Ultrasound assisted aqueous two-phase extraction of anthocyanins from blueberry and its anti-tumor activity

Abstract

Blueberry contains abundant anthocyanins, polyphenols, polysaccharides, and other active compounds. Anthocyanins are one of the most principal active components in blueberry, which have high medical and medicinal values. Consequently, the aim of this paper is to optimize the ultrasound assisted aqueous two-phase extraction (UATPE) of anthocyanins from blueberry by response surface methodology (RSM). The optimal extraction process to achieve the highest yield of anthocyanins (5.96 ± 0.04) mg/g from blueberry via UATPE was obtained under the ultrasound power of 269 W, extraction temperature of 56 °C, ammonium sulfate mass fraction of 23 %, and ethanol concentration of 25%. Subsequently, we evaluated the anti-tumor activity of blueberry anthocyanins extract (BAE) obtained under the optimal process on breast cancer by MTT, confocal laser microscopy, and flow cytometer. The results show that BAE could memorably inhibit MCF-7 cells viability, increase the intensity of nuclear blue fluorescence, and accelerate the apoptosis of MCF-7 cells. The findings provide an effective and feasible method for the extraction of anthocyanins. Moreover, these results provide a material basis for the development of natural anti-tumor drugs.

Introduction

Blueberry (Vaccinium spp.), as a small berry, is widely distributed in North America, Russia, and Northeast China (Duan et al., 2022). Blueberry has high medical and medicinal values due to its anthocyanins, polyphenols, flavonoids, and other active ingredients (Kalt et al., 2020). Hence, blueberry has been widely used in the fields of food, medicine, and health products. Anthocyanins are one of the most important active components in blueberry, which belong to a water-soluble natural pigment, safe, and non-toxic (Wang et al., 2022). Increasing researches have indicated that anthocyanins showed various biological activities such as antioxidant, antitumor, anti-inflammatory, visual enhancement, immune regulation, anti-fatigue, and other activities (Mattioli et al., 2020; Yang et al., 2021; Correa-Betanzo et al., 2014). High efficient extraction of anthocyanins is the premise for theirs development and utilization. Consequently, how to green and efficiently extract anthocyanins from blueberry has become a research hotspot.

Currently, anthocyanins are mainly extracted by traditional solvent extraction method. This method is simple and does not require special equipment. However, This method exists some disadvantage including low extraction efficiency, large solvent consumption, and time-consuming (Silva et al., 2017). Hence, the traditional solvent extraction method has not been used for extraction anthocyanins from natural resources on a large scale. With the continuous development of extraction technology, numerous potential extraction methods, namely, ultrasound assisted extraction (UAE), microwave assisted extraction (MAE), and ultra high pressure assisted extraction (UHPAE) were proposed. UAE, as a novel extraction technology, uses the cavitation, thermal, and mechanical effects produced by ultrasound to accelerate the rupture of plant cell wall and reduce the resistance of mass transfer in cells, which ultimately achieves the effect of enhancing the extraction anthocyanins (Liu et al., 2020). Therefore, UAE has been widely used in the extraction of polyphenols, total flavonoids, polysaccharides, and other active ingredients (Chmelova et al., 2020; Liu et al., 2021a; Meng et al., 2021; Zeng et al., 2022). MAE uses microwave selective heating to quickly heat the extraction solution, and the solvent in cells evaporates rapidly to generate pressure. Finally, the cell wall of blueberry breaks to enhance the extraction of anthocyanins (Pham et al., 2022). Microwave selective heating leads to uneven temperature distribution in the extract, which ultimately leads to a large amount degradation of anthocyanins (Kurtulbas Sahin et al., 2021). UHPAE, as a new non thermal processing technology, forms a greater force on the raw material cells by increasing the pressure, causing the rupture of the cell wall, accelerating the contact between the active components in the matrix and the solvent, which makes the active components more soluble in the extraction solvent (He et al., 2018). UHPAE has the advantages of short time, energy saving, simple operation, and high efficiency, which can well maintain the heat sensitivity and the biological activities of small molecules (Kitryte et al., 2020). Presently, UHPAE is not only used for sterilization of jam and wine. In addition, UHPAE is also used to extract the active components from traditional Chinese medicine and berries (Tan et al., 2018; Julia and Asuero, 2019). However, the investment cost of this extraction method is relatively high (Chen et al., 2022). Therefore, this method is not suitable for large-scale extraction anthocyanins from natural resources. To sum up, it is found that the above extraction methods use a single extraction solvent, and the yield of anthocyanins is relatively low. Currently, aqueous two-phase extraction (ATPE) is considered as a green, efficient, and universal extraction method for natural active ingredients, and it can achieve high product purity and high yield, and maintain the biological activities of active ingredients, which has been widely used in the separation of proteins, polysaccharides, and antibiotics (Zeng et al., 2016; Zhu et al., 2020; Diaz-Quiroz et al., 2020). In recent years, it has been found that the combination of multiple extraction methods can obtain more substances than a single one. To promote the mass-transfer, ultrasound was used to assist the ATPE extraction of anthocyanins (UATPE). This method have several advantages including high extraction efficiency, high product purity, high yield, low extraction cost, good resolution, and simple scale-up (Ji et al., 2018). Moreover, this method tries to combine the advantages of ATPE and UAE, and integrate the isolation, purification, and enrichment in a one-step procedure (Zhu et al., 2022). Compared with UAE and ATPE, this method has higher efficiency, less extraction time, lower energy consumption, and higher purity and higher yield (Ji et al., 2018). To data, there are limited reports on UATPE anthocyanins from blueberry. At present, the biological activities of anthocyanins mainly focus on the antioxidant activity of anthocyanins (Liu et al., 2021b). However, there are limited reports on the anti-tumor activity of anthocyanins extracts obtained by UATPE. Hence, this study aimed to: 1) evaluate the influence of four factors (ultrasound power, extraction temperature, ammonium sulfate mass fraction, and ethanol concentration) on anthocyanins yield; 2) optimize the extraction parameters of UATPE anthocyanins from blueberry by RSM; 3) evaluate the anti-tumor activity of anthocyanins extract obtained under the optimal extraction process. The findings provide an efficient extraction method for anthocyanins extraction from natural resources.

Materials and Methods

Experimental materials  Rabbiteye blueberry was purchased from in Heilongjiang Youxian Ecological Agriculture Co., Ltd (Harbin, China). Blueberries were pureed by a beating engine (XY-8688, Demas Instrument Co., Ltd, Shanghai, China), and dried in a freezing-vacuum dryer (BLK-FD, Bolaike Refrigeration Technology Development Co., Ltd, Jiangsu, China) at −20 °C for 72 h, and the dehydrated blueberries were milled into powdered particles smaller than 0.45 mm to obtain blueberry powder. Cyanidin-3-O-glucoside (C3G) was provided from Gepute Biotechnology Co., Ltd (Chendu, China). Human breast cancer MCF-7 cells were purchased from Union Medical College Cell Bank (Beijing, China). High sugar medium (DMEM), phosphate buffer, and fetal bovine serum (FBS) are from Sigma Company of the United States. Reactive oxygen species (ROS) and Annexin V-FITC/PI cell apoptosis test kits were obtained Yuanye Biotechnology Co., Ltd (Shanghai, China).

Preparation of ATPS  The ATPS is prepared with reference to the method of Liu et al. (2013). In short, accurately weighed a certain amount of ammonium sulfate and put it into a conical flask, then added an appropriate amount of deionized water to fully dissolve it. We adjusted the above solution to pH 3.0 with 1 mol/L HCl, and then a certain proportion of ethanol was added. The aqueous two-phase was formed after fully mixing.

UATPE procedure  Blueberry powder (5.0 g) was dissolved in 150 mL of ATPS, and then put it into the ultrasound equipment (TL-650CT, Tianling Instrument Co., Ltd, Jiangsu, China). According to the results of previous pre-experiments, the ultrasound power, extraction temperature, and extraction time were set as 400 W, 50 °, and 30 min, respectively. The filtrates were centrifuged (8 000 g for 15 min) by a high speed centrifuge (TD5KR, Shunzhi Instrument Manufacturing Co., Ltd, Shanghai, China). The upper and lower phases were separated by a separating funnel when the two phases reached equilibrium, and then the anthocyanins yield in the upper phase was determined by pH differential method.

Anthocyanins yield  The pH differential method was employed to measure the anthocyanins yield. The specific operation steps were performed based on the description of Tan et al. (2020). The absorbance of sample was determined via the UV-Vis spectrophotometer (SH-6600, Shengaohua Environmental Protection Technology Co., Ltd, Changzhou, China) at 510 and 700 nm, respectively. The yield of anthocyanins was calculated by using equation 1.

  

Where, M_ω is the molecular weight of C3G, g/mol; DF is the dilution factory;ε is the molar extinction coefficient of C3G, L/cm mol; L is the path length, cm; V is the total volume of extraction solvent, mL; m is the weight of blueberry powder, g.

Experimental design Single factor experiment  In this study, the UATPE method was selected to extract anthocyanins from blueberry. The appropriate experimental factors are helpful to improve the yield of anthocyanins. According to the results before our preliminary experiment, the experimental factors and their ranges are selected as follows. The ultrasound power of 80–400 W, extraction temperature of 40–60 °C, ammonium sulfate mass fraction of 20 %–28 %, and ethanol concentration of 22 %–26 % were firstly applied to single- factor experiment. Each experiment was repeated thrice, and experimental data were described as mean ± standard deviation.

Box–Behnken design  In this study, the combined experiments were designed by Box-Behnken design (BBD) based on RSM with four factors and three levels. According to the previous single factor experimental results, four experimental variables and their ranges were set as follows: ultrasound power (X₁, 160–320 W), extraction temperature (X₂, 50–60 °C), ammonium sulfate mass fraction (X₃, 20 %–24 %), and ethanol concentration (X₄, 24 %–26 %). X₁, X₂, X₃, and X₄ were set as independent experimental variables, and Y was set as dependent variable. A total of 29 randomized trials were conducted to reduce the impact of external factors on anthocyanins yield (Table 1).

Table 1 Experimental design and results of RSM.

No.	Variable				Anthocyanins yield/(mg/g)	Predicted anthocyanins yield/(mg/g)
	X1 /W	X2 /°C	X3 /%	X4 /%
1	−1 (200)	−1 (40)	0 (21)	0 (24)	4.09	4.12
2	1 (400)	−1 (40)	0 (21)	0 (24)	5.03	4.94
3	−1 (200)	1 (60)	0 (21)	0 (24)	4.65	4.89
4	1 (400)	1 (60)	0 (21)	0 (24)	5.01	5.13
5	0 (300)	0 (50)	−1 (20)	−1 (22)	4.07	4.14
6	0 (300)	0 (50)	1 (22)	−1 (22)	5.36	5.49
7	0 (300)	0 (50)	−1 (20)	1 (26)	5.55	5.58
8	0 (300)	0 (50)	1 (22)	1 (26)	5.03	5.12
9	−1 (200)	0 (50)	0 (21)	−1 (22)	4.08	4.09
10	1 (400)	0 (50)	0 (21)	−1 (22)	5.34	5.33
11	−1 (200)	0 (50)	0 (21)	1 (26)	5.31	5.34
12	1 (400)	0 (50)	0 (21)	1 (26)	5.14	5.16
13	0 (300)	−1 (40)	−1 (20)	0 (24)	4.04	4.21
14	0 (300)	1 (60)	−1 (20)	0 (24)	5.14	5.09
15	0 (300)	−1 (40)	1 (22)	0 (24)	4.97	5.05
16	0 (300)	1 (60)	1 (22)	0 (24)	5.28	5.14
17	−1 (200)	0 (50)	−1 (20)	0 (24)	4.68	4.51
18	1 (400)	0 (50)	−1 (20)	0 (24)	5.03	4.99
19	−1 (200)	0 (50)	1 (22)	0 (24)	5.05	4.91
20	1 (400)	0 (50)	1 (22)	0 (24)	5.49	5.48
21	0 (300)	−1 (40)	0 (21)	−1 (22)	4.08	3.98
22	0 (300)	1 (60)	0 (21)	−1 (22)	5.33	5.24
23	0 (300)	−1 (40)	0 (21)	1 (26)	5.37	5.29
24	0 (300)	1 (60)	0 (21)	1 (26)	5.08	5.00
25	0 (300)	0 (50)	0 (21)	0 (24)	5.85	5.74
26	0 (300)	0 (50)	0 (21)	0 (24)	5.97	5.74
27	0 (300)	0 (50)	0 (21)	0 (24)	5.91	5.74
28	0 (300)	0 (50)	0 (21)	0 (24)	6.03	5.74
29	0 (300)	0 (50)	0 (21)	0 (24)	4.93	5.74

RSM model  Design Expert version 8 was employed to analyze the relationship between four experimental factors (X₁, X₂, X₃, and X₄) and anthocyanins yield (Y). The regression coefficient of the model is obtained by fitting the experimental results with equation 2.

  

The RSM model was analyzed by using the analysis of variance (ANOVA). The coefficient of determination (R²) and lack-of-fit were used to estimate the adequacy of RSM model. In addition, the coefficient of variation (C.V.) was used to evaluate the relative dispersion of experimental points.

Models analysis  The indexes were used to evaluate the prediction performance of RSM, such as R², MSE, RMSE, SSE, and ADD (Tan et al., 2022).

  

  

  

  

  

Anti-tumor activity Cell viability  MTT assay was used to determine the effect of blueberry anthocyanins extract (BAE) obtained under the optimal process on the viability of MCF-7 cells. The MCF-7 cells (1 × 10⁴ cells/mL) were cultured in 96-well plates at 37 °C in a humidified 5 % CO₂ incubator for 24 h. MCF-7 cells were intervened with different concentrations of BAE (0, 1.0, 10.0, and 100.0 µg/mL) for 24, 48, and 72 h, and 6 multiple holes are set for each concentration. After the above intervention, the supernatant of MCF-7 cells was removed, and then added 20 µL MTT (5 mg/mL) and cultured for 4 h. Subsequently, the culture medium was replaced and added 150 µL DMSO in each well. The absorption of each sample was measured through the enzyme label analyzer (HBS-1096A, Detie Experimental Equipment Co., Ltd, Nanjing, China) at 490 nm. The equation 8 was used to calculate cell viability (Tan et al., 2020).

  

where, A₀ is the average absorbance of the blank group. A₁ is the average absorbance of treatment groups with different concentrations of BAE.

Hoechst 33342 staining  The apoptosis morphology of MCF-7 cells was observed by the confocal microscope (FV3000, Olympus, Japan). MCF-7 cells (2 × 10⁵ cells/mL) were cultured in 6-well plates and incubated 48 h at 37 °C in a humidified 5 % CO₂ incubator. After intervention, MCF-7 cells were stained with Hoechst 33342 (1 mL/well) for 20 min. The 6-well plates were washed 2–3 times with PBS, and then observed morphological changes.

Apoptosis assay  The multicolor flow cytometer (Attune NxT, Thermo Fisher Technology Co., Ltd) was used to determine the apoptosis of MCF-7 cells. Based on the method described by Tan et al. (2020), the above treated MCF-7 cells were washed by ice-cold PBS and collected, and then resuspended in Annexin V binding buffer. Interventional MCF-7 cells were added with 5 µL Annexin V-FITC and 10 µL propidium iodide (PI) dye solution in dark at 4 °C for 30 min. Subsequently, the samples were determined via the multicolor flow cytometer.

Statistical analysis  The experimental data are showed as the means ± standard deviation. Statistical 8.0 software is used to analyze the variance of each group of experimental data. p < 0.05 shows the experimental results with statistical significance. The extraction process of anthocyanins is optimized through Design Expert 8.0 software.

Results and Discussion

Effect of extraction conditions on anthocyanins yield  When studied the effect of ultrasound power on anthocyanins yield, the levels of extraction temperature, ammonium sulfate mass fraction, and ethanol concentration were fixed at 50 °C, 24 %, and 25 %, respectively. The results are displayed in Fig. 1A. Fig. 1A shows that the anthocyanins yield initially raised dramatically with increasing of ultrasound power, and then markedly decreased (p < 0.05). The maximum anthocyanins yield (5.72 ± 0.03) mg/g was obtained when the ultrasonic power was 240 W. The reason was that the “cavitation effect” and “mechanical oscillation effect” produced by ultrasound enhanced with the increase of ultrasound power, which promoted the destruction of cell wall, reduced the mass transfer resistance of anthocyanins in cells, and improved the diffusion coefficient and yield of anthocyanins from blueberry (Liu et al., 2020a). However, excessive high ultrasound power might destroy the structure of anthocyanins, which was not conducive to the extraction of anthocyanins (Xue et al., 2020a). Hence, 160, 240, and 320 W ultrasound power were used in the subsequent combination experiments.

Fig. 1.

Effect of different extraction factors on the anthocyanins yield from blueberry: (A) ultrasound power, (B) extraction temperature, (C) ammonium sulfate mass fraction, (D) ethanol concentration. Note: different letters suggest remarkable difference (p < 0.05).

When investigated the effect of extraction temperature on anthocyanins yield, the levels of ultrasound power, ammonium sulfate mass fraction, and ethanol concentration were fixed at 240 W, 24 %, and 25 %, respectively. The results are shown in Fig. 1B. Fig. 1B displays that the anthocyanins yield increased substantially with the elevating of extraction temperature when the extraction temperature was lower than 50 °C (p < 0.05). This phenomenon was attributed to the fact that the increase of extraction temperature improved the solubility of anthocyanins, enhanced extraction capacity, and promoted anthocyanins extraction (Belwal et al., 2020). Nevertheless, high temperature might cause the degradation of anthocyanins due to theirs thermal susceptibility. The similar results were obtained by other authors in the case of anthocyanins from raspberry under different extraction temperatures (Xue et al., 2020b). Consequently, 50, 55, and 60 ° extraction temperature were used in follow-up combination experiments.

When investigated the effect of ammonium sulfate mass fraction on anthocyanins yield, the levels of ultrasound power, extraction temperature, and ethanol concentration were fixed at 240 W, 50 °C, and 25 %, respectively. The results are shown in Fig. 1C. Fig. 1C describes that the anthocyanins yield memorably improved with the increase of ammonium sulfate mass fraction (p < 0.05). This reason was attributed to the increase of ammonium sulfate mass fraction, which increased its water absorption capacity, resulting in the decrease of water molecules in the upper phase and the enhancement of its polarity, which promoted the gradual dissolution of anthocyanins (Liu et al., 2022). When the ammonium sulfate mass fraction exceeded 24 %, the anthocyanins yield decreased significantly with the increase of ammonium sulfate mass fraction (p < 0.05). This result was consistent with Zhai et al., (2017) results of extraction anthocyanins from black bean skin by aqueous two-phase extraction. Therefore, the ammonium sulfate mass fraction of 20 %, 22 %, and 24 % were used in follow-up combination experiments.

When investigated the effect of ethanol concentration on anthocyanins yield, the levels of ultrasound power, extraction temperature, and ammonium sulfate mass fraction were fixed at 240 W, 50 °, and 24%, respectively. The results are described in Fig. 1D. Fig. 1D shows that the anthocyanins yield substantially enhanced with the increase of ethanol concentration from 22 % to 25 % (p < 0.05) and reached the highest anthocyanins yield (5.76 ± 0.03) mg/g at 25 %. This phenomenon was attributed to the fact that the increase of ethanol concentration improved extraction capacity and anthocyanins solubility, which was helpful to the extraction of anthocyanins (Xue et al., 2021a). However, high concentration ethanol could dissolve alcohol soluble impurities, reduce anthocyanins solubility, which was not conducive to the extraction of anthocyanins (Xue et al., 2021b). The similar results are found by other authors in the case of anthocyanins from purple sweet potatoes and roselle by aqueous two-phase extraction extraction (Liu et al., 2013; Liu et al., 2022). Thus, the ethanol concentration used for the follow-up experiments were 24 %, 25 %, and 26 %.

RSM modeling  Taking four independent variables (X₁, X₂, X₃, and X₄) and anthocyanins yield (Y) as dependent variables, the extraction anthocyanins from blueberry was investigated by BBD method based on RSM. Table 1 shows the experimental design and results of anthocyanins yield under different combined experimental conditions, and Table 2 shows the ANOVA of equation 9 for anthocyanins yield. The values of p and F are used to analyze the importance of experimental factors, and the low p values and high F values imply that the corresponding experimental factors can dominantly affect the response values. The results display that the regression model of anthocyanins yield was extremely remarkable at a level of p < 0.0001. Nevertheless, the lack of fit of regression model with anthocyanins yield was not prominent (p = 0.1603 > 0.05). In the case of anthocyanins yield, X₁, X₂, X₄, X₃X₄, X₁², X₂², X₃², and X₄² regression model parameters were found highly notable (p < 0.01), and X₁X₄ and X₂X₄ were marked (p < 0.05), whereas other experimental variables had no remarkable effect on the anthocyanins yield (p > 0.05). Moreover, the values of R² and C.V. were 0.8823 and 0.7007, respectively.

Table 2 ANVOA analysis for RSM model.

Source of variance	SQ	df	MS	F value	P value
Model	8.14	14	0.58	7.49	0.0003**
X1	0.84	1	0.84	10.85	0.0053**
X2	0.71	1	0.71	9.09	0.0093**
X3	0.59	1	0.59	7.65	0.0152*
X4	0.86	1	0.86	11.13	0.0049**
X1X2	0.084	1	0.084	1.08	0.3157
X1X3	2.025E-003	1	2.025E-003	0.026	0.8740
X1X4	0.51	1	0.51	6.58	0.0224*
X2X3	0.16	1	0.16	2.01	0.1782
X2X4	0.59	1	0.59	7.64	0.0152*
X3X4	0.82	1	0.82	10.55	0.0058**
X12	1.21	1	1.21	15.55	0.0015**
X22	1.86	1	1.86	23.93	0.0002**
X32	0.72	1	0.72	9.25	0.0088**
X42	0.69	1	0.69	8.90	0.0099**
Residual	1.09	14	0.078
Lack of fit	0.25	10	0.025	0.12	0.9967
Pure error	0.83	4	0.21
Sum	9.23	28
	R2 = 0.8823		R2Adj = 0.8043	C.V. = 0.7007

In this study, the factors in the RSM model that have no significant impact on the anthocyanins yield are eliminated, and then the experimental data are analyzed by multiple regression based on the results of the combined experiments to obtain the regression model of different factors on the anthocyanins yield. The regression model of anthocyanins yield (Y) is shown in equation 9.

  

The adjusted R²_adj was close to R² of the regression model of anthocyanins yield. In addition, non-significant lack of fit indicated that the developed regression model could predict the anthocyanins yield under different experimental conditions.

Effect of interaction of experimental factors on anthocyanins yield  According to equation 9, the 2D contour and 3D response surface charts are drawn. Figs. 2A, 2C, and 2E show that the interaction of ultrasound power (X₁) and ethanol concentration (X₄), extraction temperature (X₂) and ethanol concentration (X₄), ammonium sulfate mass fraction (X₃) and ethanol concentration (X₄) significantly affected the anthocyanins yield (p < 0.05) when the other two factors are set to zero level. Increasing studies have confirmed that the 2D contour graph of the interaction of two factors was oval, indicating that the interaction of two factors could significantly affect the response value. Figs. 2B, 2D, and 2F display oval, suggesting that the interaction of X₁X₄, X₂X₄, and X₃X₄ substantially influenced the anthocyanins yield. The results were consistent with the result of analysis of variance (Table 2).

Fig. 2.

Effect of interaction of experimental variables on anthocyanins yield: 3D response surface charts (A, C, and E) and corresponding 2D contour plots (B, D, and F).

Performance evaluation of RSM  A perfect match was obtained by plotting the experimental values and prediction results generated by RSM model, implying that the performance of the model was very good (Fig. 3A). The R² value of RSM model was 0.8823. In addition, the MSE, SSE, RMSE, and ADD values of RSM model were 0.0016, 0.0004, 0.0400, 2.7152 %, respectively (Fig. 3B), which indicated that RSM model showed good prediction ability and could predict anthocyanins yield under different extraction conditions. Hence, RSM model was used to optimize the extraction process of anthocyanins from blueberry.

Fig. 3.

Relationship between the actual experimental results vs. predicted results by RSM (A) and matching between all the datasets (B).

Optimization of the process  The extraction parameters of anthocyanins from blueberry were optimized by using RSM model. The optimized extraction parameters obtained by RSM model were ultrasound power of 268.8 W, extraction temperature of 55.8 °C, ammonium sulfate mass fraction of 22.82 %, and ethanol concentration of 24.83 %. Under the combination of the above parameters, the anthocyanins yield was 5.80 mg/g. Considering the actual situation, the ultrasound power, extraction temperature, ammonium sulfate mass fraction, and ethanol concentration were modified as 269 W, 56 °C, 23 %, and 25 %, respectively. Three repeated experiments were performed under the above parameters, and the anthocyanins yield was 5.96 ± 0.04 mg/g. The relative error between the experimental value and the theoretical value was 2.76 %, indicating that the RSM model optimization method was reliable and reasonable.

Effect of BAE and treatment time on the cell viability  First, we determined the effect of BAE on cell viability by the MTT assay. The experimental data are shown in Fig. 4. Fig. 4 displays that the cell viability decreased significantly with the increase of BAE concentration from 1.0 to 100.0 µg/mL at 24, 48, and 72 h in a concentration dependent manner (p < 0.05). When the concentration of BAE was in the range of 1–100 µg/mL, the cell viability treated with the same concentration of BAE for 24 h was significantly lower than that of 48 h and 72 h (p < 0.05). However, there was no significant difference in the cell viability after 48 h and 72 h treatment (p > 0.05). Therefore, MCF-7 cells were treated with BAE for 48 h for further analysis. The MTT results indicate that BAE can effectively inhibit the growth of human breast cancer MCF-7 cells in a concentration dependent manner.

Fig. 4.

Effect of different concentrations of BAE on the cell viability. Note: Different lowercase letters imply markedly differences among different sample concentrations (p < 0.05), and different capital letters suggest that there are remarkable differences among different samples of the same concentration (p < 0.05).

Effect of BAE on apoptosis morphology of MCF-7 cells  In this experiment, we first observed the apoptotic morphology of MCF-7 cells by Hoechst 33342 staining, and the results are shown in Fig. 5. Compared with the control group (0 µg/mL BAE), the number of MCF-7 cells in BAE treatment groups with different concentrations (1.0–100.0 µg/mL) decreased significantly, while the number of irregular MCF-7 cells increased. In addition, when MCF-7 cells were treated with different concentrations of BAE (1.0–100.0 µ/mL), the intensity of nuclear blue fluorescence of MCF-7 cells significantly enhanced in a concentration dependent manner. This phenomenon is attributed to the fact that the hydroxyl group in BAE accelerated the cell apoptosis and improved the intensity of nuclear blue fluorescence of MCF-7 cells (Xue et al., 2020a). The findings show that BAE can induce apoptosis of MCF-7 cells, and then inhibit the growth of MCF-7 cells.

Fig. 5.

Changes of apoptosis morphology of MCF-7 cells at different concentrations of BAE (×100).

Effect of BAE on apoptosis rate of MCF-7 cells  At present, numerous studies have indicated that anthocyains from natural resources showed a strong antioxidant and free radical scavenging capacity (Wu et al., 2011; Vidana Gamage et al., 2021). Moreover, BAE also showed significant inhibitory effect on the proliferation of cancer cells, and accelerated the apoptosis of cancer cells (Li et al., 2019). Therefore, on the basis of MTT and Hoechst 33342 staining to observe the apoptotic morphology of MCF-7 cells, flow analyzer was used to further explore the effect of BAE on the apoptotic rate of MCF-7 cells. The results are shown in Fig. 6. Fig. 6B describes that the apoptosis rate of MCF-7 cells enhanced significantly with increasing of BAE concentration in a concentration dependent manner (p < 0.05). When the concentration of BAE was 1.0, 10.0, and 100.0 µg/mL, the apoptosis rates of MCF-7 cells were 11.28 % ± 0.15 %, 35.25 % ± 1.12 %, and 55.23 % ± 2.14 %, respectively. Compared with the control group (0 µg/mL BAE), the apoptosis rate of MCF-7 cells in BAE treatment groups with different concentrations (1.0, 10.0, and 100.0 µg/mL) increased by 6.43 %, 30.40 %, and 50.38 %, respectively. The reason for this phenomenon was that there was a close relationship between the existence of o-diphenol in the ring of anthocyanin B and the hydroxyl radical at position 3 (Xue et al., 2021b). The results show that BAE can inhibit the proliferation of MCF-7 cells and accelerate the apoptosis in a concentration dependent manner.

Fig. 6.

Effect of BAE on apoptosis in MCF-7 cells for 48 h. (A) Apoptosis flow plots of MCF-7 cells, and (B) Analysis of results of cell apoptosis.

Conclusion

This paper studied the UATPE anthocyanins from blueberry and optimized the extraction condition by RSM model. RSM model was established and showed sufficient reliability in predicting the anthocyanins yield. The optimum extraction parameters to achieve the highest yield of anthocyanins (5.96 ± 0.04) mg/g from blueberry through UATPE was obtained under the ultrasound power of 269 W, extraction temperature of 56 °C, ammonium sulfate mass fraction of 23 %, and ethanol concentration of 25 %. In addition, BAE could memorably inhibit MCF-7 cells viability, increase the intensity of nuclear blue fluorescence, and accelerate the apoptosis of MCF-7 cells. The research results provide important material resources and research basis for the development of natural anti breast cancer drugs.

Acknowledgements  The authors gratefully thank the financial support provided by University level scientific research project of Guangzhou City Vocational College in 2022 (2022JKY04010) for this research project.

Conflict of interest  There are no conflicts of interest to declare.

CRediT authorship contribution statement  Huan Wang: Writing-original draft, Writing-review & editing, Data curation, Visualization. Qiang Jia: Methodology, Formal analysis. Jinjin Jiang: Formal analysis and Software. Lihua Huang: Conceptualization, Resources, Writing-review & editing, Supervision.

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