1. Introduction
Lotus leaves are the leaves of Nelumbo nucifera Gaertn., which are considered to be both foods and traditional medicines in China [1]. Lotus leaves have had a great vogue as drinks such as tea or in herbal formulations and have had a lot of recognition [2]. As reported by many papers, they contain a variety of alkaloids and flavonoids, which made them possess antioxidant, antiviral, anti-obesity, and bacteriostatic functions [3,4]. To develop better lotus-leaf-based products, extraction of lotus leaves could provide more specific materials (such as flavonoids) for research and industrial purposes [5]. Hence, it is necessary to apply a novel extraction method to promote research on lotus leaves.
As in Abbott’s first report, transparent and uniform liquids called deep eutectic solvents (DES) could be formed by mixing ammonium salts and amides at low temperatures [6]. After a series of research, DES are typically composed of a hydrogen bond acceptor (HBA) and a hydrogen bond donor (HBD) by intermolecular hydrogen bonding [7]. The resulting DES showed a low melting point, which was lower than that of each pure component. Compared with traditional organic solvents, DES exhibited various characteristics such as low cost, simple preparation, less volatility, and non-toxicity, which made it widely used in the extraction of natural, biologically active components in recent years [8,9]. Choline chloride has often been applied as an HBA in DES for the extraction of flavonoids [10]. In Jalel Labidi’s report, choline chloride/1,4-butanediol (75 wt%) showed the highest extraction ability for flavonoids (383 mg CE/g dried bark extract) and the highest antioxidant capacity [11]. In addition, a study optimized a ternary combination in DES with choline chloride/betaine hydrochloride/ethylene glycol with the mole ratio of 1:1:2 and 20% water content in a negative pressure cavitation-assisted extraction method for flavonoids from Equisetum palustre L. [12]. In Weng’s research, the flavonoids in Spirodela polyrrhiza were rapidly extracted by a DES-based microwave-assisted extraction method using choline chloride/levulinic acid with the mole ratio of 1:2 as the DES [13].
The DES could be combined with some assisted extraction technology such as microwave, ultrasonic, and vortex technology [14,15]. These combination methods could improve extraction efficiency and capacity. Among them, ultrasound-assisted extraction (UAE) could promote the solvent to quickly penetrate the plant material and enhance the diffusion of compounds [16]. The instrument condition of UAE was relatively easy to control in the lab compared to the other assisted methods. In recent years, UAE-DES has been applied in the extraction of many natural plants [17,18,19]. However, these methods still needed selection and optimization for different plant samples due to their complexity and composition. Considering the many benefits of flavonoids and their abundance in lotus leaves and the fact that very little research has been devoted to the utilization of UAE-DES in lotus leaves, the present research aimed to extract flavonoids by an ultrasonic-assisted DES method.
For this purpose, choline chloride, benzyltriethylammonium chloride, betaine, and guanidine-hydrochloride-based DES were prepared to extract flavonoids from lotus leaves to the maximum extent with the assistance of ultrasonic technology (UAE-DES). Then, the flavonoid extract was separated by macroporous resin from the DES, and the antioxidant capacities and antibacterial activities of the extracted flavonoids were systematically studied with beneficial results. Through this study, an effective strategy was provided to extract flavonoids in lotus leaves, which had green and convenient advantages compared to traditional methods. The lotus leaf flavonoids also exhibited antioxidant activities, making them promising in the healthcare area.
2. Materials and Methods
2.1. Materials
Dried lotus leaves were bought from Mingxing Biotechnology Co., LTD. in Bozhou, Anhui. The dried leaves were pulverized and sieved in 40 mesh sieves before use. Choline chloride, benzyltriethylammonium chloride, betaine, guanidine hydrochloride, ethylene glycol, triethylene glycol, glycerol, 2-chloropropionic acid, malonic acid, lactic acid, formic acid, acetic acid, and urea were purchased from Macklin Chemical Reagent Co. (Shanghai, China). 2,2-Diphenyl-1-picrylhydrazyl (DPPH), Diammonium 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), tri-2-pyridyl-s-triazine (TPTZ), and Folin–Ciocalteu were acquired from Sigma-Aldrich Chemicals (St. Louis, MO, USA). The Staphylococcus aureus and Escherichia coli bacterial strains were provided by Professor Qi Yang from the Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences (Changsha, China). All of the other chemicals were of analytical grade.
2.2. Preparation of DES
Choline chloride, benzyltriethylammonium chloride, betaine, and guanidine hydrochloride were selected as the HBA to synthesize DES with different HBDs (ethylene glycol, triethylene glycol, glycerol, 2-chloropropionic acid, malonic acid, lactic acid, formic acid, acetic acid, urea, oxalic acid, and acetamide). The reagents were weighed with the molar ratio at 1:2 (0.2 mol:0.4 mol) and manually stirred at 80 °C for 2 h until a homogeneous liquid formed. When the DES cooled to room temperature, they were stored at ambient temperature.
2.3. Extraction of Flavonoids by the UAE-DES Method
A dried lotus leaf (1.0 g) was powdered and added to the DES (20.0 mL) in a centrifuge tube. The sample was ultrasonically treated by an ultrasonic cleaner (Kunshan Ultrasonic Instrument, Suzhou, China) for 5 min and then extracted in an oscillator at 60 °C under shaking for 60 min. After extraction, the mixture was centrifuged at 4000 r/min for 15 min. The supernatant was taken for further analysis.
To investigate the effect of factors on the extraction efficiency of the flavonoids, the detailed conditions including the ultrasonic time (0 to 30 min), extraction time (10 to 140 min), solid–liquid ratio (1:20 to 1:80), and extraction temperature (40 to 120 °C) were optimized in single-factor experiments. All of the experiments were carried out in triplicate and the values were expressed as means ± SD.
2.4. Determination of the Total Flavonoids Content
The total flavonoid content of the extract was determined by a colorimetric method with some modifications [20]. Briefly, 10 μL of DES extract was mixed with 25 μL of sodium acetate solution (1.0 mol/L), 25 μL of aluminum chloride solution (10%, wt%), and 1940 μL of ethanol solution (50%, v:v). The mixture was thoroughly mixed and incubated at 25 °C for 30 min. After that, the absorbance of the mixture was measured at 415 nm by a UV-2700 UV–Vis spectrophotometer (Shimadzu, Kyoto, Japan). Rutin was used as the standard in this analysis and the results were expressed as milligrams of rutin equivalent per gram of extract.
2.5. Recovery of Flavonoids from the DES Extract
The recovery of flavonoids from the DES extract was carried out by adsorption and desorption by macroporous resins. The pretreatment of macroporous resin was performed before usage as in Li’s report [21]. Ten mL of DES extracts were put into a flask containing 2.0 g D101 macroporous resin. The adsorption was conducted in an oscillator under shaking (200 rpm) at 25 °C for 6 h. Then, the resin remained, was washed with water, and then desorbed with 10 mL of ethanol at 25 °C and 200 rpm for 2 h in an oscillator under shaking.
2.6. Evaluation of Antioxidant Activities
2.6.1. DPPH Assay
In brief, fresh DPPH solution (0.1 mmol/L) was prepared in ethanol. Two mL of sample solution (1–20 mg/mL) was mixed with DPPH solution and incubated in a 25 °C constant temperature water bath for 30 min. Then, the absorbance of the mixture at 517 nm was measured with a spectrophotometer. The scavenging rate of DPPH radicals was calculated as in the previously reported equation [22].
2.6.2. ABTS Assay
The ABTS assay was measured as previously described [22]. ABTS stock solution (7 mmol/L of ABTS solution mixed in 2.45 mmol/L of K2S2O8 solution with equal volume) was incubated for 12 h in the dark and diluted for use. One mL of sample solution (1–20 mg/mL) was mixed with 2 mL of ABTS working solution and incubated in the dark for 6 min. After that, the absorbance of the mixture at 734 nm was measured. The scavenging rate of ABTS radicals was calculated as the equation reported by the reference.
2.6.3. Fe3+ Reducing Assay
The Fe3+ reducing ability of the extract was investigated by the reduction of K3[Fe(CN)6] method [23]. One mL of the sample solution (1–20 mg/mL) was added into 2 mL of phosphate buffer (0.2 M, pH 6.6) and 2 mL of potassium ferricyanide solution (1%, w/v) and then incubated at 50 °C for 20 min. Then, 2 mL of water, 2 mL of trichloroacetic acid (10%, w/v), and 0.1 mL of FeCl3 (0.1%, w/v) were respectively added into the mixture. The absorbance of the final solution was recorded at 700 nm in a spectrophotometer. Rutin was used as standard (0.1–0.5 mg/mL) and the results were expressed as milligrams of rutin equivalents concentration (mg RE/mL).
2.6.4. Ferric Ion Reducing Antioxidant Power (FRAP) Assay
According to Tungjai’s report, TPTZ solution (10 mmol/L), sodium acetate solution (300 mmol/L), and FeCl3 solution (20 mmol/L) were mixed in the proportion of 10:1:1 (v:v:v) as the working solution. Then, 0.1 mL of the sample solution (1–20 mg/mL) was mixed in 2 mL of water and 3 mL of the FRAP working solution and incubated at 37 °C for 50 min. After that, the absorbance of the mixture was measured at 596 nm. FeSO4 solution was used for the calibration curve and the results were expressed as the FRAP value (mmol/L Fe2+) [24].
2.6.5. Fe2+ Chelating Assay
The metal chelating ability of the sample was conducted according to Buravlev et al. with some modifications [25]. A measurement of 0.1 mL of FeSO4 solution (2 mmol/L) and 1 mL of sample solution (1–20 mg/mL) were added to 4 mL of water. Two hundred μL of ferrozine solution (5 mmol/L) was added to start the reaction. The mixture was incubated at room temperature for 10 min and monitored the absorption at 562 nm. The results were given as the percentage of inhibition.
2.7. Antibacterial Activity
The diameter of the inhibition zone (DIZ) was determined by the paper disc diffusion method as previous reports have described [26]. Bacteria suspensions were cultured in a nutrient broth at 37 °C for 48 h and diluted to about 1 × 10−6 CFU. One hundred μL of bacterial suspensions was evenly spread on an agar plate. Then, sterilized filter paper discs (Φ = 6 mm) were soaked in samples (50 mg/mL and 100 mg/mL) and then carefully placed on the plates. The plates were incubated at 37 °C for 24 h, and the DIZ was measured by a vernier caliper. Rutin was used as a positive control, and a solvent without samples was used as a negative control. The DIZ values were expressed in millimeters and values < 6.00 mm were considered as no inhibition zone (NIZ).
The minimal inhibitory concentration (MIC) was determined by the micro-dilution broth method with some modification [26]. five hundred μL of nutrient broth and 50 μL of samples were mixed in a tube. Then, the bacterial solution (1 × 10−6 CFU) was added to the tube and incubated at 37 °C for 12 h. The absorbance at 600 nm of the bacterial suspensions was monitored by a spectrophotometer at predetermined time intervals. Rutin was used as the positive control and the nutrient broth was used as a negative control. The lowest concentration of the sample inhibiting the growth of bacteria was defined as the MIC value.
2.8. HPLC Analysis
The components analysis of the DES extract was carried out on an Agilent 1260 HPLC system (Agilent Technologies Inc., Santa Clara, CA, USA). The chromatographic separation was performed on an Agilent ZORBAX C18 column (150 mm × 4.6 mm i.d.; 5 µm, Santa Clara, CA, USA). The mobile phase consists of solvent A (0.1% v/v acetic acid solution) and solvent B (acetonitrile containing 0.1% v/v acetic acid) with gradient elution mode: 0–4 min, 10% B; 4–25 min, 10–20% B; and 25–30 min, 20–20% B. The flow rate, temperature, injection volume, and detection wavelength were controlled at 1.0 mL/min, 25 °C, 5 μL, and 254 nm, respectively.
2.9. Statistical Analysis
The statistical analysis was performed by SPSS V20 software (IBM SPSS Statistics 20, New York, NY, USA). The data were then compared using Duncan’s multiple range tests at 5% significance levels, and one-way ANOVA with Tukey’s post-hoc test [27,28]. Values followed by different letters in the same column are significantly different according to Duncan’s test (p < 0.05).
3. Results and Discussion
3.1. Selection of DES
The extraction of active compounds using DES was easily influenced by many conditions related to the target compounds and the composition of DES. A lot of research used one or two HBAs for selection [29,30]. While to select appropriate DES on a larger scale for the full extraction and dissolution of flavonoids in lotus leaves, the extraction efficiencies of 34 DES belonging to four kinds of HBA were investigated. The extraction was completed in the same ultrasonic condition and molar ratio of HBA/HBD, and the results are recorded in Table 1. The highest flavonoid yield by DES was achieved when the DES was prepared by choline chloride and urea as in HBA and HBD. The DES system using choline chloride and urea often exhibited the best extraction capacity for flavonoids and polyphenols in natural products. Choline chloride–lactic acid had the best extraction effect among forty groups of DES in the extraction of flavonoids from Dendrobium officinale [31]. Choline chloride/levulinic acid (1:2, mol/mol) could be applied as the solvent for extracting the flavonoids in Spirodela polyrrhiza [13]. Choline chloride/1,4-butanediol exhibited the best extraction efficiency from seven different DES for extracting valuable bioactive compounds from Chinese wild rice [32]. The finding in this study was also in accordance with several reports [11,33,34]. Therefore, the optimal DES type for the extraction of flavonoids in lotus leaves was set as choline chloride and urea.
3.2. Optimization of Extraction Parameters
3.2.1. Effect of the HBA/HDB Ratio
The variation of the HBA/HBD ratio would affect the viscosity and surface tension of the DES solution and the resulting different extraction capacities of flavonoids. To improve the extraction ability of DES, the extraction efficiencies of flavonoids with DES in different HBA/HBD ratios were investigated (Figure 1a). With the increasing HBA/HBD ratio, the extraction efficiencies of flavonoids first increased and then decreased. The highest extraction efficiency of 195.2 mg/g was obtained when the ratio was 1:2, which was then set as the optimal HBA/HBD ratio for preparing DES. This molar ratio (1:2) of HBA/HBD has often been selected in a lot of research and can be supported [29,35,36].
3.2.2. Effect of Water Content in DES
The water content in DES could change the viscosity and polarity of DES. The appropriate water content of DES would decrease the solvent viscosity, improve mass transfer efficiency, and increase the extraction yield of polyphenol and flavonoids [37]. In this study, the extraction efficiencies of various DES with water contents from 10% to 50% were conducted. With the results shown in Figure 1b, when the water content increased up to 40%, the extracted flavonoid amount improved, which was because of the reduction of the viscosity of solvents and the weakening of the hydrogen bonds [38]. When the water content surpassed this value, the extracted flavonoid amount started to drop off. Hence, 40% water content in DES was chosen since this was the highest extraction efficiency for the flavonoids. In Gan’s research, 40% water content in DES was chosen for the extraction of polyphenols from green tea as well [39].
3.2.3. Effect of Ultrasonic Time
The influence of ultrasonic time ranged from 0 to 30 min on the extraction efficiency was monitored. It was observed in Figure 1c that the extraction amount of flavonoids increased with the increasing of the ultrasonic time before 10 min and then was not improved with longer ultrasonic time, which indicated that a dynamic balance was reached during the ultrasonic-assisted extraction. Thus, 10 min was chosen for the extraction in further experiments taking into account the extraction efficiency and energy saving.
3.2.4. Effect of the Solid–Liquid Ratio
The effect of the solid–liquid ratio (g/mL) on the extraction efficiency of total flavonoids was monitored. As indicated in Figure 1d, the extraction efficiency of flavonoids increased with an increasing solid–liquid ratio from 1:20 to 1:80 and then decreased slightly. As a result, a 1:80 solid–liquid ratio was selected for later study.
3.2.5. Effect of Temperature
As the literature has reported, high temperatures could reduce the viscosity and surface tension of DES as well, and then improve the mobility and solubility of compounds [40]. To obtain the optimal extraction ability, the effect of different temperatures in the range of 40–120 °C on the extraction efficiency of flavonoids was studied (Figure 1e). As the temperature increased from 40 °C to 60 °C, the extraction amount of flavonoids increased up to a maximum value, but a decrease was then observed at temperatures higher than 60 °C. The decrease in high temperature might be attributed to the elevated temperature increasing the oxidation of flavonoid compounds. This trend can be observed in Wang’s and Weng’s reports as well [13,41]. Accordingly, a moderate temperature (60 °C) was set for the extraction.
3.2.6. Effect of Extraction Time
To optimize the extraction equilibrium, the extraction of flavonoids in lotus leaves was performed at different times from 10 min to 140 min using the UAE-DES method. As indicated in Figure 1f, the extraction efficiency increased as the extraction time increased from 10 to 60 min. However, the value kept stable after 60 min, which could be considered as the extraction having achieved balance. Typically, long-term extraction was not suitable for natural active components, which would affect the chemical structures and activities [13]. Accordingly, the DES extraction time was confirmed at 60 min.
3.2.7. Comparison to Conventional Solvents
To evaluate the extraction efficiency of UAE-DES, conventional solvents were used for comparison in the same extraction condition and procedure [42]. The used conventional solvents included water, 50% methanol, 95% methanol, and 95% ethanol. Under the same treatment, 50% methanol solvent exhibited the highest extraction efficiency (246.9 mg/g), which was higher than that using DES (236.6 mg/g) by 4% (Figure 2), while water demonstrated the worst extraction efficiency for target compounds. Methanol was a good organic solvent in the extraction of natural products including flavonoids. However, its toxicity to the human body and harm to the environment could not be ignored. The extraction efficiency of DES is the second-best solvent in this comparison, which was slightly lower than that of 50% methanol. Considering the ease of recycling, low cost in preparation, and environmentally friendliness, DES were selected as the extraction solvent because of its satisfactory extraction ability and sustainable properties [43].
3.3. Recovery of Flavonoids and Reusability of DES
As studied in many pieces of research, macroporous resin has often been used to enrich natural active compounds, especially for flavonoids, and then target compounds could be eluted using organic solutions [44]. In this study, four different resins (HPD-500, AB-8, D-101, and NKA-9) were selected to investigate. Through the comparison, D-101 showed the best adsorption ability for flavonoids with 90% during the same conditions (Figure 3a). In many pieces of research, D-101 has also been selected for the collection of coumarins and flavonoids [45,46]. However, AB-8 and other kinds of resin have been used for the recovery of target compounds, which exhibited the importance of selection [29,47]. Then, different concentrations of ethanol solutions have been used for desorption. As shown in Figure 3b, the desorption ratio reached 95% when ethanol concentrations were higher than 60%, and the optimal desorption ratio (99.5%) was obtained by 100% ethanol. Moreover, 100% ethanol solution was easier for removing solvent. Therefore, D-101 was used for the recovery of flavonoids, and 100% ethanol was chosen as the optimal desorption solvent.
Since the flavonoids in the solvent were adsorbed by macroporous resin, the DES could be reused for the next round of extraction [48]. After the evaporation of water from DES through rotary evaporators, a new solvent was prepared based on the appropriate water content and applied for the next cycle of extraction. As shown in Figure 4, the extraction efficiency of DES for flavonoids was decreased with increasing reuse of recovered DES. The extraction efficiency of the fifth cycle was only 78% of the initial cycle. The results demonstrated that DES could be reused four times leading to cost savings.
3.4. Antioxidant Activities of the DES Extract
Antioxidant activity was an important bioactivity for natural components, and five assays were introduced to multi-aspect evaluate the antioxidant capacity [49]. The antioxidant activities including DPPH, ABTS, Fe3+ reducing, FRAP, and Fe2+ chelating of the DES extracts are presented in Figure 5. As a result, the DES extract ranged from 1 mg/mL to 20 mg/mL and expressed gradually increased antioxidant activities in different degrees. For the DPPH and ABTS radical scavenging abilities (Figure 5a), the DES extract showed the highest inhibition at 10 mg/mL (94.1% and 94.7%). These results showed that the DES extract had strong free radical scavenging activities, although it was lower than that of rutin. The iron chelating activity was significant of active compounds in the food and cosmetic industries, which meant the protection of the metabolism, skin, and environment against heavy metals [50]. The DES extract exhibited an increasing inhibition ability with increasing concentrations and reached 41.3% at 20 mg/mL (Figure 5a), while rutin showed no activity and only 0.11% at 0.5 mg/mL. In the Fe3+ reduction and FRAP assays, the values were expressed as the equivalent concentration of standards. The higher the values, the better the antioxidant activities the sample exhibited. The measured Fe3+ reduction and FRAP values reached 0.73 mg RE /mL and 0.71 mmol/L Fe2+ (Figure 5b), while the FRAP value of 0.1 mg/mL rutin was 0.74 mmol/L Fe2+, which could be referred to as a reference. As some reports have shown, the ethanol extract of lotus leaves exhibited antioxidant activities in DPPH scavenging activity, ABTS scavenging activity, and the FRAP assay [51]. Compared to water extraction, methanol extraction had higher antioxidant activities [52]. In conclusion, the DES extract showed dose-dependent antioxidant activities in these five assays, and it could be further developed as a crude antioxidant in many areas.
3.5. Antibacterial Activity of the DES Extract
Antibacterial evaluation is a rapid, simple, and effective method to determine antimicrobial activity [53]. The antibacterial potentials of the DES extract against Staphylococcus aureus and Escherichia coli bacterial strains were investigated and are shown in Table 2. The DIZ of the DES extract at the concentration of 100 mg/mL was 8.49 ± 0.01 mm and 12.29 ± 0.01 mm for S. aureus and E. coli. It could be found that the antibacterial effect of the DES extract on two bacteria was confirmed, although the activities were lower than rutin. The antibacterial ability of the DES extract was further verified by the determination of the MIC value. As revealed in Table 2, the MIC values of the DES extract to S. aureus and E. coli were 1666 and 208 μg/mL, respectively, while the MIC values of rutin were 100 and 400 μg/mL, which was consistent with the results of DIZ. Antibacterial activity tests showed that the DES extract had a good inhibitory effect on E. coli at a low concentration, which made the potential usage of the DES extract an antibacterial agent [54]. The antibacterial testing of lotus leaf extracts is rare. Abderrahim Benslama reported that methanol extract has a moderate antibacterial activity against Staphylococcus aureus, Micrococcus luteus, and Bacillus subtilis [55]. The antibacterial ability of the DES extract in this study could expand the application range of lotus leaves.
3.6. HPLC Analysis of the DES Extract
For a better understanding of the extraction of DES, the composition of the DES extract was analyzed by HPLC. The typical chromatogram of the DES extract is shown in Figure 6. As shown in the Figure, the peaks were well separated, and three peaks at 17.3 min, 20.1 min, and 21.6 min could be found. In the previous study in our group, many flavonoid standards were acquired and analyzed [56]. Through the comparison of the UV spectra and retention time in the same HPLC analysis conditions between the DES extract and the standards, these three main peaks in the DES extract were identified as astragalin, hyperoside, and isoquercitrin. The structures and UV spectra of them are shown in the Supplementary File. The chromatograms of the three standards are also illustrated in Figure 6 (colorful lines). It could be found that the retention times of these standards were the same as those peaks in the DES extract. Astragalin was identified from the receptacles of N. nucifera as the main component in the inhibition of tyrosinase activity [57]. In other groups’ reports, hyperoside and isoquercitrin were also identified from lotus leaves, which could confirm their existence in lotus leaves [2,58]. Based on these data and analyses, three peaks in the DES extract were identified as astragalin, hyperoside, and isoquercitrin, which made further utilization of the DES extract easier.
4. Conclusions
In this study, the flavonoids from lotus leaves were extracted using UAE-DES. The choline chloride/urea system was selected from 34 kinds of DES compositions, and the factors related to the preparation of the DES and the extraction process were optimized. Through the following purification by D-101 macroporous resin, the flavonoid extract was concentrated and the solvent could be recycled. The antioxidant and antibacterial tests confirmed that the extracted lotus leaf flavonoids had good activities in dose-dependent manners, which has promising applications in food additives and food preservations. Through the HPLC analysis of the DES extract, three peaks were positively identified as astragalin, hyperoside, and isoquercitrin by comparing the UV and retention time data. Based on the findings in this study, a proper deep eutectic solvent was optimized for the extraction of flavonoids from lotus leaves using UAE-DES, which gave a more eco-friendly method for the development of lotus-based products. To meet the challenge, further research in the future could be focused on research of specific flavonoids and the industrial application of DES in the separation of natural compounds.
Data curation, Y.Z.; formal analysis, Y.Z.; methodology, L.L. and S.D.; project administration, S.D.; writing—original draft, L.L.; writing—review and editing, A.X. All authors have read and agreed to the published version of the manuscript.
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to security requirements in the institute of the corresponding author.
This work was supported by the Natural Science Foundation of Changsha, China (Grant No. kq2202323) and the Chinese Agricultural Science and Technology Innovation Project (No. ASTIP-IBFC05).
The authors declare no conflict of interest.
Footnotes
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
Enlarge this image.Figure 1. Optimization of extraction parameters in the UAE-DES method. (a) Effect of the HBA/HBD ratio on extraction efficiency; (b) effect of water content on extraction efficiency; (c) effect of ultrasonic time on extraction efficiency; (d) effect of the solid–liquid ratio on extraction efficiency; (e) effect of temperature on extraction efficiency; (f) effect of extraction time on extraction efficiency. Values followed by different letters (a–d) are significantly different according to Duncan’s test (p < 0.05).
Enlarge this image.Figure 2. Extraction efficiencies of prepared DES and conventional solvents (50%me-50% methanol, 95%me-95% methanol, and 95%et-95% ethanol). Values followed by different letters (a–d) are significantly different according to Duncan’s test (p < 0.05).
Enlarge this image.Figure 3. (a) The adsorption rate of four different macroporous resins (HPD-500, AB-8, D-101, and NKA-9); (b) The desorption rate obtained by different ethanol concentrations. Values followed by different letters (a–d) are significantly different according to Duncan’s test (p < 0.05).
Enlarge this image.Figure 4. The recycling of recovered DES. One g of powdered dried lotus leaf was added to 20.0 mL of the DES. After extraction by the UAE-DES method, the DES extracts were adsorbed with 4.0 g of D101. After the adsorption procedure, the resin was desorbed and the DES were reused. The DES were evaporated to remove water and an appropriate amount of water was added for the next cycle of extraction.
Enlarge this image.Figure 5. The antioxidant activities of DES extract in different concentrations. (a) DPPH, ABTS, and Fe2+ chelating assay. (b) Fe3+ reducing and FRAP assay.
Enlarge this image.Figure 6. The chromatograms of the DES extract (black line), hyperoside (blue line), isoquercitrin (violet line), and astragalin (dark yellow line).
The extraction efficiencies of flavonoids with different DES types.
| No. | HBA | HBD | Molar Ratio | Extraction Efficiency (mg/g) |
|---|---|---|---|---|
| 1 | Choline chloride | Ethylene glycol | 1:2 | 114.9 ± 2.5 d |
| 2 | Triethylene glycol | 1:2 | 91.5 ± 1.2 h | |
| 3 | Glycerol | 1:2 | 49.6 ± 1.2 p | |
| 4 | 2-Chloropropionic acid | 1:2 | 33.7 ± 0.1 rs | |
| 5 | Malonic acid | 1:2 | 36.3 ± 0.4 r | |
| 6 | Lactic acid | 1:2 | 54.1 ± 1.5 o | |
| 7 | Formic acid | 1:2 | 81.4 ± 0.7 l | |
| 8 | Acetic acid | 1:2 | 88.3 ± 0.8 ij | |
| 9 | Urea | 1:2 | 178.9 ± 2.8 a | |
| 10 | Benzyltriethylammonium chloride | Ethylene glycol | 1:2 | 82.1 ± 0.9 kl |
| 11 | Triethylene glycol | 1:2 | 100.9 ± 1.7 g | |
| 12 | Glycerol | 1:2 | 92.2 ± 1.3 h | |
| 13 | 2-Chloropropionic acid | 1:2 | 49.6 ± 0.4 p | |
| 14 | Malonic acid | 1:2 | 49.3 ± 0.5 p | |
| 15 | Lactic acid | 1:2 | 83.4 ± 1.4 kl | |
| 16 | Formic acid | 1:2 | 154.9 ± 2.7 b | |
| 17 | Acetic acid | 1:2 | 48.0 ± 0.9 p | |
| 18 | Oxalic acid | 1:2 | 107.4 ± 0.8 f | |
| 19 | Acetamide | 1:2 | 30.7 ± 0.2 st | |
| 20 | Betaine | Glycerol | 1:2 | 87.6 ± 0.6 ij |
| 21 | Malonic acid | 1:2 | 73.3 ± 0.4 m | |
| 22 | Lactic acid | 1:2 | 97.7 ± 1.0 g | |
| 23 | Formic acid | 1:2 | 84.4 ± 0.5 g | |
| 24 | Acetic acid | 1:2 | 113.3 ± 1.1 d | |
| 25 | Urea | 1:2 | 59.3 ± 0.4 n | |
| 26 | Oxalic acid | 1:2 | 39.8 ± 0.1 q | |
| 27 | Guanidine hydrochloride | Ethylene glycol | 1:2 | 99.3 ± 0.9 g |
| 28 | Triethylene glycol | 1:2 | 105.2 ± 1.1 f | |
| 29 | Glycerol | 1:2 | 85.3 ± 0.7 jk | |
| 30 | 2-Chloropropionic acid | 1:2 | 83.7 ± 0.4 kl | |
| 31 | Lactic acid | 1:2 | 111.4 ± 3.0 e | |
| 32 | Formic acid | 1:2 | 98.7 ± 1.0 g | |
| 33 | Acetic acid | 1:2 | 29.1 ± 0.8 t | |
| 34 | Acetamide | 1:2 | 138.9 ± 2.5 c |
Values followed by different letters (a–t) in the same column are significantly different according to Duncan’s test (p < 0.05).
The antibacterial activities of DES extracts.
| Antibacterial Properties | Samples | S. aureus | E. coli |
|---|---|---|---|
| DIZ (mm) | DES extract (50 mg/mL) | 7.29 ± 0.01 | 9.02 ± 0.02 |
| DES extract (100 mg/mL) | 8.49 ± 0.01 | 12.29 ± 0.01 | |
| Rutin (1 mg/mL) | 7.21 ± 0.01 | 7.11 ± 0.01 | |
| MIC (μg/mL) | DES extract | 1666 | 208 |
| Rutin | 100 | 400 |
Supplementary Materials
The following supporting information can be downloaded at:
References
1. Liu, J.; Shi, K.; Shi, J.; Feng, Y.; Hao, C.; Peng, J.; Chen, S. A simple strategy to monitor the temporal and spatial distribution of alkaloids in sacred lotus leaves. Biosci. Biotechnol. Biochem.; 2021; 85, pp. 1332-1340. [DOI: https://dx.doi.org/10.1093/bbb/zbab038] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33713113]
2. Chen, S.; Zheng, Y.; Fang, J.-B.; Liu, Y.-L.; Li, S.-H. Flavonoids in lotus (Nelumbo) leaves evaluated by HPLC–MSn at the germplasm level. Food Res. Int.; 2013; 54, pp. 796-803. [DOI: https://dx.doi.org/10.1016/j.foodres.2013.08.031]
3. Limwachiranon, J.; Huang, H.; Shi, Z.; Li, L.; Luo, Z. Lotus Flavonoids and Phenolic Acids: Health Promotion and Safe Consumption Dosages. Compr. Rev. Food Sci. Food Saf.; 2018; 17, pp. 458-471. [DOI: https://dx.doi.org/10.1111/1541-4337.12333]
4. Wang, Z.; Li, Y.; Ma, D.; Zeng, M.; Wang, Z.; Qin, F.; Chen, J.; Christian, M.; He, Z. Alkaloids from lotus (Nelumbo nucifera): Recent advances in biosynthesis, pharmacokinetics, bioactivity, safety, and industrial applications. Crit. Rev. Food Sci. Nutr.; 2021; 2021, pp. 1-34. [DOI: https://dx.doi.org/10.1080/10408398.2021.2009436] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/34845950]
5. Yaneva, Z.; Simeonov, E.; Rusenova, N.; Ivanova, D.; Nikolova, G.; Karamalakova, Y.; Chilev, C.; Beev, G. Flavonoids Extraction Kinetics, Antimicrobial Activity and Radical Scavenging Potential of Bulgarian Woundwort (Solidago virgaurea L.). Separations; 2022; 9, 27. [DOI: https://dx.doi.org/10.3390/separations9020027]
6. Abbott, A.P.; Capper, G.; Davies, D.L.; Rasheed, R.K.; Tambyrajah, V. Novel solvent properties of choline chloride/urea mixtures. Chem. Commun.; 2003; 39, pp. 70-71. [DOI: https://dx.doi.org/10.1039/b210714g]
7. Dheyab, A.S.; Kanaan, M.Q.; Hussein, N.A.; AlOmar, M.K.; Sabran, S.F.; Abu Bakar, M.F. Antimycobacterial Activity of Rosmarinus officinalis (Rosemary) Extracted by Deep Eutectic Solvents. Separations; 2022; 9, 271. [DOI: https://dx.doi.org/10.3390/separations9100271]
8. Dheyab, A.S.; Abu Bakar, M.F.; AlOmar, M.; Sabran, S.F.; Muhamad Hanafi, A.F.; Mohamad, A. Deep Eutectic Solvents (DESs) as Green Extraction Media of Beneficial Bioactive Phytochemicals. Separations; 2021; 8, 176. [DOI: https://dx.doi.org/10.3390/separations8100176]
9. Dwamena, A.K. Recent Advances in Hydrophobic Deep Eutectic Solvents for Extraction. Separations; 2019; 6, 9. [DOI: https://dx.doi.org/10.3390/separations6010009]
10. Amoroso, R.; Hollmann, F.; Maccallini, C. Choline Chloride-Based DES as Solvents/Catalysts/Chemical Donors in Pharmaceutical Synthesis. Molecules; 2021; 26, 6286. [DOI: https://dx.doi.org/10.3390/molecules26206286]
11. Sillero, L.; Prado, R.; Welton, T.; Labidi, J. Extraction of flavonoid compounds from bark using sustainable deep eutectic solvents. Sustain. Chem. Pharm.; 2021; 24, 100544. [DOI: https://dx.doi.org/10.1016/j.scp.2021.100544]
12. Qi, X.-L.; Peng, X.; Huang, Y.-Y.; Li, L.; Wei, Z.-F.; Zu, Y.-G.; Fu, Y.-J. Green and efficient extraction of bioactive flavonoids from Equisetum palustre L. by deep eutectic solvents-based negative pressure cavitation method combined with macroporous resin enrichment. Ind. Crops Prod.; 2015; 70, pp. 142-148. [DOI: https://dx.doi.org/10.1016/j.indcrop.2015.03.026]
13. Hao, H.; Lin, L.; Liu, S.; Kang, Y.; Wang, Y.; Huang, J.; Weng, W. Deep Eutectic Solvent-Based Microwave-Assisted Extraction for the Chromatographic Analysis of Bioactive Flavonoids in Spirodela polyrrhiza. J. Chromatogr. Sci.; 2022; 60, pp. 501-510. [DOI: https://dx.doi.org/10.1093/chromsci/bmab092] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/34313295]
14. Balanescu, F.; Busuioc, A.C.; Botezatu, A.V.; Gosav, S.; Avramescu, S.M.; Furdui, B.; Dinica, R.M. Comparative Study of Natural Antioxidants from Glycine max, Anethum graveolensand Pimpinella anisum Seed and Sprout Extracts Obtained by Ultrasound-Assisted Extraction. Separations; 2022; 9, 152. [DOI: https://dx.doi.org/10.3390/separations9060152]
15. Boli, E.; Prinos, N.; Louli, V.; Pappa, G.; Stamatis, H.; Magoulas, K.; Voutsas, E. Recovery of Bioactive Extracts from Olive Leaves Using Conventional and Microwave-Assisted Extraction with Classical and Deep Eutectic Solvents. Separations; 2022; 9, 255. [DOI: https://dx.doi.org/10.3390/separations9090255]
16. Tsvetov, N.; Sereda, L.; Korovkina, A.; Artemkina, N.; Kozerozhets, I.; Samarov, A. Ultrasound-assisted extraction of phytochemicals from Empetrum hermafroditum Hager. using acid-based deep eutectic solvent: Kinetics and optimization. Biomass Convers. Biorefinery; 2022; 12, pp. 145-156. [DOI: https://dx.doi.org/10.1007/s13399-022-02299-2]
17. Xue, H.; Li, J.; Wang, G.; Zuo, W.; Zeng, Y.; Liu, L. Ultrasound-Assisted Extraction of Flavonoids from Potentilla fruticosa L. Using Natural Deep Eutectic Solvents. Molecules; 2022; 27, 5794. [DOI: https://dx.doi.org/10.3390/molecules27185794] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/36144529]
18. Gao, H.; Wang, Y.; Guo, Z.; Liu, Y.; Wu, Q.; Xiao, J. Optimization of ultrasound-assisted extraction of phenolics from Asparagopsis taxiformis with deep eutectic solvent and their characterization by ultra-high-performance liquid chromatography-mass spectrometry. Front. Nutr.; 2022; 9, 1036436. [DOI: https://dx.doi.org/10.3389/fnut.2022.1036436]
19. Qin, G.; Lei, J.; Li, S.; Jiang, Y.; Qiao, L.; Ren, M.; Gao, Q.; Song, C.; Fu, S.; Zhou, J. et al. Efficient, green extraction of two biflavonoids from Selaginella uncinata with deep eutectic solvents. Microchem. J.; 2022; 183, 108085. [DOI: https://dx.doi.org/10.1016/j.microc.2022.108085]
20. Aouam, I.; El Atki, Y.; Taleb, M.; Taroq, A.; El Kamari, F.; Lyoussi, B.; Abdellaoui, A. Antioxidant Capacities and Total Phenolic Contents of Thymus riatarum. Mater. Today Proc.; 2019; 13, pp. 579-586. [DOI: https://dx.doi.org/10.1016/j.matpr.2019.04.016]
21. Sang, J.; Liu, K.; Ma, Q.; Li, B.; Li, C.-Q. Combination of a deep eutectic solvent and macroporous resin for green recovery of anthocyanins from Nitraria tangutorun Bobr. fruit. Sep. Sci. Technol.; 2019; 54, pp. 3082-3090. [DOI: https://dx.doi.org/10.1080/01496395.2018.1559190]
22. Zhang, L.; Liu, L.; Xiao, A.; Huang, S.; Li, D. Screening and analysis of xanthine oxidase inhibitors in jute leaves and their protective effects against hydrogen peroxide-induced oxidative stress in cells. Open Chem.; 2020; 18, pp. 1481-1494. [DOI: https://dx.doi.org/10.1515/chem-2020-0178]
23. Mansinhos, I.; Gonçalves, S.; Rodríguez-Solana, R.; Ordóñez-Díaz, J.L.; Moreno-Rojas, J.M.; Romano, A. Ultrasonic-Assisted Extraction and Natural Deep Eutectic Solvents Combination: A Green Strategy to Improve the Recovery of Phenolic Compounds from Lavandula pedunculata subsp. lusitanica (Chaytor) Franco. Antioxidants; 2021; 10, 582. [DOI: https://dx.doi.org/10.3390/antiox10040582]
24. Huang, Q.; Zhang, H.; Xue, D. Enhancement of antioxidant activity of Radix Puerariae and red yeast rice by mixed fermentation with Monascus purpureus. Food Chem.; 2017; 226, pp. 89-94. [DOI: https://dx.doi.org/10.1016/j.foodchem.2017.01.021] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/28254024]
25. Buravlev, E.V.; Shevehenko, O.G. 2-Hydroxy-3-isobornyl-5-methylbenzaldehyde derivatives: Synthesis and antioxidant activity in vitro. Russ. Chem. Bull.; 2019; 68, pp. 79-85. [DOI: https://dx.doi.org/10.1007/s11172-019-2419-1]
26. Xie, Y.; Chen, J.; Xiao, A.; Liu, L. Antibacterial Activity of Polyphenols: Structure-Activity Relationship and Influence of Hyperglycemic Condition. Molecules; 2017; 22, 1913. [DOI: https://dx.doi.org/10.3390/molecules22111913] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/29113147]
27. Alanazi, A.K.; Alqasmi, M.H.; Alrouji, M.; Kuriri, F.A.; Almuhanna, Y.; Joseph, B.; Asad, M. Antibacterial Activity of Syzygium aromaticum (Clove) Bud Oil and Its Interaction with Imipenem in Controlling Wound Infections in Rats Caused by Methicillin-Resistant Staphylococcus aureus. Molecules; 2022; 27, 8551. [DOI: https://dx.doi.org/10.3390/molecules27238551]
28. Naim, N.; Bouymajane, A.; Oulad El Majdoub, Y.; Ezrari, S.; Lahlali, R.; Tahiri, A.; Ennahli, S.; Laganà Vinci, R.; Cacciola, F.; Mondello, L. et al. Flavonoid Composition and Antibacterial Properties of Crocus sativus L. Petal Extracts. Molecules; 2023; 28, 186. [DOI: https://dx.doi.org/10.3390/molecules28010186] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/36615378]
29. Wang, G.; Cui, Q.; Yin, L.-J.; Zheng, X.; Gao, M.-Z.; Meng, Y.; Wang, W. Efficient extraction of flavonoids from Flos Sophorae Immaturus by tailored and sustainable deep eutectic solvent as green extraction media. J. Pharm. Biomed. Anal.; 2019; 170, pp. 285-294. [DOI: https://dx.doi.org/10.1016/j.jpba.2018.12.032] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30951994]
30. Wang, X.; Wu, Y.; Li, J.; Wang, A.; Li, G.; Ren, X.; Yin, W. Ultrasound-assisted deep eutectic solvent extraction of echinacoside and oleuropein from Syringa pubescens Turcz. Ind. Crops Prod.; 2020; 151, 112442. [DOI: https://dx.doi.org/10.1016/j.indcrop.2020.112442]
31. Sui, M.; Feng, S.; Liu, G.; Chen, B.; Li, Z.; Shao, P. Deep eutectic solvent on extraction of flavonoid glycosides from Dendrobium officinale and rapid identification with UPLC-triple-TOF/MS. Food Chem.; 2023; 401, 134054. [DOI: https://dx.doi.org/10.1016/j.foodchem.2022.134054] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/36103742]
32. Zeng, J.; Dou, Y.; Yan, N.; Li, N.; Zhang, H.; Tan, J.-N. Optimizing Ultrasound-Assisted Deep Eutectic Solvent Extraction of Bioactive Compounds from Chinese Wild Rice. Molecules; 2019; 24, 2718. [DOI: https://dx.doi.org/10.3390/molecules24152718] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31357469]
33. Jakovljević, M.; Vladić, J.; Vidović, S.; Pastor, K.; Jokić, S.; Molnar, M.; Jerković, I. Application of Deep Eutectic Solvents for the Extraction of Rutin and Rosmarinic Acid from Satureja montana L. and Evaluation of the Extracts Antiradical Activity. Plants; 2020; 9, 153. [DOI: https://dx.doi.org/10.3390/plants9020153]
34. Pal, C.B.T.; Jadeja, G.C. Deep eutectic solvent-based extraction of polyphenolic antioxidants from onion (Allium cepa L.) peel. J. Sci. Food Agric.; 2019; 99, pp. 1969-1979. [DOI: https://dx.doi.org/10.1002/jsfa.9395] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30270562]
35. Zuo, J.; Ma, P.; Geng, S.; Kong, Y.; Li, X.; Fan, Z.; Zhang, Y.; Dong, A.; Zhou, Q. Optimization of the extraction process of flavonoids from Trollius ledebouri with natural deep eutectic solvents. J. Sep. Sci.; 2022; 45, pp. 717-727. [DOI: https://dx.doi.org/10.1002/jssc.202100802]
36. Ali, M.C.; Chen, J.; Zhang, H.; Li, Z.; Zhao, L.; Qiu, H. Effective extraction of flavonoids from Lycium barbarum L. fruits by deep eutectic solvents-based ultrasound-assisted extraction. Talanta; 2019; 203, pp. 16-22. [DOI: https://dx.doi.org/10.1016/j.talanta.2019.05.012]
37. Huang, J.; Guo, X.; Xu, T.; Fan, L.; Zhou, X.; Wu, S. Ionic deep eutectic solvents for the extraction and separation of natural products. J. Chromatogr. A; 2019; 1598, pp. 1-19. [DOI: https://dx.doi.org/10.1016/j.chroma.2019.03.046]
38. Zheng, B.; Yuan, Y.; Xiang, J.; Jin, W.; Johnson, J.B.; Li, Z.; Wang, C.; Luo, D. Green extraction of phenolic compounds from foxtail millet bran by ultrasonic-assisted deep eutectic solvent extraction: Optimization, comparison and bioactivities. LWT; 2022; 154, 112740. [DOI: https://dx.doi.org/10.1016/j.lwt.2021.112740]
39. Luo, Q.; Zhang, J.-R.; Li, H.-B.; Wu, D.-T.; Geng, F.; Corke, H.; Wei, X.-L.; Gan, R.-Y. Green Extraction of Antioxidant Polyphenols from Green Tea (Camellia sinensis). Antioxidants; 2020; 9, 785. [DOI: https://dx.doi.org/10.3390/antiox9090785]
40. Liu, Y.; Li, J.; Fu, R.; Zhang, L.; Wang, D.; Wang, S. Enhanced extraction of natural pigments from Curcuma longa L. using natural deep eutectic solvents. Ind. Crops Prod.; 2019; 140, 111620. [DOI: https://dx.doi.org/10.1016/j.indcrop.2019.111620]
41. Zhang, H.; Hao, F.; Yao, Z.; Zhu, J.; Jing, X.; Wang, X. Efficient extraction of flavonoids from Polygonatum sibiricum using a deep eutectic solvent as a green extraction solvent. Microchem. J.; 2022; 175, 107168. [DOI: https://dx.doi.org/10.1016/j.microc.2021.107168]
42. Zhu, S.; Liu, D.; Zhu, X.; Su, A.; Zhang, H. Extraction of Illegal Dyes from Red Chili Peppers with Cholinium-Based Deep Eutectic Solvents. J. Anal. Methods Chem.; 2017; 2017, 2753752. [DOI: https://dx.doi.org/10.1155/2017/2753752] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/28831327]
43. Meng, Z.; Zhao, J.; Duan, H.; Guan, Y.; Zhao, L. Green and efficient extraction of four bioactive flavonoids from Pollen Typhae by ultrasound-assisted deep eutectic solvents extraction. J. Pharm. Biomed. Anal.; 2018; 161, pp. 246-253. [DOI: https://dx.doi.org/10.1016/j.jpba.2018.08.048] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30172879]
44. Wang, X.-H.; Wang, J.-P. Effective extraction with deep eutectic solvents and enrichment by macroporous adsorption resin of flavonoids from Carthamus tinctorius L. J. Pharm. Biomed. Anal.; 2019; 176, 112804. [DOI: https://dx.doi.org/10.1016/j.jpba.2019.112804]
45. Wang, Y.; Hu, Y.; Wang, H.; Tong, M.; Gong, Y. Green and enhanced extraction of coumarins from Cortex Fraxini by ultrasound-assisted deep eutectic solvent extraction. J. Sep. Sci.; 2020; 43, pp. 3441-3448. [DOI: https://dx.doi.org/10.1002/jssc.202000334]
46. Xia, G.-H.; Li, X.-H.; Jiang, Y.-H. Deep eutectic solvents as green media for flavonoids extraction from the rhizomes of Polygonatum odoratum. Alex. Eng. J.; 2021; 60, pp. 1991-2000. [DOI: https://dx.doi.org/10.1016/j.aej.2020.12.008]
47. Zhang, X.; Su, J.; Chu, X.; Wang, X. A Green Method of Extracting and Recovering Flavonoids from Acanthopanax senticosus Using Deep Eutectic Solvents. Molecules; 2022; 27, 923. [DOI: https://dx.doi.org/10.3390/molecules27030923]
48. Gao, M.; Wang, D.; Deng, L.; Liu, S.; Zhang, K.; Quan, T.; Yang, L.; Kang, X.; Xia, Z.; Gao, D. High-Crystallinity Covalent Organic Framework Synthesized in Deep Eutectic Solvent: Potentially Effective Adsorbents Alternative to Macroporous Resin for Flavonoids. Chem. Mater.; 2021; 33, pp. 8036-8051. [DOI: https://dx.doi.org/10.1021/acs.chemmater.1c02344]
49. Al-Azzawi, A.; Al Dibsawi, A.; Talath, S.; Wali, A.F.; Sarheed, O. Method Development: The Antioxidant and Antifungal Activity of the Isolated Component from the Ethanolic Extract of Tecoma stans Leaves Using Flash Chromatography. Separations; 2022; 9, 317. [DOI: https://dx.doi.org/10.3390/separations9100317]
50. Oliveira, G.; Marques, C.; de Oliveira, A.; de Almeida dos Santos, A.; do Amaral, W.; Ineu, R.P.; Leimann, F.V.; Peron, A.P.; Igarashi-Mafra, L.; Mafra, M.R. Extraction of bioactive compounds from Curcuma longa L. using deep eutectic solvents: In vitro and in vivo biological activities. Innov. Food Sci. Emerg. Technol.; 2021; 70, 102697. [DOI: https://dx.doi.org/10.1016/j.ifset.2021.102697]
51. Zhu, M.-Z.; Wu, W.; Jiao, L.-L.; Yang, P.-F.; Guo, M.-Q. Analysis of Flavonoids in Lotus (Nelumbo nucifera) Leaves and Their Antioxidant Activity Using Macroporous Resin Chromatography Coupled with LC-MS/MS and Antioxidant Biochemical Assays. Molecules; 2015; 20, pp. 10553-10565. [DOI: https://dx.doi.org/10.3390/molecules200610553] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/26060918]
52. Su, D.; Li, N.; Chen, M.; Yuan, Y.; He, S.; Wang, Y.; Wu, Q.; Li, L.; Yang, H.; Zeng, Q. Effects of in vitro digestion on the composition of flavonoids and antioxidant activities of the lotus leaf at different growth stages. Int. J. Food Sci. Technol.; 2018; 53, pp. 1631-1639. [DOI: https://dx.doi.org/10.1111/ijfs.13746]
53. An, J.-Y.; Wang, L.-T.; Lv, M.-J.; Wang, J.-D.; Cai, Z.-H.; Wang, Y.-Q.; Zhang, S.; Yang, Q.; Fu, Y.-J. An efficiency strategy for extraction and recovery of ellagic acid from waste chestnut shell and its biological activity evaluation. Microchem. J.; 2021; 160, 105616. [DOI: https://dx.doi.org/10.1016/j.microc.2020.105616]
54. Naseem, Z.; Zahid, M.; Hanif, M.A.; Shahid, M. Green extraction of ethnomedicinal compounds from Cymbopogon citratus Stapf using hydrogen-bonded supramolecular network. Sep. Sci. Technol.; 2021; 56, pp. 1520-1533. [DOI: https://dx.doi.org/10.1080/01496395.2020.1781894]
55. Benslama, A.; Harrar, A.; Gul, F.; Demirtas, I. Phenolic Compounds, Antioxidant and Antibacterial Activities of Zizyphus lotus L. Leaves Extracts. Nat. Prod. J.; 2017; 7, pp. 316-322. [DOI: https://dx.doi.org/10.2174/2210315507666170530090957]
56. Tang, X.; Tang, P.; Liu, L. Molecular Structure–Affinity Relationship of Flavonoids in Lotus Leaf (Nelumbo nucifera Gaertn.) on Binding to Human Serum Albumin and Bovine Serum Albumin by Spectroscopic Method. Molecules; 2017; 22, 1036. [DOI: https://dx.doi.org/10.3390/molecules22071036]
57. Jung, S.Y.; Jung, W.S.; Jung, H.K.; Lee, G.H.; Cho, J.H.; Cho, H.W.; Choi, I.Y. The mixture of different parts of Nelumbo nucifera and two bioactive components inhibited tyrosinase activity and melanogenesis. J. Cosmet. Sci.; 2014; 65, pp. 377-388.
58. Cho, W.-K.; Yang, H.J.; Ma, J.Y. Lotus (Nelumbo nucifera Gaertn.) leaf water extracts suppress influenza a viral infection via inhibition of neuraminidase and hemagglutinin. J. Funct. Foods; 2022; 91, 105019. [DOI: https://dx.doi.org/10.1016/j.jff.2022.105019]
© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.
Details
; Xiao, Aiping; Zhang, Yi; Duan, Shengwen