Microwave-assisted Aqueous Two-phase Extraction Research Articles

Optimization of Microwave Assisted Aqueous Two‐Phase Extraction of Polysaccharides from Malus hupehensis and Evaluation Its Antioxidant Activity

AbstractThe two models of microwave assisted aqueous two‐phase extraction (MATPE) of polysaccharides from Malus hupehensis are established by response surface methodology (RSM) and back propagation neural network (BP‐NN), compared their prediction ability, and comparatively optimized extraction process by using RSM and BP‐NN coupled genetic algorithm (GA), and the antioxidant activity of M. hupehensis polysaccharides (MHPs) is evaluated by the scavenging ability of free radicals. The results show that the predictive ability of BP‐NN is significantly better than that of RSM. The optimal extraction parameters to achieve the highest MHPs yield (4.71 ± 0.04)% by MATPE are obtained through BP‐NN coupled GA method under the microwave power of 337 W, mass fraction of ammonium sulfate of 23%, ethanol concentration of 29%, and liquid‐to‐solid ratio of 22 mL g−1. The IC50 values of 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) and hydroxyl radicals (OH) scavenging capacities for MHPs achieve 5.13 and 3.29 mg mL−1, respectively. The findings provide important references for the development and utilization of M. hupehensis.

Microwave-assisted aqueous two-phase extraction of polysaccharides from Hippophae rhamnoide L.: Modeling, characterization and hypoglycemic activity

Natural polysaccharides are concerned for their high biological activity and low toxicity. Two kinds of polysaccharides were extracted from Hippophae rhamnoide L. by microwave-assisted aqueous two-phase system. Under the optimal conditions predicted by RSM model (K2HPO4/ethanol (18.93 %/28.29 %), liquid to material ratio 77 mL/g, power 625 W and temperature 61 °C), the yield of total polysaccharides reached 35.91 ± 0.76 %. Moreover, the polysaccharides extraction was well fitted to the Weibull model. After purification by Sepharose-6B, the polysaccharides from top phase (PHTP, purity of 81.44 ± 1.25 %) and bottom phase (PHBP, purity of 88.85 ± 1.40 %) were obtained. GC, FT-IR, methylation and NMR analyses confirmed the backbone of PHTP was composed of a repeated unit →4)-β-D-Glcp-(1 → 2)-α-L-Rhap-(1 → 4)-β-D-Galp-(1 → 4)-α-D-GalAp-(1 → 3)-α-L-Araf-(1 → 3)-α-D-Manp-(1→, while the repeated unit in PHBP was →3)-α-L-Araf-(1 → 2)-α-L-Rhap-(1 → 4)-β-D-Glcp-(1 → 3)-α-D-Manp-(1 → 4)-β-D-Galp-(1 → 4)-α-D-GalAp-(1→. Compared with PHTP (6.46 × 106 g/mol), PHBP with relatively low molecular weight (8.2 × 105 g/mol) exhibited the smaller particle size, better water-solubility, thermal and rheological property, stronger anti-glycosylation and α-amylase inhibitory effects. Moreover, PHTP and PHBP displayed a reversible inhibition on α-amylase in a competitive manner. This study provides a high-efficient and eco-friendly method for polysaccharides extraction, and lays a foundation for sea buckthorn polysaccharides as potential therapeutic agents in preventing and ameliorating diabetes.

Effect of Microwave-Assisted Aqueous Two-Phase Extraction of α-Solanine from S. retroflexum and Analysis on UHPLC-qTOF-MS

Abstract A new, fast and efficient method, hyphenated microwave-assisted aqueous two-phase extraction (MA-ATPE) was applied in the extraction of α-solanine from Solanum retroflexum. This environmentally friendly extraction method applied water and ethanol as extraction solvents. Central composite design (CCD) was performed which included numerical parameters such as time, mass of plant powder and microwave power. The categorical factors included the chaotrope — NaCl or the kosmotrope — Na2CO3. Fitting the central composite design response surface model to the data generated a quadratic model with a good fit (R2 = 0.920). The statistically significant (p < 0.05) parameters such as time and mass of plant powder were influential in the extraction of α-solanine. Quantification of α-solanine was achieved using a robust and sensitive feature of the ultra-high performance quadrupole time of flight mass spectrometer (UHPLC-qTOF-MS), multiple reaction monitoring (MRM). The optimized condition for the extraction of α-solanine in the presence of NaCl and Na2CO3 was a period of 1 min at a mass of 1.2 g using a microwave power of 40%. Maximal extraction of α-solanine was 93.50 mg kg−1 and 72.16 mg kg−1 for Na2CO3 and NaCl, respectively. The synergistic effect of salting-out and microwave extraction was influential in extraction of α-solanine. Furthermore, the higher negative charge density of the kosmotrope (Na2CO3) was responsible for its greater extraction of α-solanine than chaotrope (NaCl). The shorter optimal extraction times of MA-ATPE make it a potential technique that could meet market demand as it is a quick, green and efficient method for removal of toxic metabolites in nutraceuticals.

Hyphenation of aqueous two-phase and microwave extraction of solasonine and solamargine from leaves of Solanum mauritianum characterized by UHPLC-qTOF-MS

The biomass Solanum mauritianum (S. mauritianum) is an invasive weed specie; however, it is a source of medicinally important metabolites, as reported in literature, such as solasonine and solamargine. The study was directed at the optimization of microwave and aqueous two-phase-based extraction techniques which involved microwave-assisted extraction (MAE), aqueous two-phase extraction followed by microwave-assisted extraction (ATPE + MAE), and the “one-pot” microwave-assisted aqueous two-phase extraction (MA-ATPE) for extraction of solasonine and solamargine from leaves of S. mauritianum which was evaluated. The microwave-assisted extraction of solasonine and solamargine yielded optimums at 5.00 min, microwave power of 270 W, and solid/liquid of 0.1 g L−1 at an ethanol concentration of 60%. Application of a two-stage extraction (MAE + ATPE) in CaO-dried alcohol resulted in decreased amounts of solasonine and solamargine extracted. The best yields of solasonine and solamargine were achieved in the MA-ATPE method. Extraction of solamargine and solasonine using Na2CO3 in CaO-dried ethanol during MA-ATPE was approximately threefold and twofold greater than that of MAE + ATPE, respectively. Furthermore, extraction of solamargine and solasonine using NaCl in CaO-dried ethanol during MA-ATPE was approximately twofold greater than that of MAE + ATPE. The synergy of microwaves and salting-out in the “one-pot” MA-ATPE technique was shown to be a contributing factor for enhanced extraction of solamargine and solasonine from leaves of S. mauritianum. Application of this time- and energy-efficient extraction method could potentially be expanded for enrichment of medicinally important compounds from biomass of other medicinal plants.

Simultaneous separation and preliminary purification of anthocyanins from Rosa pimpinellifolia L. fruits by microwave assisted aqueous two-phase extraction

Rosa pimpinellifolia L. fruits (RPF) have great industrial potential due to the high presence of anthocyanins. In this work, microwave-assisted aqueous two-phase extraction (MA-ATPE) was applied to simultaneous extraction and purification of anthocyanins from RPF with ethanol/ammonium sulfate aqueous two-phase system (ATPS). MA-ATPE process was optimized by single-factor experiments and response surface methodology. The optimal anthocyanin yields were achieved in conditions of 0% HCl (w:w), 26.85% ethanol (w:w), 19.15% ammonium sulfate (w:w), liquid to solid ratio 40, 17.29 min, 60 °C and 400 W. Predicted anthocyanin yield of MA-ATPE at optimum conditions were determined as 1373.04 mg cyanidin 3-glucoside equivalent/g dry RPF. The purity of anthocyanin rich extract obtained by MA-ATPE was 1.65-fold greater than microwave assisted extraction using 80% ethanol (MAE). The compositional characterization of anthocyanins were carried out with HPLC-QTOF-MS. MA-ATPE had no obvious changes on anthocyanin composition when compared to MAE. Results indicated that the MA-ATPE could be a promising method for the extraction and preliminary purification of anthocyanins from different plant materials.

Microwave-assisted aqueous two-phase extraction of phenolic compounds from Ribes nigrum L. and its antibacterial effect on foodborne pathogens

Microwave-assisted ammonium sulfate/ethanol aqueous two-phase system was used to extract the phenolic compounds from blackcurrant (Ribes nigrum L.) fruits. After purification with AB-8 macroporous resin, the phenolics (RMAP) was obtained with a purity of 80.50 ± 0.26%. The minimum inhibitory concentration (MIC) and inhibition zone tests showed that RMAP exhibited more potent antibacterial properties against Staphylococcus aureus than against Escherichia coli and Salmonella typhimurium. Additionally, high-performance liquid chromatography-electrospray ionization-mass spectrometry analysis confirmed RMAP was mainly composed of four phenolic compounds including kaempferol-3-O-rutinoside, rutin, cyanidin-3-O-glucoside (C3G) and hyperoside. MIC and time-kill curve assays indicated that C3G displayed the highest inhibitory activity on S. aureus among four compounds. Moreover, crystal violet and MTT assays showed that C3G exhibited obviously inhibitory effect on fresh biofilm formation of S. aureus. Transmission electron microscopy observation, alkaline phosphatase, lactate dehydrogenase, protein and DNA leakage assays verified that C3G damaged cell wall and membrane of S. aureus. On the other hand, UV absorption spectroscopy and DNA cleavage assay revealed that C3G was able to interact with DNA in the form of chimera, but had no direct effect on DNA cleavage. Whereas, DNA topoisomerase IV (DNA Topo IV) inhibition assay and molecular docking model manifested that C3G could combine with DNA Topo IV to affect its activity and further restrained the synthesis of DNA. These results indicated that the antibacterial mechanism of C3G against S. aureus was mainly related to its inhibiting biofilm formation, disrupting cell structure and resisting DNA Topo IV activity.

Development of an SVR model for microwave-assisted aqueous two-phase extraction of isoflavonoids from Radix Puerariae

In this study, simultaneous extraction and purification of isoflavonoids from Radix Puerariae was carried out by the microwave-assisted aqueous two-phase extraction technique (MAATPE). An ethanol/K2HPO4 system was used to form an aqueous two-phase system. The critical factors, namely, the concentrations of K2HPO4 and ethanol, solid–liquid ratio, extraction temperature, extraction time, and microwave power were investigated. Subsequently, the least squares support vector regression (LS-SVR) as a machine learning method was used to establish the model and predict extraction yield. The final conditions were concluded as follows: ethanol concentration 29.5% (w/w) and K2HPO4 concentration 24.9% (w/w), extraction temperature 79 °C, extraction time 8 min, and microwave irradiation power 600 W. Under the optimal condition, the extraction yield of total isoflavonoids reached 13.39%, which was similar to the predicted value of 13.48%. The recovery of isoflavonoids was 94.6%, and the yield of puerarin was 49.5 mg/g.

Concurrent Extraction and Purification of Gentiopicroside from Gentiana scabra Bunge Using Microwave-Assisted Ethanol-Salt Aqueous Two-Phase Systems.

A potential method called microwave-assisted aqueous two-phase extraction (MA-ATPE) was developed for concurrent extraction and purification of gentiopicroside from Gentiana scabra Bunge. Formation characteristics of aqueous two-phase system (ATPS) composed of ethanol and 25 kinds of salts were investigated; K2HPO4 (w/w, 21.71%) and ethanol (w/w, 40.72%) were determined to be the optimal compositions of ATPS. Response surface methodology based on Box-Behnken design was used to investigate the extraction conditions, the optimal parameters were summarized as follows: 80°C of extraction temperature, 31s of extraction time, 11:1 (mL/g) of liquid-to-solid ratio, 100 meshes of particle size and 806W of microwave power. Under these conditions, the extraction yield of gentiopicroside was 65.32±0.24mg/g with a recovery of 96.51%. Compared with other four methods, the purity of gentiopicroside in the crude extracts reached 17.16±0.25%, which was significantly higher than that of smashing tissue extraction, microwave assisted-extraction, ultrasonic-assisted extraction and heat reflux extraction, respectively. In addition, the phase-forming salt can be recyclable. Therefore, MA-ATPE was an excellent and alternative technique to the conventional extraction approaches of gentiopicroside.

Enhancement of Solasodine Extracted from Fruits of Solanum nigrum L. by Microwave-Assisted Aqueous Two-Phase Extraction and Analysis by High-Performance Liquid Chromatography.

Background: Solasodine is a major bioactive ingredient in Solanum nigrum L. that has strong pharmacological characteristics. Therefore, the development of a simple and effective extraction method for obtaining solasodine is highly important. This study aims to provide a rapid and effective method for extracting solasodine from Solanum nigrum L. by microwave-assisted aqueous two-phase extraction (MAATPE). Methods: First, the high-performance liquid chromatography (HPLC) conditions were established for the detection of solasodine. Then, the aqueous two-phase system (ATPS) compositions were examined. On the basis of the results of single-factor experiments, for a better yield, response surface methodology (RSM) was used to optimize influential factors including the extraction temperature, extraction time and liquid-to-solid ratio. Results: The maximum extraction yield of 7.11 ± 0.08 mg/g was obtained at 44 °C, an extraction time of 15 min, and a liquid-to-solid ratio of 42:1 mL/g in the ATPS consisting of EtOH solvent, (NH4)2SO4, and water (28:16:56, w/w/w). The extraction yield of the alkaloid obtained using this method was markedly higher than those of microwave-assisted extraction (MAE) and ultrasonic-assisted extraction (UAE). Conclusions: In this work, solasodine was extracted by MAATPE for the first time and a high yield was obtained. MAATPE is a simple, rapid, and green technique for extraction from medical plants. Thus, the present study will enable the development of a feasible extraction method of active alkaloids from Solanum nigrum L.

Microwave-assisted aqueous two-phase extraction of diverse polysaccharides from Lentinus edodes: Process optimization, structure characterization and antioxidant activity

Microwave-assisted aqueous two-phase extraction (MAATPE) was applied to simultaneous extraction of diverse polysaccharides from Lentinus edodes with aqueous two-phase extraction system (ATPS) of ethanol/ammonium sulfate as the biphasic extractant. Key factors in MAATPE process were optimized by response surface methodology, and the optimum conditions were concluded as follows: the ATPS composition of ethanol concentration 26.0% (w/w) and (NH4)2SO4 concentration 19.58% (w/w), extraction temperature 78.7 °C, extraction time 19.55 min and solid-to-solvent ratio 1:50. The extraction yields of the polysaccharides from the top phase (PTP) and the bottom phase (PBP) were 2.12 ± 0.21% and 11.16 ± 0.28%, respectively. By means of FT-IR, HPGPC and HPLC analysis, PTP and PBP were different from each other in chemical structure, their average molecular weight (MW) were respectively 119,533 Da and 3906 Da. Also, the mechanism of MAATPE process was explored by composition analysis and scanning electron microscopy. Both polysaccharides exhibited stronger antioxidant activities, EC50 values of scavenging DPPH, hydroxyl and superoxide radicals for PBP achieved 580 ± 3, 83.1 ± 3.6 and 91.1 ± 0.5 μg/mL as against 560 ± 4, 76.3 ± 2.2 and 89.3 ± 2.4 μg/mL of PTP, respectively. Compared with conventional methods using a mono-phase solvent, MAATPE can not only greatly improve extraction efficiency, but also selectively extract diverse polysaccharides.

Microwave and enzyme co-assisted aqueous two-phase extraction of polyphenol and lutein from marigold (Tagetes erecta L.) flower

In this study, a novel method of simultaneous extraction and enrichment of total polyphenol (TP) and lutein from marigold flower was developed by microwave and enzyme co-assisted aqueous two-phase extraction (ATPE) (MEAATPE). In this study, ethanol/ammonium sulfate was chosen to construct aqueous two-phase system (ATPS) due to its fine partition and recycling behaviors. The optimal MEAATPE condition was obtained, that is, 28% (w/w), ethanol/20% (w/w) ammonium sulfate, 0.45 U/g enzyme concentration, enzymolysis time of 150 min, enzymolysis temperature of 45 °C, microwave time of 120 s, and microwave power of 270 W, by investigating the effects of ATPS composition and other factors on the yield of target products. Under this optimal condition, the yields of TP and lutein reached 84.61 and 7.32 mg/g with the corresponding recovery rates of 95.35% and 99.85%, respectively. Compared with enzyme-assisted ATPE, microwave-assisted ATPE, ATPE, and Soxhlet extraction (SE), the extraction yield of TP by MEAATPE was the highest, which is 71.14% higher than that of SE. Scanning electron microscopy analysis further showed an optative pretreatment effect of marigold flower powders. Thus, MEAATPE was an efficient, rapid, and environmental-friendly method used for the extraction of natural products.

Microwave-assisted aqueous two-phase extraction of alkaloids from Radix Sophorae Tonkinensis with an ethanol/Na2HPO4 system: Process optimization, composition identification and quantification analysis

A rapid method for simultaneous extraction and separation of multiple alkaloids from Radix Sophorae Tonkinensis (RST) was developed by microwave-assisted aqueous two-phase extraction (MAATPE) using the aqueous two-phase extraction system (ATPS) of ethanol/Na2HPO4 as the extraction solvent. The effects of key factors on extraction yield were investigated by utilizing single-factor experiment coupled to response surface methodology (RSM). The regression model by RSM was significant (p < 0.0001) and adequate for prediction of process efficacy, the optimized conditions were successfully validated by the parallel experiments with the yield very close to the predicted value. The optimum conditions were summarized as follows: the phase ratio of 2.60 for the ATPS, the particle size of 100 mesh, the liquid-to-material ratio of 75:1, the extraction temperature of 90 °C and the extraction time of 5 min, respectively. In MAATPE process, alkaloids were extracted preferentially from RST in the top phase with a higher yield and shorter extraction time than those of heating reflux extraction (HRE) and ultrasonic-assisted extraction (UAE). Nine alkaloids extracted were identified and quantified by high-resolution ultra-performance liquid chromatography-quadrupole-orbitrap mass spectrometry (UPLC-Q-Orbitrap/MS) and HPLC with UV detection. The contents of matrine, sophocarpine, oxymatrin, sophoranol, oxysophocarpine, 5α-hydroxysophocarpine, sophoridine, cytisine and N-methylcytisine in RST were quantified in range of 0.493–10.284 mg/g with recoveries of 90.26–106.3% and RSD’s of 0.8–2.1%, respectively. Moreover, the MAATPE mechanism was explored using the different extraction systems in combination of scanning electron microscopy (SEM) morphological studies. Significant differences in extraction yield and cell rupture exhibited that the addition of the salt in the ethanol-water mixture not only improved the thermal and demixing effects, but also accelerated the mass transfer and biphasic extraction processes. MAATPE integrated the advantages of microwave-assisted extraction (MAE) and aqueous two-phase extraction (ATPE) was proved as a green, efficient and promising alternative to extraction of alkaloids from RST.

Biphasic extraction of different polysaccharides from Radix Sophorae Tonkinensis by microwave-assisted aqueous two-phase extraction: Process optimization, structural characterization and mechanism exploration

Abstract A novel method for biphasic extraction and separation of different polysaccharides from Radix Sophorae Tonkinensis (RST) was developed by microwave-assisted aqueous two-phase extraction (MAATPE) in a one-step procedure. Using ethanol/Na2HPO4 system as a green extractant, the effects of the composition of the aqueous two-phase system (ATPS), extraction temperature and extraction time were investigated by single-factor experiments coupled to response surface methodology (RSM) through UV–vis analysis. Under the optimum conditions, extraction yields of two polysaccharides from top and bottom phases were 8.52 ± 0.21% (w/w) and 2.18 ± 0.08% (w/w), respectively. Compared with heating solvent extraction (HSE) and ultrasonic assisted extraction (UAE), MAATPE exhibited higher extraction efficiency. By means of Fourier transform infrared spectroscopy (FT-IR) and high-performance gel permeation chromatography (HPGPC), two polysaccharides obtained from both phases had the characteristics of homogenous heteropolysaccharides, but there were some differences in chemical structures and the molecular weight from each other. Both polysaccharides could be extracted respectively from RST to the top ethanol-rich phase and the bottom salt-rich phase due to their polarity difference. Furthermore, the chemical compositions of two polysaccharides were confirmed by high-performance liquid chromatography (HPLC) analysis after hydrolysis and derivatization, the molar ratios of monosaccharides were glucose: arabinose: galactose: rhamnose: mannose: glucuronic acid: aminogalactose = 63.70: 18.21: 7.00: 5.10: 3.13: 1.78: 1.07 for the polysaccharide in the top phase and 75.24: 3.79: 3.71: 3.37: 2.51: 1.43: 9.95 for those in the bottom phase, respectively. By means of extraction process analysis and scanning electron microscopy (SEM), the exportation of MAATPE mechanism revealed that combining microwave-assisted extraction (MAE) with aqueous two-phase extraction (ATPE) not only improved the extraction yields, but also obtained at least two different polysaccharides. Under microwave field, the ATPS could make the herb cell ruptured, and multiphase mass-transfer was accelerated. The proposed method is a rapid, simple and efficient alternative to biphasic extraction of diverse polysaccharides from natural plants.

The neuroprotective and antioxidant profiles of selenium-containing polysaccharides from the fruit of Rosa laevigata.

Rosa laevigata fruit has been known as a functional foodstuff for a long time. Recently, increasing attention has been given to polysaccharides from R. laevigata fruit due to their numerous medicinal and nutritional properties. In this study, a rapid and effective approach for the extraction and separation of polysaccharides from the title fruit was developed using microwave-assisted aqueous two-phase extraction (MA-ATPE) with a PEG/ammonium sulfate system. After analysis of the response surface methodology (RSM) data based on a Box-Behnken design (BBD), a model was proposed and was found to predict an optimum yield value of 258.99 mg g-1 which is in good agreement with the experimental value (258.59 mg g-1). Two selenium (Se)-containing polysaccharides, Se-RLFP-1 and Se-RLFP-2, were isolated from R. laevigata fruit. Their chemical structures were elucidated by acid hydrolysis, weight-average molecular mass and Se-content analysis, along with UV, FT-IR, 1H and 13C NMR spectroscopy. As a result, Se-RLFP-1 was found to be mainly composed of mannose, glucose, galactose and xylose in a molar ratio of 1.4 : 7.9 : 1.0 : 1.5, while Se-RLFP-2 was composed of mannose, rhamnose, glucose, galactose and xylose (12.6 : 1.0 : 38.3 : 5.6 : 19.6). Furthermore, the antioxidant properties of the polysaccharides were investigated on the basis of FRAP, ABTS and DPPH radical scavenging assays. The results showed that the two polysaccharides had a noticeable effect on the radical scavenging of ABTS and DPPH, especially at high concentrations. In addition, the neuroprotective effect of Se-RLFP-1 and Se-RLFP-2 against oxidative stress induced by H2O2 in SH-SY5Y neuroblastoma cells was also investigated. In particular, Se-RLFP-1 exhibited obvious neuroprotective activity at a concentration of 100 μg mL-1.

Microwave-Assisted Aqueous Two-Phase Extraction of Cordycepin and Cordycepic Acid from Cordyceps militaris Mycelium Grown in Submerged Culture

Microwave-Assisted Aqueous Two-Phase Extraction of Cordycepin and Cordycepic Acid from Cordyceps militaris Mycelium Grown in Submerged Culture

Physicochemical characterization and in vitro hypoglycemic activities of polysaccharides from Sargassum pallidum by microwave-assisted aqueous two-phase extraction

Microwave-assisted aqueous two-phase extraction (MAATPE) was applied for simultaneous extraction and separation of polysaccharides from Sargassum pallidum (SPPs). The optimal extraction parameters, physicochemical properties, and hypoglycemic activities in vitro of SPPs were investigated. The results revealed that the optimal extraction conditions were as follows: 21.0% ethanol (w/w) and 22.0% ammonium sulfate (w/w) for ATPS, ratio of material to liquid 1:60 (g/mL), extraction time 15 min, microwave power 830 W, and extraction temperature 95 °C. Under the optimal these conditions, the maximum yields of SPPs were 0.75 ± 0.04% of the top phase (SPP-1) and 6.81 ± 0.33% of the bottom phase (SPP-2). SPP-1 and SPP-2 were homogeneous with molecular weights of 1518.6 and 50.6 kDa, respectively. SPP-1 mainly consisted of fucose, galactose, mannose, and glucuronic acid with a molar ratio of 4.97:9.75:6.44:6.07, whereas SPP-2 was mainly composed of fucose, galactose, glucose, and mannose with a molar ratio of 4.20:2.88:18.05:7.83. SPP-1 and SPP-2 exhibited favorable α-amylase and α-glucosidase inhibitory activities, and could remarkably improve glucose consumption in insulin resistance (IR) model cells. Notably, SPP-1 exhibited stronger α-glucosidase inhibitory activity than SPP-2, and even was comparable with acarbose.

Simultaneous extraction and purification of polysaccharides from Gentiana scabra Bunge by microwave-assisted ethanol-salt aqueous two-phase system

An effective method of concurrent extraction and purification for polysaccharides from Gentiana scabra Bunge stems was developed by microwave-assisted aqueous two-phase extraction (MA-ATPE). Ethanol/sodium dihydrogen phosphate was employed to form aqueous two-phase system (ATPS) by evaluating the extraction properties of five combinations of ethanol/salt systems. The important factors, namely, the composition of ATPS, extraction temperature, solvent-to-material ratio, extraction time and the particle size were investigated by single factor experiment and response surface methodology (RSM) combined with Box–Behnken Design (BBD). The final extraction conditions were concluded as follows: ethanol concentration 21.73% (w/w) and NaH2PO4 concentration 23.27% (w/w) for the ATPS, extraction temperature 95°C, extraction time 5.8min, solvent-to-material ratio 21:1 (mL/g) and particle size 80mesh. Under the optimum conditions, the extraction yield of polysaccharides were 15.97±0.32% for the top phase, 0.58±0.26% for the bottom phase and 16.55±0.13% for total polysaccharides, and the purity of water-soluble polysaccharides was 78.19±0.42%. Compared with smashing tissue extraction (STE), microwave assisted-extraction (MAE), ultrasonic-assisted extraction (UAE) and heat reflux extraction (HRE), microwave-assisted aqueous two-phase extraction not only improved the yield and purity of water-soluble polysaccharides, but also obtained a variety of polysaccharides. In addition, the chemical characterization of polysaccahrides from both the two phases were carried out by Fourier transforms infrared spectrum (FT-IR) and High performance liquid chromatography derivatization with 1-phenyl-3-methy-5-pyrazolone (HPLC-PMP). The extraction mechanism was also discussed by scanning electron microscope (SEM). Therefore, as an efficient and promising technique, microwave-assisted aqueous two-phase extraction may be a viable option for the simultaneous extraction and purification of polysaccharides from plants.

Microwave-assisted aqueous two-phase extraction coupled with high performance liquid chromatography for simultaneous extraction and determination of four flavonoids in Crotalaria sessiliflora L.

A novel method of simultaneous extraction and determination of vitexin, isovitexin, orientin and isoorientin in Crotalaria sessiliflora L. was developed by microwave-assisted aqueous two-phase extraction (MAATPE) coupled with high performance liquid chromatography (HPLC). With an aqueous two-phase system (ATPS) of ethanol/(NH4)2SO4 as extracting agent, the effects of the composition of the ATPS, extraction temperature, extraction time, particle size and solvent-to-material ratio on extraction yields of the flavonoids were investigated by single-factor experiment and response surface methodology (RSM). Optimum conditions were concluded as follows: ethanol concentration 32% and (NH4)2SO4 concentration 22% for the ATPS, extraction temperature 80°C, extraction time 8min, particle size 80 mesh and solvent-to-material ratio 50:1. With the help of MAATPE, HPLC analysis of four flavonoids above has been successfully conducted with UV detection at 340nm with linear ranges (0.20–20.00mg/L), correlation coefficients (R2>0.9996) and limits of detection (0.0309–0.0492mg/L) using acetonitrile-water containing 0.5% phosphoric acid solution (14:86) as the mobile phase. Under the optimum conditions, MAATPE coupled with HPLC was applied to simultaneous determination of vitexin, isovitexin, orientin and isoorientin in Crotalaria sessiliflora L. Their extraction yields and recoveries were in range of 162.7–240.0μg/g and 94.14%–105.5%, respectively. The extraction process was explored by comparative study. The results demonstrated that addition of (NH4)2SO4 in the water-ethanol mixture improved not only demixing effect but also microwave action, and extraction yields of the ATPS were superior to that of mono-phase solvent such as water, ethanol and the combination of both. Thus, MAATPE was a rapid and efficient alternative to the conventional extraction approaches for herb analysis and quality control.

Separation and purification of alkaloids from Sophora flavescens Ait. by focused microwave-assisted aqueous two-phase extraction coupled with reversed micellar extraction

Based on advantages of microwave-assisted extraction (MAE), aqueous two-phase extraction (ATPE) and reversed micellar extraction (RME), a novel method of extraction and purification of alkaloids from Sophora flavescens Ait. was put forward by using focused microwave-assisted aqueous two-phase extraction (FMAATPE) coupled with the reversed micellar extraction (RME). The aqueous two-phase system (ATPS) consisted of ethanol and ammonia sulfate was used as extracting solvent, FMAATPE conditions such as the compositions of ATPS, and the extraction temperature, extraction time and solvent-to-materials ratio were optimized by orthogonal test through HPLC analysis. Then alkaloids extracted by FMAATPE were further purified with reverse micelles of sodium dodecyl benzene sulfonate/isooctane/n-octanol. The relative purity of total alkaloids in the product was improved greatly by RME from 58.30% to 88.75% (w/w), and purification recovery was higher than that of solvent extraction, ion exchange resin and macroporous adsorption resin. Moreover, the reverse micelles used could be readily recycled by adding salt and water, keeping better extraction performance with about 80% (w/w) of recovery. Thus, the proposed method is easy to adjust, operate and enlarge for better application prospect; the purity of alkaloids obtained meets the requirements of the compound drug preparation.

Extraction and characterization of polysaccharides from Semen Cassiae by microwave-assisted aqueous two-phase extraction coupled with spectroscopy and HPLC

A novel and rapid method for simultaneous extraction and separation of the different polysaccharides from Semen Cassiae (SC) was developed by microwave-assisted aqueous two-phase extraction (MAATPE) in a one-step procedure. Using ethanol/ammonium sulfate system as a multiphase solvent, the effects of MAATPE on the extraction of polysaccharides from SC such as the composition of the ATPS, extraction time, temperature and solvent-to-material ratio were investigated by UV–vis analysis. Under the optimum conditions, the yields of polysaccharides were 4.49% for the top phase, 8.80% for the bottom phase and 13.29% for total polysaccharides, respectively. Compared with heating solvent extraction and ultrasonic assisted extraction, MAATPE exhibited the higher extraction yields in shorter time. Fourier-transform infrared spectra showed that two polysaccharides extracted from SC to the top and bottom phases by MAATPE were different from each other in their chemical structures. Through acid hydrolysis and PMP derivatization prior to HPLC, analytical results by indicated that a polysaccharide of the top phases was a relatively homogeneous homepolysaccharide composed of dominant gucose glucose while that of the bottom phase was a water-soluble heteropolysaccharide with multiple components of glucose, xylose, arabinose, galactose, mannose and glucuronic acid. Molar ratios of monosaccharides were 95.13:4.27:0.60 of glucose: arabinose: galactose for the polysaccharide from the top phase and 62.96:14.07:6.67: 6.67:5.19:4.44 of glucose: xylose: arabinose: galactose: mannose: glucuronic acid for that from the bottom phase, respectively. The mechanism for MAATPE process was also discussed in detail. MAATPE with the aid of microwave and the selectivity of the ATPS not only improved yields of the extraction, but also obtained a variety of polysaccharides. Hence, it was proved as a green, efficient and promising alternative to simultaneous extraction of polysaccharides from SC.