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Optimization of Ultrasound-Assisted Extraction of Phenolic Compounds from Walnut Shells and Characterization of Their Antioxidant Activities

Shusheng Wang, Wenyi Fu, Hannah Han, Milan Rakita, Qingyou Han, Qin Xu*

Department of Food Science, Purdue University, W. Lafayette, USA

Abstract: Million tons of walnut shells, the waste by-product from walnut industries, are produced annually worldwide and are under-utilized in kernel manufacturing. Walnut shells are rich in phenolic compounds thus are valuable sources for antioxidants. In this study, the optimization of ultrasound-assisted extraction of phenolic compounds from walnut shells was performed with response surface methodology (RSM). The maximum yield of total phenolic compounds from walnut shells was 92.96±1.47 mg GAE/g DW under the optimum extracting conditions of 10 min at 0°C with 43.71% ethanol and 53.93% ultrasound amplitude. Analysis of antioxidant activities of the extracts found that the radical scavenging capacity of DPPH from the walnut shell extracts (10 - 500 μg/mL) was 4.05 – 88.59%, while the ferric reducing antioxidant power (FRAP) (10 - 100 μg/mL) was 119.64-1278.95 μM Trolox equivalent (TE). The results indicate that phenolic extracts from walnut shells are a good natural source of antioxidants, making this process a strong contender for future applications in the food and agricultural industries.

Keywords: walnut shell, ultrasound-assisted extraction, phenolic compounds, antioxidant, DPPH

Received December 05, 2019; Revised January 06, 2020; Accepted January 24, 2020

Citation: Shusheng Wang, Wenyi Fu, Hannah Han, Milan Rakita, Qingyou Han, Qin Xu, Optimization of Ultrasound-Assisted Extraction of Phenolic Compounds from Walnut Shells and Characterization of Their Antioxidant Activities. Journal of Food and Nutrition Research, 8 (1). 50-57. January 2020. doi:10.12691/jfnr-8-1-7

1. Introduction

Walnut kernels are a staple in the human diet, but the kernel is enclosed by a hard, outer shell [1]. Since the walnut shell that protects the kernel comprises 67% of the total weight, a majority of the walnut becomes a waste byproduct in walnut kernel processing [2]. That is, millions tons of walnut shells become waste annually. Typically, the shells are burned as fuel or buried for disposal, leading to environmental concerns. Therefore, the walnut industries are seeking better applications of this low value waste in hopes of increasing its economic value while protecting the environment.

Walnut shells are mainly composed of cellulose, hemicellulose, and lignin. The cellulose and hemicellulose can be hydrolyzed to produce sugars for the production of bioethanol [3]. On the other hand, the production of charcoal and activated carbon by pyrolysis is another way to utilize the walnut shells [4,5,6,7,8,9,10]. In recent years, the production of pyroligneous acid in pyrolysis could result in a product consisting of mainly phenols and organic acids which bear high antioxidant and antimicrobial activities [11,12]. However, both the production of activated carbon and the pyrolysis of walnut shells are a complicated process. Therefore, it is urgent to find a valuable and simplified way of utilizing of walnut shells.

Besides the edible kernel, the shell and husk of the walnut are also valuable sources of bioactive compounds [13,14]. This fact has further motivated researchers to study the biological activities and functional properties in order to gain more usage of walnut waste [15]. Amariei et. al [16] reported that infusions from walnut shells and onion peels can be utilized for their antioxidant properties [3]. Other studies demonstrated that walnut shells have health-promoting effects because they are rich in phenolic acids and derivative polyphenols [6]. As the demand for natural sources of antioxidants is quickly increasing in the food industry, agricultural and food wastes are becoming an ideal substance from which to extract phenolic compounds as natural antioxidants [17]. Nowadays, increasing research efforts have been focused on the reutilization of low value by-products and waste from the food, forestry, and agricultural industries due to economic and environmental benefits [18,19].

As a natural antioxidant, phenolic compounds extracted from plant materials are gaining concern due to their positive effects on health such as reducing the risk of degenerative diseases and promoting the inhibition of macromolecular oxidation [1,20]. To generate nutraceuticals from plants, novel extraction methods such as microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE), and supercritical fluid extraction (SCFE) are typically applied [19,21]. Compared to other extraction methods, UAE is relatively efficient with low costs. In addition, this extraction method is very simple as it uses mechanical deformation and ultrasonic waves to break down particles in a suspension in order to increase the contact surface area between the solid and liquid phases. The UAE method also allows for the penetration of solvent into the sample matrix which speeds up the diffusion rate from solid into solvent [22,23]. The ultrasonic waves create cavitation bubbles to generate high temperatures and pressures which escalate solubility, diffusivity, and penetration of solvent into plant material for better extraction efficacy. Furthermore, the collapse of the cavitation bubbles can cause the sample to swell, leading to an expansion of the pores for the disruption of cell walls and release of cellular material into the suspension [24].

Finally, in order to determine the maximum phenolic yield, response surface methodology (RSM), a mathematical and statistical model, is most commonly used due to its ability to analyze the optimal combinations of extraction factors. Specifically, the Box-Behnken design (BBD) method is more effective than the general experimental design because it simplifies the experimental tests required for the analyses of the variables and their effects [25].

The aim of this study was to optimize the ultrasound-assisted extraction method to attain the maximum yield of phenolic compounds from walnut shells and to quantify the antioxidant activities of the extracts for potential future applications.

2. Materials and Methods

2.1. Materials and Preparation

Walnut shell chips were provided by Sierra Orchards in California, and the chips were air dried in an oven at 45 °C for 48 h. Then they were ground into powder using a spice grinder (Waring spice grinder, model-WSG 30, Waring Products, Torrington, CT, U.S.A.) and screened through a 20-mesh screen. The walnut shell powder was sealed in Ziploc® bags and stored in a freezer controlled at −18 °C for further use.

All of the chemicals were reagent grade, purchased from either Sigma-Aldrich Co. (St. Louis, MO, U.S.A.) or Fisher Scientific (Pittsburgh, PA, U.S.A.), and used without further purification.

2.2. Ultrasound Assisted Extraction (UAE)

The UAE method was modeled after the techniques utilized in the study conducted by Han et. al [26]. Briefly, 1.5 g of walnut shell powder was mixed with 30 mL of solvent to create a suspension with a 20:1 ratio of solvent to solid. The suspension was oscillated using an ultrasonic probe (Sonics and Materials, Newtown, CT, USA) in the dark at various times, temperatures, and amplitudes by an ultrasonic machine (Model VCF1500, Sonics & Materials, Newtown, CT, USA) with a drive frequency of 23 kHz and amplitude of 1500 W. The extract was immediately centrifuged (Allegra® 6, Beckman Coulter, Inc., Indianapolis, IN, U.S.A.) at 3,300 rpm for 10 min. The supernatant was collected as crude extract of phenolic compounds for further analysis.

2.3. Quantification of Total Phenolic Compounds

The total phenolic content (TPC) of the walnut shell extract was determined using a Folin-Ciocalteu method [20] with some modifications provided by Han et al [26]. Briefly, 0.5 mL of sample solution was mixed with 2.5 mL of 10-fold diluted Folin-Ciocalteu reagent and 2 mL of 7.5% sodium carbonate solution. After being heated at 45 °C for 20 min, the absorbance was read at 765 nm by a UV-visible spectrophotometer (Genesys-10S, Thermo Fisher Scientific, Waltham, MA, U.S.A.). The results were calculated as mg gallic acid equivalents per gram of dried weight of walnut shells using the equation as follows:

[picture] | (1)

where A is the absorbance and C is the concentration (mg GAE/DW).

2.4. Response Surface Methodology Design

In order to determine the range of extraction variation, single factor experiments were conducted. Every test was repeated three times, and the results were reported as the mean ± one standard deviation. Based on the preliminary data from previous experiments, the optimization was performed with a four-variable, three-level experiment and 29-run Box-Behnken Design (BBD). The high, middle and low levels were marked as +1, 0 and −1, respectively. Detailed parameters are shown in Table 1. The analysis of variance (ANOVA) for the response was implemented by Design Expert Software (version 10.0.3, State-Ease Inc., Minneapolis, MN). The significance of every response, setting p<0.05, was evaluated by means of ANOVA [25]. The interaction of independent variation on response was analyzed by three-dimensional (3D) response surface plots. F-values and p-values were used to check the significance of the regression coefficient. The coefficient of determination (R²) and the adjusted coefficient of determination (R²Adj) were employed to assess the adequacies of models. The verification of the experiment was conducted to confirm the predicted optimum conditions.

2.5. Antioxidant Activities of Phenolic Compounds Extracted from Walnut Shells

2.5.1. DPPH Radical-Scavenging Activity Assay

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of walnut shell extracts was measured following the method by Singh et al. [27]. Briefly, 50 μL of walnut shell extracts was added into 1.5 mL of DPPH solution (3.94 mg/100 mL in Ethanol), and the mixture was set in the dark for 20 min at room temperature. The absorbance at 517 nm was used to determine the concentration of the remaining DPPH by an UV-VIS spectrophotometer. The analysis was performed three times, and scavenging activity on DPPH radical was expressed as the percentage (%) inhibition with the use of the following equation [28]:

2.5.2. Ferric Reducing Antioxidant Power (FRAP)

The ferric reducing antioxidant (FRAP) assay was conducted using a modified method of Benzie and Strain [29]. Briefly, the walnut shell extracts (150 μL) was mixed with 2,850 μL of the FRAP solution for 30 min at 37 ○C in the dark. The absorbance was read at 593 nm and the results were expressed in μM Trolox equivalent (TE).

Table 1. Levels and code of variable used for BBD and observed responses for yields of TPC

3. Results and Discussions

3.1. Optimization of Phenolic Compounds Extraction from Walnut Shells

3.1.1. Effects of Extraction Parameters on Phenolic Compounds Yields from Walnut Shells

The results of four parameters in the extraction process of phenolic compounds from walnut shells were shown in Figure 1.

Extraction time was varied while the temperature, amplitude of ultrasound, and concentration of ethanol were fixed at 20°C, 60% and 50%, respectively. When the extraction time was increased from 1 to 45 min, the yield of TPC increased correspondingly and the highest extraction yield (83.01 ± 1.20 mg GAE/g DW, as shown by Figure 1a) was obtained at 5 min. In fact, even at an extraction time of 1 min, there was a relatively high yield of TPC (75.64 ± 3.38 mg GAE/g DW) because the ultrasonic probe sped up the mass transmission and diffusion of the phenolic compounds from the walnut shells into the solvent [26]. When extraction duration increased, the amount of transferred matter also increased, but the diffusion rate gradually decreased because of the increasing viscosity of the solution. Nevertheless, an excessive extraction time (>5 min) might lead to the degradation or the conversion of phenolic compounds. Therefore, the extraction time varying between 1 to 10 min was selected for the optimal extraction of phenolic compounds from walnut shells.

Different extraction temperatures were then tested when the time, ultrasound amplitude, and concentration of ethanol were set as 5 min with 60% amplitude and 50% of ethanol, respectively. As shown by Figure 1b, when the extraction temperature was increased from 0 to 20 ○C, the yield of TPC increased from 74.89 ± 6.69 mg GAE/g DW to 83.01 ± 1.20 mg GAE/g DW. However, the TPC dropped to 68.01 ± 1.12 mg GAE/g DW when the temperature was above 40°C. Celestino Santos-Buelga et al. [30] suggested that the extraction temperature should not be higher than 25°C. On the other hand, the conditions definitely affected the extraction process. Ultrasound increased the extraction temperature but decreased both surface tension and viscosity, resulting in soaring solubility and diffusion coefficient of the extractable matter. Even though the ultrasonic waves increased vapor pressure which aided in the penetration, transportation, and promotion of the phenolic compounds diffusion into solvent, the structure of the phenolic compounds could have been damaged and degraded at high temperatures [30]. Because of sonochemical effects at higher temperatures, the ultrasound-assist extraction was favored at low temperatures to increase the extraction yield of TPC [23]. Hence, the temperatures of 0, 20, and 40°C were chosen for extraction.

The influences of amplitude were investigated when the extraction time, temperature, and concentration of ethanol were set at 5 min, 20°C, and 50%, respectively. The increase in amplitude of the ultrasonic probe had a positive impact on the yield of TPC. The yield of total phenolic compounds increased to 83.21 ± 1.10 mg GAE/g DW with the amplitude set at 60%, and then decreased to 69.80 ± 3.10 mg GAE/g DW with the amplitude set at 100% (Figure 1c). The high amplitudes of ultrasound delivered more power and created more cavitation damage to the cell walls, thus releasing more phenolic compounds into the solvent [31]. However, Capelo-Martinez [32] reported that high amplitudes could degrade the extracts, thus decreasing the yields. In this case, no further increases in TPC yields would be observed when the amplitude reaches a level at which the extraction and degradation offset each other. Therefore, the amplitude range of 40-80% were chosen to extract phenolic compounds.

Ethanol and water were selected as the extraction solvents not only because they result in higher extraction yields of the TPC but also because they are safer and less toxic compared to methanol and other organic solvents [33]. Different concentrations of the ethanol were utilized while the extraction time, temperature, and amplitude were set at 5 min, 20 °C, and 60%, respectively. As shown by Figure 1d, when the ethanol concentration increased from 0% to 50%, the yield of the TPC also increased from 60.20 ± 6.06 mg GAE/g DW to 84.75 ± 8.25 mg GAE/g DW, surpassing the maximum at 50%, and then decreased when the solvent concentration was ≥70%. Using pure water (0% ethanol concentration) as a solvent produced about a 16.8% higher yield than 100% ethanol. The characteristics of phenolic compounds that exist in raw material affect the efficiency of the extraction process because the four hydroxyl groups and a carboxyl group make gallic acid readily soluble in polar solvents. Therefore, a mixture of water and ethanol allowed for higher polyphenol extraction efficiency than the usage of the solvents separately [34]. High ethanol concentrations stimulated the phenolic compounds to move from the walnut shell into solvent, which significantly assisted in the dissolution of phenolic compounds and increased the yield of the extraction. However, excessively high concentrations of ≥70% (see Figure 1d) decreased the solubility of phenolic compounds in ethanol, leading to a lower yield. The phenolics extracted from the walnut shells were a mixture of different classes of phenolic compounds, which were soluble in the solvent system. Other compounds such as sugars, organic acids, fats, and etc. can affect the yields of TPC and its antioxidant activities. Various concentrations of solvents have different polarity which also affects the yields of TPC [35]. Consequently, the appropriate solvent increases the yields of TPC and reduces the energy consumption during the extraction process, thus achieving significance for sustainable application as an energy source in the agriculture and food area [34]. Therefore, 50% ethanol was chosen as the central point of Box-Behnken Design (BBD) experiment.

Figure 1. Effect of extraction parameters (a) time, (b) temperature, (c) amplitude, and (d) ethanol concentration on TPC yields from walnut shells

3.1.2. Analysis of Predictive Model

The levels and codes of variables used for BBD and the observed responses for the yields of TPC were shown in Table 1. The analysis of variance for RSM experiments were presented in Table 2. The yields of TPC could be predicted by a second-order polynomial equation, which was attained by multiple regression analysis, as follows:

[picture] | (3)

where Y stand for the predicted yields of TPC, and A, B, C and D were coded as the variates of the extraction of temperature, time, ultrasonic amplitude and ethanol concentration, respectively.

Table 2. Analysis of variance for response surface quadratic model

Table 2 displayed the interaction of different factors on the yields of TPC, which were assessed by analysis of variance. The prediction model was analyzed comprehensively. A high F-value (F=13.43) meant that the quadratic regression equation of the predictive model was extremely significant (p<0.0001). The probability that this large F-value (F=13.43) occurred due to chance was only 0.01%. The low p-value (p<0.05) also indicated that the significance of the corresponding coefficient was high. Table 2 also showed that the linear coefficients of the ethanol concentration (D), the quadratic coefficients of the ethanol concentration (D²), and the cross-product coefficients of the extraction time versus the extraction temperature (AB) were statistically significant with p-values less than 0.05. The highly significant corresponding coefficient implies that the prediction model could be appropriately applied for the extraction progress. However, other coefficients were not significant. The F-value of 1.89 implied that the “lack of fit” was not significant relative to the pure error. The non-significant “lack of fit” is good for the conformity of the model because it can estimate the predicted response more properly [36]. Moreover, a high value of the determination coefficient (R²=0.9307) indicates that four independent parameters only had 6.93% probability unmatched by the model. Therefore, the model could be applied for the prediction of the response. The low coefficient of the variation value (C.V.=13.53) demonstrated that the model had a preferable accuracy. Thus, it could provide reliable experimental data.

3.1.3. Analysis of Response Surface and Contour Plot

To understand both the main effects and interaction effects of the factors for TPC extraction, the response surface plot was utilized. The contour plot considers the two factors simultaneously while maintaining all other factors at the settled level [37]. Figure 2 illustrated the plotted extraction time, temperature, ultrasonic amplitude, and ethanol concentration with contours, and the yield of total phenolic compounds was set as the response. The influence of the two factors on the response were assessed each time while the other two factors were kept at a central level.

Figure 2a represented TPC production in the presence of two variables: extraction time (A) and extraction temperature (B). It was observed that the yields of TPC gradually increased as the extraction time (A) increased from 1 to 10 min, while the trend was opposite as the extraction temperature (B) increased from 0 to 40 °C. Figure 2b showed that the yields of TPC increased along with ascending extraction time (A) and the ultrasound amplitude (C). Meanwhile, the mutual effects of the extraction time (A) and the ethanol concentration (D) on the yields of TPC were revealed in Figure 2c. The yields of TPC increased as the extraction time (A) increased from 1 to 10 min. The maximum yields of TPC was achieved with the long extraction times (approximately 10 min) and the high ethanol concentrations (D) (approximately 50%). Therefore, a greater yield could be obtained with longer extraction times and appropriate levels of ethanol concentration. Figure 2d showed that the effect of the extraction temperature (B) and extraction amplitude (C) on TPC yield was not evident in other factors. Figure 2e showed that the response reach peak at 50% ethanol concentration (D), along with the increasing of extraction temperature (B). However, the TPC production decreased as the temperature rose. The ultrasound amplitude (C) and the ethanol concentration (D) were determined to have quadric influence on the yield of TPC as shown by Figure 2f.

Figure 2. Response surface plots (a–f) showing the effects of the extraction time, temperatures, amplitude, and concentration of solvent on the yields of the total phenolic content (TPC) from walnut shells

According to the result from the regression analysis and the response surface plots, the highest yield of the TPC was attained at 10 min, 0°C, 53.93% ultrasound amplitude, and 43.71% ethanol concentration. Under those optimal variables, the highest predicted yield of TPC was 95.49 mg GAE/g DW.

3.1.4. Verification Test

As mentioned previously, three confirmatory experiments were conducted with the optimal variables to verify the optimal parameters obtained from the model equations. The maximum yield of TPC was 92.96±1.47 mg GAE/g DW, which was close to the predicted value. Hence, the experimental data showed that the response model was reliable and could be effectively employed to establish an UAE system for the recovery process of the phenolic compounds from walnut shells.

3.2. Antioxidant Activities of Phenolic Extracts from Walnut Shells

3.2.1. Scavenging Activity of DPPH

DPPH is a stable organic nitrogen free radical, and its scavenging capability has been widely used to evaluate the antioxidant capacity of extracts from plant materials [13].

The antioxidant activity of ultrasonically extracted phenolic compounds from the walnut shells was compared against antioxidant activity of two commonly used synthetic antioxidants, TBHQ and BHA, as seen in Figure 3. At lower concentrations of phenolic compounds (10 to 30 µg/mL), the scavenging activities were all similar and within the range of 4.05 – 13.25 %. Between concentrations of 125 and 250 µg/mL, the scavenging activities of the walnut shell extracts were greater than TBHQ and BHA. At a concentration of 500 µg/mL, the scavenging activity of walnut shell extracts was close to TBHQ but higher than BHA, which was 88.59, 88.22, and 76.95 %, respectively. DPPH data showed that the phenolic compounds from walnut shells could applied as a natural antioxidant replacement of TBHQ and BHA.

3.2.2. Ferric Reducing Antioxidant Power (FRAP)

The measurement of the antioxidant capacity in the natural complex of plant materials usually uses more than one single method. Therefore, in addition to DPPH radical scavenging assay, the ferric reducing antioxidant power (FRAP) assay was used to check the antioxidant activity of the walnut shell extracts [38]. FRAP reveals the ability of samples to bind to metal ions for reducing metals and thus inhibit the metal ion catalyzed generation of reactive species [39,40].

Figure 3. Comparison of the antioxidant activity of the phenolic extract from walnut shells with BHA, and TBHQ by DPPH assay

Figure 4 showed the FRAP of the phenolic compounds from walnut shells, TBHQ and BHA at concentrations of 10-100 µM, expressed as µM of Trolox equivalent. The results indicate that the FRAPs of the phenolic extracts from the walnut shells were lower than those of both TBHQ and BHA. The phenolic compounds extracted from walnut shells are crude without further purification. Therefore, although the behavior of the antioxidant power in the extract was different compared to the behavior of the artificial antioxidants, the FRAP values of the walnut shell extracts were highly correlated with the total phenolic compounds.

Despite the fact that the FRAP values of the walnut shell extracts were lower compared to BHA and TBHQ, the antioxidant activity of the extract could be modified by doubling or tripling its concentration to match the antioxidant power of BHA and TBHQ. Thus, the natural, plant-derived antioxidant can be utilized as a replacement for synthetic antioxidants that bear many negative effects to human health [39].

Figure 4. Comparison of the antioxidant activity of the phenolic extract from walnut shells with BHA, and TBHQ by FRAP assay

4. Conclusion

Walnut shells are by-products in the walnut processing industries, most of which are deposited in a landfill or burned. Thus, researchers have taken various approaches in order to recycle walnut shells. In this study, the application of an ultrasonic probe for extraction was evaluated, and the optimal operating conditions were determined by surface response methodology. The antioxidant activities of the walnut shell extract, as measured by DPPH radical scavenging capacity, was determined to be equivalent and even potentially better than the activities of synthetic antioxidants (BHA and TBHQ). However, the ferric reducing antioxidant power of the walnut shell extracts was lower than BHA and TBHQ. Despite this, the antioxidant activity of walnut shell extracts can be enhanced by increasing the concentration to match the antioxidant power of BHA and TBHQ. Therefore, the phenolic extracts from walnut shells can be recognized as a natural source of antioxidants. Since the phenolic extracts of plant materials are usually a mixture of different types of phenolic compounds and thus soluble in an organic solvent system, ethanol was used the solvent for the production of crude phenolic extract. To further investigate the mechanism of the antioxidant activity of phenolic extracts from walnut shells, additional purification steps are required to remove nonphenolic substances. This study demonstrated a potential way to increase profitability for the walnut industry by simplifying the process of producing antioxidants from walnut shells.

Acknowledgements

The authors are grateful for the financial support from John & Emma Tse through Li-Fu Chen Memorial Laboratory Fund, Center for Materials Processing Research, and the Science and Technology Department of Jilin Province (No. 20170101108JC) for supporting Wang’s visit to Purdue University.

Statement of Competing Interests

The authors have no competing interests.

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