Establishment and Application of Method for Concentration Determination of Anlotinib in Human Plasma / 中国药房

OBJECTIVE：To establish a method for concentration determination of anlotinib in human plasma and apply it in the clinic. METHODS ：The plasma samples were pretreated by salting-out assisted with liquid-liquid extraction with ammonium acetate as salting out assistant and acetonitrile as solvent. Using voriconazole as internal standard ，LC-MS/MS method was adopted. The separation was performed on Waters X Bridge C 18 column with mobile phase consisting of 0.2％ formic acid solution- acetonitrile（gradient elution ）at the flow rate of 1 mL/min. The column temperature was set at 40 ℃，and sample size was 10 μL. The split ratio was 3∶7. The electrospray ion source and multiple reaction monitoring mode were used for the analysis. The ion pair of anlotinib and internal standard under positive ion mode were m/z 408.3→339.3 and m/z 350.2→281.3，respectively. RESULTS ： Anlotinib showed a good linear relationship in the concentration range of 0.2-200 ng/mL（R2＞0.996 7）. The lowest limit of quantitation was 0.2 ng/mL. Intra-day and inter-day RSDs were no more than 12％ （n＝6 or n＝3）. Accuracies were 90.92％-108.00％（n＝6 or n＝3）. The average extraction recoveries were 87.51％-100.00％（RSD＜8％，n＝6）. The average matrix effects were 96.66％-99.93％（RSD＜5％，n＝6）. The plasma concentration of 3 patients with NSCLC treated with anlotinib was 8.74-65.60 ng/mL. CONCLUSIONS ：The method is simple ，accurate and specific ，and is suitable for the plasma concentration monitoring of anlotinib in NSCLC patients.

Saline Water Salting and Drying Technology of Gastrodiae Rhizoma / 中国实验方剂学杂志

Objective:Fresh tubers of Gastrodiae Rhizoma were harvested at the right time. A saline water salting and drying technology was developed for obtaining the medicinal materials of Gastrodiae Rhizoma in the place of origin and avoiding rot and mildew. Method:Fresh tubers of Gastrodiae Rhizoma were dug in Yiliang,Yunnan,Dejiang,Guizhou,and Chenggu,Shaanxi,the experiments of natural drying,and saline water salting and drying were carried out in the place of origin and Beijing. After the dirt was removed,the samples were tiled in a container immediately,added with varied proportions of saline water (0.03-0.10 g·mL-1 NaCl in water),hermetically pickled for 6-15 d. after being soaked and rinsed with water,the samples were put in a cool ventilated place or under sunshine to prepare dried medicinal materials of Gastrodiae Rhizoma. We described the appearance characteristics,measured the moisture content,gastrodin and nitrite. And the appearance was observed after storage in a simple warehouse for one year later. Result:Fresh tubers of Gastrodiae Rhizoma from three origins were naturally dried,the surface of gastrodia tubers became black,decayed and moldy,then we could not get dried medicinal materials. The appearance and the content of gastrodins in the medicinal materials of Gastrodiae Rhizoma processed by saline water salting and drying technology met the requirements for Gastrodiae Rhizoma in the Pharmacopoeia of the People's Republic of China 2015 and relevant standards of nitrite in salted food in National Food Safety Standard Determination of Nitrite and Nitrate in Foods,Hygienic Standard for Preserved Vegetables,Green Food-Soybean Paste and Salted Vegetable. Conclusion:The saline water salting and drying technology is developed to make medicinal materials of Gastrodiae Rhizoma quickly from fresh tubers of Gastrodia elata in the place of origin and Beijing. The metamorphism had not been observed after being stored in simple warehouses for one year. This technology can guarantee the quality of Gastrodiae Rhizoma, and provide a new method for the filed processing of Gastrodiae Rhizoma.

Effect of added halide ions and pH on the solubility of nebivolol hydrochloride and the evaluation of selected, discriminating dissolution media / 药学学报

The solubility of nebivolol hydrochloride was determined in acidic aqueous media in the absence and presence of different concentration of NaCl, NaBr, or NaI at 37 ℃ in order to facilitate proper selection of dissolution media that have adequate discriminating power for enhancing the likelihood of a generic drug product to successfully pass in-vivo bioequivalence test. In the range of pH 5.0 to pH 1.0, the solubility of nebivolol hydrochloride decreased with the decrease in the pH of aqueous solution, and the solubility of nebivolol hydrochloride further decreased with the increase in the concentration of added sodium chloride. The solubility decrease of a few weakly basic drug molecules in acidic media and in higher concentration of added chloride was published previously by other researchers, and the observed decrease in the solubility in the presence of higher chloride concentration was interpreted in terms of common-ion effect. However, the results in this paper showed that the solubility of nebivolol hydrochloride also decreased when sodium chloride was replaced with sodium bromide or iodide. The approach described in this paper (i.e. substituting sodium chloride with sodium bromide or iodide) provides an effective method to verify whether common-ion effect is the true (or at least the sole) driving force behind the observed decrease in the solubility of nebivolol hydrochloride in the presence of sodium chloride. The solubility decrease reported in this paper can be interpreted in terms of salting-out effect of sodium chloride, bromide, and iodide. For hydrochloride salt of a weakly basic drug molecule like nebivolol hydrochloride, its solubility in an acidic dissolution medium can be purposely decreased to the lower end of sink condition by adding sodium chloride to make the resulting medium more discriminating. As shown in this paper, a medium at pH 1.2 with added sodium chloride is discriminating and this medium is shown to be bio-relevant to the in-vivo data collected under fasting condition (in-vivo study protocol was approved by Institutional Review Board).

Influences of Different Coating Materials on the Quality Changes of Hardboiled Salted Duck Eggs under Ambient Storage

Abstract The present study investigated the influences of selected coatings (paraffin wax (PW), chitosan (CH), whey protein isolate (WPI), and soy protein isolate (SPI)) on the quality changes of hardboiled salted duck eggs when kept under ambient temperature (30±2 oC). At 5-day intervals for 15 days, samples were tested for color (L*, C*, and h˚), shell strength, weight loss, microbial analysis, water activity, moisture, pH, salt content, TBARS, and sensory analysis. L*, C* and h˚ gradually decreased in egg white, whereas C* and h˚ gradually increased in egg yolk. Shell strength gradually decreased in all cases, and weight loss similarly increased throughout the storage. PW and WPI coatings gave the best shell strengths and the least weight loss. The aw was not significantly different between the treatments. WPI and PW retained the most moisture. A slight decrease in pH was observed in all the samples (P ≥ 0.05). On the other hand, salt content gradually increased with storage time, and the WPI and SPI treatments gave < 2% salt accumulation. TBARS steadily increased throughout storage, and the WPI samples had the least lipid oxidation. TPC, mold, and yeast at the end of storage were the least with the WPI treatment. Both the storage period and coating material choice significantly influenced the sensory scores that declined throughout the storage. Overall, the WPI coating treatment gave the best results.

Estudio molecular del exón 3 del gen atp7b en pacientes cubanos con Enfermedad de Wilson / Molecular genetic analysis of exon 3 of the ATP7B gene in Cuban patients with Wilson's disease

Introducción: La Enfermedad de Wilson es una enfermedad con patrón de herencia autosómico recesivo. Es causada por las mutaciones en el gen atp7b. El exón 3 del gen atp7b es polimórfico y se informan más de 120 polimorfismos en el gen atp7b. Objetivo: Identificar los cambios conformacionales en el exón 3 del gen atp7b y detectar polimorfismos en pacientes cubanos con diagnóstico clínico presuntivo de la enfermedad de Wilson. Materiales y Métodos: Se realizó un estudio descriptivo, en el Centro Nacional de Genética Médica y en el Instituto Nacional de Gastroenterología, durante el período 2007-2013, que incluyó 105 pacientes con diagnóstico clínico presuntivo de la enfermedad de Wilson. La extracción del ADN fue por la técnica de precipitación salina. Se utilizó la técnica de Reacción en Cadena de la Polimerasa para la amplificación del fragmento de interés, y para detectar los cambios conformacionales y la presencia del polimorfismo p.L456V, se usó la técnica de Polimorfismo Conformacional de Simple Cadena, en el exón 3 del gen atp7b. Resultados: En el exón 3 se detectan los cambios conformacionales denominados b y c que correspondieron al polimorfismo p.L456V en estado heterocigótico y homocigótico respectivamente. La frecuencia alélica del polimorfismo p.L456V es de 41 por ciento. Las manifestaciones más frecuentes en los pacientes que presentaron este polimorfismo son las hepáticas. Conclusiones: Se identificó el polimorfismo p.L456V en 64 pacientes cubanos con diagnóstico clínico de la enfermedad de Wilson, lo cual posibilitará hacer estudios moleculares por métodos indirectos(AU)

Introduction: Wilson's disease is a rare inherited autosomal recessive disorder caused by mutations in the ATP7B gene. The exon 3 of the ATP7B gene is polymorphic, and more than 120 polymorphisms of this type have been reported in the literature. Objective: To identify conformational band shifts in exon 3 and detect polymorphisms of the ATP7B gene in Cuban patients, clinically diagnosed with Wilson's disease. Materials and Methods: A descriptive study including 105 patients with the clinical diagnosis of Wilson's disease was conducted at the National Center for Medical Genetics and the National Institute of Gastroenterology from 2007 to 2013. Salting-out protocol was used for DNA extraction. The Polymerase Chain Reaction was used to amplify the fragment of interest and the Single-Strand Conformational Polymorphism was applied in the region of exon 3 of the ATP7B gene to identify conformational changes and the presence of the polymorphism p.L456V. Results: The conformational change called B and C corresponded to the p.L456V polymorphism in the heterozygous and homozygous states, respectively. The allelic frequency of the p.L456V polymorphism in 105 Cuban patients clinically diagnosed with Wilson's disease was 41 percent. The most common manifestations in patients with this polymorphism were related to the liver. Conclusion: The p.L456V polymorphism was identified in 64 Cuban patients with Wilson disease, which will enable us to conduct molecular studies by indirect methods(AU)

Influence of Prolonged Salting on the Physicochemical Properties of Duck Egg White

ABSTRACT Salted duck eggs are unique among the important authentic foods of Thailand. They are mainly produced in Chaiya district, Surat Thani province, in the southern part of Thailand. The egg whites of salted duck eggs are normally unusable due to heavily salty taste upon prolonged storage. The present study was aimed to examine the foaming characteristics of egg white after being cured for a prolonged period (30 days). At 5-day intervals the egg whites were measured for salt, moisture, water activity, hydrophobicity, pH, zeta potential, particle size, SH groups, surface tension and foaming properties (index of whipping, foam durability, specific density, overrun, air phase and yield stress). The results showed that prolonged curing significantly affected egg white and its foam. Salt content, hydrophobicity, particle size, and exposed SH groups gradually increased with storage period. On the other hand, moisture, water activity, pH, zeta potential, surface tension and total SH groups decreased continuously throughout the storage. In addition, foaming properties slightly declined due to accumulation of salt content with extended storage. Conversely, the yield stress - an important indicator of foam quality - increased from 32.33 to 70.55 % during storage. Overall, the egg whites could possibly serve as a key and/or a substitute ingredient in foam-based food products.

Avaliação da qualidade da salmoura durante seu período de utilização em indústria de laticínios / Evaluation of the quality of the brine during its period of use in the dairying

A etapa de salga é de fundamental importância na produção de queijos por contribuir com diversos parâmetros como: formação do sabor, maturação, controle microbiológico, controle de umidade, dentre outros. O processo de salga de queijos em salmoura é largamente utilizado no Brasil, podendo acarretar problemas de contaminação microbiana nos produtos em virtude de falhas em sua correção ou recuperação. Este trabalho teve por objetivo acompanhar a qualidade da salmoura em uma pequena indústria de laticínios situada em Minas Gerais, durante seu período de utilização. Foram realizadas determinações de temperatura, acidez titulável, concentração de NaCl, além de contagem padrão de micro-organismos mesófilos aeróbios, determinação do número mais provável de coliformes totais e termotolerantes e contagem de fungos filamentosos e leveduras. O experimento foi conduzido em três repetições, sendo as análises realizadas em duplicata nos tempos 0, 7, 14, 21 e 30 dias de armazenamento. Os valores encontrados para as análises de temperatura e cloreto de sódio apresentaram-se dentro dos padrões estabelecidos pela literatura. O valor de acidez titulável manteve-se constante em 0,08% de ácido lático durante todo o período analisado, indicando necessidade de correção. A contagem de mesófilos aeróbios variou entre 1,23 log UFC.mL-1 a 3,64 log UFC. mL-1, coliformes totais e termotolerantes entre <0,47 log NMP. mL-1 a 1,17 log NMP. mL-1 e fungos filamentosos e leveduras 1 log UFC. mL-1 (est.) a 2,9 log UFC. mL-1 (est.). A salmoura apresentou boa qualidade microbiológica e físico-química, exceto quanto à acidez titulável, que deve ser corrigida. Foi verificado aumento da carga microbiana no momento da correção da concentração de sal, o que pode estar relacionado à qualidade deste produto.(AU)

Conventional and volatile salts as precipitating agents for recombinant antiviral protein / Sais convencionais e voláteis como agentes de precipitação para proteína recombinante com ação antiviral

Recombinant proteins expressed in cell culture have been shown to be relevant in the biopharmaceutical production focusing human health. The current work investigated the precipitation process of recAVLOEc protein, synthesized by E. coli BL21 (DE3) pLysS cells. The system is used for the AVLO expression that shown antiviral activity and it was found in the hemolymph of Lonomia obliqua caterpillar. The precipitation was conducted by the use of conventional salts (ammonium sulfate and sodium sulfate) and the volatile ammonium carbamate salt. Initially, the precipitated protein obtained from bacterial lysate was added to L929 cells to evaluate the cytotoxic effect; and besides Vero cells were infected with measles virus to verify the antiviral action of the precipitated recombinant protein. Toxic effect on the culture of L929 cells was observed for the precipitate obtained by the use of ammonium sulfate and sodium sulfate. In addition, tests in L929 cell cultures infected with EMC virus showed that samples of precipitated protein by salts did not show antiviral action. In Vero cell cultures, the precipitated protein by sodium sulfate showed antiviral action for measles virus.

Proteínas recombinantes expressas em culturas celulares têm se mostrado importantes na produção de fármacos de interesse para a saúde humana. Este estudo investigou a precipitação da proteína recAVLOEc, sintetizada por células de E. coli BL21 (DE3) pLysS, utilizadas como sistema de expressão da AVLO, proteína com atividade antiviral, originalmente encontrada na hemolinfa da lagarta Lonomia obliqua. A precipitação foi conduzida por meio do uso de sais convencionais (sulfato de amônio e de sódio) e do sal volátil carbamato de amônio. Inicialmente o precipitado proteico obtido do lisado bacteriano foi administrado em culturas de células L929 para avaliar o efeito citotóxico e posteriormente em células Vero infectadas com o vírus do sarampo, para a verificação da ação antiviral. Um efeito tóxico em culturas de L929 foi observado para os precipitados obtidos pelo uso de sulfato de amônio e de sódio. Testes em culturas de L929 infectadas com o vírus EMC foram também efetuados e as amostras de proteínas precipitadas com os sais convencionais e o sal volátil não resultaram em ação antiviral. Em culturas de células Vero, o uso do sulfato de sódio como agente de precipitação das proteínas contidas no lisado bacteriano resultou em ação antiviral para o sarampo.

Purification of metallo-B-lactamase CcrA from Bacteroides fragilis with salting-out method

A simple and effective salting-out method was developed for the purification of the metallo-β-lactamase CcrA from Bacteroides fragilis based on the plasmid pMSZ02, in which the crude protein secreted into growth medium was precipitated by 80% sulfate saturation of the medium and purified with Q-Sepharose to offer pure CcrA with yield of 20.1 mg per litter medium. The dependence of the amount of protein precipitation on sulfate saturation was investigated, which showed that more than 80% sulfate saturation resulted the maximum protein precipitated. The purified CcrA was evaluated by steady-state kinetics using penicillin G and cephalothin V as substrates, which showed the Km values of 68±2 and 17±2 µM and Kcat values of 63±1 and 102±3 S-1, respectively. The comparison with the data of the protein from literature method showed that the salting-out method was viable, and it could be useful for the purification of other proteins secreted into growth medium.

A rapid salting out method for DNA extraction from buccal swabs / 第二军医大学学报

Objective To establish a rapid salting out method for extraction of genomic DNA from buccal swabs. Methods Buccal epithelial cells were digested with cell lysate solution and proteinase K solution. Then the proteins were removed by salting out and centrifugation and DNA was precipitated with isopropyl alcohol. Finally, the precipitations of DNA were washed with 70% ethanol and were resuspended in T. The rsl04252 loc o TP5 gene and rsl291098 loc o CHRNA gene were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. The samples with different genotypes were confirmed by direct sequencing analysis. Results The DNA yield of single buccal swab ranged from 0. 68 to 2. 56 μg; the D260/D280 value ranged from 1. 77 to 1. 94. After PCR amplification and enzyme digestion, two single nucleotide polymorphisms (SNPs) of 10 samples were clearly genotyped. The results of PCR-RFLP agreed well with the results of direct sequencing. Conclusion The present salting out method is rapid, simple, and economical for DNA extraction from buccal swabs. The obtained genomic DNA is of high quality.

Avaliação físico-química, microbiológica e sensorial de tilápias do nilo (Oreochromis niloticus) inteiras evisceradas submetidas a salga e secagem natural / Evaluación físico-química, microbiológica y sensorial de tilapias del nilo (Oreochromis niloticus) enteras evisceradas sometidas a sal y secado natural / Physicochemical, microbiological and sensorial evaluation of clean whole Nile tilapias (Oreochromis niloticus) submitted to salting and natural drying

Os peixes são produtos de grande valor nutritivo, além de ser uma atividade econômica, para produção de alimentos que mais cresce no mundo.Assim,o presente trabalho teve como objetivo avaliar físico quimicamente, microbiologicamente e sensorialmente amostras de tilápias após a salga e secagem natural seguidas de armazenamento à temperatura ambiente por 30 dias. Os resultados indicam que o processo de salga e secagem foi efetivo, resultando na diminuição da umidade e na penetração do sal para o interior do pescado. Microbiologicamente o produto obtido foi estável e obedecia aos padrões da ANVISA. Os provadores não foram capazes de identificar a amostra contendo o produto, quando comparadas com formulação contendo bacalhau comercial e batatas, sendo ainda que sensorialmente o produto obteve uma boa aceitação para os atributos de textura, cor, aroma e sabor.

Fishes are products of great nutritional value and an economical activity for production of the fastest growing food in the world. This work had as objective evaluates physical-chemically, microbiologically and sensorially tilapias samples after salting and natural drying following by storage to room temperature for 30 days. The results indicate that the process of salting and drying was effective, resulting in the decrease of the water contents and in the penetration of the salt for the interior of the fish, microbiologically the obtained product was stable and it obeyed the patterns of ANVISA. The painelists were not capable to identify the sample containing the product when compared with formulation containing commercial cod and potatoes, being although sensorially the product obtained a good acceptance for the texture, color, aroma and flavor atributes.

Los peces son productos de gran valor nutritivo, además de ser una actividad económica para producción de alimentos que más crece en el mundo. Así, esta investigación tuvo el objetivo de evaluar físico, químicamente, microbiológicamente y sensorialmente muestras de tilapias después de saladas y secados natural, seguidas de almacenaje a temperatura ambiente por 30 días. Los resultados indican que el proceso de salado y secado fue efectivo, resultando en la disminución de la humedad y en la penetración del sal para el interior del pescado. Microbiológicamente el producto obtenido fue estable y obedecía las normas de la ANVISA. Los probadores no fueron capaces de identificar la muestra conteniendo el producto, cuando comparadas con formulación conteniendo bacalao comercial y patatas, siendo que sensorialmente el producto obtuvo buena aceptación para los atributos de textura, color, aroma y sabor.

Conservação de filés de tilápia-do-nilo (Oreochromis niloticus) em salga seca e salga úmida / Nile tilapia (Oreochromis niloticus) preservation by means of dry salting and saturated brine

Acompanhou-se o desenvolvimento dos processos da salga em salmoura saturada (salga úmida) e salga seca de filés de tilápia-do-nilo (Oreochromis niloticus) e avaliaram-se algumas características indicativas de qualidade do produto durante a estocagem. Os processos foram acompanhados por 156 horas na salga úmida e por 96 horas na salga seca, e os filés salgados foram estocados, respectivamente, por 60 e 45 dias à temperatura ambiente. Os teores máximos de cloreto nos filés (14 por cento) foram atingidos com 72 horas na salga úmida e com 36 horas na salga seca. Os filés de tilápia salgados em salmoura mantiveram as características próprias do produto por um período de 45 dias, e os submetidos à salga seca apresentaram baixo teor de umidade (6 por cento) e alta concentração de extrato etéreo (4,6 por cento). Recomenda-se somente o processo de salga em salmoura saturada como forma de conservação dos filés de tilápia-do-nilo.

The processes of salting of Nile tilapia fillets (Oreochromis niloticus) submitted to saturated brine and dry salting were observed, and some characteristics that indicate the quality of the product during the storage were evaluated. The brine saturated process was followed up to 156 hours and the dry salting was followed up to 96 hours. When the salting finished, fillets were stored for 45 (dry salting) and 60 days (saturated brine), respectively. The highest values for chloride in fillets (14 percent) were reached within 72 hours in brine salting and 36 hours in dry salting. The tilapia fillets salted in brine kept the proper characteristics of the product for a period of 45 days and the fillets submitted to dry salting showed low moisture ratios (6 percent) and a high concentration of lipids (4.6 percent). Thereby, it is only recommended the salting process in saturated brine to be used as a mean of conservation for Nile tilapia fillets.

Preparation of enteric microsphere of oleanolic acid dihemiphthalate sodium by salting-out action using spherical crystallization technique / 药学学报

Aim Enteric microspheres were prepared to prevent the interaction of drug with gastric acid and to improve its bioavailability. Methods The enteric microspheres with a matrix structure were successfully produced using a spherical crystallization technique. Hydroxypropyl methylcellulose phthalate ( HP-55 ), an enteric material, was coprecipitated with the drug by salting-out effect during the preparation process. A mixture of water and ethanol was chosen as a good solvent and dichloromethane was used as a the first time to prepare microspheres by making the water-soluble drug and water-insoluble excipient coprecipitated. In vivo test demonstrated that the drug absorption from the enteric oleanolic acid dihemiphthalate sodium (OADHPS) microspheres was significantly prolonged compared to that with OADHPS powder after a lag-time. Furthermore, the drug bioavailability was 181.6% greater than that with the OADHPS powder. Conclusion The microspheres of water soluble drug could be prepared by using water phase replacing organic phase as poor solvent which decrease the quantity of organic solvent and benefit the environment prevention.

Gradient pH co-precipitation to recover proteins from plasma discarded in artificial-liver treatment / 中华传染病杂志

Objective To set up a system in vitro to rapidly recover plasma proteins lost during artificial-liver treatment.Methods The polyprotein precipitation was obtained by all proteins whose isoelectric point pH value were between 7.3 and 5.1,which collided with each other and aggregated using gradient pH co-precipitation(adding 1 mol/L citric acid slowly in the plasma solution to change the pH values gradually from 7.3 to 5.1 in 5 h)combined with salting out(degree of saturation of NaCl is 33%,reacted for 5.5 h at 4℃)or low-temperature ethanol precipitation(40% ethanol, reacted for 5.5 h at -7℃)so that to get rid of toxicants by discarding the supernatant.Results In the range of pH 5.1-7.3,50%(29g/57g)of the total plasma proteins had been recovered by the gradient pH salting out and 41%(25 g/61g)by the gradient pH low-temperature ethanol co-precipi- tation.The protein remained in the supernatant was mostly albumin and its combined bilirubin.The levels of total bilirubin decreased to 0.07% and 0.06% of the original levels by these two methods respectively and the serum HBV DNA level decreased to be undetected(quantitative PCR).Conclu- sions The proteins with close isoelectric point can co-precipitated with the presence of high concen- tration of NaCl or low-temperature ethanol and by changing the pH value gradually.The total protein in the discarded plasma during artificial-liver treatment can be recovered rapidly using the gradient pH coprecipitation.