Effects of Monoammonium Glycyrrhizinate on Stem Cell-like Characteristics, Oxidative Stress and Mitochondrial Function of Acute Promyelocytic Leukemia NB4 Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of monoammonium glycyrrhizinate on the stem cell-like characteristics, oxidative stress and mitochondrial function of acute promyelocytic leukemia cells NB4.@*METHODS@#CCK-8 method was used to detect the viability of acute promyelocytic leukemia cells NB4, and the appropriate dose was screened; Cloning method was used to detect the proliferation rate of NB4 cell; Western blot was used to detect the expression of cell cycle-related protein; flow cytometry was used to detect cell apoptosis and sort NB4 stem cells positive (CD133+); Stem cell markers (Oct4, ABCG2, Dclk1) were detected by RT-PCR; ROS was detected by fluorescence; The kit was used to detect the level of oxidative stress markers (MDA); The flow cytometry was used to detect the change of mitochondrial membrane potential; Western blot was used to detect the expression of mitochondrial damage index-related proteins (Bax/BCL-2).@*RESULTS@#Compared with the control group, if the concentration of MAG was less than 5 μmol/L, the cell NB4 viability showed no significant difference; if the concentration was higher than 5 μmol/L, the inhibitory effect on the growth of cell NB4 increased and showed significant difference (P<0.05), according to the results of CCK-8 experiment, four groups were set based on the concentration of MAG 0 μmol/L, MAG 5 μmol/L, MAG 10 μmol/L, and MAG 20 μmol/L; compared with the control group (MAG 0 μmol/L), the cells in MAG 5 μmol/L group showed no significant difference, while the proliferation rate, cyclin expression, mitochondrial membrane potential, stem cell CD133+ ratio, and marker mRNA level ( Oct4, ABCG2, Dclk1) of NB4 cell were significantly reduced (P<0.05); the apoptosis rate, reactive oxygen species, MDA content and Bax/BCL-2 expression of NB4 cell significantly increased (P<0.05).@*CONCLUSION@#Monoammonium glycyrrhizinate has a significant inhibitory effect on acute promyelocytic leukemia cells NB4, which may be related to the regulation of stem cell-like characteristics, oxidative stress and mitochondrial function.

Simultaneous determination of seven constituents in Chenxiang Lubailu tablet by HPLC / 药学实践与服务

Objective To establish the method for the simultaneous determination of liquiritin, ammonium glycyrrhizinate, hesperidin, nobiletin;tangeretin;costunolide, dehydrocostuslactone in Chenxiang Lubailu tablet by HPLC. Methods ZORBAX Eclipse XDB-C18 chromatographic column (4.6 mm×250 mm, 5 μm) was used. The mobile phase was methanol-0.1% phosphoric acid solution. Gradient elution with flow rate of 1.0 ml/min was used. Column temperature was 35 ℃. Detection wavelength for liquiritin, ammonium, tangeretin, and costunolide was at 237 nm. Detection wavelength for glycyrrhizinate was at 283 nm. Detection wavelength for hesperidin and nobiletin was at 330 nm. Injection volume was 10 μl. 16 batches of samples were tested. Results The linear ranges for the detection of liquiritin, ammonium, glycyrrhizinate, hesperidin, nobiletin, tangeretin, and costunolide were 1.110 - 55.72 (r=0.9992), 22.15 - 1108 (r=0.9999), 6.140 - 307.2 (r=0.9995), 1.130 - 56.25 (r=0.9997), 0.3700 - 18.75 (r=0.9982), 0.5200 - 26.01 (r=0.9991), and 1.180 - 58.95 (r=0.9999) μg/ml respectively. The average recoveries were 98.71%, 98.12%, 98.44%, 98.22%, 99.17%, 99.18%, and 97.93%, and the RSDs were 0.16%, 0.67%, 0.57%, 0.62%, 0.48%, 0.56%, and 0.58% respectively. The contents of the seven components in 16 batches of samples were 0.1250 - 1.174, 2.354 - 7.426, 1.822 - 27.21, 0.0370 - 1.399, 0.0723 - 0.4433, 0.0140 - 0.1990, and 0.2207 - 1.407 mg/g respectively. Conclusion The method is accurate, reproducible and durable, which could be used to the quality control and evaluation of Chenxiang Lubailu tablet.

Quality control of Tibetan medicine Baimai Ointment based on multi-component determination, fingerprint and multivariate statistical analysis / 中草药

Objective: To establish a fingerprint method of Baimai Ointment (BO) and determine the content of its main components. The BO of 14 batches from two production areas was scientifically and comprehensively evaluated based on multivariate statistical analysis, which provided the basis for the quality control. Methods: HPLC method was used to determine the content of liquiritin, ammonium glycyrrhizinate, nardosinone and curcumin in BO, and the fingerprint of BO was established. The fingerprint similarity analysis, cluster analysis, principal component analysis (PCA) and factor analysis were performed to comprehensively evaluate the different batches of BO in two producing areas. Results: The methodological determination of liquiritin, ammonium glycyrrhizinate, nardosinone and curcumin met the requirements, and the content was 0.051-0.200 mg/g, 0.136-0.622 mg/g, 0.030-0.345 mg/g, 0.001-0.069 mg/g, respectively. The established fingerprints of BO were calibrated with 17 common peaks. Three chromatographic peaks of liquiritin, ammonium glycyrrhizinate and nardosinone were identified by reference. The similarity of 14 batches of sample was greater than 0.975. In the cluster analysis, 14 batches of BO from two producing areas can be divided into four categories, among which batches S1-S11 produced by Linzhi City of Tibet were grouped into one category. And S12 produced in Lanzhou City of Gansu Province was clustered into one class, and S13 was clustered into one class, S14 was grouped into one class. The results of PCA and factor analysis showed that the comprehensive scores of the three batches of S12-S14 produced in Lanzhou City of Gansu Province were higher than the 11 batches of S1-S11 produced by Linzhi City of Tibet, presumably because of the changes in production conditions or sources of medicinal materials. The result was consistent with cluster analysis. Conclusion: This study is the first to establish a scientific and reliable quality control method of Tibetan medicine BO based on multi-component determination, fingerprint and multivariate statistical analysis. It can be used not only for the quality control of Baimai Ointment, but also for the comprehensive evaluation of batch quality consistency. It provides reference for the improvement of the quality standard of BO and the quality evaluation among Chinese medicine batches.

Diammonium glycyrrhizinate promotes the regeneration and repair of central nervous system in rats with severe traumatic brain injury by Wnt/β-catenin signaling pathway / 中华危重病急救医学

Objective@#To observe the effects of diammonium glycyrrhizinate (DG) on nerve regeneration repair in rats with severe traumatic brain injury (STBI) from the perspective of Wnt/β-catenin signaling pathway.@*Methods@#Seventy-two Sprague-Dawle (SD) male rats were randomly divided into normal group, STBI model group, ganglioside (GA) treatment group and DG treatment group. The STBI animal model was reproduced referring to modified Feeney free fall impact model. No injury was made in normal group. Six hours after modeling, monosialotetrahexosylganglioside sodium injection and DG injection were injected via tail vein of rats in GA treatment group and DG treatment group respectively, once a day for 7 days. Normal group and STBI model group were given the same amount of normal saline. Six rats in each group were sacrificed on the 1st, 3rd and 7th day after the challenge for neurological severity score (NSS), and then the blood of abdominal aorta was drawn and brain tissue was harvested. The contents of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in serum were detected by enzyme linked immunosorbent assay (ELISA). The pathological changes of sub-granular zone (SGZ) were observed under light microscope after hematoxylin eosin (HE) staining. Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the mRNA expressions of Wnt3a, β-catenin, glycogen synthetase kinase-3β (GSK-3β) and Axin.@*Results@#① There was no neurological deficit in the normal group and NSS was 0. NSS score of rats increased significantly on the first day after modeling, and then decreased gradually over time. NSS of the rats treated with GA and DG were significantly lower than that of the STBI model rats (score: 7.33±2.07, 6.17±2.23 vs. 9.33±1.63, both P < 0.01). Though NSS gradually decreased over time, the differences were still statistically significant on the 7th day (score: 2.67±0.82, 1.00±0.00 vs. 6.17±2.23, both P < 0.01), and NSS of DG treatment group was significantly lower than that of GA treatment group. ② In SGZ of rats, cells were arranged in a compact and orderly way in the normal group, but neurons and tissues were damaged and destroyed at different time points in the STBI model group. After either GA or DG treatment, the damage of nerve tissue was improved gradually over time, and the effect of DG was more obvious.③ In the normal group, the mRNA expressions of Wnt3a and β- catenin were almost not expressed, the mRNA expressions of GSK-3β and Axin were higher, and the contents of BDNF and NGF in serum were less. On the 1st day after STBI, the mRNA expressions of Wnt3a and β- catenin in hippocampus, the contents of BDNF and NGF in serum were significantly increased, and the mRNA expressions of GSK-3βand Axin were significantly decreased. The mRNA expressions of Wnt3a and β- catenin in the hippocampus and the contents of BDNF and NGF in serum were significantly higher than those in the model group 1 day after GA or DG was added, the mRNA expressions of GSK-3β and Axin were significantly decreased, and the effect of DG was more significant than that of GA [Wnt3a mRNA (2-ΔΔCt): 3.51±0.14 vs. 2.93±0.05, β- catenin mRNA (2-ΔΔCt): 1.90±0.08 vs. 1.75±0.04, BDNF (ng/L): 4.06±0.55 vs. 3.16±0.64, NGF (ng/L): 9.53±1.08 vs. 7.26±0.43, GSK-3β mRNA (2-ΔΔCt): 0.75±0.01 vs. 0.79±0.01, Axin mRNA (2-ΔΔCt): 0.74±0.02 vs. 0.76±0.02, all P < 0.05]. It was gradually increasing or decreasing over time and the difference was still statistically significant up to the 7th day.@*Conclusion@#DG can promote the recovery of nerve function in rats with STBI, and its mechanism may be related to the regeneration of nerve cells proliferation and differentiation by Wnt/β-catenin signaling pathway and the reconstruction of nerve tissue in SGZ of hippocampus.

Establishment of HPLC fingerprint and determination of three components of classical Linggui Zhugan Decoction / 中草药

Objective: To establish an HPLC fingerprint of classical named Linggui Zhugan Decoction and determination method of the content of three compounds, and provide reference for the study of the material basis of the classical Linggui Zhugan Decoction. Methods: The method was performed by high performance liquid chromatography with CNW Athena C18 (250 mm × 4.6 mm, 5 μm) and gradient elution with acetonitrile (A)-0.1% phosphoric acid (B) as the mobile phase, gradient elution: 0-10 min, 5%-10%; 10-20 min, 10%-15% A; 20-50 min, 15%-30% A; 50-75 min, 30%-40% A; 75-95 min, 40%-50% A; 95-111 min, 50%-95% A. The flow rate was 1.0 mL/min, the injection volume was 10 μL, and the column temperature is 30 ℃. The fingerprint and the detection wavelength of glycyrrhizin and ammonium glycyrrhizinate were 230 nm, and cinnamic aldehyde was 290 nm. The chromatographic fingerprint evaluation system published by the State Pharmacopoeia Commission (2012 Edition) was used to establish the fingerprint of 10 batches of Lingguizhugan decoction, and the content of three kinds of index components were determined simultaneously by the established HPLC method. Results: The research on the 10 batches of Lingguizhugan soup showed that the fingerprint similarity was greater than 0.9, and 24 common peaks were calibrated, and the peak resolution was good. The content determination results showed that the content of glycyrrhizin and ammonium glycyrrhizinate was high. According to the methodological investigation, the three components showed a good linear relationship within a certain concentration range; The precision RSD values were all less than 1.0%; The average recovery rates of glycyrrhizin, cinnamaldehyde and ammonium glycyrrhizinate were 98.35%, 101.51%, and 102.59%, respectively. The RSD is less than 2.5%; The sample is stable within 48 h, and the method has good repeatability. Conclusion: The fingerprint method and the determination method of three components in the traditional Chinese medicine classical prescription named Linggui Zhugan Decoction established in this study are simple, stable, accurate and reliable, and can be used for the quality control of the Linggui Zhugan Decoction.

Effect of different decocting methods on active ingredients in Banxia Xiexin Decoction / 中草药

Objective To investigate the effects of the three methods of decocting with deslag, decocting without deslag, and double decocting on the content of nine ingredients baicalin, baicalein, ginsenoside Re, ginsenoside Rb1, monoammonium glycyrrhizinate hydrate, liquiritin, 6-gingerol, berberine hydrochloride, palmatine hydrochloride, and total flavonoids in Banxia Xiexin Decoction (BXD). Methods Nine index components were determined by HPLC. The HPLC analysis was performed on Welch Ultimate XB-C18 column (250 mm × 4.6 mm, 5 μm) with mobile phase of acetonitrile-0.1% phosphate aqueous solution for gradient elution; And carried out at column temperature of 28 ℃, volume flow of 0.9 mL/min, and detection wavelength of 203, 252, 280, and 355 nm. The total flavonoids were determined by colorimetry. Results Nine kinds of ingredients and total flavonoids could be detected in three different decoctions. In the method of decocting with deslag, baicalin, baicalein, ginsenoside Rb1, monoammonium glycyrrhizinate hydrate, and liquiritin increased by 10.01%, 12.88%, 29.09%, 16.75%, and 15.02%, respectively, compared with decocting without deslag; It decreased by 5.54%, 4.15%, 14.49%, 7.85%, and 9.18%, respectively compared with double decocting; Ginsenoside Re, 6-gingerol, berberine hydrochloride, and palmatine hydrochloride increased by 37.90%, 3.78%, 5.33%, and 5.99% compared with decocting without deslag, respectively; compared to the double decocting methods, it increased by 1.07%, 11.57%, 3.41%, and 1.93%. The total flavonoids increased 22.61% higher than decocting without deslag and 6.54% higher than double decocting. Conclusion: The results can effectively reflect the quality difference of different decocting methods. Among the three methods of decoction, the method of decocting without deslag has significantly improved the dissolution of the active ingredients of each component in the decoction, and improve the clinical efficacy of BXD to a certain extent. It provides a good experimental basis for the decocting without deslag method used in Zhang Zhongjing’s Treatise on Febrile Diseases.

Diammonium glycyrrhizinate promotes the regeneration and repair of central nervous system in rats with severe traumatic brain injury by Wnt/β-catenin signaling pathway / 中华危重病急救医学

Objective To observe the effects of diammonium glycyrrhizinate (DG) on nerve regeneration repair in rats with severe traumatic brain injury (STBI) from the perspective of Wnt/β-catenin signaling pathway. Methods Seventy-two Sprague-Dawle (SD) male rats were randomly divided into normal group, STBI model group, ganglioside (GA) treatment group and DG treatment group. The STBI animal model was reproduced referring to modified Feeney free fall impact model. No injury was made in normal group. Six hours after modeling, monosialotetrahexosylganglioside sodium injection and DG injection were injected via tail vein of rats in GA treatment group and DG treatment group respectively, once a day for 7 days. Normal group and STBI model group were given the same amount of normal saline. Six rats in each group were sacrificed on the 1st, 3rd and 7th day after the challenge for neurological severity score (NSS), and then the blood of abdominal aorta was drawn and brain tissue was harvested. The contents of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in serum were detected by enzyme linked immunosorbent assay (ELISA). The pathological changes of sub-granular zone (SGZ) were observed under light microscope after hematoxylin eosin (HE) staining. Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the mRNA expressions of Wnt3a, β-catenin, glycogen synthetase kinase-3β (GSK-3β) and Axin. Results ① There was no neurological deficit in the normal group and NSS was 0. NSS score of rats increased significantly on the first day after modeling, and then decreased gradually over time. NSS of the rats treated with GA and DG were significantly lower than that of the STBI model rats (score: 7.33±2.07, 6.17±2.23 vs. 9.33±1.63, both P < 0.01). Though NSS gradually decreased over time, the differences were still statistically significant on the 7th day (score: 2.67±0.82, 1.00±0.00 vs. 6.17±2.23, both P < 0.01), and NSS of DG treatment group was significantly lower than that of GA treatment group. ② In SGZ of rats, cells were arranged in a compact and orderly way in the normal group, but neurons and tissues were damaged and destroyed at different time points in the STBI model group. After either GA or DG treatment, the damage of nerve tissue was improved gradually over time, and the effect of DG was more obvious.③ In the normal group, the mRNA expressions of Wnt3a and β-catenin were almost not expressed, the mRNA expressions of GSK-3β and Axin were higher, and the contents of BDNF and NGF in serum were less. On the 1st day after STBI, the mRNA expressions of Wnt3a and β-catenin in hippocampus, the contents of BDNF and NGF in serum were significantly increased, and the mRNA expressions of GSK-3βand Axin were significantly decreased. The mRNA expressions of Wnt3a and β-catenin in the hippocampus and the contents of BDNF and NGF in serum were significantly higher than those in the model group 1 day after GA or DG was added, the mRNA expressions of GSK-3β and Axin were significantly decreased, and the effect of DG was more significant than that of GA [Wnt3a mRNA (2-ΔΔCt):3.51±0.14 vs. 2.93±0.05, β-catenin mRNA (2-ΔΔCt): 1.90±0.08 vs. 1.75±0.04, BDNF (ng/L): 4.06±0.55 vs. 3.16±0.64, NGF (ng/L): 9.53±1.08 vs. 7.26±0.43, GSK-3βmRNA (2-ΔΔCt): 0.75±0.01 vs. 0.79±0.01, Axin mRNA (2-ΔΔCt): 0.74±0.02 vs. 0.76±0.02, all P < 0.05]. It was gradually increasing or decreasing over time and the difference was still statistically significant up to the 7th day. Conclusion DG can promote the recovery of nerve function in rats with STBI, and its mechanism may be related to the regeneration of nerve cells proliferation and differentiation by Wnt/β-catenin signaling pathway and the reconstruction of nerve tissue in SGZ of hippocampus.

Simultaneous Determination of 4 Components in Xinyi Biyan Pills by HPLC-switching Wavelength Method / 中国药房

OBJECTIVE: To establish the method for simultaneous determination of pim-O-glucosylcimifugin, liquiritin, 5-0-methylvisammioside and amonium glycyrrhizinate in Xinyi byan pills.METHODS: HPLC-switching wavelength method was used for content determination of 52 batches of Xinyi byan pills sample from enterprises A, B, C. The determination was performed on Kromasil C18 column with mobile phase consisted of acetonitrile-0. 1 ％ phosphoric acid at the flow rate of 1. 0 mL/min. The detection wavelengths were set at 220 nm (pim-O-glucosylcimifugin, liquiritin, 5-O-methylvisammioside) and 250 nm (amonium glycyrrhizinate). The column temperature was 30℃, and sample size was 10 μ L. RESULTS: The linear ranges of prim-O-glucosylcimifugin, liquiritin, 5-O-methylvisammioside and amonium glycyrrhizinate were 6. 138-122. 77 μg/mL (r=0. 999 9), 2. 502-50. 03 μg/mL (r=0. 999 9), 5. 988-119. 75 μg/mL (r=0. 999 9) and 12. 788-255. 76 μg/mL (r=0. 999 9), respectively. RSDs of precision, stability and reproducibility tests were all lower than 2. 0％ (n=6). The recovery rate were 100. 32％ (RSD=0. 58％, n=6), 100. 24％ (RSD=0. 56％, n=6), 101. 28％ (RSD=0. 91％, n=6) and 101. 48％ (RSD=0. 79％, n=6), respectively. Total contents of 4 components in enterprise A were generally higher than enterprises B, C, among which the difference of liquiritin was significant; the content of prim-O-glucosylcimifugin in enterprise B was higher than enterprises A, C, while the content of 5-O-methylvisammioside was lower than enterprises A, C. The content of liquiritin in enterprise B was outlier. CONCLUSIONS: This method is simple, reproducible and can provide reference for quality control of Xinyi biyan pills.

Simultaneous Determination of 5 Constituents in Xiao'er Magan Granule by HPLC-QAMS / 中国药师

Objective:To develop a method of quantitative analysis of multi-components by single marker(QAMS)for the determi-nation of five constituents(ephedrine hydrochloride,amygdalin,liquiritin, baicalin and ammonium glycyrrhizinate)in Xiao'er Magan granule. Methods:Amygdalin was used as the internal reference substance, and the relative correlation factors(RCF) of ephedrine hydrochloride,liquiritin,baicalin and ammonium glycyrrhizinate to amygdalin were calculated and evaluated. The contents of the five constituents were determined by the external standard method(ESM) and QAMS,respectively. The content results determined by the two methods were compared and the feasibility of QAMS method was verified. Results:The RCF between amygdalin and the other con-tents was 1.237,1.318,1.327 and 0.884,respectively. There were no significant differences in the results between QAMS and ESM with the relative errors less than 0.3%. Conclusion:The QAMS method is accurate and feasible for the simultaneous determination of Xiao'er Magan granule.

Quaily Analysis of Weiling Granules / 中国药师

Objective: To analyze 167 batches of Weiling granules according to the quality standard and exploratory research, and evaluate the overall quality and standard condition of the preparation. Methods: A TLC method was established for the identification of Atractylodis macrocephalae Rhizoma;an HPLC method was established for the fingerprint and the content determination of paeoniflorin, tetrahydropalmatine and ammonium glycyrrhizinate. An Agilent Poroshell120,SB-C18analytical column (100 mm×4. 6 mm, 2. 7 μm) was employed with gradient elution of acetonitrile-0. 1% phosphoric acid as the mobile phase at the flow rate of 1. 8 ml·min-1, and the sample size was 3 μl. Acid base titration method was used for measuring acid-neutralizing capacity. Results: No interference from the negative controls was shown to the TLC identification of Atractylodis macrocephalae Rhizoma. The fingerprint exhibited better separation of each peak. The precision, reproducibility and stability of the method were good,and the RSDs of the relative retention time and rela-tive peak area were less than 3. 0% . The linear range of paeoniflorin, tetrahydropalmatine and ammonium glycyrrhizinate was 0. 057-0. 568 μg(r=0. 999 9), 0. 035-0. 353 μg(r=0. 999 9)and 4. 244×10 -3-42. 44×10 -3μg(r=0. 999 9), respectively, and the av-eragerecoverywas99.3%(RSD=1.0%,n=6),100.0%(RSD=0.8%,n=6) and99.8%(RSD=1.2%,n=6),respectively. The average recovery of acid-neutralizing capacity was 99. 5% (RSD=0. 5% ,n=6). Conclusion: Exploratory research increases the specificity, controllability and safety of the standards, which provides reference for the further drug standards revision and the drug quality control.

Research on HPLC Fingerprint of Jinsangqi Kangdu Dropping Pills / 中国中医药信息杂志

Objective To establish fingerprint of Jinsangqi Kangdu Dropping Pills by HPLC; To control the quality of the preparation. Methods Waters XSELECT CSH-C18 chromatographic column (4.6 mm × 150 mm, 5 μm) was used and eluted with acetonitrile - 0.1% phosphoric acid solution gradient at the flow rate of 1.0 mL/min. The detection wavelength was 260 nm with column temperature of 30 ℃. Using calycosin-7-O-β-D-glucopyranoside, rutin, liquiritin, hyperoside, quercetin and ammonium glycyrrhizinate as the object references, ten batches of Jinsangqi Kangdu Dropping Pills were tested and analyzed by similarity comparison.Results Fringerprint spectrum of Jinsangqi Kangdu Dropping Pills had 24 common peaks in total, and characteristic spectrums of Hypericum Perforatum, Mori Cortex, Astragali Radix and Glycyrrhizae Radix et Rhizoma had been found, while similarity of HPLC fingerprint was more than 0.9 among those batches of samples. Conclusion Using HPLC fingerprint can evaluate the Jinsangqi Kangdu Dropping Pills quality totality,which can provide references for improving the quality control of the preparation.

In vitro effect of diammonium glycyrrhizinate on the growth of human hair follicles and Wnt/β-catenin signaling pathway / 中华皮肤科杂志

Objective To evaluate the effect of diammonium glycyrrhizinate extracted from the Chinese traditional medicine licorice root on the growth of human hair follicles cultured in vitro,and to detect the expression of wnt/β-catenin signaling pathway-related molecules.Methods Isolated hair follicles were cultured with diammonium glycyrrhizinate at different concentrations of 0.1,0.01,0.001 and 0.000 1 μmol/L for 10 days,and the hair follicles cultured in Williams' E medium without diammonium glycyrrhizinate served as a control group.The length of hair follicles was measured under a microscope every day,the morphologic changes of hair follicles were observed,and photos were taken.Immunofluorescence assay was performed to assess the proliferation of hair matrix cells,as well as to determine the expression of β-catenin,glycogen synthase kinase 3β (GSK3β),phosphorylated GSK3β (p-GSK3β) and lymphoid enhancer factor-1 (Lef1) in the Wnt/β-catenin signaling pathway.Statistical analysis was carried out by repeated-measures analysis of variance and one-way analysis of variance.Results As repeated-measures analysis of variance showed,only 0.01 μmol/L diammonium glycyrrhetate showed significantly promotive effect on the growth of hair follicles compared with the medium alone (P ＜ 0.05),and there were no significant differences in the length of hair follicles between the other concentration groups and the control group.Compared with the control group,the transition to the catagen phase of human hair cycle was delayed in the 0.01-μmol/L diammonium glycyrrhetate group,while it did not change in the other diammonium glycyrrhetate groups and control group.Immunofluorescence assay showed that the number of ki67-positive hair matrix cells was obviously increased in the 0.1-,0.01-,0.001-μmol/L diammonium glycyrrhizinate groups compared with the control group,while there was no difference between the 0.000 1-μmol/L diammonium glycyrrhizinate group and the control group.One-way analysis of variance revealed that the expression of β-catenin,p-GSK3β and Lef1 significantly differed among all the groups (F =12.604,16.65,15.266 respectively,P ＜ 0.05),while no significant difference in the expression of GSK3β was found among these groups (F =1.472,P ＞ 0.05).Least significant difference (LSD)-t test revealed that the expre-ssion of β-catenin,p-GSK3β and Lef1 in the hair matrix cells was significantly higher in the 0.1-,0.01-,0.001-μmol/L diammonium glycyrrhizinate groups than in the control group (all P ＜ 0.05),but there was no significant difference between the 0.000 1-μmol/L diammonium glycyrrhizinate group and the control group (P ＞ 0.05).Conclusion Diammonium glycyrrhetate at the concentration of 0.01 μmol/L shows markedly promotive effect on the in vitro growth of hair follicles,and can increase the proliferative activity of hair matrix cells and delay the transition to the catagen phase,which may be associated with the activation of Wnt/β-catenin signaling pathway.

Difference of chemical constituents and efficacy between crude and processed Pinelliae Rhizoma / 中草药

Objective To study the chemical constituents and effects of crude and processed Pinelliae Rhizoma. Methods The contents of inosine, guanosine, adenosine, succinic acid, ephedrine hydrochloride, liquiritin, glyeyrrhizie acid, and 6-gingerol of Pinelliae Rhizoma, Pinelliae Rhizoma Praeparatum cum Alumine, Pinelliae Rhizoma Praeparatum cum Zingibere et Alumine, and Pinelliae Rhizoma Praeparatum were detected by HPLC. The pharmacodynamics of the traditional efficacy of expectorant and cough relieving was studied by stimulating mice with phenol red and concentrated ammonia in the trachea of mice. Results The contents of inosine, guanosine, adenosine, succinic acid, and ephedrine hydrochloride decreased significantly after processing, and inosine was not detected in Pinelliae Rhizoma Praeparatum cum Alumine. Compared with the three processed products, the content of inosine, guanosine, adenosine and succinic acid was the highest in the Pinelliae Rhizoma Praeparatum cum Alumine, the lowest was in Pinelliae Rhizoma Praeparatum cum Zingibere et Alumine, and in consistent with the effect of resolving phlegm. The four components were the active components of resolving phlegm effect. Adding alumen during Pinelliae Rhizoma Praeparatum cum Alumine processing has also enhanced its effectiveness. Pinelliae Rhizoma Praeparatum has the strongest antitussive effect, followed by Pinelliae Rhizoma, Pinelliae Rhizoma Praeparatum cum Alumine and Pinelliae Rhizoma Praeparatum cum Zingibere et Alumine. Adding licorice and lime water during Pinelliae Rhizoma Praeparatum processing, licorice (peak 6: liquiritin, peak 7: ammonium glycyrrhizinate) had a powerful antitussive effect and enhanced its antitussive effect. After processing by ginger and white peony, ginger (peak 8: 6-gingerol) is good at warming middle energizer to arrest vomiting, thus enhance antiemetic effect and weaken phlegm, cough effect of Pinelliae Rhizoma Praeparatum cum Zingibere et Alumine. Conclusion The chemical composition and efficacy of Pinelliae Rhizoma have changed after being processed, and different processing methods have different effects on its chemical composition and efficacy.

Clinical effect of Yinchenhao Decoction in the treatment of icteric hepatitis / 中国现代医生

Objective To study the clinical effect of Yinchenhao Decoction in the treatment of icteric hepatitis. Methods 100 cases with icteric hepatitis diagnosed and treated in our hospital from July 2010 to June 2017 were selected as the research subjects and were randomly divided into observation group and control group, with 50 cases in each group. Both the two groups were given oral antiviral treatment with nucleoside analogues and drip therapy of reduced glutathione, diammonium glycyrrhizinate and potassium aspartate magnesium. The observation group added Yinchenhao Decoction in the foundation of the control group. The levels of transaminase and bilirubin before and after treatment in two groups, and the effects after treatment of the two groups were compared. Results The transaminase levels in the two groups after treatment were significantly lower than those before treatment (P＜0. 05). The transaminase level in observation group was lower than that in the control group after treatment, and the difference was significant (P＜0. 05). The total bilirubin and direct bilirubin in the two groups were significantly lower than those before treatment (P＜0. 05), and the total bilirubin and direct bilirubin in the observation group were significantly lower than those in the control group, the differences were statistically significant(P＜0. 05). The total effective rate of the observation group was 98％, which was significantly higher than that of the control group at 74％, the difference was statistically significant (P＜0. 05). Conclusion Yinchenhao Decoction in the foundation of regular treatment in the treatment of icteric hepatitis can significantly promote the clinical effect, improve liver function and reduce bilirubin level.

Preparation of thermo-sensitive diammonium glycyrrhizinate binary liposome gel and the in vitro drug-release behaviors / 中成药

AIM To prepare thermo-sensitive diammonium glycyrrhizinate binary liposome gel and to evaluate the in vitro drug-release behaviors.METHODS Cold dissolution method was adopted in the preparation of gel.With gel transition temperature as an evaluation index,amounts of Poloxamer 407 (P407),Poloxamer 188 (P188) and polysaccharides from Bletillae Rhizoma as influencing factors,central composite design-response surface method was applied to optimizing the formulation.Then the in vitro drug-release behaviors were evaluated by HPLC and Franz vertical diffusion cell method.RESULTS The optimal formulation was determined to be 18％ for P407 amount,4％ for P188 amount,and 0.6％ for polysaccharides' amount,the gel transition temperature was (37.5 ± 0.3) ℃.The accumulative release rate of obtained thermo-sensitive binary liposome gel was (65.52 ± 0.63) ％ within 48 h,which showed more obvious sustained-release effect as compared with liposome and thermosensitive gel.CONCLUSION Thermo-sensitive diammonium glycyrrhizinate binary liposome gel can reduce drug-release rate and increase its retention time in the rectum.

Simultaneous Determination of 3 Components in Xiaoer Huadu Powder by HPLC / 中国药房

OBJECTIVE:To establish a method for simultaneous determination of paeoniflorin,berberine hydrochloride and ammonium glycyrrhizinate in Xiaoer huadu powder.METHODS:HPLC method was adopted.The separation was performed on Waters SunFireTM-C18 column with mobile phase consisted of acetonitrile-0.1％ phosphoric acid (gradient elution) at the flow rate of 1.0 mL/min.The detection wavelength was set at 238 nm,and column temperature was 25 ℃.The sample size was 10 μL.RESULTS:The linear ranges of paeoniflorin,berberine hydrochloride and ammonium glycyrrhizinate were 8.808-88.08 μg/mL(r=0.999 8),1.778-17.78 μg/mL(r=0.999 6),2.533-25.33 μg/mL(r=0.999 9),respectively.LOQ were 4.404,0.889,2.533 μg/mL;LOD were 1.101,0.445,1.267 μg/mL.RSDs of precision,stability and reproducibility tests were all lower than 2％.The recoveries were 95.08％-99.61％ (RSD=1.77％,n =9),96.93％-99.94％ (RSD=0.92％,n=9),98.33％-102.05％ (RSD=1.27％,n=9).CONCLUSIONS:The method is simple,accurate and reproducible,and can be used for simultaneous determination of paeoniflorin,berberine hydrochloride and ammonium glycyrrhizinate in Xiaoer huadu powder.

Establishment of Quality Standard for Extractum Glycyrrhizae Capsules / 中国药师

Objective:To establish the quality standard for two effective components in extractum glycyrrhizae capsules. Methods:Radix glycyrrhizae was identified by a TLC method. The contents of liquiritin and ammonium glycyrrhizinate in extractum glycyrrhizae capsules were determined by HPLC. An Inertsil C18 (150 mm × 4. 6 mm, 5μm) column was used. The mobile phase consisted of ace-tonitrile (A)-0. 2％ phosphoric acid (B) (0-8 min: 20％A-20％A;8-34 min: 20％A-50％A;34-35 min: 50％A-100％A;35-40 min:100％A-20％A) at a flow rate of 1. 0 ml·min-1 . The detection wavelength was at 237 nm under 25℃. Results: The spots in TLC were clear. Liquiritin showed a good linear relationship within the range of 0. 0020-0. 1000 mg·ml-1(r=0. 9995). The aver-age recovery was 100. 29％, and the RSD was 2. 94％(n=6). Ammonium glycyrrhizinate showed a good linear relationship within the range of 0. 0020-0. 1000 mg·ml-1(r=0. 9998). The average recovery was 101. 46％, and the RSD was 2. 33％(n=6). Conclu-sion:The method is simple,reliable and reproducible, which can be used for the quality control of the preparation.

Simultaneous Determination of Five Active Components in Pifukang Lotion by HPLC with Multiple UV Wavelengths / 中国药师

Objective: To establish an HPLC method for the simultaneous determination of five active components (paeoniflorin, liquiritin, ammonium glycyrrhizinate, edomin and osthole)in Pifukang lotion with multiple UV wavelengths.Methods: A Diamonsil C18 (200 mm×4.6 mm,5 μm) column was used with the mobile phase consisting of 0.1% hydrochloric acid and acetonitrile with gradient elution.The flow rate was 1.5 ml·min-1, and the column temperature was 30℃.Paeoniflorin was detected at 232 nm(0-6 min), liquiritin was detected at 277 nm(6-10 min),and ammonium glycyrrhizinate, edomin and osthole were detected at 254 nm(after 10 min).Results: The linear range of paeoniflorin,liquiritin,ammonium glycyrrhizinate, edomin and osthole was 28.200-282.000 μg·mL-1(r=0.999 7),12.150-121.500 μg·ml-1(r=0.998 8),13.420-134.200 μg·ml-1(r=0.999 5),0.047-0.466 μg·ml-1(r=0.999 9) and 2.38-23.8 μg·ml-1(r=0.999 9), respectively.The average recovery (RSD) was 98.49%(0.71%), 99.00%(0.62%), 98.38%(0.85%), 97.36%(0.92%) and 97.70%(0.78%), respectively (n=6).Conclusion: The established method is simple, accurate, highly sensitive and well reproducible, which can be used for the determination of the five active ingredients in Pifukang lotion.

Simultaneous Determination of 6 Kinds of Components in Yindan Pinggan Capsules by HPLC / 中国药房

OBJECTIVE:To develop a method for simultaneous determination of chlorogenic acid,geniposide,gentiopicro-side,ferulic acid,baicalin and ammonium glycyrrhizinate in Yindan pinggan capsule. METHODS:HPLC method was adopted. The separation was performed on Wonda SilTM-C18 column with mobile phase consisting of acetonitrile-0.4% phosphoric acid solu-tion(gradient elution)at the flow rate of 0.8 mL/min. The detection wavelength were set at 325 nm(chlorogenic acid,ferulic ac-id),250 nm (geniposide,ammonium glycyrrhizinate) and 275 nm (gentiopicroside,baicalin). The column temperature was 30 ℃. RESULTS:The linear ranges were 0.087-3.480 μg for chlorogenic acid(r=0.9998),0.201-8.040 μg for geniposide(r=0.9997),0.200-8.000 μg for gentiopicroside(r=0.9995),0.016-0.640 μg for ferulic acid(r=0.9999),0.105-4.200 μg for ba-icalin (r=0.9999) and 0.028-1.120 μg for ammonium glycyrrhizinate (r=0.9995),respectively. The limits of quantitation were 1.31,0.75,1.14,1.25,0.94,0.98 ng,and the limits of detection were 0.87,0.67,0.96,0.93,0.60,0.88 ng,respectively. RSDs of precision,stability and reproducibility tests were lower than 2.0%;recoveries were 99.9%-101.9%(RSD=0.7%,n=6), 98.7%-100.9%(RSD=0.9%,n=6),98.1%-101.5%(RSD=1.4%,n=6),98.5%-101.3%(RSD=1.3%,n=6),98.5%-101.7%(RSD=1.2%,n=6),98.2%-101.4%(RSD=1.2%,n=6),respectively. CONCLUSIONS:The method is simple and accurate , can be used for simultaneous determination of chlorogenic acid,geniposide,gentiopicroside,ferulic acid,baicalin and ammonium glycyrrhizinate in Yindan pinggan capsule.

Effects of diammonium glycyrrhizinate on expressions of Rac-1, Claudin-5 and vessel endothelium-Cadherin in rats after cerebral ischemic reperfusion / 中华神经医学杂志

Objective To investigate the effect ofdiammonium glycyrrhizinate on neurovascular units in rats after cerebral ischemia reperfusion (IR) injury.Methods Two hundred and forty health SD rats were randomly assigned into normal control group (n=30),sham-operated group (n=30),IR group (n=90) and diammonium glycyrrhizinate group (DG,n=90).The rats in the IR group and DG group were divided into 2,6 and 12 h subgroups after modeling,respectively (n=30).The rats in the IR group and DG group were induced middle cerebral artery occlusion (MCAO) models,and after the models were successfully established,9.11 mL DG sodium chloride injection was given to DG group,while equal saline to normal group,sham-operated group and IR group via the tail vein.The brain tissues of each group were harvested 2,6 and 12 h,resperctively,after modeling.The infraction rate was measured by TTC staining;immunohistochemistry was employed to detect the expresions of Claudin-5 and vessel endothelium (VE)-Cadherin;Western blotting was used to detect the protein expression levels of Rac-1 and Claudin-5.Results The DG group had signficantly lower infarction rate than IR group 2,6 and 12 h after modeling (P＜0.05).The Claudin-5 expression rates in the 6 h and 12 h DG subgroups were signficantly higher than those in the 6 h and 12 h IR subgroups (P＜0.05).The VE-Cadherin expression rates in the DG group were significantly higher than that in IR group at 2,6 and 12 h after modeling (P＜0.05).Samely,the Claudin-5 relative quantity in DG group was significantly higher than that in IR group at 2,6 and 12 h after modeling (P＜0.05).The Rac-1 quantity in DG group was only statistically higher than IR group at 2 h after modeling (P＜0.05).Conclusion The DG can upregulate the Rac-1,VE-Cadherin and Claduin-5 expressions in neurovascutar units,and partly protect neurovascular units after cere bral acute IR injury.