Overview of AlphaFold2 and breakthroughs in overcoming its limitations.

Predicting three-dimensional (3D) protein structures has been challenging for decades. The emergence of AlphaFold2 (AF2), a deep learning-based machine learning method developed by DeepMind, became a game changer in the protein folding community. AF2 can predict a protein's three-dimensional structure with high confidence based on its amino acid sequence. Accurate prediction of protein structures can dramatically accelerate our understanding of biological mechanisms and provide a solid foundation for reliable drug design. Although AF2 breaks through the barriers in predicting protein structures, many rooms remain to be further studied. This review provides a brief historical overview of the development of protein structure prediction, covering template-based, template-free, and machine learning-based methods. In addition to reviewing the potential benefits (Pros) and considerations (Cons) of using AF2, this review summarizes the diverse applications, including protein structure predictions, dynamic changes, point mutation, integration of language model and experimental data, protein complex, and protein-peptide interaction. It underscores recent advancements in efficiency, reliability, and broad application of AF2. This comprehensive review offers valuable insights into the applications of AF2 and AF2-inspired AI methods in structural biology and its potential for clinically significant drug target discovery.

Combinatorial preparation and structural characterization of anthocyanins and aglycones from Purple-heart Radish for evaluation of physicochemical stability and pancreatic lipase inhibitory activity.

In this study, an efficient approach to preparation of different anthocyanins from Purple-heart Radish was developed by combining microwave-assisted extraction (MAE), macroporous resin purification (MRP) and ultrasound-assisted acid hydrolysis (UAAH) for evaluation of physicochemical stability and pancreatic lipase (PL) inhibitory activity. By optimization of MAE, MRP and UAAH processes, the anthocyanins reached the yield of 6.081 ± 0.106 mg/g, the purity of 78.54 ± 0.62 % (w/w) and the content of 76.29 ± 1.31 % (w/w), respectively. With high-resolution UHPLC-Q-Orbitrap/MS, 15 anthocyanins were identified as pelargonins with diverse glucosides and confirmed by pelargonidin standard. By glycosylation, pelargonins exhibited higher stability in different pH, temperature, light, metal ions environments than that of pelargonidin. However, PL inhibitory assay, kinetic analysis and molecular docking demonstrated that pelargonidin had higher PL inhibitory activity than pelargonins even though with similar binding sites and a dose-effect relationship. The above results revealed that the effect of glycosylation and deglycosylation on PL inhibitory activity and physicochemical stability.

Method for determination of elemental impurities in metronidazole benzoate using inductively coupled plasma mass spectrometry.

The elemental impurities in pharmaceutical products have aroused widespread concern among respective supervising authorities and official pharmacopoeias since they are harmful and have no therapeutic effects. Metronidazole benzoate is used extensively to treat a variety of infections. However, impurities will inevitably be introduced in the manufacturing process of metronidazole benzoate. Hence, in this study, a sensitive method was developed for trace determination of elemental impurities in metronidazole benzoate active pharmaceutical ingredients by using inductively coupled plasma mass spectrometry in kinetic energy discrimination mode. The method was validated for system suitability, specificity, linearity, sensitivity, accuracy, and precision according to USP chapter <233> Elemental Impurities-Procedure. The method had good linearity with correlation coefficients > 0.99. The limits of detection were in the range of 0.0003-0.1411 µg/g, which was lower than the acceptable limit and indicated the high sensitivity of the method. The method was accurate with the recoveries in the range of 92%-107%. Moreover, the content of seven elemental impurities in the three batches of metronidazole benzoate active pharmaceutical ingredients by this method was originally below their limits and less than 30% of permitted daily exposure, meeting the requirement of International Council for Harmonization Q3D guidelines. Thus, this newly developed and validated method for estimating elemental impurities in metronidazole benzoate active pharmaceutical ingredients was within the permitted limit and suitable for routine use.

Simultaneous separation and determination of several chiral antidepressants and their enantiomers in wastewater by online heart-cutting two-dimensional liquid chromatography.

A novel method for simultaneous separation and detection of the racemates and the enantiomers of common chiral antidepressants in wastewater matrix was developed by online heart-cutting two-dimensional liquid chromatography (2D-LC) coupled to solid-phase extraction (SPE). Screening of chiral stationary phases (CSPs) and chromatographic conditions was investigated for complete enantioseparation to be compatible with RP-HPLC in 1st D-LC. Using methanol-0.1 % (v/v) ammonia solution as mobile phase, a 2D-LC system was configured by reversed mode with a combination of C18 column and the serially CPS columns as 2D-LC stationary phases respectively. The target analytes could achieve satisfactory transformation between 2D-LCs with transfer rate of 90.57-98.58 %. By means of freeze-drying and SPE, three antidepressants in wastewater were greatly preconcentrated under the optimized conditions, improving the method performance. The racemates and the enantiomers of mirtazapine, bupropion and fluoxetine exhibited good linearity in the range of 0.10-30.00 ng/mL (R2≥0.9986), and LODs and LOQs ranged in 0.0183-0.0549 ng/mL and 0.0661-0.1831 ng/mL, respectively. By this way, the method was successfully applied to simultaneous determination of the racemates and the enantiomers of mirtazapine, bupropion and fluoxetine in wastewater samples. Among them, three samples contained bupropion at level of 0.401-0.822 ng/mL, and mirtazapine at level of 0.328 and fluoxetine at level of 0.381 ng/mL were detected respectively in the other two samples. The enantiomers were at level of 0.140-0.189 ng/mL for mirtazapine, 0.182-0.419 ng/mL for bupropion and 0.179-0.204 ng/mL for fluoxetine, respectively. The proposed method providing an efficient approach to monitoring chiral drugs and their enantiomers in wastewater, facilitating to pollution assessment of chiral drugs in the environment and regional survey of illicit abuse in drug control.

Efficient Removal of Hexavalent Chromium (Cr(VI)) from Wastewater Using Amide-Modified Biochar.

The utilization of biochar, derived from agricultural waste, has garnered attention as a valuable material for enhancing soil properties and serving as a substitute adsorbent for the elimination of hazardous heavy metals and organic contaminants from wastewater. In the present investigation, amide-modified biochar was synthesized via low-temperature pyrolysis of rice husk and was harnessed for the removal of Cr(VI) from wastewater. The resultant biochar was treated with 1-[3-(trimethoxysilyl) propyl] urea to incorporate an amide group. The amide-modified biochar was characterized by employing Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and X-ray diffraction (XRD) techniques. During batch experiments, the effect of various parameters, such as adsorbent dosage, metal concentration, time duration, and pH, on Cr(VI) removal was investigated. The optimal conditions for achieving maximum adsorption of Cr(VI) were observed at a pH 2, an adsorbent time of 60 min, an adsorbent dosage of 2 g/L, and a metal concentration of 100 mg/L. The percent removal efficiency of 97% was recorded for the removal of Cr(VI) under optimal conditions using amide-modified biochar. Freundlich, Langmuir, and Temkin isotherm models were utilized to calculate the adsorption data and determine the optimal fitting model. It was found that the adsorption data fitted well with the Langmuir isotherm model. A kinetics study revealed that the Cr(VI) adsorption onto ABC followed a pseudo-second-order kinetic model. The findings of this study indicate that amide-functionalized biochar has the potential to serve as an economically viable substitute adsorbent for the efficient removal of Cr(VI) from wastewater.

Pharmacophore Oriented MP2 Characterization of Charge Distribution for Anti-SARS-CoV-2 Inhibitor Nirmatrelvir.

Quantum mechanical second order Møller-Plesset (MP2) perturbation theory and density functional theory (DFT) Becke, 3-parameter, Lee-Yang-Parr (B3LYP) and Minnesota 2006 local functional (M06L) calculations were performed to optimize structure of nirmatrelvir and compute the Merz-Kollman electrostatic potential (MK ESP), natural population analysis (NPA), Hirshfeld, charge model 5 (CM5), and mulliken partial charges. The mulliken partial charge distribution of nirmatrelvir exhibits a poor correlation with the MK ESP charges in MP2, B3LYP, and M06L calculations respectively. The NPA, Hirshfeld, and CM5 partial charge scheme of nirmatrelvir indicate a reasonable correlation with MK ESP charge assignments in B3LYP and M06L calculations. The above correlations were not improved by the inclusion of implicit solvation model. The MK ESP and CM5 partial charges show a strong correlation between the results of MP2 and two DFT methods. The three optimized structures present a certain degree of differences from the crystal bioactive conformation of nirmatrelvir, suggesting the nirmatrelvir-enzyme complex is formed in the induced-fit model. The Reactivity of warhead electrophilic nitrile is justified by the relatively weaker strength of π bonds in the MP2 calculations. The nirmatrelvir hydrogen bond acceptors consistently show strong delocalization of lone pair electrons in three calculations, whereas hydrogen bond donors are found to have high polarization on the heavy nitrogen atoms in MP2 computations. This work helps to parametrize the force field of nirmatrelvir and improve accuracy of molecular docking and rational inhibitor design.

Kinetics of Catalytic Oxidation of Methylene Blue with La/Cu Co-Doped in Attapulgite.

Methylene blue (MB) is a common pollutant in wastewater from the printing and dyeing industries. In this study, attapulgite (ATP) was modified with La3+/Cu2+, using the method of equivolumetric impregnation. The La3+/Cu2+ -ATP nanocomposites were characterized using X-ray diffraction (XRD) and scanning electron microscopy (SEM). The catalytic properties of the modified ATP and the original ATP were compared. At the same time, the influence of the reaction temperature, concentration of methylene blue and pH on the reaction rate were investigated. The optimal reaction conditions are as follows: MB concentration is 80 mg/L, the dosage of the catalyst is 0.30 g, the dosage of hydrogen peroxide is 2 mL, the pH is 10 and the reaction temperature is 50 °C. Under these conditions, the degradation rate of MB can reach 98%. The recatalysis experiment was carried out reusing the catalyst, and the experimental results showed that the degradation rate could reach 65% after three uses, indicating that the catalyst could be recycled many times and costs could be reduced. Finally, the degradation mechanism of MB was speculated, and the reaction kinetic equation was obtained as follows: -dc/dt = 14,044 exp(-3598.34/T)C(O)0.28.

TsOH-catalyzed acyl migration reaction of the Bz-group: innovative assembly of various building blocks for the synthesis of saccharides.

We developed an efficient method to achieve the regioselective acyl migration of benzoyl ester. In all the cases, the reactions required only the commercially available organic acid catalyst TsOH·H2O. This method enables the benzoyl group to migrate from secondary groups to primary hydroxyl groups, or from equatorial secondary hydroxyl groups to axial hydroxyl groups. The 1,2 or 1,3 acyl migration would potentially occur via five- and six-membered cyclic ortho acid intermediates. A wide range of orthogonally protected monosaccharides, which are useful intermediates for the synthesis of natural oligosaccharides, were synthesized. Finally, to demonstrate the utility of the method, a tetrasaccharide portion from a mycobacterial cell wall polysaccharide was assembled.

QM/MM study of N501 involved intermolecular interaction between SARS-CoV-2 receptor binding domain and antibody of human origin.

Intermolecular interaction between key residue N501 of the epitope on SARS-CoV-2 RBD and screening antibody B38 was studied using the QM/MM and QM approach. The QM/MM optimized geometry shows that angle X-H---Y is 165° for O-H---O between mAb light chain S30 and RBD N501. High level MP2 calculations indicated the interaction between RBD N501 and S30 of B38 Fab light chain provide a relatively strong attractive force of - 3.32 kcal/mol, whereas the hydrogen bond between RBD Q498 and S30 was quantified as 0.10 kcal/mol. The decrease in ESP partial charge on hydrogen atom of hydroxyl group on S30 drops from 0.38 a.u. to 0.31 a.u., exhibiting the sharing of 0.07 a.u. from the lone pair electron oxygen of N501 due to hydrogen bond formation. The NBO occupancy of hydrogen atom also decreases from 25.79 % to 22.93 % in the hydroxyl H-O NBO bond of S30. However, the minor change of NBO hybridization of hydroxyl oxygen of S30 from sp3.00 to sp3.05 implies the rigidity of hydrogen bond tetrahedral geometry in the relative dynamic protein complex. The O-H---O angle is 165° which is close but not exactly linear. The structural requirement for sp3 hybridization of oxygen for hydroxyl group on S30 and dimension of protein likely prevent O-H---O from adopting linear geometry. The hydrogen bond strengths were also calculated using a variety of DFT methods, and the result of - 3.33 kcal/mol from the M06L method is the closest to that of the MP2 calculation. Results of this work may aid in the COVID-19 vaccine and drug screening.

TopClosure® tension­relief system improves clinical outcomes of patients with breast cancer undergoing mastectomy.

The present randomized controlled study aimed to investigate the effects of TopClosure® tension-relief system (TRS) on patients with breast cancer undergoing mastectomy. A total of 402 female patients with breast cancer who came to the Renmin Hospital of Wuhan University between March 2014 and June 2018 were involved in the present study. All patients receiving mastectomy were randomly divided into the TRS group (n=201) and the control group (n=201). Serum levels of high-sensitivity C-reactive protein, TNF-α, IL-6 and procalcitonin were measured using ELISA. Vancouver Scar Scale was recorded at 2 weeks and 1-3 and 6 months following the operation. The 36-Item Health Survey Scales were performed for all patients at 1 month after surgery. The TRS reduced the incidence of flap necrosis, infection and the duration of hospital stay. In addition, the TRS was found to attenuate inflammation and improve scar outcomes as well as the quality of life. It was concluded that the TRS could significantly improve the clinical outcomes.

Screening and identification of lipase inhibitors extracted from Dioscorea nipponica Makino by UV-vis and HPLC coupled to UPLC-Q-TOF-MS/MS.

Dioscoreae nipponica Makino (D. nipponica) as the rhizome of dioscoreaceae rich in steroidal saponins, has been reported to have the hypolipidemic effects etc. However, it is still unclear which exact active components are primary responsible for the beneficial effects. This study was conducted to fish out the lipase inhibitors from D. nipponica, and evaluate the inhibitory activity on porcine pancreatic lipase (PPL) through in vitro kinetic assay using p-nitrophenyl palmitate as substrate. Accordingly, the ethanolic extract was subjected to D101 macroporous resin purification for spectrophotometric screening, high performance liquid chromatography (HPLC) separation and structural characterization by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Through orlistat validation, the PPL inhibitory activity and IC50 value of the extract were respectively 68.34 ± 1.47 % and 107.05 µg/mL under the optimized inhibition conditions. From 6 steroidal saponins identified, the inhibitory components named the protodioscin, protogracillin, dioscin and gracillin were fished out by grouping separation and HPLC analysis. Furthermore, dioscin and gracillin with the parent structure of diogenin were confirmed as the major inhibitors by virtue of stability tests based on transformation of protodioscin and protogracillin. Finally, the inhibitory mechanism of the major inhibitors toward PPL was further clarified by kinetic analysis and molecular docking analysis. The proposed method not only revealed the PPL inhibitory components in D. nipponica, but also provided an effective approach to hierarchical screening of PPL inhibitors from natural plants.

Method for trace determination of N-nitrosamines impurities in metronidazole benzoate using high-performance liquid chromatography coupled with atmospheric-pressure chemical ionization tandem mass spectrometry.

Genotoxic impurity control has been a great concern in the pharmaceutical industry since the recall of the large round of sartans worldwide in 2018. In these sartans, N-nitrosamines were the main contaminants in active pharmaceutical ingredients and formulations. Numerous analytical methods have been developed to detect N-nitrosamines in food, drugs, and environmental samples. In this study, a sensitive method is developed for the trace determination of N-nitrosamine impurities in metronidazole benzoate pharmaceuticals using high-performance liquid chromatography/atmospheric-pressure chemical ionization tandem mass spectrometry in the multiple reaction monitoring mode. The method was validated regarding system suitability, selectivity, linearity, accuracy, precision, sensitivity, solution stability, and robustness. The method showed good linearity with R2 ≥ 0.999 and FMandel  < Ftab(95%) ranging from 0.33 to 8.00 ng/ml. The low limits of detection of N-nitrosamines were in the range of 0.22-0.80 ng/ml (0.0014-0.0050 ppm). The low limits of quantification were in the range of 0.33-1.20 ng/ml (0.0021-0.0075 ppm), which were lower than the acceptable limits in metronidazole benzoate pharmaceuticals and indicated the high sensitivity of the method. The recoveries of N-nitrosamines ranged from 84% to 97%. Thus, this method exhibits good selectivity, sensitivity, and accuracy. Moreover, it is a simple, convenient, and scientific strategy for detecting N-nitrosamine impurities in pharmaceuticals to support the development of the pharmaceutical industry.

Ultrasonic Enhancement of Aqueous Two-Phase Extraction and Acid Hydrolysis of Flavonoids from Malvaviscus arboreus Cav. Flower for Evaluation of Antioxidant Activity.

The ultrasonic-assisted aqueous two-phase extraction (UAATPE) of flavonoid glycosides from Malvaviscus arboreous Cav. flower (MACF) was developed using ethanol/ammonia sulfate systems, followed by the ultrasonic-assisted acid hydrolysis (UAAH) of the top extract with HCl solution. The optimization of UAATPE and UAAH processes was accomplished by single-factor experiments and response surface methodology. As a result, the flavonoid glycosides enriched in the top phase could achieve a maximum yield of 35.9 ± 1.1 mg/g by UAATPE and were completely hydrolyzed by UAAH deglycosylation. The flavonoid glycosides and their hydrolyzates were separated and characterized by high-performance liquid chromatography and ultra-high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry. Ultrasonic enhancement of the extraction and hydrolysis was explored by comparative study. Furthermore, the in vitro activity of the flavonoid glycosides and the aglycones were comprehensively evaluated by antioxidant activity assays, including ferric-reducing antioxidant power and scavenging DPPH, hydroxyl, and superoxide radicals. All of the IC50 values suggest that the antioxidant activity of flavonoid aglycones was stronger than that of their glucosides and even vitamin C, revealing that the deglycosylated flavonoids from MACF were the more powerful antioxidants. This study provided an effective and eco-friendly strategy for the extraction, separation, and purification of flavonoids from MACF, as well as for the development of the potential flavonoid antioxidants.

Enzyme and microwave co-assisted extraction, structural characterization and antioxidant activity of polysaccharides from Purple-heart Radish.

A novel method of simultaneous extraction and separation of diverse polysaccharides from Purple-heart Radish was developed by integrating EAE with MAATPE. The effects of different enzymes, the ATPS composition, extraction temperature, time etc. were investigated by single-factor experiments and RSM. Under the optimum conditions, the extraction yields of PTP, PBP and total polysaccharides were 9.107 ± 0.391%, 32.506 ± 0.046% and 41.613 ± 0.437%, respectively. By means of HPGPC and PMP-HPLC, Mw of PTP and Mw of PBP were 15935 Da and 27962 Da, respectively. PTP and PBP were mainly composed of mannose, glucuronic acid, aminogalactose, glucose, galactose and arabinose. Moreover, both polysaccharides exhibited stronger antioxidant activities for scavenging multiple radicals and anti-lipid peroxidation. Compared to the conventional extraction methods, EAE-MAATPE achieved higher extraction efficiency due to the synergistic effect between EAE and MAATPE leading to rupture and enzymolysis of cell. Thus, EAE-MAATPE provided an efficient alternative to simultaneous extraction of different polysaccharides from natural products.

Ciprofloxacin removal by ultrasound-enhanced carbon nanotubes/permanganate process: In situ generation of free reactive manganese species via electron transfer.

Recently, free reactive manganese species (RMnS) generated via permanganate catalytic oxidation technology has been applied to contaminants abatement and sludge dewatering. This study proposed a novel free RMnS generation method in ultrasound enhanced carbon nanotube (CNTs)/permanganate process (UCP) for organics removal. Taking ciprofloxacin as a target contaminant, the removal efficiency in the UCP process (9.78 s-1) was remarkably higher than that of the permanganate (0.71 s-1) and CNTs/permanganate (2.57 s-1) processes. CNTs could enrich manganese compounds and ciprofloxacin, and act as an electronic platform for the electronic transfer from ciprofloxacin to manganese compounds for free RMnS generation, which was revealed by DFT calculation and spectrum analysis. Meanwhile, ultrasound further regulated the generation of RMnS as it could transform the inactive solid Mn(IV) into free RMnS. In the UCP process, non-free radical modes including RMnS oxidation (49.8%) and electron transfer (23.5%) were the dominant processes for ciprofloxacin removal in the UCP process, and hydroxyl radical oxidation (13.2%), CNTs adsorption (5.5%), and PM oxidation (8.0%) also contributed to ciprofloxacin removal. Interestingly, CNTs could be well reused in the UCP process as more than 88.75% of ciprofloxacin was removed after five times reuse of CNTs. The UCP process provides a novel strategy for rapid contaminants removal in water treatment via continuous generation of free RMnS.

Preparation and characterization of a chiral molecularly imprinted polymer with a novel functional monomer for controlled release of S-sulpiride.

A novel molecularly imprinted polymer (MIP) with chiral recognition affinity to S-sulpiride (S-SUL) enantiomer was prepared by using newly synthesized N-acryloyl-tryptophan (ATrp) as function monomer, S-SUL as the template molecule, and ethyleneglycol dimethacrylate (EGDMA) as the cross linker. Under the optimized synthesis conditions, the MIP was synthesized by bulk polymerization according to the molar ratio of 1:4 of S-SUL to ATrp, and structurally characterized by Fourier transform infrared spectroscopy (FT-IR), scanning electron microscope (SEM) and laser particle analysis. The results illustrated that the MIP offered uniform, loose and porous structure. The adsorption performance of the MIP was evaluated by the isotherm and kinetic models, and the adsorption isotherm conformed to the Freundlich model. The maximum adsorption capacity, selectivity factor and enantioselectivity coefficient to S-SUL were respectively 226.2389 µmol/g, 2.34 and 11.66. Based on the chiral recognition specificity, the drug release experiments demonstrated that the MIP as controlled and sustained release carrier could inhibit the release rate of S-enantiomer compared to the tablet without the MIP, exhibiting the potential of the MIP synthesized in chiral drug delivery.

4,6-Di-O-Benzylidenyl group-directed preparation of 2-deoxy-2-azido-α-d-galactopyranosides promoted by 3-O-TBDPS.

In this study, we designed a method to prepare 2-deoxy-2-azido-α-d-galactopyranosidic bonds using 4,6-di-O-benzylidenyl-3-O-t-butyldiphenylsilyl protected 2-deoxy-2-azido-1-thio-d-galactopyranoside 5 as donors. The donor 5 gives a good to excellent α-selectivity in the glycosylation with secondary alcohols, which was found to be associated with the benzylidenyl on 4,6-di-O and TBDPS on 3-O of the donor 5. Compared with results of the donor 6 and 7, the 3-O-TBDPS could increase the activity of the thioglycoside, and the lone pairs on 4,6-di-O-benzylidenyl group enhanced the gg-cofnormation, which plays a role in improving the stereoselectivity. Finally, this method was demonstrated through the synthesis of a α-galactosamine -containing pentasaccharide 26.

Microwave-assisted extraction followed by salting-out phase separation for hierarchical screening of illegal adulterants in aphrodisiac health products by multi-dimensional fingerprint profiling analysis.

A novel method for hierarchical screening of illegal adulterants in Fur seal ginseng pills (FSGP) products was developed by microwave-assisted extraction (MAE) coupled to salting-out assisted liquid-liquid extraction (SALLE) with multi-dimensional fingerprint profiling analysis. Using a homogeneous system formed by dimethyl carbonate (DMC) and water as the extractant, the MAE conditions were investigated to maximize extraction recoveries, followed by addition of ammonium sulfate to induce DMC phase separation for SALLE enrichment of 16 potentially illegal adulterants such as phosphodiesterase type-5 inhibitors, androgens, α receptor antagonists and yohimbine etc. By means of high-performance liquid chromatography (HPLC) with diode array detection (DAD) and fluorescence detection (FLD), multi-dimensional fingerprints were acquired by multi-wavelength detection to highlight the signals of the potentially illegal adulterants and reduce or remove interferences from the sample matrix. For high accuracy and reliability, a hierarchical screening strategy was designed by multi-dimensional fingerprinting profiling analysis (MDFPA). The method exhibited proper identification and quantification performance, and it was successfully applied to screening of illegal adulterants in 18 batches of the samples through the step-by-step MDFPA. Also, the results were further confirmed by ultra high-performance liquid chromatography-quadrupole-orbitrap mass spectrometry (UHPLC-Q-Orbitrap/MS). The proposed method was proved to be a green, efficient and reliable alternative to monitoring aphrodisiac health products.

Multi-dimensional fingerprint profiling analysis for screening and quantification of illegal adulterated antidiabetics in hypoglycemic health products by aqueous two-phase extraction and multi-wavelength detection.

A novel method for screening and quantification of illegal adulterated antidiabetics in hypoglycemic health products was developed by multi-dimensional fingerprint profiling analysis (MDFPA). By means of aqueous two-phase extraction (ATPE), using aqueous two-phase system (ATPS) of butanol-water as the extractant, 11 common antidiabetics could be effectively extracted to the upper and lower phases, respectively. HPLC separation conditions for the extracts from two phases were investigated by multi-wavelength detection before and after p-nitrobenzoyl chloride (p-NBC) and 2,4-dinitrofluorobenzene (DNBF) derivatizations to establish multi-dimensional fingerprints. For high accuracy and reliability, a hierarchical screening approach to screening illegal adulterated antidiabetics in samples was established by MDFPA and spectral purity examination. Meanwhile, detection limits of identification for illegal adulterants were defined by detection limits of spectra (SLOD). The proposed method exhibited good identification and quantification performances. SLODs, LODs and LOQs of 11 antidiabetics were 1.22-8.37 µg/g, 0.225-4.23 µg/g and 0.755-14.10 µg/g, respectively. They had good linearity ranged from 2.0 µg/g to 300.0 µg/g (R2 ≥ 0.9978). The recoveries and RSDs were 76.83-109.6% and 0.50-6.5%, respectively. The method was successfully applied to screening of 15 batches of samples in different forms. Among them, four samples were detected to contain 5.47 µg/g of metformin, 6.50 µg/g of phenformin, 3.69 µg/g of glibenclamide and 9.11 µg/g of glimepiride, respectively. The results proved that it was an efficient and feasible alternative to screening and detection of illegal adulterated antidiabetics in hypoglycemic health products.

Sequential extraction and enrichment of flavonoids from Euonymus alatus by ultrasonic-assisted polyethylene glycol-based extraction coupled to temperature-induced cloud point extraction.

A green method for simultaneous extraction and enrichment of flavonoids from Euonymus alatus was developed by ultrasonic-assisted extraction (UAE) and temperature-induced cloud point extraction (TICPE) using PEG-base aqueous solution as the extractant. Based on screening different molecular weights of PEGs, PEG-400/water was used as the extractant, and the effects of key factors on extraction yields of flavonoids were investigated by single-factor experiments and response surface methodology (RSM). The optimum conditions of UAE were as follows: PEG-400 concentration of 16% (w/w), particle size of 80 mesh, solvent-to-material ratio of 60:1, extraction temperature of 90 °C and extraction time of 15 min. The results obtained by validation experiments were consistent with the values predicted by RSM. Temperature-induced formation of the aqueous two-phase system (ATPS) and TICPE process were further investigated by controlling temperature and adding (NH4)2SO4. In the presence of (NH4)2SO4, the ATPS formed at 75 â„ƒ and pH 3.5 could effectively improve separation and recovery of flavonoids with enrichment factor of above five times. Gallic acid, catechin, dihydromyricetin and ellagic acid in the extract were identified and confirmed by UPLC-Q-TOF-MS and the corresponding standards. The UAE-TICPE coupled to HPLC was successfully applied for extraction and determination of flavonoids in two batches of Euonymus alatus. The extraction yields of catechin, dihydromyricetin and total flavonoids were 0.377-0.684 mg/g, 1.091-1.353 mg/g and 2.612-3.146 mg/g, respectively. Compared to conventional extraction methods, PEG-based UAE integrated with TICPE in one-step procedure exhibited higher extraction efficiency and better extraction selectivity.