Application of Two Dimensional Liquid Chromatography in Drug Analysis / 中国药学杂志

Two dimensional liquid chromatography (2D-LC) has become an important way to achieve the separation of complex samples due to higher peak capacity, higher selectivity and better separation ability compared with one-dimensional liquid chromatography(1D-LC). 2D-LC has been developing rapidly in many fields, such as medicine, food, metabolites in vivo or in vitro and so on. This review illustrates various separation modes of 2D-LC and its applications and developments, with emphases on the research of quality control of drugs.

HPLC determination of terbutaline sulfate impurity I / 中国药学杂志

OBJECTIVE: To establish a qualitative and quantitative HPLC method for the determination of impurity Iin tebutaline sulfate. METHODS: The Kromasil C18 column(4.6 mm×150 mm,5 μm) was used as the analysis column;buffer solution [dissolving 4.23 g of sodium hexanesulfonate in 770 mL of 0.050 mol·L-1 ammonium formate solution (pH 3)]-methanol (77:23) was used as the mobile phase. The flow rate was 1.0 mL·min-1, the column temperature was maitained at 30℃, the detection wavelength was set at 276 nm,and the injectiong volume was 20 μL. Area normalization method with correction factor was used for the quantitative analysis of the impurity I. RESULTS: Under the separation condition, the impurity I was completely separated from the principal components. The calibration curve showed good linearity in the concentration range of 0.10-579 μg·mL-1(r=1.000 0). The correction factor was 3.6. CONCLUSION: The area normalization method with correction factor developed in the paper can be used for the qualitative and quantitative analysis of the impurity Iin terbutaline sulfate, which can not only solve the problem of the availability of impurity reference standards, but also reflect the actual contents of impurity. The method provides an efficient and convenient method for quality control of terbutaline sulfate.

Analysis of related substances in valsartan capsules / 中国药学杂志

OBJECTIVE: To evaluate the status of related substances in valsartan capsules and find the existing problems. METHODS: One hundred and seventy-five batches of samples from eight manufacturers were tested using statutory methods combined with exploratory research,and the results were evaluated by statistical analysis. RESULTS: The related substances in 174 batches of valsartan capsules met the requirement of current specification, while one batch did not. For the batch of substandard sample, the amount of an individual impurity was 1.0% and the total amount was 1.1%, exceeding the corresponding limits of 0.2% and 0.7%, respectively. The unknown impurity was detected in samples from two manufacturers in the exploratory studies,but not detected in valsartan API, suggesting that the unknown impurity may be degradation product, rather than process impurity. The structure of the unknown impurity was identified by LC-MS/MS, and the reason for its generation was elucidated through preparation process research. CONCLUSION: The related substances in valsartan capsules are in good control overall and have met the requirement of current statutory specification. The individual manufacturer should strengthen process control to ensure the stability of the products by inference of unqualified samples.

Analysis of TERT and TIN2 Gene Expression in Patients with Acquired Aplastic Anemia and Their Correlation with Pathogenesis / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To detect the mRNA expression levels of TERT and TIN2 in peripheral blood mononuclear cells of acquired aplastic anemia(AAA) patients, and to explore their correlation with pathogenesis of acquired aplastic anemia.</p><p><b>METHODS</b>Peripheral blood mononuclear cells of 40 cases of AAA including 33 cases of non-severe aplastic anemia(NSAA), 7 cases of severe aplastic anemia (SAA) and 20 subjects as control group were collected to detect mRNA expression of TERT and TIN2 by using real-time quantitative polymerase chain reaction(RT-qPCR), the correlation of TERT and TIN2 mRNA expression levels with classification of peripheral blood cells were analyzed.</p><p><b>RESULTS</b>The expression levels of TERT and TIN2 mRNA in patients with AAA were lower significantly than those in control group (P<0.05), and the SAA (P<0.01). The expression levels of TERT and TIN2 mRNA in patients with SAA were all lower significantly than those in patients with NSAA (P<0.05). The expression levels of TIN2 mRNA in patients with NSAA were lower significantly than those in control group (P<0.01). There were no significant difference in the expression level of TERT mRNA between patients with NSAA and control group (P=0.082). There was significant correlation between the expression level of TERT mRNA and red blood cells count (r=0.437, P=0.029), and hemoglobin level (r=0.522, P=0.007). There was significant correlation between the expression levels of TIN2 mRNA and the lymphocyte percentage (r=-0.404, P=0.045).</p><p><b>CONCLUSION</b>The expression level of TERT mRNA may be associated with the red blood cells and hemoglobin level. The expression level of TIN2 mRNA may be associated with the lymphocyte percentage.</p>

Holotransferrin enhances selective anticancer activity of artemisinin against human hepatocellular carcinoma cells / 华中科技大学学报（医学）（英德文版）

Artemisinin, also termed qinghaosu, is extracted from the traditional Chinese medicine artemesia annua L. (the blue-green herb) in the early 1970s, which has been confirmed for effectively treating malaria. Additionally, emerging data prove that artemisinin exhibits anti-cancer effects against many types of cancers such as leukemia, melanoma, etc. Artemisinin becomes cytotoxic in the presence of ferrous iron. Since iron influx is high in cancer cells, artemisinin and its analogs selectively kill cancer cells with increased intracellular iron concentrations. This study is aimed to investigate the selective inhibitory effects of artemisinin on SMMC-7721 cells in vitro and determine the effect of holotransferrin, which increases the concentration of ferrous iron in cancer cells, combined with artemisinin on the anticancer activity. MTT assay was used for assessing the proliferation of SMMC-7721 cells treated with artemisinin. The induction of apoptosis and inhibition of colony formation in SMMC-7721 cells treated with artemisinin were determined by TdT-mediated dUTP nick end labeling (TUNEL) and colony formation assay, respectively. The results showed that artemisinin at various concentrations significantly inhibited growth, colony formation and cell viability of SMMC-7721 cells (P<0.05), likely due to induction of apoptosis of SMMC-7721 cells. Of interest, it was found that incubation of artemisinin combined with holotransferrin sensitized the growth inhibitory effect of artemisinin on SMMC-7721 cells (P<0.01). Our data suggest that treatment with artemisinin leads to inhibition of viability and proliferation, and apoptosis of SMMC-7721 cells. Furthermore, we observed that holotransferrin significantly enhanced the anti-cancer activity of artemisinin. This study may provide a potential therapeutic choice for liver cancer.

Holotransferrin enhances selective anticancer activity of artemisinin against human hepatocellular carcinoma cells / 华中科技大学学报（医学）（英德文版）

Artemisinin, also termed qinghaosu, is extracted from the traditional Chinese medicine artemesia annua L. (the blue-green herb) in the early 1970s, which has been confirmed for effectively treating malaria. Additionally, emerging data prove that artemisinin exhibits anti-cancer effects against many types of cancers such as leukemia, melanoma, etc. Artemisinin becomes cytotoxic in the presence of ferrous iron. Since iron influx is high in cancer cells, artemisinin and its analogs selectively kill cancer cells with increased intracellular iron concentrations. This study is aimed to investigate the selective inhibitory effects of artemisinin on SMMC-7721 cells in vitro and determine the effect of holotransferrin, which increases the concentration of ferrous iron in cancer cells, combined with artemisinin on the anticancer activity. MTT assay was used for assessing the proliferation of SMMC-7721 cells treated with artemisinin. The induction of apoptosis and inhibition of colony formation in SMMC-7721 cells treated with artemisinin were determined by TdT-mediated dUTP nick end labeling (TUNEL) and colony formation assay, respectively. The results showed that artemisinin at various concentrations significantly inhibited growth, colony formation and cell viability of SMMC-7721 cells (P<0.05), likely due to induction of apoptosis of SMMC-7721 cells. Of interest, it was found that incubation of artemisinin combined with holotransferrin sensitized the growth inhibitory effect of artemisinin on SMMC-7721 cells (P<0.01). Our data suggest that treatment with artemisinin leads to inhibition of viability and proliferation, and apoptosis of SMMC-7721 cells. Furthermore, we observed that holotransferrin significantly enhanced the anti-cancer activity of artemisinin. This study may provide a potential therapeutic choice for liver cancer.

Clinicopathologic features of papillary tumors of the pineal region / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the clinicopathologic features of papillary tumor of the pineal region (PTPR).</p><p><b>METHOD</b>Three hundred and eighty six cases of pineal region and posterior third ventricle tumors, two newborn and two adult pineal glands were analyzed by HE, PAS and immunohistochemistry of 16 antibodies (EnVision method).</p><p><b>RESULTS</b>Five cases of PTPR were diagnosed with mixed papillary features and densely cellular areas, and included one recurrent case. In the papillary areas, the vessels were lined by one or several layers of cuboidal/columnar cells; the vessel wall was hyalinized. In the densely cellular areas, sheets or nests of tumor cells were seen. The tumor cells of these five cases were immunoreactive to CK, CK8/18, synaptophysin, MAP2, nestin, S-100, and vimentin. Four cases were immunoreactive to NSE and CgA; and 2 cases were immunoreactive to NF. All five cases were negative for EMA, CK5/6, CEA, and NeuN. Ki-67 labeling index ranged from 1% to 6%.Three patients were alive, and the recurrent one died.</p><p><b>CONCLUSIONS</b>PTPR occurs in patients with over a wide age range, from children to adults, and is more commonly found in male than female. PTPR is composed of both papillary and solid areas, characterized by epithelial cytology, and needs to be differentiated from ependymoma. PTPR may originate from the specialized ependymocytes of the subcommissural organ. The prognostic factors are early diagnosis, complete surgical resection and radiotherapy.</p>

Expression of TRF1, TRF2 and RAP1 mRNA in peripheral blood mononuclear cells of patients with acquired aplastic anemia / 中国实验血液学杂志

This study was aimed to investigate the expression levels of TRF1, TRF2 and RAP1 mRNA in peripheral blood mononuclear cells of patients with acquired aplastic anemia, and to explore their relation with onset of acquired aplastic anemia. Peripheral blood mononuclear cells of 40 patients with acquired aplastic anemia and 20 normal subjects as control were collected to detect mRNA expression of TRF1, TRF2 and RAP1 by using real-time quantitative polymerase chain reaction. The results showed that the expression levels of TRF1 and RAP1 in peripheral blood mononuclear cells of patients with acquired aplastic anemia were significantly higher than that in normal controls (P < 0.05), while the expression level of TRF2 was lower than that in normal controls (P < 0.01). There was significant correlation between TRF2 and RAP1 expressions level (r = 0.522, P = 0.001). It is concluded that the changes in expression levels of TRF1, TRF2 and RAP1 may play a role in the pathogenesis of acquired aplastic anemia.

Expression and significance of P53 protein and MDM-2 protein in human gliomas / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>P53 is one of the most studied tumor suppressors in the cancer research, and over 50% of human tumors carry P53 mutations. MDM-2 is amplified and/or overexpressed in a variety of human tumors of diverse tissue origin. The aim of this study was to examine the expression of P53 protein and MDM-2 protein in gliomas, and to investigate the relationship between the expression of the two proteins and the histopathological grades of glioma. The relationship between MDM-2 protein expression and P53 protein expression was also analyzed.</p><p><b>METHODS</b>The expression of P53 protein and MDM-2 protein was immunohistochemically detected using monoclonal antibodies in 242 paraffin embedded tissues, including 30 normal brain tissues from patients with craniocerebral injury and 212 tissues from patients with primary glioma (grade I - II group: 5 cases of grade I, 119 cases of grade II; and grade III--IV group: 53 cases of grade III, and 35 cases of grade IV).</p><p><b>RESULTS</b>The P53 positive rate was significantly higher in the glioma groups than in the control group (P < 0.0001). The P53 positive rate was significantly higher in glioma tissues of grade III - IV than in glioma tissues of grade I - II group (P = 0.001). The MDM-2 positive rate was significantly higher in glioma groups than in the control group (P < 0.0001). There was no significant difference in the MDM-2 positive rate between the two glioma groups (P = 0.936). The expression of P53 protein was not related to expression of MDM-2 protein (P = 0.069)</p><p><b>CONCLUSIONS</b>Overexpression of P53 protein might be related to the occurrence and progression of glioma. Overexpression of MDM-2 protein may play an important role in glioma tumorigenesis, but may not be involved in glioma progression. The overexpression of MDM-2 protein was an early event in malignant transformation of glioma. MDM-2 may be a key player in glioma in its own right.</p>

Regulation mechanism of breast cancer resistance protein by toremifene to reverse BCRP-mediated multidrug resistance in breast cancer cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To explore the regulation mechanism of the reversal of breast cancer resistance protein-mediated multidrug resistance by toremifene.</p><p><b>METHODS</b>Two recombinant plasmids (pcDNA3-promoter-BCRP and pcDNA3-CMV-BCRP) were designed to express the wild-type full-length BCRP cDNA enforced driven by its endogenous promoter containing a functional ERE and a CMV promoter as control, respectively. Two recombinant plasmids were transfected into ERα-positive MCF-7 and ERα-negative MDA-MB-231 breast cancer cell lines. Four kinds of BCRP expressing cell lines of MCF-7/Promoter-BCRP, MCF-7/CMV-BCRP, MDA-MB-231/Promoter-BCRP and MDA-MB-231/CMV-BCRP were established in which BCRP was promoted by the BCRP promoter and a CMV promoter as control, respectively. The drug resistant cells were treated with toremifene. Then RT-PCR, Western blot, mitoxantrone efflux assays and cytotoxicity assay were performed to detect the reversal function of BCRP by toremifene on the drug resistance cell lines.</p><p><b>RESULTS</b>Toremifene significantly downregulated BCRP mRNA levels in a dose-dependent manner in ERα-positive MCF-7/Promoter-BCRP cells than that of untreated control cells. In MCF-7/Promoter-BCRP cells, toremifene at the dose of 0.1, 1 and 10 µmol/L decreased BCRP mRNA expression by 29.5% (P < 0.05), 68.1% (P < 0.01) and 97.4% (P < 0.01), respectively. After being treated with toremifene and 17β-estradiol, the BCRP mRNA level in MCF-7/Promoter-BCRP cells was 64.2% ± 1.3%, significantly higher than that of toremifene treatment control cells (3.8% ± 0.2%,P < 0.01). Furthermore, the effect of toremifene on BCRP protein is similar in BCRP mRNA. Toremifene obviously increased the mitoxantrone fluorescence intensity and decreased the efflux activity by 47.3% (P < 0.05) in MCF-7/promoter-BCRP cells when compared with the untreated control, whereas intracellular accumulation of mitoxantrone obviously decreased and the efflux activity increased by 61.5% were observed in combination with 17β-estradiol when compared with toremifene treatment alone. The results therefore suggested that toremifene reversed mitoxantrone resistance in MCF-7/Promoter-BCRP cells. However, in MCF-7/CMV-BCRP, MDA-MB-231/Promoter-BCRP and MDA-MB-231/CMV-BCRP cells, toremifene or in combination with 17β-estradiol did not affect intracellular mitoxantrone uptake.</p><p><b>CONCLUSION</b>Taken together, our findings indicate that expression of BCRP is downregulated by toremifene, via a novel transcriptional mechanism which might be involved in the ERE of BCRP promoter through ER-mediated to inactivate the transcription of BCRP gene.</p>

Research of ginsenosides in kou zi qi using HPLC-MS-MS / 药学学报

<p><b>AIM</b>To analyze the ginsenosides in Kou zi qi (rhizomes of Panax japonicus C. A. Mey. var. major (Burkill) C. Y. Wu et K. M. Feng), and to supply evidences for chemotaxanology of Panax species and clinical uses of Kou zi qi.</p><p><b>METHODS</b>The ginsenosides were isolated by HPLC, then the positive- and negative-ion API-MS/MS of constituents collected from HPLC were measured.</p><p><b>RESULTS</b>Eight ginsenosides were identified as ginsenoside Re, ginsenoside Ro, chikuseksusaponins IV, IVa, notoginsenoside R2, ginsenosides Rb1, Rc and Rd, respectively, based on comparison of retention time with those of standards by HPLC, and analysis on their API-MS/MS data. Ginsenoside Ro and chikuseksusaponin IVa are the major components of Kou zi qi.</p><p><b>CONCLUSION</b>This plant had a close relationship to P. stipuleanatus, P. zinginensis and P. japonicus var major; a relatively remote relationship to P. ginseng and P. quinquefolius, in a view of chemotaxanology. Ginsenoside Ro and chikuseksusaponin IVa might be the anti-inflammatory constituents of Kou zi qi.</p>

Clinical observation on treatment of acute myelocytic leukemia by supplementing qi, nourishing yin and clearing heat principle / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the clinical efficacy of TCM with supplementing Qi, nourishing Yin and clearing heat principle (SQNYCH) combined with chemotherapy in treating myelocytic leukemia.</p><p><b>METHODS</b>One hundred and fourteen patients were randomly divided into the treated group (n = 68) and the control group (n = 46). To the treated group, SQNYCH was applied as the basic treatment, with combined chemotherapeutic protocol, using DA, HA and IA, to induce remission, and to the M3 patients, all-trans retinoic acid and arsenic trioxide were given. As for patients in the control group, only western medicine was administered.</p><p><b>RESULTS</b>In the treated group 49 patients (72.1%) were completely remitted, 9 (13.2%) partially remitted and the total remission rate being 85.3%, which was significantly different from that in the control group. After treatment, the blood and bone marrow picture were obviously improved in both groups, but the increase of hemoglobin and platelet were better in the treated group than in the control group (P < 0.05 or P < 0.01). Immune functions were enhanced in both groups, but the elevation of CD4, CD4/CD8 ratio and NK cells were higher in the treated group than in the control group (P < 0.05 and P < 0.01).</p><p><b>CONCLUSION</b>Application of SQNYCH principle in treating acute myelocytic leukemia could elevate the clinical efficacy, which is of great value in clinical practice.</p>