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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 study the efficacy enhancing effect of compatibility of Aconitum carmichaeli liposoluble alkaloids and total glucosides of peony in the treatment of wind-cold-dampness type bi-syndrome model rats. METHODS： Totally 100 SD male rats were randomly divided into blank group （water）， model group （water）， rotundine group [positive control， 10.0 mg/（kg·d）]， dexamethasone group [positive control， 0.3 mg/（kg·d）]，A. carmichaeli alkaloids low-dose and high-dose groups [A. carmichaeli liposoluble alkaloid extract 12.5， 25.0 mg/（kg·d）]， total glucosides of peony low-dose and high-dose groups [total glucosides of peony powder 200.0，400.0 mg/（kg·d）]， compatibility low-dose and high-dose groups [A. carmichaeli liposoluble alkaloid+total glucosides of peony powder were 12.5 mg/（kg·d）+400.0 mg/（kg·d）， 25.0 mg/（kg·d）+800.0 mg/（kg·d） respectively]， with 10 rats in each group. Except for blank group， other groups were given complete Freund’s adjuvant 0.1 mL on the right hind paw and wind-cold-dampness stimulation to induce wind-cold-dampness type bi-syndrome model. Nineteen days after modeling， they were given relevant medicine or water intragastrically， once a day， for consecutive 14 d. The joint tenderness threshold （except for dexamethasone group） and joint swelling index （except for rotundine group） were measured at 0 （before medication）， 3rd and 6th day after administration respectively. HE staining was used to observe the pathological changes of synovial membrane of ankle joint in rats （except for rotundine group）. ELISA assay was used to determine anti-cyclic citrullinated peptide （CCP） antibody in serum of rats （except for rotundine group）， and compatibility index （CI） were calculated. RESULTS： Compared with blank group， joint tenderness threshold of rats were decreased significantly in model group at different time points， while joint swelling index was increased significantly （P＜0.01）. Inflammatory lesions in synovial tissue were obvious， and the content of anti-CCP antibody in serum was significantly increased （P＜0.01）. Compared with model group， the joint tenderness threshold， joint swelling index， synovial tissue pathological changes and anti-CCP antibody content in serum of rats in medication groups were improved in varying degrees， most of the indicators had significant difference （P＜0.05 or P＜0.01）， and the improvement effect of the compatibility groups was better than single component treatment groups. CI value calculated based on anti-CCP antibody content was 0.213，suggesting that the compatibility of the two components had synergistic effect. CONCLUSIONS： The compatibility of A. carmichaeli liposoluble alkaloids and total glucosides of peony in the treatment of wind-cold-dampness type bi-syndrome model rats has analgesia，anti-inflammation and synovial tissue protection， with synergistic efficacy-enhancing effect.
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OBJECTIVE： To study the diterpenoid alkaloids in Aconitum carmichaeli from Guizhou province， and to provide reference for the further enrichment of chemical constituents of plants from the genus Aconitum and the development and utilization of A. carmichaeli from Guizhou province. METHODS： The root tuber of A. carmichaeli from Guizhou province was collected. The total alkaloids of A. carmichaeli roots were extracted by acid extraction and alkali precipitation method. The diterpenoid alkaloids were obtained by isolating and purifying total alkaloids extract with silica gel column chromatography. The structures of the isolated compounds were elucidated on the basis of NMR and MS analysis. RESULTS & CONCLUSIONS： Fifteen diterpenoid alkaloids， including 12-epi-15-O-acetyl-17-benzoyl-16-hydroxy-16，17-dihydronapelline（Ⅰ），songorine（Ⅱ），songoramine（Ⅲ），10-aconifine（Ⅳ），hypaconitine（Ⅴ），deoxyaconitine（Ⅵ），aconitine（Ⅶ），mesaconitine（Ⅷ），14-benzoyltalasamine（Ⅸ），talatisamine（Ⅹ），neoline（ⅩⅠ），6-epi-chasmanine（ⅩⅡ），isotalatizidine（ⅩⅢ），sachaconitine（ⅩⅣ）and kalakoline（ⅩⅤ） were identified form A. carmichaeli roots from Guizhou Province. Compounds Ⅸ and ⅩⅡ were isolated from this specie for the first time.
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OBJECTIVE：To study the toxicity mechanism of lipid-soluble alkaloids of Aconitum carmichaeli to adjuvant-induced arthritis （AIA） model rats. METHODS： Totally 40 rats were randomly divided into blank group （ultrapure water）， model group （ultrapure water） and A. carmichaeli lipid-soluble alkaloids low-dose and high-dose groups （12.5， 35 mg/kg）， with 10 rats in each group. Except for blank group， rats in other groups were given complete Freund’s adjuvant 0.1 mL on the right hind paw to induce AIA model. 19 d after modeling， they were given relevant medicine intragastrically， once a day. After 14 d of administration， endogenous metabolites were separated and identified from plasma by UPLC-LTQ/Orbitrap MS. Then， the collected data were analyzed by principal component analysis （PCA） and partial least squares-discriminant analysis （PLS-DA）. Variable importance projection （VIP）＞1 and P value （＜0.05） were used to screen differential metabolites in plasma. Retrieving the database of Kyoto Encyclopedia of Genes and Genomes according to the differential metabolites，the toxic mechanism of A. carmichaeli liposoluble alkaloids to AIA rats were speculated. RESULTS： A total of 57 plasma metabolites were indentified， and 11 differential metabolites such as L-proline， 6-hydroxynicotinic acid and adenosine were identified. After inducing AIA model， the plasma contents of L-proline and uridylic acid were decreased significantly （P＜0.05 or P＜0.01）， and the content of deoxycytidine was increased significantly （P＜0.01）. Low dose of A. carmichaeli lipid-soluble alkaloids could decrease the plasma contents of adenosine and L-proline in rats （P＜0.05 or P＜0.01）， while the plasma contents of deoxycholic acid was increased significantly （P＜0.05）. High dose of A. carmichaeli lipid-soluble alkaloids could decrease the plasma contents of 6-hydroxynicotinic acid， adenosine， carnitine， L-proline， N-formylaminobenzoic acid were decreased significantly （P＜0.05 or P＜0.01）， while the plasma contents of deoxycholic acid， L-arginine， deoxycytidine and L-lysine were increased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS： The toxicity of low-dose of A. carmichaeli lipid-soluble alkaloids to AIA model rats is less； the toxicity of high-dose of A. carmichaeli lipid-soluble alkaloids to AIA model rats may be related to abnormal bile secretion， lysine biosynthesis and metabolic disorders of purine， pyrimidine， tryptophan， proline and arginine.
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Four compounds were isolated from the lateral roots of Aconitum carmichaeli by Al2O3,silica gel and preparative HPLC.Their structures were identified as:(13R,15S,19S)-13,15,19-triol-hetisan (1),fuziline (2),neoline (3) and beiwutine (4),by methods of NMR,MS spectroscopy as well as single crystal X-ray crystallography.Among them,compound 1 was considered as a new diterpenoid alkaloid.
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The recognition and use of Aconitum carmichaeli have undergone various changes from the pre-Qin period to Tang dynasty. From "the drug of evil" to "the leader of hundreds of medicine ", people's understanding of A. carmichaeli is more and more deep and specific. The medicinal values of A. Carmichaeli have been excavated, and it is widely used in brewing skill, agricultural production, Taoism, Buddhism and festival customs. Its function is becoming more and more diversified and routinized. By studying it, we can glimpse some rules in the evolution of traditional Chinese medicine, for instance relationships between drugs and poisons, drugs and non-drug; the inheritance and integration of medical books' recording; the generalization and routinization of Chinese medicinal medicines' functions, and so on.
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Objective: To define sequences about rDNA-ITS and 28 S rDNA D1/D2 of Peronospora aconiti separated from cultivated Aconitum carmichaeli in Jiangyou area of Sichuan province and provide a theoretical basis for the diagnosis and prevention of downy mildew disease. Methods: Spores and hyphae of P. aconiti from diseased plants were collected and total genomic DNA of pathogen were extracted and then rDNA-ITS and 28 S rDNA D1/D2 fragment were amplified and sequenced. According to the above results, the abutment (Neighbor-joining, NJ) phylogenetic tree of pathogen was constructed and analyzed. Results: The rDNA-ITS and 28 S rDNA D1/D2 sequences of P. aconiti were sequenced and compared according to the database from NCBI. Compared that with P. pulveracea and P. aparines, the similarity of rDNA-ITS sequences of P. aconiti was 94%. The similarity of 28 S rDNA D1/D2 sequences of P. aconiti was 97% compared that with P. pulveracea, P. ficariae and P. bulbocapni. Conclusion: The results of morphological identification of downy mildew pathogen separated from A. carmichaeli are consistent with those from molecular identification (rDNA-ITS and 28 S rDNA D1/D2 sequences) and the pathogen of Aconitum downy mildew should be P. aconiti. Therefore, rDNA-ITS and 28 S rDNA D1/D2 sequences constructed in this paper can be used to identify downy mildew pathogen from Aconitum carmichaeli Debx.
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An MSAP analysis method was established for detecting DNA methylation of Aconitum carmichaeli leaves, and the DNA methylation of different leaf shapes and different leaf position was analyzed by MSAP. The study made experiments on the leaves of different position of mosaic and moxa leaf type A. carmichaeli, researched the effects of restriction digestion of genomic DNA by using two restriction enzymes, screened the suitable selective amplification primers, and analyzed the methylation differences of leaves by calculating the 6% acrylamide gel electrophoresis bands and lane. The best reaction system of MSAP was obtained, under the conditions of 37 ℃, the 16 h incubated time was more suitable for 150 ng DNA, and 25 pairs of selective amplification primers were selected from 256 pairs. Totally, 273 electrophoresis bands were obtained by 25 pairs of selective primers, including 228 non methylation or single chain methylation bands,27 double chain methylation bands,and 18 single stranded methylation bands, the total methylation rate was 16.48%. The methylation rate was slightly different in mosaic and moxa leaf type A. carmichaeli leaf, which were 15.36%, 14.34%, respectively, and article 8, article 6 nucleotide fragments of genome methylation modification differences were obtained, accounted for 3%, 2.26% of the total number of bands. Based on this study it can provide new ideas for molecular identification, breeding and cultivation, and genetic evolution of A. carmichaeli.
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An offline two-dimensional system combining a rat cardiac muscle cell membrane chromatography time-of-flight mass spectrometry (CMC-TOF/MS) with a high performance liquid chromatography time-of-flight mass spectrometry (HPLC-TOF/MS) was established for investigating the parent components and metabolites in rat urine samples after administration of the roots of Aconitum carmichaeli.On the basis of the analysis of the first dimension,retention components of the mine sample were collected into 30 fractions (one fraction per minute).Then offline analysis of the second dimension was carried out.34 compounds including 24 parent alkaloids and 10 potential metabolites were identified from the dosed rat urine,and then binding affinities of different compounds on cell membranes were compared and influences of some functional groups on activity were estimated with the semi-quantification and curve fitting method.As a result,binding affinities decreased along with the process of deacylation,debenzoylation and demethylation,which may be related to the alleviation of toxicity in the procedure of herb processing or metabolism.Moreover,some minor components in rat urine (Songorine,14-benzoylneoline,Deoxyaconitine,etc.) exerted relatively strong affinity on cell membranes are worth exploring.The results delivered by the system suggest that the CMC can be applied to in vivo study.
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An offline two-dimensional system combining a rat cardiac mascle cell membrane chromatography time-of-flight mass spectrometry （CMC-TOF/MS） with a high performance liquid chromatography time-of-flight mass spectrometry （HPLC-TOF/MS） was established for investigating the parent components and metabolites in rat urine samples after administration of the roots of Aconitum carmichaeli. On the basis of the analysis of the first dimension, retention components of the urine sample were collected into 30 fractions （one fraction per minute）. Then offline analysis of the second dimension was carried out. 34 compounds including 24 parent alkaloids and 10 potential metabolites were identified from the dosed rat urine, and then binding affinities of different compounds on cell membranes were compared and influences of some functional groups on activity were estimated with the semi-quantification and curve fitting method. As a result, binding affinities decreased along with the process of deacylation, debenzoylation and demethylation, which may be related to the alleviation of toxicity in the procedure of herb processing or metabolism. Moreover, some minor components in rat urine （Songorine, 14-benzoylneoline, Deoxyaconitine, etc. ） exerted relatively strong affinity on cell membranes are worth exploring. The results delivered by the system suggest that the CMC can be applied to in vivo study.
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OBJECTIVE: To research the content of alkaloids and polysaccharides in different varieties of Aconitum carmichaeli. METHODS: The contents of total alkaloids, diester diterpenoid alkaloids and polysaccharides were measured by acid dye colorimetry, HPLC and sulphuric acid-anthrone colorimetric method respectively. RESULTS: In different varieties, the differences of total alkaloids and diester diterpenoid alkaloids were significant. However, the difference of polysaccharides wasn’t significant. The content of total alkaloids and diester diterpenoid alkaloids in processed A. carmichaeli reduced significantly while the content of polysaccharides increased slightly. CONCLUSION: The results of study could provide a scientific basis for rational breeding of A. carmichaeli.
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OBJECTIVE:To compare the different sample preparation and optimize the isolation of three kinds of alkaloids in Aconitum carmichaeli.METHODS:RP-HPLC was applied and performed on Zorbax extend-C18 column (150 mm?4.6 mm,5 ?m) column.The differences among the content of alkaloids prepared by preparation technologies were compared.RESULTS:The best preparation was aether ultrasonic extraction.The separation condition was as follows:mobile phase consisted of acetonitrile-0.1% ethylenediamine (gradient elution) with flow rate of 1 mL?min-1.The detection wavelength was set at 240 nm and column temperature was 30 ℃.CONCLUSION:Aether ultrasonic extraction is simple with ideal separation,extraction and re-covery to provide scientific basis for the quality control of A.carmichaeli.
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Object To establish an effective plantlet regeneration system of Aconitum carmichaeli Debx. for the purpose to obtain a large number of high quality seedling in a short time. Methods Leaves in vitro were tried as the explants and cultivated in different media with the various portion of hormones. Results The medium of MS+BA 1.0 mg/L+KT 0.5 mg/L+NAA 0.2 mg/L was the most suitable one for the induction of shoots; the medium of MS+BA 1.5 mg/L+NAA 0.1 mg/L was beneficial to the propagation of shoots; the medium of MS+BA 0.5 mg/L+NAA 0.1 mg/L was good for the elongation of shoots; and the medium of 1/2 MS without any hormone was suitable for the induction of roots. Conclusion Rapid propagation of A. carmichaeli could be achieved by tissue culture and this will lead to the possibility for its seedling in the industrial production.
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Objective ISSR Identification of genetic diversity in Aconitum carmichaeli by ISSR marker technique. Methods Genetic diversity between Jiangyou Radix Aconiti Lateralis Preparata and nine wild A. carmichaeli populations was determined by ISSR technique. Results Eight primers were selected to produce highly reproducible ISSR bands. Among 98 amplified bands, 68 showed polymorphism, the percentage of polymorphic bands (PPB) reached to 69.39%. Observed number of alleles (Na), effective number of alleles (Ne), Nei′s gene diversity index (H), and Shannon information index (I) were 1.693 9, 1.371 5, 0.230 8, and 0.353 0, respectively. A DNA profile was discovered with a single primer, ISSR 855, in which each of ten tested populations had its unique patterns and was distinguished from each other. Conclusion ISSR Method is suitable for DNA fingerprinting, identification , and genetic diversity analysis of A. carmichaeli.
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AIM: Radix Aconite is unprocessed parent root tuber of Aconitum carmichaeli Debx.containing toxic diterpenoid alkaloids such as mesaconitine,aconitine and hypaconitine.To establish the quality standard for this potent herb. METHODS: The microscopic examination technique,HPLC-UV and LC-MS-MS methods had been used. RESULTS: These methods can be used to analyse the quality of this potent herb. CONCLUSION: This paper proposes microscopic examination technique and physio-chemical methods such as HPLC-UV and LC-MS-MS for the identification and quantitative analysis of the herbs.
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Uracil (I) (2,4-Diketo-pyrimidine) was isolated from the radix aconiti collected from Jiangyou region of Sichuan Province.The chemical structure of this principle was determined by elemental analysis,IR,UV,MS,'H and 13C-NMR spectral data. The experiments were made to study the effect of uracil (I) on the cardiac contractile force and beating rate of the isolated toad hearts in comparison with uracil (II) (artificial synthesis). The results indicated that the 5?mol/L of uracil (I) and uracil (II) both had the effect of increasing the amplitude of myocardial contraction (P0.05) at 3 and 5 min. When the concentration was increased to 10 and 20?mol/L, the amplitudes of myocardial contraction were more incremental and 3 min later,the estimated value of uracil (I) and uracil (II) to increase the amplitude of myocardial contraction by 50% approached to 4.17 and 4.68, respectively. As described above, uracil (I) is a new cardiotonic in Jiangyou Fuzi.