1. Introduction
Small berries constitute important sources of potential health-promoting phytochemicals. These fruits are rich sources of polyphenols, such as phenolic acids (PAs), anthocyanins, and other flavonoids [
1]. Among them, PAs constitute a large group of secondary plant products with an aromatic ring bearing one or more hydroxyl substituents [
2]. Epidemiological and laboratory studies show a convincing link between the antioxidant and anti-inflammatory properties of plant-derived polyphenolic compounds and their health-promoting and/or disease-preventing effects, such as anti-atherosclerosis [
3,
4], anti-aging [
5], and improvement of metabolic syndrome [
6]. In our previous study, seven PAs identified in the serum of rats fed a 10% lowbush blueberry diet were found to promote bone growth [
7], to show potential athero-protective effects [
8], and to attenuate mammosphere formation [
9], which suggested that PAs may be important in vivo bioactive compounds in blueberries. In this study, PAs were firstly analyzed and quantified in polyphenol fractions of blueberry cultivars from China by high performance liquid chromatography/tandem mass spectrometry (HPLC/MS
2).
The blueberry is one of the few fruits native to North America. Highbush, lowbush, and rabbiteye blueberries are the three major species in the US market. They have all found their way into agricultural practices worldwide and are part of the cuisine in areas ranging from Asia to the Mediterranean. Blueberries have been found to contain extremely high levels of polyphenols, including anthocyanins, procyanins, flavonols, and PAs, resulting in antioxidant and anti-inflammatory effects in vitro/vivo that provide health benefits [
10,
11,
12,
13,
14,
15,
16], such as the protective effect against atherosclerosis in the apoE
−/− mouse model [
17,
18]. However, the mechanism that links polyphenols and the atherosclerosis-protective effects in blueberry cultivars in China are rarely reported.
MicroRNAs (miRNAs) are a class of endogenous, small, noncoding RNAs involved in post-transcriptional gene repression. They play critical roles in several different physiological processes. Interfering with miRNA expression in the artery wall is a potential approach toward addressing atherosclerotic plaques and cardiovascular disease development [
19,
20]. Specific miRNAs, such as miR-21, miR-146, and miR-125, have been found to be responsible for vascular inflammation and disease [
21,
22,
23].
Since the 1980s, many blueberry cultivars have been introduced in China due to the very few species of blueberry available there. However, up to now, not much research has been conducted on the blueberry’s PAs levels, antioxidant and anti-inflammatory activities, and inhibition of atherosclerosis-related miRNAs in different imported (commercialized) blueberry cultivars grown in China [
24,
25,
26,
27]. Many factors, including climatic conditions, growing locations, harvest seasons, and cultivars can affect the contents and the bioactivities of blueberries [
28,
29,
30].
The aim of the present work was to quantify the PAs contents in the polyphenol fractions of different Chinese blueberry cultivars, and to study the relationship between PAs contents and antioxidant and anti-inflammatory activities, and the inhibition of atherosclerosis-related miRNAs of the blueberry polyphenol fractions.
3. Materials and Methods
3.1. Chemicals and Reagents
GA, 3,4-DHBA, VA, CGA, CA, SGA, FA, benzoic acid-d5, DPPH, methanol (MeOH), DMSO, formic acid, phosphate-buffered saline (PBS), LPS from Escherichia coli O111:B4, and Histopaque 1077 were all obtained from Sigma-Aldrich (Milwaukee, WI, USA). Fetal bovine serum (FBS) was bought from Hyclone (Logan, UT, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), RPMI-1640 culture medium, and 2′,7′-dichlorofluoresceindiacetate (DCFDA) were obtained from Invitrogen (Carlsbad, CA, USA).
3.2. Plant Materials
Sixteen fresh blueberry samples of highbush (North and South high) (
V. corymbosum) and half-highbush (
V. corymbosum/
V. anugustifolium) were obtained from Dandong, Liaoning Province (samples
1–
4,
6,
9, and
11–
14) and Lijiang, Yunnan Province (samples
5,
7,
8,
10,
15, and
16), in China (
Table 1). They were provided by two main Chinese blueberry companies, Jiawo and Pulan. The imported (commercialized) blueberry cultivars (Reka, Patriot, Brigitta, Bluecrop, Berkeley, Duke, Darrow, Northland, Northblue, Northcountry, Bluesource, Southgood, O’Neal, and Misty) were identified by Prof. Jiali An (Jiawo) and Prof. Lin Wu (Pulan). Blueberries were all collected during the harvest period (April and June) in 2015 (
Table 1). After the fresh blueberries were harvested, they were frozen and stored at −20 °C until transport to the laboratory. The frozen samples were lyophilized and ground into powder; they were kept at −40 °C until analysis. The moister index of blueberry was calculated to be 86%.
3.3. Polyphenol-Enriched Extraction Procedure
The lyophilized blueberry powders were subjected to extraction with a mixed solvent (MeOH:H2O:acetic acid = 85:15:0.5); then, the extracts were purified using solid-phase extraction (SPE) cartridges with a 60-mL tube (Supelco, Milwaukee, WI, USA) filled with C-18 (Cosmosil 75 C18-PREP; Nacalai Tesque, Kyoto, Japan). The cartridge was equilibrated with 100 mL MeOH and washed with 100 mL 0.2% formic acid in H2O. The blueberry extract was loaded onto the column and washed with 100 mL 0.2% formic acid in H2O. Then, the polyphenol-enriched fraction was collected by washing the column with 100 mL 0.2% formic acid in MeOH. Finally, the sample was dried under nitrogen flow.
The dried sample was ready for DPPH assay, enzyme-linked immunosorbent assay, and the expression of miR-21, miR-125b, and miR-146a.
For the HPLC/MS2 analyses, the dried sample was reconstituted with 0.2 mg extract/mL in MeOH.
3.4. HPLC/MS2 Analysis
The HPLC/MS
2 analysis was performed using an Agilent 1100 HPLC system including an autosampler, a binary pump, and a diode array detector (Agilent Technologies, Palo Alto, CA, USA), which was coupled to a 4000 Q TRAP mass spectrometer (Applied Biosystems, Foster City, CA, USA). Separation was carried on an Agilent Poroshell 120 SB-C18 column (50 × 3.0 mm, 2.7 μm) with a flow rate of 0.4 mL/min. The solvent consisted of (A) 0.2% (
v/
v) formic acid in water and (B) methanol. The 16 min gradient was as follows: 10%–14% B (0–2 min); 14%–18% B (2–4 min); 18%–25% B (4–6 min); 25%–27% B (6–8 min); 27%–30% B (8–10 min); 30%–35% B (10–15 min); 35%–35% B (15–16 min); and 10 min of re-equilibration of the column before the next run. The multi-reaction monitoring (MRM) mode scan was used for quantitation. A mass spectrometer equipped with an ESI-Turbo V source was operated in negative ion mode. The major parameters were optimized as follows: ion spray voltage, −4.5 kV; curtain gas (CUR), 50 psi; source temperature, 400 °C; nebulizing (GS1) and turbo spray gas (GS2), 30 and 50 psi, respectively. The entrance potential (EP), declustering potential (DP), collision energy (CE), and collision cell exit potential (CXP) were optimized individually with each standard. The postcolumn splitting ratio was 3:1. The analysis was controlled with Analyst v1.4.2 (Applied Biosystems, Forest City, CA, USA). The quantitative parameters for the PAs standards (1–7) and benzoic acid-
d5(internal standard) (8) are shown below (
Table 5).
3.5. DPPH Antioxidant Assay
The antiradical activity was determined using the DPPH method described by Grajeda-Iglesias [
37]. Briefly, each sample was diluted in MeOH and then mixed with an equal volume of 0.25 mg/mL DPPH MeOH solution. The mixtures were added to a 96-well microplate and absorbance was read at 525 nm after 30min. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was used as the internal control, and for the blank, the sample was substituted with MeOH. The concentration range of Trolox is 1, 10, 20, 40, 60, and 100 µM. Because the moister index of blueberry was calculated to be 86%, the results were expressed in terms of µmol Trolox equivalent (TE) per 100 g fresh blueberries (IC
50 (Trolox)/IC
50 (sample) ×100 g) [
32].
3.6. Enzyme-Linked Immunosorbent Assay Analysis of Interleukin-6 and Tumor Necrosis Factor-α Expression
The enzyme-linked immunosorbent assay was conducted based on the method previously reported [
8]. RAW 264.7 cells (Invivogen, San Diego, CA, USA) were cultured in DMEM supplemented with penicillin (100 units/mL), streptomycin sulfate (100 µg/mL),
l-glutamine (4 mM), and 10% (
v/
v) FBS (Hyclone, Logan, UT, USA). The cells (1 × 10
5 cells/well) were then pretreated with various concentrations of polyphenol-enriched fractions of the 16 blueberry samples for 24 h before LPS (100 ng/mL) stimulation. After 18 h of LPS stimulation, the supernatant was collected. The TNF-α and IL-6 levels in the supernatant were determined with an enzyme-linked immunosorbent assay (ELISA) performed using Duoset ELISA kits (R&D, Minneapolis, MN, USA) according to the manufacturer’s instructions. The optical density was determined using a BMG Polar Star microplate reader (BMG Labtech, Durham, NC, USA) at 450 nm.
3.7. Analysis of miR-21, miR-125b, and miR-146a Expression
RAW 264.7 cells (Invivogen, San Diego, CA, USA) were cultured in DMEM supplemented with penicillin (100 units/mL), streptomycin sulfate (100 µg/mL), l-glutamine (4 mM) and 10% (v/v) FBS (Hyclone, Logan, UT, USA). The cells (1 × 105 cells/well) were then pretreated with various concentrations of polyphenol-enriched fractions of the 16 blueberry samples for 24 h before LPS (100 ng/mL) stimulation. After 18 h of LPS stimulation, the supernatant was collected. For the detection of miRNA expression, the miScript Reverse Transcription Kit (Qiagen, Valencia, CA, USA) was used for cDNA synthesis. The miScript SYBR Green PCR Kit (Qiagen) was used in combination with a pair of miRNA-specific primers for the detection of mature miRNAs. RNU6B was used as an internal control.
3.8. Statistical Analysis
The post-hoc test was applied to perform ANOVA analysis, which was used to evaluate whether differences were significant. Pearson’s correlation coefficient was used to analyze the correlation between the content of phenolic acids and antioxidant and anti-inflammatory activities, as well as miRNA inhibitions. A value of p < 0.05 was considered as a significant difference. Statistical analyses were performed using SPSS 13.0 (IBM, New York, NY, USA).
4. Conclusions
To the best of our knowledge, this is the first report regarding the relationship between PAs contents and antioxidant and anti-inflammatory activities, and the inhibition of atherosclerosis-related miRNAs of blueberry polyphenol fractions. Sixteen blueberry samples belonging to 14 commercialized cultivars collected during the harvest time in China were used in this study. Correlation analysis showed that blueberries that had higher levels of PAs displayed stronger antioxidant and anti-inflammatory capacities. Most of the blueberry samples originating from Lijiang, Yunnan Province had better results than those from Dandong, Liaoning Province. The polyphenol-enriched fractions of the 16 blueberry samples inhibited three atherosclerosis related miRNAs, but the results did not reveal a positive relationship between the PAs contents and miRNAs inhibitions. Therefore, further study is required to determine which kinds of polyphenols play a role in the inhibition of miRNAs.
