Scopoletin is a coumarin compound with various biological activities including detumescence and analgesic, insecticidal, antibacterial and acaricidal effects. However, interference with scopolin and other components often leads to difficulties in purification of scopoletin with low extraction rates from plant resource. In this paper, heterologous expression of the gene encoding β-glucosidase An-bgl3 derived from Aspergillus niger were carried out. The expression product was purified and characterized with further structure-activity relationship between it and β-glucosidase analyzed. Subsequently, its ability for transforming scopolin from plant extract was studied. The results showed that the specific activity of the purified β-glucosidase An-bgl3 was 15.22 IU/mg, the apparent molecular weight was about 120 kDa. The optimum reaction temperature and pH were 55 ℃ and 4.0, respectively. Moreover, 10 mmol/L metal ions Fe²⁺ and Mn²⁺ increased the enzyme activity by 1.74-fold and 1.20-fold, respectively. A 10 mmol/L solution containing Tween-20, Tween-80 and Triton X-100 all inhibited the enzyme activity by 30%. The enzyme showed affinity towards scopolin and tolerated 10% methanol and 10% ethanol solution, respectively. The enzyme specifically hydrolyzed scopolin into scopoletin from the extract of Erycibe obtusifolia Benth with a 47.8% increase of scopoletin. This demonstrated that the β-glucosidase An-bgl3 from A. niger shows specificity on scopolin with good activities, thus providing an alternative method for increasing the extraction efficiency of scopoletin from plant material.
Aspergillus niger/genetics* ; beta-Glucosidase/chemistry* ; Scopoletin ; Polysorbates ; Coumarins

The effective components of flavonoids in the "Pueraria lobata-Hovenia dulcis" drug pair have low bioavailability in vivo due to their unstable characteristics. This study used microemulsions with amphoteric carrier properties to solve this problem. The study drew pseudo-ternary phase diagrams through titration compatibility experiments of the oil phase with emulsifiers and co-emulsifiers and screened the prescription composition of blank microemulsions. The study used average particle size and PDI as evaluation indicators, and the central composite design-response surface method(CCD-RSM) was used to optimize the prescription; high-dosage drug-loaded microemulsions were obtained, and their physicochemical properties, appearance, and stability were evaluated. The results showed that when ethyl butyrate was used as the oil phase, polysorbate 80(tween 80) as the surfactant, and anhydrous ethanol as the cosurfactant, the maximum microemulsion area was obtained. When the difference in results was small, K_(m )of 1∶4 was chosen to ensure the safety of the prescription. The prescription composition optimized by the CCD-RSM was ethyl butyrate(16.28%), tween 80(9.59%), and anhydrous ethanol(38.34%). When the dosage reached 3% of the system mass, the total flavonoid microemulsion prepared had a clear and transparent appearance, with average particle size, PDI, and potential of(74.25±1.58)nm, 0.277±0.043, and(-0.08±0.07) mV, respectively. The microemulsion was spherical and evenly distributed under transmission electron microscopy. The centrifugal stability and temperature stability were good, and there was no layering or demulsification phenomenon, which significantly improved the in vitro dissolution of total flavonoids.
Polysorbates/chemistry* ; Flavonoids ; Pueraria ; Surface-Active Agents/chemistry* ; Ethanol ; Emulsions ; Particle Size ; Solubility

Based on the fact that chromogenic reaction of blue complex, a reaction product which can be dissolved in organic solvents, can be realized by polyethoxy and ammonium thiocyanate in tween 80, a rapid and accurate way for the determination for tween 80 in pharmaceutical adjuvant was established in this study, providing reliable technical means for quality control of traditional Chinese medicine injections. Based on the study of reaction kinetics, chromogenic reaction temperature and time, as well as extraction of organic solvents and other key conditions were optimized, and Kumu injection was used as the test material for method validation and applicability investigation. It was finally determined that 3 mL ammonium thiocyanate solution was added in the sample solution, and the reaction was carried out in a boiling water bath for 2 h. After cooling to room temperature, 5 mL of dichloromethane was added to extract the chromogenic product. The absorbance value was measured at the wavelength of 623 nm to calculate the tween 80 content in the sample. Under optimized conditions, tween 80 solution showed a good linear relationship with the absorbance in the range from 0.8 mg to 3.0 mg. The linear regression equation was ＝0.258-0.047. The correlation coefficient was 0.999 6. Under the experimental conditions, the average recovery was 99.66%, and the precision RSD was less than 2.0%. The results showed that this method can quickly and accurately determine the content of tween 80 in Kumu injection, and it could be applicable to the quality control of traditional Chinese medicine injections.
Adjuvants, Pharmaceutic ; chemistry ; Medicine, Chinese Traditional ; Polysorbates ; chemistry ; Quality Control ; Solvents ; Temperature

OBJECTIVETo establish the method for determining polysorbate 80 in Reduning injection by HPLC-ELSD, and to control the mass of polysorbate 80 in Reduning injection.

METHODIt was performed by HGPC-ELSD with TOSHTSK-GEL G4000PWxl (7.8 mm x 300 mm, 10 μm). Water was used as mobile phase, the flow rate was 0.7 mL x min(-1), and the temperature was set at 30°C. The evaporated light scattering detector was adopted. The drift tube temperature was 55°C, and nitrogen was used as carrier gas, with the flow rate of 2.0 L x min(-1) and gain of 1.0.

RESULTThe calibration curve showed good linearity of polysorbate 80 in the test range from 1.01 to 15.20 g x L(-1) (r2 = 0.999 3). The recovery rate was 98.10% with RSD of 2.0%.

CONCLUSIONThe method is simple, rapid, accurate and reliable and suitable for the determination of polysorbate 80 in Reduning injection.

Calibration ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; chemistry ; Injections ; Polysorbates ; analysis ; Reproducibility of Results ; Time Factors

OBJECTIVETo research the effect of polysorbate 80 (Tween 80) on Yuxingcao injection and volatile oils from Houttuynia cordata.

METHOD1H-NMR spectra of aldehydic and new matter in Yuxingcao injection, volatile oils of H. cordata, and solutions of Tween 80 and volatile oil of H. cordata are determined and compared from various angles of growing origin, storage temperature, and storage time.

RESULTThree aldehydic singlets in 1H-NMR spectra of every volatile oil from 4 aerial part of H. cordata were observed. These aldehydic peaks were basically disappeared and a new peak at delta 8.30 was found in 1H-NMR spectra of the volatile oil solutions in tween 80. Any obvious aldehydic peak in 1H-NMR spectra did not be observed in Yuxincao injection. A weak peak at 8 8.30 was found in 1H-NMR spectra in Yuxincao injection, and the peak high of delta 8.30 was remarked gone up when the injection was stored in 40 degrees C for 1 to 3 months.

CONCLUSIONTween 80 might cause the obvious reduce of aldehydic compounds contents and the production of a novel singal at delta 8.30 in 1H-NMR spectra when it was mixed with the volatile oil from the aerial part of H. cordata. The novel signal at delta 8.30 in 1H-NMR spectra existed in Yuxincao injection and was very small, but was increased remarkably when the Yuxincao injection was stored at 40 degrees C for 1 month at least.

Chemistry, Pharmaceutical ; instrumentation ; Drugs, Chinese Herbal ; chemistry ; Houttuynia ; chemistry ; Oils, Volatile ; chemistry ; Plant Oils ; chemistry ; Polysorbates ; Temperature

This study is to prepare the W/O microemulsion containing NaCl and fluorouracil (5-Fu) as a model drug to investigate the transdermal characteristics and skin irritation of the microemulsion in vitro. Isopropylmyristate (IPM) acting as oil phase, Aerosol-OT (AOT) as surfactant, Tween 85 as cosurfactant, NaCl solution was added dropwise to the oil phase to prepare W/O microemulsion at room temperature using magnetic stirring, and then 5-Fu powder was added. According to the area of microemulsion based on the pseudo-tertiary phase diagrams, the optimum formulation was screened initially. And the permeation flux of fluorouracil across excised mice skin was determined in vitro using Franz diffusion cells to study the influence of the amount of water and the drug loading capacity and optimize the formulation further. Refer to 5-Fu cream, the irritation of microemulsion on the rat skin was studied. The optimum formulation was composed of 0.7% (w/v) 5-Fu, 50% NaCl solution (0.05 mol x L(-1)), 20% mix-surfactant (AOT/Tween 85, K(m) = 2) and 29.3% oil (IPM). The cumulative amount of fluorouracil permeated in 12 h was (2 013.4 +/- 41.6) microg x cm(-2), 20.23 folds and 10.38 folds more than 0.7% fluorouracil aqueous solution and 2.5% (w/w) fluorouracil cream, respectively. Microemulsion exhibited some irritation, but could be reversed after drug withdrawal. The addition of NaCl significantly increased the content of water and the drug loading in microemulsion systems. The NaCl/AOT-Tween 85/IPM microemulsion system promoted the permeation of fluorouracil greatly, which may be a promising vehicle for the transdermal delivery of fluorouracil and other hydrophilic drug.
Administration, Cutaneous ; Animals ; Antimetabolites, Antineoplastic ; administration & dosage ; adverse effects ; pharmacokinetics ; Dioctyl Sulfosuccinic Acid ; chemistry ; Drug Carriers ; Drug Delivery Systems ; Emulsions ; Exanthema ; chemically induced ; Fluorouracil ; administration & dosage ; adverse effects ; pharmacokinetics ; In Vitro Techniques ; Male ; Mice ; Myristates ; chemistry ; Oils ; chemistry ; Polysorbates ; chemistry ; Rats ; Rats, Sprague-Dawley ; Skin Absorption ; Sodium Chloride ; chemistry ; Surface-Active Agents ; chemistry ; Water

OBJECTIVETo observe the anaphylaxis and hemocytolysis of 6 kinds of tween-80 injection from different source.

METHODThe Hartley albinism guinea pig was used to carry on the active systematic anaphylaxis (ASA) and the passive cutaneous anaphylaxis (PCA). The in vitro hemolytic experiment and the hemocytolysis was observed by means of spectrophotometry on the domestic rabbit.

RESULTThe result of ASA and PLA of 6 kinds of tween-80 injection from different source assumed the negative. In observation time, the temolysis rate of 3 kinds of tween-80 injection are more than 5%, while the others are also more than 5% only in the highly concentrated test tube.

CONCLUSIONSix kinds of tween-80 injection from different source have not caused the immune-mediated anaphylaxis, but it may have hematolysis tendency on intravenous injection. The hemocytolysis of tween-80 may not be entirely caused by the impurities. It is worthy of further study that the physical and chemical properties of the product itself and the undeserved concentration is doubtful whether there is also some internal relations with the generation of hemolytic.

Anaphylaxis ; chemically induced ; Animals ; Chemistry, Pharmaceutical ; Female ; Guinea Pigs ; Hemolysis ; drug effects ; Injections ; Male ; Passive Cutaneous Anaphylaxis ; drug effects ; Polysorbates ; administration & dosage ; adverse effects ; chemistry ; Rabbits

This study was aimed at the transport across blood-brain barrier (BBB) of polysorbate-80 modified neurotoxin loaded polybutylcyanoacrylate nanoparticle (P-80-NT-NP) and its cytotoxicity. An in vitro model of BBB using rat brain microvascular endothelial cells (rBMECs) was established. The cytotoxicity of P-80-NT-NP was measured by the MTT assays, where neurotoxin (NT), nanoparticle (NP), neurotoxin nanoparticle (NT-NP) as control, and the permeability of P-80-NT-NP was determined by using of Millicell insert coculture with rBMECs and fluorescence spectrophotometry. MTT results showed that NT, NP, NT-NP and P-80-NT-NP were avirulent to rBMECs when the concentration of NT was lower than 200 ng x mL(-1). But the cytotoxicity of NP, NT-NP and P-80-NT-NP would be augmented accordingly as concentration increased (P < 0.01), causing obvious reductions of cell survival rate, with no significant difference between them (P > 0.05). When the concentration of NT was 150 ng x mL(-1), the permeability on rBMECs of P-80-NT-NP and NT-NP were both significantly higher than that of NT (P < 0.01), and the permeability of P-80-NT-NP was greater than that of NT-NP (P < 0.05). In conclusion, polysorbate-80 modified neurotoxin nanoparticles can transport across the BBB, while concentration of NT is greater than 200 ng x mL(-1), P-80-NT-NP has a little cytotoxicity against rBMECs.
Animals ; Biological Transport ; Blood-Brain Barrier ; Brain ; blood supply ; Capillary Permeability ; Cell Survival ; drug effects ; Cells, Cultured ; Drug Carriers ; Electric Impedance ; Enbucrilate ; chemistry ; toxicity ; Endothelial Cells ; cytology ; metabolism ; Female ; Male ; Nanoparticles ; Neurotoxins ; administration & dosage ; pharmacokinetics ; Particle Size ; Polysorbates ; chemistry ; toxicity ; Rats ; Rats, Sprague-Dawley

This study is to prepare the microemulsion-based gel based on the W/O microemulsion and fluorouracil (5-Fu) as a model drug to study the transdermal characterization and observe its skin irritation of the microemulsion-based gel in vitro. IPM acted as oil phase, AOT as surfactant, Tween 85 as cosurfactant, water was added dropwise to the oil phase to prepare W/O microemulsion at room temperature using magnetic stirring, then 5-Fu powder was added. The gelatin was used as substrate to prepare 5-Fu microemulsion-based gel. The permeation flux of 5-Fu from 5-Fu microemulsion-based gel across excised mice skin was determined in vitro using Franz diffusion cell to study the influence of the amount of gelatin and the drug loading capacity. Refer to 5-Fu cream, the irritation of microemulsion and microemulsion-based gel on the rat skin was studied. Based on the water/AOT/Tween 85/IPM microemulsion, only the gelatin can form the microemulsion-based gel. At 25 degrees C, 32 degrees C and 40 degrees C, the amount of gelatin required for the formation of microemulsion-based gel were 7%, 14% and more than 17%, respectively. The 12 h transdermal cumulated permeation amount of 5-Fu from microemulsion-based gel containing 14% gelatin and 0.5% drug loading were (876.5 +/- 29.1) microg x cm(-2), 12.3 folds and 4.5 folds more than 0.5% 5-Fu aqueous solution and 2.5% (w/w) 5-Fu cream, respectively. Microemulsion-based gel exhibited some irritation, but could be subsided after drug withdrawal. Microemulsion-based gel may be a promising vehicle for transdermal delivery of 5-Fu and other hydrophilic drug.
Administration, Cutaneous ; Animals ; Antimetabolites, Antineoplastic ; administration & dosage ; adverse effects ; pharmacokinetics ; Dioctyl Sulfosuccinic Acid ; Drug Carriers ; Drug Delivery Systems ; Emulsions ; Exanthema ; chemically induced ; Fluorouracil ; administration & dosage ; adverse effects ; pharmacokinetics ; Gelatin ; chemistry ; Male ; Mice ; Myristates ; chemistry ; Polysorbates ; chemistry ; Skin ; pathology ; Skin Absorption ; Succinates ; chemistry ; Surface-Active Agents ; Viscosity

To establish kinetic assay method for the analysis of hemolysis and to investigate dynamic hemolytic process of polysorbate 80. The UV-VIS spectrum of heme changes when hemoglobin is released continuously during the hemolytic process. Therefore, dynamic hemolytic curve was determined as a new way to characterize the kinetic process of interaction between polysorbate 80 and red blood cells. The effect of polysorbate 80 on blood cells could be perfectly investigated by the hemolytic dynamics. Dynamic hemolytic parameters of polysorbate 80 were calculated according to the hemolytic curves. The constants of hemolytic rate and maximum hemolytic rate of polysorbate 80 had fine linear relationships at the range of 1-20 mg x mL(-1) and 5-20 mg x mL(-1), respectively. In comparison with the present official method such as macroscopic observation and static spectrophotometric methods, kinetic spectrophotometry has the advantages of real time, on-line determination, sensitive, objective, good reproducibility and 2-dimensional information acquired. Therefore, as a biological technique, kinetic spectrophotometry could be applied to evaluate the quality of polysorbate 80 and to screen other solubilizing excipients.
Animals ; Drug Stability ; Erythrocytes ; chemistry ; Excipients ; chemistry ; Hemoglobins ; analysis ; Hemolysis ; Kinetics ; Polysorbates ; chemistry ; Rabbits ; Reproducibility of Results ; Spectrophotometry, Ultraviolet ; methods