Ultrasound-Assisted Extraction of Flavonoids from Potentilla fruticosa L. Using Natural Deep Eutectic Solvents

A series of natural deep eutectic solvents (NADESs) were prepared with choline chloride, betaine, and a variety of natural organic acids in order to find new environmentally-friendly green solvents to replace the traditional solvents. The NADESs were employed to extract flavonoids from Potentilla fruticosa L. (PFL) with the help of ultrasound. The eutectic solvent diluted with an appropriate amount of water improved the extraction ability of flavonoids due to the decrease of solution viscosity. The microstructure of the raw sample and the samples subjected to ultrasonic bath in different solutions were observed using scanning electron microscope (SEM) to determine the role of the NADESs in the extraction process. The DPPH method and glucose consumption method were used to study the antioxidant and hypoglycemic ability of flavonoid compounds in PFL. Single factor method and response surface methodology (RSM) were designed to analyze the effects of three extraction parameters, including solvent/solid ratio, ultrasonic power, and extraction time, on the extraction yield, antioxidant capacity, and hypoglycemic capacity, and the corresponding second-order polynomial prediction models were established. The optimal extraction conditions for the maximum extraction yield, antioxidant capacity, and hypoglycemic capacity were predicted by RSM, and the reliability of RSM simulation results was verified by a one-off experiment.     

Lactic Acid-Based Natural Deep Eutectic Solvents to Extract Bioactives from Marine By-Products

Natural deep eutectic solvents (NaDES) were used to extract bioactive compounds from marine by-products: codfish bones, mussel meat, and tuna vitreous humor. NaDES were prepared using natural compounds, including lactic acid (Lac), fructose (Fru), and urea (Ur), and were characterized to define their physicochemical properties, including the viscosity, density, surface tension, and refractive index. FTIR and NMR analysis confirmed the presence of intermolecular hydrogen bonding in NaDES. The extracts obtained using these NaDES were characterized to define their composition. Results demonstrated that the extract’s composition differed highly, depending not only on the DES used, but also on the structure and composition of the raw material. Proteins and lipids were mainly present in extracts obtained from mussels, while ash content was highest in the extracts obtained from codfish bones. The biocompatibility of NaDES and the soluble fractions (SF) of the raw materials in NaDES was evaluated, and it was possible to conclude that the soluble ingredients obtained from the raw materials improved the biocompatibility of NaDES.     

Extraction of Bioactive Components from Chamaenerion angustifolium (L.) Scop. with Choline Chloride and Organic Acids Natural Deep Eutectic Solvents

Chamaenerion angustifolium (L.) Scop. (fireweed) is a perennial herbaceous plant of the Onagraceae family widely used in folk and scientific medicine. It is a promising source of bioactive components. One of the modern trends in extraction is the use of natural deep eutectic solvents (NADESs) combined with ultrasound-assisted extraction (UAE). However, works devoted to the extraction of biologically active substances from C. angustifolium using NADESs are scarce. The aim of this work is a comprehensive study of UAE of bioactive components from C. angustifolium using NADESs based on choline chloride and malonic, malic, tartaric, and citric acids. The antioxidative properties, total phenols, and flavonoids content were estimated for NADES-based extracts. The reference solvents were water and 90% v/v ethanol. Volatile extracted components were identified using GC-MS. The kinetics of the UAE were studied at 45 °C for 20–180 min with water added to 30 wt% NADES. The power of the ultrasound was 120 W, and the frequency was 40 kHz. It was found that NADES choline chloride + citric acid is more effective for the extraction of bioactive components. For this, NADES UAE conditions were optimized following a Box–Behnken design of the experiment and a response surface methodology. The temperature ranged from 30 to 60 °C, the time of extraction ranged from 20 to 60, and the addition of water ranged from 30 to 70 wt%. We established the optimal extraction conditions: temperature 58 °C, time of extraction 35 min, and 70 wt% water. The obtained results expand the knowledge about the use of NADES for the extraction of biologically active compounds from cheap and available plant raw materials.     

Natural Deep Eutectic Solvents for the Extraction of Bioactive Steroidal Saponins from Dioscoreae Nipponicae Rhizoma

In the present study, a simple and environmentally friendly extraction method based on natural deep eutectic solvents (NADESs) was established to extract four bioactive steroidal saponins from Dioscoreae Nipponicae Rhizoma (DNR). A total of twenty-one types of choline chloride, betaine, and L-proline based NADESs were tailored, and the NADES composed of 1:1 molar ratio of choline chloride and malonic acid showed the best extraction efficiency for the four steroidal saponins compared with other NADESs. Then, the extraction parameters for extraction of steroidal saponins by selected tailor-made NADES were optimized using response surface methodology and the optimal extraction conditions are extraction time, 23.5 min; liquid–solid ratio, 57.5 mL/g; and water content, 54%. The microstructure of the DNR powder before and after ultrasonic extraction by conventional solvents (water and methanol) and the selected NADES were observed using field emission scanning electron microscope. In addition, the four steroidal saponins were recovered from NADESs by D101 macroporous resin with a satisfactory recovery yield between 67.27% and 79.90%. The present research demonstrates that NADESs are a suitable green media for the extraction of the bioactive steroidal saponins from DNR, and have a great potential as possible alternatives to organic solvents for efficiently extracting bioactive compounds from natural products.     

Natural Deep Eutectic Solvent-Based Microwave-Assisted Extraction of Total Flavonoid Compounds from Spent Sweet Potato (Ipomoea batatas L.) Leaves: Optimization and Antioxidant and Bacteriostatic Activity

Natural deep eutectic solvents (NADESs) coupled with microwave-assisted extraction (MAE) were applied to extract total flavonoid compounds from spent sweet potato (Ipomoea batatas L.) leaves. In this study, ten different NADESs were successfully synthesized for the MAE. Based on single-factor experiments, the response surface methodology (RSM) was applied, and the microwave power, extraction temperature, extraction time, and solid–liquid ratio were further evaluated in order to optimize the yields of total flavonoid compounds. Besides, the extracts were recovered by macroporous resin for the biological activity detection of flavonoid compounds. As a result, NADES-2, synthesized by choline chloride and malic acid (molar ratio 1:2), exhibited the highest extraction yield. After that, the NADES-2-based MAE process was optimized and the optimal conditions were as follows: microwave power of 470 W, extraction temperature of 54 °C, extraction time of 21 min, and solid–liquid ratio of 70 mg/mL. The extraction yield (40.21 ± 0.23 mg rutin equivalents/g sweet potato leaves) of the model validation experiment was demonstrated to be in accordance with the predicted value (40.49 mg rutin equivalents/g sweet potato leaves). In addition, flavonoid compounds were efficiently recovered from NADES-extracts with a high recovery yield (>85%) using AB-8 macroporous resin. The bioactivity experiments in vitro confirmed that total flavonoid compounds had good DPPH and O₂⁻· radical-scavenging activity, as well as inhibitory effects on E. coli, S. aureus, E. carotovora, and B. subtilis. In conclusion, this study provides a green and efficient method to extract flavonoid compounds from spent sweet potato leaves, providing technical support for the development and utilization of sweet potato leaves’ waste.     

Deep Eutectic Solvents Based on Natural Ascorbic Acid Analogues and Choline Chloride

Deep eutectic solvents (DES) are one of the most promising green technologies to emerge in recent years given their combination of environmentally friendly credentials and useful functionalities. Considering the continued search for new DES – especially those that exemplify the aforementioned characteristics, we report the preparation of DES based on natural analogues of l -ascorbic acid for the first time. The onset of eutectic melting occurred at temperatures far below the melting point of the individual components and resulted in the generation of glass forming fluids with glass transition temperatures, viscosities and flow behavior that are comparable to similar systems. This work expands the current array of DES that can be produced using naturally occurring components, which given their potential to be bio-derived, interesting physicochemical properties (e. g. propensity to supercool and vitrify) and apparent antibacterial nature, may provide utility within a range of applications.     

Bioassay-Guided Isolation of Two Eudesmane Sesquiterpenes from Lindera strychnifolia Using Centrifugal Partition Chromatography

In this study, a centrifugal partition chromatography (CPC) separation was applied to identify antioxidant-responsive element (ARE) induction molecules from the crude extract of Lindera strychnifolia roots. CPC was operated with a two-phase solvent system composed of n-hexane-methanol-water (10:8.5:1.5, v/v/v) in dual mode (descending to ascending), which provided a high recovery rate (>95.5%) with high resolution. Then, ARE induction activity of obtained CPC fractions was examined in ARE-transfected HepG2 cells according to the weight ratios of the obtained fractions. The fraction exhibiting ARE-inducing activity was further purified by preparative HPLC that led to isolation of two eudesmane type sesquiterpenes as active compounds. The chemical structures were elucidated as linderolide U (1) and a new sesquiterpene named as linderolide V (2) by spectroscopic data. Further bioactivity test demonstrated that compounds 1 and 2 enhanced ARE activity by 22.4-fold and 7.6-fold, respectively, at 100 μM concentration while 5 μM of sulforaphane induced ARE activity 24.8-fold compared to the control.     

Solubility and Stability of Some Pharmaceuticals in Natural Deep Eutectic Solvents-Based Formulations

Some medicines are poorly soluble in water. For tube feeding and parenteral administration, liquid formulations are required. The discovery of natural deep eutectic solvents (NADES) opened the way to potential applications for liquid drug formulations. NADES consists of a mixture of two or more simple natural products such as sugars, amino acids, organic acids, choline/betaine, and poly-alcohols in certain molar ratios. A series of NADES with a water content of 0–30% (w/w) was screened for the ability to solubilize (in a stable way) some poorly water-soluble pharmaceuticals at a concentration of 5 mg/mL. The results showed that NADES selectively dissolved the tested drugs. Some mixtures of choline-based NADES, acid-neutral or sugars-based NADES could dissolve chloral hydrate (dissociated in water), ranitidine·HCl (polymorphism), and methylphenidate (water insoluble), at a concentration of up to 250 mg/mL, the highest concentration tested. Whereas a mixture of lactic-acid–propyleneglycol could dissolve spironolacton and trimethoprim at a concentration up to 50 and 100 mg/mL, respectively. The results showed that NADES are promising solvents for formulation of poorly water-soluble medicines for the development of parenteral and tube feeding administration of non-water-soluble medicines. The chemical stability and bioavailability of these drug in NADES needs further studies.     

Extraction and Stabilization of Betalains from Beetroot (Beta vulgaris) Wastes Using Deep Eutectic Solvents

Deep eutectic solvents (DES) using magnesium chloride hexahydrate [MgCl₂·6H₂O] and urea [U] proportions (1:1) and (2:1), were prepared for their use as extracting and stabilizer agents for red and violet betalains from beetroot (Beta vulgaris) waste. The synthetized DES [MgCl₂·6H₂O] [U] showed similar properties to eutectic mixtures, such as, liquid phase, low melting points and conductivity, thermal stability, and variable viscosity. In turn, betalain DES extracts (2:1) exhibited compatibility in the extraction and recovery of betalains from beetroot wastes, showing a betalain content comparable to that of betalain extracts. Betalain stability was determined by degradation tests; the exposure conditions were visible light (12 h), molecular oxygen from atmospheric air and environmental temperature (20–27 °C) for 40 days. The kinetic curves of the betalain degradation of water samples depicted a first-order model, indicating the alteration of a violet colouration of betalains from beetroot waste for 5–7 days. However, betalains from DES extracts were kept under visible light for 150 days, and for 340 days in storage (amber vessels), achieving a stability of 75% in comparison with initial beet extracts.     

Ultrasound-Assisted Dispersive Liquid-Liquid Microextraction Using Deep Eutectic Solvents (DESs) for Neutral Red Dye Spectrophotometric Determination

Deep eutectic solvents (DES), which have low toxicity and are low cost, biodegradable, and easily synthesized, were used for the extraction of neutral red (NR) dye before its spectrophotometric analysis. DES, containing choline chloride as a hydrogen bond acceptor and phenol as a hydrogen bond donor with a molar ratio of 1:2, was used for the extraction of NR dye from aqueous media. The possible interaction of different DESs with NR was studied using density functional theory (DFT) calculations. Experimentally, a UV-visible spectrophotometer was used for the quantitative analysis. The most important parameters affecting method performance, such as pH, extraction temperature, DES type, its volume, THF volume, sonication time, and centrifugation time, were optimized. The developed method provides exceptional sensitivity in terms of LOD and LOQ, which were 2.2 and 7.3 µg/L respectively. The relative standard deviation was 1.35–1.5% (n = 10), and the pre-concentration factor was 40. The method was found to be linear in the range of 2–300 µg/L (R2 = 0.9967). The method was successfully used for the determination of NR in wastewater samples. Finally, the DES-based method presents operational simplicity, high sensitivity, and rapid determination (<5 min) compared with other analytical procedures.     

Optimization of Ultrasonic—Assisted Extraction (UAE) Method Using Natural Deep Eutectic Solvent (NADES) to Increase Curcuminoid Yield from Curcuma longa L., Curcuma xanthorrhiza,and Curcuma mangga Val.

This study aims to optimize ultrasonic-assisted natural deep eutectic solvents (NADES) based extraction from C. longa. Choline chloride-lactic acid (CCLA-H₂O = 1:1, b/v) was used to investigate the impact of various process parameters such as solvent’s water content, solid loading, temperature, and extraction time. The optimal yield of 79.635 mg/g of C. longa was achieved from extraction in 20% water content NADES with a 4% solid loading in 35 °C temperature for 1 h. Peleg’s model was used to describe the kinetics of the optimized ultrasonic-assisted extraction (UAE) method, and the results were found to be compatible with experimental data. The optimum conditions obtained from C. longa extraction were then used for the extraction of C. xanthorriza and C. mangga, which give yields of 2.056 and 31.322 mg/g, respectively. Furthermore, n-hexane was utilized as an anti-solvent in the separation process of curcuminoids extract from C. longa, C. xanthorriza, and C. mangga, which gave curcuminoid recovery of 39%, 0.74%, and 27%, respectively. Solidification of curcuminoids was also carried out using the crystallization method with n-hexane and isopropanol. However, the solution of CCLA and curcuminoids formed a homogeneous mixture with isopropanol. Hence, the curcuminoids could not be solidified due to the presence of NADES in the extract solution.     

Development of Green and Efficient Extraction of Bioactive Ginsenosides from Panax ginseng with Deep Eutectic Solvents

The extraction of active constituents from natural sources in a green and efficient manner is considered an important field in the pharmaceutical industry. In recent years, deep eutectic solvents (DESs), a new type of green solvent, have attracted increasing attention. Therefore, we aimed to establish a green and high-efficiency extraction method for ginsenosides based on DESs. This study takes Panax ginseng as a model sample. Eighteen different DESs were produced to extract polar ginsenosides. Ultrasound-assisted extraction (UAE) was applied for simplicity and efficiency. A binary DES synthesized using choline chloride and urea at a proportion of 1:2 prepared by a heating stirring method is proven to be more effective than other solvents, such as the widely used 70% ethanol for the extraction of ginsenosides. Three variables that might affect the extraction, including the DES content in the extraction solvent, liquid/solid ratio, and ultrasound extraction time, were evaluated for optimization. The optimum extraction conditions for ginsenosides were determined as follows: DES water content of 20 wt%, liquid/solid ratio of 15 mL g⁻¹, and an ultrasonic extraction time of 15 min. The extraction yield for the optimized method is found to be 31% higher than that for 70% ethanol, which achieves efficient extraction. This study shows that DESs are available to extract ginsenosides for use in traditional Chinese medicine. The discovery also contributes to further research into the green extraction of ginsenosides.     

The Role of Hydrogen Bond Donor on the Extraction of Phenolic Compounds from Natural Matrices Using Deep Eutectic Systems

Recently, deep eutectic systems (DESs) as extraction techniques for bioactive compounds have surfaced as a greener alternative to common organic solvents. In order to study the effect of these systems on the extraction of phenolic compounds from different natural sources, a comprehensive review of the state of the art was carried out. In a first approach, the addition of water to these systems and its effect on DES physicochemical properties such as polarity, viscosity, and acidity was investigated. This review studied the effect of the hydrogen bond donor (HBD) on the nature of the extracted phenolics. The effects of the nature of the HBD, namely carbon chain length as well as the number of hydroxyl, methyl, and carbonyl groups, have shown to play a critical role in the extraction of different phenolic compounds. This review highlights the differences between DES systems and systematizes the results published in the literature, so that a more comprehensive evaluation of the systems can be carried out before any experimental trial.     

Ultrasonic-Assisted Efficient Extraction of Coumarins from Peucedanum decursivum (Miq.) Maxim Using Deep Eutectic Solvents Combined with an Enzyme Pretreatment

In this study, the ultrasonic-assisted extraction of total coumarins from Peucedanum decursivum (Miq.) Maxim (P. decursivum) via the combination of deep eutectic solvents (DESs) with cellulase pretreatment was carried out. Among the 15 kinds of DESs with choline chloride as hydrogen bond acceptors, the DES system of choline chloride/1,4-butanediol with a molar ratio of 1:4 showed the best extraction effect. First, single-factor experiments were performed using the following factors: liquid–solid ratio, pH, enzyme dosage and ultrasonic temperature. The Box–Behnken design (BBD) and response surface methodology (RSM) were employed to optimize the extraction conditions and obtain the following optimal parameter values for the extraction of coumarins from P. decursivum: liquid–solid ratio 14:1 mL/g, pH 5.0, enzyme dosage 0.2%, ultrasonic temperature 60 °C and ultrasonic time 50 min. Under these conditions, the extraction yield of total coumarins from P. decursivum could reach 2.65%, which was close to the predicted extraction yield of 2.68%. Furthermore, the contents of six coumarins, namely, umbelliferone, nodakenin, xanthotoxin, bergapten, imperatorin and decursin were determined to be 0.707 mg·g⁻¹, 0.085 mg·g⁻¹, 1.651 mg·g⁻¹, 2.806 mg·g⁻¹, 0.570 mg·g⁻¹ and 0.449 mg·g⁻¹, respectively, using HPLC-MS after the optimization. In addition, the cell fragmentation of P. decursivum powder obtained using ultrasonic-assisted DES extraction with enzyme pretreatment was found to be the most comprehensive using scanning electron microscopy (SEM), which indicated the highest extraction efficiency for P. decursivum. Finally, the in vitro antioxidant activity of the extracts was evaluated via radical scavenging with 1,1-diphenyl-2-picrylhydrazyl (DPPH), which showed that ultrasonic-assisted DES extraction with enzyme pretreatment exhibited significant antioxidant activity with DPPH radical scavenging of up to 97.90%. This work developed a new and efficient extraction method for coumarins.     

Latest Insights on Novel Deep Eutectic Solvents (DES) for Sustainable Extraction of Phenolic Compounds from Natural Sources

Phenolic compounds have long been of great importance in the pharmaceutical, food, and cosmetic industries. Unfortunately, conventional extraction procedures have a high cost and are time consuming, and the solvents used can represent a safety risk for operators, consumers, and the environment. Deep eutectic solvents (DESs) are green alternatives for extraction processes, given their low or non-toxicity, biodegradability, and reusability. This review discusses the latest research (in the last two years) employing DESs for phenolic extraction, solvent components, extraction yields, extraction method characteristics, and reviewing the phenolic sources (natural products, by-products, wastes, etc.). This work also analyzes and discusses the most relevant DES-based studies for phenolic extraction from natural sources, their extraction strategies using DESs, their molecular mechanisms, and potential applications.     

Effect of Natural Deep Eutectic Solvents on trans-Resveratrol Photo-Chemical Induced Isomerization and 2,4,6-Trihydroxyphenanthrene Electro-Cyclic Formation

trans-Resveratrol is a natural bioactive compound with well-recognized health promoting effects. When exposed to UV light, this compound can undergo a photochemically induced trans/cis isomerization and a 6π electrochemical cyclization with the subsequent formation of 2,4,6-trihydroxyphenanthrene (THP). THP is a potentially harmful compound which can exert genotoxic effects. In this work we improved the chromatographic separation and determination of the two resveratrol isomers and of THP by using a non-commercial pentafluorophenyl stationary phase. We assessed the effect of natural deep eutectic solvents (NaDES) as possible photo-protective agents by evaluating cis-resveratrol isomer and THP formation under different UV-light exposure conditions with the aim of enhancing resveratrol photostability and inhibiting THP production. Our results demonstrate a marked photoprotective effect exerted by glycerol-containing NaDES, and in particular by proline/glycerol NaDES, which exerts a strong inhibitory effect on the photochemical isomerization of resveratrol and significantly limits the formation of the toxic derivative THP. Considering the presence of resveratrol in various commercial products, these results are of note in view of the potential genotoxic risk associated with its photochemical degradation products and in view of the need for the development of green, eco-sustainable and biocompatible resveratrol photo-stable formulations.     

A Green Method of Extracting and Recovering Flavonoids from Acanthopanax senticosus Using Deep Eutectic Solvents

In recent years, green extraction of bioactive compounds from herbal medicines has generated widespread interest. Deep eutectic solvents (DES) have widely replaced traditional organic solvents in the extraction process. In this study, the efficiencies of eight DESs in extracting flavonoids from Acanthopanax senticosus (AS) were compared. Response surface methodology (RSM) was employed to optimize the independent variable including ultrasonic power, water content, solid-liquid ratio, extraction temperature, and extraction time. DES composed of glycerol and levulinic acid (1:1) was chosen as the most suitable extraction medium. Optimal conditions were ultrasonic power of 500 W, water content of 28%, solid-liquid ratio of 1:18 g·mL⁻¹, extraction temperature of 55 °C, and extraction time of 73 min. The extraction yield of total flavonoids reached 23.928 ± 0.071 mg·g⁻¹, which was 40.7% higher compared with ultrasonic-assisted ethanol extraction. Macroporous resin (D-101, HPD-600, S-8 and AB-8) was used to recover flavonoids from extracts. The AB-8 resin showed higher adsorption/desorption performance, with a recovery rate of total flavonoids of up to 71.56 ± 0.256%. In addition, DES solvent could efficiently be reused twice. In summary, ultrasonic-assisted DES combined with the macroporous resin enrichment method is exceptionally effective in recovering flavonoids from AS, and provides a promising environmentally friendly and recyclable strategy for flavonoid extraction from natural plant sources.     

Isolation of Mirificin and Other Bioactive Isoflavone Glycosides from the Kudzu Root Lyophilisate Using Centrifugal Partition and Flash Chromatographic Techniques

Pueraria lobata (Willd.) Ohwi is a legume taxon native to Southeast Asia and widely used in traditional medicine systems of that region. The therapeutic applications of the underground parts of this species (known as kudzu root) are related to its high content of isoflavones, mainly the characteristic C-glycoside derivatives. Within this group, the most explored compound both phytochemically and pharmacologically is puerarin. However, current scientific findings document important anti-biodegenerative effects for some of the minor isoflavones from kudzu roots. Therefore, the main objective of the study was to develop an original preparative method that allowed the efficient isolation of closely related hydrophilic daidzein C-glycosides, including mirificin, from vacuum-dried aqueous-ethanolic extracts of kudzu roots. For this purpose, the combined centrifugal partition (CPC) and flash chromatographic (FC) techniques were elaborated and used. The optimized biphasic solvent system in CPC, with ethyl acetate, ethanol, water, and 0.5% (V/V) acetic acid as a mobile phase modifier, enabled the purification and separation of the polar fraction containing bioactive isoflavones and ultimately the isolation of mirificin, 3′-hydroxy- and 3′-methoxypuerarin, puerarin, and daidzin using FC. The identity of isoflavones was confirmed using spectroscopic (UV absorption and nuclear magnetic resonance) and mass spectrometric methods. The determined purity of isolated mirificin was 63%.