Abstract
Gallic acid is recognized as a notable bioactive compound among secondary polyphenolic metabolites. In the current study, gallic acid-enriched extracts were obtained from mango peels using different solvents (ethanol or water) via ultrasound-assisted extraction, and optimized yields were compared with the conventional extraction technique (decoction). Independent variables for the optimization through response surface methodology were ethanol concentration (0–60%), solvent ratio (10–50 mL/g), temperature (30–60℃), and time (10–30 min) for ethanolic extraction. However, extraction carried out by using water had extraction conditions of pH (2–8), solvent ratio (20–0 mL/g), extraction temperature (40–70℃), and time (30–60 min). The optimized yield of gallic acid obtained through ethanol was 5.75 ± 0.21 mg/g, whereas 3.14 ± 0.24 mg/g of gallic acid was quantified in extraction through water. The results were compared with the aforementioned conventional method of decoction, and it was concluded that the ethanolic extracts of mango peels showed the highest gallic acid yield and total flavonoid contents. The obtained extracts could be a potential source of polyphenolics, especially gallic acid, for use in nutraceuticals as well as in other food applications.
1 Introduction
Phenolic compounds have gained a prominent place in the food, cosmetic, and pharmaceutical industries. These compounds possess bioactive properties that can positively impact the immune systems of both humans and animals [1]. Fruits, vegetables, and their agro-waste are the resort of bioactive compounds, and their extraction becomes, even more, cost-effective if extracted for the valorization of agricultural waste since it is economically beneficial as well as reduces pollution in the environment that is caused by this waste [2]. Regarding agro-waste, mango waste has also attracted researchers toward its bioactive compound profile and multiple polyphenols [3]. Mango processing generates huge quantities (40–60%) of mango waste, including seed kernels and peels, which are a great source of bioactive compounds but go to waste [3,4]. Mango waste consists of numerous effective bioactive compounds, which confirms that it is a coin dumped under rocks, not mere waste. This waste contains high levels of phenolics and can be utilized in food and feed applications if these active compounds are correctly extracted [5].
The composition of mango peels comprises 45–80% dietary fiber content, 16–30% soluble and 30–50% insoluble fractions, and it varies based on the cultivar. Apart from this, various proteins, celluloses, hemicelluloses, and polyphenols are also present abundantly [6]. Polyphenolics such as gallotannins, alkyl resorcinol, xanthones, flavonols, and derivatives of benzophenones [7,8], as well as mangiferin, anthocyanins, gallic acid, kaempferol, catechin, quercetin, tannins, and ellagic acid are also present in mango peels [9]. Among all, gallic acid is the most abundant phenolic compound of mango waste [10], followed by p-OH-benzoic acid, m-coumaric acid, p-coumaric acid, and ferulic acid [1,5]. It has major therapeutic benefits such as antioxidant, antibacterial and antiviral, anti-inflammatory, antiallergic, anticarcinogenic and antimutagenic properties [11,12].
The gallic acid content of mango peel differs according to variety, origin of the fruit, and method of extraction. Its concentration was reported to be 0.08–0.59 mg/g when extracted with 80% ethanol [13], 4.54 mg/g of extract by using 70% methanol and acetone [14], and 12.5–25.6 mg/100 g of dry matter when extracted using 80% methanol [15]. Two other studies reported it as 18 µg/100 mL and 20.21 µg/g of extract as a result of extraction with 65% and 50% ethanol, respectively, with a combination of different enzymes [16,17]. However, several studies report the yield of gallic acid from mango peels with different extraction solvents but none has studied the optimization of gallic acid. Furthermore, the extraction processes reported in the studies have higher concentrations of organic solvents such as methanol, acetone, and ethanol. The utilization of organic solvents (ethanol, methanol, diethyl ether, acetone etc.) with or without combination with water has been widely reported for the extraction of bioactive compounds with multiple extraction techniques [2,9,16,18,19,20]. Even so, there are some health and environmental concerns related to the safety of these extraction solvents, which is why it is necessary to adopt green extraction solvents and methods [2]. However, among the organic solvents, ethanol is labeled as generally recognized as safe, by US food and drug administration, to be used in food products [21].
Green extraction principles have been developed to promote the use of viable plant resources and varieties, water as an alternative solvent, reduced energy consumption, safe and speedy processes, and the production of biodegradable and uncontaminated plant extracts [22]. These are also known as “cold extraction techniques,” where the stability of the extracted compounds is not compromised as well as the amount of time or energy required for extraction is kept to a minimum [23]. However, the use of solvents such as methanol, ethanol, acetone, and their aqueous combinations in extraction has been a prominent characteristic of recovering bioactive compounds from plants [24]. Despite the higher extraction efficiency of organic solvents, water has remained the commonly used solvent in industries because it is safe for the environment, cheap, and non-toxic. Water can be the most environment-friendly solvent when used in conjunction with modern extraction techniques to produce sustainable nutraceuticals [25,26].
The extraction process can be significantly improved by utilizing modern techniques and applying mechanical and physical actions to the material. Those that use less solvent and for a shorter period of time have a positive impact on the environment. High hydrostatic pressure, supercritical fluid extraction, microwave hydrodiffusion and gravity, pulsed electric field, ohmic heat, radio frequency, microwave-assisted extraction, and ultrasound-assisted extraction (UAE) are the most commonly used unconventional methods for effective and sustainable extraction of polyphenols from plant matrices [19,27]. Among these, UAE is a successful extraction approach due to the feature of acoustic cavitation, which is caused by the passage of an ultrasound wave produced in the solvent, and may improve extraction capability in plants [28]. UAE is regarded as a good extraction method due to its ease of adoption and application, as well as the lower cost of developing a workable setup when compared to other current procedures [21]. In this line, a study was conducted by our research team to optimize extraction of gallic acid from mango seed kernel via UAE with 19.4% ethanol and the resulting yields were comparable to that of high-solvent extraction systems [29].
The current study was planned to utilize mango peels for extraction of gallic acid by following the same methodology. The study aimed at evaluating the extraction efficiency of two different extractions (with or without ethanol) described in methodology. The obtained extracts were subjected to high-performance liquid chromatography (HPLC) analysis to quantify gallic acid. Both extracts obtained after optimization of extraction conditions were compared for the yield of gallic acid and total flavonoid content (TFC) with conventional extraction of decoction. This meticulous comparison aimed to provide a comprehensive understanding of the characteristics of the extracts obtained from mango peels with different solvents to enable its utilization in food and nutraceutical applications.
2 Materials and methods
2.1 Chemicals
For this experiment, gallic acid standard was acquired from Sigma Aldrich, Germany. Acetonitrile, ethanol, formic acid, methanol, and water, all of HPLC grade, were purchased from Merck, Germany. Additionally, we procured 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Alfa Aesar, USA) and Folin–Ciocalteu (Scharlau, Spain); sodium hydroxide, aluminum chloride, and sodium nitrite were procured from Daejung, South Korea.
2.2 Collection and extraction of mango peels
Mango peels (Chaunsa variety) were procured in June 2021 from Iftekhar Ahmed & Co., a fruit processing unit located in District Sargodha, Punjab, Pakistan. The peels were then air dried until they reached a moisture level of <10%, ground into a fine powder, and screened through a 40-mesh sieve to obtain a particle size of 0.4 mm on the basis of a preliminary analysis. Samples were then stored in labeled plastic bags and stored at 4℃, maintaining their quality until they are needed.
Extraction was executed in an ultrasonic bath (E30H, Elma, Germany) having a frequency of 37 kHz, with an adjustable time and temperature controller. One gram of mango peel powder was weighed precisely and mixed in the extraction solvent in a beaker. After proper mixing, the sample was transferred to an ultrasonic bath and subjected to predetermined irradiation conditions as described in Section 2.3.1. Next, the samples were filtered using Whatmann filter paper (No. 1, England), followed by concentration in a water bath (Memmert, Germany) at 50℃ till it reached one-third of the actual volume. The extracts were then stored at 4℃ until next analysis.
2.3 Experiment design
2.3.1 Single-factor experiments of ethanolic extraction
In order to determine the working ranges for independent variables, initial experiments were conducted. These independent variables included solvent concentration (ethanol), solvent ratio, extraction time, and extraction temperature. The extracts were prepared and tested at different ethanol concentrations (0, 20, 40, and 60%), solvent ratios of 10, 20, 30, 40, and 50 mL/g, temperature (30, 40, 50, and 60℃), and extraction time (10, 15, 20, 25, and 30 min). When not being evaluated, all extraction variables were fixed at 40℃, 10 mL/g, and 10 min. HPLC analysis was performed on the prepared extracts to determine the gallic acid content. Based on the results of these single-factor experiments, the working ranges (maximum and minimum values) were selected.
2.3.2 Single-factor experiments of aqueous extraction
In the second extraction procedure, only water was used for the extraction by changing its pH. Solutions of NaOH and HCl were used to set the pH. The effect of pH along with solvent ratio, temperature, and time of extraction was studied. Selected pH levels were 4, 6, and 8, the solvent ratios at 20, 30, 40, 50, and 60 mL/g, extraction temperatures of 40, 50, 60, and 70℃, and the extraction time range was 30, 40, 50, and 60 min. Whenever not being evaluated, the extraction variables were fixed at 50℃, 30 mL/g, and 40 min. The prepared extracts were then analyzed on HPLC to determine the yield of gallic acid. Selection of the extraction variable ranges (minimum and maximum values) for optimization was done on results of single-factor experiments.
2.3.3 Multiple-factor experiments
After the selection of extraction ranges (highest, middle, and lowest) of all four variables for response surface methodology, three central composite design levels for both extraction procedures were used for extraction optimization [29]. These variables included ethanol concentration and pH (X 1) for both procedures, respectively. The other extraction variables for both procedures were solvent ratio (X 2), temperature (X 3), and time (X 4), with a focus on the yield of gallic acid as response variable. Table 1 shows the actual and coded levels of each variable for extraction procedures based on our preliminary study of single-factor extraction. Twenty-seven experimental runs were carried out with three replications at center point in order to establish the method repeatability index. A second-order polynomial regression model was used to accurately express the contents of gallic acid.
Working ranges of experimental variables
| Factors | −1 | 0 | 1 |
|---|---|---|---|
| (a) Ethanolic extraction | |||
| Ethanol concentration (%) | 10 | 20 | 30 |
| Solvent ratio (mL/g) | 20 | 30 | 40 |
| Temperature (℃) | 30 | 40 | 50 |
| Time (min) | 15 | 20 | 25 |
| (b) Aqueous extraction | |||
| pH | 3 | 4 | 5 |
| Solvent ratio (mL/g) | 20 | 30 | 40 |
| Temperature (℃) | 30 | 40 | 50 |
| Time (min) | 30 | 40 | 50 |
2.4 HPLC analysis
HPLC analysis of extracts was carried out by partitioning of the extracts with n-hexane as previously described by Hayat et al. [29]. Briefly, the extracts underwent a filtration process using a 0.45 μm syringe filter for analysis. The Agilent HPLC (1260 Infinity II, Agilent Technologies Inc., USA) with a VWD-UV visible detector was used to obtain the phenolic profiles of extracts. The analysis was conducted on a Zorbax Eclipse plus C18 analytical column measuring 4.6 mm × 150 mm with a particle size of 5 µm, manufactured by Agilent, USA. The mobile phase consisted of solvent A (distilled water: formic acid, 99:1, v/v) and solvent B (acetonitrile: formic acid, 99:1, v/v), with a flow rate of 0.6 mL/min and following a linear gradient scheme of t in min; %B: (0; 0%), (5; 20%), (10; 50%), (15; 100%), and (20; 0%). Chromatograms were recorded at a wavelength of 280 nm using 20 µL of injection volume at 25°C temperature. Quantification of gallic acid was done based on standard curve comparisons and peak area. Results were presented as a mean value ± SD of the assay for each experiment which was conducted in triplicates.
2.5 Comparison of optimized ethanolic and aqueous extracts of mango peels
The gallic acid-rich extracts obtained after optimization were compared with each other as well as with a conventional extraction method of decoction in order to evaluate the efficacy of the methods. Decoction extracts were prepared by mixing mango peel powder with water and incubating it in boiling water for 30 min, as previously described by Chanda et al. [30]. The extracts were filtered, concentrated, and stored as mentioned in Section 2.2.
The extracts were freeze-dried (−50℃, 24 h) and were labeled as MP-Eth. (ethanolic extracts of mango peels), MP-Aq. (aqueous extracts of mango peels), and MP-Dec. (decoction extracts of mango peels), respectively. To calculate TFC, 1 mL of extracts (at a concentration of 0.01/1 mL) was combined with 5 mL of distilled water as previously described [29]. A volume of 0.3 mL of 5% sodium nitrite was added, followed by the addition of 0.6 mL of 10% aluminum chloride, 2 mL of 1 M sodium hydroxide, and 2.4 mL of distilled water. The reagents were mixed thoroughly and allowed a 5 min interval before adding the next reagent. Analysis was performed in triplicate, and absorbance was recorded at 510 nm via spectrophotometer. TFC was calculated and represented as milligrams of catechin equivalent per gram of dried extract using the catechin calibration curve.
2.6 Statistical analysis
All the experiments were conducted in triplicates and results were presented as mean ± SD. Analysis of variance (ANOVA) was applied to detect the statistical differences (p < 0.05) through SPSS (version 25) on the data generated by the comparison study of extraction techniques. Data analysis for model construction, predicting values, and visualizing the influence of independent variables were all done through Design Expert Software (Version 12, Stat-Ease, Inc., Minneapolis, MN, USA). The three-dimensional graphs were used to plot the yields of gallic acid as a response variable. The non-significant lack of fit was calculated to best fit the model’s regression equation. The coefficient of determination of R 2, lack of fit, and Fisher value test (F-value) were assessed to determine the sufficiency and quality of the model.
3 Results and discussion
3.1 Single-factor experiments of ethanolic and aqueous extraction
3.1.1 Effect of ethanol concentration
The study analyzed the effect of varying ethanol concentrations (0, 20, 40, and 60%) on gallic acid yields from mango peels. The solvent ratio was fixed at 10 mL/g, temperature at 40℃, and time at 10 min. The results showed a notable increase in gallic acid yield (4.21 mg/g) as ethanol concentration went up from 0 to 20% (Figure 1a). However, a gradual decline in yield was observed as the concentration increased to 40 or 60%, unlike the studies that reported the highest phenolic content obtained at 40–60% of aqueous ethanol through UAE [19,27,28,31]. The results suggest that increase in ethanol concentration has a negative effect on the extraction of gallic acid. Polarity of solvents plays an important role in extraction of bioactive compounds; since water is a polar solvent, increase in water percentage with ethanol allowed to extract hydrophilic phenolic or non-phenolic compounds, efficiently [32]. Hence, 20% ethanol concentration was used in successive experiments.
Single-factor experiments of ultrasound-assisted ethanolic extraction of mango peels (a) ethanol concentration (%); (b) solvent ratio (mL/g); (c) temperature (℃); (d) time (min).
3.1.2 Effect of pH
Aqueous extraction of mango peel powder was carried out by adjusting the pH levels of distilled water to 2, 4, 6, and 8. Other extraction parameters were kept constant at pre-determined levels. Results showed that the yield of gallic acid was higher at pH 4 (4.14 mg/g). The yield gradually declined with the increase in pH of the extraction solvent as shown in Figure 2(a). The results are in agreement with the study reporting effect of pH of total phenolic contents extracted from mango peels ranging from pH 2 to 8. The phenolic content increased as the pH increased up to 4 but declined when a pH higher than 4 was used [33]. Acidic pH facilitates the gallic acid yield which is basic in nature as the non-charged form of gallic acid transfers to the ionic liquid-rich phase due to low pH, leading toward the increased solubility of the gallic acid in extraction solvent [34]. As the solubility increases, the concentration of the extractable also increases until the total pool of extractable in the sample is exhausted or the solubility of dissociated form of the extractable exceeded [25]. Based on the results, pH 4 was selected to be used in subsequent experiments.
Single-factor experiments of ultrasound-assisted aqueous extraction of mango peels (a) pH; (b) solvent ratio (mL/g); (c) temperature (℃); (d) time (min).
3.1.3 Effect of solvent ratio
Various solvent ratios were tested, including 10, 20, 30, 40, and 50 mL/g, while adjusting other variables such as 20% solvent concentration, 40℃ temperature, and 10 min. Figure 1(b) illustrates that gallic acid content increased as the ratio increased to 30 mL/g. However, a gradual decline in yield was seen with further increases to 40 or 50 mL/g. The maximum yield was observed at a solvent ratio of 30 mL/g, which was 6.27 mg/g for the ethanolic extraction procedure. A similar effect of solvent ratios ranging from 20 to 60 mL/g for aqueous extraction was observed as the gallic acid yield was highest at 30 mL/g (4.13 mg/g) as depicted in Figure 2(b). There was a gradual decline in gallic acid with the increase in ratio of more than 30 mL/g as similar results were reported for extraction of total phenolic content from mango peels when extracted with 50% ethanol at 30 mL/g solvent ratio [19]. Another study reported the same liquid-to-solid ratio suitable for the maximum extraction yield of mangiferin from mango peels [28]. There are some other studies that reported high solvent ratios ranging from 40 to 50 mL/g to give maximum phenolic recovery [32,33]. As the ratio increases, the compounds present in plants are more efficiently dissolved, which results in increasing the extraction yield [35]. The principle of mass transfer is consistent with raise in yield of gallic acid. The main driving force behind this process is the gradient in sample and solvent concentration, which becomes stronger with a higher solvent ratio [33]. The highest gallic acid yield was observed at a solvent ratio of 30 mL/g, which was used further.
3.1.4 Effect of extraction temperature
Different temperature levels (30, 40, 50, and 60℃) were tested, with other extraction parameters set at 20% ethanol concentration, 30 mL/g of solvent ratio, and 10 min of time. The overall gallic acid yield was increased as the extraction temperature rose to 40℃, then reduced at 50 and 60℃ (Figure 1c). The highest yield, 6.19 mg/g, was achieved at 40℃. Likewise, aqueous extraction was performed on temperatures from 40 to 70℃ by keeping other factors constant or fixed at determined points. Upon analysis of extracts, it was revealed that the extraction temperature at 40℃ was the most efficient one to yield the highest gallic acid (4.22 mg/g) among all (Figure 2c). Similar findings were reported in a study where phenolic contents extracted from mango peels were highest between 35 and 45℃ [19]. The yield was reduced at higher temperatures as higher extraction temperatures increase solvent diffusion into cells, improving desorption and solubility of compounds, and facilitates splitting up of compounds. However, bioactive compounds are often denatured at high temperatures, as observed in the decrease in yield of gallic acid at temperatures between 40 and 60℃ [36]. The results suggest that gallic acid has the highest solubility and desorption equilibrium at 40℃. Additionally, it was found to be thermally stable between 30 and 50℃. These findings have significant implications for the potential applications of gallic acid in various industries [37]. However, higher temperatures have been found to reduce the viscosity of solvent and enhance the plant cell wall permeability, resulting in a positive impact on overall yield. It is important to note that excessively high temperatures can have adverse effects on the quantity and quality of extracted phenolic compounds [28,33]. Thus, for optimization purposes, an extraction temperature of 40℃ was selected for both methods.
3.1.5 Effect of extraction time
The study involved processing samples using UAE for varying time periods (10, 15, 20, 25, and 30 min) at 20% of solvent concentration, 30 mL/g of solvent ratio extracted at 40℃. Based on the results (Figure 1d), the yield gradually increased from 10 to 20 min and decreased thereafter. The significantly higher yield of 6.24 mg/g was obtained at 20 min. Similar time period was reported to gain maximum polyphenols and flavonoids in a study where peels were treated with UAE for 20 min [27,28]. However, gallic acid yield was higher at 40 min in aqueous extraction when different time intervals (30, 40, 50, and 60 min) were studied to determine a center point for the single-factor extraction optimization of gallic acid from mango peels (Figure 2d). It was observed that gallic acid yield was reduced with the increase in extraction time. The study suggests that ultrasonic waves enhance the equilibrium coefficient between the cell walls of plants and solvent, thereby dissolving target compounds more quickly. This makes UAE a more efficient extraction method than conventional approaches [39]. However, the prolonged exposure to ultrasonic waves may lead to the degradation of bioactive compounds [28]. While the extraction time of 10–20 min increased the yield of gallic acid, further increases beyond 20 min yielded diminishing returns. Hence, 20 and 40 min were chosen as extraction times for optimization of MP-Eth. and MP-Aq., respectively.
3.2 Multiple-factor ethanolic extraction optimization of gallic acid-enriched extracts
The gallic acid yield was obtained after running 27 experiments in triplicates (Table 2). The extracts were prepared and analyzed according to the experimental variable combinations generated by the design. The gallic acid content was observed between 4.16 and 5.95 mg/g. The highest gallic acid yield was obtained at the extraction variables of X 1 = 20%, X 2 = 30 mL/g, X 3 = 40℃, and X 4 = 20 min.
Gallic acid yield of ethanolic extraction of mango peels for the experimental design
| Run | X 1 Ethanol concentration | X 2 Solvent ratio | X 3 Temperature | X 4 Time | Gallic acid (mg/g)* |
|---|---|---|---|---|---|
| 1 | 10(−1) | 40(1) | 30(−1) | 15(−1) | 5.25 ± 0.03 |
| 2 | 20(0) | 30(0) | 40(0) | 20(0) | 5.80 ± 0.03 |
| 3 | 20(0) | 30(0) | 40(0) | 20(0) | 5.72 ± 0.02 |
| 4 | 10(−1) | 20(−1) | 50(1) | 25(1) | 4.71 ± 0.02 |
| 5 | 30(1) | 40(1) | 50(1) | 25(1) | 5.03 ± 0.02 |
| 6 | 10(−1) | 40(1) | 50(1) | 25(1) | 4.38 ± 0.07 |
| 7 | 40(2) | 30(0) | 40(0) | 20(0) | 5.76 ± 0.03 |
| 8 | 20(0) | 50(2) | 40(0) | 20(0) | 5.95 ± 0.03 |
| 9 | 30(1) | 20(−1) | 50(1) | 15−1) | 4.77 ± 0.03 |
| 10 | 20(0) | 30(0) | 40(0) | 20(0) | 5.80 ± 0.02 |
| 11 | 30(1) | 20(−1) | 30(−1) | 25(1) | 4.85 ± 0.04 |
| 12 | 10(−1) | 20(−1) | 30(−1) | 25(1) | 4.85 ± 0.03 |
| 13 | 20(0) | 30(0) | 40 (0) | 30(2) | 4.65 ± 0.06 |
| 14 | 20(0) | 30(0) | 60(2) | 20(0) | 4.24 ± 0.03 |
| 15 | 30(1) | 40(1) | 30(−1) | 25(1) | 5.73 ± 0.04 |
| 16 | 10(−1) | 40(1) | 50(1) | 15(−1) | 5.33 ± 0.05 |
| 17 | 30(1) | 40(1) | 50(1) | 15(−1) | 5.38 ± 0.01 |
| 18 | 30(1) | 40(1) | 30(−1) | 15(−1) | 5.24 ± 0.03 |
| 19 | 10(−1) | 20(−1) | 30 (−1) | 15(−1) | 4.45 ± 0.04 |
| 20 | 30(1) | 20(−1) | 50(1) | 25(1) | 4.74 ± 0.03 |
| 21 | 20(0) | 30(0) | 40(0) | 10(−2) | 4.16 ± 0.03 |
| 22 | 10(−1) | 40(1) | 30(−1) | 25(1) | 5.54 ± 0.03 |
| 23 | 0(−2) | 30(0) | 40(0) | 20(0) | 5.64 ± 0.01 |
| 24 | 30(1) | 20(−1) | 30(−1) | 15(−1) | 4.56 ± 0.03 |
| 25 | 10(−1) | 20(−1) | 50(1) | 15(−1) | 5.03 ± 0.04 |
| 26 | 20(0) | 30(0) | 20 (−2) | 20(0) | 4.78 ± 0.05 |
| 27 | 20(0) | 10(−2) | 40(0) | 20(0) | 5.40 ± 0.02 |
*Results are presented as mean ± SD.
Test and response variables were checked via multiple regression analysis of experimental data by following equation (1).
where Y is the gallic acid yield which is the response variable while X 1, X 2, X 3, and X 4 are the factors for extraction optimization.
ANOVA for the regression equation is given in Table 3. The analysis confirmed that lack of fit helped us ensure the model’s adequacy. We found a lack of fit that was not statistically significant (p > 0.05), indicating that the model was fitting the experiment appropriately. Moreover, we accurately measured the signal-to-noise ratio, which was 12.962, a preferred value as it is higher than 4. This indicated a definite design space, implying that this model could be of great help. Additionally, we noticed that the plotted points were clustered finely around the diagonal line, demonstrating the connection between experimental and predicted values. This is a sign of the model’s fitness, as the predicted R-squared value of 0.6883 was in reasonable agreement with the R-squared adjusted value of 0.8820.
ANOVA for the fitted quadratic model
| Source | Sum of square | Degree of freedom | Mean square | F-value | p-value |
|---|---|---|---|---|---|
| Model | 6.95 | 14 | 0.4967 | 14.88 | <0.0001 |
| X 1 | 0.0417 | 1 | 0.0417 | 1.25 | 0.2857 |
| X 2 | 1.05 | 1 | 1.05 | 31.46 | 0.0001 |
| X 3 | 0.1980 | 1 | 0.1980 | 5.93 | 0.0314 |
| X 4 | 0.0267 | 1 | 0.0267 | 0.7990 | 0.3890 |
| X 1 × X 2 | 0.0625 | 1 | 0.0625 | 1.87 | 0.1962 |
| X 1 × X 3 | 0.0020 | 1 | 0.0020 | 0.0607 | 0.8096 |
| X 1 × X 4 | 0.0600 | 1 | 0.0600 | 1.80 | 0.2047 |
| X 2 × X 3 | 0.2970 | 1 | 0.2970 | 8.90 | 0.0114 |
| X 2 × X 4 | 0.0462 | 1 | 0.0462 | 1.39 | 0.2621 |
| X 3 × X 4 | 0.6084 | 1 | 0.6084 | 18.23 | 0.0011 |
| X 1 2 | 0.0220 | 1 | 0.0220 | 0.6580 | 0.4331 |
| X 2 2 | 0.0313 | 1 | 0.0313 | 0.9393 | 0.3516 |
| X 3 2 | 2.32 | 1 | 2.32 | 69.43 | <0.0001 |
| X 4 2 | 2.70 | 1 | 2.70 | 80.93 | <0.0001 |
| Residual | 0.4005 | 12 | 0.0334 | ||
| Lack of fit | 0.3962 | 10 | 0.0396 | 18.57 | 0.0521 |
The three-dimensional surface plots of the model exhibit the optimized values to predict and display the relationship of all the processed variables. An elliptical shape of the plot shows a significant interaction of the variables, whereas the circular one depicts the insignificance of interaction between variables [38]. The results obtained from this study of gallic acid extraction optimization from peels of mango showed that ultrasonic treatment during 15–20 min and temperature ranging between 30 and 35°C with the least ethanol concentration proves to be an effective technique to extract bioactive compounds. Furthermore, the results indicate a significant interaction of time of UAE treatment and temperature that can enhance the gallic acid yield (Figure 3). Optimal values for the extraction variables were achieved by solving the regression equation. After running the experiment on the software, the optimal conditions for gallic acid extraction were as follows: 21.2% of ethanol concentration, 30.2 mL/g of solvent ratio, with 33.4°C extraction temperature, and 17.6 min of extraction time, with the corresponding Y = 5.77 ± 0.18 mg/g. Extraction was carried out in triplicates under the optimized conditions to recheck the results further. Gallic acid obtained from the extracts was 5.75 ± 0.21 mg/g, indicating that the model fitted the experimental data very well. Hence, optimization to obtain gallic acid-enriched extracts from mango peels through ethanol was completed (Table 6).
Response surface plots as effected by extraction variables of ethanolic extraction of gallic acid-enriched extracts from mango peels (a) ethanol concentration and solvent ratio; (b) ethanol concentration and temperature; (c) ethanol concentration and time; (d) solvent ratio and temperature; (e) solvent ratio and time; (f) temperature and time.
3.3 Multiple-factor aqueous extraction optimization of gallic acid-enriched extracts
The total number of runs generated was 27, so extracts were prepared according to the variables (Table 4) and gallic acid content was analyzed. The yields of gallic acid ranged from 0.65 to 3.15 mg/g. The highest gallic acid yield was obtained with the extraction variables of X 1 = 4, X 2 = 30 mL/g, X 3 = 40℃, and X 4 = 40 min.
Gallic acid yield of aqueous extraction of mango peels for the experimental design
| Run | X 1 pH | X 2 Solvent ratio | X 3 Temperature | X 4 Time | Gallic acid (mg/g)* |
|---|---|---|---|---|---|
| 1 | 3(−1) | 40(+1) | 50(+1) | 30(−1) | 2.15 ± 0.02 |
| 2 | 4(0) | 30(0) | 20(−2) | 40(0) | 2.41 ± 0.02 |
| 3 | 3(−1) | 20(−1) | 50(+1) | 50(+1) | 1.53 ± 0.04 |
| 4 | 3(−1) | 40(+1) | 30(−1) | 30(−1) | 2.08 ± 0.03 |
| 5 | 2(−2) | 30(0) | 40(0) | 40(0) | 2.10 ± 0.02 |
| 6 | 5(+1) | 40(+1) | 50(+1) | 30(−1) | 2.05 ± 0.03 |
| 7 | 5(+1) | 20(−1) | 50(+1) | 30(−1) | 1.47 ± 0.02 |
| 8 | 3(−1) | 40(+1) | 50(+1) | 50(+1) | 2.57 ± 0.04 |
| 9 | 5(+1) | 40(+1) | 30(−1) | 50(+1) | 2.33 ± 0.02 |
| 10 | 3(−1) | 20(−1) | 30(−1) | 30(−1) | 1.17 ± 0.03 |
| 11 | 3(−1) | 20(−1) | 50(+1) | 30(−1) | 1.70 ± 0.04 |
| 12 | 4(0) | 10(−2) | 40(0) | 40(0) | 0.65 ± 0.05 |
| 13 | 4(0) | 30(0) | 40(0) | 40(0) | 3.14 ± 0.02 |
| 14 | 4(0) | 30(0) | 40(0) | 40(0) | 3.15 ± 0.03 |
| 15 | 5(+1) | 20(−1) | 50(+1) | 50(+1) | 1.30 ± 0.03 |
| 16 | 4(0) | 30(0) | 40(0) | 60(+2) | 1.79 ± 0.02 |
| 17 | 4(0) | 30(0) | 60(+2) | 40(0) | 2.76 ± 0.04 |
| 18 | 4(0) | 50(+2) | 40(0) | 40(0) | 1.66 ± 0.03 |
| 19 | 5(+1) | 40(+1) | 50(+1) | 50(+1) | 1.75 ± 0.03 |
| 20 | 5(+1) | 40(+1) | 30(−1) | 30(−1) | 2.00 ± 0.04 |
| 21 | 3(−1) | 40(+1) | 30(−1) | 50(+1) | 1.64 ± 0.02 |
| 22 | 4(0) | 30(0) | 40(0) | 20(−2) | 1.22 ± 0.09 |
| 23 | 3(−1) | 20(−1) | 30(−1) | 50(+1) | 1.26 ± 0.04 |
| 24 | 5(+1) | 20(−1) | 30(−1) | 30(−1) | 1.5 ± 0.02 |
| 25 | 4(0) | 30(0) | 40(0) | 40(0) | 2.97 ± 0.02 |
| 26 | 6(+2) | 30(0) | 40(0) | 40(0) | 2.03 ± 0.04 |
| 27 | 5(+1) | 20(−1) | 30(−1) | 50(+1) | 1.50 ± 0.02 |
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*Results are presented as mean ± SD.
Experimental data of extraction variable and gallic acid yield were evaluated through multiple regression analysis using equation (2).
where Y is the gallic acid yield which is the response variable while X 1, X 2, X 3, and X 4 are the factors for extraction optimization.
Table 5 shows the ANOVA for the regression equation. The analysis presented that lack of fit helped in ensuring the model’s adequacy. A lack of fit was found that was not statistically significant (p > 0.05), indicating that the model was fitting the experiment appropriately. Moreover, we accurately measured a higher than 4 signal-to-noise ratio, which was 16.387, a preferred value. This indicated a definite design space, implying that this model could be of great help. Additionally, we noticed that the plotted points were clustered finely around the diagonal line, demonstrating the connection between experimental and predicted values. This is a sign of the model’s fitness, as the predicted R-squared value of 0.7052 was in reasonable agreement with the R-squared adjusted value of 0.8865.
ANOVA for the fitted quadratic model
| Source | Sum of square | Degree of freedom | Mean square | F-value | p-value |
|---|---|---|---|---|---|
| Model | 9.68 | 14 | 0.6917 | 15.50 | <0.0001 |
| X1 | 0.0048 | 1 | 0.0048 | 0.1079 | 0.7482 |
| X2 | 2.14 | 1 | 2.14 | 47.87 | <0.0001 |
| X 3 | 0.1261 | 1 | 0.1261 | 2.83 | 0.1185 |
| X 4 | 0.0337 | 1 | 0.0337 | 0.7564 | 0.4015 |
| X1 × X2 | 0.0110 | 1 | 0.0110 | 0.2471 | 0.6281 |
| X1 × X3 | 0.4096 | 1 | 0.4096 | 9.18 | 0.0105 |
| X1 × X4 | 0.0001 | 1 | 0.0001 | 0.0022 | 0.9630 |
| X2 × X3 | 0.0006 | 1 | 0.0006 | 0.0140 | 0.9077 |
| X2 × X4 | 0.0042 | 1 | 0.0042 | 0.0947 | 0.7636 |
| X3 × X4 | 0.0025 | 1 | 0.0025 | 0.0560 | 0.8169 |
| X12 | 1.54 | 1 | 1.54 | 34.43 | <0.0001 |
| X22 | 5.24 | 1 | 5.24 | 117.54 | <0.0001 |
| X32 | 0.4082 | 1 | 0.4082 | 9.15 | 0.0106 |
| X42 | 3.56 | 1 | 3.56 | 79.72 | <0.0001 |
| Residual | 0.5354 | 12 | 0.0446 | ||
| Lack of fit | 0.5150 | 10 | 0.0515 | 5.03 | 0.1771 |
| Pure error | 0.0205 | 2 | 0.0102 |
The three-dimensional surface plots obtained by the model exhibited the optimized values that showed the interaction between the variables explored in optimization process. The appearance of the plot helps in identifying these interactions between variables as the circular one exhibits the insignificant interaction while an elliptical shape depicts that there is a significant interaction between variables [38]. The three-dimensional surface plots in Figure 4 represent the interaction of extraction factors effecting the yield of gallic acid for aqueous extraction of mango peels. Quadratic polynomial equation was established to quantify the relationship between the extraction parameters and response. The optimized values for extraction were obtained by solving the regression equation.
Response surface plots as effected by extraction variables of aqueous extraction of gallic acid-enriched extracts from mango peels (a) pH and solvent ratio; (b) pH and temperature; (c) pH and time; (d) solvent ratio and temperature; (e) solvent ratio and time; (f) temperature and time.
After running the experiment, the common regions for optimized conditions of gallic acid extraction from mango peels were obtained with the corresponding Y = 3.14 ± 0.21 mg/g. Optimized conditions were used to perform triplicate extraction tests in order to validate the optimization. The gallic acid yield was found to be 3.14 ± 0.24 mg/g indicating that the extraction procedure for gallic acid was fully optimized (Table 6).
Predicted and experimental values of gallic acid yield for both extraction methods
| Extraction method | Optimum conditions | Extraction yield (mg/g) | ||||
|---|---|---|---|---|---|---|
| X 1 | X 2 | X 3 | X 4 | Predicted | Experimental* | |
| UAE-ethanolic | 21.2 | 30.2 | 33.4 | 17.6 | 5.77 ± 0.18 | 5.75 ± 0.21 |
| UAE-aqueous | 3.8 | 33.02 | 43.35 | 40.47 | 3.14 ± 0.21 | 3.14 ± 0.24 |
*Results are presented as mean ± SD.
Hence, the extraction of gallic acid from mango peels through UAE was an efficient green extraction method with lesser power consumption. It yielded comparable amounts of gallic acid to those obtained using organic solvents in previous studies [13–18]. In the current study, the extraction done with 21% ethanol showed significantly higher yield of gallic acid (5.77 ± 0.18 mg/g) as compared to water (3.14 ± 0.21 mg/g) within a lesser time of 17.6 min. Gallic acid yields were quite higher in the current study than the reported studies. A combination of different enzymes with 65% alcohol and 50% ethanol resulted in a concentration of 18 µg/100 mL and 20.21 µg/g of extracts, respectively [15,39]. However, the gallic acid yield obtained with ethanolic extraction was comparable with another study where 80% acetone was used to extract bioactive compounds and reported 4.54–6.29 mg/g of extract gallic acid from mango peels [14]. The solvent concentration to obtain this yield is too high as compared to the solvent percentages used in the current study. Palafox-Carlos et al. [39] studied the major phenolic compounds of mango fruit that contribute to antioxidant activity and reported that mango peels yield in a range from 40 to 60 µg/mL of gallic acid at different maturity stages and showed highest antioxidant potential. In another study, the reported gallic acid values were 12.5, 13, and 25.6 mg/100 g of extract and concluded that the antioxidant potential of mango peel extracts is greatly contributed by gallic acid and makes it useful as a functional ingredient and nutraceutical [6]. It is important to note that mango peels, typically thrown away during fruit juice processing, are actually a rich source of phenolic compounds. The peels contain even more phenolic compounds than the mango pulp itself and gallic acid is one of the most prominent phenolic compounds found in mango peels having reported antioxidant potential [32,37].
3.4 Comparison of optimized ethanolic and aqueous extracts of mango peels
The extracts were compared for their gallic acid content and TFC in order to determine the quality of extraction methods. MP-Eth. showed significantly higher (p < 0.05) gallic acid yield as well as TFC in contrast to MP-Aq. and MP-Dec (Table 7). Gallic acid content extracted from mango peels with 50% ethanol was reported to be 144.18 µg/g through UAE [40] and reported as the abundantly found phenolic acid in mango peels. Similarly, another study reported gallic acid content of three different varieties of mango peels ranging from 12.51 to 25.62 mg/100 g dry matter when the peels were irradiated for 15 min with 80% methanol through UAE [15]. However, comparing the extraction time and temperature to the UAE-optimized conditions in current study obtained higher amount of gallic acid (5.75 ± 0.21 mg/g) and TFC (9.46 ± 0.03 mg/g) in a much shorter time of 17.6 min with a temperature of 33.4℃. Total flavonoids extracted from mango peels through UAE were reported as 556.6 ± 0.3 µg/g with 45% ethanol in a 60 min period of time [21]. Another study reported 18 mg/100 g fresh weight TFC of mango peels when extracted with methanol using UAE [41]. However, Borrás-Enríquez et al. [27] reported a higher TFC (12.28 mg/g) when mango peels were subjected to UAE with 50 mL of 99% HPLC-grade ethanol, which is a higher concentration as compared to the observed optimized variables of current study.
Comparison of extraction methods
| Extraction method | Gallic acid (mg/g) | TFC as CE (mg/g) |
|---|---|---|
| MP-Eth. | 5.75 ± 0.21a | 9.46 ± 0.03a |
| MP-Aq. | 3.14 ± 0.02b | 7.08 ± 0.01b |
| MP-Dec. | 2.57 ± 0.02c | 6.71 ± 0.02c |
Extraction with ethanol (MP-Eth.), water (MP-Aq.), and decoction (MP-Dec.) Results are provided as mean ± SD. Significance (p < 0.05) is depicted by superscripts.
4 Conclusion
The results of the current study report the optimization of extraction conditions to obtain gallic acid-enriched extract from mango peels by using ethanol and water, where extraction using ethanol as a solvent gave preferred results for gallic acid yields and TFCs. The optimized conditions of ethanolic extraction were 21.2% ethanol concentration, 30.3 mL/g of solvent ratio at 33.4℃ for 17.6 min, whereas extraction done with water was optimized at pH 3.8, solvent ratio of 33.02 mL/g at 43.35℃ for 40.47 min with a gallic acid yield of 5.75 ± 0.21 mg/g and 3.14 ± 0.02 mg/g, respectively. Both extraction methods showed higher gallic acid and TFC when compared with conventional extraction of decoction. The reported UAE method may help in designing more efficient extraction methods to extract valuable compounds from mango peels and enable its utilization.
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Funding information: This work was carried out with financial support from the UK Government’s Department of Health and Social Care, Global AMR Innovation Fund (GAMRIF), and the International Development Research Centre (IDRC), Ottawa, Canada (Grant No. 109051–003). The views expressed herein do not necessarily represent those of IDRC or its Board of Governors.
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Author contributions: Conceptualization, T.R., Z.H., and K.A.; Formal analysis, T.R., K.S., M.A., and Z.T.; Investigation, Z.H. and S.U.R.; Methodology, Z.H., T.R., K.A., K.S., and A.S.; Project administration, Z.H., S.U.R., A.M., and U.F.; Software, T.R., H.U.R., and A.S.; Supervision, Z.H. and S.U.R.; Validation, T.R., H.U.R., and K.A.; Writing – original draft, T.R.; Writing – review and editing, T.R., Z.H., and K.A.; Funding acquisition, Z.H. and S.U.R.
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Conflict of interest: The authors declare no conflict of interest.
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Ethical approval: The conducted research is not related to either human or animal use.
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Data availability statement: All data generated or analyzed during this study are included in this published article.
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