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Article

Phenolic Profiles, Antioxidant, and Hypoglycemic Activities of Ribes meyeri Fruits

by
Le Zhang
1,†,
Qiang Wang
1,†,
Yayun Zhao
1,
Juan Ge
1,* and
Dajun He
1,2,*
1
College of Life Science, Shihezi University, Shihezi 832003, China
2
Analysis and Testing Center, Shihezi University, Shihezi 832003, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Foods 2023, 12(12), 2406; https://doi.org/10.3390/foods12122406
Submission received: 28 April 2023 / Revised: 15 June 2023 / Accepted: 16 June 2023 / Published: 18 June 2023
(This article belongs to the Special Issue Nutritional Composition, Bioactive Compounds and Health Benefits of Edible Plants)
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Abstract

:
Ribes meyeri is a Ribes genus in the Saxifragaceae family, which is used as both medicine and food. However, the active components and biological activities of R. meyeri fruits are still unknown. In this paper, the phenolic components and their antioxidant and hypoglycemic activities of R. meyeri fruits were studied. Firstly, a total of 42 phenolic components of R. meyeri fruits, including 26 anthocyanins, 9 flavonoids, and 7 phenolic acids, were tentatively identified using HPLC-QTOF-MS/MS, and the main four anthocyanins were quantified using UPLC-MS/MS. The result indicated that cyanidin-3-O-rutinoside is the main anthocyanin in the R. meyeri fruits. The anthocyanin fraction of R. meyeri fruits exhibited significant inhibitory activity on α-amylase and α-glucosidase. The anthocyanin fraction from R. meyeri fruits significantly increased the glucose uptake of 3T3-L1 adipocytes. This is the first study of a qualitative and quantitative analysis of the phenolics of R. meyeri fruits.

1. Introduction

In the human diet, berries are the most commonly consumed fruit. There is growing evidence that the edible and colorful fruits have various health-promoting and disease-preventing benefits, which are related to a variety of bioactive phytochemicals, including phenolic compounds [1,2].
Black currants and red currants are favorite Ribes berries. Researchers have found that currants contain a lot of polyphenols, particularly anthocyanins. The main anthocyanins of black currants are glucosides and rutinosides of cyanidin and delphinidin [3]; those of red currants are the cyanidin-diglycosides [4], including phenolic acid derivatives, flavonols, as well as proanthocyanidins [5]. Increasing evidence suggests that black currant fruit has a variety of bioactive activity, such as antioxidant, anti-inflammatory, anti-tumor, antidiabetic, and so on [6,7,8,9].
Unlike the mainstream berries on the market, which might contain residues of pesticides and chemicals, wild plants require no irrigation water or fertilizers, and their wild berries are considered a healthy alternative [10,11]. Ribes meyeri fruit is a unique, small, black edible berry that is widely distributed in Xinjiang, China, as shown in Figure 1, and is used in Uyghur medicine to reduce blood pressure and blood lipids and to treat menstrual disorders [12]. In our previous study, we found that Ribes meyeri fruit is a rich source of phenolics, specifically anthocyanin [13], and shows a good potential to become an alternative for commercial Ribes fruits such as Ribes nigrum. However, research on the chemical constituents and biological activities of Ribes meyeri fruits is scarce. In order to make better use of Ribes meyeri fruits, it is essential to establish a rapid and accurate method to discover the active ingredients.
α-Glucosidase is an important target for screening hypoglycemic activity that catalyzes the final stage of carbohydrate digestion. α-Glucosidase inhibitors can slow glucose release from complex carbohydrates and delay glucose absorption, resulting in lower postprandial plasma glucose levels [14]. α-Amylase is one of the key enzymes involved in the carbohydrate digestion process by catalyzing the hydrolysis of the α-D-1,4-glucosidic bond in starch [6]. Therefore, the inhibition of α-amylase activity is considered an effective strategy to regulate blood sugar levels and manage food-linked hyperglycemia [15,16]. In addition to the two conventional screening targets for hypoglycemic activity mentioned above, protein tyrosine phosphatase 1B (PTP1B) is currently one of the most promising targets for the treatment of type 2 diabetes (T2D). PTP1B is a negative modulator of insulin signaling and plays an important role in insulin resistance. However, most PTP1B inhibitors failed in clinical trials because of poor clinical efficacy and severe side effects. Therefore, it is essential to discover novel PTP1B inhibitors for the development of new drugs [17,18]. In this study, we screened the hypoglycemic activity of Ribes meyeri fruits using these three important targets.
The combination of high-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (HPLC-QTOF/MS) has been recognized as a highly sensitive and accurate analytical tool to identify the phenolics of medicinal and edible plants [19]. In this study, the phenolics of R. meyeri fruits were characterized using HPLC-QTOF/MS; meanwhile, their antioxidative and antidiabetic activities were determined at the molecular and cellular levels. To date, this is the first comprehensive study to analyze the phenolics and evaluate the antioxidative and hypoglycemic properties of R. meyeri berries. It will provide a reference for enriching edible Ribes resources.

2. Materials and Methods

2.1. Plant Material and Reagents

In August 2021, Ribes meyeri fruits were collected in Manasi County South Mountain Forestry, Xinjiang, China, and identified by Associate Prof. Wenbin XU. Then they were stored at −20 °C. The Ribes meyeri fruit specimen (RML210811) was coded and maintained in the botany laboratory of Shihezi University.
Four standards of anthocyanin, cyanidin-3-O-rutinoside, delphinidin-3-O-rutinoside, cyanidin-3-O-glucoside, and delphinidin-3-O-glucoside, were purchased from Chengdu Durst Biotechnology Co. (Chengdu, China). Formic acid, acetonitrile, and methanol (Optima™ LC/MS Grade) were purchased from Fisher Scientific (Waltham, MA, USA). The following analytical purity chemicals were provided by Adamas Co. (Shanghai, China): methanol, hydrochloric acid, potassium chloride, ethanol, potassium persulfate, and sodium potassium tartrate. A macroporous resin, AB-8, was obtained from Cangzhou Bao’en Adsorption Material Technology Co. (Cangzhou, China).
Protein tyrosine phosphatase 1B (PTP1B, EC 3.1.3.48) from human recombinant protein, α-glucosidase (EC 3.2.1.20) from Saccharomyces cerevisiae, α-amylase (EC 3.2.1.1) from porcine pancreas, p-nitrophenyl-β-D-glucoside (pNPG), Na3VO4, ascorbic acid, acarbose, 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were provided by Sigma-Aldrich Chemical Co. (St Louis, MO, USA).

2.2. Extraction Procedure

One hundred grams of R. meyeri fruits were homogenized with a homogenizer, added to 1000 mL methanol (including 0.1% hydrochloric acid), and then ultrasonically extracted at room temperature for 30 min. The extraction procedure was repeated three times. Then the extracts were filtered, and the filtrate was centrifuged at 10,000 rpm for 20 min. Subsequently, the solvent of the supernatant was removed using a rotary evaporator at 40 °C to obtain the extract (7.9 g). For LC-MS analysis, each sample was dissolved in methanol at the appropriate concentration and filtered with Acrodisc syringe filters (0.22 μm, Pall Corporation, GA, USA).

2.3. Purification of Anthocyanin from R. meyeri Fruits

In order to purify anthocyanin from R. meyeri fruits, 1.0 g of methanol extract was dissolved in 200 mL ultrapure water and added to a pretreated AB-8 macroporous resin column (100 × 5 cm). Firstly, the flow rate of the resin column was 5.0 mL/min, and the anthocyanins were absorbed on the AB-8 macroporous resin. Then, 500 mL ultrapure water was added to remove impurities. Finally, a 100 mL methanol solution containing 0.1% HCl was added to elute the anthocyanins, at a flow rate of 1.0 mL/min. The methanol eluent was combined and concentrated using a rotary evaporator at 40 °C to obtain the anthocyanin fraction (0.74 g), which was saved at −20 °C for quantitative analysis and activity evaluation.

2.4. Qualitative Analysis of Phenolics from R. meyeri Fruits

Chromatographic conditions: Shimadzu UFLC liquid system was equipped with SIL-20AC autosampler, two LC-20AD pumps, CTO-20AC column temperature box, DGU-20A degassing device, and CBM-20A communication bus module. A Prodigy Phenyl-3 column (150 mm × 4.6 mm, 5 μM, Phenomenex, Torrance, CA, USA) was used for chromatographic separation at a column temperature of 40 °C and a flow rate of 0.5 mL/min; 20 μL of sample were injected. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid/methanol (B), where the gradient elution was 95–5% A from 0 to 95 min, and 5% A isocratic from 95 to 110 min.
MS conditions: a four-stage rod time-of-flight MS TOF® 4600 (Applied Biosystems/MDS Sciex, Framingham, MA, USA) with ESI interface was used. The MS analysis was recorded in both positive and negative ionization modes. The MS parameters were as follows: electrospray ionization (ESI) voltage of −4500 V, auxiliary gas of 50 psi, nebulizer gas of 35 psi, curtain gas of 25 psi, turbine gas temperature of 450 °C, decluster voltage of −80 V, and collision energy of 50 eV. The quality scan range was m/z 100-1100. The Analyst TF 1.7 software was used to obtain and process the MS data.

2.5. Quantitative Analysis of Anthocyanin from R. meyeri Fruits

2.5.1. Determination of Anthocyanin Content

The anthocyanin content was determined according to the pH-differential method [20] with some modifications. In brief, the absorbance of a cyanidin-3-glucoside solution (potassium chloride–hydrochloric acid buffer solution, pH = 1.0 and 24.8 mM sodium acetate buffer solution, pH = 4.5) was recorded at 510 nm and 700 nm using a spectrophotometer. The content of anthocyanin was expressed as milligrams cyanidin-3-glucoside equivalents per gram of dry weight (mg C-3-G/g DW).

2.5.2. UPLC-MS/MS Analysis

The four main anthocyanins of the methanol extract and anthocyanin fraction from R. meyeri fruits were quantitatively analyzed with the UPLC-MS/MS method [21]. A UPLC system and a triple quadrupole mass spectrometry with electrospray ionization (ESI) source (Xevo TOS, Waters Corp., Milford, MA, USA) was run with MassLynx software v.4.1. An ACQUITY UPLC BEH C18 column (50 mm × 5 mm, 1.7 μm, Waters, Milford, MA, USA) was used for the anthocyanin separation at 30 °C with a flow rate of 0.3 mL/min, and the injection volume was 1.0 µL. In order to optimize the simultaneous quantitative analysis method for the four major anthocyanins of R. meyeri berries with high resolution and narrow peaks, different UPLC conditions were compared, including different mobile phases (MeOH/H2O, ACN/H2O) and different modifiers (no modifier, 0.1%, 1%, and 5% formic acid). The optimized conditions were as follows: the mobile phase consisted of 5% formic acid (A) and acetonitrile (B), and the gradient elution was 0–1.0 min, 98% A; 1.0–3.0 min, 98–91% A; 3.0–4.0 min, 91–85% A; 4.0–6.0 min, 85–0% A; 6.0–6.2 min, 0–98% A; and 6.2–8.0 min, 98% A.
The positive ion mode with the multiple reaction monitoring (MRM) mode was used in MS, and the optimized MS parameters were as follows: source temperature of 150 °C, desolvation temperature of 450 °C, capillary voltage of 2.0 KV, nebulizer pressure of 7.0 bar, desolvation gas flow of 800 L/h, and cone gas flow of 150 L/h. The MS parameters of each component are shown in Table 1.

2.6. Determination of Antioxidant Activity

The antioxidant activity of the methanol extract and anthocyanin fraction from the R. meyeri fruits was determined with two colorimetric methods, the radical DPPH and ABTS radical scavenging methods, according to the previous report [22]. The absorbance was recorded at 510 nm with a Spectra Max MD5 microplate reader (Molecular Devices, San Jose, CA, USA). Ascorbic acid was the positive control. The IC50 was calculated using linear regression.

2.7. Antidiabetic Activity Assay

The α-amylase inhibitory activity was assayed according to the method in [22,23]. In brief, the sample solution (including the methanol extract and anthocyanin fraction from R. meyeri fruits, 10 μL dissolved in DMSO) and 0.5 U/mL of the α-amylase solution (10 μL) were mixed and incubated at 37 °C for 15 min. Subsequently, 500 μL of 1% starch solution was added and incubated at 37 °C for 10 min. The reaction was terminated by adding the DNS reagent (300 μL). The absorbance was determined at 540 nm with a Spectra Max MD5 microplate reader. Acarbose was the positive control.
The α-glycosidase inhibitory activity was determined according to the previous report [23]. Briefly, 20 μL of the sample solution, including the methanol extract and anthocyanin fraction from R. meyeri fruits, were combined with 0.22 U/mL α-glucosidase (20 μL) in 140 μL of phosphate buffer (0.1 M) and incubated at 37 °C for 20 min. Then, 10 mmol/L of pNPG solution (50 μL) as reaction substrate was added, followed by a 20-min incubation at 37 °C. The reaction was terminated by adding 80 μL of Na2CO3 solution (0.4 M). The absorbance of released p-nitrophenol at 405 nm was recorded with a microplate reader. Acarbose was the positive control.
The inhibitory activity of protein tyrosine phosphatase 1B (PTP1B) was measured using the color reaction of PTP1B hydrolyzing the phosphate group of p-nitrophenyl disodium phosphate (p-NPP) according to the reported method in [22]. In brief, 1 μL sample solution (dissolved in DMSO); 1 μL of PTP1B (0.115 mg/mL); and 96 μL of buffer containing 100 mM NaCl, 0.1% BSA, dithiothreitol (DTT, 1 mM), and 4-(2-hydroxyerhyl) piperazine-1-erhanesulfonic acid (HEPES, 50 mM, pH 7.3) were mixed and incubated at 37 °C for 10 min. Subsequently, 2 μL p-NPP (2 mM) was added to start the enzyme reaction at 37 °C. After 30 min, the reaction was terminated by adding 5 μL of NaOH (3 mol/L). Sodium vanadate was the positive control. The absorbance of the released p-nitrophenyl was measured at 405 nm. The inhibition rate was calculated as follows:
Inhibition rate of PTP1B (%) = (Ablank − Asample)/Ablank × 100%.
The effect of the R. meyeri fruits’ anthocyanin on the glucose consumption of 3T3-L1 adipocytes was evaluated according to a previous method [22]. The Shanghai Institute of Biochemistry and Cell Biology provided 3T3-L1 preadipocytes, which were routinely cultured and differentiated in DMEM and the differentiation medium (provided by Gibco1 Life Technologies, Carlsbad, CA, USA). After the 3T3-L1 adipocytes were fully differentiated and the cells’ density reached 8 × 103 cells/well, they continued to be cultured under starvation conditions for 5 h. Finally, the 3T3-L1 adipocytes were exposed to the blank control (0.1% DMSO), insulin (reference compound, 100 nM), and the anthocyanin fractions of 12.5 μg/mL and 25 μg/mL at 37 °C for 18 h. The content of the glucose uptake was quantified with a glucose oxidase assay kit from Applygen Co. (Beijing, China). The results were expressed as the mean ± SD (n = 3).

2.8. Statistical Analysis

The data were presented as the mean ± SD (n = 3). To analyze the correlation between the anthocyanin content and the antioxidant hypoglycemic activities, the Pearson’s correlation coefficient was calculated with the statistical software R version 3.5.3, and results with p < 0.05 were considered to be significant.

3. Results

3.1. Qualitative Identification of the Chemical Composition of R. meyeri Fruits

The phenolics of the methanol extract from the R. meyeri fruits were characterized with HPLC-QTOF-MS/MS. A strategy was proposed (Figure S1) for identification of the chemical composition of R. meyeri fruits. Firstly, the methanol extract of R. meyeri fruits was analyzed in both positive and negative ion modes. Then, the important information, including accurate mass, the matched molecular formula, the pseudo-molecular ions, and the MS/MS product ions, was acquired. Finally, the chemical composition of R. meyeri fruits was qualitatively identified based on the typical product ions, fragment ions search, and the typical neutral losses. With this strategy, 42 chemical compositions were tentatively characterized from the methanol extract from R. meyeri fruits. In the positive ion mode, 26 anthocyanins were tentatively characterized using TIC chromatograms as shown in Figure 2. In the negative ion mode, nine flavonoids and seven phenolic acids were tentatively characterized. The TIC chromatogram is shown in Figure 2. The molecular formula, error, main product ions, and retention time of the flavonoids, phenolic acids, and anthocyanins are shown in Table 2 and Table 3.
Identification of anthocyanins. Compound 42 shows its molecular ion at m/z 287.0563. The molecular formula is C15H11O6, calculated using accurate mass data. Compound 42 was identified as cyanidin by comparison of the retention time, precursor ions, high-resolution MS data, and fragmentation ions’ MS/MS data with those of the reference standard. The typical fragmentation ions of m/z 241 and m/z 213 show in its MS/MS spectra. Compound 22 shows its precursor ion at m/z 449.1097; its intense fragmentation ion of m/z 287 [M-C6H10O5]+ indicates the loss of a hexosyl (162 Da). The typical MS/MS fragmentation ions of m/z 287, 241, and 213 suggest the existence of a cyanidin moiety. Then, compound 22 was deduced to be cyanidin hexoside. Finally, compound 22 was confirmed as cyanidin-3-glucoside by comparison of the chromatographic and high-resolution MS data with those of the authentic standard. Thus, based on the fragmentation ion search of cyanidin, the typical fragmentation ions of cyanidin (m/z 287, 241, and 213), and the typical losses of glycosyls (C6H10O5 for hexosyl, C6H10O4 for deoxyhexosyl, and C5H8O4 for pentosyl), a series of cyanidin glycosides was identified. Similarly, other anthocyanins with aglycones of delphinidin and pelargonidin were identified with this strategy.
Identification of other phenolic glycosides. Other phenolic glycosides with various aglycones were similarly identified with this strategy.
Identification of phenolic acids. The typical fragmental pathway of the phenolic acids was the neutral loss of a CO2 (44 Da) in ESI negative ion mode. Herein, neutral loss of CO2 was detected in the MS/MS spectral data of caffeic acid and coumaric acid [24].

3.2. Quantitative Analysis of the Anthocyanins of R. meyeri Fruits

The content of the total anthocyanins was determined with a spectrophotometer and calculated using linear regression. The anthocyanins showed good linear relationships (R2 ≥ 0.9941) over the tested concentration range. The content of the methanol extract from the R. meyeri fruits was 34.37 ± 0.01 mg/g. The content of the anthocyanin fraction reached 137.65 ± 0.68 mg/g, and its content increased by nearly four times compared with the methanol extract. The result indicates that AB-8 macroporous resin can quickly and efficiently enrich and purify the anthocyanins of R. meyeri fruits.
The four main anthocyanins of the methanol extract and the anthocyanin fraction from R. meyeri fruits were quantified with UPLC-MS/MS. In the optimized chromatographic conditions, the four anthocyanins had the better resolution and peak shape, as shown in Figure 3.
The four anthocyanins show good linear relationships (R2 ≥ 0.9941) in the measured concentration ranges. The limit of quantification (LOQ) and limit of detection (LOD) of the four anthocyanins were calculated based on the signal-to-noise ratios of 10 and 3, respectively. Their LOQ and LOD ranged from 7.09 to 18.44 ng/mL and 2.36 to 5.38 ng/mL, respectively. The intra-day precision was calculated by measuring six replicates of the mixture solution containing four anthocyanins standard in a day. The inter-day precision was calculated by measuring the four anthocyanin standards for three consecutive days. The RSD values of the intra-day ranged from 1.09 to 4.65, and the RSD values of the inter-day were in the range of 1.17–5.57%. The six samples of R. meyeri fruits were prepared using the same method, and their repeatability was evaluated. The RSD values of repeatability ranged from 3.80% to 5.91%. The sample stability was determined for 0, 2, 4, 8, 12, and 24 h at room temperature, and the RSD values were in the range of 2.68–5.95%. The spiked recoveries were determined by adding the four anthocyanin mixed standards to a sample of known concentration. The recoveries were between 83% and 94%, and their RSD values ranged from 4.27 to 5.77%. The validation results show that the above UPLC-MS/MS method can determine the four anthocyanins efficiently, sensitively, and accurately, as shown in Table 4 and Table 5. Among the four anthocyanins, the content of cyanidin-3-O-rutinoside was the highest, at amounts of 22.01 ± 0.37 mg/g, while the content of delphindin-3-O-rutinoside was the lowest, at amounts of 0.06 mg/g. In a previous study, delphindin-3-O-rutinoside was the major anthocyanin in the black currant (Ribes nigrum L.) fruits, comprising 40.0–49.4% of the total anthocyanins [2,25]. Though the berries of the two Ribes plants are dark black fruits and are very similar, their anthocyanin contents differ significantly, which will impact their biological properties.

3.3. Antioxidant Activities

The antioxidative activities of R. meyeri fruits were investigated using ABTS and DPPH radical scavenging methods, as shown in Table 6. The result showed that the anthocyanin fraction of R. meyeri fruits had a higher DPPH radical scavenging rate than the methanol extract. The correlation coefficient between the anthocyanin content and the DPPH radical scavenging rate was 0.804. The results suggest that anthocyanins may be the active component in scavenging DPPH free radicals. For the ABTS radical, the methanol extract had a higher scavenging rate than the anthocyanin fraction, and the correlation between the anthocyanin content and the ABTS free radical scavenging rate was relatively low (correlation coefficients of 0.690). As previously reported, ascorbic acid, phenolic compounds, and anthocyanin are the most widespread antioxidants in berry fruits, particularly in Ribes nigrum [26,27]. For ABTS radicals, the methanol extract of R. meyeri fruits may have lost some antioxidant ingredients, such as ascorbic acid or other phenolics, during the enrichment of the anthocyanins with the AB-8 macroporous resin. In vitro analyses such as DPPH and ABTS radical scavenging are widely used to evaluate the antioxidant potentials of foods due to their speed and low cost. However, these in vitro methods have limitations. As previously indicated, in vitro tests for antioxidant capacity of different fruits showed poor correlation with in vivo radical scavenging capacity or physiological effects due to poor absorption and metabolism [28,29]. In future research, we will further investigate the in vivo antioxidant activity of phenolic compounds from R. meyeri fruits to clarify their mechanism of action.

3.4. α-Amylase, α-Glucosidase, and PTP1B Inhibitory Activities

The inhibitory activities of R. meyeri fruits on α-glucosidase, α-amylase, and PTP1B are shown in Table 6. For α-glucosidase, the methanol extract does not show inhibitory activity, but the anthocyanin fraction shows higher α-glucosidase inhibitory activity than acarbose. The correlation coefficient between the anthocyanin content and the α-glucosidase inhibitory activity is 0.874, which is probably due to the higher content of anthocyanin (in particular, cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside) in the anthocyanin fraction. These results suggest that anthocyanins, especially cyanidin-glycoside, are efficient α-glucosidase inhibitors. The methanol extract and the anthocyanin fraction exhibit significant α-amylase inhibitory activities, and their activities are higher than acarbose. Although the anthocyanin content of the anthocyanin fraction is much higher than that of the methanol extract, their amylase inhibitory activities are very similar. The correlation coefficient between the anthocyanin content and the α-amylase inhibitory activity is 0.773. This may suggest that other phenolics of the R. meyeri fruit play a role in the amylase inhibitory activity. These results are similar to the previous study, which indicated that the anthocyanins in black currants regulate postprandial hyperglycemia primarily by inhibiting α-glucosidase, while other phenolics modulate salivary α-amylase, glucose uptake, and sugar transporters [6]. The previous study indicated that the excessive inhibition of pancreatic α-amylase could result in the abnormal bacterial fermentation of undigested carbohydrates in the colon, which could be responsible in part for the side effects of acarbose [30]. Therefore, combinations of R. meyeri fruit extract with low-dose acarbose could be employed as an alternative antidiabetic therapy, reducing the side effects of acarbose. Although there is a high correlation coefficient (0.810) between the anthocyanin content and the PTP1B inhibitory activity, the methanol extract and the anthocyanin fraction show lower PTP1B inhibitory activity than sodium vanadate. The inhibitory activity of phenolics, especially anthocyanins, against α-amylase and α-glucosidase has been reported to be highly related to their binding affinity to the enzymes. The structure of phenolic compounds, including aglycone structures, substitution groups, and sugar side chains, can affect their enzymatic activity [31,32,33]. The results of this study suggest that the phenolics of R. meyeri fruits exhibit antidiabetic effects through different targets. The anthocyanins had the better effect on hypoglycemic activity by inhibiting α-glucosidase, and some of the other phenolics may be the active components of α-amylase inhibition.

3.5. The Effect of R. meyeri Fruits Anthocyanin on Glucose Consumption of 3T3-L1 Adipocytes

The effect of R. meyeri fruits’ anthocyanin fractions on the glucose uptake of 3T3-L1 preadipocytes is shown in Figure 4. Compared with the control group, the anthocyanin fractions (12.5 and 25 μg/mL) promoted glucose uptake in 3T3-L1 adipocytes in the conditions without insulin. After adding insulin (100 nM), the anthocyanin fraction showed an antagonistic action on insulin and inhibited glucose consumption of the 3T3-L1 adipocytes. These results indicate that the anthocyanin fractions of R. meyeri fruits could significantly promote the glucose uptake of 3T3-L1 adipocytes.

4. Conclusions

In this study, 26 anthocyanins, 7 phenolic acids, and 9 flavonoids were identified from R. meyeri fruits using HPLC-QTOF-MS/MS. The contents of four anthocyanins were determined under optimized mass spectrometry conditions. Among the four anthocyanins, the content of cyanidin-3-O-rutinoside was the highest. After purification with AB-8 macroporous resin, the anthocyanin content in the anthocyanin fraction increased by approximately four times, indicating that AB-8 macroporous resin can quickly and efficiently purify and enrich the anthocyanins of R. meyeri fruits. In ABTS and DPPH radical scavenging assays, the activities of the methanol extract and anthocyanin fraction from R. meyeri fruits were lower than that of the ascorbic acid, and their PTP1B inhibitive effects were lower than that of sodium vanadate. However, they exhibited significant inhibitory activity on α-amylase and α-glucosidase. The anthocyanin fraction from R. meyeri fruits significantly promoted glucose consumption in 3T3-L1 adipocytes. R. meyeri fruits may play a hypoglycemic role in different targets, and the anthocyanin fraction has the better activity, which may be related to the high content of anthocyanin in them. Therefore, R. meyeri fruits are potential hypoglycemic small berries. It is necessary to elucidate the hypoglycemic mechanisms of the bioactive compounds from R. meyeri fruits, especially in in vivo studies using animal models.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/foods12122406/s1, Figure S1: Compound identification scheme of the research methods.

Author Contributions

Conceptualization, L.Z. and Q.W.; experiments, L.Z., Q.W. and Y.Z.; data analysis, L.Z. and Q.W.; writing—original draft preparation, L.Z. and Q.W.; writing—review and editing, J.G. and Y.Z.; supervision, D.H. and J.G.; project administration, D.H.; funding acquisition, D.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by National Natural Science Foundation of China (grant number 31660093) and the Natural Science Foundation of Shihezi University (grant number ZZZC2022115).

Data Availability Statement

Data are contained within the article.

Acknowledgments

We are very grateful to Wenbin Xu for his help in Ribes meyeri fruits identification.

Conflicts of Interest

The authors declare no conflict of interest.

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Foods 12 02406 g001 550
Figure 1. Ribes meyeri and fruits.
Figure 1. Ribes meyeri and fruits.
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Foods 12 02406 g002 550
Figure 2. TIC chromatogram of R. meyeri fruits extract: (A) negative ion model, (B) positive ion model.
Figure 2. TIC chromatogram of R. meyeri fruits extract: (A) negative ion model, (B) positive ion model.
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Foods 12 02406 g003 550
Figure 3. The MRM; chromatograms of (1) Delphinidin-3-O-glucoside; (2) Delphinidin-3-O-rutinoside; (3) Cyanidin-3-O-glucoside; (4) Cyanidin-3-O-rutinoside; (A) Standard; (B) The extract of R. meyeri fruits.
Figure 3. The MRM; chromatograms of (1) Delphinidin-3-O-glucoside; (2) Delphinidin-3-O-rutinoside; (3) Cyanidin-3-O-glucoside; (4) Cyanidin-3-O-rutinoside; (A) Standard; (B) The extract of R. meyeri fruits.
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Foods 12 02406 g004 550
Figure 4. Effect of R. meyeri fruits’ anthocyanins on glucose consumption of 3T3-L1 preadipocytes.
Figure 4. Effect of R. meyeri fruits’ anthocyanins on glucose consumption of 3T3-L1 preadipocytes.
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Table
Table 1. List of quantitative compounds and MRM parameters.
Table 1. List of quantitative compounds and MRM parameters.
CompoundMolecular FormulaRelative Molecular MassIon ModeRetention Time (min)Parent Ion (m/z)Daughter Ion (m/z)Cone Hole Voltage (V)Collision Energy (eV)
Delphinidin-3-O-glucosideC27H31O15465.1ESI+3.89465.20303.1 *3520
Delphinidin-3-O-rutinosideC27H31O16611.1ESI+4.13611.01302.9 *5834
228.95868
Cyanidin-3-O-
glucoside		C21H21O12449.1ESI+4.47449.04136.9 *3450
286.83466
Cyanidin-3-O-
rutinoside		C21H21O11595.0ESI+4.72595.01286.9 *5826
136.95870
* mean quantitative ions.
Table
Table 2. Identification of the phenolics of R. meyeri fruits.
Table 2. Identification of the phenolics of R. meyeri fruits.
No.tR (min)Molecular Formula[M-H]−Major MS2 IonsError
(ppm)		Identification
110.97C13H16O10331.0672169, 1253.70Galloyl Hex
212.18C15H14O7305.0670261, 219, 179, 167, 1254.66Gallocatechin/Epigallocatechin
313.18C13H16O8299.0775239, 179, 1374.27Benzoyl Hex
415.47C15H18O8325.0933163, 145, 1194.63Hydrocinnamoyl Hex
517.81C27H30O15593.1525285, 284, 255, 2274.05Luteolin-Hex-DeHex
618.11C15H14O6289.0720245, 205, 203, 1094.62Catechin
718.91C15H18O8325.0934163, 145, 1194.94Hydrocinnamoyl Hex
819.96C15H18O8325.0934163, 145, 1194.94Hydrocinnamoyl Hex
920.34C9H8O4179.03471354.55Caffeic acid
1021.15C15H14O6289.0719245, 205, 203, 1094.62Epicatechin
1123.23C33H40O20755.2058609, 447, 301, 300, 271, 255, 243, 179, 1513.81Quercetin-DeHex-Hex-DeHex
1224.87C9H8O3163.03971194.47Coumaric acid
1325.73C27H30O16609.1474301, 300, 271, 255, 243, 229, 179, 1513.90Quercetin-3-O-Hex-DeHex
1426.80C21H20O12463.0891301, 300, 283, 271, 2554.31Quercetin-3-O-Hex
1529.69C15H12O8319.0463183, 153, 1393.56Dihydromyricetin
1636.73C34H24O22301.0353271, 255, 229, 179, 1513.39Quercetin
Hex: hexose; DeHex: deoxyhexose; Pen: pentose.
Table
Table 3. Identification of the anthocyanins of R. meyeri fruits.
Table 3. Identification of the anthocyanins of R. meyeri fruits.
No.tR (min)Molecular Formula[M+H]−Major MS2 IonsError
(ppm)		Identification
1715.77C33H41O20757.2218611, 449, 433, 287, 241, 2134.26Cyanidin-Hex-Hex-DeHex
1815.81C33H41O20757.2222611, 449, 433, 287, 241, 2134.79Cyanidin-Hex-Hex-DeHex
1915.88C32H39O20743.2065581, 449, 303, 287, 241, 2134.82Cyanidin-Hex-Pen-Hex
2016.47C27H31O16611.1633449, 287, 241, 2134.32Cyanidin-Hex-Hex
2116.72C32H39O19727.2116581, 449, 287, 241, 2134.94Cyanidin-Hex-Pen-DeHex
2217.00C21H21O11449.1097287, 241, 2134.15Cyanidin-3-glucoside
2317.18C26H29O15581.1528449, 287, 241, 2134.65Cyanidin-Hex-Pen
2417.48C32H39O19727.2112581, 449, 287, 241, 2134.39Cyanidin-Hex-Pen-DeHex
2517.70C27H31O15595.1683449, 287, 241, 2134.29Cyanidin-Hex-DeHex
2618.81C21H21O10433.1148271, 225, 1974.33Pelargonidin-Hex
2718.83C33H41O20757.2220611, 449, 433, 287, 241, 2134.53Cyanidin-Hex-Hex-DeHex
2819.91C20H19O10419.0985287, 241, 2132.93Cyanidin-Pen
2922.03C21H21O10433.1145287, 241, 2133.64Cyanidin-DeHex
3023.13C32H39O20743.2069611, 465, 449, 303, 257, 2295.40Cyanidin-Hex-Hex-Pen
3123.16C33H41O20757.2223611, 465, 303, 257, 2294.92Delphinidin-Hex-DeHex-DeHex
3224.30C26H29O16597.1478465, 303, 257, 2294.67Delphinidin-Hex-Pen
3325.71C27H31O16611.1632465, 303, 257, 2294.15Delphinidin-Hex-DeHex
3426.30C21H21O12465.1045303, 257, 2293.76Delphinidin-Hex
3526.70C21H21O12465.1046303, 257, 2293.97Delphinidin-Hex
3627.69C15H11O5271.0613253, 225, 197, 1504.43Pelargonidin isomer
3729.33C15H11O5271.0608253, 225, 197, 1502.58Pelargonidin isomer
3829.70C15H11O5271.0611253, 225, 197, 1503.69Pelargonidin isomer
3931.30C15H11O5271.0611253, 225, 197, 1503.69Pelargonidin isomer
4036.54C15H11O7303.0511257, 229, 201, 165, 1533.86Delphinidin
4136.93C15H11O5271.0610253, 225, 197, 1503.32Pelargonidin isomer
4241.71C15H11O6287.0563241, 2134.48Cyanidin
Hex: hexose; DeHex: deoxyhexose; Pen: pentose.
Table
Table 4. Linear regression equation, limit of quantification (LOQ), and limit of detection (LOD) for four anthocyanins.
Table 4. Linear regression equation, limit of quantification (LOQ), and limit of detection (LOD) for four anthocyanins.
AnthocyaninsRegression EquationR2LOQ
(ng/mL)		LOD
(ng/mL)		Linear Range
(ng/mL)
Delphinidin-3-O-glucosidey = 26.0x − 322.60.994116.983.0110–1500
Cyanidin-3-O-glucosidey = 312.6x − 3160.10.99807.092.3610–1500
Delphinidin-3-O-rutinosidey = 6260.1x − 24904.00.996913.864.5510–1500
Cyanidin-3-O-rutinosidey = 3216.0x − 8827.90.995718.445.3810–1500
In the linear regression equation, y represents the peak area of quantitative ions, and x represents the concentration of anthocyanins.
Table
Table 5. The four anthocyanin contents of R. meyeri fruits (mg/g, n = 3).
Table 5. The four anthocyanin contents of R. meyeri fruits (mg/g, n = 3).
SampleDelphinidin-3-O-glucosideCyanidin-3-O-glucosideDelphinidin-3-O-rutinosideCyanidin-3-O-rutinoside
Ribes meyeri fruits methanol extract2.27 ± 0.054.76 ± 0.020.06 ± 0.0022.01 ± 0.37
Ribes meyeri fruits anthocyanin fraction5.99 ± 0.0926.70 ± 0.590.09 ± 0.0079.36 ± 1.01
Table
Table 6. Antioxidative and antidiabetic activities of R. meyeri fruits (Expressed as IC50, μg/mL, n = 3).
Table 6. Antioxidative and antidiabetic activities of R. meyeri fruits (Expressed as IC50, μg/mL, n = 3).
SampleDPPH Radical Scavenging RateABTS Radical Scavenging Rateα-Glucosidase Inhibitory
Activity		α-Amylase
Inhibitory Activity		PTP1B
Inhibitory
Activity
R. meyeri fruits
methanol extract		111.22 ± 2.284.06 ± 0.32-0.35 ± 0.01744.60 ± 6.00
R. meyeri fruits anthocyanin fraction74.22 ± 8.259.89 ± 0.8756.67 ± 0.560.30 ± 0.01218.72 ± 0.49
Positive controlAscorbic acid5.97 ± 0.062.61 ± 0.12
Acarbose 330.23 ± 1.033.91 ± 0.05
Sodium vanadate 1.46 ± 0.40
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Zhang, L.; Wang, Q.; Zhao, Y.; Ge, J.; He, D. Phenolic Profiles, Antioxidant, and Hypoglycemic Activities of Ribes meyeri Fruits. Foods 2023, 12, 2406. https://doi.org/10.3390/foods12122406

AMA Style

Zhang L, Wang Q, Zhao Y, Ge J, He D. Phenolic Profiles, Antioxidant, and Hypoglycemic Activities of Ribes meyeri Fruits. Foods. 2023; 12(12):2406. https://doi.org/10.3390/foods12122406

Chicago/Turabian Style

Zhang, Le, Qiang Wang, Yayun Zhao, Juan Ge, and Dajun He. 2023. "Phenolic Profiles, Antioxidant, and Hypoglycemic Activities of Ribes meyeri Fruits" Foods 12, no. 12: 2406. https://doi.org/10.3390/foods12122406

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