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OBJECTIVE To establish the fingerprint and multi-component content determination method of Crataegus pinnatifida leaves from different producing areas， and to evaluate the quality of C. pinnatifida leaves and screen the differential markers. METHODS Seventy-eight batches of C. pinnatifida leaves were collected from Chengde of Hebei Province， Huludao of Liaoning Province， Yuncheng of Shanxi Province and Linyi of Shandong Province. High-performance liquid chromatography （HPLC） and Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints （2012 edition） were used to draw the fingerprints and conduct similarity evaluation. Grey correlation analysis， cluster analysis （CA）， principal component analysis （PCA） and orthogonal partial least squares-discriminant analysis （OPLS-DA） were performed by using SPSS 19.0， MetaboAnalyst 5.0 and SIMCA 14.1 software. The differential markers affecting the quality of C. pinnatifida leaves were screened with variable importance in the projection （VIP） value greater than 1 and the error line not exceeding the origin as the criterion. Using vitexin rhamnoside as an internal reference， the contents of chlorogenic acid， glucosylvitexin， hypericin and isoquercetin in 78 batches of C. pinnatifida leaves were determined by the same HPLC combined with quantitative analysis of multi- components by single-marker （QAMS）， and the results were compared with external standard method. RESULTS Eight common peaks were calibrated in the fingerprints for 78 batches of C. pinnatifida leaves from 4 producing areas. Five known components were identified， including chlorogenic acid （peak 1）， glucosylvitexin （peak 3）， vitexin rhamnoside （peak 4）， hypericin （peak 7） and isoquercetin （peak 8）； their similarities ranged from 0.871 to 0.998. Average relative correlations of samples from Chengde of Hebei Province， Huludao of Liaoning Province， Yuncheng of Shanxi Province and Linyi of Shandong Province were 0.538， 0.528， 0.462 and 0.435， respectively. CA and PCA showed that the samples from Chengde of Hebei Province and Huludao of Liaoning Province were roughly classified into one category， while the samples from Linyi of Shandong Province and Yuncheng of Shanxi Province were roughly classified into one category； VIP values of peak 1， 2， 3 and 5 were all greater than 1. By QAMS， the relative correction factors of chlorogenic acid， glucosylvitexin， hypericin and isoquercetin were 0.401， 0.993， 1.670 and 1.615 （RSD＜2%）. Compared with external standard method， except for isoquercetin in the two batches of samples （S39 and S41）， there was no significant difference in the content of each component in other batches of samples （the relative deviations≤ 5%）. CONCLUSIONS The established fingerprint and QAMS method are simple to operate and can be used to evaluate the quality of C. pinnatifida leaves. The sample from Chengde of Hebei Province is relatively good in quality. Chlorogenic acid （peak 1）， glucosylvitexin （peak 3）， and the corresponding components of peaks 2 and 5 may be differential markers affecting the quality of C. pinnatifida leaves.
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OBJECTIVE To determi ne the contents of total fla vonoids i n Scutellaria barbata standard decoction ，evaluate in vitro antioxidant activity ，establish the fingerprint and conduct chemical pattern recognition analysis. METHODS The contents of total flavonoids in S. barbata standard decoction （calculated by scutellarein ）were determined by ultraviet-visible spectrophotometry. In vitro antioxidant activity of S. barbata standard decoction was investigated by free radical scavenging tests of 1，1-diphenyl- 2-trinitrophenylhydrazine（DPPH）and 2，2′-azino-bis（3-ethylbenzothiazoline-6-sulfonic acid ）ammonium salt （ABTS）；HPLC method was adopted. Using scutellarin as reference ，the fingerprints of 16 batches of S. barbata standard decoction were drawn and evaluated by Similarity Evaluation System of TCM Chromatogram Fingerprint （2004 A edition ），and the common peaks were determined；Pearson correlation analysis was carried out by using SPSS 24.0 software to screen substances with in vitro antioxidant activity. Taking them as variables ，cluster analysis and principal component analysis were carried out by using SPSS 24.0 and SIMCA 14.1 software. RESULTS The linear range of total flavonoids were 2.106-21.06 μg/mL（R2＝0.999 3）；RSDs of precision ， reproducibility and stability tests （120 min）were all lower than 2％；the recovery was 100.62％（RSD＝0.55％，n＝6）；the contents of total flavonoids were 0.634-1.053 mg/mL. Median inhibitory concentration （IC50） of DPPH radical scavenging experiment ranged 1.120-3.602 mg/mL，and IC 50 of ABTS radical scavenging e xperiment range d 0.684-1.327 mg/mL. The results of correlation analysis showed that the content of total flavonoids Δ 基金项目 ：河北省高校省级重点学科建设项目 （No.冀教 in S. barbata standard decoction was negatively correlated 高〔2013〕4号）；承德医学院自然科学研究计划项目（No.201824） ＊讲师，硕士。研究方向：中药质量控制 。电话：0314-2291186。 with the IC 50 of DPPH free radical and ABTS free radical E-mail：duyilongww@sina.com scavenging experiment ，and the correlation coefficients were # 通信作者 ：教授，硕士。研究方向 ：中药质量控制 。电话： －0.976 and －0.940 respectively（P＜0.01）. There were 18 0314-2291186。E-mail：phf2301@163.com common peaks in the fin gerprints of 16 batches of S. barbata 中国药房 2022年第33卷第4期 China Pharmacy 2022Vol. 33 No. 4 ·425· standard decoction ；the s imilarities were 0.964-0.997. A total of 4 common peaks were identified ，such as scutellarin （peak 8）， scutellarein（peak 14），luteolin（peak 15），apigenin（peak 17）.In the HPLC fingerprints of S. barbata standard decoction ，the peak areas of peak 3-4，8-9，12-15 and 17 were significantly negatively correlated with the IC 50 of DPPH free radical and ABTS free radical scavenging experiment （P＜0.05）. The results of cluster analysis showed that 16 batches of S. barbata standard decoction could be clustered into two categories ，of which S 2，S7-S8 and S 14-S16 were clustered into one category ，S1，S3-S6 and S 9-S13 were clustered into one category. By principal component analysis ，16 batches of S. barbata standard decoction were divided into two categories ，of which S 2，S4，S7 and S 14-S16 were clustered into one category ，and S 1，S3，S5-S6 and S 8-S13 were clustered into one. The comprehensive scores were high in the samples of S 4，S13，S15. CONCLUSIONS Established HPLC fingerprint and chemical pattern recognition analysis method can be used to evaluate the quality of S. barbata standard decoction ； peak 3-4，8-9，12-15 and 17 and total flavonoids are the potential material basis for S. barbata standard decoction to scavenge DPPH free radical and ABTS free radical.
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OBJE CTIVE To establish a method for quality evaluation of Xin ’an capsule by combining fingerprint ， multi-component quantitative analysis and chemical pattern recognition analysis. METHODS High performance liquid chromatography（HPLC）combined with Similarity Evaluation System of TCM Chromatogram Fingerprint （2012 edition）were used to establish the fingerprints of 24 batches of Xin ’an capsules and evaluate the similarity. The common peaks were determined. The contents of glucosylvitexin ，rhamnosylvitexin，vitexin，hyperoside and isoquercetin in Xin ’an capsules were determined by the same HPLC method. Taking the common peak area of fingerprint as the variable ，MetaboAnalyst 5.0 tool was used to draw the cluster analysis （CA）heat map. SIMCA 14.1 software was used to perform principle component analysis （PCA）and partial least squares-discriminant analysis （PLS-DA）. RESULTS Twelve common peaks were identified with the similarity greater than 0.97. Six common peaks were identified as chlorogenic acid ，glucosylvitexin，rhamnosylvitexin，vitexin，hyperoside and isoquercetin.The linear range of glucosylvitexin ，rhamnosylvitexin，vitexin， hyperoside and isoquercetin were 2.36-151.35，9.15-585.20， 1.20-76.50， 0.68-43.20， 0.44-27.90 µg/mL（all r＞0.999）.RSDs of precision ，repeatability and stability （24 h）tests were 163.com all less than 2.00％ . The average recoveries were 95.80％（RSD＝0.96％ ，n＝6），102.10％ （RSD＝0.93％ ，n＝6）， 103.26％（RSD＝1.28％，n＝6），103.89％（RSD＝0.73％，n＝6） and 102.09％（RSD＝1.79％，n＝6），respectively. The contents of the five components were 0.988 8-1.559 1，4.336 6-11.220 1， 0.065 1-0.830 5，0.043 8-0.692 5 and 0.023 2-0.427 2 mg/grain，respectively. The results of CA and PCA showed that 24 batches of samples could be divided into three categories ，i.e. S 1-S15，S16-S18 and S 19-S24. PLS-DA showed that variable importance in projection values of the corresponding component of peak 6 and glucosylvitexin （peak 7），rhamnosylvitexin（peak 8），hyperoside （peak 10） and isoquercetin （peak 11） were greater than 1. CONCLUSIONS The established HPLC fingerprint and multi-component quantitative method are simple and feasible. Combined with chemical pattern recognition analysis ，it can be used for the quality control of Xin ’an capsules. Glucosylvitexin ，rhamnosylvitexin and other components may be differentital markers affecting the quality of each batch of samples.
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Objective To explore whether tumor necrosis factor receptor-associated protein 1 (TRAP1)gene copy number variation was associated with susceptibility and clinical characteristics of systemic lupus erythematosus (SLE).Methods The study enrolled 304 SLE patients and 391 healthy controls.They were used to investigate the association between TRAP1 gene copy number variation and SLE susceptibility.Then,304 SLE patients were divided into copy number=2 group and copy number＞2 group to study the association between TRAP1 gene copy number variation and disease activity or clinical characteristics of SLE.AccuCopyTM Kit was used to detect the TRAP1 gene copy number.Data analyses were performed by SPSS 10.01 software.The suitable method was selected among t test,rank sum test and x2 test for analysis based on the data type and distribution,univariate and multivariate logistic regression analysis were performed to investigate the associ-ation between TRAP1 gene copy number variation and susceptibility and clinical characteristics of SLE.Results The copy number variation of TRAP1 gene showed an association with the susceptibility to SLE crude OR=5.257,95％CI (1.108,24.937),P=0.037;the adjusted OR=5.578,95％CI (1.172,26.556),P=0.031].There was no association between TRAP1 gene copy number variation and SLE disease activity index (SLEDAI) score (Z=-0.117,P=0.907).The copy number variation of TRAP1 gene had a marginal association with skin lesions in SLE [OR=0.130,95％CI (0.016,1.069),P=0.058],but it disappeared after adjusting for potential confounders [OR=0.288,95％CI (0.029,2.831),P=0.286,PBH=0.808].There was no correlation between TRAP1 gene copy number variation and arthritis,alopecia,oral ulcers,fever,hematologic disorder,lupus nephritis as well as photosensitivity in SLE [x2=0.751,OR=1.234,95％CI (0.767,1.988),P=0.386].No multiplicative interaction was found between TRAP1 gene copy number variation and age or body mass index (BMI) [age:x2=0.751,OR=1.234,95％CI (0.767,1.988),P=0.386;BMI:x2=0.282,OR=1.172,95％CI(0.652,2.109),P=0.596].Conclusions The copy number variation of TRAP1 gene may be associated with susceptibility to SLE.Increased TRAP1 gene copy number may be a risk factor for SLE.
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Objective To investigate the effect of urate-lowering therapy on renal function in chronic kidney disease (CKD) stages 2-5 patients with hyperuricemia (HUA).Methods A total of 132 patients of CKD stages 2-5 with HUA between July 2016 and December 2017 in Department of Nephrology of the Second Affiliated Hospital of Anhui Medical University were prospectively and self-controlled analyzed.Serum uric acid (SUA),estimated glomerular filtration rate (eGFR) and other clinical parameters were measured at baseline and after 1-6 months treatment.The patients were divided into group A (CKD stages 2-3a) and group B (CKD stages 3b-5) on the baseline value of eGFR.The changes of SUA and eGFR before and after treatment were compared.According to the level of SUA after 6 months treatment,patients were divided into attainment group (SUA ＜ 360 μmol/L) and nonattainment group (SUA ≥ 360 μmol/L).The difference of renal function in pre-treatment and post-treatment was compared.Multiple stepwise linear regression was used to analyze the relationship among the change of eGFR after receiving 6 months' treatment (deGFR) and SUA level,baseline eGFR and other indexes.Results After 1,3,6 months treatment,the average levels of SUA,Scr and urea nitrogen of all patients were decreased significantly while eGFR value was increased significantly (all P ＜ 0.050) than those in pre-treatment period.After six-month-therapy,proteinuria and hematuria were improved significantly in all patients (P ＜ 0.001,P=0.001).Compared with pre-treatment period,both the SUA levels of group A and group B were declined significantly while eGFR had a significant rise after treatment (P ＜ 0.001).The change of eGFR post-treatment in group A was significantly higher than that of group B [(13.64±15.35) vs (8.97±9.79) ml· min-1· (1.73 m2)-1,P=0.044].At 6 months after treatment,the eGFR value increased markedly in both attainment group and nonattainment group compared with pre-treatment period (P ＜ 0.001).After six-month-therapy,the eGFR value in attainment group was increased more obviously than that of nonattainment group [(13.96 ± 14.64) vs (8.03±9.69) ml· min-1· (1.73 m2)-1,P=0.021].Multiple stepwise linear regression analysis showed that the baseline eGFR value was an influencing factor of deGFR (b=0.161,P=0.020).Conclusions The renal function of CKD stages 2-5 patients with HUA can be significantly improved by urate-lowering therapy,which can effectively reduce proteinuria and hematuria.
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Objective:To study the HPLC fingerprint of Xinan capsules from different manufacturers, and establish the chemical pattern recognition method by using principal component analysis and cluster analysis in order to provide reference for the quality con-trol of Xinan capsules. Methods:The HPLC chromatographic column was Agilent ZORBAX SB-C18 (250 mm × 4. 6 mm, 5 μm);the mobile phase was 0.1% formic acid(A)-acetonitrile(B)– tetrahydrofuran(C) with gradient elution, the flow rate was 1.0 ml· min-1;the detection wavelength was 350 nm and the column temperature was 30 ℃. Totally 15 batches of samples were analyzed by the Evaluation System of Traditional Chinese Medicine Chromatographic Fingerprint Similarity (2004A version) and SPSS 19. 0 statisti-cal software. Results:According to the results of cluster analysis and principal component analysis, 10 batches of Xinan capsules were screened out, and the fingerprint common pattern was established. Conclusion:The method is accurate and reliable, and can be used to control the quality of Xinan capsules.
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OBJECTIVE:To establish a method for the simultaneous determination of chlorogenic acid,vitexin glucoside,vi-texin rhamnoside,vitexin,rutin and hyperoside in Crataegus pinnatifida. METHODS:With reference peak of vitexin glucoside, HPLC was conducted to calculate the relative correction factor(RCF)of chlorogenic acid,vitexin glucoside,vitexin rhamnoside, vitexin,rutin and hyperoside,then the contents of above-mentioned 5 components in C. pinnatifida were calculated. The column was Agilent ZORBAX SB C18 with mobile phase of 0.1% formic acid-acetonitrile-tetrahydrofuran (gradient elution) at a flow rate of 1.0 ml/min,the detection wavelength was 350 nm,column temperature was 30 ℃,and the injection volume was 10 μl. RE-SULTS:The linear range was 12.50-400.0 μg for chlorogenic acid(r=0.999 8),25.00-800.0 μg for vitexin glucoside(r=0.999 9), 31.25-1 000.0 μg for vitexin rhamnoside(r=0.999 9),6.470-260.0 μg for vitexin(r=0.999 9),2.50-80.0 μg for rutin(r=0.999 8) and 9.375-300.0 μg for hyperoside(r=0.999 9);RSDs of precision,stability and reproducibility tests were lower than 2.0%;re-coveries were 99.2%-103.9%(RSD=1.6%,n=6),97.9%-100.8%(RSD=1.2%,n=6),99.2%-100.8%(RSD=0.5%,n=6), 97.3%-101.3%(RSD=1.5%,n=6),98.0%-103.0%(RSD=1.9%,n=6)and 95.5%-101.5%(RSD=2.2%,n=6). RCFs of vitex-in glucoside with chlorogenic acid,vitexin rhamnoside,vitexin,rutin and hyperoside were 1.119,1.009,0.706,1.063 and 0.830, respectively. CONCLUSIONS:The method is simple with good precision,stability and reproducibility,and it can be sued for the simultaneous determination of 6 components in C. pinnatifida.
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Gluconobacter oxydans converts glucose to gluconic acid and subsequently to 5-keto-D-gluconic acid (5-KGA), a precursor of industrially important L(+)-tartaric acid. To increase the yield of 5-KGA, fermentation conditions of 5-KGA production was optimized. Under the optimum medium and culture conditions in the shake flask, the highest 5-KGA production reached 19.7 g/L, increased by 43.8% after optimization. In a 5-L bioreactor, the pH was controlled at 5.5 and dissolved oxygen (DO) at 15%, 5-KGA production reached 46.0 g/L, raised at least 1.3 times than in the shake flask. Glucose feeding fermentation process was further developed, and the highest 5-KGA production of 75.5 g/L with 70% of yield was obtained, 32.0% higher than the highest reported value. Therefore, this newly developed fermentation process provided a practical and effective alternative for the commercial production of 5-KGA, and further of L(+)-tartaric acid.
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Bioreactors , Fermentation , Gluconates , Metabolism , Gluconobacter oxydans , Metabolism , Glucose , Metabolism , Industrial Microbiology , Tartrates , MetabolismABSTRACT
The cis-epoxysuccinate hydrolase (CESH) from Rhizobium strain BK-20 is the key enzyme for L(+)-tartaric acid production. To establish a highly efficient and stable production process, we first optimized the enzyme production from Rhizobium strain BK-20, and then developed an immobilized cell-culture process for sustained production of L(+)-tartaric acid. The enzyme activity of free cells reached (3 498.0 +/- 142.6) U/g, and increased by 643% after optimization. The enzyme activity of immobilized cells reached (2 817.2 +/- 226.7) U/g, under the optimal condition with sodium alginate as carrier, cell concentration at 10% (W/V) and gel concentration at 1.5% (W/V). The immobilized cells preserved high enzyme activity and normal structure after 10 repeated batches. The conversion rate of the substrate was more than 98%, indicating its excellent production stability.
Subject(s)
Alginates , Chemistry , Cells, Immobilized , Glucuronic Acid , Chemistry , Hexuronic Acids , Chemistry , Hydrolases , Metabolism , Rhizobium , Metabolism , Tartrates , MetabolismABSTRACT
Objeetive To explore the differentiation of B lymphocytes and expression of B7-related protein-1 (B7RP-1)on B lymphocytes in patients with systemic lupus erythematosus(SLE).Methods Three-color immunofluorescent staining and flow cytometric assay were used to analyze the frequency of three types of B lymphocytes,I.e.,plasma cells,memory B lyphocytes and naive B lymphocytes,as well as the expression of B7RP-1 on these cells in peripheral blood from 23 patients with SLE and 16 normal human controls.Clinical data of these patients with SLE were collected.and SLE disease activi index(SLEDAI)was also evaluated.The relationship was assessed between the expression of B7RP-1 and SLEDAI.Results The frequency of plasma cells was highest in patients with active SLE.followed by patients with inactive SLE and normal human controls(P＜0.01).A significant decrease was observed in the frequency of memory B lymphocytes in patients with active SLE compared with normal controls (P＜0.01),but no significant difference was found between patients with inactive SLE and those with active SLE(P＞0.05).Regarding the frequency of naive B lymphocytes,there was no significant difierence among the three groups.Increased frequency of plasma cells was also noted in patients with lupus nephritis(LN)compared with those without LN [(6.15±3.12)%vs(3.31±1.41)%,P＜0.05 ],but no significant difierence was found with regard to the frequency of memory or naive B lymphocytes between these two groups.The expression rate Of B7RP-1 was significantly lower on total lymphocytes from patients with SLE than from normal human controls (46.51%vs 63.75%,P＜0.05),which was the case with B7RP-1 on plasma cells,memory B lyphocytes and naive B lymphocytes (all P＜0.01),whereas no significant difierence was found between patients with inactive SLE and active SLE or between patients with and without LN.In addition.no correlation was found between the expression of B7RP-1 and SLEDAI(r=0.035,P＞0.05).Conclusions In peripheral blood of patients with SLE,the frequency of plasma cells is increased,while the expression of B7RP-1 on lymphocytes is decreased,which may be relevant to the pathogenesis of SLE.
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Objective: To establish a determination for psoralen and isopsoralen in Longbishu Capsules (Fructus Psoraleae, Herba Leonuri, Succinum, Pseudobulbus Cremastrae seu pleiones, Herba Lysimachiae, Spora Lygodii). Methods : TLC-Scanning was used. Results : The average recovery of psoralen was 97.4% and RSD was 3.06%,respectively, and isopsoralen was 97.6% and RSD was 2.72%,respectively. Conclusion : The method can be used for quality control of Longbishu Capsules.