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OBJECTIVE：To establish and optimize a extraction method of Fructus Gleditsiae Abnormallis ，and to analyze and identify chemical components of the extract simultaneously. METHODS ：Fructus Gleditsiae Abnormallis was extracted with CO 2 supercritical fluid extraction （SFE）method. Based on single factor tests ，using extraction yield as index ，extraction temperature ， extraction pressure and extraction time as investigation factors ，SFE technology was optimized with orthogonal test ，and validation test was performed. Chemical components in the extract were identified by GC-MS. Relative percentage of each component was calculated with area normalization method. RESULTS ：The optimal SFE extraction technology of Fructus Gleditsiae Abnormallis was extraction temperature of 60 ℃，extraction pressure of 300 MPa and pression time of 15 min. Average extraction of 3 times of validation tests was 1.73％（RSD＝1.78％，n＝3）. The 48 components in the extracts of Fructus Gleditsiae Abnormallis were identified，which accounted for 98.31％ of the total amount of the extracts. The extracts of Fructus Gleditisae Abnormalis mainly included organic acids ，accounting for 36.99％，followed by alkaloids ，accounting for 12.59％ in total. Main components were palmitic acid （16.62％），oleic acid （14.12％），N-aminotetrahydropyrrole（9.79％），2，6-dimethyloctane-1，7-dien-3-ol（5.95％）， tetrahydropyran（3.83％），vanillin（3.39％），etc. CONCLUSIONS ：SFE method of Fructus Gleditisae Abnormalis is established successfully，and the extract is mainly organic acids.
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OBJECTIVE： To optimize the ethanol extraction technology of total alkaloids from Shuanghu capsules. METHODS： Using dendrobine as control， the contents of total alkaloids from Dendrobium nobile and Dendrobium officinale in Shuanghu capsules were determined by acidic dyes colorimetry. Using comprehensive scores calculated by the yield of the extract and the contents of total alkaloids as evaluation indexes， the effects of soaking time， ethanol volume fraction， extraction time， solid-liquid ratio and extraction times were investigated with single factor tests. L9（34） orthogonal test was used to optimize ethanol volume fraction， extraction time， solid-liquid ratio and extraction times according to the results of single factor test. The optimized technology was validated. RESULTS： The linear range of dendrobine were 4.16-14.56 μg/mL （r＝0.999 2）. RSDs of repeatability and precision tests were all lower than 5％. Average recovery tests were 93.01％ （RSD＝1.97％， n＝6）. The optimal ethanol extraction technology included soaking for 12 h， ethanol volume fraction of 70％， solid-liquid ratio of 1 ∶ 12 （g/mL）， extracting for 28 min， extracting 3 times. Results of validation test showed that the average yield of extract was 12.80％ （RSD＝4.39％， n＝3）， and the content of alkaloids was 0.359 0 mg/g（RSD＝0.66％， n＝3）. CONCLUSIONS： Established acidic dyes colorimetry is simple， precise and accurate， which can be used for the content determination of total alkaloids. The optimized ethanol extraction technology is stable and feasible， and can be used for the extraction of total alkaloids from Shuanghu capsules.
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OBJECTIVE： To optimize the expression induction condition of two recombinant methioninases， and to investigate their inhibitory effects on the proliferation of human lung adenocarcinoma cells GLC. METHODS： Recombinant methioninases expression plasmid PGEX-4T1-4A1-MGL and PGEX-4T1-3B8-MGL were transfected into competent Escherichia coli Dh5α， and induced by isopropyl-β-D-thiogalactoside. Using the expression level of target protein as index， the initial OD600 nm value before induction， culture temperature and induction time were optimized by single factor test. The recombinant methioninase 4A1-MGL and 3B8-MGL were purified by affinity chromatography. The concentration of recombinant methioninase was detected by Coomassie blue method. The purity of the product was detected by sodium lauryl benzene sulfonate-polyacrylamide gel electrophoresis； its activity was detected by spectrophotometry. The proliferation of cells was detected by MTT assay after treated with low-dose， medium-dose and high-dose of recombinant methioninases （4A1-MGL or 3B8-MGL was 0.1， 0.2， 0.4 U/mL） for 24， 48， 74 h. Inhibitory rate of cells were calculated. RESULTS： The optimal induction condition of two recombinant methioninases included that initial OD600 nm of 0.9， culture temperature of 37 ℃， induction time of 5 h. The results of validation test showed that protein expression level of 4A1-MGL was 1.52±0.04， that of 3B8-MGL was 1.28±0.03 （RSD＜3％，n＝3）. After purification， the concentration， purity and activity of 4A1-MGL were （0.70±0.02）mg/mL， （96.42±3.15）％ and （0.45±0.02） U/mg； and those of 3B8-MGL were （0.56±0.02）mg/mL， （97.43±2.96）％ and （0.91±0.03）U/mg. After treated with low-dose and medium-dose of 4A1-MGL and 3B8-MGL for 48 and 72 h， treated with high-dose of 4A1-MGL and 3B8-MGL for 24， 48 and 72 h， inhibitory rate of GLC cell was increased significantly， and high-dose group for 72 h was significantly higher than low-dose and medium-dose groups at same time point （P＜0.05）. CONCLUSIONS： The induction conditions of recombinant methioninase expression are successfully optimized in this study. The obtained 4A1-MGL and 3B8-MGL could inhibit the proliferation of GLC cells in a dose-dependent manner.
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The single-factor test was used to optimize the high-pressure homogenization method to prepare the phenolic extract nanosuspensions(DBNs). The physicochemical properties of the obtained nanosuspensions were characterized and the cumulative release in vitro was evaluated. The results showed that the drug concentration was 0.5 g·L~(-1), the mass concentrations of PVPK30 and SDS were 0.5 and 0.25 g·L~(-1), respectively, the probe ultrasonic time was 5 min, the homogenization pressure was 900 bar, and the number of homogenization was 2 times. The prepared DBNs had an average particle size of(168.80±0.36) nm, polydispersity index(PDI) of 0.09±0.04, stability index(SI) of 0.85, and DBNs were stable for storage within 30 days. Scanning electron microscopy showed that the particle size of the dragon's blood extract was reduced and the uniformity was improved in the obtained nanosuspensions. X-ray diffraction pattern and differential scanning calorimetry showed that the phenolic extract of dragon's blood was still in an amorphous state after being prepared into nanosuspensions. The results of saturated solubility measurement showed that the solubility of DBNs lyophilized powder reached 6.25 g·L~(-1), while the solubility of DB raw powder was only 28.67 mg·L~(-1). The in vitro dissolution experiments showed that DBNs lyophilized powder accumulated in gastrointestinal fluid for 8 h. The release amount was 90%,the cumulative release of the raw powder in the gastrointestinal fluid for 24 h was less than 1%, and the solubility and dissolution rate of the DBNs lyophilized powder were significantly higher than the DB raw powder. The method is simple in process and convenient in operation, and can successfully prepare uniform and stable nanosuspensions to improve its solubility, and provides a research basis for solving the application limitation of dragon's blood extract.
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Calorimetry, Differential Scanning , Nanoparticles , Particle Size , Plant Extracts , Chemistry , Solubility , Suspensions , X-Ray DiffractionABSTRACT
AIM To prepare Guizhi Fuling transdermal patch.METHODS With kinds of pressure-sensitive adhesive and penetration enhancer,kind and consumption of solvent,drug loading as influencing factors,appearance,formability and viscidity of patch,extract dispersion as evaluation indices,single factor test was applied to optimizing the preparation.And subsequent in vitro transdermal absorption test was performed to investigate the steady-state permeation rates of paeoniflorin,cinnamic acid and paeonol onto rat skins.RESULTS The optimal conditions were determined to be Duro-Tak 87-2677 polyacrylate pressure-sensitive adhesive (matrix),1 ∶ 0.5 for ratio of extract to propanediol (solvent),3％ azone as a penetration enhancer,and 20％ for drug loading,the obtained transdermal patch demonstrated both ideal initial adhesion force and holding adhesion force.These three constituents' average transdermal rates were 34.32,1.684,72.90 μg/(cm2 · h) with the average release rates of 26.81,1.523,111.8 μg/(cm2 · h),respectively,whose in vitro transdermal permeation curves conformed to Higuchi equation.CONCLUSION Guizhi Fuling transdermal patch processed with simple and stable preparation technique exhibits good in vitro transdermal permeation performance.
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Objective:To optimize the forming technology of Zuojin concentrated pills. Methods: Single factor test was used to optimize the types of excipients and wetting agents with the shaping result as the index. Orthogonal test was used to optimize the forming technology taking the dissolution time,shaping rate and appearance quality as the evaluation indices,and the proportion of excipients, the rate of drugs to excipients and wetting agent amount as the investigation factors. Results: The best forming technology of Zuojin concentrated pills was as follows:MCC and PVP-K30 were used as the excipients with the ratio of 3:1,the ratio of drugs to excipients was 1:1,5% water was used as the wetting agent to obtain the damp mass,and then Zuojin concentrated pills were prepared in a pill machine. Conclusion:The optimized forming technology is stable with good reproducibility,and the pills are round and smooth with u-niform color,whose quality meets the requirements described in Chinese Pharmacopoeia(2015 edition,partⅣ,page 0108 for concen-trated pills). The study provides reference for the further study.
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OBJECTIVE: To optimize reflux extraction technology of total flavonoids from the roots and stems of Angelica sinensis. METHODS: The reflux extraction technology of total flavonoids from the roots and stems of A. sinensis was optimized by Box-Behnken design-response methodology based on single factor test using volume fraction of extraction solvent ethanol, solide-liquid ration, extraction time, extraction times as investigation factors, the content of total flavonoids in extract as evaluation index. RESULTS: The optimal extraction technology of total flavonoids from the roots and stems of A. sinensis was that volume fractions of ethanol were 70％ and 50％; solid-liquid ratios were 1: 40 and 1: 30; extraction time were 1. 3 h and 1. 7 h; The number of extraction times is two times. In verification test, the contents of total flavonoids were 7. 253 6, 25. 144 1 mg/g (RSD= 1. 57％, 1. 49％, n = 3); relative errors of those to predicted value (6. 942 8, 25. 703 5 mg/g) were 4. 28％, 2. 24％. CONCLUSIONS: Optimized extraction technology for total flavonoids from the roots and stems of A. sinensis is simple, reproducible and predictable.
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Objective: To optimize the matrix prescription of Compound Qingfengteng Cataplasm (CQC). Methods: Taking the comprehensive senses as evaluation index, the types and dosage of the matrix were investigated by single factor. Further combined with the initial viscosity and holding viscosity index to optimize the matrix prescription of CQC by orthogonal test, and in vitro transdermal absorption experiments were carried out by Franz diffusion pool method to screen penetration enhancers. Results: The optimal formulation of CQC was as follows: 2.5 g of NP-700, 11 g of glycerin, 0.08 g of glycine aluminum, 0.08 g of tartaric acid, 0.8 g of PVP K90, 0.5 g of PVPP, 20 g of water, and 4.2 g of dry extract. 3% nitrile as a penetration agent could enhance penetration action. Conclusion: The preferred matrix prescription is practicable; CQC adhesion is moderate, no residue, paste traits, skin followability are good.
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AIM To prepare diosgenin nanosuspensions.METHODS The nanosuspensions prepared by media milling method were solidificated by freeze drying method.With particle size and polydispersity index (PDI) as evaluation indices,stabilizer kind,ratio of diosgenin to stabilizer,ratio of preliminary nanosuspension volume to grinding bead amount,milling time,lyoprotectant kind and its amount as influencing factors,single factor test was applied to screening preparation and solidification processes.The morphology of nanosuspensions was observed,then the particle sizes and polydispersity indices of nanosuspensions and freeze-dried powder were determined.RESULTS The optimal conditions were determined to be 6:1 for ratio of diosgenin to Pluronic F127 (stabilizer Ⅰ),50:1 for ratio of diogenin to sodium dodecyl sulfate (SDS,stabilizer Ⅱ),1:4 for ratio of preliminary nanosuspension volume to grinding bead amount,120 min for milling time,and 8％ PEG-6000 and 2％ mannitol as lyoprotectants.The average particle size and polydispersity index of rod-like or flaky nanosuspensions were (348.1 ±14.2) nm and 0.244 ± 0.059,respectively,which were lower than those of freeze-dried powder.At room temperature,the particle sizes of nanosuspensions and freeze-dried powder remained stable within 35 d and 3 months,respectively.CONCLUSION The physical stability of diosgenin freeze-dried powder is superior to that of its nanosuspensions,which can be used after being reconstituted.
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AIM To prepare diosgenin nanosuspensions.METHODS The nanosuspensions prepared by media milling method were solidificated by freeze drying method.With particle size and polydispersity index (PDI) as evaluation indices,stabilizer kind,ratio of diosgenin to stabilizer,ratio of preliminary nanosuspension volume to grinding bead amount,milling time,lyoprotectant kind and its amount as influencing factors,single factor test was applied to screening preparation and solidification processes.The morphology of nanosuspensions was observed,then the particle sizes and polydispersity indices of nanosuspensions and freeze-dried powder were determined.RESULTS The optimal conditions were determined to be 6:1 for ratio of diosgenin to Pluronic F127 (stabilizer Ⅰ),50:1 for ratio of diogenin to sodium dodecyl sulfate (SDS,stabilizer Ⅱ),1:4 for ratio of preliminary nanosuspension volume to grinding bead amount,120 min for milling time,and 8％ PEG-6000 and 2％ mannitol as lyoprotectants.The average particle size and polydispersity index of rod-like or flaky nanosuspensions were (348.1 ±14.2) nm and 0.244 ± 0.059,respectively,which were lower than those of freeze-dried powder.At room temperature,the particle sizes of nanosuspensions and freeze-dried powder remained stable within 35 d and 3 months,respectively.CONCLUSION The physical stability of diosgenin freeze-dried powder is superior to that of its nanosuspensions,which can be used after being reconstituted.
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OBJECTIVE:To prepare Frovatriptan succinate film-coated tablet,and investigate its in vitro dissolution behavior. METHODS:Using lactose monohydrate,microcrystalline cellulose,dioxide,silica,sodium carboxymethyl starch and magnesium stearate as accessories,Frovatriptan succinate tablet was prepared. Using opadry premix spray-coating liquid,Frovatriptan succinate film-coated tablet was prepared. Single factor test was used,using moisture,angle of repose,rigidity,friability,disintegration time and dissolution rate as indexes,to screen the formulation;using dissolution degree as index,coating material dosage was screened. The dissolution curves in vitro of self-made tablets and imported tablets in water,0.1 mol/L HCL,pH of 5.5,6.8 phos-phate buffer solutions were compared. RESULTS:The optimal formulation of Frovatriptan succinate uncoated tablet was as follow as frovatriptan succinate 3.91 mg,lactose monohydrate 99.18 mg,microcrystalline cellulose 33.06 mg,magnesium stearate 1.40 mg,sodium carboxymethyl starch 1.05 mg,silica 1.40 mg;optimal coating weighed quality was 2.0%-4.0%. In the 4 mediums, the dissolution behavior of self-made tablets and imported tablets were similar. CONCLUSIONS:Frovatriptan succinate film-coated tablet is prepared successfully,and its in vitro dissolution behavior is similar to the imported preparations.
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OBJECTIVE:To prepare the betulinic acid nanoparticles,and characterize them. METHODS:Using ethanol as sol-vent and water as anti-solvent,anti-solvent recrystallization method was used to prepare betulinic acid nanoparticles. Using particle size as indicator,single factor test and orthogonal test were adopted to optimize the mass concentration of betulinic acid solution, anti-solvent-solvent volume ratio,anti-solvent drip rate,reaction temperature and stirring speed in formulation technology of betulin-ic acid nanoparticles,and verification test was conducted. The betulinic acid nanoparticles were characterized by scanning electron microscopy,laser particle size analyzer,Fourier infrared spectrometer and mass spectrum analyzer. RESULTS:The optimal technol-ogy was as follow as betulinic acid solution mass concentration of 3 mg/mL,anti-solvent-solvent volume ratio of 1:1,anti-solvent drip rate of 8 mL/min,reaction temperature of 20 ℃ and stirring speed of 900 r/min. The average size of prepared betulinic acid nanosuspension was(156.0±8.6)nm(n=3)and the particle size was(235.0±12.2)nm(n=3)after freeze-drying,with nearly spherical appearance,uniform size and regular form. Compared with raw material of betulinic acid,the chemical structure of pre-pared betulinic acid nanoparticles did not change,and there were no significant changes in molecular weight and mass ratio. CON-CLUSIONS:Betulinic acid nanoparticles are successfully prepared.
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OBJECTIVE:To optimize the flash extraction technology for Guizhi fuling capsule. METHODS:Using blade speed,liquid-solid ratio,extraction time as investigation factors,using the comprehensive evaluation values consisting of transfer rates of gallic acid,paeoniflorin,benzoic acid,cinnamic acid,benzoyl paeoniflorin,amygdalin as investigation index,single fac-tor test was combined with Box-Behnken design-response surface method to optimize the flash extraction technology for Guizhi ful-ing capsule. Verification test was conducted and compared with conventional decoction extraction method(extracting twice,totally 240 min). RESULTS:The optimal flash extraction technology for Guizhi fuling capsule was using water as extraction solvent with blade speed of 5600 r/min and liquid-solid ratio of 10.5:1,extracting for 60 s. The average comprehensive evaluation value of veri-fication test was 85.40%(n=3),with relative error of 0.15% to the predicted value(85.55%),higher than decoction extraction method(72.65%). CONCLUSIONS:Optimized flash extraction technology for Guizhi fuling capsule is efficient and rapid.
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Objective: To explore the optimal separation of polypeptides from Periplaneta americana by selecting appropriate macroporous resins and optimizing purification technology. Methods: Taking the index component test and inhibition experiment of tumor cells in vitro method as indexes, macroporous resins type was chosen and single factor test was adopted to evaluate some factors affecting separating technology. The elution position was analyzed by Automatic amino acid analyzer. Results: Macroporous resin HP20 had the best separating efficiency and the best purification conditions were as follows: the content in P. americana liquid 0.3 g/mL equivalent to raw material, the volume of drug 2 BV, the adsorption rate 2 BV/h, the volume of 70% ethanol as eluant 2 BV with elution rate of 2 BV/h. The recovery rate of polypeptides of elution position could reach 88.53% and the bound amino acids (polypeptides) was 55.64%. Conclusion: This technology is reasonable and feasible and has good separation effect, which could be used for enrichment and purification of antineoplastic compounds from P. americana.
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OBJECTIVE:To optimize ultrasonic extraction and purification technology of solanesol from tobacco leaf. METH-ODS:Using extraction rate and transport rate of solanesol as indexes,single factor test was used to investigate liquid-solid ratio, ultrasonic extraction temperature and time,ultrasonic power and extraction times,and the amount of soap alkali lye(volume ratio of soap alkali lye to extraction liquid),acidizing fluids (volume ratio of acidizing fluids to soap alkali lye extract),extraction times of purification technology. Optimized technology was validated,and the purity of solanesol was calculated;the amount of ex-tracted solanesol was compared between this method and traditional extraction method (spending 30 h),solvent continuous cyclic extraction (spending 5-6 h). RESULTS:Optimized extraction technology was as follows as volume ratio of soap alkali lye to ex-traction liquid 1∶14,ultrasonic extraction temperature 70 ℃,ultrasonic extraction time 60 min,ultrasonic power 120 W,extract-ing for 3 times;optimized purification technology was as follows as volume ratio of soap alkali lye to extraction liquid 2∶35,vol-ume ratio of acidizing fluids to soap alkali lye extract 2∶14,extracting for 4 times. In validation test,extraction rate,transport rate and purity were 92.45%(RSD=0.46%,n=3),79.88%(RSD=0.30%,n=3)and 55.86%(RSD=0.40%,n=3). The amount of solanesol extracted with 3 methods were 52.22,45.22 and 26.10 mg/g. CONCLUSIONS:The optimized technology is simple and stable,costs less time and saves source with high extraction amount and purity,which is suitable for production,extraction and purification of solanesol from tobacco leaf.
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Objective: To synthesize asialoglycoprotein receptor (ASGPR) ligand cholesterol-vinyl sebacate-lactitol (CH-VS-LA) by using enzymatic reaction in organic phase and to optimize its synthesis process. Methods: MS and 13C-NMR were used to identify the structure of product, enzymatic synthesis conditions were optimized via single-factor test and orthogonal experimental design. Results: The enzymatic optimum conditions were as following: the molar ratio of cholesterol-vinyl sebacate (CH-VS) and lactitol (LA) was 4:1, the amount of lipase Novozym 435 was 25 mg, reactional time was 32 h, and productive rate was 92%. Conclusion: The method is highly effecient, the reaction conditions are reasonable, and the process produces no by-product.
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OBJECTIVE:To optimize the extraction technology of polysaccharides from Gossypium herbaceum L. by pectinase hydrolysis. METHODS:Polysaccharides were extracted from G. herbaceum L. by pectinase hydrolysis combined with traditional hot water extraction. With the extraction rate of polysaccharides as the evaluated index,single factor test and orthogonal design were applied to investigate the effects of 4 factors including enzymolysis temperature,enzymolysis time,the amount of enzyme and pH value on polysaccharides extraction rate,and verification tests were conducted. RESULTS:The optimal extraction technolo-gy was as follows as enzymolysis temperature of 40 ℃,enzymolysis time of 150 min,enzyme accounting for 2.0%,pH value for enzymolysis of 4.6. Under the above conditions,the average extraction rate of polysaccharides from G. herbaceum L. was 2.474%(RSD=3.34%,n=5). CONCLUSIONS:Pectinase hydrolysis is an effective method to extract polysaccharides from G. herbace-um L.. The optimal extraction technology is reasonable and feasible.
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OBJECTIVE:To optimize the extraction technology of glucosinolates from Uighur medicine Brassica rapa L.. METHODS:Ethanol refluxing method was adopted to extract glucosinolates from Uighur medicine B. rapa L.. With the comprehen-sive score of glucosinolates and dry extract yield as the index,L9(34)orthogonal test based on the single factor test was adopted to observe the effects of ethanol volume fraction,extraction time and material-liquid ratio on the extraction technology of glucosino-lates from B. rapa L.,and verification test was conducted. RESULTS:The optimal extraction technology was to add 95% ethanol 8 times as much as the amount of the herbs,twice for 1.0 h reflux extractions. The extraction amount of glucosinolates and dry ex-tract yield were 7.36 mg/g and 25.29% at average,respectively.The comprehensive score of RSD was 0.52%(n=3). CONCLU-SIONS:The optimal extraction technology is stable and feasible and can be used for the extraction of glucosinolates from B. rapa L..
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This study was aimed to optimize optimum preparation process of 5,7,3',4'-O-tetramethylquercetin (TMQ). With 5,7,3',4'-O-tetramethylrutin as raw material, TMQ was prepared by acidic aqueous solution method. The preparation process of TMQ was optimized by single factor test and orthogonal test. The results showed that the optimum technology conditions were ethanol concentration at 0, temperature at 80℃, hydrochloric acid concentration at 0.50%, and the hydrolysis time for 1.0 h. It was concluded that the optimum preparation process had the charac-teristics of high yield, simple process, stable and feasible.
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Objective: To optimize the matrix recipe of Compound Analgesic Cataplasm. Methods: Taking the early adhesion and appearance (paste traits, residue, and skin adhesive ability) as evaluation indexes, the optimal matrix recipe of Compound Analgesic Cataplasm was optimized by single factor test and central composite design-response surface method, and the optimal preparation process was determined at the same time. Results: The optimal ratio of the matrix is as follows: NP-700-carbopol 940-aluminium glycinate-silica-glycerin (5.34:0.63:0.2:6:30). Conclusion: The optimal matrix has the moderate adhesion without residue but with better paste traits and skin adhesive ability under the ideal preparation technology.