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To optimize the formulation and technology of oxymatrine-astragaloside IV coloaded liposomes (Om-As-Lip) based on quality by design (QbD) principles, and further to verify the feasibility of its amplification process, Om-As-Lip was prepared by ethanol injection combined with pH gradient method. The critical material attributions of Om-As-Lip were evaluated by dual-risk analysis tools and Plackett-Burman design (PBD). The formulation of Om-As-Lip was further optimized with the Box-Behnken design (BBD). The design space was also established based on the contour plots of BBD. In order to further investigate the amplification process of Om-As-Lip, the critical process parameters of high-pressure homogenization (HPH) were optimized by single-factor test, and the quality of the final product was also evaluated. The results of risk analysis and PBD confirmed that the astragaloside concentration, cholesterol concentration, and phospholipid ratio (HSPC∶SPC) were the ctitical material attributes. The model established by BBD had a good predictability, and the optimized mass ratio of As to phospholipids was 1∶40, cholesterol to phospholipids was 1∶10, HSPC to SPC was 51∶9. The design space of Om-As-Lip was as follows: the ratio of cholesterol to phospholipids was 1∶12-1∶5 and HSPC to SPC was 1∶7-17∶3. The optimized high-pressure homogenization pressure was 600 bar, temperature was 4 ℃, and cycle times was 6 times for HPH-Om-As-Lip. The quality of Om-As-Lip prepared based on the QbD concept can meet the expected CQAs, and the formulation and technology established can provide a reliable experimental basis for its future development and applications.
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Age-related macular degeneration (AMD) is one of the main causes of vision loss among middle-aged and elderly people worldwide. The prevention and treatment of AMD is a current topic of interest in ophthalmology but remains challenging. Oxidative stress-induced retinal pigment epithelial cell autophagic dysfunction, cellular senescence, and an abnormal immune inflammatory response are key pathogenic factors for AMD. Many bioactive ingredients of traditional Chinese medicine not only exert anti-oxidative, anti-inflammatory, anti-aging, and anti-apoptotic effects, but also prevent/block the occurrence of AMD through different pathways. This review summarizes our current understanding of the pathogenesis of AMD, the types of natural bioactive ingredients capable of treating AMD, as well as the known mechanisms by which these agents act, and may provide new strategies for the prevention and treatment of AMD.
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Objective::To compare the adsorption characteristics of different macroporous adsorption resins for the total flavonoids in Epimedii Folium, clarify the adsorption mechanism, and screen the optimal resin for the purification of total flavonoids in Epimedii Folium. Method::Taking the adsorption and desorption capacities of the total flavonoids in Epimedii Folium and five representative flavonoids (epimedin A, epimendin B, epimendin C, icariin, baohuoside Ⅰ) as indexes, static adsorption and dynamic adsorption experiments were conducted to compare the adsorption characteristics of five macroporous including HPD100, HPD600, AB-8, X-5 and D101.The adsorption kinetics of the selected resin was studied by using the pseudo-first-order, pseudo-second-order kinetic models and intraparticle diffusion model, and the thermodynamic process was analyzed by using the Langmuir and Freundlich isothermal adsorption models, which explored the adsorption mechanism of resin from the perspective of physical chemistry. Result::HPD100 macroporous resin had a better adsorption and desorption properties than the others. The adsorption process of HPD100 macroporous resin for total flavonoids in Epimedii Folium and five representative flavonoids conformed to the pseudo-second-order kinetic model. The adsorption thermodynamic process of HPD100 resin for total flavonoids of Epimedii Folium conformed to the Freundlich model, and for the sum of five representative flavonoids conformed to the Langmuir model. The adsorption process of HPD100 resin for total flavonoids in Epimedii Folium was the exothermic process dominated by physical adsorption, and the optimal adsorption temperature was 25 ℃. Conclusion::HPD100 macroporous resin has large adsorption capacity, easy desorption and clear adsorption mechanism, it is suitable for isolation and purification of total flavonoids in Epimedii Folium.
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To optimize the formulation and preparation process of icaritin-coix seed oil microemulsion(IC-MEs) based on quality by design(QbD) concept. IC-MEs were prepared by water titration. Firstly, the risk factors that may affect the quality of IC-MEs were evaluated. Then Plackett-Burman design was used to screen out prescription factors and process parameters that had a significant effect on the indicators. Finally, Box-Behnken design was used to optimize the prescription ratio of IC-MEs. Through the risk assessment and Plackett-Burman design, three formulation factors [drug loading efficiency, the ratio of mixed-oil(coix seed oil-Glycerol tributyrate) to mixed-surfactant(HS15-RH40) and water addition] were determined as the key factors affecting IC-MEs. The regression model established by Box-Behnken design had a good predictability. The optimal formula was as following: the drug loading efficiency of 0.92%, the ratio of mixed-oil(coix seed oil-glycerol tributyrate) to mixed-surfactant(HS15-RH40) of 4∶6, and the water addition of 5.7 mL. According to this prescription, IC-MEs were prepared, and its encapsulation efficiency after 1 week was 92.45%±1.00%. Therefore, the stability of IC-MEs could be improved by optimizing prescription and process parameters of IC-MEs based on the QbD concept, which can provide certain reference value for the future development of IC-MEs.
Subject(s)
Coix , Emulsions , Flavonoids , Plant OilsABSTRACT
AIM To prepare the liposomes loaded with celastrol,tanshinone Ⅱ A and tanshinone Ⅱ A sodium sulfonate and to evaluate the effects of preparation technologies and entrapment materials on the encapsulation efficiencies.METHODS The liposomes for three constituents were prepared by film dispersion method and reverse phase evaporation method,respectively.50% Lecithin high potency,80% egg yolk lecithin,92% lecithin high potency and complex phospholipid (5 ∶ 1 for ratio of dipalmitoyl phosphatidylcholine to 50% lecithin high potency) were served as entrapment materials.The encapsulation efficiencies were thus calculated based on the measured ean sizes and polydispersity of the liposomes.RESULTS Celastrol and tanshinone Ⅱ A sodium sulfonate liposomes accomplished by reverse phase evaporation method got the highest encapsulation efficiency when entrapped with 80% egg yolk lecithin and 92% lecithin high potency,respectively,which were higher than those prepared by film dispersion method.And celastrol liposomes exhibited an even superior performance than tanshinone Ⅱ A sodium sulfonate liposomes.Tanshinone Ⅱ A lipisome prepared by film dispersion method and then entrapped with 50% lecithin high potency obtained its the highest value of encapsulation efficiency,which was much lower than those of another two liposomes.CONCLUSION Reverse phase evaporation method is appropriate for hydrophobic celastrol with multiple hydrogen bonds and hydrophilic sodium tanshinone Ⅱ A without hydrogen bonds to prepare their liposomes,while film dispersion method is applicable to hydrophobic tanshinone Ⅱ A without hydrogen bonds.
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AIM To determine the contents of five marker components and total flavonoids in Paishi Granules (a medication to remove stones in the body,containing Glechomae Herba,Akebiae Caulis,Cynanchi Paniculati Radix et Rhizoma,etc.).METHODS The content determination of various components in 50% methanol extract of Paishi Granules was accomplished by RP-HPLC.The analysis was performed on a 30 ℃ thermostatic Aglient SBC1s column (250 mm ×4.6 mm,5 μm),with the mobile phase comprising of acetonitrile-0.05% phosphoric acid flowing at 1.0 mL/min in a gradient elution manner,and the detection wavelength was set at 240 nm.The content of total flavonoids was detected by ultraviolet spectrophotometry.RESULTS Chlorogenic acid,loganin,rutin,rosmarinic acid and ammoninm glycyrrhizinate showed good linear relationships within the ranges of 18.88-604.00,18.50-592.00,20.25-648.00,18.75-600.00 and 18.81-602.00 μg/mL,whose average recoveries were 97.9%,100.7%,103.3%,96.4% and 102.4% with theRSDsof1.4%,0.4%,2.7%,1.2% and 2.3%,respectively.The contents of total flavonoids in twenty batches of samples all met pharmacopeia requirements,while those of marker components were found to be different,among which loganin displayed the most significant change,followed by rutin and ammoninm glycyrrhizinate.CONCLUSION This simple,accurate and reliable method can be used for the quality control of Paishi Granules.
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<p><b>OBJECTIVE</b>To explore the clinical value of PKC412 (midostaurin) in treatment of AML patients with FLT3.</p><p><b>METHODS</b>The bone marrow or peripheral blood were collected and heparinized from 21 newly diagnosed FLT3AML patients, then the mononuclear cells from bone marrow or peripheral blood were isolated by density-gradient method. The sensitivity of leukemia cells to PKC412 of 8 concentration in vitro was detected by ATP-bioluminescence-tumor chemosensitivity assay (ATP-TCA), and the relationship among sensitivity results in vitro, risk stratification and therapeutic efficacy was analyzed.</p><p><b>RESULTS</b>The leukemia cells of 21 patients with AML displayed different sensitivities to PKC412 in vitro. The rate of sensitivity in vitro was 42.9%, and sensitive concentration in vitro were between 1 µmol/L and 5 µmol/L. There was no significant relationship between risk stratification and sensitivity results of PKC412 in vitro. There was also no significant relationship between clinical efficacy and sensitivity results of PKC412 in vitro. The survival of patients in low-risk and intermediate-risk groups was better than that of patients in high-risk groups (P=0.015).</p><p><b>CONCLUSION</b>PKC412 can be one of the effective therapeutic method for AML patients without FLT3 mutation. The sensitivity of leukemia cells to PKC412 may become a prognostic marker for evaluating clinical efficacy of PKC412, which is independent of other factors.</p>
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Objective: To prepare and evaluate bionic enzymatic Epimedium flavonoids-loaded enteric-coated capsules, composed of Epimedium flavonoids as drug, snail enzyme as hydrolase and common formulation accessories, which is in favor of in vivo real-time enzymolysis and real-time absorption of Epimedium flavonoids. Methods: The UV method and single factor experiment were applied to investigating the effects of key factors such as filling agents species and dosage, disintegrating agents species and dosage, wetting agents species and drying temperature on the cumulative release degree of Epimedium flavonoids. Response surface method was used to optimize the prescription for bionic enzymatic Epimedium flavonoids-loaded enteric-coated capsules, and the enzymatic process of the four Epimedium flavonoids (icariin and epimendin A, B, C) was also evaluated by HPLC. Results: The optimal formulation for the bionic enzymatic Epimedium flavonoids-loaded enteric-coated capsules was composed of 100 mg Epimedium flavonoids extract, 68 mg snail enzyme, 65 mg α-lactose, and 11.7 mg low-substituted hydroxypropyl cellulose. This enteric-coated capsules showed a cumulative Epimedium flavonoids release rate of 85.43% within 45 min in simulated intestinal fluid and a complete enzymolysis within the intestine emptying time (3-6 h). Conclusion: The bionic enzymatic Epimedium flavonoids-loaded enteric-coated capsules show an advantage in improving hydrolysis and anti-osteoporosis efficacy of Epimedium flavonoids, providing some research ideas for improving the oral bioavailability of other flavonoids.
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<p><b>OBJECTIVE</b>To screen signaling pathway proteins in myelodysplastic/myeloproliferative neoplasms-unclassifiable (MDS/MPN-U), and to explore the possible role of the differentially expressed signaling pathway proteins in pathogenesis of MDS/MPN-U.</p><p><b>METHODS</b>Protein Pathway Array (PPA) was applied to analyze the differential expression levels of signaling pathway proteins in 10 patients with MDS/MPN-U and normal controls, and furthermore to identify the signaling pathways and network in which these proteins were analyzed by Ingenuity pathway analysis program.</p><p><b>RESULTS</b>The expressions of 25 signaling proteins in MDS/MPN-U were significantly different, compared with the control group. Among them 15 proteins were upregulated in MDS/MPN-U patients, while 10 proteins were downregulated. These dysregulated proteins were involved in 10 major signaling pathways related with cell proliferation and immunity. The complicated interactive network was established by these proteins and pathways.</p><p><b>CONCLUSION</b>The differentially expressed signaling proteins screened from the MDS/MPN-U patients by PPA might be helpful to reveal the pathogenesis of MDS/MPN-U and to discover the therapeutic targets.</p>
Subject(s)
Humans , Cell Proliferation , Hematologic Neoplasms , Myelodysplastic-Myeloproliferative Diseases , Signal TransductionABSTRACT
This study was performed to prepare immobilized β-glucosidase and snailase, then optimize and compare the process conditions for conversion of icariin. Immobilized β-glucosidase and snailase were prepared using crosslink-embedding method. The best conditions of the preparation process were optimized by single factor analysis and the properties of immobilized β-glucosidase and snailase were investigated. The reaction conditions including temperature, pH, substrate ratio, substrate concentration, reaction time and reusing times of the conversion of icariin using immobilized β-glucosidase or snailase were optimized. Immobilized β-glucosidase and snailase exhibited better heat stabilities and could remain about 60% activity after storage at 4 degrees C for 4 weeks. The optimized conditions for the conversion of icariin were as follows, the temperature of 50 degrees C, pH of 5.0, enzyme and substrate ratio of 1 : 1, substrate concentration of 0.1 mg x mL(-1), reaction time of 6 h for β-glucosidase and 2 h for snailase, respectively. In 5 experiments, the average conversion ratio of immobilized β-glucosidase and snailase was 70.76% and 74.97%. The results suggest an effect of promoted stabilities, prolonged lifetimes in both β-glucosidase and snailase after immobilization. The immobilized β-glucosidase and snailase exhibited a higher conversion rate and reusability compared to the free β-glucosidase and snailase. Moreover, the conversion rate of immobilized snailase was higher than that of immobilized β-glucosidase. The process of icariin conversion using immobilized β-glucosidase and snailase was moderate and feasible, which suggests that immobilized enzymes may hold a promise for industrial usage.
Subject(s)
Enzymes, Immobilized , Chemistry , Flavonoids , Chemistry , Hydrolysis , Temperature , beta-Glucosidase , ChemistryABSTRACT
This study was aimed to investigate the genetic characteristics of human acute lymphoblastic leukemia cell line Molt-4, and evaluate its application in measuring telomere length by Flow-FISH. Molt-4 cell line was cultured in suspension and subcultured regularly. Eight different passages of Molt-4 cells in exponential stage were selected.The growth curves were drawn by cell counting method, meanwhile calculating the population doubling times of cells,DNA ploidies were determined by flow cytometry,karyotypes were analyzed by G-banding and telomere lengths were measured by Southern blot. The results showed that the population doubling time of Molt-4 cell line was (1.315 ± 0.062) d, DNA ploidy index was (2.085 ± 0.0093) , and the telomere length was (32.05 ± 5.27) kb. There were no significant difference among different passages (P = 0.931,0.888 and 0.935 separately). The karyotypes showed that the chromosome numbers of Molt-4 cell line were from 91 to 99 in different metaphases, and the majority of them were hypertetraploid, and stable and recurrent structural abnormalities of chromosomes could be kept. It is concluded that the stable genetic characteristics and the longer telomere length of Molt-4 cell line makes it be a feasible control cells in measurement of telomere length by Flow-FISH.
Subject(s)
Humans , Cell Line, Tumor , DNA, Neoplasm , Flow Cytometry , Karyotyping , Ploidies , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Genetics , Telomere , GeneticsABSTRACT
This study was aimed to investigate the feasibility of flow cytometry-fluorescence in situ hybridization (Flow-FISH) in measuring the telomere length of bone marrow cell subgroups in patients with myelodysplastic syndrome (MDS). Seven newly diagnosed patients with low-risk MDS and seven nutritional anemia patients who were matched with age and sex, were enrolled in this study. Heparinized bone marrow were sampled. Taking Molt-4 cell line as internal control cells, leukocytes isolated from whole bone marrow were labeled with CD34-Alexa Fluork ® 647, then denatured by high temperature and hybridized with FITC-conjugated telomere probe. The DNA was counterstained and the relative telomere length (RTL) of nucleated cells and CD34(+) cells in bone marrow were measured by four-color flow cytometry. The results showed that CD34(+) cells could be gated for the measurement of RTL in both groups, undergoing the denaturation and hybridization. Primary analysis indicated that the RTL of bone marrow CD34(+) cells in MDS patients was significantly shorter than that of bone marrow nucleated cells (P = 0.001), and the RTL of both CD34(+) cells and nucleated cells in bone marrow of MDS patients were significantly shorter than that of control group (P = 0.020, 0.002). It is concluded that the application of Flow-FISH in the measurement of RTL of certain cell subgroup is feasible by labeling the cell with thermostable fluorescence-conjugated antibody, and this technique is worthy to be investigated further.
Subject(s)
Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Antigens, CD34 , Allergy and Immunology , Bone Marrow Cells , Cell Biology , Allergy and Immunology , DNA , Flow Cytometry , Methods , In Situ Hybridization, Fluorescence , Methods , Myelodysplastic Syndromes , Genetics , Telomere , GeneticsABSTRACT
The objective of study was to evaluate the clinical values of multiparameter flow cytometry (MPFC) and cytomorphology of bone marrow aspiration(BMA) in detecting bone marrow involvement in patients with B cell Non-Hodgkin's lymphoma (B-NHL). 96 bone marrow samples from the patients with B-NHL were measured by MPFC using CD45/SSC and CD20/SSC gating strategy combined with anti-kappa and anti-lamda monoclonal antibodies, and then compared with results acquired by cytomorphologic analysis of BMA. The results showed that the bone marrow involvement was confirmed by MPFC in 38 cases (39.6%), while it was detected by cytomorphologic analysis of BMA only in 12 cases (12.5%). There was a significant difference between the two methods (p<0.05). 12 positive cases detected by cytomorphologic analysis of BMA were also positive by MPFC. There was no difference of 3-year overall survival rate between negative and positive cases detected by MPFC, but their 4-year overall survival rate was 73.18+/-6.65% and 44.13%+/-19.55% respectively (p<0.05). It is concluded that the MPFC is a more sensitive method for detecting bone marrow involvement in patients with B-NHL than cytomorphologic analysis of BMA. The 4-year overall survival rate of the patients without bone marrow involvement was significant higher than those of patients with bone marrow involvement. Bone marrow involvement in B-NHL detected by MPFC can be useful for clinical evaluation and prognosis prediction.
Subject(s)
Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Young Adult , Bone Marrow , Pathology , Flow Cytometry , Methods , Lymphoma, B-Cell , Pathology , Lymphoma, Non-Hodgkin , Pathology , Neoplasm Staging , Prognosis , Survival RateABSTRACT
The study was aimed to investigate the application value of interphase fluorescence in situ hybridization (FISH) on cell smears in hematological diseases. Both interphase FISH on peripheral blood smears and bone marrow smears treated by methanol/acetic acid, and routine interphase FISH of bone marrow cells dropped on slides were done at the same time, in order to detect Ph chromosome by BCR/ABL dual color, dual fusion probe in 20 patients with chronic myelogenous leukemia or acute lymphoblastic leukemia which had been proven to display Ph chromosome positive. The results indicated that as compared with routine interphase FISH, the interphase FISH on cell smears could also offer reliable result. It is concluded that interphase FISH on cell smears is a kind of reliable and time-saving technique, which is also suitable for retrospective research and worthy to further apply in clinic.
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Young Adult , Cytogenetic Analysis , Methods , Hematologic Diseases , Diagnosis , Genetics , In Situ Hybridization, Fluorescence , Methods , Interphase , GeneticsABSTRACT
This study was purposed to investigate the megakaryocytic dysplasia and leukemia-associated phenotypes (LAP) of acute myeloid leukemia (AML) in the elderly. The megakaryocytic dysplasia, lineage infidelity, asynchronous antigen expression, total WBC count, and karyotypes were observed in the 147 none M(3)-AML patients. Logistic regression were used to analyzed the difference between the elderly (age > or = 60) and the control. The results showed that out of the total 147 patients (66 elderly patients, and 81 younger patients) 124 patients accepted induction chemotherapy, in which 70 cases achieved complete remission (elderly 18, younger 52, p = 0.008); megakaryocytic dysplasia was found in 32 patients (21.8%); CD33 and CD19/CD7 (lineage infidelity) was co-expressed in 55 patients (37.4%), CD34 and CD11b (asynchronous antigen expression) was co-expressed in 65 patients (44.2%); white blood cell count > 25 x 10(9)/L was found in 52 patients (35.4%). By the Logistic regression, compared with the control, in the elderly patients there was difference in the megakaryocytic dysplasia, and the co-expression of CD33/CD19/CD7 and CD34/CD11b (OR = 4.315, 2.761, 0.397; p = 0.001, 0.006, 0.020), but there was no difference in the total WBC count and karyotypes (OR = 0.802, 1.096; p = 0.646, 0.813). It is concluded that the incidence of megakaryocytic dysplasia, such as lineage infidelity, and asynchronous antigen expression, in elderly patients is higher than that in younger patients.
Subject(s)
Adolescent , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Young Adult , Age Factors , Immunophenotyping , Leukemia, Myeloid, Acute , Allergy and Immunology , Pathology , Logistic Models , Megakaryocytes , Pathology , PrognosisABSTRACT
<p><b>BACKGROUND</b>Multiple sclerosis (MS) is a continuously disabling disease and it is unresponsive to high dose steroid and immunomodulation with disease progression. The autologous haematopoietic stem cell transplantation (ASCT) has been introduced in the treatment of refractory forms of multiple sclerosis. In this study, the clinical outcomes followed by ASCT were evaluated for patients with progressive MS.</p><p><b>METHODS</b>Twenty-two patients with secondary progressive MS were treated with ASCT. Peripheral blood stem cells were obtained by leukapheresis after mobilization with granulocyte colony stimulating factor. Etoposide, melphalan, carmustin and cytosine arabinoside were administered as conditioning regimen. Outcomes were evaluated by the expanded disability status scale and progression free survival. No maintenance treatment was administered during a median follow-up of 39 months (range, 6 to 59 months).</p><p><b>RESULTS</b>No death occurred following the treatment. The overall confirmed progression free survival rate was 77% up to 59 months after transplantation which was significantly higher compared with pre-transplantation (P = 0.000). Thirteen patients (59%) had remarkable improvement in neurological manifestations, four (18%) stabilized their disability status and five (23%) showed clinical recurrence of active symptoms.</p><p><b>CONCLUSIONS</b>ASCT as a therapy is safe and available. It can improve or stabilize neurological manifestations in most patients with progressive MS following failure of conventional therapy.</p>
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Hematopoietic Stem Cell Transplantation , Leukapheresis , Multiple Sclerosis , Therapeutics , Transplantation Conditioning , Transplantation, AutologousABSTRACT
The aim was to study the roles that the bone marrow mesenchymal stem cells (MSC) and cytokines play in cord blood CD34(+) cell expansion ex vivo and the influence of culture ex vivo on expression of the adhesive molecule of CD44. CD34(+) cells sorted from cord blood cells had been cultured in each well of 24 well culture plates containing culture medium supplemented with mesenchymal stem cells layer or/and cytokines for a week, and then all kinds of indexes of different groups were compared. The results showed that as for cord blood cell expansion, there was no significant difference between the groups with cytokines SDF-1alpha + SCF + TPO + FL and SCF + TPO + FL no matter if MSC layer existed or not. The groups with MSC layer and cytokines were superior to the corresponding groups without MSC layer. In addition, the expression of the adhesion molecule CD44 had no distinct change after culture. It is concluded that SDF-1alpha has no distinct influence on the effect of cytokines SCF + TPO + FL on cord blood cell expansion ex vivo. MSC enhance the effect of cytokines on cord blood cell expansion ex vivo. Such expansion ex vivo may not influence the expression of the adhesive molecule CD44 on cord blood cells.