Abstract
This study aimed to optimize the extraction of phenolic compounds from jujube by conventional (CE), ultrasound (UAE), and microwave-assisted (MAE) extractions using the response surface methodology, compare the extraction techniques and determine some bioactive properties of the phenolic extracts. In addition to time and solvent concentration, temperature for CE, sonicator power and duty cycle for UAE and microwave power for MAE were optimized. The optimum point to recover phenolic extract from jujube by CE was 1410 min for extraction time, 80℃ for extraction temperature, and 40% for methanol concentration. The optimal extraction conditions for phenolic compounds of jujube by MAE were determined to be as follows: 300 W microwave power, 127 s extraction time, and 43% methanol concentration. The optimum values of jujube extract by UAE are 20 min, 10:7, 130 W, and 40% for extraction time, duty cycle, sonicator power, and methanol concentration, respectively. It was found that the total phenolic compounds (TPC) in CE, MAE, and UAE extracts were 2.7552mgGAE/g, 3.3455mgGAE/g, and 1.9416mgGAE/g, respectively. The highest TPC was in MAE as 3.3455mgGAE/g, the highest TFC was in UAE as 1671.1111mgQE/kg, the highest ascorbic acid was in UAE as 2.2489 mg/100 g, the highest TEAC was found in MAE as 3449.79mgTE/kg, and the highest FRAP was found in CE as 670mgTE/kg. Different amounts of catechin, succinic acid, cyanidin chloride, rosmarinic acid, salicylic acid, routine, oxalic acid, and quercetin dihydrate were detected in the extracts.
Supplementary Information
The online version contains supplementary material available at 10.1038/s41598-025-07277-y.
Keywords: Jujube, Phenolic component, Extraction, Ultrasound, Microwave
Subject terms: Biochemistry, Plant sciences
Introduction
Nowadays, consumers’ tendency towards fruits that contain anthocyanins, are rich in phenolic compounds, have antioxidant properties and have positive effects on health has increased. One of the fruits with these important bioactive properties is Jujube (Ziziphus jujuba). Jujube has gained popularity in recent years by demonstrating its bioactive properties. Jujube is among the foods with high nutritional value, with its high content of phenolic compounds, alkaloids, vitamins, minerals, fatty acids, carbohydrates, and proteins1. Jujube contain different types of phenolic compounds, including hydroxycinnamates (caffeic, ferulic, cinnamic, and p-coumaric acids), flavanols (quercetin and kaempferol), and flavan-3-ols (catechin and epicatechin)2–5.
One of the most critical stages in isolating and identifying phenolic compounds from plants is extraction. There is no standard technique for the extraction of phenolic compounds6. The most common factors affecting extraction processes are matrix properties of the plant part, solvent, temperature, pressure, and time. Conventional extraction techniques, which include solid-liquid extraction, liquid-liquid extraction, and maceration, are not consistent techniques as they generally use larger extraction solvent volumes, and manual procedures that are often researcher-dependent, and labor-intensive. For these reasons, the use of advanced extraction techniques has increased today to fill the gaps left by conventional extraction techniques. The use of non-conventional techniques, which are more environmentally friendly due to decreased use of synthetic and organic chemicals, reduced extraction time, better yield, better process repeatability and quality of extract, have increased in recent years7–9. Supercritical fluid extraction10,11, microwave-assisted extraction12,13, ultrasound-assisted extraction14,15, pressurized liquid extraction16,17 and enzyme-assisted extraction18,19 are the most used non-conventional techniques for extraction of bioactive compounds from plant materials.
Microwaves are non-ionizing electromagnetic waves located between the radio-frequency range at the lower frequency and infrared at the higher frequency in the electromagnetic spectrum, within the frequency band of 300 MHz to 300 GHz20. Microwave-assisted extraction is a technique that involves the use of microwave radiation energy to heat the solute-solvent mixture. The heat generated facilitates the diffusion of solvents into the sample to improve the diffusion of target phytochemicals through the sample. Diffusion of the solvent through the sample increases the disruption of hydrogen bonds holding the sample, thus allowing target compounds to dissolve in the extraction liquid8. Ballard et al.21 from peanut shells, Rafiee et al.22 from olive leaves, Setyaningsih et al.23 from rice grain, Araujo et al.12 from avocado peels, Dairi et al.24 from red onion, and Kurtulbaş Şahin et al.25 from sour cherry peels successfully extracted phenolic compounds using microwave-assisted extraction.
Ultrasound, which has frequencies from 20 kHz to 10 MHz, is a simple method used in many ways for cell disruption and extraction in food processing26. Ultrasound can produce cavitation and vibration in media. The cavitation effect, thermal effect, and mechanical effect of ultrasound have a significant impact on the extraction process. These effects lead to cell wall destruction, particle size reduction, and an increasing rate of reaction through mass transfer of the cell wall without causing the changes in the structure and function of the extracts27,28. This mechanism offers shorter repeatability, lower solvent consumption and temperature, and lower energy input29. In the literature, the extraction of phenolic compounds by ultrasound-assisted extraction has been reported from grapes30, tamarillo fruit14, Pistacia lentiscus leaves31, olive leaves32, Clinacanthus nutans33, Alpiniae oxyphyllae Fructus34, and jujube seeds15.
The efficiency of the recovery of phenolic compounds depends on various factors such as matrix properties of the plant part, applied extraction method, solid-liquid ratio, type of solvent, concentration of solvent, the temperature employed, the time of the extraction process, etc. Therefore, optimization of extraction parameters for each extraction method is crucial to achieve maximum recovery of phenolic compounds in the extract. To our knowledge, no study compares CE, MAE, and UAE for extraction of phenolic compounds from jujube in the literature. This study aimed to optimize the extraction of phenolic compounds from jujube, which has high phenolic compound content, by CE, UAE, and MAE, compare the extraction techniques and determine some bioactive properties of the obtained phenolic extracts.
Materials and methods
Material
The commercially available jujube fruits harvested from the province of Manisa (Turkey) in October 2022 were used. After the jujube was separated from their seeds, they were dried in an oven (Mikrotest MST120, Turkey) for 12 h at 50℃, and the dried fruits were ground with the help of a mill (Demsan, Turkey) and kept at + 4℃ until analysis. We confirm that all methods in this study were performed in accordance with the relevant guidelines/regulations/legislation.
Conventional extraction (CE) and experimental design
CE of phenolic compounds from jujube was carried out in an oven using the solid-liquid extraction technique. Methanol was used as the extraction solvent and the sample-solvent ratio was 1:9 w/v. After the CE process, the extract was filtered using the Whatman filter paper 42 by a vacuum pump (DRVAC 400, Taiwan) and the solvent in the filtrate was removed from the rotary evaporator (IKA RV 3 V, Germany). For the optimization of CE conditions of phenolic compounds of jujube, time (120–1440 min), temperature (40–80℃) and solvent concentration (40–100%) were taken as independent variables and the optimum process conditions providing the maximum TPC content were determined using the response surface methodology. In creating the model, extracts were produced at 17 different experimental points with five repetitions in the center point, created with three independent variables, using the Box-Behnken experimental design.
Ultrasound-assisted extraction (UAE) and experimental design
A laboratory-scale ultrasonic homogenizer (Nanolinker NL200, China) with a 3 mm probe was used for UAE. Methanol was used as the extraction solvent and the sample-solvent ratio was 1:9 w/v (This ratio was adopted after preliminary experiments). After the UAE process, the extract was centrifuged at 4000 rpm for five minutes (NUVE NF800, Türkiye), then supernatant was filtered using the Whatman filter paper 42 with a vacuum pump. The solvent in the filtrate was removed in the rotary evaporator. In optimizing the extraction conditions, extraction time (5–20 min), sonicator power (50–200 W), duty cycle (10:6–10:10 s: sec) and solvent concentration (40–100%) were determined as independent variables for the UAE process. The response was based on the extraction conditions of phenolic compounds from jujube, ensuring the highest TPC. In creating the model, extracts were produced at 27 different experimental points with three repetitions in the center point, created with four independent variables, using the Box-Behnken experimental design.
Microwave-assisted extraction (MAE) and experimental design
MAE from jujube was performed using a household microwave oven (Samsung MS23 F300EEW, Malaysia). The sample-solvent ratio was 1:19 w/v and methanol was used as extraction solvent. After the MAE process, the extract was filtered using the Whatman filter paper 42 with a vacuum pump and the solvent in the filtrate was removed in the rotary evaporator. The process of extraction was carried out by using three different parameters: (1) the microwave power (300–600 W), (2) extraction time (30–180 s), and (3) solvent concentration (40–70%). The optimum conditions that ensured the highest TPC was determined using response surface methodology. Using the Box-Behnken experimental design, a model was created with 17 different experimental points with three independent variables and three repetitions in the center point.
Analytical methods
pH value
The pH value of the extracts was measured using a WTW Inolab pH 7110 Set 2 (Germany) model pH-meter35.
Total phenolic compounds (TPC)
The TPC of the extracts were determined by the Folin-Ciocalteau method described by Franke et al.36. 5mL of Folin-Ciocalteau reagent (10%; v/v) and 15mL NaHCO3 (20%; w/v) were added to 1mL of the extract and making up to 100mL with distilled water. After incubation at 25 °C for 2 h in the dark, the absorbance of samples was measured at 760 nm. The TPC is presented as mgGAE/g.
Total flavonoid content (TFC)
TFC of the extracts was determined according to Almaraz-Abarca et al.37. 4 mL of distilled water and 0.3 mL of 5% NaNO2 were added to 1 mL of the sample, mixed and incubated for five minutes. After incubation, 0.3 mL of 10% AlCl3 and 2 mL of 1 M NaOH were added. After mixing, distilled water was added to make the final volume 10 mL. Absorbance was measured at 510 nm using a UV/VIS spectrophotometer (T80+, PG Instruments, United Kingdom). Quercetin was used as a standard and the results are expressed as mg QE/kg.
Ascorbic acid content
Ascorbic acid values of the samples were determined by the spectrophotometric method according to Hışıl38. One milliliter of extract was mixed with 9 mL 2,6 dichlorophenol-indophenol, and immediately, the absorbance was measured with a spectrophotometer at 518 nm against the one milliliter extract mixed with 9 mL distilled water. The ascorbic acid values were calculated with the calibration curve that was made using different concentrations of L(+) ascorbic acid standard and the results were expressed in mg/100 g extract.
Antioxidant capacity
The antioxidant capacities of the samples were determined using TEAC (trolox equivalent antioxidant capacity) and FRAP (the ferric reducing antioxidant power) assays. The TEAC method was evaluated in accordance with the method described by Re et al.39. ABTS radical cation was produced by mixing 2.5 mL of 7 mmol L–1 ABTS stock solution and 44 µL of 140 mmol L–1 K2S2O8, both diluted with distilled water. This mixture was stored 12–16 h in darkness and then diluted with ethanol to an absorbance of 0.70 ± 0.02 at 734 nm. After the addition of 300 µL of Trolox or sample to 3 mL of diluted ABTS solution, absorbance readings were taken after 6 min of the initial mixing. Trolox (± -6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) was used for the standard and the results were expressed in µmol Trolox/mL.
The spectrophotometric method described by Benzie and Strain40 was used for the FRAP method. The stock solution consisting of 300 mM acetate buffer, 10 mM TPTZ solution in 40 mM HCl and 20 mM FeCl3 solution in proportions of 10:1:1 (v/v) respectively was prepared. After that 20 µL of sample and allowed to react with 2.98 mL of the FRAP solution for 30 min in darkness. Absorbance was measured at 593 nm and expressed in µmol Troloks /mL.
Identification and quantification of phenolic compounds
The compositions of phenolic and flavonoid components of phenolic extracts obtained at optimum points with three different extraction techniques were carried out with the LC-MSMS analysis system at Tübitak Marmara Research Center. The chromatographic separation was performed using an Eclipse Plus Phenyl Hexyl column (150 mm×4.6 mm, 3.5 μm) at 40℃. Mobile phase A was water with 0.1% acetic acid, and mobile phase B was 100% methanol. The gradient elusion started with 5% mobile phase B at the beginning, increased linearly to 100% at the 30th minute and decreased to 5% at the 31st minute and held for 10 min. The injection volume was 20 µL and the flow rate was 0.5 mL/min. MS experiments were conducted on an Agilent 6460 triple quadrupole mass spectrometer equipped with electrospray ionization source (Agilent Technologies, Palo Alto, CA, USA). The MS conditions were as follows: drying gas temperature, 350 °C; drying gas flow, 10 L/min; sheath gas temperature, 400 °C; sheath gas flow, 10 L/min; nebulizer pressure, 45 psi.
Statistical analysis
The significant difference between the means was established by ANOVA variance analysis and Tukey tests. The results were performed with the SPSS statistical package program (SPSS 17.0 for Windows Evaluation Version (17.0.3); SPSS Inc., Chicago, USA).
Design Expert software (Version 7.0.0, Stat-Ease Inc., Minneapolis, USA) was used for the construction of experimental designs and regression analysis of experimental data. The ANOVA test was used to assess the statistical significance of the regression coefficient by F-test at 95% confidence level. Model fitting was performed by R2, adjusted R2, the adequate precision value and Lack of fit test.
Results and discussion
Optimization of CE of jujube by RSM
CE of phenolic compounds from jujube was optimized using Box-Behnken experimental design, 17 experiments were conducted, and then, the TPC of CE extracts were calculated as the response variable, as shown in Table 1. In the experiments, the highest TPC amount was 3.0128 mgGAE/g where the extraction time was 780 min, the extraction temperature was 80℃ and the methanol concentration was 40%, while the lowest TPC amount was 0.3634 mgGAE/g where the extraction time was 120 min, the extraction temperature was 60℃ and the methanol concentration was 100%. In all experiments where 40% methanol was used as solvent, the highest TPC amounts were obtained under all conditions, and in all experiments where 100% methanol was used, the lowest TPC amounts were obtained.
Table 1.
The Box-Behnken design matrix of three variables. Factors and TPC of CE of jujube extracts.
| Run | Extraction time (minute) (x1) |
Extraction temperature (oC) (x2) |
Methanol concentration (%) (x3) |
TPC (mgGAE/g) |
|---|---|---|---|---|
| 1 | 120 | 60 | 100 | 0.3634 |
| 2 | 780 | 40 | 100 | 0.7143 |
| 3 | 120 | 60 | 40 | 2.6563 |
| 4 | 780 | 60 | 70 | 1.8171 |
| 5 | 1440 | 80 | 70 | 1.6029 |
| 6 | 780 | 80 | 40 | 3.0128 |
| 7 | 780 | 60 | 70 | 1.5447 |
| 8 | 780 | 60 | 70 | 1.6308 |
| 9 | 780 | 60 | 70 | 1.8107 |
| 10 | 1440 | 60 | 40 | 2.8667 |
| 11 | 780 | 40 | 40 | 1.9715 |
| 12 | 780 | 60 | 70 | 1.8159 |
| 13 | 1440 | 40 | 70 | 1.6166 |
| 14 | 120 | 40 | 70 | 1.5508 |
| 15 | 780 | 80 | 100 | 0.9136 |
| 16 | 1440 | 60 | 100 | 0.8144 |
| 17 | 120 | 80 | 70 | 1.4444 |
While the linear effect of the methanol concentration (x3) used in extraction was statistically significant (p > 0.05), the linear effects of extraction time (x1) and extraction temperature (x2) were statistically insignificant (p > 0.05). Model fitting was performed by R2 (0.9160), adjusted R2 (0.8966), and the adequate precision value (19.549). Lack of fit of the experimental data, which indicates a variation in the data around the fitted model, was not significant (p > 0.05), and the regression model was statistically significant at a 95% confidence level.
It was determined that the optimum point to produce phenolic extract from jujube through CE was 1410 min for extraction time, 80℃ for the extraction temperature, and 40% for methanol concentration. While the estimated TPC at the determined optimum point was 2.8640 mgGAE/g, the experimental TPC was calculated as 2.8653 mgGAE/g. The multiple regression equation showing the coded levels of the variables of Jujube extract by CE is given as follows:
 |
Where x1: extraction time (second), x2: extraction temperature (℃), x3: methanol concentration (%).
Optimization of UAE of jujube by RSM
The experiments were performed as per the Box-Behnken using RSM for identification of an optimum condition of UAE for TPC of jujube. The design of the experimental plan and response are presented in Table 2. The linear effect of the solvent concentration used in extraction was statistically significant (p < 0.05). In all experiments where 100% methanol was used as solvent, the lowest TPC amounts were obtained under all conditions. With this, when the solvent concentration was increased from 40 to 100% the TPC of jujube extract was decreased. The responses, TPC, were in the range of 0.3523–2.2289 mg GAE/g in experiments.
Table 2.
The experimental plan of UAE of jujube extracts.
| Run | Extraction time (minute) (x1) | Duty cycle (sec: sec) (x2) | Sonicator power (Watt) (x3) | Methanol concentration (%) (x4) | TPC (mgGAE/g) |
|---|---|---|---|---|---|
| 1 | 12.50 | 10:10 | 50 | 70 | 1.4201 |
| 2 | 12.50 | 10:8 | 125 | 70 | 1.5378 |
| 3 | 12.50 | 10:6 | 125 | 100 | 0.4386 |
| 4 | 12.50 | 10:8 | 50 | 100 | 0.4619 |
| 5 | 20.00 | 10:8 | 125 | 100 | 0.5313 |
| 6 | 20.00 | 10:6 | 125 | 70 | 1.6840 |
| 7 | 5.00 | 10:8 | 200 | 70 | 1.5695 |
| 8 | 12.50 | 10:6 | 200 | 70 | 1.3928 |
| 9 | 20.00 | 10:10 | 125 | 70 | 1.5165 |
| 10 | 12.50 | 10:8 | 125 | 70 | 1.7787 |
| 11 | 5.00 | 10:8 | 50 | 70 | 1.5048 |
| 12 | 12.50 | 10:8 | 50 | 40 | 1.6871 |
| 13 | 5.00 | 10:6 | 125 | 70 | 1.4678 |
| 14 | 12.50 | 10:6 | 50 | 70 | 1.3074 |
| 15 | 20.00 | 10:8 | 50 | 70 | 1.4342 |
| 16 | 12.50 | 10:6 | 125 | 40 | 1.7988 |
| 17 | 5.00 | 10:8 | 125 | 40 | 1.7846 |
| 18 | 5.00 | 10:8 | 125 | 100 | 0.7941 |
| 19 | 20.00 | 10:8 | 125 | 40 | 2.2289 |
| 20 | 12.50 | 10:10 | 200 | 70 | 1.2398 |
| 21 | 12.50 | 10:10 | 125 | 100 | 0.4121 |
| 22 | 12.50 | 10:8 | 200 | 100 | 0.3523 |
| 23 | 12.50 | 10:10 | 125 | 40 | 1.6644 |
| 24 | 12.50 | 10:8 | 200 | 40 | 1.7556 |
| 25 | 5.00 | 10:10 | 125 | 70 | 1.5562 |
| 26 | 20.00 | 10:8 | 200 | 70 | 1.3732 |
| 27 | 12.50 | 10:8 | 125 | 70 | 1.4455 |
The results of the analysis of variance (ANOVA) are shown in Table 3. F-value is used to determine significance of the model. In this model the F-value was 49.07, which indicated that the model is significant. The p-value of the model terms was < 0.0001 indicating that the item is highly significant. In addition, the coefficient of determination R2 is used to evaluate the quality of the model. When the value of R2 is closer to 1, it indicates that the regression equation fits the observations better. The R2values and adjusted R2values in this model were 0.9828 and 0.9628, respectively. The coefficient of variation (C.V.) is a measure of deviation from the mean values, which shows the reliability of the experiment. In general, CV < 10% indicates better reliability. CV value is 7.22 for UAE as seen in Table 3.
Table 3.
ANOVA table and statistical values of jujube extracts by UAE.
| Source | Sum of squares | Df | Mean square | F value | p value |
|---|---|---|---|---|---|
| Model | 6.42 | 14 | 0.46 | 49.07 | < 0.0001 |
| x 1 | 6.916E-004 | 1 | 6.916E-004 | 0.074 | 0.7902 |
| x 2 | 6.547E-003 | 1 | 6.547E-003 | 0.70 | 0.4190 |
| x 3 | 1.459E-003 | 1 | 1.459E-003 | 0.16 | 0.6998 |
| x 4 | 5.24 | 1 | 5.24 | 560.47 | < 0.0001 |
| x 1 x 2 | 0.016 | 1 | 0.016 | 1.75 | 0.2104 |
| x 2 x 3 | 3.950E-003 | 1 | 3.950E-003 | 0.42 | 0.5279 |
| x 1 x 4 | 0.12 | 1 | 0.12 | 13.37 | 0.0033 |
| x 2 x 3 | 0.018 | 1 | 0.018 | 1.89 | 0.1945 |
| x 2 x 4 | 2.911E-003 | 1 | 2.911E-003 | 0.31 | 0.5871 |
| x 3 x 4 | 7.930E-003 | 1 | 7.930E-003 | 0.85 | 0.3752 |
| x 1 2 | 0.034 | 1 | 0.034 | 3.62 | 0.0814 |
| x 2 2 | 0.069 | 1 | 0.069 | 7.37 | 0.0188 |
| x 3 2 | 0.14 | 1 | 0.14 | 15.28 | 0.0021 |
| x 4 2 | 0.70 | 1 | 0.70 | 74.88 | < 0.0001 |
| Residual | 0.11 | 12 | 9.348E-003 | ||
| Lack of Fit | 0.053 | 10 | 5.298E-003 | 0.18 | 0.9765 |
| Pure error | 0.059 | 2 | 0.030 | ||
| Cor total | 6.53 | 26 | |||
| R2 | 0.9828 | ||||
| Adj-R2 | 0.9628 | ||||
| Pred-R2 | 0.9329 | ||||
| C.V. % | 7.22 | ||||
| Adeq Precision | 25.043 |
The efficacy of extracting phenolic compounds from plant materials heavily relies on the type and concentration of solvent utilized. In our study, the linear effect of the solvent concentration used in extraction, the interaction effect of extraction time-solvent concentration, the quadratic effects of duty cycle, sonicator power, and solvent concentration were statistically significant (p < 0.05). The response surface graph showing the effects of independent variables as methanol concentration and extraction time (Fig. 1). Lima et al.41 mentioned that the linear effect of solvent concentration is significant for the extraction of phenolics from eucalyptus leaves by ultrasound-assisted extraction. Alasalvar42 studied extracting phenolic compounds from immortelle (Helichrysum italicum) flowers by UAE reported TPC significantly increased after 15 min of extraction time, but prolonging extraction time showed an insignificant effect on TPC.
Fig. 1.
3D response surface graphics of UAE.
The optimal control variable values for UAE of jujube extract were 20 min, 10:7, 130 W, and 40% for extraction time, duty cycle, sonicator power, and methanol concentration, respectively. The temperature of the extract produced under these optimum conditions was measured as 60℃. Experimental value of TPC was 2.1729 mgGAE/g. Predictive value of TPC was 2.1801 mgGAE/g. The experimentally determined value was in good agreement with those indicated by the RSM and hence the model fitness was highly relevant for the specified range of variables. The quadratic model expressions for TPC have been presented as follows:
 |
Where x1: extraction time (minute), x2: duty cycle (sec: sec), x3: sonicator power (Watt), x4: methanol concentration (%).
Optimization of MAE of jujube by RSM
A Box-Behnken RSM model with three input variables at three levels was applied to investigate the effect of extraction factors (microwave power, extraction time, and solvent concentration) on the TPC of MAE. Table 4 shows the design of the experimental plan and the experimental results for the extraction. To obtain optimal conditions, the extractions were conducted based on experimental design.
Table 4.
Experimental response values of Box-Behnken design of jujube extracts by MAE.
| Run | Microwave power (Watt) (x1) | Extraction time (second) (x2) |
Methanol concentration (%) (x3) | TPC (mgGAE/g) |
|---|---|---|---|---|
| 1 | 450 | 105 | 55 | 3.1736 |
| 2 | 600 | 30 | 55 | 2.6682 |
| 3 | 600 | 105 | 40 | 2.9926 |
| 4 | 450 | 105 | 55 | 3.1289 |
| 5 | 300 | 105 | 70 | 2.6628 |
| 6 | 300 | 105 | 40 | 3.4608 |
| 7 | 450 | 180 | 70 | 2.6239 |
| 8 | 450 | 30 | 70 | 2.3847 |
| 9 | 450 | 180 | 40 | 3.3657 |
| 10 | 600 | 105 | 70 | 2.6342 |
| 11 | 450 | 30 | 40 | 2.9917 |
| 12 | 300 | 30 | 55 | 2.7727 |
| 13 | 450 | 105 | 55 | 3.1502 |
| 14 | 450 | 105 | 55 | 3.2269 |
| 15 | 300 | 180 | 55 | 3.2390 |
| 16 | 450 | 105 | 55 | 3.1465 |
| 17 | 600 | 180 | 55 | 2.9121 |
It was observed that the lowest TPC was 2.3847 mgGAE/g under the conditions of 450 W microwave power, 30 s extraction time and 70% methanol concentration. According to the results, the highest TPC was achieved with 3.4608 mgGAE/g under the conditions of 300 W microwave power, 105 s extraction time and 40% methanol concentration. Higher microwave power, which causes an increase in temperature, can accelerate the extraction by disrupting hydrogen bonds, increasing solvent penetration into the matrix, or can further degrade target compounds43,44. Therefore, it is important to optimize the microwave power for the extraction of phenolic compounds from jujube.
On the basis of the RSM analysis, the predicted optimum conditions for extraction of Jujube by MAE were as follows: microwave power = 300 W, extraction time = 127 s, and methanol concentration = 43%, the theoretical TPC was 3.5035 mg/g. The actual TPC was 3.3981 mgGAE/g. After microwave assisted extraction, the temperature of the extract was recorded as 70℃.
As shown in Table 5, the ANOVA results exhibit that the quadratic model is greatly significant (p < 0.0001) for TPC which was mainly influenced by microwave power (x1), extraction time (x2) and methanol concentration (x3). With this the interaction effect of microwave power-extraction time, microwave power-solvent concentration, and the quadratic effects of microwave power, extraction time and solvent concentration were statistically significant (p < 0.05) as seen in Table 5. The low value of the coefficient of variation (C.V., 1.40%) indicated the similarity between predicted and experimental values, suggesting that the model had a high degree of reliability. The response surface graph showing the effects of independent variables as methanol concentration and extraction time (Fig. 2). Similar to our study, Berkani et al.45 optimize the extraction conditions of TPC from Zizyphus lotus seeds by microwave procedure using response surface methodology and found that phenolic compounds extracted from Zizyphus lotus seeds using MAE are mainly dependent on solvent concentration, microwave power, and extraction time.
Table 5.
ANOVA table and statistical values of jujube extracts by MAE.
| Source | Sum of squares | Df | Mean square | F value | p value |
|---|---|---|---|---|---|
| Model | 1.46 | 9 | 0.16 | 93.64 | < 0.0001 |
| x 1 | 0.11 | 1 | 0.11 | 62.26 | < 0.0001 |
| x 2 | 0.22 | 1 | 0.22 | 126.57 | < 0.0001 |
| x 3 | 0.78 | 1 | 0.78 | 453.55 | < 0.0001 |
| x 1 x 2 | 0.012 | 1 | 0.012 | 7.15 | 0.0318 |
| x 1 x 3 | 0.048 | 1 | 0.048 | 27.93 | 0.0011 |
| x 2 x 3 | 4.543E-003 | 1 | 4.543E-003 | 2.63 | 0.1491 |
| x 1 2 | 0.031 | 1 | 0.031 | 17.82 | 0.0039 |
| x 2 2 | 0.14 | 1 | 0.14 | 80.33 | < 0.0001 |
| x 3 2 | 0.085 | 1 | 0.085 | 49.13 | 0.002 |
| Residual | 0.012 | 7 | 1.730E-003 | ||
| Lack of Fit | 6.338E-003 | 3 | 2.113E-003 | 1.46 | 0.3506 |
| Pure Error | 5.770E-003 | 4 | 1.442E-003 | ||
| Cor Total | 1.47 | 16 | |||
| R2 | 0.9918 | ||||
| Adj-R2 | 0.9812 | ||||
| Pred-R2 | 0.9249 | ||||
| C.V. % | 1.40 | ||||
| Adeq Precision | 33.860 |
Fig. 2.
3D response surface graphics of MAE.
The quadratic model expressions of TPC according to three selected parameters are following:
 |
Where x1: microwave power (Watt) x2 : extraction time (second) x3 : methanol concentration (%).
Characterization of jujube extracts
pH
The pH value of the extracts was measured at 5.05 in CE, 5.27 in MAE, and 5.19 in UAE. The difference between the pH values in the extracts obtained by CE, UAE and MAE was statistically significant (p < 0.05). The fact that the extraction time of CE was longer than the extraction time of MAE and UAE may have caused a further decrease in pH value. The extracts obtained by CE, UAE, and MAE may have different usage possibilities in the food industry, especially in the production of slightly acidic food products. The results obtained in our study were higher than the pH values of extracts produced from different sources with different techniques in literature. Kutlu Kantar46 determined the pH values of cranberry extracts by ultrasonic, ohmic heating assisted ultrasonic, microwave, and ohmic heating assisted methods as 2.95, 3.06, 2.30 and 3.03, respectively. The pH values of the extracts obtained from sour cherry pulp by conventional and ultrasound-assisted extraction methods at 40 and 60℃ were in the range of 2.37–2.4447. Dinçer et al.48 reported that the pH values of the hibiscus extracts were in the range of 2.413–2.420 as a result of the extraction by traditional thermal and thermosonication methods at 40℃ and 75℃.
TPC
Phenolic compounds are crucial because they contribute to the color, taste, aroma, and flavor of the fruit, as well as having antimicrobial and antioxidant effects. These compounds may also be beneficial for health by improving the quality of the fruit49,50. The TPC of jujube extracts are given in Table 6. The highest value of TPC was found in the extract by MAE with 3.3455 mg GAE/g. The difference between the TPC in the extracts obtained by the three techniques was statistically significant (p < 0.05). Although the solvent concentrations used in all techniques were similar (CE: 40% methanol; UAE: 40% methanol; MAE: 43% methanol), there were large differences between the extraction times. The extraction of phenolic compounds was also affected by extraction time which prolonged extraction time can negatively impact on extracted compound as its degradation51. The TPC of the extract by MAE performed in 127 s was 3.3455 mg GAE/g, while it was determined as 2.1729 mg GAE/g in the UAE performed in 20 min and 2.8653 mg GAE/g in CE performed in 1410 min. It was determined that the total phenolics obtained by MAE from jujube were higher than other extraction techniques. Thus, in this study, it was defined that the use of microwave provides higher recovery with shorter processing time. TPC results of EC, MAE, and UAE were lower than other studies conducted with jujube extract in the literature. Han et al.52 who examined the optimum conventional extraction conditions for the highest TPC of jujube pulp and seeds. It was determined that optimum extraction conditions were 61.2 °C, 38 h, and 60.4% ethanol for jujube pulp and 58 °C, 34 h, and 59.2% ethanol for jujube seed. The TPC of the extracts were determined as 18 and 18.3 µg GAE/mg, respectively. Berkani et al.15 extracted phenolic compounds from jujube seeds by ultrasound-assisted extraction, determined the TPC as 2383.10 mgGAE/100 g with 50.16% ethanol, 29.01℃ sonication temperature, 34.1:1 solvent: sample ratio and 15.94 min sonication time. Berkani et al.45 determined the TPC of jujube fruit seeds by using microwave-assisted extraction with conditions of 60% ethanol, 210 s, and 600 W microwave power as 6709.01 GAE mg/100 g.
Table 6.
Total phenolic, total flavonoid and ascorbic acid compound values of jujube extracts.
| TPC, mgGAE/g |
TFC, mg QE/kg |
Ascorbic acid, mg/100 g |
|
|---|---|---|---|
| CE | 2.7552 ± 0.1051b | 133.67 ± 4.41a | 1.1577 ± 0.1093a |
| MAE | 3.3455 ± 0.0843c | 758.44 ± 29.13b | 0.9852 ± 0.2248a |
| UAE | 1.9416 ± 0.2058a | 1671.11 ± 25.46c | 2.2489 ± 0.0589b |
CE conventional extraction, MAE microwave-assisted extraction, UAE ultrasound-assisted extraction.
a, b different lowercase letters indicate statistical differences of the samples at the same column (p < 0.05).
TFC
The highest TFC in the extracts (Table 6) was obtained by UAE and was 1671.11 mgQE/kg. The performance of UAE depends on the phenomenon of cavitation. This initiates the formation of micropores in the matrix cell and accelerates the passage of the solvent to the flavonoid compound in the intracellular component53. The lowest TFC was recorded as 133.67 mgQE/kg in CE extract which has long extraction time (1410 min.). The extraction time is a crucial factor in the extraction process of target compounds from plants samples. While a moderate increase in extraction time causes an increase in flavonoid content, an excessive increase in extraction time may negatively affect the flavonoid content. This could be because after a long extraction time, components other than flavonoids in the sample tissue attached to the sample surface possibly inhibiting the contact between sample and solvent and extraction efficiency54. The difference between the TFC in the extracts was statistically significant (p < 0.05). These results are in agreement with results reported by Çiftçi et al.55 and Yılmaz56. Çiftçi et al.55 found TFC values in the range of 872.5-5037.5 mg CE/kg from jujube (Z. jujube) using conventional extraction method with different solvent types [ethanol, methanol, acetone, and aqueous mixtures of water and organic solvents (1:1)]. Yılmaz56 studied that the TFC of the extracts from jujube tree leaves, unripe jujube, and ripe jujube by traditional extraction using methanol and water as solvent. It was in the range of 53.74–932.60 mg CE/L in the extraction with water and in the range of 98.66–1047.00 mg CE/L in the extraction with methanol. However, Rajaei et al.57 have quantified a higher TFC value (71.53 mg QE/g in jujube pulp extract and 9.77 mg QE/g in jujube seed extract) from jujube pulp and seed extracts by UAE than our results.
Ascorbic acid content
Ascorbic acid is a very important component as it acts as both a reducing and chelating agent that scavenges free radicals. Ascorbic acid content of jujube extracts by CE, MAE, and UAE are given in Table 6. UAE extract showed high ascorbic acid content with 2.2489 mg/100 g and MAE extract showed the lowest ascorbic acid content with 0.9852 mg/100 g. However, there was no significant difference in the ascorbic acid content between the CE and MAE extract (p > 0.05). A similar trend was reported by Temizsoy58 who carried out the extraction of vitamin C from the spindle (Elaeagnus Angustı̇folı̇a L.) using traditional and ultrasound-assisted extraction. It was reported that ascorbic acid value was calculated as 39.79 mg/100 g in the ultrasound extraction performed at 60 °C at 60% amplitude for 10 min was approximately three times higher than that of the extract obtained by traditional extraction (12.34 mg/100 g).
Antioxidant capacity
Antioxidants are molecules that prevent cell damage by preventing the formation of free radicals or scavenging existing radicals. They generally contain phenolic functions in their structure. The antioxidant activities of jujube mainly depend on the contents of different species of phenolic acids and flavonoids, which may differ with the genotypes, geographical environment, and growing conditions59. Jujube extract by UAE exhibited a significant Trolox equivalent antioxidant capacity (3449.79 mgTE/kg) and was higher than those obtained by CE as 3040.44 mgTE/kg and by UAE as 698.67 mgTE/kg. This may be related to the higher amount of catechin contained in the UAE extract than other extracts. Generally, extracts containing high amounts of polyphenols also exhibit high antioxidant activity. The antioxidant power of flavonoids is generally proportional to the total number of hydroxyl groups39.
The antioxidant activities of CE, MAE, and UAE jujube extracts were quantified by also using the ferric reducing activity power (FRAP) assay. The FRAP assay evaluates antioxidants present in samples as reductants in a redox-linked colorimetric reaction and reflects the reducing power of antioxidants. As shown in Table 7, it was found that the antioxidant capacity in CE, MAE, and UAE extracts was 670.000 mgTE/kg, 351.984 mgTE/kg, and 292.237 mgTE/kg, respectively. There was no correlation between the results of TEAC and FRAP assays.
Table 7.
Antioxidant capacity values of jujube extracts.
| Antioxidant capacity | ||
|---|---|---|
| TEAC, mgTE/kg | FRAP, mgTE/kg | |
| CE | 3040.44 ± 0.042b | 670 ± 10.18c |
| MAE | 3449.79 ± 133.16c | 351.984 ± 25.66b |
| UAE | 698.67 ± 93.07a | 292.237 ± 14.95a |
CE conventional extraction, MAE microwave-assisted extraction, UAE ultrasound-assisted extraction.
a, b different lowercase letters indicate statistical differences of the samples at the same column (p < 0.05).
The antioxidant activities of jujube extracts produced by different extraction techniques, determined by different antioxidant assays, vary in literature. Han et al.52 examined the traditional extraction of jujube pulp and seeds. It was determined the antioxidant activity of jujube pulp and seed as 379.3 and 343.24 µg extract/mL by DPPH and 28.5 µmol trolox/g and 29.6 µmol trolox/g by ABTS, respectively. Rajaei et al.57 reported that the DPPH radical scavenging activity of jujube pulp and seed extracts by ultrasound-assisted extraction was 53.97 µg/ml and 88.68 µg/ml, respectively. Çiftçi et al.55 examined the effect of extraction with different solvent types on the bioactive properties of jujube. DPPH and ABTS.+ radical scavenging activities of extracts were determined in the range of 1.49–63.7% and 0.38–13.65 µg Trolox/g, respectively, and the highest values were detected in the extraction with acetone: water (1:1).
Identification and quantification of phenolic compounds
Table 8 shows the phenolic profile of CE, UAE, and MAE extracts. The results from jujube extracts, evidenced the presence of 18 phenolic compounds in CE extract, 17 phenolic compounds in MAE extract, and 18 phenolic compounds in UAE extract within the 31 standard phenolics (clorogenic acid, pelargonidin HCL, quercetin dihydrate, gallic acid, oxalic acid, four hydroxybenzoic acid, apigenin, cathechin, cyanidin chloride, naringenin, quercetin three beta D glycoside, rosmarinic acid, rutin, succinic acid, tartaric acid, (+)-l-alliin, cyanin chloride, M P coumaric acid 10–12, T-3-OH cinnamic acid, pelargonin chloride syringic acid, cafeic acid, salicylic acid, cinnamic acid, kaempherol, luteolin, ferulic acid, vanilic acid, sinapic acid, genistisie acid, ellagic acid) tested in the analysis. As shown in Table 8, it was found that the total amounts of tested individual phenolics in CE, MAE, and UAE extracts were 161.425, 272.140, and 470.936 ppm, respectively. With this, TPC in CE, UAE, and MAE extracts were 2755.52, 3345.50, and 1941.60 ppm, respectively. No correlation was found between the total amount of individual phenolics tested and the TPC of the extracts. This is thought to be due to the fact that only 31 standard phenolic standards were examined.
Table 8.
Phenolic and flavonoid compound compositions (ppm) of jujube extracts.
| Phenolic/Flavonoid | CE | MAE | UAE |
|---|---|---|---|
| Clorogenic acid | 0.060 | 0.023 | 0.094 |
| Pelargonidin HCL | 1.917 | 0.383 | 1.176 |
| Quercetin dihydrate | 9.539 | 2.343 | 2.729 |
| Gallic acid | nd | nd | nd |
| Oxalic acid | 5.756 | 15.090 | 7.240 |
| 4 Hydroxybenzoic acid | nd | nd | nd |
| Apigenin | nd | nd | nd |
| Cathechin | 36.663 | 13.672 | 209.682 |
| Cyanidin chloride | 16.921 | 47.446 | 35.524 |
| Naringenin | nd | nd | nd |
| Quercetin 3 Beta D Glycoside | 6.099 | 4.388 | 6.792 |
| Rosmarinic acid | 25.227 | 9.704 | 16.824 |
| Rutin | 14.412 | 10.287 | 16.238 |
| Succinic acid | 11.353 | 158.155 | 156.179 |
| Tartaric acid | nd | nd | nd |
| (+)-l-Alliin | nd | nd | nd |
| Cyanin chloride | nd | nd | nd |
| M P Coumaric acid 10–12 | 0.124 | 0.082 | 0.114 |
| T-3-OH Cinnamic acid | 0.124 | 0.082 | 0.114 |
| Pelargonin chloride | 0.351 | 1.373 | 0.550 |
| Syringic acid | nd | nd | nd |
| Cafeic acid | 0.057 | nd | nd |
| Salicylic acid | 31.398 | 7.860 | 16.453 |
| Cinnamic acid | 0.401 | 0.281 | 0.351 |
| Kaempherol | nd | nd | nd |
| Luteolin | nd | nd | nd |
| Ferulic acid | 0.481 | 0.451 | 0.603 |
| Vanilic acid | 0.542 | 0.429 | 0.032 |
| Sinapic acid | nd | nd | 0.241 |
| Genistisie acid | nd | 0.091 | nd |
| Ellagic acid | nd | nd | nd |
| Total | 161.425 | 272.140 | 470.936 |
CE conventional extraction, MAE microwave-assisted extraction, UAE ultrasound-assisted extraction.
nd not detected.
The most abundant phenolic compounds in the extracts were cathechin (UAE), succinic acid (UAE), cyanidin chloride (MAE), salicylic acid (CE), rosmarinic acid (CE), rutin (UAE), oxalic acid (MAE), quercetin dihydrate (CE), quercetin 3 beta D glycoside (UAE) and pelargonin chloride (MAE). 209.682 ppm catechin was detected in the UAE extract. This value was approximately 15 times higher than that of the MAE extract and approximately six times higher than that of the CE extract. Succinic acid, the second most abundant phenolic component in the extracts, was 158.155 ppm in the extract obtained by MAE, 156.179 ppm in the extract obtained by UAE, and 11.353 ppm in the extract obtained by CE. The third most abundant phenolic compound in the extracts was cyanidin chloride. It was detected as 47.446ppm in the MAE extract, 35.524ppm in the UAE extract, and 16.921ppm in the CE extract.
Sinapic acid was detected only in the UAE extract as 0.241ppm, genistisie acid was detected only in the MAE extract as 0.091ppm, and cafeic acid was detected only in the CE extract as 0.057ppm. The amounts of gallic acid, 4 hydroxybenzoic acid, apigenin, naringenin, tartaric acid, (+)-l-alliin, cyanidin chloride, syringic acid, kaempherol, luteolin and ellagic acid in all extracts were below detectable limits. Phenolic component distributions and amounts of the extracts differed according to the extraction techniques applied. This may arise from differences of extraction temperature, extraction time, cavitation effect of ultrasound or microwave power on the release of phenolic compounds from the cell.
The amounts and types of phenolic compounds of jujube extracted by different extraction techniques vary in the literature. A previous study showed that the jujube seeds extract by microwave procedure contents of significant major compounds comprised 6-methyl-2-O-glucoside xanthone, jasminoside isomer, citric acid, gallocatechin, imidazole carboxylate derivative, kaempferol-3-O-robinobioside, 6-gingerol, 2-hydroxy-2-methyl-1-[4-[3-(2,4,5-trihydroxyhexan-3-yloxy)propyl]phenyl]propan-1-one, 3-(decyloxy)-2-hydroxy-propyl prop-2-enoate, tenasogenin and some small peptides45. Yılmaz56 identified 14 phenolic compounds, including gallic acid, 3–4 hydroxybenzoic acid, catechin, vanillic acid, syringic acid, epicatechin, caustic acid, chlorogenic acid, caffeic acid, coumaric acid, ferulic acid, routine trihydrate, kaempferol 3-glucoside, and quarantine from jujube tree leaves and fruit extracts using water-methanol extraction method. Liu et al.54 identified fifteen types (catechin, dihydromyricetin, L-epicatechin, rutin, vitexin, quercetin 3-β-D-glucoside, taxifolin, quercitrin, (+)-dihydrokaempferol, luteolin, quercetin, chalcone, apigenin, kaempferol, isorhamnetin) of flavonoids from jujube extracts with different extraction methods including traditional extraction, ultrasound-assisted extraction, microwave-assisted extraction, and enzyme-assisted extraction. Rutin concentration was the highest for all extraction techniques. Catechin and epicatechin were detected in all extracts except ultrasonic-assisted extraction. Catechin exhibited the second highest concentration for the extraction techniques, with its content being significantly higher in traditional and ultrasound-assisted extraction.
Conclusions
In this study, phenolic compounds were extracted from jujube by conventional, microwave, and ultrasound-assisted extraction techniques, compared extraction techniques and evaluated some bioactive properties of phenolic extracts. The Box-Behnken experimental design approach using RSM was successfully applied to optimize phenolic compounds extracted from jujube by CE, MAE, and UAE. When the bioactive properties of extracts produced at optimum extraction conditions were examined, MAE showed the highest TPC and the highest Trolox equivalent antioxidant capacity; UAE demonstrated the highest TFC and the highest ascorbic acid value; CE showed the highest iron (III) reducing antioxidant power. This study showed that innovative techniques such as microwave and ultrasound-assisted extraction might be an alternative technique to recover phenolic compounds from jujube and in a short period compared to the conventional one.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Acknowledgements
IHK: Methodology, Investigations, Data collections, Visualization. KT: Visualization, Conceptualization, Methodology, Data analyzing, Supervision, Writing - original draft, Review, and editing.
Author contributions
IHK: Methodology, Investigations, Data collections, Visualization. KT: Visualization, Conceptualization, Methodology, Data analyzing, Supervision, Writing - original draft, Review, and editing.
Funding
This work was supported by the Tokat Gaziosmanpaşa University-Scientific Research Projects (grant number: 2022-13).
Data availability
The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. Should any raw data files be needed in another format they are available from the corresponding author upon reasonable request. Source data are provided with this paper.
Declarations
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Data Availability Statement
The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. Should any raw data files be needed in another format they are available from the corresponding author upon reasonable request. Source data are provided with this paper.