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ObjectiveThrough the correlation analysis between intestinal absorption profile and inhibition of macrophage foaming， the pharmacodynamic components of Zhuriheng dripping pills（ZRH） were explored to provide a basis for establishing its quality standard. MethodIntestinal absorption fluids with 0， 5， 10， 15， 20 times clinical equivalent doses were prepared by a rat everted gut sac（EGS）， and the oxidized low density lipoprotein（ox-LDL）-induced RAW264.7 macrophage foaming model was used to investigate the effect of intestinal absorption fluid with different doses on the accumulation of lipids in RAW264.7 cells by oil red O staining and cholesterol content determination， and to screen for the optimal dose. Ultra performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry（UPLC-Q-Exactive Orbitrap MS） was used to analyze and identify intestinal absorption fractions of ZRH intestinal absorption fluids， and partial least squares-discriminant analysis（PLS-DA） and orthogonal partial least squares-discriminant analysis（OPLS-DA） were performed on different doses of ZRH intestinal absorption fluids using SIMCA 13.0 with peak area as the independent variable and the pharmacodynamic indicators as the dependent variables to screen the compounds with variable importance in the projection（VIP） value>1.0 as contributing components， and Pearson correlation analysis was used to determine the spectral effect relationship， determined the compounds and positive correlation with pharmacodynamic were as active ingredients. Molecular docking was used to verify the binding energy of peroxisome proliferator-activated receptor α（PPARα）， PPARγ， PPARβ， human retinoid X receptor α（RXRA） and nuclear transcription factor-κB（NF-κB） with the active ingredients in ZRH intestinal absorption fluids. Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） was performed to detect the mRNA levels of PPARγ， scavenger receptor A1（SRA1） and adenosine triphosphate-binding cassette transporter A1（ABCA1） in RAW264.7 cells， Westen blot was used to detect the expression level of PPARγ protein in RAW264.7 cells， and enzyme-linked immunosorbent assay（ELISA） was used to detect the levels of interleukin（IL）-1β and NF-κB in RAW264.7 cells. ResultAccording to the results of oil red O staining and cholesterol content determination， the ZRH intestinal absorption fluids could significantly reduce macrophage foaming， and intestinal absorption fluids with 15， 20 times clinical equivalent doses had the best effect， the 15-fold ZRH intestinal absorption fluid was finally determined as the study subject. Spectral effect relationship showed that 52 corresponding peaks in the ZRH-containing intestinal fluid were positively correlated with the efficacy， including organic acids， phenylpropanoids， iridoids， flavonoids， bile acids， coumarins and chromones. Target validation results showed that 86.9%-96.2% of the total components processed good binding activities with the key targets of PPARα， PPARγ， PPARβ， RXRA and NF-κB， and the docking energy values were all less than -6.0 kcal·mol-1（1 cal≈4.19 J）. The results of validation showed that， compared with the normal group， the model group showed a significant increase in the levels of SRA1 and PPARγ mRNA expression， a significant decrease in ABCA1 mRNA expression， a significant increase in the level of PPARγ protein expression， and a significant increase in the levels of IL-1β and NF-κB（P<0.01）， compared with the model group， the 15-fold intestinal absorption fluid group showed a significant decrease in the levels of SRA1 and PPARγ mRNA expression（P<0.05， P<0.01）， ABCA1 mRNA expression level was significantly up-regulated， the levels of IL-1β and NF-κB were significantly reduced（P<0.01）， and PPARγ protein expression level was significantly reduced（P<0.05）. ConclusionThis study identifies 52 components and their metabolites in ZRH intestinal absorption fluid that are positively correlated with the inhibition of macrophage foaming， which may be related to the regulation of the PPARs pathway in cells and the reduction of the levels of inflammatory factors， and can provide a reference for the quality control and clinical application of ZRH.
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OBJECTIVE To establish the methods to identify the chemical components of Ixeris chinensis， and determine the contents of 7 components （chlorogenic acid， luteolin， quercetin， rutin， protocatechuic acid， isochlorogenic acid A， luteoloside）. METHODS HPLC-Q-Exactive-MS was used to identify the chemical components of I. chinensis. The contents of 7 components in I. chinensis， including chlorogenic acid， were determined by HPLC-MS/MS. RESULTS A total of 45 components were identified in I. chinensis， including 20 organic acids， 13 flavonoids， 4 fatty acids， 4 amino acids， 3 nucleosides， and 1 coumarin. The linear range of chlorogenic acid， luteolin， quercetin， rutin， protocatechuic acid， isochlorogenic acid A and luteoloside were 503.00- 25 150.00， 42.00-2 100.00， 5.05-252.50， 20.05-1 002.50， 25.10-1 255.00， 750.00-37 500.00， 196.00-9 800.00 ng/mL （r≥0.999 2）， respectively. RSDs of precision， stability and reproducibility tests were all less than 3.00% （n＝6）， and average recovery ranged from 96.72% to 105.84% （all RSD＜4.00%， n＝6）. The contents of 7 components in 3 batches of I. chinensis were 1 145.77- 3 261.25， 23.75-97.90， 0.92-2.12， 1.06-23.18， 9.35-21.85， 833.25-1 045.58， 199.56-1 869.78 μg/g， respectively. CONCLUSIONS The established methods for identification and content determination are rapid and simple， and can be used for the identification of chemical components and the content determination of 7 components in I. chinensis.
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OBJECTIVE To systematic ally stu dy the chemic al components of ethanol extract from Sanzi san ，and to provide reference for clarifying the pharmacodynamic material basis of the formulation. METHODS HPLC-Q-Exactive-MS technology was adopted. The determination was performed on Shim-pack GIST-HP C 18 column with mobile phase consisted of acetonitrile- 0.1％ formic acid aqueous solution for gradient elution at the flow rate of 1 mL/min. The column temperature was 40 ℃，and sample size was 10 μL. Mass spectrometry conditions included the electrospray spray ion source was used for detection in positive and negative ion detection modes. Full MS/dd-MS 2 detection mode was adopted ，the resolution of Full MS was 70 000 and the resolution of dd-MS2 was 17 500. The scanning range was m/z 110-1 200. The ion peaks were identified by comparing with the information of control substances ，literature references and self-built database. RESULTS A total of 64 components were identified in the ethanol extract of Mongolian medicine Sanzi san ， including 9 flavonoids，13 iridoids，14 organic acids ，18 tannins，3 triterpenes，3 amino acids and 4 fatty acids. CONCLUSIONS The ethanol extract of Mongolian medicine Sanzi san mainly include iridoids ，tannins and flavonoids ，which might be the pharmacodynamic material basis of Sanzi san.
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BACKGROUND:It has been proved that bone marrow mononuclear cel transplantation can obviously improve neurological function of rats with cerebral hemorrhage. OBJECTIVE:To investigate the effects of transplanted bone marrow mononuclear cel s on the neurological function and apoptosis in perihematomal brain tissues fol owing cerebral hemorrhage in a rat model. METHODS:Twenty-four Sprague-Dawley rats were given stereotaxical injection of col agenase IV into the caudate nucleus to establish cerebral hemorrhage models in transplantation group (n=12) and model group (n=12), and then at 6 hours after cerebral hemorrhage, rats in these two groups were administrated 3x1010/L al ograft bone marrow mononuclear cel s and the same amount of PBS, respectively. Another 12 rats were given no interventions as control group. Neurological functions of rats were assessed at 1, 4, 8, 16 days after cerebral hemorrhage;pathological changes of the injury sites were observed at 16 days after transplantation;neuronal apoptosis rates in the perihematomal brain tissue were detected by flow cytometry at 2 and 4 days after transplantation. RESULTS AND CONCLUSION:The modified neurologic severity scores in the transplantation group were significantly lower than those in the model group at 8 and 16 days after cerebral hemorrhage (P<0.05). In the control group, cel s in each layer arranged closely with complete structure, and neurons and glial cel s were in good shape;in the model group, perihematomal brain tissues were loose with intercel ular gap, in which most neurons and glial cel s became necrotic;in the transplantation group, cel s in each layer arranged closely and regularly, and glial cel proliferation occurred. Besides, compared with the model group, the neuronal apoptosis rate in the transplantation group was significantly lower (P<0.05). To conclude, bone marrow mononuclear cel s can significantly enhance the neurological function recovery and reduce neuronal apoptosis in the brain of cerebral hemorrhage rats.
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The medium for fermentation has been selected by means of orthogonal designs. The number of cell has reached 10~(11)/ml, and spore forming rate is 70—80%. LC_(50) of mosquito larvicied is lower than 0.125 ppm. Three targets are obviously better than those of previous report. The stable results are proved by studying of fermentation in shaking flask and further in 50L~3-bench fermenter. The production cost of per ton is decreased by 33%.