Central composite design (CCD) is one of the most commonly used design methods in response surface optimization and has been widely applied in the field of pharmaceutics to optimize preparations. On the 20th anniversary of the introduction of CCD into China, the paper reviews its application in domestic pharmaceutical researches. Based on the brief introduction of basic principle and operation steps of CCD, the mistakes emerging in the application of CCD are summarized, including conceptual confusion with Box-Behnken design and face-centered CCD as well as wrong designs. Besides, the issues concerning the selection of factors and responses are discussed. The article is helpful for researchers to comprehensively understand the CCD and facilitates the rational application of this method.

Bone Density ; Bone and Bones ; metabolism ; Hepatitis B ; complications ; Humans ; Liver Cirrhosis ; metabolism ; Male ; Tumor Necrosis Factor-alpha ; analysis

The biological properties of cultured mesenchymal stem cells (MSC) have been intensively investigated, while there is still a paucity of information about the definite in vivo sites that harbor these stem cells due to the lack of specific surface markers. Previous data have demonstrated that human and murine MSC can be isolated from the compact bones. To investigate if it is the case for other species, the femurs from Wistar rats, Beagles, C57 mice and New Zealand rabbits were collected, minced and digested with collagenase type I. The digested bone fragments were seeded into the medium for human bone marrow culture after removal of the suspended cells in the digestion. The results showed that the fibroblast-like cells were observed to migrate from the bone fragments after several days of culture, and they gradually formed an adherent confluent layer. The adherent cells could be passaged and expressed homogenously the mesenchymal cell marker vimentin. Differentiation assays showed that these cells had the capacity to differentiate into osteoblasts and adipocytes. In conclusion, the results here provide new information for the further investigations on the in vivo biological features of MSC in the context of the simplicity of the compact bone structure.
Animals ; Bone and Bones ; cytology ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Dogs ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Inbred C57BL ; Rabbits ; Rats ; Rats, Wistar

In the present study, the effects of murine bone marrow endothelial cell conditioned medium (mBMEC-CM) on the growth of yolk sac and bone marrow hematopoietic stem/progenitor cells (HSPC) were investigated. Nonadherent cells of yolk sac and bone marrow were collected for semisolid culture assay of CFU-GM and HPP-CFC after being cultured in DMEM with 10% FBS, 10% mBMEC-CM and/or FL (5 ng/ml), TPO (2 ng/ml) for 24 hours. The number of CFU-GM and HPP-CFC was counted by day 7 and 14 respectively. Atlas cDNA Expression Array was used for analysis of cytokine receptor expression of yolk sac and bone marrow HSPC. The results showed that mBMEC-CM could support the expansion of CFU-GM and HPP-CFC in liquid culture system. The expansion effects of mBMEC-CM were enhanced by combination with FL and TPO. mBMEC-CM was more effective on expansion of bone marrow CFU-GM and HPP-CFC than that of yolk sac CFU-GM and HPP-CFC. The differential expression of cytokine receptors were detected between yolk sac and bone marrow HSPC. PDGF-Rbeta, PDGF-Ralpha and corticotropin releasing factor receptor (CRFR) were only expressed in yolk sac hematopoietic cells while IFN-gammaR, GM-CSFR, Dopamine D2R and follicle-stimulating hormone receptor were only expressed in bone marrow hematopoietic cells. In conclusion, mBMEC-CM could support the growth and proliferation of yolk sac and bone marrow HSPC, and this effect was further enhanced by addition of FL and TPO. mBMEC-CM was more effective on expansion of bone marrow HSPC than on expansion of yolk sac HSPC. The comparative study indicated that the different expressions of cytokine receptors existed between yolk sac and bone marrow hematopoietic cells, which might lead to the difference in expansion in vitro between embryonic and adult HSPC.
Animals ; Bone Marrow Cells ; physiology ; Cell Division ; Cells, Cultured ; Culture Media, Conditioned ; Endothelial Cells ; physiology ; Female ; Hematopoiesis ; Hematopoietic Stem Cells ; physiology ; Male ; Mice ; Receptors, Cytokine ; analysis ; Thrombopoietin ; pharmacology ; Yolk Sac ; cytology

OBJECTIVETo investigate the role of sulfated tyrosine in regulating the activity of tyrosylprotein sulfotransferases (TPST) 1 and TPST2.

METHODSConstructs of TPST 1 and TPST2 were amplified by polymerase chain reaction (PCR), then fused into immunoglobulin G1 Fc region. All the variants in which sulfated tyrosines were mutated to phenylalanine were made by the PCR-based Quick Change method and confirmed by sequencing the entire reading frame. Small hairpin RNA (shRNA) constructs-targeting nucleotides 259-275 of TPST1 and nucleotides 73-94 of TPST2 were generated and subcloned into pBluescript. Human embryonic kidney (HEK) 293T cells were transfected with these plasmids. One day later, cells were split: one part was labeled with 35S-cysteine and methionine or 35S-Na2SO3 overnight, the second part was used for 125I labeled binding experiment, and the third part was retained for binding and flow cytometry.

RESULTSTyrosines at position 326 of TPST1 and position 325 of TPST2 were sulfated posttranslationally. Tyrosine sulfation of TPSTs was effectively inhibited by sulfation inhibitors, including specific shRNAs and non-specific NaCIO3. shRNAs reduced the sulfation of C3a receptor and C5a receptor, and partially blocked the binding of these two receptors to their respective ligands.

CONCLUSIONSThe activities of TPSTs were regulated by tyrosine sulfation. Inhibition of sulfotyrosine decreases the binding ability of C3a receptor and C5a receptor to their respective ligands.

Cell Line ; Complement C3a ; metabolism ; Complement C5a ; metabolism ; Humans ; Protein Binding ; Protein Processing, Post-Translational ; Receptor, Anaphylatoxin C5a ; metabolism ; Receptors, Complement ; metabolism ; Sulfotransferases ; genetics ; metabolism ; Transfection ; Tyrosine ; analogs & derivatives ; metabolism

OBJECTIVETo evaluate the antitumor activity of a novel class of 4, 8-Disubstituted-8, 9-dihydropyrazine[2, 3-g]quinazoline-7(6H)-ketones in vitro, and to screen potential anticancer compounds for further study.

METHODSSeventeen compounds of 4, 8-Disubstituted-8, 9-dihydropyrazine[2, 3-g]quinazoline-7(6H)-ketones were synthesized with solid-phase method for biological evaluation of EGFR tyrosine kinase. MTT method was used to evaluate the cytotoxic activity in vitro against three human cancer cell lines (human lung carcinoma cell line A549, human leukemia cell lines K562 and human gastric carcinoma cell line SGC7901).

RESULTSCompound 7-13 and 7-14 showed potent antitumor activities against A549 cells, with IC(50) values of 8.10 and 8.12 mol/L, respectively. Eight compounds showed proliferative inhibition effect on K562 cells, especially 7-2, 7-13 and 7-17, with IC(50) values of 2.22,0.57 and 7.20 mol/L,respectively.And compound 7-13 and 7-3 showed potent antitumor activity against SGC7901 cells, with IC(50) values of 4.20 and 9.71 mol/L, respectively.

CONCLUSIONThe synthesized compounds 4, 8-Disubstituted-8, 9-dihydropyrazine[2, 3-g] quinazoline-7(6H)-ketones show inhibition effects on human cancer cell lines in vitro. Compound 7-13 has anticancer activity in all three cancer cell lines, which might be used as a potential antitumor drug for further study.

Antineoplastic Agents ; chemical synthesis ; chemistry ; pharmacology ; Cell Line, Tumor ; Drug Screening Assays, Antitumor ; Humans ; K562 Cells ; Lung Neoplasms ; pathology ; Molecular Structure ; Pyrazines ; chemical synthesis ; chemistry ; pharmacology ; Quinazolines ; chemical synthesis ; chemistry ; pharmacology ; Receptor, Epidermal Growth Factor ; antagonists & inhibitors ; Stomach Neoplasms ; pathology ; Structure-Activity Relationship

OBJECTIVETo study the pathogenesis of abnormal bone metabolism in patients with HBV liver cirrhosis.

METHODSNM-300 signal-energy X-ray absorptiometry system was used to measure the bone mineral density (BMD) in 61 liver cirrhosis patients and 30 age-matched healthy controls. The serum levels of 1,25(OH)2D3, parathyroid hormone (PTH), calcitonin (CT), bone gamma-carboxyglutamic acid-containing protein (BGP), IL-1beta, IL-6, tumor necrosis factor (TNF)alpha and urine crosslaps were also detected in these patients.

RESULTSBMD in patients with HBV liver cirrhosis was lower than those of the controls. The serum levels of 1,25(OH)2D3 and BGP in cirrhosis patients were lower than those in the controls, and they were much lower in the osteoporosis (OP) group than in the non-osteoporosis (NOP) group. The PTH and CT were higher significantly in the patients than in the controls. The changes of serum 1,25(OH)2D3 and BGP were correlated with the changes of BMD. The serum levels of IL-1beta, IL-6, TNFalpha and urine crosslaps in cirrhosis patients were higher than those of the controls, and they were much higher in the OP group than in the NOP group. We also found that the serum levels of IL-1beta, IL-6, TNFalpha and urine crosslaps had a negative correlation with BMD.

CONCLUSIONSThese data suggest that bone formation is weakened and bone resorption is increased in patients with HBV liver cirrhosis, 1,25(OH)2D3 plays an important role in abnormal bone formation. Elevation of serum IL-1beta, IL-6, TNFalpha can accelerate bone resorption and cause hepatic bone disease (HBD). Taking 1,25(OH)2D3 and reducing the level of IL-1beta, IL-6, TNFalpha may be very important in preventing and treating HBD.

Adult ; Bone Density ; Bone and Bones ; metabolism ; Calcitriol ; pharmacology ; Hepatitis B, Chronic ; complications ; Humans ; Liver Cirrhosis ; complications ; Male ; Middle Aged ; Osteoporosis ; etiology

Soxhlet extraction is a common method of sample preparation. However, there has been no discussion about the efficiency of Soxhlet extraction from different batches and the factors that cause content fluctuation. In this study, Panax ginseng was selected as a model sample. Soxhlet extraction by means of a water bath, which has always been neglected, was identified as a novel key factor in the poor repeat-ability in different batches of Soxhlet extraction, as it can affect the siphon times and reflux time, which have been positively correlated with the ginsenoside contents. By substituting round bottom flasks in the same column, the relative standard deviation of the most fluctuated compound, ginsenoside Rb1, was decreased from 24.6% to 5.02%. Scanning electron microscopy analysis confirmed that the breakdown of the surface of the ginseng powder in the Soxhlet extraction led to a better dissolution of ginsenosides, indicating that chloroform may promote the extraction of ginsenosides by disrupting the cell structure. Moreover, 70% methanol was regarded as the better solvent for extracting the ginsenosides. Overall, this work offers a practical and effective protocol for improving the accuracy and repeatability of Soxhlet extraction methodology for ginsenosides and other analytes.

Background: Colorectal cancer (CRC) is the second most common cancer in Malaysia. Awareness of risk factors, symptoms and warning signs of CRC will help in early detection. This paper presents the level of CRC awareness among the urban population in Malaysia. Method: A cross-sectional study was conducted from November 2015 till December 2016 at three government clinics in the Klang Valley. The validated Bowel Cancer Awareness Measure questionnaire in both English and Malay was used. The mean knowledge scores for the warning signs and risk factors of CRC in different socio-demographic groups were compared using ANOVA in SPSS version 23. Statistical significance was set at p<0.05 and a 95% confidence level. Results: Of the 426 respondents, 29.1% were unable to recall the warning signs and symptoms of CRC. Average recall was less than two warning signs and symptoms (mean 1.62, SD 1.33). The mean total knowledge score for CRC was 9.91 (SD 4.78), with a mean knowledge scores for warning signs and risk factors at 5.27 (SD 2.74) and 4.64 (SD 2.78), respectively. Respondents with a higher level of education were found to have higher level of knowledge regarding the warning signs of CRC. There was a significant positive association between knowledge score for warning signs and level of confidence in detecting warning signs. Regarding the total knowledge score for CRC, 3.3% of respondents scored zero. For warning signs and risk factors, 8.2% and 8.5% of respondents had zero knowledge scores, respectively. Conclusions: Generally, awareness of CRC is poor among the urban population of Klang Valley. Greater education and more confidence in detecting warning signs are significantly associated with better knowledge of warning signs. CRC awareness programs should be increased to improve awareness.

Objective To optimize the extraction technology for Yangxin Anshen Granules. Methods With yield of volatile oil as index, single factor tests were adopted to investigate effects of water, soaking time and distillation time on extraction technology of volatile oil. Using water amount, extraction time and extraction times as factors, the contents of paeoniflorin and total solid as indexes, orthogonal test was employed to optimize the extraction technology of Yangxin Anshen Granules. Results The optimical extraction technology conditions were as follows:Cinnamomi Ramulus, Saposhkoviae Radix, and Citri Sarcodactylis Fructus were extracted to get volatile oil with eight-folds amount water of herbs for 6 hours; other herbs were boiled with eight-folds amount water of herbs and extrancted for two times, 1 h each time. Conclusion This extraction process is reasonable and practical, and can well guarantee the quality of preparation.