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ObjectiveTo develop a quality control method for the simultaneous determination of multiple active components in Nymphaeae Flos aiming at the problems of the single index for quality control and the relatively low overall quality control level. MethodUltra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS/MS）was used to identify and select the index components for quality control with the mobile phase of 0.1% formic acid aqueous solution（A）-acetonitrile（B）for gradient elution （0-2 min， 3%-8%B； 2-4 min， 8%-10%B； 4-13 min， 10%-15%B； 13-19 min， 15%-20%B； 19-26 min， 20%-45%B） at a flow rate of 0.4 mL·min-1， detection wavelength of 350 nm， electrospray ionization（ESI）， negative ion scanning mode， ion source temperature of 120 ℃， scanning range of m/z 100-1 200， transmit collision energy of 6 eV for low-energy scanning and 25-50 eV for high-energy scanning. High performance liquid chromatography（HPLC）was used to establish the quality control method for the simultaneous determination of multi-index components with the mobile phase of 0.2% phosphoric acid aqueous solution（A）-acetonitrile（B） for gradient elution（0-30 min， 12%-15%B； 30-60 min， 15%-22%B； 60-90 min， 22%-40%B）and detection wavelength of 350 nm. The preparation method of the test solution for content determination was refluxing extraction for 60 min with 80 times the amount of 70% methanol. ResultBy comparing the retention time， ultraviolet absorption characteristics， MS and MS/MS spectrometric signals in the samples with the reference substances， 8 active components with high contents， including brevifolincarboxylic acid， ellagic acid， rutin， nicotiflorin， astragalin， quercetin， quercetin-3-methylether and kaempferol， were identified qualitatively from Nymphaeae Flos， which were selected as the index components for quality control. Under the established HPLC conditions， the above 8 components could be well separated（resolution>1.5）， and showed good linearity（r=0.999 9）between the concentration ranges of 1.99-99.6， 1.76-176， 1.52-75.8， 3.60-180， 0.964-96.4， 1.18-118， 1.94-96.8， 1.04-104 mg·L-1 and the peak areas， respectively. The detection limits of them were 10-49 μg·L-1， and the limits of quantitation were 34-164 μg·L-1. The average recoveries were 97.12%-103.1% with the relative standard deviations （RSDs） were 1.1%-2.2%. ConclusionA quality control method for simultaneous determination of the multiple active components in Nymphaeae Flos have been developed， which is simple， accurate and reproducible， and it can provide a scientific basis for the formulation of quality standard of this herb and lay a research foundation for the transformation of Uygur hospital preparations containing Nymphaeae Flos into new drugs.
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ObjectiveTo explore the antidepressant effect of Sophora flavescens seed extract and its molecular mechanism. MethodA mouse depression model was established by intraperitoneal injection of lipopolysaccharide（LPS）， and normal group， model group， fluoxetine group（2.5 mg·kg-1）， and S. flavescens seed low， medium and high dose groups（200， 400， 800 mg·kg-1） were set up for 7 d of consecutive gavage. Then the antidepressant effect of S. flavescens seed extract was evaluated by using open field test， elevated plus maze test and forced swimming test. Pathological morphological changes in the hippocampal tissue was observed by hematoxylin-eosin（HE） staining. Protein expression levels of G1/S-specific cyclin D1（Cyclin D1）， Wnt1， β-catenin and phosphorylated glycogen synthase kinase-3β（p-GSK-3β） in mouse brain tissues were detected by Western blot. Hippocampal cell apoptosis was detected by terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate（dUTP） nick end labeling（TUNEL）. ResultThe results of mouse behavioral experiments showed that compared with the normal group， the speed of movement in the open field and the distance of movement in the central area of the open field， and the time spent on the open arms of the elevated plus maze were significantly reduced in the model group（P<0.01）， while immobility time in the forced swimming test was significantly increased（P<0.05）. Compared with the model group， the S. flavescens seed medium and high dose groups had increased speed of movement in the open field test and time spent on the open arms of the elevated plus maze test（P<0.05， P<0.01）， and decreased immobility time in the forced swimming test（P<0.05）， the distance of movement in the central area of the open field test increased in the high dose group（P<0.05）. HE staining results showed that compared with the normal group， the hippocampal neuron structure of mice in the model group was damaged. Compared with the model group， after treatment of S. flavescens seed extract， the pathological state of the mouse hippocampal neuron structure was alleviated， and the neurons increased， were neatly arranged， and the cytoplasm was clear. Western blot results showed that the protein expression levels of Wnt1 and β-catenin in mouse brain tissue were significantly decreased（P<0.01）， while the protein expression levels of Cyclin D1 and p-GSK-3β were significantly increased（P<0.01） after LPS injection. Compared with the model group， protein expression levels of Wnt1 and β-catenin in brain tissue of S. flavescens seed medium and high dose groups were significantly increased（P<0.01）， while the protein expression levels of Cyclin D1 and p-GSK-3β were significantly decreased（P<0.01）. TUNEL staining results showed that the hippocampal cell apoptosis rate in the model group was significantly increased compared with that of the normal group（P<0.01）， while the hippocampal cell apoptosis rate in the S. flavescens seed medium and high dose groups was significantly decreased compared with that of the model group（P<0.01）. ConclusionS. flavescens seed extract can effectively improve the severity of depression in LPS-induced depressed mice， and its molecular mechanism is related to the regulation of neuroinflammation and hippocampal neuronal apoptosis mediated by Wnt/β-catenin signaling pathway.
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<p><b>OBJECTIVE</b>To establish a method for quick finding of the absorption ingredients of Paeoniae Radix Alba in order to select the index of quality control.</p><p><b>METHOD</b>The absorption ingredients of three concentration of Paeoniae Radix Alba were investigated with the in vitro-everted intestinal sac (VEIS) model. The intestinal sac fluids of jejunum and ileum were collected in different time and detected by HPLC. The accumulative absorption quantity of albiflorin and paeoniflorin were calculated, respectively.</p><p><b>RESULT</b>Five ingredients could be detected. In different concentrations of Paeoniae Radix Alba, albiflorin and paeoniflorin in various intestinal sections were the linear absorption (R2 > 0.9), conformed to the zero order absorption rate. The values of Ka in the jejunum and ileum were increased along with the raised dosage of the Paeoniae Radix Alba (P < 0.05), indicating a passive absorption manner.</p><p><b>CONCLUSION</b>SEMAC could be used as a tool to find the absorption ingredients of Paeoniae Radix Alba. Compared with the jejunum, the ileum could provide the more absorption information. It was showed that the optimal detecting time was 60 min.</p>
Assuntos
Animais , Masculino , Ratos , Absorção Intestinal , Paeonia , Ratos WistarRESUMO
<p><b>OBJECTIVE</b>To evaluate the inhibitory effect of total bufadienolides from toad venom against H22 tumor in mice and preliminarily analyze the structures of the metabolites in tissues.</p><p><b>METHOD</b>HPLC and LC-MS were used for analysis of the chemical composition of TBFs. High, middle and low dosages of TBFs were orally administered or intra-peritoneally injected to H22 tumor-bearing mice for thirteen days. The animals were killed and the tumors were stripped and weighed. The metabolites in the tissues such as heart, liver, spleen, lung and kidney, were analyzed by HPLC and LC-MS.</p><p><b>RESULT</b>The chemical composition of TBFs were identified by comparison of the retention times with those of reference substances, on-line UV spectra and MS data. Its main components are concerned with gamabufotalin, arenobufagin, bufotalin, resibufagin, cinobufotalin, bufalin, cinobufagin and resibufogenin. TBFs had no obvious influence on body weight of H-22 tumor-bearing mice orally administered and the inhibition rate against tumor were 14.76%, 16.38% and 10.32% for low (5 mg x kg(-1)), middle (10 mg x kg(-1)) and high dosage (20 mg x kg(-1)), respectively. The mice intra-peritoneally injected with middle and high-dose of TBFs gained body weight slower than the control mice on the 5th day and recovered on the 13th day. The inhibition rate against tumor were 17.30%, 19.80% and 40.95% for low (1.5 mg x kg(-1)), middle (3 mg x kg(-1)) and high dose (6 mg x kg(-1)), respectively. The inhibitory effect took on dose-dependent manner. Based on the HPLC analyses on heart, liver, spleen, lung and kidney, bufadienolides were found in the liver tissue and 11 compounds of them were tentatively identified by LC-DAD-MS.</p><p><b>CONCLUSION</b>TBFs by oral administration had no inhibitory effect against H22 tumor in mice, however, TBFs by intra-peritoneal injection displayed the significantly inhibitory effect, accompanying some toxicity for early duration of the study. The identification of bufadienolides in the liver provides a good basis for the further investigation of the metabolic pathways of TBFs in vivo.</p>