Extraction optimization, antioxidants, and lipid-lowering activities of polygonatum cyrtonema Hua polysaccharides

ABSTRACT

Polygonatum cyrtonema Hua polysaccharides (PCP) have beneficial effects on lipid metabolism and oxidative stress. Here, PCP extraction was optimized using response surface methodology (RSM), and the antioxidant and lipid-lowering effects of its main component, PCP1, were evaluated in vitro. The maximum PCP yield was 11.05% at an extraction temperature was 72°C, liquid-to-solid ratio of 21, extraction time of 96 min, and two extraction rounds. PCP1 was purified by column chromatography and showed significant scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, and 2, 2'-azino-bis 3-ethylbenzthiazo-line-6-sulfonic acid (ABTS) radicals at concentration of 0.2–1.0 mg/mL. At a concentration of 200 $\mathit{\mu }$g/mL, PCP1 significantly improved lipid disorder and damage induced by palmitic acid in HepG2 cells. These results show that PCP has potent antioxidant and lipid-lowering effects in vitro; moreover, the PCP extraction rate is significantly increased using RSM. Therefore, PCP is an effective antioxidant and lipid-lowering product with potential medicinal applications.

KEYWORDS:

• Polygonatum cyrtonema Hua
• polysaccharide
• extraction optimization
• antioxidant activity
• lipid-lowering effect

1. Introduction

Polygonatum cyrtonema Hua (PCH), a perennial herb in the family Liliaceae, is often used as a tonic for kidney disorders, a yin nourishing food, and a tissue moistener (J. Li et al., 2022). PCH contains natural substances, such as polysaccharides, saponins, flavonoids, and amino acids, and has various biological activities (L. Li et al., 2018; Tang et al., 2019). As the main natural active substance in PCH, PCH polysaccharide (PCP) has hypoglycemic, anti-inflammatory, anti-oxidation, anti-fatigue, and anti-depression effects (Cui et al., 2018; J. Li et al., 2022; S. Liu et al., 2022; F. Shen et al., 2022; W. D. Shen et al., 2021). Polysaccharides extracted from PCH cultivated on Mount Jiuhua are considered high quality. At present, PCH from Mount Jiuhua is not only an important source of income for residents, but has also been developed into tea, medicinal wine, and other products. Therefore, the enormous medicinal and economic value of PCH requires further research (J. Hu et al., 2021; Y. Hu et al., 2022).

The dysregulation of glucose and lipid metabolism can result in various diseases and poses a significant threat to human health (Ding, Wang, et al., 2021; Sun et al., 2023). Oxidative stress, insulin resistance, and inflammatory damage are considered as important mechanisms for the occurrence and development of glycolipid metabolic disorder (Ding, Tang, et al., 2021; Zhou et al., 2023). In a preliminary experiment, we found that a neutral polysaccharide, PCP1, is the main component of PCP. Chemical analysis showed that the molecular weight of PCP1 is 4,650 g/mol, and that it is mainly composed of fructose, glucose, and galactose. Pharmacological studies showed that PCP1 has the effect of improving lipid metabolism disorders, oxidative stress, and structural damage to the intestinal microflora in non-alcoholic fatty liver disease (S. Liu et al., 2022). However, we also found that PCP has a low yield and is difficult to separate in the extraction process, which may be related to the solution viscosity and other physical properties of the high-molecular-weight polysaccharide. Response surface methodology (RSM) is often used to identify optimal extraction process parameters and can systematically provide people with effective production processes (Dai et al., 2020; Tang et al., 2023). Thus, RSM was used to optimize the conditions for PCP extraction.

In this study, hot water extraction and ethanol precipitation were used to extract PCP to ensure the integrity of the polysaccharides. DEAE-52 cellulose and Sephadex G-75 dextran gels were used for the separation and purification of PCP, respectively. Then, the homogeneous component PCP1, as determined by liquid chromatography, was evaluated for in vitro antioxidant and lipid-lowering activities. The objective of this study was to explore the optimal extraction process, and antioxidant and lipid-lowering effects of PCP. The results of this study provide a theoretical reference for the development and utilization of PCH in the Mount Jiuhua area, and for the development of safe and effective drugs for non-alcoholic fatty liver disease prevention and treatment.

2. Materials and methods

2.1 Materials and chemicals

The rhizome of PCH was collected from Mount Jiuhua (30.5° N, 117.8° E) and authenticated by Professor Chunyan Liu (Wannan Medical College, Wuhu, China). The DEAE-52 cellulose and Sephadex G-75 dextran gels were supplied by Solarbio (Beijing, China). Kits for measuring the levels of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydroxyl radical, 2, 2'-azino-bis 3-ethylbenzthiazo-line-6-sulfonic acid (ABTS) radical, triglycerides (TG), total cholesterol (TC), alanine transaminase (ALT), and aspartate transaminase (AST) were procured from the Nanjing Jiancheng Institute of Biotechnology (Nanjing, China).

2.2 Optimization of PCP extraction

The extraction procedure followed the method described by W. Liu et al. (2022). The PCH rhizome was washed, cut into 6–8 mm slices, baked at 60°C for 72 h, dried, and crushed into a powder with a ~250 μm particle size. Then, 4 g of powder was added to deionized water for extraction according to the experimental design requirements. The cooled extract was centrifuged at 8,000 rpm for 15 min to concentrate the supernatant to 1/10 of the original volume under reduced pressure. Then, we slowly added 3 × the volume of a concentrated solution of anhydrous ethanol, precipitated it in a 4°C freezer for 12 h, and freeze-dried the material to obtain a PCP dry powder. Finally, the PCP yield (%) was calculated using Equation.1:

(1) $\mathit{PCP}\mathit{yield}\left(\mathrm{\%}\right)=\frac{\mathit{W}\times 100}{W_m}$(1)

where W is the weight (g) of PCP and W_m is the weight (g) of the dried PCH powdered sample.

To investigate the effects of extraction temperature (X₁: 50, 60, 70, 80, 90°C), liquid-to-solid ratio (X₂: 10, 15, 20, 25, 30 mL/g), extraction time (X₃: 30, 60, 90, 120, 150 min), and number of extractions (one, two, or three) on PCP yield, we first performed single-factor experiments for which the parameter settings are shown in Table 1.

Table 1. Single-factor experimental design.

Level	Extraction temperature (°C)	Liquid-to-solid ratio (mL/g)	Extraction time (min)	Number of extractions
1	50	10	30	1
2	60	15	60	2
3	70	20	90	3
4	80	25	120
5	90	30	150

After determining the range of preliminary extraction parameters using a single-factor experiment, a three-level, three-variable Box – Behnken design (BBD) was applied to statistically optimize the yield of PCP. Three independent variables were tested, including extraction temperature (X₁: 60–80°C), liquid-to-solid ratio (X₂: 15–25 mL/g), and extraction time (X₃: 60–120 min); the response variable was the yield of PCP. The Design-expert 12.0.3 software was employed for statistical analysis of experimental data and 17-run extraction experimental designs were conducted.

2.3 PCP1 isolation and purification

Briefly, 100 mg of PCP powder, from which pigments and protein were previously removed by macroporous resin and Sevag reagent, respectively, was dissolved in 10 mL of deionized water, loaded with a DEAE-52 cellulose column (2.5 × 40 cm), eluted with deionized water at a flow rate of 0.6 mL/min, and collected in a test tube. The anthrone-sulfuric acid reagent was used to track the degree of elution of the polysaccharides. The eluate was collected according to absorbance and subsequently concentrated, dialyzed, and freeze-dried to obtain the PCP1 polysaccharide components.

A Sephadex-75 dextran gel-chromatography column (1.6 × 60 cm) was then used to purify PCP1. Briefly, 30 mg of PCP1 was loaded onto the Sephadex-75 chromatography column and eluted with deionized water at a flow rate of 0.4 mL/min. Lastly, the main peak of the elution curve was collected to obtain the purified PCP1 component.

The Lc-l0Avp high performance gel permeation chromatography (HPGPC) system (Shimadzu, Japan) equipped with a RID-10A refractive index detector and TSKgel G3000PWXL column (7.8 × 300 mm, Tosoh Corp., Japan) was used to determine the homogeneity of PCP1. Parameter settings were as follows: PCP1 concentration, 5 mg/mL; mobile phase, deionized water; flow rate, 0.7 mL/min; injection amount, 20 μL; column temperature, 40°C.

2.4 PCP1 antioxidant activity

2.4.1 PCP1 DPPH radical-scavenging capacity

The DPPH radical-scavenging capacity assay was based on a previously reported method with slight modifications (Souza et al., 2012). Briefly, PCP1 was dissolved in deionized water to prepare solutions with concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL. After reacting a mixture of 0.6 mL DPPH reagent and 0.4 mL PCP1 solution under dark conditions at 25°C for 30 min, the absorbance of the mixture was measured at 517 nm and Vc was used as a positive control. The scavenging ability was calculated using Equation. 2:

(2) $\mathrm{DPPH}\mathrm{radical}-\mathrm{scavenging}\mathrm{rate}\left(\mathrm{\%}\right)\mathit{\hspace{0.17em}}=\frac{\left[A_b\mathit{\hspace{0.17em}}-\mathit{\hspace{0.17em}}\left(A_s\mathit{\hspace{0.17em}}-A_c\right)\right]\mathit{\hspace{0.17em}}\times \mathit{\hspace{0.17em}}100}{A_b}$(2)

where A_b is the absorbance measured using ethanol instead of the sample, A_s is the absorbance of the sample reacting with the DPPH reagent, and A_c is the absorbance of the DPPH reagent replaced by deionized water.

2.4.2 PCP1 hydroxyl radical-scavenging capacity

The Fenton-type reaction (Pan & Wu, 2014) was used for the determination of PCP1 hydroxyl radical-scavenging capacity. Briefly, 0.1 mL of different concentrations (0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL) of PCP1 solution were mixed with 1 mL of 9 mM salicylic acid and 1 mL of 9 mM ferrous sulfate solution, and deionized water was added to 10 mL. Then, 10 mL of 9 mM hydrogen peroxide solution was added and the absorbance of the mixed solution was measured at 510 nm after 1 h at 37°C. Vc was used as a positive control and the scavenging capacity was calculated using Equation. 3:

(3) $\mathrm{Hydroxyl}\mathrm{radical}-\mathrm{scavenging}\mathrm{rate}\left(\mathrm{\%}\right)\mathit{\hspace{0.17em}}=\frac{\left[A_b\mathit{\hspace{0.17em}}-\mathit{\hspace{0.17em}}\left(A_s\mathit{\hspace{0.17em}}-\mathit{\hspace{0.17em}}{A}_c\right)\right]\mathit{\hspace{0.17em}}\times 100}{A_b}$(3)

where A_b is the absorbance measured by replacing the sample with deionized water, A_s is the absorbance of the test sample reaction, and A_c is the absorbance of the salicylic acid – ethanol solution replaced with deionized water.

2.4.3 PCP1 ABTS radical-scavenging capacity

The reaction mixture solution underwent oxidation by oxidizing enzymes to produce ABTS free radicals (Zhu et al., 2016). Then, 10 μL of PCP1 solution at different concentrations was mixed with 20 mL of peroxidase solution and 170 μL of ABTS radical solution at 25°C for 6 min and the absorbance was measured at 405 nm; Vc was used as a control group and the scavenging ability of ABTS radicals was calculated using Equation. 4:

(4) $\mathrm{ABTS}\mathrm{radical}-\mathrm{scavenging}\mathrm{rate}\left(\mathrm{\%}\right)\mathit{\hspace{0.17em}}=\mathit{\hspace{0.17em}}\frac{\left[A_b\mathit{\hspace{0.17em}}-\mathit{\hspace{0.17em}}\left(A_s\mathit{\hspace{0.17em}}-\mathit{\hspace{0.17em}}{A}_c\right)\right]\mathit{\hspace{0.17em}}\times 100}{A_b}$(4)

where A_b is the absorbance measured by replacing the sample with deionized water, A_s is the absorbance of the sample reacting with ABTS radical solution, and A_c is the absorbance of ABTS radical solution replaced with deionized water.

2.5 In vitro PCP1 lipid-lowering activity

2.5.1 Cell culture

Human hepatocellular carcinoma cell line HepG2 was purchased from the Cell Bank of the Chinese Academy of Sciences and cultured at 37°C in a humidified thermostatic incubator in the presence of 5% CO₂. The culture was performed in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, CA, U.S.A.) containing 10% fetal bovine serum and 1% penicillin – streptomycin. Palmitic acid (PA, 0.2 mM) incubation for 48 h was used to induce cellular lipid accumulation, and different concentrations (100 and 200 μg/mL) of PCP1 solution were added for the last 24 h of the incubation period. PCP1 showed high solubility in the above culture medium and was readily dissolved by slight shaking at 37°C.

2.5.2 CCK-8 assay

HepG2 cells were cultured in 96-well plates, incubated with different concentrations of PA and PCP1 solution, and then detected using CCK-8 reagents.

2.5.3 Oil red O staining

Lipid droplets in HepG2 cells were observed using Oil Red O staining. After the cells cultured in 6-well plates were incubated in PA and PCP1 solution, 4% paraformaldehyde was fixed for 10 min. Then, cells were stained with Oil Red O staining solution for 10 min followed by staining with hematoxylin solution for 3 min, before finally being washed with phosphate buffer saline (PBS) and observed under a light microscope (OLYMPUS IX51, OLYMPUS, U.S.A.).

2.5.4 Biochemical index detection

Cells were cultured in a 6-well cell plate, incubated with PA and PCP1 solutions, and the levels of TG, TC, ALT, and AST were measured using the corresponding commercial kits.

2.6 Statistical analysis

All experimental data were expressed as the mean ± standard deviation (SD). The GraphdPad 8.0.1 software was used for data analysis. Design Expert (Version 12.0.3.0; Stat-Ease Inc., Minneapolis, MN, U.S.A.) was used for the design and data analysis of RSM. Treatment effects were compared using analysis of variance (ANOVA), and the significance of treatment differences was evaluated using the least significant difference (LSD) multi-scope test at a p < .05 significance level.

3. Results and discussion

3.1 Single-factor experiments to determine optimum conditions for PCP extraction yield

To maximize the PCP yield, single-factor experiments were performed using the parameter settings listed in Table 1. The extraction process was repeated three times for each set of parameters. The PCP yield increased slightly with increases in the extraction temperature from 40 to 70°C (Figure 1a), the liquid-to-solid ratio from 10 to 20 mL/g (Figure 1b), time from 30 to 90 min (Figure 1c), and extraction rounds from one to two (Figure 1d). Therefore, 70°C, 20 mL/g, 90 min, and two rounds were selected for subsequent analyses.

Figure 1. Effects of (a) extraction temperature, (b) liquid-to-solid ratio, (c) extraction time, and (d) number of extraction rounds on polygonatum cyrtonema Hua polysaccharide (PCP) extraction yield. Data are shown as mean ± standard deviation (S.D.).

3.2 Statistical analysis and model fitting

A BBD was applied using the PCP yield as the response variable, and 17 runs were conducted to optimize individual parameters (Table 2). In a multiple regression analysis, a quadratic polynomial regression model was fitted and the relationship between PCP yield (Y) and test variables (X₁, X₂, X₃) was defined by Equation. 5, as follows:

(5) $\mathit{Y}=10.98+0.7738{\mathrm{X}}_1+0.2388{\mathrm{X}}_2+0.2875{\mathrm{X}}_3-0.29{\mathrm{X}}_1{\mathrm{X}}_2+0.0625{\mathrm{X}}_1{\mathrm{X}}_3+0.4775{\mathrm{X}}_2{\mathrm{X}}_3-2.38{{\mathrm{X}}_1}^2-0.8785{{\mathrm{X}}_2}^2-0.941{{\mathrm{X}}_3}^2$(5)

Table 2. Box – Behnken design and response value of polygonatum cyrtonema Hua polysaccharide (PCP) extraction.

Run	X1 extraction temperature (°C)	X2 liquid-to-solid ratio (mL/g)	X3 extraction time (min)	Yield (%)
1	60	15	90	6.23
2	80	15	90	8.39
3	60	25	90	7.63
4	80	25	90	8.63
5	60	20	60	6.52
6	80	20	60	7.91
7	60	20	120	7.28
8	80	20	120	8.92
9	70	15	60	9.44
10	70	25	60	8.62
11	70	15	120	8.75
12	70	25	120	9.84
13	70	20	90	10.95
14	70	20	90	10.04
15	70	20	90	10.88
16	70	20	90	11.89
17	70	20	90	11.06

where X₁, X₂, and X₃ are the extraction temperature, liquid-to-solid ratio, and extraction time, respectively.

ANOVA results are shown in Table 3. The F-statistic (13.58) and p-value (0.0012) indicated a good model fit. A lack-of-fit F-statistic of 0.3009 and high p-value (0.8243) indicated that a lack of fitting was not significant compared with the error; therefore, the model was suitable for accurately predicting variation. In addition, the coefficient of determination (R² = 0.8762) and adjusted coefficient of determination (R²_Adj = 0.7714) indicated a high correlation between the independent and response variables (Tian et al., 2021; Wang et al., 2020).

Table 3. Analysis of variance (ANOVA) of a fitted response-surface quadratic polynomial model.

Source	Sum of squares	Degrees of freedom	Mean squares	F-value	p-value
Model	40.68	9	4.52	13.58	.0012
X1	4.79	1	4.79	14.39	.0068
X2	0.456	1	0.456	1.37	.2801
X3	0.6613	1	0.6613	1.99	.2015
X1X2	0.3364	1	0.3364	1.01	.3482
X1X3	0.0156	1	0.0156	0.0469	.8346
X2X3	0.912	1	0.912	2.74	.1418
X1^2	23.92	1	23.92	71.87	<.0001
X2^2	3.25	1	3.25	9.76	.0167
X3^2	3.73	1	3.73	11.2	.0123
Residual	2.33	7	0.3328
Lack-of-fit	0.4289	3	0.143	0.3009	.8243
Pure error	1.9	4	0.4752
Cor total	43.01	16

3.3 Response surface analysis

The Design Expert software was used to describe the interaction between the extraction process and PCP yield, and to estimate the yield over ranges of values for independent variables. The three dimensional (3D) response-surface plots and two dimensional (2D) contour maps are shown in Figure 2. The PCP yield increased as the liquid-to-solid ratio (X₂) increased from 15 to 20 mL/g and the temperature (X₁) increased from 60 to 70°C. When X₁ or X₂ exceeded this range, the PCP yield decreased gradually (Figure 2a,b). The PCP yield increased as the temperature (X₁) increased from 60 to 70°C and time (X₃) increased from 60 to 90 min, but decreased gradually when X₁ or X₃ exceeded these levels (Figure 2c,d). Finally, the PCP yield increased as X₂ increased from 15 to 20 mL/g and X₃ increased from 60 to 90 min but decreased as X₂ and X₃ exceeded these values (Figure 2e,f).

Figure 2. Response-surface three-dimensional (3D) plots and corresponding two dimensional (2D) contour plots of the effects of various variables on polygonatum cyrtonema Hua polysaccharide (PCP) yield.

3.4 Model verification

Using the Box – Behnken design, the theoretical optimal combination of variables in the extraction process was as follows: extraction temperature of 71.547°C, liquid-to-solid ratio of 20.821 mL/g, extraction time of 95.961 min, and two extraction rounds. The theoretical PCP yield was 11.09%. To ensure the operability of the extraction process, the parameters were adjusted as follows: extraction temperature, 72°C; liquid-to-solid ratio, 21 mL/g; extraction time, 96 min, and rounds of extraction, two. An actual yield of 11.05% ± 0.13% was obtained, which was in good agreement with the predicted value (p > .05).

3.5 Isolation and purification of PCP1

As shown in Figure 3a,b, the main polysaccharide component of PCP, PCP1, was obtained after the separation of PCP using a DEAE-52 cellulose column and purification using a Sephadex-75 chromatography column. HPGPC (Figure 3c) revealed that PCP1 is a polysaccharide with a narrow molecular weight distribution.

Figure 3. Isolation, purification, and homogeneity analysis of polygonatum cyrtonema Hua polysaccharide 1 (PCP1). (A) DEDE-52 elution curve, (b) sephadex-G-75 elution curve, and (c) high performance gel permeation chromatography (HPGPC) spectrogram.

3.6 PCP1 antioxidant activity

As shown in Figure 4, in the concentration range of 0.2 to 1.0 mg/mL, the scavenging ability of PCP1 against DPPH, hydroxyl, and ABTS radicals increased gradually with increasing concentrations. Furthermore, PCP1 was more sensitive to DPPH radicals than to hydroxyl or ABTS radicals. At a concentration of 1.0 mg/mL, the maximum scavenging rate (94.7% ± 2.9%) was close to that of Vitamin C (Vc). Reducing excessive free radicals can delay aging and prevent cancer, cardiovascular, and cerebrovascular diseases (Shi et al., 2023; Venkatesan et al., 2019). These data demonstrate that PCP1 has a high antioxidant capacity and the potential to be used for disease prevention.

Figure 4. In vitro antioxidant activity analysis of polygonatum cyrtonema Hua polysaccharide 1 (PCP1). (a) 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity. (b) Hydroxyl radical-scavenging activity. (c) 2, 2'-azino-bis 3-ethylbenzthiazo-line-6-sulfonic acid (ABTS) radical-scavenging activity. Data are shown as mean ± standard deviation (S.D.).

3.7 PCP1 in vitro lipid-lowering activity

Long-term overload with fatty acids and/or sugars can lead to an increase in lipid accumulation in cells, leading to cytotoxicity (Gan et al., 2022; Park et al., 2014). As shown in Figure 5a, incubation with 0.2 mmol/L PA for 48 h significantly reduced the activity of HepG2 cells and was used to construct the model (Yao et al., 2011). As shown in Figure 5b, PCP1 significantly increased cell activity in the PA-treated group. However, at 400 μg/mL, cell activity decreased significantly, presumably owing to the inhibitory effect of PCP1 on cell activity. Therefore, PCP1 concentrations of 100 and 200 μg/mL were selected for subsequent testing.

Figure 5. Lipid-lowering effect of polygonatum cyrtonema Hua polysaccharide 1 (PCP1) in vitro. (a) Effect of palmitic acid (PA) concentration on the activity of HepG2 cells. (b) Effect of PCP1 on the activity of HepG2 cells incubated with PA. (c) Intracellular triglycerides (TG) level. (d) Intracellular total cholesterol (TC) level. (e) Intracellular alanine transaminase (ALT) level. (f) Intracellular aspartate transaminase (AST) level. (g) Oil red O staining diagram. Data are shown as mean ± standard deviation (S.D.) **p < .01 versus normal group, ^##p < .01 versus PA incubation group.

The effects of PCP1 on liver cell damage and fat accumulation are summarized in Figure 5. Incubation with PCP1 can significantly reduce PA-related increases in cellular TG, TC, ALT, and AST levels, particularly at a PCP1 concentration of 200 µg/mL (Figure 5c–f). In addition, Oil Red O staining (Figure 5g) showed that PCP1 could reduce the significant increases in intracellular lipid droplets and aggregation caused by PA stimulation. These findings indicate that PCP1 can significantly reduce PA-induced cellular fat accumulation in vitro.

4. Conclusions

In this study, PCP was extracted from PCH using hot water extraction and ethanol precipitation, and its yield was optimized using BBD. The optimum extraction process parameters for PCP were extraction temperature of 72°C, liquid-to-solid ratio of 21 mL/g, extraction time of 96 min, and two rounds of extraction. Under these conditions, the extraction yield of PCP was 11.05%, which was consistent with the predicted results. Furthermore, PCP1, a homogeneous polysaccharide component that was isolated and purified from PCP, exhibited a strong ability to scavenge DPPH, hydroxyl, and ABTS radicals in vitro at a concentration of 0.2 to 1.0 mg/mL, and was more responsive to DPPH radicals with a rate of 94.7%. Moreover, PCP1 showed a good alleviating effect on HepG2 cell damage and lipid metabolism disorder induced by palmitic acid. These findings support the development and use of PCP as a natural antioxidant, and provide a theoretical reference for further research on its lipid-lowering and liver protective mechanism.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was financially supported by the Anhui Provincial Natural Science Foundation under grant [1908085MH248], Natural Science Foundation of the Education Department of Anhui Province under grant [KJ2021ZD0101], Science and Technology Project of Wuhu under grant 2022cg20, and Academic Funding Project for Top-notch Talents in Disciplines (Majors) of the Universities in Anhui Province under grant [gxbjZD2022043].