Effects of different extraction methods on antioxidant properties of blueberry anthocyanins

2.1.1 Experimental materials

The main experimental material is blueberries from the small berry garden of Jilin Agricultural University. The selected blueberry fruit (the variety is patriot) is provided by Liaoning Dandong Organic Food Co., Ltd, which has reached the fruit period of 8 weeks and is in a mature state, with a diameter of 5–10 mm, purple red, moisture content of 88%, half height clumped blueberry variety. Fresh fruits are picked and stored in a refrigerator at −20°C. During the experiment, the frozen fruits were taken out of the refrigerator at −20°C and thawed in the refrigerator at 4°C.

2.1.2 Experimental reagents

The preparation and the selection of experimental reagents mainly include two aspects, namely, the reagents used in the determination process and the relevant reagents used in the anthocyanin extraction process. The reagents required for the experiment are formic acid, anhydrous methanol, sodium acetate, ferric trichloride, DPPH, ABTS (Sinopharm Chemical Reagent Co., Ltd), potassium chloride, potassium hydroxide, aluminum trichloride, lead acetate (Xilong Chemical Co., Ltd), hydrochloric acid, sodium dihydrogen phosphate (Beijing Chemical Plant), ferric trichloride, potassium persulfate, ascorbic acid, trihydroxymethyl aminomethane, soybean lecithin (Sinopharm Chemical Reagent Co., Ltd), cyanidin-3-glucoside standard, and standard of mallow pigment-3-galactoside (Sigma Company).

2.1.3 Experimental instruments

As with the experimental reagents, the instruments used in this experiment are also divided into three aspects: anthocyanin extraction, anthocyanin content identification, and antioxidant performance testing. The equipment and models required for the experiment include LBI206 Ultrasonic Chinese medicine processor (Jining Aobo ultrasonic electric Co., Ltd), UV-6100 Ultraviolet visible spectrophotometer, L3600D Low speed, PHSJ-3F Precision PH meter (Shanghai Yuan Analytical Instrument Co., Ltd), XDB-C18 chromatographic column, LCMS-IT/TOF High performance liquid phase ion trap/time of flight tandem mass spectrometer, LC-20AD High resolution fast liquid chromatography system (SHIMADZU Company), AL104 Electronic balance (Mettler Toledo), and XMTB Electrothermal constant temperature Water bath (Tianjin Zhonghuan Experimental Electric Furnace Co., Ltd).

2.2.1 Preparation of blueberry crude extract

Five solvents containing 3% trifluoroacetic acid were used, including (water, n-butanol, methanol, 70% ethanol, ethyl acetate), with a solid–liquid ratio of 1:5 (g/mL) and ultrasonic-assisted extraction at 30°C (Power 180 W) three times, each time 30 min, the material liquid ratio of the second and third times is halved. The extracts were combined and centrifuged at 4,500 rpm for 5 min, the supernatant was taken, and the solvent was concentrated under reduced pressure at 40–60°C, and the extract was freeze-dried to obtain a sample water extract. A 60% ethanol solution was used to mix uniformly at a solid–liquid ratio of 1:20 (m/V), and the mixture was extracted in a constant temperature water bath at 50°C for 90 min.

2.2.2 Preparation of blueberry extract

Using methanol containing 0.3% trifluoroacetic acid as a solvent, the aforementioned extraction steps are followed to obtain an extract-like extract, dissolve with 500 mL of purified water, filter, and take 300 mL of the filtrate onto a LS-305 macroporous resin column with a 0.3% trifluoroacetic acid aqueous solution (approximately 400 mL). Impurities such as sugar and acid are removed by rinsing and then eluted with 0.3% trifluoroacetic acid methanol solution (approximately 100 mL). The eluate was collected and concentrated under reduced pressure at 40°C to extract, dissolve with purified water and make up 150 mL. 25 mL of this sample solution was taken, concentrated under reduced pressure at 40°C to extract, and freeze-dried to obtain blueberry total extract (RP). The remaining 125 mL of the sample was sequentially extracted with ethyl acetate and n-butanol. The sample was extracted using ethyl acetate extraction three times, the first time with an equal volume and the next two times with a half volume, and combined extracts were concentrated under reduced pressure at 40°C to remove the solvent and freeze-dried to obtain blueberry ethyl acetate extract (ARP). The sample was again extracted with n-butanol four times, the first time with an equal volume and the next three times with a half volume, and the extract was concentrated under reduced pressure at 60°C to remove the solvent and freeze-dried to obtain blueberry n-butanol extract (BRP). The solvent was concentrated under reduced pressure at 50°C, and blueberry water extract was obtained after freeze-drying.

2.3.1 Solvent extraction method

Anthocyanins are less stable in neutral and weakly alkaline solutions, and so the extraction process is usually performed under acidic conditions. Steps of extraction method are as follows: Configure the solvent buffer pH 1.0 buffer: 0.2 mol/L KCl–0.2 mol/L HCl, and the pH 4.5 buffer is 1 mol/L NaAc–1 mol/L HCl. Weigh blueberries accurately, add 15 times the amount (v/w) of acidic methanol with a concentration of 80% after mechanical crushing, extract at 40°C, and determine the proportion of the solvent to collect the supernatant. Using acidic ethanol as the extractant and optimizing each factor by response surface analysis, the best extraction parameters were obtained: ethanol extract volume fraction, 60.65%; material–liquid ratio, 1:20.65(g/mL); extraction time, 122.53 min; pH, 3.0; the extraction temperature, 50°C, and the extraction was performed twice to determine the content of anthocyanins in the frozen blueberries, which was about 3.264 mg/g.

2.3.2 Enzymatic extraction

Citric acid and sodium citrate were mixed at a certain ratio to adjust the pH. 5 g of blueberry was crushed, and 5 mg/g pectinase was used for enzymolysis at 45°C, pH 4.5, and the duration of enzymatic hydrolysis with the material–liquid ratio of 1 g:8 mL was 60 min and 90 min, and after centrifugation at 2,000 rpm in 20 min, the blueberry anthocyanin extract was collected.

2.3.3 Ultrasonic extraction method

5 g of blue poisonous fruit was weighed and 60% acidified methanol with a ratio of 1:10 (g/mL) was used as the extractant and sonicated at 50°C for 50 min to obtain blueberry anthocyanin extract [13,14]. The actual quantity of extracted blueberry anthocyanin was 3.927 mg/g.

2.3.4 Extraction and purification

Three different anthocyanin extraction methods were used for purification. The purification and separation method used was centrifugation. Generally, the temperature increases during centrifugation, which has a great impact on heat-sensitive substances. Therefore, this experiment uses a refrigerated centrifuge, which can ensure that the sample is centrifuged at low temperature, so that the substance does not lose its activity [15]. The effect of centrifugation is affected by factors such as solution viscosity, centrifugation time, and centrifugation speed. The change of the absorbance value of the solution before and after centrifugation is used as an index for analysis, and the conditions close to or greater than the absorbance value of the solution before centrifugation are preferred.

2.4.1 Identification of anthocyanins

After analyzing the anthocyanins of blueberry by HPLC-DAD-MS, the experimental results were compared with the UV spectrum, mass spectrum, and retention time in the blueberry anthocyanin database to identify the anthocyanins in blueberry [16,17]. Blueberry anthocyanin identification criteria are presented in Table 1.

2.4.2 Anthocyanin monomer composition analysis

Anthocyanin composition of the blueberry extract was detected and analyzed by high-performance liquid chromatography (HPLC)-mass spectrometry. The HPLC conditions are set similar to Kromasil C18 reversed-phase column, mobile phase A: water:formic acid:acetonitrile = 92:2:6 v/v; mobile phase B: water:formic acid:acetonitrile = 44:2:54 v/v; flow rate, 1 m/min; column temperature, 50°C; detection wavelength, 525 nm; injection volume, 30 µL; gradient elution procedure: 0–4 min: 6–10% B, 4–12 min: 10–25% B, 12–13 min: 25% B, 13–20 min: 25–40% B, 20–35 min: 40–60% B, 35–40 min: 60–100% B, 40–45 min: 100–6% B. The HPLC of the obtained blueberry anthocyanins is shown in Figure 1. The results of anthocyanin monomer composition analysis are presented in Table 2.

Figure 1

HPLC spectrum of blueberry anthocyanins.

There is a high molecular conjugation system in anthocyanins, which contains acid and alkaline groups, and is easily soluble in polar solvents such as water, methanol, ethanol, dilute alkali, and dilute acid. It has strong absorption in the UV and visible region, the maximum absorption wavelength in the UV region is around 280 nm and the maximum absorption wavelength in the visible region is in the range of 500–550 nm. Anthocyanins belong to bioflavonoids, and the main physiological activity of flavonoids is free radical scavenging ability and antioxidant ability. It has been proved that anthocyanin is the most effective antioxidant and the most powerful free radical scavenger found by human beings. The antioxidant performance of anthocyanin is 50 times higher than VE and 20 times higher than VC.

2.4.3 Calculation of blueberry anthocyanin content

The color scale of blueberry anthocyanins is calculated as shown in equation (1):

In equation (1), A is the absorbance, W is the mass of the blueberry anthocyanin extracted, and r is the dilution factor of the sample taken when measuring the absorbance [18]. The color value of the extract was measured 36. The formula for calculating total anthocyanin content in the solution by wavelength scanning using pH difference method is as follows:

where and are the absorbance values of anthocyanins at 520 nm at pH 1.0 and pH 4.5, V is the total volume of the extract, n is the dilution factor, and M is the relative molecular mass of anthocyanins. In addition, ε is the extinction coefficient of anthocyanins and m is the sample mass.

2.5.1 Determination of anthocyanin resistance to lipid oxidation

50 g of lard was taken in an iodine volumetric flask, and then, blueberry anthocyanin and 0.2% ascorbic acid from different extraction methods were added to different iodine volumetric flasks. After being stirred evenly, flasks were placed in an oven at 70°C, and the peroxide value was measured every 2 days. 3 g of fat was taken for testing; 30 mL of chloroform-glacial acetic acid and 1 mL of saturated potassium iodide were added to an iodine volumetric flask, shaked for 30 s, and placed in dark for 3 min; then 100 mL distilled water was added and shaked well. When titrating with sodium thiosulfate standard solution to light yellow, two drops of starch indicator were added, and titration was continued until the blue color disappears, and a blank test was carried out at the same time. The formula for the inhibition rate of blueberry anthocyanin lipid peroxidation is as follows:

The conversion formula of the calculation result is as follows:

In equations (3) and (4), V, respectively, represent the volume of the sodium thiosulfate standard titration solution consumed in the measurement sample and the blank experiment, C is the concentration of the sodium thiosulfate standard titration solution, and M is the mass of the measurement sample.

2.5.2 Determination of anthocyanin reducing power

The reaction of divalent iron ions and ferric chloride produces Prussian blue, which has a maximum absorption at 700 nm. The reaction formula is as follows:

Therefore, the reduction ability of the antioxidant can be indirectly evaluated by measuring the absorbance at 700 nm. To eliminate the effect of yellow potassium ferricyanide on the absorbance value, after the completion of reaction, trichloroacetic acid is added to the system to react with the remaining potassium ferricyanide to form a precipitate [21]. After centrifugation, the absorbance of the supernatant and ferric chloride was measured. The stronger the reducing ability of the sample to be tested, the greater the measured absorbance value.

2.5.3 Anthocyanin clearance ˙OH determination

1 mL of blueberry anthocyanin, ferric sulfate, and salicylic acid–ethanol solution was added to the test tube, 1 mL of hydrogen peroxide was added, and then water was added in a 37°C water bath and was kept for 30 min. Distilled water was used as a blank and the absorbance was measured at 510 nm. Hydrogen peroxide was replaced with distilled water and the same method was used to measure the corresponding absorbance. The hydroxyl scavenging rate of anthocyanin from blueberry obtained by different extraction methods was calculated using equation (6).

where A X 0 is the absorbance of the background of the sample solution without the addition of hydrogen peroxide.

2.5.4 Ability of anthocyanins to remove O₂ ⁻˙

Pyrogallol’s auto-oxidation process is a chain reaction. In an alkaline environment, auto-oxidation can quickly occur to generate a superoxide anion, which in turn can accelerate the rate of pyrogel’s auto-oxidation and generate colored intermediates. The accumulation of intermediate products has a good linear relationship with time, generally maintained for about 4 min, and then gradually slowed down. When a superoxide anion scavenger is present in the system, the scavenger causes the disproportionation reaction of O₂ ⁻ to be converted into O₂ and hydrogen peroxide [22], thereby reducing the pyrogallol auto-oxidation rate and preventing the accumulation of intermediate products. The auto-oxidation rate of catechol is positively correlated with the concentration of superoxide anions in the system, so the ability of antioxidants to remove superoxide anions can be indirectly evaluated by measuring the auto-oxidation rate of catechol in the presence of antioxidants [23].

2.5.5 Anthocyanin clearance of DPPH˙

The characteristic purple-red group of 1,1-diphenyl-2-picrylhydrazyl (DPPH) solution has the maximum light absorption at 517 nm. The reduction of the absorption value after the addition of antioxidants indicates its scavenging effect on free radicals. The principle of anthocyanin clearance of DPPH˙ is shown in Figure 2 [24].

Figure 2

Reaction equation of DPPH and antioxidant.

The blueberry anthocyanin ethanol solution obtained by different extraction methods and the same volume of 0.2 mmol L⁻¹ DPPH ethanol solution were to the test tube. Absorbance was measured at 517 nm using absolute ethanol as a reference, and three measurements were taken in parallel to obtain the average value. The DPPH free radical scavenging rate is calculated as follows [25]:

where A q is 2 mL of absolute ethanol + 2 mL of DPPH solution, A p is 2 mL of anthocyanin ethanol solution + 2 mL of DPPH solution, and A w represents 2 mL of anthocyanin ethanol solution + 2 mL of absolute ethanol. The relationship between the blueberry flower color and the antioxidant activity of DPPH was obtained through testing (Table 3).