Purification and component identification of total proanthocyanidins in Choerospondias axillaris pericarp / 中国中药杂志

The present study determined the quantitative markers of total proanthocyanidins in the purification of the industrial waste Choerospondias axillaris pericarp based on the comparison results of high-performance liquid chromatography(HPLC) and mass spectrometry(MS) and optimized the purification process with two stable procyanidins as markers. The adsorption and desorption of five different macroporous adsorption resins, the static adsorption kinetics curve of NKA-Ⅱ resin, the maximum sample load, and the gradient elution were investigated. The UPLC-Q-TOF-MS/MS was employed for qualitative analysis of the newly-prepared total proanthocyanidins of C. axillaris pericarp. As revealed by the results, NKA-Ⅱ resin displayed strong adsorption and desorption toward total proanthocyanidins. The sample solution(50 mg·mL~(-1)) was prepared from 70% ethanol crude extract of C. axillaris pericarp dissolved in water and 7-fold BV of the sample solution was loaded, followed by static adsorption for 12 h. After 8-fold BV of distilled water and 6-fold BV of 10% ethanol were employed to remove impurities, the solution was eluted with 8-fold BV of 50% ethanol, concentrated, and dried under reduced pressure, and purified total proanthocyanidin powder was therefore obtained. Measured by vanillin-hydrochloric acid method, the purity and transfer rate of total proanthocyanidins were 47.67% and 59.92%, respectively, indicating the feasibi-lity of the optimized process. UPLC-Q-TOF-MS/MS qualitative analysis identified 16 procyanidins in C. axillaris total proanthocyanidins. The optimized purification process is simple in operation and accurate in component identification, and it can be applied to the process investigation of a class of components that are difficult to be separated and purified. It can also provide technical support and research ideas for the comprehensive utilization of industrial waste.

Comparative of functional components, antioxidant and α-glucosidase inhibition activities between Choerospondias axillaris fruit peel vinegar and apple vinegar / 中国中药杂志

Based on the idea of plant metabolomics, ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS/MS) was used to compare the chemical composition between 6 batches of fruit vinegar brewed from Choerospondias axillaris fruit peel and 6 batches of apple vinegar purchased from 3 companies. Antioxidant and α-glucosidase inhibition activities were also tested in vitro. A total of 43 compounds were identified by reference substance, liquid chromatography-mass spectrometry(LC-MS/MS) fragmentation information or literature data. A total of 40 compounds were identified in the C. axillaris fruit peel vinegar. A total of 16 compounds were identified in apple vinegar. There were 13 common ingredients including organic acids and esters such as citric acid, 2-isopropyl malic acid, and triethyl citrate. The results of partial leastsquares-discriminant analysis(PLS-DA) indicated that they had 33 significantly different compounds such as proanthocyanidin oligomer, quercetin-3-O-rhamnoside and heptadecanoic acid. The proanthocyanidins and flavonoid glycosides in C. axillaris peel vinegar were more abundant than apple vinegar, so it had better health function than ordinary fruit vinegar. The results showed that C. axillaris fruit peel vinegar had stronger antioxidant and α-glucosidase inhibition activities in vitro. The vinegar brewed from waste C. axillaris fruit peel had more chemical ingredients than the apple vinegar. C. axillaris fruit peel vinegar had better biological activity and health function, so it had good development prospect. This study provided the scientific evidence for exploiting the C. axillaris fruit peel into high value-added products. It also provided ideas for the comprehensive development and utilization of similar Chinese medicine waste.

Screening Serum Differential Proteins for Childhood Asthma at Different Control Levels by Isobaric Tags for Relative and Absolute Quantification-based Proteomic Technology / 中国医学科学院学报

Objective To screen serum differential proteins for childhood asthma at different control levels,which provided the basis for the prevention and treatment of childhood asthma. Methods Isobaric tags for relative and absolute quantification,two-dimensional liquid chromatography,nanoelectrospray ionization,and high-resolution tandem mass spectrometry using the hybrid quadrupole time-of-flight platform was used to screen the differential proteins in serum samples from pediatric patients with controlled,partly controlled,or uncontrolled childhood asthma. Differential proteins were validated using enzyme-linked immunosorbent assay (ELISA). Results A total of 260 expressed proteins were identified. Among them 57 differentially expressed proteins were found among the different control levels of childhood asthma (fold<0.8 or fold>1.2). The differentially expressed proteins were involved mainly in 21 biological processes and 8 molecular functions and were located in 17 cellular components. ELISA showed that the serum vitronectin level was significantly higher in controlled group [(573.92±412.43) μg/ml] than in uncontrolled group[(382.27±238.64)]μg/ml (P=0.0399). Conclusion We identified 57 differential proteins for childhood asthma at different control levels,which may be used as potential biological targets for the control of childhood asthma.

In Vivo and In Vitro Inhibitory Effects of Icariin on Angiogenesis / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the inhibitory effects of icariin(ICA),an active ingredient of Herb Epimedii,on angiogenesis.</p><p><b>METHODS</b>The chick chorioallantoic membrane(CAM)assay was adopted to evaluate the effects of various doses of the ICA on the angiogenesis. The cell growth inhibitory effect of ICA on human umbilical vein endothelial cells(HUVEC)was measured by MTT assay. Cell cycle arrest and the induction of apoptosis were evaluated by flow cytometry. The effect of ICA on the migration of HUVEC cells was measured on Transwell model.</p><p><b>RESULTS</b>ICA remarkably inhibited angiogenesis in CAM in a concentration-dependent manner. The proliferation of HUVEC cells was inhibited by ICA, and the effect was time-and concentration-dependent. ICA-treated HUVEC cells showed cell cycle arrest;180 μg/ml of ICA decreased the percentage of migrating HUVEC cells by 78.0%.</p><p><b>CONCLUSION</b>ICA can effectively suppress angiogenesis;however,its in vivo inhibitory effect on angiogenesis warrants further investigations.</p>

Prokaryotic expression of extracellular ligand binding domains of chick tie-2 and its anti-angiogenesis effect / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the prokaryotic expression of extracellular ligand binding domains of chick tie-2, the purification, refolding conditions of the recombinant protein, and its anti-angiogeneic effect.</p><p><b>METHODS</b>A DNA fragment encoding extracellular ligand binding domains of chick tie-2 was obtained by PCR amplification using a previous constructed plasmid as a template. The amplified fragment was then inserted into prokaryotic expression vector pQE30, and was expressed in E.Coli XL-1 blue by adding isopropyl-beta-D-thiogalactoside(IPTG). The recombinant protein in inclusion bodies was purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography under denatured conditions. Then the refolding of the purified protein was performed with gradient dialysis. The target protein was injected s.c. into mouse, and the antibody was detected by ELISA and Western blot analysis. The antibody was purified from the antiserum and then incubated with human umbilical endothelial vein cell (HUEVC) to find its anti-angiogenesis in vitro by using propidium iodide(PI) dying through FACS. Alginate encapsulated tumor cell assays were performed and micro-vessel density was determined by counting per high power field in the sections stained with an antibody reactive to CD31 to test its inhibition of angiogenesis.</p><p><b>RESULTS</b>The recombinant protein was highly expressed in E.Coli XL-1 blue, and the antibody produced in mouse could specifically recognize the recombinant protein. The purified antibody could induce apoptosis of HUEVC in vitro. The anti-angiogenic effect of the antibody could also be found in alginate-encapsulate tumor cell assay and by counting micro-vessel density.</p><p><b>CONCLUSION</b>The protein of extracellular ligand binding domains of chick tie-2 can be expressed at high level in the prokaryotic expression system, and the expressed protein can induce immune response in mouse. Furthermore, the antibody can induce the anti-angiogenic effect.</p>

Inhibition of tumor growth and metastasis via local administration of recombinant human endostatin adenovirus / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>The growth and metastasis of solid tumors are dependent on angiogenesis. Endostatin, the C-terminal proteolytic fragment of collagen XVIII, is a potent endogenous angiogenesis inhibitor. The authors designed a topical antiangiogenic gene therapy with recombinant human endostatin adenovirus (Ad-hEndo) and assessed its effects on the inhibition of angiogenesis in vitro, and tumor growth and metastasis in vivo.</p><p><b>METHODS</b>Malignant cells (A549) were infected with Ad-hEndo. The expression of recombinant protein and the inhibition of cultured human umbilical vein endothelial were investigated. Immunodeficient A549 nude mice were treated with intratumoral injection of Ad-hEndo, the empty vector Ad-control or saline (NS). The dose-response, side effects, and serum concentration of endostatin were observed.</p><p><b>RESULTS</b>Recombinant endostatin protein was detected in the infected tumor cells with different MOI Ad-hEndo and its inhibitory effect on endothelial cells growth was shown. In animal study, the volume of tumor and the number of pulmonary metastatic lesions in the Ad-hEndo treatment group were significantly smaller than those in the control groups (P<0.05).</p><p><b>CONCLUSION</b>The present findings provide evidence of the anti-tumor effects of the endostatin and may be important for the further use of it in topical antiangiogenic gene therapy of cancer.</p>

Diagnosis and Treatment of 82 Children with Ovarian Disease / 实用儿科临床杂志

Objective To investigate the morbidity,clinical feature,diagnosis and therapy of ovarian disease in children.Methods Eighty-two children with ovarian disease were admitted and treated in Nanjing children's hospital from Jan.1992 to Jan.2007,were analyzed retrospectively with age,emergency admissions or not,dwell,pathology and method of operation.Results The age of 82 patients ranged from 1 day to 14 years old and the mean age was 6.7 years old.Thirty-one cases(37.8%) were emergency admissions and 51 cases(62.2%) were routine admissions.Twenty-seven cases(32.9%) were rural patients and 55 cases(67.1%) were urban patients.Forty-five cases(54.8%) were nontumorous disorder,31 cases(37.8%) were benign tumor and only 6 cases(7.4%) were malignant tumor.About the morbidity,12 patients(14.6%)were admitted from 1992 to 1996,24 patients(29.5%) from 1997 to 2001 and 46 patients(55.9%) from 2002 to 2007.Chemotherapy were carried out in 6 cases with malignant tumor in internal medicine,2 cases with sexual precosity kept observation,the others were cured.Conclusions Ovarian disease can occur at any age in children.The clinical manifestation is characterized mainly by acute abdomen.The incidence of ovarian disease of children in urban areas is higher than that children in rural areas.The morbidity continues to show an upward tendency and the pathologic manifestations are mostly benign,laparoscopic operation has obviously superiority.