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Objective: To clone the full-length cDNA sequence of CoDXR, a key enzyme gene of Cornus officinalis, and provide a basis for further study of C. officinalis. Methods: In this study, we used the transcript sequence c147202_g1 from the transcriptome data of C. officinalis obtained in our laboratory as template, designed specific primers through Primer Premier 5.0, cloned the full-length cDNA sequence of C. officinalis DXR gene by RT-PCR technology, and the bioinformatics analysis and function prediction were carried out through the relevant bioinformatics software. Results: The results showed that the CoDXR gene was 1 505 bp in length and the ORF was 729 bp in length, encoding 242 amino acids. The results of predictive analysis of CoDXR protein by SignalP4.0Server and HMMTOP showed that the protein was a hydrophobic protein without signal peptide and transmembrane region. Phylogenetic tree analysis showed that the CoDXR protein had the highest similarity to the DXR protein sequence of Camellia sinensis. Conclusion: In this study, the key enzyme gene CoDXR was successfully cloned based on the sequencing of the C. officinalis transcriptome, and related bioinformatics analysis was carried out. The results of this study laid the foundation for further study on the function of CoDXR gene in the terpenoid synthesis pathway of C. officinalis.
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Objective: To clone and analyze the cDNA sequence of 3-Hydroxy-3-methylglutaryl coenzyme-a reductase (HMGR1) of Cornus officinalis. Methods: In this study, specific primers were designed at both ends of the open reading frame (ORF) based on the unigene (c100572_g1) in the transcriptome data from C. officinalis. Subsequently, the cDNA sequence of CoHMGR1 gene was amplified by RT-PCR, cloned into the pTOPO-T vector and sequenced. This gene and its encoded protein were analyzed using bioinformatics methods. Results: The results suggested that CoHMGR1 was 2 116 bp in length andthe ORF was 1 338 bp in length, which encodes 445 amino acids and is a hydrophobic protein. Multiple sequence alignment and phylogenetic analysis showed that the amino acid sequence encoded by this gene has a high similarity with that of Camptotheca acuminate, reaching 79.56%. Based on the first comparison of transcriptome sequencing from leaves and fruits of C. officinalis, the CoHMGR1 gene was successfully cloned and analyzed. Conclusion: This study laid a foundation for studying the function of CoHMGR1 protein and the molecular mechanism of terpene biosynthesis pathway of C. officinalis.
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Objective: Through network pharmacology, the network relationship between the active component of Sanqi Mixture, the target of hepatic ischemia- reperfusion injury(HIRI), and biological pathway was constructed to explore the key target and mechanism of effect of Sanqi Mixture on HIRI. Method: Through literature research at home and abroad, Traditional Chinese Medicine Systems Pharmacology (TCMSP) platform, Pharm Mapper, Swiss Target Prediction and other servers, oral availability (OB) and drug-likeness (DL) were selected as the limited conditions to collect the relevant targets for Sanqi Mixture for intervention in HIRI. The OMIM database was used to screen and collate HIRI related genes and protein targets. Excel table was used to merge and sort the intersection between disease and targets through Cytoscape3.7.2 software plug-ins Network Analyzer, with topological parameters (degree) ≥ 5 (average degrees of freedom 4.5) for the filter to find the core targets; And the intersection targets were imported to the server STRING, and with Confidence Score of 0.85 or higher for the filter conditions to build the core protein interactions (Hub-PPI) network. The intersection target was introduced into FunRich 3.0 software for biological process and biological pathway analysis, and Cytoscape3.7.2 was used to construct the network of "traditional Chinese medicine-active ingredient-HIRI target-biological pathway". Result: Sanqi mixture could reduce the expression of Aspartate aminotransferase (AST) and glutamate transaminase (ALT) in HIRI mice (P < 0.01). After screening, 45 active components of Sanqi Mixture were obtained, corresponding to 3 273 targets, and the main compounds included ursolic acid, oleanolic acid, brucine, quercetin, ginsenoside F2, paeoniflorin, etc. Among the 196 targets obtained by HIRI, 46 targets were intersected with components, including 11-β-hydroxysteroid dehydrogenase (HSD11B1), adenosine receptor A3 (ADORA3), cyclooxygenase 2 (PTGS2), adenosine receptor A1 (ADORA1), protein kinase C-ε (PKC), etc. With the STRING server setting the qualified condition of Confidence Score ≥ 0.85, the PPI network with high Confidence was obtained and clustered into three categories through cluster processing. Five biological processes including protein metabolism, signal transduction, negative regulation of enzyme activity, inflammatory response and transmembrane receptor protein tyrosine kinase signal pathway were analyzed by FunRich software (P < 0.05). 16 biological pathways including integrin-linked kinase signal, TNF receptor signaling pathway, P38 mitogen-activated protein kinase signaling pathway, and TRAIL signaling pathway (P < 0.01). Conclusion: It is preliminarily discussed that Sanqi Mixture intervenes HIRI through the interaction of multiple components and multiple targets, as well as the regulation of multiple biological pathways and biological processes. However, the key core targets and the specific regulation mechanism still need further experimental verification.
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Objective To study the efficacy enhancing and toxicity reducing effects of compatibility of Aconitum carmichaeli and Cornus officinalis on chronic heart failure (CHF) rats. Methods The CHF rats was established by ip injection of adriamycin (ADM), the CHF rats were administrated tested drugs for three weeks by means of ig administration, the tested drugs included extracts of A. carmichaeli, C. officinalis, and Compound. The serum brain natriuretic peptide (BNP) level, activity of Ca2+-ATP and Na+, K+-ATP enzymes in cardiac myocytes, and cardiac histopathology were measured. Results After three weeks of modeling, the CHF rats showed signs of ascites, loss of weight, loose stool, hogback, etc. The left ventricular ejection fraction (EF) and fraction shortening (FS) decreased significantly, and the level of BNP in serum was significantly improved; Pathological changes of ventricular tissue included rupture of myocardial fibers, degeneration and necrosis of cardiomyocytes, etc. After three weeks of gavage compatibility of A. carmichaeli and C. officinalis, the general state and cardiac histopathology of the animal was obviously improved, the level of BNP in serum was reduced significantly, the activity of Na+, K+-ATP enzymes was increased significantly. No notable improvement in the above indexes was obtained after administration of A. carmichaeli and C. officinalis alone. Conclusion The compatibility of A. carmichaeli and C. officinalis can increase the activity of Na+, K+-ATP enzyme in cardiac myocytes, and improve the energy metabolism and activity of cardiac myocytes in chronic heart failure. The compatibility of A. carmichaeli and C. officinalis play the key role of enhancing efficacy and reducing toxicity.
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Objective To investigate the anti-inflammatory effect of Cornus officinalis (CO) Decotion and its refined solutions from membrane separation by using 0.05 μm inorganic ceramic membrane (CO-0.05) and 10K polysulfone hollow fiber membrane (CO-10K), and evaluate the applicability of the membrane separation technique for concentrating the anti-inflammatory compounds of C. officinalis Decotion. Methods Inflammatory model of interleukin (IL)-1β and tumor necrosis factor (TNF)-α stimulated human fibroblast-like synoviocytes (HFLS) was prepared. The CCK-8 assay and ELISA were applied to detect the effects of C. officinalis Decotion and its refined solutions on the viability of HFLS and the secretion of inflammatory cytokines, respectively. Moreover, the animal model of adjuvant arthritis (AA) was used. The SD rats were divided into six groups: control group, model (AA) group and AA groups intragastrically receiving CO (120 mg/kg), CO-0.05 (120 mg/kg), CO-10K (120 mg/kg) and TGP (0.125 mg/kg) with daily treatments for 23 days. The weight and paw swelling of rats in different groups were detected. The ELISA was used to detect secretion levels of prostaglandin E2 (PGE2), IL-1, IL-6, and TNF-α in serum. Results The production of vascular endothelial growth factor (VEGF) induced by IL-1β/TNF-α were significantly inhibited with C. officinalis Decotion and its refined solutions by membrane separation treatment (P < 0.05, 0.01, 0.001). C. officinalis Decoction and the refined solutions significantly ameliorated paw swelling and increased weight gain of AA rats (P < 0.05, 0.01, 0.001), and reduced the secretion of TNF-α, PGE2, IL-1, IL-6 in serum (P < 0.001). By comparing the inhibition efficiency of inflammatory cytokines by inorganic ceramic membrane refined solution and polysulfone hollow fiber membrane refined solution, the polysulfone hollow fiber membrane refined solution exhibited better anti-inflammatory activity. Conclusion Both of refined solutions of C. officinalis Decotion from inorganic ceramic membrane and polysulfone hollow fiber membrane separation exhibited dramatically anti-inflammtory activity. Moreover, the polysulfone hollow fiber membrane was more applicable for concentrating the anti-inflammatory compounds of C. officinalis Decotion.
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Objective: Cornus officinalis is a commonly used medicinal material in our country. In the study, the CobHLH7 was cloned and analyzed, which might be closely related to the iridoid glycosides synthesis, based on the transcriptome sequencing of Cornus officinalis fruits. Methods The special primers were designed from the both sides of open reading frame of unigene c15204_g1. The total RNA was extracted and reversed transcribed into cDNA. The transcription factor CobHLH7 was cloned through RT-PCR method, and the pMD19-T cloning vector were used for sequencing. A series of bioinformatics analysis of CobHLH7 was performed by software Protparatam, ProtScale, and SOPMA etc. Results The cDNA of CobHLH7 gene was 941bp in length, encoding 266 amino acids with a molecular weight of 29 220 and isoelectric point of 6.32. The analysis of bioinformatics through SOPMA showed that the protein was a neutral unstable protein. Its advanced structure mainly was alpha helix and random coil, and the content of beta turn and extended strand were less. Multiple sequence alignments and phylogenetic trees showed that CobHLH7 protein has high homology with ILR3 of Solanum tuberosum, ILR3-like of model organism Nicotiana attenuate. Conclusion For the first time, the CobHLH7 cDNA sequence was cloned and analyzed successfully from C. officinalis, which would lay the foundation for studying its biological functions deeply.
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Objective: To investigate the effects of a herb complex extract (HCE) prepared from Cornus officinalis Sieb. Et Zucc., Eriobotrya japonica Lindley, and olive leaves on immune response of mouse spleen NK cells in vitro and in vivo analysis. Methods: The activity of natural killer (NK) cells was measured in splenocytes and YAC-1 cells. Mice were immunosuppressed using cyclophosphamide (5 mg/kg body weight). Three different doses of HCE (200, 400, and 800 mg/kg body weight) and red ginseng extract (800 mg/kg body weight) which was used as standard immunomodulatory herb were administered orally for 4 weeks. The body weight, dietary, water intake, organs (liver, thymus, and spleen) weight, completed blood count, and cytokines (tumor necrosis factor alpha, interferon gamma, and interleukin-2) production was measured. Results: At the maximum concentration of HCE, the activity of NK cells was increased by 48.5%. HCE increased liver, spleen, and thymus weights without altering numbers of white blood cells, lymphocytes, and neutrophils in a cyclophosphamide-induced immunosuppression rat model. However, HCE recovered the inhibited cytokine expression; HCE (800 mg/kg) increased cytokines levels. The results indicate the immune enhancement potential of this HCE. Conclusion: The HCE enhances immunity by increasing NK cell activity, regulating cytokine levels, and maintaining spleen weight. Therefore, it may be used as a potential immunity enhancer.
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Objective:To investigate the effects of a herb complex extract (HCE) prepared from Cornus officinalis Sieb. Et Zucc., Eriobotrya japonica Lindley, and olive leaves on immune response of mouse spleen NK cells in vitro and in vivo analysis.Methods:The activity of natural killer (NK) cells was measured in splenocytes and YAC-1 cells. Mice were immunosuppressed using cyclophosphamide (5 mg/kg body weight). Three different doses of HCE (200, 400, and 800 mg/kg body weight) and red ginseng extract (800 mg/kg body weight) which was used as standard immunomodulatory herb were administered orally for 4 weeks. The body weight, dietary, water intake, organs (liver, thymus, and spleen) weight, completed blood count, and cytokines (tumor necrosis factor alpha, interferon gamma, and interleukin-2) production was measured.Results:At the maximum concentration of HCE, the activity of NK cells was increased by 48.5%. HCE increased liver, spleen, and thymus weights without altering numbers of white blood cells, lymphocytes, and neutrophils in a cyclophosphamide-induced immunosuppression rat model. However, HCE recovered the inhibited cytokine expression; HCE (800 mg/kg) increased cytokines levels. The results indicate the immune enhancement potential of this HCE.Conclusion:The HCE enhances immunity by increasing NK cell activity, regulating cytokine levels, and maintaining spleen weight. Therefore, it may be used as a potential immunity enhancer.
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Objective: To establish a method of quantitative analysis of multi-components by single marker (QAMS) for medicinal materials and pieces of Cornus officinalis. This method was used in combination with electronic-eye and electronic-tongue technique, and the best steaming time of Cornus officinalis was selected. Methods: Medicinal materials and pieces of C. officinalis were used as the research objects. The contents of five components were determined by establishing the relative correction factor (RCF) of gallic acid, 5-hydroxymethyl furfural (5-HMF), morroniside, cornuside, and internal reference loganin in C. officinalis. Color and taste were measured by electronic eye and electronic tongue technique. The data were analyzed by principal component analysis (PCA), and the best steaming time was optimized by analyzing the results of three methods. Results: The five compounds were well separated. The RSD values of precision and reproducibility were all less than 2%. The stability was good in 24 h. The linear relationship among the concentration and peak areas of the five compounds was all linear (r ≥ 0.999 6). The average recoveries were between 98% and 100.1% and the RSD values were all less than 2%; The RCFs of loganin with the other four compounds were 0.560, 1.344, 1.255, and 0.972 in a linear range. In the principal component analysis (PCA), the sums of main components were 94.618% and 94.98% and the discrimination indexes (DI) were 98 and 93, which indicated that all the samples of C. officinalis could be distinguished well by the electronic-eye and the electronic-tongue. The results showed that the optimum steaming time of C. officinalis was 4 h. Conclusion: The best steaming time of C. officinalis can be optimized by the combination of QAMS with electronic-eye and electronic-tongue techniques.
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Aim To observe the protective mechanism of loganinand morroniside ( active components in Cornus officinalis) on HUVEC injury induced by advanced glycation end products ( AGEs ) .Methods HUVECs were cultured in vitro and divided into control group , model group ( AGEs group ) , loganin group , morroni-side group and aminoguanidine group ( set as positive control).After being incubated with loganin and mor-roniside( final concentrations were 100,10,1 μmol?L-1 ) for 1 h, HUVECs were stimulated by AGEs of 200 mg? L-1 for 24 h.Then, the cell viability was measured by using MTT method .The supernatant was extracted and the levels of NO ,ET-1,MCP-1,VCAM-1 were measured by the corresponding kits .Receptors of advanced glycation end products ( RAGE ) and NF-κB in HUVEC were detected by Western blot .Results Loganin and morroniside could inhibit HUVEC injury induced by AGEs .In model group ,the contents of ET-1,MCP-1,VCAM-1 increased(P<0.01),the content of NO decreased ( P <0.01 ) and the expression of RAGE and NF-κB increased(P<0.01); however,lo-ganin and morronside could reduce the ET-1,MCP-1, VCAM-1contents,increase the NO content and down-regulate the expression of RAGE and NF-κB to differ-ent extents .Conclusion Loganin and morroniside could ameliorate HUVEC injury , and its mechanism may be related to inhibit inflammation , the improve-ment of endothelial cell function , and the decrease of the expression of RAGE .
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Aim To explore the protective effect of lo-ganin ( an active component in Cornus officinalis ) on podocyte injury induced by advanced glycation end products ( AGEs) and its possible mechanism. Meth-ods Mouse podocytes were cultured in vitro and di-vided into Normal group, model group ( AGEs group) , loganin group and aminoguanidine group ( set as posi-tive control) . After being incubated with loganin( final concentrations are 0. 1, 1, 10 μmol · L-1 ) for 1 h, podocytes were stimulated by AGEs of 100 mg · L-1 for 24 h. Then, the cell viability was measured by u-sing MTT method. Podocytes apoptosis was evaluated by Hoechst33342/PI staining and flow cytometry. Re-ceptors of advanced glycation end products ( RAGE ) ,desmin and apoptosis-related protein like Bax, Bcl-2, cleaved caspase-3 in podocytes were detected by Western blot. Results Loganin ameliorated podocyte injury induced by AGEs, down-regulated the expression of desmin and RAGE. Loganin also reduced the apoptotic rate of podocytes and decreased the ratio of Bax/ Bcl-2 and the expression of pro-apoptotic protein cleaved caspase-3 in podocytes. Conclusion Loganin could ameliorate podocyte injury, and its mechanism may be related to the decrease of the expression of RAGE and inhibition of the apoptotic pathway.
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PURPOSE: Diabetes is the leading cause of end-stage renal failure. The present study was undertaken to characterize the effects of Corni Fructus on diabetic nephropathy in streptozotocin-induced diabetic rats and their mechanisms. MATERIALS AND METHODS: Streptozotocin-diabetic rats were orally administrated with Corni Fructus at a dose of 100, 200 or 400 mg/kg body mass for 40 days. RESULTS: Corni Fructus-treated diabetic rats showed significant decreases of blood glucose, urinary protein levels and water consumption. Corni Fructus also reduced serum total cholesterol, total triglyceride and low-density lipoprotein cholesterol levels, and showed a tendency of enhancing high-density lipoprotein cholesterol level. Levels of serum albumin and creatinine in diabetic rats were also significantly reduced by Corni Fructus administration at a dose of 200 and 400 mg/kg body mass compared with non-treated diabetic rats. Corni Fructus increased catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidose (GSH-px) activities in the kidneys of diabetic rats. Furthermore, Corni Fructus treatment enhanced renal peroxisome proliferator-activated receptor-gamma (PPARgamma) expression in diabetic rats. CONCLUSION: These results demonstrated that Corni Fructus may have the potential to protect the animals from diabetic nephropathy by amelioration of oxidative stress and stimulation of PPARgamma expression.
Subject(s)
Animals , Male , Rats , Blotting, Western , Body Weight/drug effects , Catalase/metabolism , Cornus/chemistry , Diabetes Mellitus, Experimental/drug therapy , Glucose Tolerance Test , Glutathione/metabolism , Hypoglycemic Agents/therapeutic use , Malondialdehyde/metabolism , Plant Extracts/therapeutic use , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , Superoxide Dismutase/metabolismABSTRACT
AIM: To investigate the chemical constituents of the plant drug Cornus officinalis Sieb et Zucc. which is widely used in Chinese traditional medicine, having actions of invigorating the liver and kidney, strengthening the body, and of an astringent, etc. METHODS: A sedoheptulose gallate was isolated from the water extracts of fruits of C.officinalis using various chromatographies. The structure determination was based on spectral (UV, IR, 1H & 13CNMR and MS) and chemical evidence. RESULTS: Its structure was characterized to be 7-O-galloyl-D-sedoheptulose (I). The proportion of its three major conformation forms of β-F, α-F and α-P in aqueous solution was estimated to be 57:24:19 according to their C-1 peak heights in the 13CNMR spectrum. CONCLUSION: I was found in natural world for the first time and its β-D-heptufuranosyl form was the predominant existing tautomer in its equilibrated aqueous solution according to NMR determination.
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Object To study the effective portion in Cornus officinalis Sieb. et Zucc. extract (COE) for the treatment of arrhythmia. Methods Effect of COE on chloroform induced ventricular fibrillation in mice and electrophysiology of isolated guinea pig papillary muscle were studied. Results Antiarrhythmic effect of COE may be related to its prolongation of action potential duration, increase of the absolute value of resting potential and a decrease of autonomy of sinus node. The effective portion in COE may be its total organic acid and a certain yet unknown trace substance, whereas its total glycosides were devoid of such activities. Conclusion Pharmacodynamic and myocardial electrophysiologic studies showed that the total organic acid and a certain unknown trace substance possessed the obvious antiarrhythmic activity.
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Objective To analyze the genetic relationship among the different cultivars of Cornus officinalis and provide some foundation for heredity breeding. Methods A modified method of extracting total DNA from the leaves of C. officinalis was selected by improving the traditional method-CTAB, the total DNA was analyzed by RAPD; the genetic similarity correlation was calculated by SPSS 10.0 DICE method, cluster analyses were carried out using Between-groups linkage method, and the genetic dendrogram was established. Results Random primers (22 10-bp) were selected to be used for the PCR, a total of 133 bands were amplified by 12 samples, among which 75 bands were polymorphic, accounting for 56.39%. The cluster analysis indicated that: spindleform itself was one group, short pear-shape and short cylindericform clustered together, the rest longer types clustered together, which reflected the result of artificial selection in the course of cultivating. Conclusion The result keeps accordance with the biologic character and territorial distribution of C. officinalis cultivars; RAPD analysis is an assistant mean for seed breeding of C. officinalis.
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Objective To study the technology of supercritical fluid CO_2 extraction(SFE CO_2) on urosolic acid from Cornus officinalis.Methods The effects of pressure,temperature,time,CO_2 flow rate types,and volume of entrainer on the urosolic acid extracts were studied. The optium conditions for SFE CO_2 was determined.The technology of SFE CO_2 was compared with that of traditional solvent extraction.Results The optium extraction conditions were as follows: pressure 35.0 MPa,temperature 318 K,absolute ethyl alcohol entrainer,content 4%,time 3 h and flow rate of CO_2 8 kg/h.Conclusion SFE CO_2 excels the traditional solvent extraction in yield,safety and efficiency.