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ABSTRACT OBJECTIVE：To study the improvement effect of Periplaneta americana extract Ento-A on damp-heat ulcerative colitis（UC）model rats. METHODS：Totally 70 rats were randomly divided into normal control group（n＝8）and modeling group （n＝62）. The damp-heat UC model was induced in modeling group by high sugar，high fat，spicy diet combined with 2，4， 6-trinitrobenzene sulfonic acid enema. 48 modelrats were randomly divided into model control group，mesalazine group （300 mg/kg），Changyanning group（300 mg/kg）and Ento-A low-dose，medium-dose and high-dose（50，100，200 mg/kg，calculated by the extract），with 8 rats in each group. Normal control group and model control group were given normal saline intrsgastrically，and other groups were given relevant medicine intragastrically，once a day，for consecutive 14 days. After last administration， disease activity index （DAI）， colonic mucosal injury index （CMDI） and histopathological score （HS）of rats were determined. The spleen index，liver index and colon index in rats were determined. The serum levels of IL-8，IL-17，SOD and MDA，colonic levels of IL-2，PGE2 and MPO were detected by ELISA. RESULTS：Compared with normal control group，the DAI score，CMDI score，HS score，colonic index，the serum levels of IL-8，IL-17 and MDA， colonic levels of MPO and PGE2 were increased significantly（P＜0.01）；serum level of SOD and colonic level of IL-2 were decreased significantly （P＜0.01）. Compared with model control group，DAI score，CMDI score，serum levels of IL-17 and MDA，colonic levels of PGE2 were decreased significantly in Ento-A high-dose groups（P＜0.05 or P＜0.01），while serum level of SOD and colonic level of IL-2 were increased significantly（P＜0.01）. CMDI score and HS score，serum levels of IL-8，IL-17 and MDA，colonic levels of PGE2 and MPO were decreased significantly in Ento-A medium-dose group（P＜0.05 or P＜0.01）， while colonic level of IL-2 was increased significantly（P＜0.01）. HS score，serum levels of IL-17 and MDA，colonic levels of MPO and PGE2 were decreased significantly in Ento-A low-dose group（P＜0.05 or P＜0.01），while serum level of IL-2 was increased significantly（P＜0.01）. CONCLUSIONS：P. americana extract Ento-A may play improvement effect on damp-heat UC rats by regulating immune system balance and reducing inflammatory damage.
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OBJECTIVE：To study the spectrum-ef fect relationship of anti-hepatoma effect of C Ⅱ-3 extract from Periplaneta americana，and to preliminarily clarify the anti-hepatoma active components of it. METHODS ：Based on UHPLC fingerprints of 10 batches of C Ⅱ-3 samples，with the help of UHPL C-Q-TOF/MS，the compounds corresponding to each chromatographic peak were identified qualitatively by standard substances and related literatures. Using the IC 50 value of each batch of C Ⅱ-3 sample against human hepatoma cells HepG 2 as anti-hepatoma activity index ，the spectrum-effect relationship of fingerprint and anti-hepatoma effect was established and analyzed by the combination of grey relational analysis （GRA）and orthogonal partial least squares（OPLS）. RESULTS ：There were 25 common peaks in UHPLC fingerprints of 10 batches of C Ⅱ-3 samples，and 10 chemical compounds were identified ，which were cyclo- （Tyr-Pro）（peak 24），cyclo-（Gly-Phe）（peak 15），hypoxanthine（peak 3）， adenine（peak 7），phenylalanine（peak 10），inosine（peak 11），N-acetyldopamine（peak 16），cyclo-（Pro-Ala）（peak 13），2-hydroxy propionyl（peak 22），cyclo-（Pro-Ser）（peak 6）. The IC 50 of 10 batches of C Ⅱ-3 samples to HepG 2 cells was 70.550-200.303 μg/mL. Among 25 common peaks ，the order of GRA correlation （r）of anti-hepatoma activity was peak 20＞23＞24＞15，all of r values were greater than 0.7；the order of variable importance projection （VIP）of OPLS analysis was peak 23＞18＞15＞24＞7＞14＞6＞ 2＞20，all of VIP values were greater than 1. The standard regression coefficients of peak 7，15，20，23，24 were all greater than 0；while the standard regression coefficients of peak 2，6，14，18 were all less than 0. Conjoint analysis shows that the order of anti-hepatoma activity was peak 20＞23＞24＞15. CONCLUSIONS：unknown chemical ingredients （peak 20， ），cyclo-（Tyr-Pro）（peak 24）and cyclo- （Gly-Phe）（peak 15） in C Ⅱ-3 may be main anti-hepatoma active components.
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OBJECTIVE： To investigate the acute toxicity， long-term toxicity， skin irritation and anaphylaxis of Bee venom （BV） plastics， and to evaluate its preclinical safety. METHODS： The acute toxicity of BV plastics to rats was investigated after administration of high-dose， medium-dose and low-dose （144， 96， 48 mg/kg） of BV plastics. The long-term toxicity of BV plastics was investigated by continuous administration of high-dose， medium-dose and low-dose （72， 48， 24 mg/kg） of BV plastics for 28 days. The irritation of intact and damaged skin in rabbits with 8 mg/kg BV plastics was investigated by using the self-control method of left and right homologous body. The skin anaphylaxis of guinea pig were investigated after sensitized with 15 mg/kg BV plastics on the left back （on 0， 7th， 14th day） and stimulated with 15 mg/kg BV plastics on the right back. RESULTS： During the acute toxicity experiment with BV plastic，the weight of rats and the changes of viscera were normal，and there was no relevant toxic reaction. Long-term toxicity test results showed that no significant pathological changes were observed at 24 h after the last administration； the spleen index of rats in BV low-dose group， testicular index in middle-dose group and epididymis index in high-dose and middle-dose groups were significantly increased， while PT in plasma of rats in BV medium-dose and low-dose groups was significantly prolonged （P＜0.05）. There were no abnormal changes in organ appearance， other organ index， coagulation index and blood biochemical index. All above indexes became normal at the end of 2-week recovery period. Skin irritation test showed that BV plastics could cause slight erythema and obvious scab on the skin of rabbits which along with little irritation on intact or damaged skin. Skin anaphylaxis test showed that BV plastics produced mild erythema in the skin of guinea pigs， belonging to light allergy. CONCLUSIONS： No acute or long-term toxicity is observed after transdermal administration of BV plastics， which is safe and only causes mild irritation and irritability to skin， indicating there is good safety of the plastic under experiment doses.
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OBJECTIVE： To study the improvement effects of Bee venom（BV） plastics on experimental cerebral thrombosis in rats. METHODS： Totally 96 SD rats were randomly divided into sham operation group （normal saline）， model group （plastics blank matrix）， Nimodipine group （positive drug， 4.00 mg/kg） and BV plastics low-dose， medium-dose， high-dose groups （1.67， 3.33， 6.67 mg/kg）， with 16 rats in each group. Rats in sham operation group and Nimodipine group were given medicine intragastrically， while rats in model group and BV plastics groups were given medicine by transdermal smearing. After 5 days of continuous administration， the experimental cerebral thrombosis model was established by ligating the right external carotid artery and pterygomandibular artery， and injecting compound thrombus inducer into the internal carotid artery. The wet mass ratio of right brain to left brain was measured to investigate the degree of brain edema on the infarcted side. The content of Evans blue （EB） in the left and right hemispheres of rats was determined by ultraviolet spectrophotometry to investigate the cerebral vascular permeability. Blood rheology and coagulation function indicators of rats were measured. The pathological changes of brain tissue in rats were observed by HE staining， and the number of survival neuron cells was counted. RESULTS： Compared with the indexes of sham operation group， the cerebral thrombosis model was established successfully. Compared with model group， the area of blue staining in the right brain （infarcted side） of rats in BV plastics groups was significantly reduced， and the right brain/left brain wet mass ratio and the content of EB in the right brain tissue were significantly reduced （P＜0.05 or P＜0.01）. The whole blood viscosity and Casson viscosity of rats in BV plastics groups， and the plasma viscosity of rats in BV plastics medium-dose and high-dose groups decreased significantly （P＜0.01）. PT and APTT of rats were prolonged significantly in BV plastics medium-dose group （P＜0.01）. The pathological changes of brain tissue in rats in BV plastics groups were significantly alleviated. The arrangement of neuron cells was more orderly， the shape and structure of cells were clear， the nucleolus was clear， the membrane was intact， and the number of survival neuron cells was significantly increased （P＜0.01）. CONCLUSIONS： BV plastics can alleviate brain edema， inhibit cerebral vascular permeability， improve hemorheology and coagulation function indicators of rats after the formation of cerebral thrombosis， and alleviate nerve cell injury after ischemia.
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Objective:To prepare mesalazineβ-cyclodextrin inclusion compound and observe its properties. Methods:The struc-ture was characterized by UV, IR and DSC. The inclusion process of mesalazine andβ-cyclodextrin was simulated using molecular doc-king technique. The solubility and dissolution of inclusion compound were determined. Results:UV, IR and DSC were used to deter-mine the formation of inclusion complex. The inclusion ratio was 1: 1 and the phase solubility diagram was AL type. The solubility of inclusion compound in deionized water (25 ℃, pH 7. 0) was 7. 8 mg·ml-1 . The preparation of mesalazine β-cyclodextrin inclusion compound could significantly increase the dissolution rate and solubility of mesalazine. Conclusion:The prepared mesalazineβ-cyclo-dextrin inclusion compound can obviously improve the poor water solubility and dissolution of mesalazine.
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This study aimed at exploring a fast method to accurately identify the medicinal insect Vespa mandarinia Smith from its adulterants using DNA barcode and COI sequences.The extracted DNAs from V.mandarinia and its adulterants V.soror were amplified by polymerase chain reaction (PCR) and sequenced bilaterally based on COI barcode sequence investigation.The information of the COI sequences of V.mandarinia and V.soror were gathered from GenBank.All the sequences were compared and analyzed,and their intraspecific and interspecific genetic distances were calculated using MEGA 6.06.In addition,the phylogenetic tree was established with neighbor-joining (NJ) method.As a result,the COI sequences of V.mandarinia and V.soror were successfully amplified.The minimum interspecific distance between V.mandarinia and its adulterants was 0.152 ± 0.017,being considerably larger than the maximal intraspecific distance between V.mandarinia,0.009±0.004.The constructed phylogenetic tree showed an independent branch for each species.It was concluded that the DNA barcode based on COI sequence can efficiently identify V.mandarinia and its adulterants.This study provided an innovative tool for the quality control and market regulation of Chinese materia medica,securing the safe medication of V.mandarinia.
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Objective Human mitochondrial transcription termination factor 3 ( MTERF3 ) is a negative regulator of mito?chondrial gene expression and energy metabolism. This study was to construct a prokaryotic expression system for MTERF3 in Esche?richia coli ( E. coli ) and prepare its mouse?anti?human polyclonal antibody. Methods The complete open reading frame ( ORF) of human MTERF3 cDNA was amplified by RT?PCR and subcloned into prokaryotic expression vector pET28b. Then the recombinant plasmid pET28b?MTERF3 was transformed into competent E.coli BL21(DE3) and IPTG induced the expression of 6×his fusion protein. The recom?binant human MTERF3 protein was purified through Ni2+?NTA agar?ose gel column affinity chromatography and the purified recombinant protein was used as immunogen to immunize the BALB/c mice to pre?pare its specific polyclonal antibody. The titer and specificity of the antibody were analyzed by ELISA, Western blot and cellular immuno?fluorescence, respectively. Results The recombinant human MTERF3 protein was successfully expressed in E. coil and the mouse?anti?human MTERF3 polyclonal antibody with high quality was successfully prepared. ELISA showed that the titer of the antibody was 1:105 . Western blot and immunofluorescence detection revealed that the mouse?anti?human MTERF3 antibody could recognize the native MTERF3 antigen specifically. Conclusion Human MTERF3 expressed in the prokaryotic system has strong immunogenicity and the polyclonal antibody obtained from immunizing mice has high titer and specificity. The prokaryotic expression of human MTERF3 and the preparation of its antibody lay the foundation for further function research of human MTERF3.
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Objective To research the protective effect of Ento-Ⅰagainst cerebral ischemia-reperfusion injury in rats,and to evaluate its analgesic and anticoagulating effects in mice. Methods The ischemic model was established with line embolism to block the middle cerebral artery of male rats. The 56 rats were randomly assigned into 7 groups of sham-operation,blank-matrix,nor? mal saline,Ento-Ⅰplastic of 3 doses(6.67,3.33,1.67 mg/kg),and ozagrel sodium(8.3 mg/kg,ip). The effect of Ento-Ⅰplastic on anti-cerebral ischemia was measured by nervous function scores and the areas of cerebral infarction were determined by TTC staining for the calculation of cerebral infarction rates. The analgesic effect of Ento-Ⅰplastic was determined with acetic acid-induced twisting experiment. Sixty KM mice were randomly allocated into blank-matrix,aspirin,aspirin-plastic,and Ento-Ⅰplastic of 3 doses(5,10 and 20 mg/kg),the number of mouse twisting were recorded right after intraperitoneal injection of 0.7%acetic acid solution at the time of 1 h after the last administration. Moreover,the anticoagulant activity of Ento-Ⅰplastic was tested by glass capillary method. Re?sults The results of acetic acid-induced twisting experiment displayed that Ento-Ⅰplastic of all 3 dose groups(5,10 and 20 mg/kg) could significantly reduce the number of body torsion and increase the inhibitory rates of twisting,compared with that of blank matrix group(the inhibitory rates of twisting for 3 dose groups were 21.79%,48.89%,and 56.15%,respectively),with dose-response man?ner. According to the results of glass capillary test,the clotting time of mouse blood could be significantly prolonged by mid-(10 mg/kg)and low-dose(5 mg/kg)of Ento-Ⅰplastic with corresponding clotting time of(155.20±54.19)s and(155.80±73.84)s,compared with normal saline group at(92.10 ± 24.61)and blank-matrix group at(80.40 ± 48.09,P<0.05). The experiment results of the isch?emia-reperfusion injury by line embolism method in rats exhibited that Ento-Ⅰplastic in mid-dose(3.33 mg/kg)could significantly re?duce the neurological scores after 24 h of reperfusion injury,from(2.33 ± 0.52)of normal saline group to(1.00 ± 0.00)of mid-dose group(P<0.01). The results from TTC staining revealed that the cerebral infarction rates of normal saline group and blank-matrix group were(24.89±7.24)%and(27.72±7.89)%,respectively,whereas those of 6.67 mg/kg and 3.33 mg/kg group of Ento-Ⅰplastic were(14.01±2.65)%and(14.73±4.94)%,respectively. Compared to the 2 negative-control groups,both the high-and mid-dose of Ento-Ⅰplastic could significantly reduce the cerebral infarction rates after ischemic reperfusion injury in rats (P<0.01). Conclu?sion Ento-Ⅰplastic demonstrates strong analgesic and anticoagulant effects,and could substantially reduce the neurological scores and reduce cerebral infarction rates for ischemia-reperfusion injured rats. These are likely to be the mechanism of action for Ento-Ⅰplastic realizing its anti-cerebral ischemia effect.