Cinnamomum camphora leaves as a source of proanthocyanidins separated using microwave-assisted extraction method and evaluation of their antioxidant activity in vitro

2.3.1 Extraction of PCs of C. camphora leaves

The de-oiled leaf powders of C. camphora leaves (5 g) were transferred into glass flask and then extracted under different conditions by MAE. The effects of MAE factors, including ethanol concentration, liquid/solid ratio, microwave time, and microwave power on the yield of PCs from C. camphora leaves were studied using single experimental trials to obtain the suitable ranges for the following optimization by BBD. Afterwards, the suspension liquids were filtered and then stored in a refrigerator at 4 °C before measuring the PCs contents in the extracts.

2.3.2 Experimental design and optimization

The independent or interactive effects of variables in the extraction process could determine the extraction efficiency (Sabino et al., 2021). Therefore, BBD was applied to investigate the factors: ethanol concentration (A), liquid/solid ratio (B), microwave time (C), and microwave power (D). Table 1 demonstrated the levels of the code and the real variables and the complete experimental design, which contains 29 runs (5 replicates are the central points). The experimental data was fitted using a second-order polynomial equation (Eq. (1)):

2.3.3 Determination of PCs

The Vanillin assay method was employed to determine PCs content with some modifications (Sun et al., 1998). The sample solution (1 mL) was blended with 1.5 mL of hydrochloric acid and 3 mL of 40 mg/mL vanillin in methanol (w/v). A control group was performed by replacing the vanillin solution with methanol to calibrate the absorbance by non-vanillin reactive substances to exclude the effect of the interference effect (e.g., anthocyanins). After incubation in water bath (20 ± 1C°) for 15 min, the absorbance of the samples and control groups was determined at 500 nm and their difference was applied to measure the PCs in C. camphora leaves, which was conveyed as mg grape PCs/g samples (dry weight). Calculation of PCs was on the basis of a generated calibration curve using grape PCs (y = 3.284 × −0.0046, R² = 0.9979).

2.5.1 Scavenging ability on DPPH free radicals of PCs

The scavenging activities on DPPH radicals of PCs were performed based on the method proposed in the previous study (Liu & Yang, 2018). A 25 mg/L DPPH solution was prepared using anhydrous ethanol. 3.9 mL of DPPH solution was mixed with 0.1 mL of sample solutions of different concentrations (0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg/mL), respectively. Afterward, the mixtures were placed at 20 °C for 30 min and then measured at 517 nm. The blank group included 3.9 mL of DPPH solution and 0.1 mL of ethanol. Vitamin C (V_C), butylated hydroxytoluene (BHT) and butylated hydroxytoluene (BHA) were investigated as the positive controls. The formula of DPPH radical scavenging activity of samples was presented as follows:

2.5.2 Ferric reducing antioxidant power (FRAP) of PCs

The FRAP assay is performed based on the previously published study (Oyaizu, 1986). The FRAP stock solution was prepared by mixing of 300 mM acetate buffer (25 mL, pH 3.6), 10 mM TPTZ solution (2.5 mL) and 20 mM FeCl₃⋅6H₂O solution (2.5 mL) and was incubated at ambient temperature (37 ± 2 ◦C) for 30 min. The sample solution (0.15 mL) was blended with the FRAP stock solution (2.85 mL), placed in the darkness for 30 min, and determined at 593 nm. VC, BHT, and BHA were used as the positive controls. Trolox was employed to plot a standard curve with concentrations ranging from 37.5 to 600 mM. The FRAP was conveyed as mM Trolox equivalents per gram PCs.

2.5.3 Reducing power of PCs

The reducing power of PCs was investigated on the basis of a previous study (Oyaizu, 1986). One milliliter of the sample solutions (0.03–0.15 mg/mL) was combined with 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% K₃Fe(CN)₆ and then the tubes were capped and incubated at 50 °C for 20 min. Afterward, 2.5 mL of 10% trichloroacetic acid was added in reaction mixtures and then the mixtures were centrifuged (3000g) for 10 min. The upper supernatant (2.5 mL) was blended with 2.5 mL of distilled water and 0.5 mL of 0.1% FeCl₃ and then measured at 707 nm. The blank groups were prepared using ethanol solution to replace the sample solutions, VC, BHT, and BHA were investigated as the positive controls.

3.1.1 Effect of ethanol concentration on PCs yield

The solubility of target compounds in the solvent strongly connects to the extraction efficiency in separation science (Lovrić et al., 2017). Hence, ethanol concentration was investigated to clarify its effect on the procedure for plant PCs extraction. Ethanol concentration was varied between 0 and 100% against water as liquid/solid ratio, microwave time, and power were kept at 20 mL/g, 15 min, and 385 W, respectively. The results demonstrated that the highest yield of PCs was obtained at an ethanol concentration of 75% (74.96 ± 2.50 mg/g) as depicted in Fig. 2a. The further increase in ethanol concentration led to a significant decrease in C. camphora leaf PCs yield. This is likely due to two reasons: on the one hand, an increase in ethanol concentration led to the decrease of polarity, and hence giving rise to the insufficient extraction of oligomeric PCs (Murga et al., 2000); on the other hand, a high concentration of ethanol can promote the conversion of proanthocyanidins into anthocyanidins (Gao et al., 2020). A similar phenomenon was also observed in the previous studies (Downey & Hanlin, 2010; Bosso et al., 2016). Therefore, the range of ethanol concentration was determined from 50% to 100% in the subsequent optimization study.

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Fig. 2

Effects of ethanol concentration (a), liquid/solid ratio (b), microwave time (c) and microwave power (d) on the yield of PCs from C. camphcra leaves.

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3.1.2 Effect of liquid/solid ratio on PCs yield

The effect of liquid/solid ratio on the C. camphora leaf PCs yield was also investigated. The yield of PCs was measured by varying liquid/solid ratio between 10 and 30 mL/g at fixed 50% of ethanol concentration, 15 min of microwave power, and 385 W of microwave power. As presented in Fig. 2b, it was seen that the PCs significantly raised with initially increasing of liquid/solid ratio. Nevertheless, when the liquid/solid ratio increased from 25 mL/g to 30 mL/g, the PCs yield showed a nonsignificant increase. Hence, the range between 15 and 25 mL/g was used for the next optimization investigations.

3.1.3 Effect of microwave time on PCs yield

Microwave time is a crucial parameter in the extraction process of MAE. Inadequate microwave time, either shorter or longer microwave time, can change the yield of target compounds from the plant matrix. This is because the underexposure or overexposure of plant materials under microwave radiation could decrease the extraction yield of target components. Thus, the effect of microwave time on C. camphora leaf PCs yield was investigated within the range of 5 to 25 min as shown in Fig. 2c; and the other variables including ethanol concentration, liquid/solid ratio, and microwave power were maintained at 50%, 20 min, and 385 W, respectively. PCs yield was observed and obviously increased as microwave time function increased from 5 to 15 min and followed by a significant decline when the microwave time exceeded 15 min. This phenomenon demonstrated the dissolution process of target compounds from solid to the solvent stopped as the longtime exposure to microwave radiation triggered degradation (Alara and Abdurahman, 2019). The range of microwave time was then determined from 10 to 20 min for further optimization.

3.1.4 Effect of microwave power on PCs yield

On the basis of the previous study (Dahmoune et al., 2014), microwave irradiation is triggered by the dissemination of electromagnetic energy and can promote the mass transfer coefficient between the extraction solvent and the plant materials. For that reason, microwave power was changed within 120 W, and 700 W was kept at 50% of ethanol concentration, 20 mL/g of liquid/solid ratio, and 20 min of microwave time. The PCs yield increased with an enhancement of microwave power from 120 W to 385 W, and a further increase in microwave power did not significantly enhance PCs yield. Still, an apparent decrease of over 540 W of microwave power (Fig. 2d). The reduction in PCs beyond 540 W could be attributed to the effect of the excessive microwave irradiation originated from improved power. Therefore, the range of microwave power from 230 to 540 W was screened for the subsequent optimization process.

3.2.1 Model fitting and regression coefficients

BBD is autonomous without any influences on the abovementioned screening proposals. Three levels of each variable are usually recommended, whereas runs in which all variables simultaneously at lower or higher points are not included, building a design with fewer trials and thus more economical (Massart et al., 1997). Twenty-nine runs associated with four variables, namely ethanol concentration (A), liquid/solid ratio (B), microwave time (C), and microwave power (D) of MAE, were carried out for the further optimization of the PCs extraction process using BBD to obtain the optimal conditions as presented in Table 1. The regression analysis was carried out to study the actual responses acquired from the experiments, and a quadratic regression model equation was finally generated as given in Eq. (4):

The model significance and suitability were tested by analysis of variance (ANOVA) for the response. The regression coefficients of the developed model including the intercept, linear, quadratic, and interaction terms were investigated and submitted to a significance test. P value was applied to examine the significance of each coefficient. P values less than 0.05 demonstrate the corresponding term is significant. The ANOVA analysis results of the PCs yield are given in Table 2. A good connection was observed between the PCs yield and the process variables as evidenced by a coefficient of determination (R²) of 0.9675; the values of adjusted R² (0.9349) also showed an excellent agreement to that of predicted R² (0.8332). Furthermore, coefficient of variance (C.V.% = 1.54) implied a good reproducibility of the developed model. The model F value of 29.72 with a P value of 0.2262 for lack of fit all implied that the developed model is significant, indicating that the model can be applied to further analyze of the correlations of variables within the boundaries used. ANOVA results revealed that all four individual factors were significant model terms, while with respect to the interactions between variables and only the interactions of liquid/solid ratio (B) versus ethanol concentration (A) and microwave time (C) were nonsignificant terms.

3.2.2 Investigation of model adequacy

Three diagnostic plots of the developed model, including normal plot, residuals vs. run number, and predicted vs. actual. Fig. 3a showed that the residuals follow a normal distribution and were distributed mainly in the straight line. Therefore, the error of the developed secondary model was normally and identically distributed (Sekhar et al., 2021). The residuals vs. run number plot (Fig. 3b) showed a random distribution of data points in the range of −3 to 3 surround the central line, which implied that the developed quadratic model built interaction between the causal parameters of the MAE method and the PCs yield. The actual responses obtained based on experimental trails were depicted against the predicted response (Fig. 3c). This plot aims to seek out a value or a cluster of values that the developed model could not easily predict. It was observed that the predicted values by the developed model have a linear relation with the actual values. Briefly, the three diagnostic plots indicated that the developed model has a good capacity to optimize the MAE process for PCs extraction from C. camphora leaves.

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Fig. 3

Diagnostic plots for model adequacy checking. Normal plot (a), residuals vs. run number (b), and predicted vs. actual (c).

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3.2.3 Effects of MAE conditions on PCs through RSM

Fig. 4a showed the 3-D response surface plots for the yield of PCs with varying ethanol concentration (A) and microwave time (C) while fixing liquid/solid ratio (B) and microwave power (D) at the middle level of 20 mL/g and 385 W, respectively. The depicted plot showed that the PCs yield increased gradually as the ethanol concentration and microwave time increased, but they all exhibited a curvature as further increased. The highest extracted PCs yield was 76.55 mg/g and occurred at an ethanol concentration of 84% and at a microwave time of 18 min. The 3-D response surface plot was obtained for the yield of PCs with varying ethanol concentration (A) and microwave power (D) at the average liquid/solid ratio (B) and microwave time (C) as shown in Fig. 4b. It can be observed that ethanol concentration and microwave power showed a quadric effect on PCs yield and the maximum yield (76.86 mg/g) of PCs can be obtained when ethanol concentration and microwave power were 75% and 472 W, respectively. Fig. 4c depicted the interactive influence of liquid/solid ratio (B) and microwave power (D) on the PCs yield; by improving microwave power from 230 to 500 W, the C. camphora leaf yield of PCs tends towards increase with liquid/solid ratio enhancing to optimum values and then gradually decreased. Fig. 4d and Table 2 showed a strong interaction between microwave time (C) and power (D) significantly influencing PCs yield. By increasing the microwave time to 18 min and microwave power to 501 W, the optimum PCs yield of 77.97 mg/g was achieved from C. camphora leaves. The synergetic effect between microwave time and power can be illustrated by improving the extraction system temperature, increasing the extraction yield. Based on the previous study (Alara et al., 2021), an increase of microwave power or microwave time enables keeping the temperature in the microwave oven at high levels, giving rise to the maximum extraction yields.

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Fig. 4

3-D response surface plots from BBD. (a) The interactive influences of ethanol concentration (A) and microwave time (C) on PCs yield from C. camphcra leaves while fixing liquid/solid ratio and microwave power at the middle level of 20 mL/g and 385 W; (b) the interactive influences of the ethanol concentration (A) and microwave power (D) on PCs yield from C. camphcra leaves while at the average liquid/solid ratio (20 mL/g) and microwave time (15 min); (c) the interactive influences of liquid/solid ratio (B) and microwave power (D) on C. camphcra leaf PCs yield while fixing ethanol concentration (A) and microwave time at the middle level of 75% and 15 min; and (d) the interactive influences of microwave time (C) and power (D) on C. camphcra leaf PCs yield while fixing ethanol concentration (A) and liquid/solid ratio (B) at the middle level of 75% and 20 mL/g.

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3.2.4 Method validation

RSM has performed the process optimization of the PCs extraction process on the developed quadric regression equation. The optimal MAE conditions with a predicted yield of 79.79 mg/g for C. camphora leaf PCs were obtained as: 76.90% of ethanol concentration, 19.68 mL/g of liquid/solid ratio, 18.39 min of microwave time, and 525.59 W of microwave power. The validation experiments were performed three times with the slight modifications of the optimal conditions (77% of ethanol concentration, 20 mL/g of liquid/solid ratio, 18 min of microwave time, and 530 W of microwave power) to verify the accuracy and acceptance of the MAE process. A mean yield of 81.56 ± 2.03 mg/g was obtained for C. camphora leaf PCs extraction, which indicated that the obtained conditions by BBD are reliable.

3.4.1 DPPH radical-scavenging activity

The DPPH free radical could receive an electron or hydrogen radical and transform into a stable diamagnetic molecule (Soares et al., 1997). The DPPH stock solution was prepared with a maximum absorbance of 0.84 at 517 nm. Its purple color usually fades when DPPH solution is mixed with the antioxidant reagents, leading to a decrease in absorbance at 517 nm (Ma et al., 2012). As depicted in Fig. 6a, the scavenging activity of all the samples increased with the increase of their concentrations, and the DPPH scavenging influence decreased in that order: BHA > PCs > V_C > BHT.

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Fig. 6

Antioxidant activity of PCs from C. camphcra leaves. (a) DPPH radical scavenging activity, (b) ferric scavenging activity, and (c) reducing power.

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3.4.2 FRAP

The antioxidant potential of C. camphora leaf PCs was investigated for their ability to convert the TPTZ-Fe³⁺ complex into the PTZ-Fe²⁺ complex. The FRAP conveyed by Trolox equivalent antioxidant capacity and the FRAP of PCs, BHA, BHT and V_C were given in Fig. 6b. The results obviously demonstrated that the PCs from C. camphora leaves possessed the highest FRAP to those of BHA, BHT, and VC. The results indicated that C. camphora leaf PCs had strong FRAP in comparison to the commercial antioxidants.

3.4.3 Reducing power

Reducing power trials were performed as an indicator to estimate the antioxidant activity of C. camphora leaf PCs. The existence of reductants in the mixed solution gives rise to the conversion of Fe³⁺ or ferricyanide into Fe²⁺ and then Fe²⁺ is detected by measuring the absorbance at 707 nm (Ardestani and Yazdanparast, 2007). As shown in Fig. 6c, the reducing power of C. camphora leaf PCs and the reference groups all improved linearly with an increase in their concentration. PCs showed a little bit weaker reducing power than the reference samples.