry leaves. Similarly, a high stability was found for the catechin nanoemulsion prepared from green tea leaf waste [38] and from avocado peel [39].

Characteristics of Rabbiteye Blueberry Nanoemulsion
Both the repeatability (intra-day variability) and intermediate precision (inter-day variability) data are shown in Table S2, with the relative standard deviation (RSD) being from 1.14-4.83% for the former and from 1.61-6.33% for the latter. All the precision data are in agreement with a report issued by TFDA [37], stating that the RSD should be <10% for the analyte concentration ≥ 1 ppm for both repeatability and intermediate precision data. Similarly, the recovery data of phenolic acid and flavonoid compounds in rabbiteye blueberry leaves ranged from 90.37-102.65% (Table S3), which also meets the requirement set by TFDA [37], stating that the recovery should be from 85-110% for the analyte concentration ≥100 ppm. Thus, all the method validation data demonstrated that a high precision and accuracy was achieved for the method employed in this study. Figure 2 shows the appearance of blueberry nanoemulsion (2A), particle size distribution (2B), and TEM images (2C). As mentioned in the Methods section, a 10 mL transparent nanoemulsion containing four phenolic acids (5 mg/mL), including 3-Ocaffeoylquinic acid, 5-O-caffeoylquinic acid, 3,5-dicaffeoylquinic acid, and 4,5dicaffeoylquinic acid, was successfully prepared, with the mean particle size being 16.0 nm as determined by DLS and 19 nm by TEM, as well as PDI being 0.214 and zeta-potential −53.8 mV. Both DLS and TEM analyses showed a similar mean particle size of the blueberry leaf nanoemulsion. Additionally, the encapsulation efficiency based on the content of four phenolic acids was calculated to be 84.13%, which should be responsible for the nanoemulsion's high stability during storage at 4℃ for 3 months (Table 4) and during heating at 40-100 °C for 2 h (Table 5), as only a slight change in mean particle size, PDI, and zeta potential was observed. However, we must point out here that the total amount of four phenolic acids was used as the indicator components instead of flavonoids for calculation of encapsulation efficiency, as the former was shown to be present at a much higher level in rabbiteye blueberry leaves. Similarly, a high stability was found for the catechin nanoemulsion prepared from green tea leaf waste [38] and from avocado peel [39].

Animal Experiment
In this study we injected D-galactose into mice to induce aging, as it has been established that the intake of D-galactose in excess can cause metabolic abnormality of non-enzymatic glycation and results in the production of a large amount of reactive oxygen species (ROS), leading to the impairment of normal cells for subsequent aging [40,41]. The effect of administration of rabbiteye blueberry leaf extract and nanoemulsion on body weight in D-galactos-induced aged mice is shown in Table 6. Following 12-week administration, the body weight of the induction group (D) declined by 3.82% compared to the control group (C). However, compared to the induction group (D), the body weight was higher by 1.66%, 1.32%, 8.28%, and 4.64% for the LE, HE, LN, and HN groups, respectively. Nevertheless, the difference in body weight among the LE, HE, LN, and HN groups remained insignificant (p > 0.05). This outcome implied that the injection of D-galactose had a mild impact on the body weight of mice for all six groups. A similar result was reported by Tsai and Yin [42], who studied the anti-glycative and anti-inflammatory effects of protocatechuic acid on brain of mice treated by D-galactose. Figure 3 shows the effect of administration of blueberry leaf extract and nanoemulsion on brain weight and liver weight in D-galactose-induced aged mice. Following 12-week administration, only a slight change in brain weight and liver weight was shown for all the treatments. This finding indicated that the D-galactose injection only had a mild impact on both brain and liver weights in mice during administration for 12 weeks.  [42], who studied the anti-glycative and anti-inflammatory effects of protocatechuic acid on brain of mice treated by D-galactose. Figure 3 shows the effect of administration of blueberry leaf extract and nanoemulsion on brain weight and liver weight in D-galactose-induced aged mice. Following 12week administration, only a slight change in brain weight and liver weight was shown for all the treatments. This finding indicated that the D-galactose injection only had a mild impact on both brain and liver weights in mice during administration for 12 weeks.  The effect of administration of blueberry leaf extract and nanoemulsion on the brain dopamine content in D-galactose-induced aged mice is shown in Figure 4. Compared to the induction group (D), the brain dopamine content of the control group (C) rose by 224.74%. Likewise, for the other groups, the brain dopamine contents increased pronouncedly by 161.38%, 170.88%, 177.27%, and 202.60% for the LE, HE, LN, and HN groups, respectively, with the HN group showing the most pronounced rise. It may be postulated that the blueberry leaf nanoemulsion at high dose was capable of crossing the blood-brain barrier for the activation of substantia nigra cells to secrete more dopamine. In several previous studies, Krzysztoforska et al. [43] reported that the dopamine contents were raised by 115%, 96%, and 596% in the striatum, hippocampus, and prefrontal cortex, respectively, following the administration of protocatechuic acid at 100 mg/kg to D-galactose-induced mice for 48 days. Likewise, Wang et al. [44] found an elevation in dopamine level by 13% and 17% following the administration of 50 and 300 mg/kg of enzyme degradation extract from Porphyra yezoensis in their study dealing with alleviation of oxidative stress and brain injury in D-galactose-induced aging mice. In addition, the anthocyanin extract prepared from black chokeberry was shown to raise dopamine contents by 32% and 47%, respectively, following administration at a dose of 15 and 30 mg/kg to D-galactose-induced mice for eight weeks. By comparison, the brain dopamine contents in D-galactose-induced mice in our study were shown to raise to a much higher level, which should be due to a higher capability of the blueberry leaf nanoemulsion crossing the blood-brain barrier as mentioned above when compared to the extract. the same column are significantly different at p < 0.05. C, control group; D, induction group; LE, low-dose extract group; HE, high-dose extract group; LN, low-dose nanoemulsion group; HN, highdose nanoemulsion group.
The effect of administration of blueberry leaf extract and nanoemulsion on the brain dopamine content in D-galactose-induced aged mice is shown in Figure 4. Compared to the induction group (D), the brain dopamine content of the control group (C) rose by 224.74%. Likewise, for the other groups, the brain dopamine contents increased pronouncedly by 161.38%, 170.88%, 177.27%, and 202.60% for the LE, HE, LN, and HN groups, respectively, with the HN group showing the most pronounced rise. It may be postulated that the blueberry leaf nanoemulsion at high dose was capable of crossing the blood-brain barrier for the activation of substantia nigra cells to secrete more dopamine. In several previous studies, Krzysztoforska et al. [43] reported that the dopamine contents were raised by 115%, 96%, and 596% in the striatum, hippocampus, and prefrontal cortex, respectively, following the administration of protocatechuic acid at 100 mg/kg to D-galactose-induced mice for 48 days. Likewise, Wang et al. [44] found an elevation in dopamine level by 13% and 17% following the administration of 50 and 300 mg/kg of enzyme degradation extract from Porphyra yezoensis in their study dealing with alleviation of oxidative stress and brain injury in D-galactose-induced aging mice. In addition, the anthocyanin extract prepared from black chokeberry was shown to raise dopamine contents by 32% and 47%, respectively, following administration at a dose of 15 and 30 mg/kg to D-galactose-induced mice for eight weeks. By comparison, the brain dopamine contents in Dgalactose-induced mice in our study were shown to raise to a much higher level, which should be due to a higher capability of the blueberry leaf nanoemulsion crossing the blood-brain barrier as mentioned above when compared to the extract.  Liver is a vital organ in the human body. However, it can be susceptible to attacks by ROS. It was reported that both sinusoidal endothelial cells and bile duct cells in the liver are highly sensitive to the impairment caused by oxidative stress, and the apoptosis of these cells can lead to liver aging [45]. Thus, the presence of enzymatic and non-enzymatic antioxidants plays a vital role in maintaining normal function of liver. Figure 4 shows the effect of administration of blueberry leaf extract and nanoemulsion on superoxide dismutase (SOD) activity in the liver of D-galactose-induced aged mice. Compared to the control group (C), the SOD activity of the induction group (D) was significantly lower (p < 0.05) by 38.98%. However, the SOD activity of the HE, LN, and HN groups were significantly higher (p < 0.05) than the D group by 59.58%, 76.56%, and 79.64%, respectively, Liver is a vital organ in the human body. However, it can be susceptible to attacks by ROS. It was reported that both sinusoidal endothelial cells and bile duct cells in the liver are highly sensitive to the impairment caused by oxidative stress, and the apoptosis of these cells can lead to liver aging [45]. Thus, the presence of enzymatic and non-enzymatic antioxidants plays a vital role in maintaining normal function of liver. Figure 4 shows the effect of administration of blueberry leaf extract and nanoemulsion on superoxide dismutase (SOD) activity in the liver of D-galactose-induced aged mice. Compared to the control group (C), the SOD activity of the induction group (D) was significantly lower (p < 0.05) by 38.98%. However, the SOD activity of the HE, LN, and HN groups were significantly higher (p < 0.05) than the D group by 59.58%, 76.56%, and 79.64%, respectively, while no significant difference (p > 0.05) in SOD activity was found between the LE and D groups. A similar trend was shown for the glutathione peroxidase (GSH-Px) activity, as evident by a rise of 3.86%, 3.73%, and 6.63% for the HE, LN, and HN groups, respectively, when compared to the D group (Figure 4). Interestingly, for catalase (CAT) activity, an increase of 9.16%, 11.26%, and 36.33% was shown for the HE, LN, and HN groups, respectively, when compared to the D group ( Figure 4). However, no significant difference (p > 0.05) in CAT activity was observed among the HE, LN, and D groups. Additionally, the CAT activity between the HN and C group was insignificant (p > 0.05). These outcomes clearly revealed that the antioxidant activities in mice liver were elevated substantially following the administration of blueberry leaf nanoemulsion at a high dose.
The effect of administration of blueberry leaf extract and nanoemulsion on malondialdehyde (MDA) content in the brain of D-galactose-induced aged mice is shown in Figure 5. Compared to the C group, the MDA content of the D group was significantly higher (p < 0.05) by 9.11%. However, compared to the D group, the MDA contents of the HE, LN and HN groups were significantly lower (p < 0.05) by 14.93%, 25.07%, and 20.69%, respectively. Like MDA, a similar outcome was observed for the protein carbonyl (PC) content in the brains of D-galactose-induced aged mice ( Figure 5). A decline in PC content of 0.77%, 7.84%, 7.69%, and 11.80% was shown for the groups of LE, HE, LN, and HN groups, respectively, when compared to the D group. However, the difference among these groups was insignificant (p > 0.05). As both MDA and PC are vital indexes of lipid peroxidation, the results obtained in our study indicated that both blueberry leaf extract and nanoemulsion may be effective in decreasing oxidative stress, with the nanoemulsion at a high dose showing the most pronounced effect.
The elevation of antioxidant enzyme activity and decrease of MDA and PC contents in liver of D-galactose-induced aged mice following administration of polyphenol-rich extracts have been well documented. For instance, Guo et al. [46] observed a raise in both SOD and GSH-Px activities in the liver by 232% and 109%, as well as a decline in MDA content in the brain by 88% following a 10-week administration of 100 mg/kg of polyphenol-rich Apocynum venetum extract in D-galactose-induced oxidative stress in mice. Similarly, a dose-dependent decrease was shown for the PC content in the brain of Dgalactose-induced aged mice, which was reduced by 51% following the administration of protocatechuic acid at 2 mg/kg for 8 weeks [42]. In a study dealing with the anti-aging and redox state regulation effects of A-type proanthocyanidins-rich cranberry concentration and its comparison with grape seed extract in mice, Jiao et al. [47] reported that, following administration of the former and the latter at 30 mg/kg for 8 weeks, the MDA contents in mice brain were respectively reduced by 25% and 30%, while the TBARS level in mice liver were diminished by 30-56% and 35-57% at low (15 mg/kg), moderate (30 mg/kg), and high (60 mg/kg) doses.
Furthermore, the hepatic GSH-Px activity in mice was enhanced by 42% for the cranberry concentrate treatment at 60 mg/kg, while the SOD activity was raised by 13% for the grape seed extract at 30 mg/kg. Surprisingly, for the cranberry concentrate treatment at 15, 30, and 60 mg/kg, the CAT activities decreased by 40%, 31%, and 33% in mice, respectively [47]. This result indicated that the antioxidant enzyme activities may be varied depending on sample variety and preparation method. In another report, the activities of SOD, GSH-Px, and CAT in D-galactose-induced mice liver were shown to increase by 30%, 63%, and 28%, respectively, following the administration of the ethanol extract from Ficus vasculosa at 50 mg/kg for 8 weeks, which can be due to the presence of polyphenols in the ethanol extract [48]. Moreover, a higher dose at 200 mg/kg was found to result in a rise of the SOD, GSH-Px, and CAT activities by 49%, 76%, and 38%, respectively, demonstrating a dose-dependent increase in the antioxidant enzyme activities of D-galactose-induced mice liver. The elevation of antioxidant enzyme activity and decrease of MDA and PC cont in liver of D-galactose-induced aged mice following administration of polyphenol extracts have been well documented. For instance, Guo et al. [46] observed a raise in SOD and GSH-Px activities in the liver by 232% and 109%, as well as a decline in M content in the brain by 88% following a 10-week administration of 100 mg/kg of poly nol-rich Apocynum venetum extract in D-galactose-induced oxidative stress in mice. S larly, a dose-dependent decrease was shown for the PC content in the brain of D-g tose-induced aged mice, which was reduced by 51% following the administration of tocatechuic acid at 2 mg/kg for 8 weeks [42]. In a study dealing with the anti-aging redox state regulation effects of A-type proanthocyanidins-rich cranberry concentra and its comparison with grape seed extract in mice, Jiao et al. [47] reported that, follow administration of the former and the latter at 30 mg/kg for 8 weeks, the MDA conten mice brain were respectively reduced by 25% and 30%, while the TBARS level in liver were diminished by 30-56% and 35-57% at low (15 mg/kg), moderate (30 mg and high (60 mg/kg) doses.
Furthermore, the hepatic GSH-Px activity in mice was enhanced by 42% for the c berry concentrate treatment at 60 mg/kg, while the SOD activity was raised by 13% fo In another study, Yu et al. [49] compared the antioxidant capacity and phenolic compounds of oat and buckwheat vinegar during the production process. The activities of SOD, GSH-Px, and CAT in D-galactose-induced mice liver were elevated by 29%, 29%, and 24%, respectively, when compared to the induction group, following administration of the oat vinegar at 5 mg/kg for 8 weeks, while for buckwheat vinegar at the same dose, the antioxidant enzyme activities were raised by 29%, 30%, and 26%, respectively. This outcome implied that both oat and buckwheat vinegars showed a similar antioxidant enzyme activity in D-galactose-induced mice liver, which can be attributed to the presence of phenolic compounds in both vinegars. Taken together, all the results showed above demonstrated that the presence of phenolic compounds in plants extracts play a vital role in decreasing oxidative stress in D-galactose-induced mice liver through enhancement of the antioxidant enzyme activities.
Theoretically, senescence can result in a decline of dopamine, norepinephrine, and serotonin in the frontal sinus of brain for a subsequent decrease of volume and function in the prefrontal cortex of brain. Furthermore, the behavior disorder expressed by the elderly is similar to that expressed by patients with frontal lobe lesion, which in turn failed to inhibit interference information, leading to a persistent error for a drop in capability of working memory [50]. Thus, it can be inferred that the defect of prefrontal lobe is the main reason responsible for behavior disorder. Additionally, the white matter of the frontal lobe of brain degenerates faster following a rise in age, which may affect the memory circuit of the prefrontal cortex. Research has also shown that the dopamine loss is partially responsible for the memory loss of patients with Alzheimer's disease [50]. More specifically, like other monoamine neurotransmitters, dopamine is a brain secretion and shows its function for regulation of rapid neurotransmission-mediated glutamic acid and r-aminobutyric acid (GABA). Additionally, dopamine shows a vital physiological function in olfactory modulation, immune system, sympathetic nervous system, cardiovascular, retina, kidney, and hormone regulation [51][52][53][54][55][56]. However, the most notable disease which can be closely associated with dopamine is Parkinson's disease, which can be due to the inadequate secretion of dopamine caused by apoptosis of substantia nigra cells [57]. The maladjustment of dopamine can lead to attention deficit hyperactivity disorder, depression, and Tourette's syndrome [58][59][60]. Huntington's disease, caused by the degeneration of the brain striatum, also plays a vital role in the pathogenesis of Parkinson's disease, as the striatum is a brain region rich in dopamine for signal transduction.
Taken together, a total of four phenolic acids and four flavonoids were extracted, separated, identified, and quantified in Rabbiteye blueberry leaves by UPLC-MS/MS in SRM mode, followed by the preparation of extract and nanoemulsion to evaluate the anti-aging activity in mice, with the latter showing a more marked effect. Of the two nanoemulsion doses (8 and 40 mg/kg bw) tested, the nanoemulsion at a high dose (40 mg/kg bw) was shown to be more effective in alleviating the D-galactose-induced aging in mice brain by elevating the dopamine level and reducing the levels of malondialdehyde and protein carbonyl, as well as increasing the activities of antioxidant enzymes, including superoxide dismutase, glutathione peroxidase, and catalase in mice liver. The observed anti-aging effect by rabbiteye blueberry leaf nanoemulsion in mice could be attributed to the facilitated uptake of nanoemulsion particles by cells. As cell membranes are mostly negatively charged, a strong binding and eventual uptake of nanoemulsion particles by cells is feasible only if the particles are positively charged [61,62]. In other words, the positively-charged nanoemulsion particles have a strong binding for efficient absorption by cells and higher bioactivity when compared to the negatively-charged and neutral ones [62]. However, the rabbiteye blueberry leaf nanoemulsion prepared in this study with a negative surface charge (zeta potential, −54 mV) has shown a pronounced anti-aging effect in mice, implying that the uptake of nanoemulsion particles by cells was not caused by electrostatic binding, but may be due to some other types of interactions. Several comprehensive review articles have highlighted that the cellular uptake of negatively-charged or neutral nanoparticles is possible through strong and non-specific interactions with the plasma membrane, followed by one or a combination of five endocytosis mechanisms including phagocytosis, clathrin-mediated endocytosis, caveolin-mediated endocytosis, clathrin/caveolae-independent endocytosis, and micropinocytosis [62,63]. Although these five different endocytosis mechanisms are the major uptake processes for nanoparticles to enter into cells, some other uptake mechanisms have also been reported, including hole formation, passive diffusion, electroporation, and direct microinjection [62]. Moreover, several studies have demonstrated the internalization of negatively-charged tanshinone nanoemulsion and lycopene-nanogold nanoemulsion particles into lung cancer cells (A549) and colon cancer cells (HT-29), respectively [64,65]. Overall, the antiaging effects demonstrated in this study may be caused by the cellular uptake of negatively-charged rabbiteye blueberry leaf nanoemulsion through non-specific interactions and subsequent endocytosis.

Conclusions
In conclusion, an UPLC-MS/MS method was developed to determine four phenolic acids and four flavonoids in rabbiteye blueberry leaves with 3-O-caffeoylquinic acid present in the highest amount, followed by quercetin-3-O-galactoside, quercetin-3-O-rutinoside, quercetin-3-O-glucoside, 5-O-caffeoylquinic acid, kaempferol-3-O-glucoside, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid. The blueberry leaf nanoemulsion was prepared by using an appropriate ratio of blueberry leaf extract, soybean oil, Tween 80, ethanol, glycerol, and deionized water, with the average particle size and zeta potential being 16 nm and −54 mV, respectively. Comparatively, the high-dose nanoemulsion was the most efficient in elevating the dopamine content in mice brains, as well as the activities of SOD, GPH-Px, and CAT in mice liver, while reducing the contents of MDA and PC in mice brain. Taken together, the high-dose rabbiteye blueberry leaf nanoemulsion possesses great potential to be developed into a health food.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/foods12101942/s1, Figure S1: UPLC-MS/MS chromatograms of phenolic acid and flavonoid standards (A) and rabbiteye blueberry leaf extract (B) as well as internal standard EGCG as detected by SRM mode; Table S1: Linear regression equation and coefficient of determination of phenolic acid and flavonoid compounds in rabbiteye blueberry leaves as determined by UPLC-MS/MS; Table S2: Quality control data of phenolic acid and flavonoid compounds in rabbiteye blueberry leaves as determined by UPLC-MS/MS; Table S3