Abstract
Flavonoids were extracted from Phyllostachys heterocycla leaves by adopting microwave-assisted extraction technology. Based on the single factor experiment and Plackett-Burman design results, the extraction process of flavonoids was further optimized using the response surface methodology. The optimum conditions were as follows: an ethanol concentration of 78.1%, an extraction time of 24.9 min, and a microwave power of 559 W. Under these conditions, the extraction yield of flavonoids was 4.67%, which was in close proximity to the predicted value (4.70%) and higher than the extraction yield from traditional Soxhlet extraction (3.35%). Moreover, the possible extraction mechanisms of these two extraction methods were further derived to explain why the microwave-assisted extraction of flavonoids was more efficient compared with traditional Soxhlet extraction. Ultimately, the antioxidant activities in vitro of flavonoids from Phyllostachys heterocycla leaves were evaluated via DPPH and ABTS radical scavenging assay. The flavonoids from Phyllostachys heterocycla leaves exhibited excellent antioxidant activities in vitro and Phyllostachys heterocycla leaves could be a new natural source for developing antioxidants. Overall, the findings of this research could provide a theoretical reference for the further comprehensive development and utilization of bamboo resources.
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Microwave-assisted Extraction of Flavonoids from Phyllostachys heterocycla Leaves: Optimization, Mechanism, and Antioxidant Activity in vitro
Jie Xu,a Jianjun Wu,a,* Juan Qi,b Juan Li,b Yongju Liu,b Zekai Miao,a Guofeng Qiu,a and Wenke Jia a
Flavonoids were extracted from Phyllostachys heterocycla leaves by adopting microwave-assisted extraction technology. Based on the single factor experiment and Plackett-Burman design results, the extraction process of flavonoids was further optimized using the response surface methodology. The optimum conditions were as follows: an ethanol concentration of 78.1%, an extraction time of 24.9 min, and a microwave power of 559 W. Under these conditions, the extraction yield of flavonoids was 4.67%, which was in close proximity to the predicted value (4.70%) and higher than the extraction yield from traditional Soxhlet extraction (3.35%). Moreover, the possible extraction mechanisms of these two extraction methods were further derived to explain why the microwave-assisted extraction of flavonoids was more efficient compared with traditional Soxhlet extraction. Ultimately, the antioxidant activities in vitro of flavonoids from Phyllostachys heterocycla leaves were evaluated via DPPH and ABTS radical scavenging assay. The flavonoids from Phyllostachys heterocycla leaves exhibited excellent antioxidant activities in vitro and Phyllostachys heterocycla leaves could be a new natural source for developing antioxidants. Overall, the findings of this research could provide a theoretical reference for the further comprehensive development and utilization of bamboo resources.
Keywords: Phyllostachys heterocycla leaves; Microwave-assisted extraction; Flavonoids; Antioxidant activity; Extraction mechanism
Contact information: a: School of Chemical Engineering and Technology, China University of Mining and Technology, Xuzhou 221116 China; b: School of Chemical Engineering, Xuzhou College of Industrial Technology, Xuzhou 221140 China;
* Corresponding author: jjuw@163.com
GRAPHICAL ABSTRACT
INTRODUCTION
Bamboo is widely distributed throughout the world and is a perennial evergreen herb that belongs to the Gramineae family and the Bambuseae subfamily (He et al. 2014). In recent years, research on promoting the utilization of bamboo resources has been a very active field of study throughout the world. Bamboo resources have been considered as an important ingredient in traditional Asian medicine, especially in China and India (Ayurveda) (Yuan et al. 2020). Bamboo leaves are a type of forestry resource with potential utilization value worldwide. It has long history of consumption in China since it contains many secondary plant metabolites with medicinal and nutritive properties, which are related to human health (Gong et al. 2015). Bamboo leaf extract can be constituted as a light brown powder extracted from bamboo leaves and contains a large amount of biologically active components, which primarily include flavonoids, phenolic acids, and coumaric lactones (Gao et al. 2019).
Phyllostachys heterocycla is native to China. It grows as a thick rod, and the surface of the stalk is covered with a layer of white powder. It is one of several vital scattered bamboo species, primarily distributed in southern China (Tao et al. 2019; Yuan et al. 2021). Flavonoids are secondary plant metabolites and present in nearly all parts of bamboo, such as leaves, roots, and xylem. Flavonoids play an important role in plants growth and development. Besides, they have been related to the defense mechanism in the aerial parts (leaves and stems) of plants (Orsat and Routray 2017). Flavonoids have attracted much attention due to their beneficial health effects, including antioxidant, antifungal, antitumor, and antidiabetic effects (Fang et al. 2020; Ma et al. 2020; Li et al. 2021). Presently, flavonoids have great application potential in chemical, pharmaceutical and other industries, and the market demand for flavonoids is gradually increasing. In addition, the research on the extraction of flavonoids from vegetal materials mostly focuses on the traditional heat reflux extraction (Liu et al. 2021; Shamsuddin et al. 2021; Xia et al. 2021). This extraction process is time-consuming, laborious, and inefficient, and the quantity and quality of the flavonoids obtained from this method are not satisfactory (Proestos and Komaitis 2008; Zhang et al. 2011). Therefore, it is urgent to exploit a high-efficiency extraction method with optimum parameters for achieving the maximum yield of flavonoids.
Microwave-assisted extraction is one of the advanced methods of extraction, which has been increasingly applied for the extraction of bioactive compounds. When compared with the traditional heat reflux extraction method, microwave-assisted extraction is more efficient in many cases and has many advantages, including a strong penetrating force, high selectivity, shorter extraction time, reduced solvent consumption, and a higher yield with a high quality of the targeted compound (Routray and Orsat 2012; Routray and Orsat 2014; Odabas and Koca 2021; Petrotos et al. 2021). Many reports have been published on the application of microwave-assisted extraction of secondary metabolites from plants (Zhong et al. 2016; Alara et al. 2019; Alirezalu et al. 2020; Carbone et al. 2020; Chaves et al. 2020; Yadav et al. 2020; Kaanin-Boudraa et al. 2021). Nevertheless, there is little information about the extraction of flavonoids from Phyllostachys heterocycla leaves using microwave-assisted extraction. Therefore, the primary purpose of this study was to optimize the microwave-assisted extraction of flavonoids using the response surface methodology and to elucidate the mechanism underlying microwave-assisted extraction of flavonoids from Phyllostachys heterocycla leaves and compare it with traditional Soxhlet extraction. In addition, the antioxidant activities of flavonoids were also investigated to expand its applications in the healthcare domain.
EXPERIMENTAL
Materials and Chemicals
Phyllostachys heterocycla leaves were collected from the Yibin region (Sichuan, China). These fresh leaves were rinsed with distilled water three times and dried in a vacuum oven at a temperature of 50 °C for 24 h. Then, the leaves were ground in a domestic mixer grinder and sieved with a 60-mesh screen. Finally, the prepared samples were stored in a desiccator for subsequent experiments. The rutin, pyrogallic, salicylic acid, vitamin C and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were purchased from Aladdin Co., while the aluminum nitrate, sodium nitrite, sodium hydroxide, and 2,2-azino-bis-3-ethylbenzo-thiazoline-6-sulfonic acid (ABTS) were procured from MacLeans Co. All chemicals were of analytical grade.
Methods
Microwave-assisted extraction
A microwave synthesis reactor (MCR-3KT, Zhengzhou Ketai Experimental Instrument Co. Ltd., Zhengzhou, China) with a maximal microwave power of 800 W at a frequency of 2450 MHz was employed to extract the flavonoids. A schematic diagram of the MCR-3KT device is presented in Fig. 1. The pre-prepared Phyllostachys heterocycla leaf powder (4 g) was transferred into a 250 mL round-bottom flask containing 120 mL of 70% (v/v) ethanol. Then, the flask was moved into the chamber of this device connected with condensing tubes. Next, the chamber door was closed, and programs of different microwave powers and extraction times were set. After the microwave-assisted extraction, the treated mixture was cooled to room temperature. Then, the supernatant was collected via centrifugation (5000×g, 15 min) and concentrated at a temperature of 50 °C using a rotary evaporator. Finally, the concentrate was made up to a volume of 100 mL by adding 30% ethanol for the subsequent determination of the total flavonoid content. All experiments were investigated in triplicate.
Traditional Soxhlet extraction
A Soxhlet apparatus was used to extract the flavonoids from Phyllostachys heterocycla leaves using the following optimal conditions: the sample (4 g) was extracted with 120 mL of ethanol (60%) for 7 h at an extraction temperature of 100 °C based on the preliminary orthogonal experiment (44). After the Soxhlet extraction, the extracts were evaporated at a temperature of 50 °C using a rotary evaporator. Then, the extracts were transferred to brown glass bottles and stored at a temperature of 4 °C until later use. All Soxhlet extractions were performed in triplicate.
Experimental Design
Single factor experiment and Plackett-Burman design
The microwave-assisted ethanol extraction method was implemented to extract the flavonoids from Phyllostachys heterocycla leaves. The reason why this approach was chosen was that ethanol as an extraction solvent can achieve efficient recovery of flavonoids and the extraction process conforms to the principle of “green” chemistry. The extraction yield is affected by several factors, including ethanol concentration, solid to liquid ratio, temperature, and time (Su et al. 2019). Considering the multiple effects of various factors on the extraction of target compounds, it is of great importance to explore the optimal extraction conditions in terms of designing a complete extraction process. For this study, the ethanol concentration, solid to liquid ratio, extraction time, and microwave power were chosen as single factors. Then, based on the results of the single factor experiment, the Plackett-Burman design with four independent variables, i.e., ethanol concentration (X1), solid to liquid ratio (X2), extraction time (X3), and microwave power (X4), at two levels (±1) was applied to design 12 experimental points to explore effects of the above independent variables on the extraction yield of flavonoids from bamboo leaves. The significance of each variable was determined via a Student’s t-test.
Experimental design of the response surface methodology (RSM)
The response surface methodology (RSM) is widely applied to explore the estimated functional relationship between a response variable and design variables as well as search for the optimal conditions of factors for desirable responses. According to the preliminary results of the single factor and Plackett-Burman design experiments, the independent variables and their ranges were chosen for the further optimization. In order to obtain the optimal extracting conditions and investigate the effects of the independent variables on the flavonoid yield (Y), a Box-Behnken design with three variables, i.e., concentration of ethanol (A), extraction time (B), and microwave power (C), at three levels (Table 4) was performed using Design Expert 8.0 software (Stat-Ease, Minneapolis, MN). In addition, Y was used as the dependent variable, and its values in each trial were the average of the triplicates.
To predict the optimal extracting conditions, a second-order polynomial model concerning the relationship between the independent and response variables was established, as shown in Eq. 1,
(1)
where Yk is the response, ak0, aki, akii, and akij are coefficients for the intercept, linear, quadratic, and interactive terms, respectively, and xi, xi2, and xixj are the linear, quadratic, and interactive terms of the coded independent variables, respectively.
Fig. 1. Schematic diagram of the experimental setup for microwave-assisted extraction
Analytical Method
Determination of total flavonoids content
The calibration curves were constructed using rutin solutions with different concentrations, i.e., 0.008, 0.016, 0.024, 0.032, 0.04, 0.048, 0.056, and 0.064 mg/mL. According to the analysis of the calibration curves, the regression model of the linear relationship between the concentration of the rutin standard solution (C) and the absorbency (A) was obtained using Eq. 2,
(2)
where R2 equals 0.9965.
The total flavonoid content was determined by the colorimetric method with some modifications (Zhu et al. 2010; Lu et al. 2013). First, 1 mL of the sample solution and 1 mL of a NaNO2 (5%) solution were accurately added to a volumetric flask (25 mL); the mixture was mixed thoroughly and kept at room temperature (25 °C) for 5 min. Second, 1 mL of an Al(NO3)3 (10%) solution was removed to the above flask and reacted for 6 min. Finally, 10 mL of a NaOH (4%) solution was added to the above volumetric flask, which was followed by adding alcohol (30%) to the scale. After 20 min, the absorbance of the resulting solution was measured at 510 nm using an ultraviolet-visible detector. The sample solution without coloration was used as a reference solution and the final data was presented as the mean of the three replicate measurements. The formula for calculating the extraction yield of flavonoids is shown in Eq. 3,
(3)
where Y (%) is the extraction yield of flavonoids, C (mg/mL) is the flavonoid content of the test solution calculated by the linear regression model, V (mL) is the volume of the test solution, N is the total dilution value, and m (g) is the mass of test sample.
Fourier-transform infrared (FTIR) analysis
Fourier-transform infrared (FTIR) spectra of the extracts were obtained using a Nicolet 6700 (Thermo Fisher Scientific, Waltham, MA) in the wavenumber range of 4000 to 400 cm-1 using a KBr disc containing approximately 1% finely ground samples.
Scanning electron micrographs
The surface morphology of the Phyllostachys heterocycla leaves after either the microwave-assisted extraction or traditional Soxhlet extraction were examined using a field-emission scanning electron microscope (SEM) (ZEISS Gemini 500, Oberkochen Germany) with InLens mode. The sample was fixed on the sample holder with conductive adhesive and a thin layer of gold was sputtered on the sample to render the surface conductive prior to examination.
Evaluation for Antioxidant Activity
DPPH radical scavenging activity assay
The scavenging effect of the extracted flavonoids on the DPPH radicals was measured according to the previous report by Ge et al. (2020), with some modifications. Next, 600 μL of different concentrations of the samples (0.018 to 0.18 mg/mL) were added to separate glass test tubes. Then, 4 mL of 0.1 mM DPPH (0.1 mM DPPH in ethanol was freshly prepared) was poured into each test tube. These two solutions were mixed thoroughly and stored at room temperature (25°C) in the dark for 30 min. Finally, the absorbance of the resulting solution was measured at 517 nm using a UV/VIS spectrophotometer (TU-1900, Beijing Purkinje General Instrument Co., Ltd, Beijing, China). Vitamin C was used as a positive control. The DPPH radical-scavenging activity (%) was calculated by Eq. 4,
(4)
where A0 is the absorbance of the DPPH-ethanol solution mixed with ethanol, Ax is the absorbance of the DPPH-ethanol solution mixed with the sample solution, and Ax0 is the absorbance of ethanol mixed with the sample solution.
ABTS radical scavenging activity assay
The scavenging effect of the samples on the ABTS radicals was determined as described previously by Hu et al. (2016), with some modifications. First, ABTS stock solution with the concentration of 7.4 mM was prepared, and then 5 mL of ABTS stock solution was mixed with 5 mL of 2.6 mM potassium persulfate to prepare the ABTS reagent solution. This solution was stored at room temperature in the dark for 12 h. In addition, the first thing to do when starting the ABTS assay is to dilute the ABTS reagent solution with a phosphate-buffered saline (pH 7.4) to maintain an absorbance of 0.70 ± 0.02 at 734 nm. Then, 400 μL of the sample of different concentrations was added to 4 mL of the diluted ABTS solution to react in the dark at a temperature of 30 ℃ for 6 min. Finally, the absorbance of the mixture was measured at a wavelength of 734 nm. Vitamin C was used as a positive control. ABTS radical-scavenging activity (%) was calculated with Eq. 5,
(5)
where A0 is the absorbance of the diluted ABTS solution mixed with ethanol, and Ax is the absorbance of the diluted ABTS solution mixed with the sample solution.
RESULTS AND DISCUSSION
Analysis of the Singal Factor and Plackett-Burman Experimental Results
The effects of different factors on the total flavonoid extraction yield are shown in Figs. 2a through 2d. For instance, in Fig. 2a, the total flavonoid extraction yield increased as the ethanol concentration increased, before achieving the highest extraction yield (4.26%) at an ethanol concentration of 70%, and then the yield rapidly decreased. A possible explanation for this phenomenon might be that the increase in the ethanol proportion in the extraction solvent will gradually reduce the solvent polarity (Butsat and Siriamornpun 2016). Furthermore, a high concentration of ethanol may hold back the dissolution of polyphenols, thereby affecting the extraction yield (Bouras et al. 2015; Ghaffar et al. 2020; Petrotos et al. 2021). In consideration to the limitation of the ethanol proportion, a 60% to 80% (v/v) ethanol concentration was used as the optimal range for the Plackett-Burman experiment. Regarding the influence of the solid to liquid ratio (as shown in Fig. 2b), the flavonoid extraction yield showed a downward trend when the solid to liquid ratio exceeded 1 to 30, which may be explained by the fact that the solid to liquid ratio of 1 to 30 was more suitable to completely extract the soluble flavonoids.
Regarding the influence of the extraction time (as shown in Fig. 2c), the extraction yield showed a prominent increase during the range of 5 to 20 min. However, the yield began to decrease when the extraction time exceeded 20 min, which indicated that too long an extraction time may lead to the decomposition of flavonoids (Su et al. 2019). In terms of microwave power (Fig. 2d), as the microwave power increased from 160 to 640 W, the flavonoid yield gradually increased until it reached its maximum value at 480 W. However, the extraction yields dropped as the microwave power was further increased. The preliminary improvement to the yield at an elevated microwave power could be attributed to the increased destruction of the cellular structure, which can be explained by the rapid pressure built up in the cellular matrix and the corresponding increase in temperature due to dielectric heating (Wang et al. 2012; Routray and Orsat 2014; He et al. 2016; Zhong et al. 2016). However, a high microwave power might increase the temperature of the flavonoids too much and decrease the extraction yield due to the breakdown of the flavonoid structure (Ma et al. 2009).
Based on the above results, the optimal conditions amongst the four parameters tested were evaluated as follows: an ethanol concentration of 70%, solid to liquid ratio of 1 to 30, extraction time of 20 min, and the microwave power of 480 W, which resulted in a flavonoid extraction yield of 4.26%. Similar studies have been investigated by Lu et al. (2013), who found that the highest flavonoid yield (2.70%) extracted from Cryptotaenia japonica Hassk was achieved at a solid to liquid ratio of 1 to 20 using 70% (v/v) ethanol for 15 min.
To further select the significant variables for the extraction of flavonoids, the ethanol concentration, solid to liquid ratio, extraction time, and microwave power were tested and identified via the Plackett-Burman design experiment. The experimental design with the name, symbol code, and actual level of the variables are presented in Table 1. The corresponding responses are shown in Table 2. The adequacy of the model was calculated, and the variables evidencing statistically significant effects were screened via Student’s t-test for ANOVA (Table 3).
In addition, factors were selected for further optimization studies with p-values of less than 0.05 suggesting significant effects on the response. The ethanol concentration was determined to be the most significant factor with a probability value of 0.000, followed by microwave power (0.004), and extraction time (0.007). A lower probability values indicates a more significant factor on the flavonoid extraction yield. The insignificant variable, i.e., the solid to liquid ratio, was neglected, and the optimum levels of the three variables, i.e., the ethanol concentration, extraction time and microwave power, were further determined via an RSM design.
The Pareto chart indicating the significant factors for the flavonoid extraction process is shown in Fig. 3. It showed the standardized effect of each parameter or the combination of parameters, as calculated by Eq. 6,
(6)
where Y is the response (the flavonoid extraction yield), and the coefficient of each parameter reveals the effect of the parameters on the extraction process. Among these parameters, the ethanol concentration and extraction time exerted positive effects, whereas the microwave power and solid to liquid ratio exerted negative effects.
Fig. 2. Effect of the ethanol concentration (a); solid to liquid ratio (b); extraction time (c); and microwave power (d) on the total flavonoid extraction yield
Fig. 3. Pareto chart indicating the significant parameters
Table 1. Experimental Variables at Different Levels Used for the Microwave-Assisted Extraction of Flavonoids Using the Plackett-Burman Design
Table 2. The Twelve-Trial Plackett-Burman Design Matrix of the Four Variables with Actual Values
Table 3. Estimated Effect, Regression Coefficient, and Corresponding t-value and p-value for the Flavonoid Extraction Yield of the Four Variables Plackett-Burman Design Experiment
Statistical Analysis and Model Fitting of the Flavonoid Extraction Process
The three independent variables, i.e., concentration of ethanol (A), extraction time (B), microwave power (C), and their levels in the response surface design are shown in the Table 4. The response surface analysis of the flavonoid yield varying with A, B, and C is presented in Table 5. The second-order polynomial model for the relationship between the independent variables and the flavonoid extraction yield (Y) was established as shown in Eq. 7,
(7)
It can be seen from the data in Table 6 that the proposed regression model for the flavonoid extraction yield had a satisfactory determination coefficient (R2) value and no significant value of the lack of fit (0.1644). The R2 value was 99.07%, which indicated a reasonable correlation between the experimental results and the theoretical values. In addition, the p-values were applied to check the significance of each variable. As shown in Table 6, the linear terms of ethanol concentration (A), extraction time (B), and microwave power (C), as well as the quadratic terms of extraction time (B2) and microwave power (C2) showed significant (p-value was less than 0.05) effects on the flavonoid extraction process. Among the interaction terms, AB and AC were significant (p-value was less than 0.05) for this extraction process.
Table 4. Independent Variables and Their Levels in the Response Surface Design
Table 5. Results of the Response Surface Analysis of the Variation in Flavonoid Yield
The scatter diagram of the predicted results versus the actual values for the flavonoid yield is shown in Fig. 4. The predicted values obtained by the regression equation matched the experimental values reasonably well with an R2 value of 99.07%. Moreover, adequate precision (AP) was applied to compare the range of the predicted values at the design points to the average prediction error (Fig. 4). According to Table 6, the AP values (24.08) higher than four confirmed that the predicted model can be used to navigate the design space. The coefficient of variance (CV) as the ratio of the estimated standard error to the average of the observed response defines the reproducibility of the model. The CV values (0.81%) in Table 6 was less than 10%, which demonstrated that the model can be considered reproducible (Ghafari et al. 2009). Similarly, the normal probability distribution diagram of the residuals is shown in Fig. 5. It was found that the externally studentized residuals were also distributed on the straight line with a 45° slope, which satisfied the normal hypothesis. From this analysis, it was found that the established regression model could approximately predict the flavonoid extraction yield.
Table 6. Variance Analysis of Regression Model
