Molecular analysis of interleukin 10 promoter region-592A/C polymorphism in hypertensive disorders of pregnancy in Qinghai Han nationality / 解剖学报

Objective To investigate the association between the interleukin 10 (IL-10) gene promoter region-592A/C (rs1800872) polymorphism and hypertensive disorders of pregnancy (HDP) in Han women of Qinghai province and to determine the expression of this gene in two groups (HDP group and healthy control group) preliminarily. Methods A total of 140 HDP patients (HDP group) and 140 normal pregnant women (control group) in Qinghai Province were selected. Using blood DNA as template, the IL-10-592A/C polymorphism typing of HDP group and control group was detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and verified by sequencing. The expression of IL-10 mRNA in the placental tissues of the two groups was detected by Real-time PCR. Plasma IL-10 levels of the two groups were detected by ELISA. Results The frequencies of AA, AC and CC genotypes of IL-10 gene in HDP group and control group were 24. 29%, 44. 29%, 31. 42% and 13. 57%, 41. 43%, 45. 00% respectively, the difference in genotype distribution between the two groups was statistically significant (P<0. 05);AA genotype frequency in HDP group(24. 29%)was higher than that of control group(13. 57%)(P<0. 05), CC genotype frequency in HDP group (31. 42%) was lower than that in control group (45. 00%) (P < 0. 05), while there was no significant difference in genotype frequency of AC between the two groups (P<0. 05); The distribution of A and C allele frequencies of IL-10592A/C polymorphism was different between the two groups, and the A allele frequency of HDP group was higher than that of control group (

Polymorphism of 5,10-methylenetetrahydrofolate reductase gene rsl801131 in Qinghai Han patients with hypertensive disorders complicating pregnancy / 解剖学报

Objective To investigate the correlation between the polymorphism of 5,10-methylenetetrahydrofolate reductase ( MTHFR) gene rs!801131 and hypertensive disorders complicating pregnancy ( HDCP ) in Qinghai Han nationality. Methods The polymorphism of MTHFR rsl801131 in 120 pregnant women with HDCP (HDCP group) and 120 normal pregnant women ( control group) were detected by restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP) and verified by sequencing. Results The frequencies of AA, AC, and CC genotype of MTHFR gene in the HDCP group were 56. 67% , 32. 50% , and 10. 83% respectively, and those in the control group were 74.17%, 23.33% and 2. 50% respectively (P<0. 05, the distribution of genotype was different significantly between the two groups). The frequency of AA genotype of HDCP group (56. 67%) was lower than that of control group (74. 17%, P<0. 05) , the frequency of CC genotype of HDCP group ( 10. 83%) was higher than that of control group ( 2. 50% , P< 0. 05) , while there was no significant difference in the frequency of AC genotype between HDCP group and control group ( P<0. 05). The frequency distribution of alleles A and C of MTHFR rsl801131 polymorphism was significantly different between the HDCP group and the control group (P<0. 001) , and the frequency of allele C in the HDCP group was significantly higher than that in the control group (X2 = 12. 229, 0R=L 574, 95% C/= 1. 181-2. 099, P<0. 001). Conclusion The polymorphism of MTHFR rsl801131 is related to the occurrence of HDCP in Qinghai Han population. The C gene might be the susceptibility gene of HDCP, and CC genotype might be the susceptibility genotype of HDCP.

Development of an eco-friendly and fast HPLC method for quantitative analysis of four nucleosides in Cordyceps and related products / 中国天然药物

An eco-friendly and fast HPLC method was developed for the determination of adenosine, inosine, guanosine and uridine in Cordyceps and related products (fermented mycelia of Hirsutella sinensis andPaecilomyces hepiali). The sample was ultrasonically extracted using 0.5% phosphoric acid solutions for 2.5 min. Sample separation was performed on a Poroshell SB-Aq column (50 mm × 4.6 mm, 2.7 μm) using eco-friendly mobile phase consisting of formic acid and ammonium formate aqueous solution at a flow rate of 1.0 mL·min

Association between the polymorphism of aldosterone synthase gene promoter-344T/C and preeclampsia in Qinghai Province / 解剖学报

Objective To investigate the relationship between preeclampsia (PE) and polymorphism of aldosterone synthase gene (CYP11B2) promoter region-344T/C in Qinghai Province. Methods A total of 120 PE subjects and 155 normal pregnancy subjects were studied. The genotype of CYP11B2 was analyzed by polymerase chain reaction fragment length polymorphism (PCR-RFLP). The mutation was confirmed by sequencing. Results The frequencies of CYP11B2 TT, CT and CC genotype in the PE group were 43.0%, 45.6%, and 11.4%, and in the control group were 51.0%, 45.1%, and 3.9%, respectively. There was difference in frequency distribution of CYP11B2 genotype between the PE and control groups. The frequency of C allele in the PE group was higher than the control group (χ

Profiling and identification of aqueous extract of Cordyceps sinensis by ultra-high performance liquid chromatography tandem quadrupole-orbitrap mass spectrometry / 中国天然药物

Characterization of aqueous extract in traditional Chinese medicine (TCM) is challenging due to the poor retention of the analytes on conventional C columns. This study presents a systematic characterization method based on a rapid chromatographic separation (8 min) on a polar-modified C (Waters Cortecs T3) column of aqueous extract of Cordyceps sinensis. UHPLC-HRMS method was used to profile components in both untargeted and targeted manners by full MS/PIL/dd-MS acquisition approach. The components were identified or tentatively identified by reference standards comparison, fragmentation rules elucidation and available databases search. A total of 91 components, including 10 nucleobases, 20 nucleosides, 39 dipeptides, 18 amino acids and derivatives and 4 other components, were characterized from the aqueous extract of C. sinensis. And this was the first time to systematically report the presence of nucleosides and dipeptides in C. sinensis, especially for modified nucleosides. The chemical basis inquiry of this work would be beneficial to mechanism exploration and quality control of C. sinensis and related products. Meanwhile, this work also provided an effective solution for characterization of aqueous extract in TCM.

Effects of different drying conditions on protein in Cordyceps / 中国中药杂志

In this study,the protein in different Cordyceps samples,which include fresh sample( S1),22 ℃ drying sample( S2),37 ℃ drying sample( S3) and 60 ℃ drying sample( S4),were analyzed by sodium dodecylsupinate-polyacrylamide gel electrophoresis( SDS-PAGE) and two-dimensional electrophoresis( 2-DE). The total protein contents in Cordyceps samples were from 1. 655-4. 493 mg·g~(-1) and the protein contents in fresh sample was the highest. The results of SDS-PAGE showed that the mainly ranges of protein molecular weight of Cordyces samples were 10-100 kDa and the numbers of protein bands were 28 to 41,the fresh sample had the maximum number of protein bands. The 2-DE profiles were analyzed by PDQuest software. The resulted indicated that 488-876 protein spots were detected in different Cordyceps samples and the isoelectric point( pI) was distributed between 4. 5 and 6. 5,the protein molecular weight was distributed in 10-20 kDa and 25-100 kDa,the fresh sample had the maximum number of protein spots. Therefore,the drying process could decrease contents and species of protein in Cordyceps,and the different drying conditions had different effects on protein. These results provide a reference for improving the drying process of Cordyceps.

Analysis of chemical components in Cordyceps by online gradient extraction-HPLC / 中国中药杂志

Online gradient extraction-high performance liquid chromatography( HPLC) method was developed for simultaneous determination of high and low polar components in Cordyceps. The sample powder of Cordyceps was uniformly mixed with diatomaceous earth,packed into extraction tank,and installed into the HPLC system. Online gradient extraction was conducted with mobile phase at 70 ℃. The separation was performed on Zorbax SB-AQ( 4. 6 mm×150 mm,5 μm) column with 0. 1% formic acid solution-methanol as the mobile phase for gradient elution at 1. 0 mL·min~(-1). The column temperature was 30 ℃,and detection wavelength was set at 260 nm. The results showed that the high and low polar components in Cordyceps could be simultaneously extracted and separated by the developed method. Meanwhile,six high polar compounds( uracil,uridine,thymine,inosine,guanosine and adenosine) and one low polar compound( ergosterol) were identified by comparison with the reference peaks. The established method is rapid,stable and environment friendly,which is helpful to improve the quality evaluation level for Cordyceps.

Rapid identification of Cordyceps and its products using DNA barcoding method / 中国中药杂志

Cordyceps is one of the most valuable traditional Chinese medicines. There are various counterfeits in markets because of high price and limited output. In this study,116 Cordyceps,146 hosts and 29 related products were collected and detected by using normal DNA barcoding technology and specific PCR method. The results indicated that Cordyceps and its adulterants could be distinguished from each other through DNA barcoding technology based on ITS and COⅠsequences. Two pairs specific primers ITSSF1/ITSSR1 and ITSSF2/ITSSR2 were developed to amplify 297 bp and 136 bp ITS regions of Cordyceps sinensis,respectively. It could be used to identify C. sinensis specifically and rapidly. Furthermore,specific primers ITSSF1/ITSSR1 and ITSSF2/ITSSR2 combined with ITS and COⅠsequences could differentiate powder Cordyceps from fermentation mycelia and could identify related products. Therefore,the method developed from this study could be applied to identify the powder of Cordyceps from fermentation mycelia and related products efficiently.

SDS-PAGE and 2-DE protein profiles of Ganoderma lucidum from different origins

Ganoderma lucidum [Chizhi in Chinese] is one of the most valuable and widely used medicinal fungi in traditional Chinese medicines [TCMs]. Most of previous studies were focused on the triterpenoids and polysaccharides of G. lucidum, whereas less attention had been paid on the protein, which is another bioactive compound in it. In the present study, protein maps of fourteen G. lucidum samples were comprehensively analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis [SDS-PAGE] and two-dimensional electrophoresis [2-DE]. The results indicated that there were significant differences in protein profiles of G. lucidum samples from different origins. Furthermore, previous reported bioactive proteins from the fruiting bodies of G. lucidum, were mainly distributed in 4 taxa [A, B, C and D] based on their molecular weights on the 2-DE maps. The proteins should be considered as marker for the quality control of G. lucidum, because the proteomic variation may affect on their pharmacological activities

Discovery of differential sequences for improving breeding and yield of cultivated Ophiocordyceps sinensis through ITS sequencing and phylogenetic analysis / 中国天然药物

To accelerate the breeding process of cultivated Ophiocordyceps sinensis and increase its yield, it is important to identify molecular fingerprint of dominant O. sinensis. In the present study, we collected 3 batches of industrially cultivated O. sinensis product with higher yield than the others and compared their internal transcribed spacer (ITS) sequences with the wild and the reported. The ITS sequence was obtained by bidirectional sequencing and analyzed with molecular systematics as a DNA barcode for rapid and accurate identification of wild and cultivated O. sinensis collected. The ITS sequences of O. sinensis with detailed collection loci on NCBI were downloaded to construct a phylogenetic tree together with the sequences obtained from the present study by using neighbor-joining method based on their evolution relationship. The information on collection loci was analyzed with ArcGIS 10.2 to demonstrate the geographic distribution of these samples and thus to determine the origin of the dominant samples. The results showed that all wild and cultivated samples were identified as O. sinensis and all sequences were divided into seven phylogenetic groups in the tree. Those groups were precisely distributed on the map and the process of their system evolution was clearly presented. The three cultivated samples were clustered into two dominant groups, showing the correlation between the industrially cultivated samples and the dominant wild samples, which can provide references for its optimized breeding in the future.

Discovery of differential sequences for improving breeding and yield of cultivated Ophiocordyceps sinensis through ITS sequencing and phylogenetic analysis / 中国天然药物

To accelerate the breeding process of cultivated Ophiocordyceps sinensis and increase its yield, it is important to identify molecular fingerprint of dominant O. sinensis. In the present study, we collected 3 batches of industrially cultivated O. sinensis product with higher yield than the others and compared their internal transcribed spacer (ITS) sequences with the wild and the reported. The ITS sequence was obtained by bidirectional sequencing and analyzed with molecular systematics as a DNA barcode for rapid and accurate identification of wild and cultivated O. sinensis collected. The ITS sequences of O. sinensis with detailed collection loci on NCBI were downloaded to construct a phylogenetic tree together with the sequences obtained from the present study by using neighbor-joining method based on their evolution relationship. The information on collection loci was analyzed with ArcGIS 10.2 to demonstrate the geographic distribution of these samples and thus to determine the origin of the dominant samples. The results showed that all wild and cultivated samples were identified as O. sinensis and all sequences were divided into seven phylogenetic groups in the tree. Those groups were precisely distributed on the map and the process of their system evolution was clearly presented. The three cultivated samples were clustered into two dominant groups, showing the correlation between the industrially cultivated samples and the dominant wild samples, which can provide references for its optimized breeding in the future.

Effects of cultivated Cordyceps sinensis on proliferation and apoptosis of human leukemia K562 cells / 中国中药杂志

The present study was designed to investigate the effect of cultivated Cordyceps sinensis (CCS) on leukemia-derived K562 cells, and further explore the underlying mechanisms. After routine culture of K562 cells, MTT assay was used to detect the effect of CCS on survivel of human leukemia cell lines K562;DAPI staining was used to observe the morphological changes of the nucleus and AO/EB staining was used to observe cell apoptosis. JC-1 staining was employed to detect the changes in mitochondrial membrane potential. Flow cytometry (FCM) was used to detect cell cycle distribution, and Western blot analysis was used to detect the expression levels of Bax, Bcl-2, caspase 3, caspase 8, cyclin D1, CDK2, and CDK4 in K562 cells. The results showed that CCS (0.345-5.524 g·L⁻¹) substantially suppressed proliferation of K562 cells and induced G₁/S phase arrest in a dose-dependent manner. DAPI and AO/EB staining indicated that cell apoptosis was significantly induced by CCS treatment, accompanied by decreased mitochondrial membrane potential demonstrated by JC-1 staining. Western blot results showed that CCS significantly increased the expression of Bax and, meanwhile, decreased the expression levels of Bcl-2, cyclin D1, CDK2, CDK4, caspase 3 and caspase 8. Collectively, our data demonstrated that CCS dose-dependently suppressed cell proliferation and induced cell apoptosis in K562 cells, and the mechanism might be associated with inducing cell cycle arrest, regulating Bcl-2/Bax ratio and activating the mitochondrial apoptosis pathway.

Simultaneous determination of cordycepin and 2'-deoxyadenosine in Cordyceps genus by online SPE-HPLC / 中国中药杂志

An online SPE-HPLC method for simultaneous determination of cordycepin (3'-deoxyadenosine) and 2'-deoxyadenosine in Cordyceps genus (C. sinensis,C. militaris,Hirsutella sinensis and C. sobolifera) was developed. The samples were enriched on a ZORBAX SB-AQ (4.6 mm×12.5 mm,5 μm) column with isocratic elution by 9% methanol solution. The separation of analytes was performed on a ZORBAX SB-AQ (4.6 mm×150 mm,5 μm) column with gradient elution by 0.1% formic acid solution and methanol (91∶9). The flow rate was 1.0 mL•min⁻¹. Column temperature was 40 ℃ and detection wavelength was 260 nm. This method has been applied for analysis of different Cordyceps genus. The 2'-deoxyadenosine was detected in C. sinensis,Hirsutella sinensis and C. sobolifera. The cordycepin was detected in C. militaris. In summary,the cordycepin chromatographic peak from C. sinensis in some past reports may be the 2'-deoxyadenosine chromatographic peak or the mixture peak of 2'-deoxyadenosine and cordycepin in which 2'-deoxyadenosine content was higher than cordycepin. The developed method is suitable for analysis of cordycepin and 2'-deoxyadenosine in Cordyceps genus.

Comparative study on specific chromatograms and main nucleosides of cultivated and wild Cordyceps sinensis / 中国中药杂志

This study is to establish the HPLC specific chromatogram and determine four main nucleosides of wild and cultivated Cordyceps sinensis. Uridine, inosine, guanosine and adenosine were selected as reference substance. HPLC analysis was performed on a Waters XSelect HSS T3 C₁₈ (4.6 mm×250 mm, 5 μm), with a mobile phase consisting of water(A)-acetonitrile (B) at a flow rate of 0.6 mL•min⁻¹ (0-5 min,0% B;5-15 min,0%-10% B, 15-30 min,10%-20% B, 30-33 min, 20%-50% B, 33-35 min, 50%-0% B, 35-40 min, 0% B). The detection wavelength was 260 nm and the column temperature was controlled at 30 ℃, and the injection volume was 5 μL. HPLC specific chromatogram of wild and cultivated C. sinensis was established and four main nucleosides were simultaneously determined by the above method. Specific chromatograms and contents of four main nucleosides showed no significant differences between cultivated and wild C. sinensis. These results can provide scientific evidences for further development and utilization of cultivated C. sinensis.

Clinical application of Acrysof Toric intraocular lens based on Sirius measurement data / 局解手术学杂志

Objective To evaluate the effect of corneal curvature based on Sirius measurement on the accuracy of implantation of Acrysof Toric intraocular lens (IOL) and astigmatism correction.Methods In this prospective study,23 patients (36 eyes) with cataract and mild to moderate corneal astigmatism were recruited and underwent phacoemulsification and Acrysof Toric IOL implantation.In the observation group,the corneal curvature value of 19 eyes measured by Sirius was used as the basis for calculating Acrysof Toric intraocular lens power.In the control group,the corneal curvature value of 17 eyes measured by manual corneal curvature was used as the basis for calculating Acrysof Toric intraocular lens power.Main outcome measurements including uncorrected visual acuity (UCVA),best corrected visual acuity (BCVA) surgically induced astigmatism(SIA),deviation of lens axis (LAD),index of corrected astigmatism (CAI) at 3 months postoperatively were observed.Results At 3 months postoperatively,the average UCVA of the observation group was (0.8 ± 0.11) and the average UCVA of the control group was (0.7 ± 0.73),there was no statistically significant differences (Z =1.277,P =0.372).The average BCVA of the observation group was (0.8 ± 0.21),and the average BCVA of the control group was (0.8 ± 0.41),there was no statistically significant differences (z =0.962,P =0.329).The deviation of lens axis of the observation group was (2.81 ± 1.72) ° and it was (3.12 ± 1.56)°in the control group,and there was no statistically significant differences (t =0.481,P =0.631).The index of corrected astigmatism of the observation group was (1.01 ± 0.21) and it was (1.03 ±0.32) in the control group,with no statistically significant differences (t =0.581,P =0.615).Conclusion Compared with the manual keratometer,Acrysof Toric IOL calculation based on Sirius measurement data is accurate and reliable.Meanwhile,Sirius has obvious advantages such as high resolution,non-contact,non-invasive and easy to operate.

Quantitative analysis of nucleosides in four Cordyceps genus by HPLC / 中国中药杂志

To compare the main nucleosides in Cordyceps genus herbs (C. sinensis, C. millitaris, Hirsutella sinensis and C. sobolifera), an HPLC method for simultaneous determination of uridine, inosine, guanosine, adenosine and cordycepine in Cordyceps genus herbs was developed. The sample was extracted with 0.5% phosphoric acid solution to prepare test solution. The separation was performed on a Zorbax SB-Aq (4.6 mm×150 mm, 5 μm) column with gradient elution by 0.04 mol•L⁻¹ potassium dihydrogen phosphate solution and acetonitrile, column temperature 30 ℃,flow rate 0.8 mL•min⁻¹,and detection wavelength 260 nm. The content of nucleosides in four Cordyceps genus herbs was evaluated by fingerprint analysis and hierarchical cluster analysis (HCA). The calibration curves of five nucleosides showed good linear regression (r>0.99) and the average recoveries were between 95.0% and 105.0%. The contents of the five nucleosides in the four Cordyceps genus herbs were different and could be obviously distinguished by HCA. The fingerprint analysis result showed that the similarity between C. sinensis and the others was less than 0.9. The method was accurate and reliable, which can be used for quality control of Cordyceps genus herbs.

Determination of ketamine and norketamine in blood and urine by high performance liquid chromatography / 法医学杂志

OBJECTIVE@#To develop a high-performance liquid chromatographic method for determination of ketamine and norketamine in blood and urine.@*METHODS@#The compounds were extracted from blood or urine by liquid-liquid extraction using toluene after blood or urine was adjusted pH to 14. The extracts were analyzed by HPLC.@*RESULTS@#Linear limits of ketamine and norketamine determination in blood ranged from 0.05 microg/mL to 10 microg/mL (R2 > 0.9993) and in urine ranged from 0.01 microg/mL to 200 microg/mL (R2 > 0.9995). Limits of detection (LODs) for ketamine and norketamine were 0.006 microg/mL and 0.003 microg/mL (S/N > or = 3), respectively. The mean extraction recovery was over 82.4% and its coefficients of variation were less than 10.0% for ketamine and norketamine. Concentration-time curves and urinary drug velocity curves of ketamine norketamine were obtained by determinations of them in blood and urine in rat using the developed method.@*CONCLUSION@#The method is sensitive, simple, rapid and suitable for determination of ketamine and norketamine in blood and urine for toxicological and clinical pharmaceutical analysis.

Analysis of paraquat in blood and urine by sodium borohydride/nickel chloride chemical reduction-gas chromatography/thermionic specific detector / 法医学杂志

OBJECTIVE@#To establish a new method for the analysis of paraquat in blood and urine by sodium borohydride/nickel chloride chemical reduction-gas chromatography/thermionic specific detector.@*METHODS@#An initial procedure of precipitation was performed by adding hydrochloric solution with sodium chloride and a mixture of chloroform and ethanol. Then the analyte contained in supernatant was reduced by a reduction system of sodium borohydride and nickel chloride and extracted by acetic ether. Ethyl paraquat (EPQ) was used as internal standard. GC/TSD was used to identify and quantify the analyte.@*RESULTS@#The limits of detection (S/N=3) in blood and urine were 0.002 and 0.004 microg/mL, respectively. The linear ranges were 0.050-30.0 microg/mL. Correlation coefficients in blood and urine were 0.999 and 0.998, respectively. The recoveries exceeded 80% both in blood and urine.@*CONCLUSION@#This method is applicable for quantification of paraquat in biological fluids.