Optimisation of ultrasonic-assisted extraction and biological activity of total flavonoids from leaves of Murrayae exotica using response surface methodology

A one-way test was used to obtain a preliminary range of extraction variables using the total flavonoid yield of M. exotica leaves as an indicator. The mixture was extracted by weighing 2 g of M. exotica leaf powder precisely in a conical flask, concomitant with the use of an ultrasonic extraction device. The experiments were carried out with the five variables of ethanol concentration (50%, 60%, 70%, 80% and 90%), sonication power (200 W, 240 W, 280 W, 320 W and 360 W), sonication temperature (30 °C, 40 °C, 50 °C, 60 °C and 70 °C), sonication time (15 min, 30 min, 45 min, 60 min and 75 min) and material–liquid ratio (1:5, 1:10, 1:15, 1:20 and 1:25). The five variables were tested in turn. The extracts were then transferred to centrifuge tubes and centrifuged at 4,390 g for 5 min and the supernatant was used to determine MELTFC.

According to the results of the single-factor test, the main influencing factors for the extraction rate of MELTF were ethanol concentration (X₁, %), sonication time (X₂, min) and liquid-to-material ratio (X₃, mL · g⁻¹). A three-factor (Table 1), three-level BBD of RSM response surface method was used for the optimisation of the extraction process of MELTF, with MELTF extraction rate as the index. Each variable was assigned three levels, coded as +1, 0 and −1 to indicate high, medium and low values, respectively.

Two grams of the required material were weighted and placed into a conical flask. The flask was then filled with 40 mL of ethanol and left to soak for 1 hr. The solution in the flask was then centrifuged at 4,390 g for 5 min to determine how many total flavonoids were present in the supernatant (MELTFC). Following response surface optimisation, the yields of the MELTF extracted by ultrasonic extraction and ethanol leaching extraction were compared.

After being soaked in 95% ethanol for 24 hr, AB-8 macroporous resin was packed onto a glass column (20 mm × 40 cm). Since the resin’s height was determined to be 18 cm, its bed volume (BV) was around 60 mL. The macroporous resin was washed with 95% ethanol until the effluent mixed with water (1:5) did not become white and turbid. After that, deionised water was used to wash the column until the smell of ethanol was completely gone.

The purification of total flavonoids was ascertained according to the method reported by Yao et al. (2013), with certain modifications. Following the packing of the column with 1 BV of the sample solution, the column was washed with 3 BV of deionised water at a flow rate of 1.5 BV · hr⁻¹. After that, a flow rate of 1.5 BV · hr⁻¹ of 60% ethanol was used to elute the column. The effluents were collected, and the quantity of flavonoids present, as well as the combined, concentrated and lyophilised effluents inclusive of the total flavonoid content, was ascertained based on studying the colour reaction between NaNO₂-Al(NO₃)₃ and NaOH. The combined, concentrated and lyophilised effluents that included total flavonoids. For the next assays, the purified total flavonoids [MELTF after purification (MELPTF)] were kept in a freezer at 4 °C. The total flavonoids purity of the leaves of M. exotica was calculated using the following formula:

2 Purity(%)=C×VM×100% ${\rm{Purity}}\left( {\rm{\% }} \right) = {{{\rm{C}} \times {\rm{V}}} \over {\rm{M}}} \times 100\% $

where C is the concentration of the total flavonoids of the leaves of M. exotica (mg · mL⁻¹); V is the volume of the effluents (mL); and M is the sample weight (g).

The literature was used to evaluate the α-glucosidase inhibitory activity with a little modification (Striegel et al., 2015). A 96-well plate was incubated for 5 min at 37 °C with a combination of 40 μL of sample at various concentrations (0, 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 mg · mL⁻¹, dissolved in 0.1 M phosphate buffer) and 40 μL of α-glucosidase solution (1 U · mL⁻¹, dissolved in 0.1 M phosphate buffer). A total of 50 μL of PNPG (10 mM, dissolved in 0.1 M phosphate buffer) was then added, and the mixture was left at 37 °C for 30 min. A total of 90 μL of Na₂CO₃ solution (0.1 M) was added to end the catalytic process. The positive control was acarbose. The absorbance was measured at 405 nm with a microplate reader, and the inhibition percentage was calculated as follows:

3 Inhibition percentage(%)=[1−AS−ABA0]×100% ${\rm{Inhibition\,percentage}}\left( {\rm{\% }} \right) = \left[ {1 - {{{\rm{A}}_{\rm{S}} - {\rm{A}}_{\rm{B}} } \over {{\rm{A}}_{\rm{0}} }}} \right] \times 100\% $

where A_S represents the absorbance of the sample reaction solution; A_B is the absorbance of the reaction system without α-glucosidase; and A₀ is the absorbance of the reaction system without sample.

The α-amylase inhibitory activity was determined according to a previously published method (Eleazu et al., 2016). A 96-well plate was incubated at 37 °C for 15 min with a combination of 40 μL of sample at various concentrations (0, 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 mg · mL⁻¹, dissolved in 0.1 M phosphate buffer) and 40 μL of α-amylase (0.5 mg · mL⁻¹, dissolved in 0.1 M phosphate buffer). Thereafter, 20 μL of 1% starch solution (dissolved in 0.1 M phosphate buffer) was added, and it was left at 37 °C for 15 min. A total of 100 μL of 3,5-dinitrophenylsalicylic acid was then added and heated for 15 min. Subsequently, the mixture was cooled to room temperature. The positive control was acarbose. The absorbance was measured at 540 nm with a microplate reader, and the inhibition percentage was calculated as follows:

4 Inhibition percentage(%)=[1−AS−ABA0]×100% ${\rm{Inhibition\,percentage}}\left( {\rm{\% }} \right) = \left[ {1 - {{{\rm{A}}_{\rm{S}} - {\rm{A}}_{\rm{B}} } \over {{\rm{A}}_{\rm{0}} }}} \right] \times 100\% $

where A_S represents the absorbance of the sample reaction solution; A_B is the absorbance of the reaction system without α-amylase; and A₀ is the absorbance of the reaction system without sample.

In assessing the DPPH-scavenging activity of MELTF and MELPTF, an approach presented in the literature (Chen et al., 2019) was adopted after carrying out a slight modification. We combined all the test samples and DPPH solution (180 μL, 0.2 mM, absolute ethanol) with vitamin C (VC, 20 μL, 0–3.2 mg · mL⁻¹, distilled water). After the reaction was allowed to take place at room temperature for 20 min in the dark, the absorbance was measured at 517 nm on a microplate reader. The formula for calculating the DPPH^· scavenging rate is as follows:

5 Scavenging rate(%)=[1−A1−A2A0]×100% ${\rm{Scavenging\,rate}}\left( {\rm{\% }} \right) = \left[ {1 - {{{\rm{A}}_{\rm{1}} - {\rm{A}}_{\rm{2}} } \over {{\rm{A}}_{\rm{0}} }}} \right] \times 100\% $

where A₀ is the absorbance of DPPH^· mixed with distilled water; A₁ is the absorbance of DPPH^· mixed with the sample; and A₂ is the absorbance of anhydrous ethanol mixed with the sample.

The literature-based methodology used to determine ABTS^·+ scavenging ability has been somewhat changed (Rozi et al., 2019). To create an ABTS^·+ stock solution, 7 mM ABTS and 2.45 mM potassium persulphate were combined in an equal volume and heated to 25 °C for 12 hr in the dark. The stock solution was diluted to have an absorbance between 0.2 and 0.8 at 734 nm. Thereafter, we combined all of the samples or the VC (100 μL, 0–3.2 mg · mL⁻¹) with the diluted ABTS^·+ solution (100 μL). The reaction was permitted to take place at 25 °C for 20 min in the dark, and upon its completion, the absorbance was measured at 734 nm. The formula for calculating the ABTS^·+ scavenging rate is as follows:

6 Scavenging rate(%)=[1−A1−A2A0]×100% ${\rm{Scavenging\,rate}}\left( {\rm{\% }} \right) = \left[ {1 - {{{\rm{A}}_{\rm{1}} - {\rm{A}}_{\rm{2}} } \over {{\rm{A}}_{\rm{0}} }}} \right] \times 100\% $

where A₀ is the absorbance of ABTS^·+ mixed with distilled water; A1 is the absorbance of ABTS^·+ mixed with the sample; and A₂ is the absorbance of anhydrous ethanol mixed with the sample.

As shown in Table 2, the extraction yield of MELTF values varied from 4.39 mg · g⁻¹ to 8.38 mg · g⁻¹. The results of extraction yield affected by solvent concentration, ultrasonic time and liquid–solid ratio were fitted to a second-order polynomial equation, and the values of regression coefficients were calculated. The effects of the three variables on the extraction yield of MELTF were highly significant (Table 3). The predicted model of the extraction yield value was obtained using the following second-order polynomial equation:

7 Y1=8.00+0.93X1+0.61X2+0.63X3+0.26X1X2−0.43X1X3+0.19X2X3−0.78X12−0.65X22−0.52X32 $$\displaylines{ {\rm{Y1 = 8}}{\rm{.00 + 0}}{\rm{.93}}{{\rm{X}}_1} + 0.61{{\rm{X}}_2} + 0.63{{\rm{X}}_3} + 0.26{{\rm{X}}_1}{{\rm{X}}_2} \cr \,\,\, - 0.43{{\rm{X}}_1}{{\rm{X}}_3} + 0.19{{\rm{X}}_2}{{\rm{X}}_3} - 0.78{\rm{X}}_1^2 - 0.65{\rm{X}}_2^2 \cr \,\,\, - 0.52{\rm{X}}_3^2 \cr} $$

According to the data obtained (Table 3), the p-value is 0.0024, indicating that the model is highly significant. The p-value of lack of fit is 0.3255, which is greater than 0.05 and not significant. In addition, the R² = 0.9329 and R²_adj. = 0.8467, which are not very different and both close to 1.0, indicating that the model is reliable and statistically significant. Secondly, when we checked the p-values, p = 0.0007 < 0.01 for variable X₁ (ethanol concentration), p = 0.0068 < 0.01 for variable X₂ (ultrasonic time) and p = 0.0060 < 0.01 for variable X₃ (liquid–solid ratio), which proved that the effects of ethanol concentration, ultrasonic time and liquid–solid ratio on the extraction yield of MELTF were highly significant; additionally, while the effects of interaction terms of variables X₁X₃ and X₂X₃ on the extraction of MELTF were significant, the effect of the interaction term X₁X₂ on the extraction of MELTF was not significant. This indicates that the degree of influence of factors on the yield was the following: ethanol concentration > liquid–solid ratio > ultrasonic time.

The graphical representations of the regression equation were the 3D response surface and the 2D contour plots. They offered a means to show the interactions between two test factors, as well as the relationship between responses and experimental levels of each variable. The circular or elliptical contour plots show whether or not there are substantial mutual interactions between the variables. The elliptical contour map shows strong interactions between the relevant variables, whereas the circular contour plot shows minor interactions between the corresponding variables. In this study, the results of extraction yield of MELTF affected by solvent concentration, liquid–solid ratio and ultrasonic time are presented in Figure 2 and Figure 3.

Figure 2.

Figure 3.

As shown in Figure 2A, the 3D response surface plot of ethanol concentration was steeper than that of ultrasonic time, indicating that the ethanol concentration has a greater influence on the extraction yield of the MELTF than that of ultrasonic time, which is consistent with the conclusion drawn by the statistical and model-fitting analyses. As shown in Figure 3A, the contour plot was similar to an ellipse, indicating that the ethanol concentration and ultrasonic time have a certain interactive effect on the extraction yield of the MELTF.

As shown in Figure 2B, the 3D response surface plot of ethanol concentration was steeper than that of the liquid–solid ratio, which shows that the influence of ethanol concentration on the extraction yield of total flavonoids is greater than that of the liquid–solid ratio, which is the same as the conclusion drawn from the statistical and model-fitting analyses. The contour plot in Figure 3B presented an ellipse, which shows that the interaction between the liquid–solid ratio and the ethanol concentration has a greater impact on the extraction yield of the total flavonoids from the leaves of M. exotica.

As shown in Figure 3C, the steepness of the 3D response surface plot of the liquid–solid ratio in Figure 2C was similar to that of ultrasonic time, which indicates that the two factors have similar effects on the MELTF, which demonstrates consistency with the conclusion drawn from the statistical analysis presented in Table 3 (liquid–solid ratio, p = 0.0068; and ultrasonic time, p = 0.0060). As shown in Figure 3C, the contour plot was slightly elliptical, indicating that the interaction between ultrasonic time and liquid–solid ratio has a certain effect on the extraction yield of the MELTF.

Since α-glucosidase inhibitors may considerably lower postprandial blood glucose levels, which is a critical component in the treatment of DM, the inhibition percentage of α-glucosidase can be used to quantify the anti-diabetic impact of medications (Ademiluyi et al., 2014). Acarbose, MELTF and MELPTF were tested for their ability to inhibit glucosidase, and the findings are displayed in Figure 5. As shown in Figure 5, the total flavonoids of the leaves of M. exotica before and after purification showed dose-dependent inhibitory effects on α-glucosidase activity when the concentration of total flavonoids was in the range of 0.1–3.2 mg · mL⁻¹. The IC₅₀ values of MELTF and MELPTF were determined to be 0.028 mg · mL⁻¹ and 0.021 mg · mL⁻¹. As compared to MELTF, MELPTF had higher α-glucosidase inhibitory activity, with an IC₅₀ value of 0.021 mg · mL⁻¹, which was 1.33 times higher than that of MELTF (0.028 mg · mL⁻¹). In the range of ≈0.1–3.2 mg · mL⁻¹, the inhibition percentage of acarbose reached more than 90%, and the inhibition percentage of MELPTF was 88.29% at a concentration of 3.2 mg · mL⁻¹, which was close to that of acarbose. After purification, the inhibition percentage of the total flavonoids from the leaves of M. exotica has shown improvement, and this percentage was relatively high, indicating that the total flavonoids from the leaves of M. exotica have development value in the direction of being an α-glucosidase inhibitor. At the same concentration, MELPTF’s α-glucosidase inhibitory activity was superior to that of the flavonoid-rich extracts made from the peel of Ficus carica (Meziant et al., 2021). The outcome indicated that MELPTF could be the active ingredient in charge of M. exotica’s anti-diabetic action.

Figure 5.

Dietary starch and glycogen can be broken down by the α-amylase to provide glucose and maltose. As a result, delaying a rise in the blood glucose level through inhibition of α-amylase activity is crucial for the treatment of DM (Lordan et al., 2013). Acarbose, MELTF and MELPTF were tested for their ability to inhibit amylase, and the findings are displayed in Figure 6. As shown in Figure 6, the α-amylase inhibitory activities of all samples correlated positively with increasing concentrations in the range of 0.1–3.2 mg · mL⁻¹. The IC₅₀ values of acarbose, MELTF and MELPTF were determined to be 0.043, 0.199 and 0.094 mg · mL⁻¹, respectively. As compared to MELTF, MELPTF had higher α-amylase inhibitory activity, with an IC₅₀ value of 0.094 mg · mL⁻¹, which was 2.12 times higher than that of MELTF (0.199 mg · mL⁻¹). MELPTF strongly inhibited α-amylase, with an IC₅₀ value of 0.094 mg · mL⁻¹, which was close to that of acarbose (0.043 mg · mL⁻¹). In the range of 0.1–3.2 mg · mL⁻¹, the inhibition percentage of acarbose reached 92.45%, and the inhibition percentage of MELPTF was 71.13% at a concentration of 3.2 mg · mL⁻¹, which was close to that of acarbose. As compared to MELTF, MELPTF had a higher α-amylase inhibitory activity, with an inhibition percentage of 71.13%, which was higher than that of MELTF (64.28%) at 3.2 mg · mL⁻¹. Following purification, the MELTF showed better inhibition percentages, and these percentages were reasonably high, indicating that the MELTF have potential for development as α-amylase inhibitors. At the same dose, MELPTF showed exceptional amylase inhibitory action, outperforming certain flavonoids that had been previously reported (Liu et al., 2013; Wang et al., 2018).

Figure 6.

A traditional technique used in the food business and agriculture to assess the antioxidant potential of foods is the DPPH-scavenging experiment. Figure 7 displays the findings from the analysis of the DPPH-scavenging abilities of VC, MELTF and MELPTF. When the concentration of total flavonoids was between 0.1 mg · mL⁻¹ and 3.2 mg · mL⁻¹, as seen in Figure 7, the total flavonoids of M. exotica leaves both before and after purification demonstrated dose-dependent scavenging effects on DPPH-scavenging activity. The IC₅₀ values of VC, MELTF and MELPTF were determined to be 0.027, 0.777 and 0.245 mg · mL⁻¹, respectively. As compared to MELTF, MELPTF had a higher DPPH^· scavenging activity, with an IC₅₀ value of 0.245 mg · mL⁻¹, which was 3.17 times higher than that of MELTF (0.777 mg · mL⁻¹). In the range of 0.1–3.2 mg · mL⁻¹, the inhibition percentage of VC reached more than 90%, and the inhibition percentage of MELPTF was 85.34% at a concentration of 3.2 mg · mL⁻¹, which was close to that of VC. The total flavonoids from M. exotica’s leaves offer potential for development as antioxidants, as shown by the enhanced scavenging rate of these compounds after purification. This scavenging rate was also rather high. MELPTF demonstrated exceptional DPPH-scavenging activity, which was even higher than that of the extracts from Sophora flavescens that were rich in flavonoids (0.984 mg · g⁻¹) (Zhou et al., 2018). The outcome indicated that MELPTF could be the active ingredient in charge of M. exotica’s antioxidant action.

Figure 7.

It is possible for flavonoids to scavenge ABTS^·+, a specific kind of free radical. VC, MELTF and MELPTF were tested for their ability to scavenge ABTS^·+, and the findings are displayed in Figure 8. All samples had ABTS^·+ scavenging activities that increased in a positive correlation with concentrations between 0.1 mg · mL⁻¹ and 3.2 mg · mL⁻¹, as shown in Figure 8. The IC₅₀ values of VC, MELTF and MELPTF were determined to be 0.047, 0.201 and 0.113 mg · mL⁻¹, respectively. As compared to MELTF, MELPTF had a higher ABTS^·+ scavenging activity, with an IC₅₀ value of 0.113 mg · mL⁻¹, which was 1.78 times higher than that of MELTF (0.201 mg · mL⁻¹). MELPTF strongly scavenged ABTS^·+, with an IC₅₀ value of 0.094 mg · mL⁻¹, which was close to that of VC (0.043 mg · mL⁻¹). In the range of 0.1–3.2 mg · mL⁻¹, the scavenging rate of VC reached 100.00%, and the scavenging rate of MELPTF was 98.95% at a concentration of 3.2 mg · mL⁻¹, which was close to that of VC. As compared to MELTF, MELPTF had higher ABTS^·+ scavenging activity, with an scavenging rate of 98.95%, which was higher than that of MELTF (87.59%) at 3.2 mg · mL⁻¹. The total flavonoids from M. exotica’s leaves offer potential for development as antioxidants, as shown by the enhanced scavenging rate of these compounds after purification. This scavenging rate was also rather high, and thus demonstrated a greater effectiveness compared with the flavonoid-rich extracts from Crataegus (0.39 mg · mL⁻¹); accordingly, we are led to infer that MELPTF is capable of providing significant ABTS^·+ scavenging activity (Čopra-Janićijević et al., 2018).

Figure 8.