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Objective: To investigate the content changes of alkaloids in stems and leaves of Aconiti Radix at different growth period. Methods: The Phenomenex C18 column (150 mm × 4.6 mm, 5 μm) was used and 0.1% formic acid aqueous solution-acetonitrile was selected as mobile phase; The mass spectrum was scanned by ESI+ multiple reaction monitoring (MRM) mode. The HPLC-MS/MS method was established for the simultaneous determination of aconitine, mesaconitine, hypaconitine, indaconitine, benzoylaconine, benzoylmesaconine, benzoylhypaconitine, aconine, fuziline, neoline, talatisamine, songorine, higenamine, and salsoline in the stems and leaves of Aconite Radix. Combined with principal component analysis (PCA), the transfer rule of various alkaoids was tracked in the growth cycle of Aconiti Radix. Results: Methodological validation results showed that the linear range of the 14 compounds was good (r2 > 0.990 0). The limit of quantification was 2.27-18.27 ng/mL, and the average recovery was 94.73%-104.50%. The results showed that there was considerable amounts of alkaloids in stems and leaves of Aconiti Radix, and the total contents of alkaloids in stems were higher than those in leaves. In May, June, July, and August, the total content of alkaloids in stems was 0.087 1%, 0.182 8%, 0.141 0%, and 0.199 4% respectively, which showed a wave-like upward trend. The total content of alkaloids in leaves was 0.074 7%, 0.075 9%, 0.081 4%, and 0.058 9% respectively, which showed a trend of rising first and then decreasing, and the total content of alkaloids in leaves was highest in July. Conclusion: PCA found that the alkaloids content in stems and leaves showed different variation trend in the different period. The considerable alkaloids in stems reached the peak value at the harvest time. The amount of alkaloids is considerable, which has the potential and development value of becoming new medicinal resources.
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This present study is to develop an HPLC method for simultaneous determination of four diester diterpenoid alkaloids, beiwutine, mesaconitine, hypaconitine and aconitine in the leaves of Aconitum kusnezoffii, so as to provide evidence of the quality control of this herb. The four constituents were measured on a Waters XBridge CC₁₈ column(4.6 mmχ250 mm, 5 μm). The mobile phase was acetonitrile-40 mmol·L⁻¹ ammonium acetate solution(adjusted pH to 10.5 with ammonia solution)(33:67) with isocratic elution at a flow rate of 1.0 mL·min⁻¹; the detection wavelength was 235 nm; the column temperature was 30 °C, and the injection volume was 10 μL. Next, this contents of the four diester diterpenoid alkaloids in 12 samples were 0.025 5-0.088 5, 0.039 1-0.071 5, 0.026 6-0.081 0 and 0.008 12-0.031 2 mg·g⁻¹, respectively. Next, this method has been successfully applied to the analysis of A. kusnezoffii folium in different harvest periods. The contents of the four alkaloids decreased primarily, and then increased with the postponing of harvest. The established method is proved to be accurate and sensitive for the determination of alkaloids in A. kusnezoffii folium, and may be useful for the quality improvement of this herbal medicine. Moreover, these results indicated the scientific significance for the toxicity and the suitable harvest time of this herb.
Subject(s)
Aconitine , Aconitum , Chemistry , Chromatography, High Pressure Liquid , Diterpene Alkaloids , Drugs, Chinese Herbal , Chemistry , Phytochemicals , Plant Leaves , Chemistry , Plants, Medicinal , ChemistryABSTRACT
Objective:To develop an HPLC gradient elution method for the simultaneous determination of fangchinoline,tetrandrine,mesaconitine,aconitine and hypaconitine in Huoxue Zhentong plaster.Methods::A Dikma-C18 (200 mm×4.6 mm,5 μm) chromatographic column was adopted,the mobile phase was methanol-acetonitrile (3∶1)(A)-0.06% diaethylamin solution (B) with gradient elution at a flow rate of 1.0 ml·min-1,the detection wavelength was 280 nm for fangchinoline and tetrandrine,and 235 nm for mesaconitine,aconitine and hypaconitine.The column temperature was set at 30 ℃,and the injection volume was 10 μl.Results::There was a good linear relationship when the content of fangchinoline,tetrandrine,mesaconitine,aconitine and hypaconitine was within the range of 7.490-149.800 μg·ml-1(r=0.999 9),14.610-292.200 μg·ml-1(r=0.999 8),4.150-83.000 μg·ml-1(r=0.999 2),5.250-105.000 μg·ml-1(r=0.999 6) and 5.140-102.800 μg·ml-1(r=0.999 9),respectively.The average recovery and the corresponding RSD were 99.87%(0.49%),97.79%(1.11%),96.97%(1.75%),98.60%(1.50%) and 97.94%(0.98%)(n=6),respectively.Conclusion:An HPLC gradient elution method is successfully established for the simultaneous determination of 5 components in Huoxue Zhentong plaster.The established method is simple,accurate and reliable,which is helpful to the quality control of Huoxue Zhentong plaster.
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Objective: To study the fragmentation pathways of six aconitine-type alkaloids (aconitine, hypaconitine, mesaconitine, benzoylaconitine, benzoylhypaconitine, and benzoylmesaconitine) in mass spectra (MS). Methods: The samples were analyzed by liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS). Results: In positive mode, The typical fragmentation pathways of the six aconitine-type alkaloids were mainly continuous loss of CH3OH and H2O. The characteristic fragmentation pathway of diester-diterpene type aconitine was losing an acetic acid molecule from C-8 position; However this fragmentation could not be observed in MS of mono-ester aconitine alkaloids. Conclusion: The study on fragmentation pathways could be adopted for the structural identification of the six aconitine-type alkaloids.
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The species in Aconitum L. are commonly used in China as well known toxic herbs. Its main components, such as aconitine, mesaconitine, and hypaconitine, have significant pharmacological activity, and are also its toxic components. As a result, the therapeutic dose and toxic dose are very close, and the clinical therapeutic window is narrow. Adverse reactions and poisoning incidents occur frequently in clinic, which limits its wide applications. Modern toxicology studies on the plants of Aconitum L. to make the toxicity and its clear mechanism have important significance for more reasonable clinical guidance and safety evaluation. This paper reviews the toxicity of the plants of Aconitum L. and its mechanism and provides a scientific basis for clinical use.
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ObjectiveTo investigate the effects of aconitine, mesaconitine and hypaconitine on calcium release in isolated adult rat cardiac myocytes.MethodsThe left ventricular cardiac myocytes isolated from adult Sprague-Dawley rats were perfused withacnitine, mesaconitine and hypaconitine at 0.3 μmol/L, 1μmol/L, 3 μmol/L for 12 min. The spontaneous calcium release (SCR) rate, the end-diastolic[Ca2+](F0) and the calcium transient amplitude (ΔF) were detected 4 min, 8 min and 12 min after the perfusion. 12 min after the perfusion with acnitine, mesaconitine and hypaconitine at 0.3 μmol/L, the changes of systolic dynamics and calcium transient were detected for the positive inotropic effect. Results Any of aconitine, mesaconitine and hypaconitine induced SCR, mesconitine-induced SCR rate was highest at low concentration (0.3 μmol/L), and aconitine-induced SCR rate highest at high concentration (3 μmol/L). Compared with the control, 12 min after the perfusion with acnitine, mesaconitine and hypaconitine at 3 μmol/L elevated F0 (1.459 ± 0.379, 1.585 ± 0.493, 1.213 ± 0.254vs.1.079 ± 0.108, allP<0.05) and ΔF(1.615 ± 0.455, 2.210 ± 0.756, 1.528 ± 0.422vs. 1.036 ± 0.125, allP<0.05), mesaconitine with ΔF higher than aconitine and hypaconitine. At low concentration (0.3 μmol/L), compared with control, aconitine, mesaconitine and hypaconitine increased ΔF (0.409 ± 0.127, 0.423 ± 0.107, 0.414 ± 0.118vs.0.260 ± 0.065;P<0.05 orP<0.01) and contraction amplitudes (5.464% ± 2.239%, 7.449% ± 2.548%, 5.524% ± 1.645%vs.3.428% ± 0.911%;P<0.05 orP<0.01), prolonged the time to peak of calcium transient (0.041 ± 0.016 s, 0.039 ± 0.009 s, 0.038 ± 0.011 svs.0.032 ± 0.007 s;P<0.05 or P<0.01); compared with aconitine, mesaconitine and hypaconitine decreased calcium transient time constant (0.301 ± 0.054 s, 0.324 ± 0.064 svs.0.361 ± 0.076 s;P<0.05 orP<0.01) and diastolic t50 (0.124 ± 0.035 s, 0.126 ± 0.040 svs.0.157 ± 0.056 s;P<0.05 orP<0.01).ConclusionsAconitine, mesaconitine and hypaconitine reveal the positive inotropic effects couple with the toxic effects. Increased[Ca2+]in cardiac myocytes is the key factor for the positive inotropic effects, but also the risk factor for SCR.
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Aim: To study pharmacokinetics of aconitine,mesaconitine and hypaconitine in rats after single oral administration of decoctions composed with Radix Aconiti Lateralis.Methods: Four groups of rats were orally administered four decoctions including decoction a(Sini decoction),decoction b(decoction composed with Radix Aconiti Lateralis),decoction c(decoction composed with Radix Aconiti Laterali and Radix et Rhizoma Glycyrrhizae Praeparata Cum Melle)and decoction d(decoction composed with Radix Aconiti Laterali and Rhizoma Zingiberis),respectively.Quantitative analysis of aconitine,mesaconitine and hypaconitine in rat plasma was achieved using a liquid chromatography-electrospray ionization/tandem mass spectrometry method.Pharmacokinetic parameters were estimated using DAS 2.0.Results: Pharmacokinetic parameters of aconitine,mesaconitine and hypaconitine were different after oral administration of four decoctions according to Radix Aconiti Laterali combined with different herbal medicines.Multiple peaks were observed in plasma concentration-time curve after oral administration of the decoction of herb couple Radix Aconiti Laterali and Radix et Rhizoma Glycyrrhizae Praeparata Cum Melle,and the results showed a delay in tmax and a prolonger in MRT0-t compared with the decoction of Radix Aconiti Latera-lis.When Radix Aconiti Lateralis was combined with Radix et Rhizoma Glycyrrhizae Praeparata Cum Melle and Rhizoma Zingiberis at the same time in Sini decoction,tmax was delayed too but MRT0-t,was shorten than that of the group of Radix Aconiti Lateralis.Conclusion: The pharmacokinetic parameters of the three compounds obtained in this work shows that the pharmacokinetics of aconitine,mesaconitine and hypaconitine were influenced diversely when Radix Aconiti Laterali was combined with different herbal medicines.
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Objective To establish a method for the determination of Alkaloid in cut crude drug of aconite root.Method Contents of aconitine,hypaconitine and mesaconitine incrude drug of aconite root were determined by LC-MS.It was carried out on Inertsil ODS-3 column(150 mm?4.6 mm,5 ?m),with acetonitrile-ammonium acetate buffer solution(60:40) as mobile phase,at a flow rate of 0.2 mL/min and temperature of 30 ℃.Result The method of determining Alkaloid content in crude drug of aconite root was established and 25 lots of crude drugs were determined.Conclusion The method was sensitive and specifical,can be used to control the quality of cut crude drug from aconite root.
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Objective To observe the poisoning components in plasma and histological changes of rabbits with acute toxicity of aconitum kusnezoffii (草乌).Methods Eight rabbits were garaged with aconitum kusnezoffii liquor,aconitum poisoning model was reproduced,electrocardiogram (ECG) and blood pressure were recorded,the concentrations of aconitine,hypaconitine and mesaconitine in plasma after 0.5,1, 2,3 and 6 hours were measured,and the pathological changes of heart,liver and cerebral cortex were observed.Results After garage with poisoning liquor,arrhythmias and the declination of blood pressure, presenting a tendency of progressive aggravation [before garage:(121.98?16.77)/(110.66?8.78) mm Hg, 1 hour after garage:(102.98?8.34)/(90.22?5.85) mm Hg,2 hours after garage:(66.81?9.13)/ (53.40?6.32) mm Hg,1 mm Hg=0.133 kPa,all P
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Objective:To optimize the extraction procedure of total alkaloid in Radix Aconiti.Methods:The orthogonal design(L9(34) ) was carried out for the optimum extraction conditions guided by yield of the extract and the content of aconitine,hypaconitine and mesaconitine determined with RP-HPLC method,the mobile phase was MeOH-H2O2-CHCl3-DEA(70∶30∶ 2∶0.1).Results:The best extraction process was as follows:moisten with ammonia test solution,then leach for 24 hours with 15 times of ether.Conclusion:The optimum extraction methods are rational.
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Objective To establish a method used for determination of Aconitine and Hypaconitine and Mesaconitine in blood by LC/MS/MS.Methods Extraction of blood sample was conducted by use of 1% thirdchloroactic acid and acetonitrile liquid,and the electrospray fog ionic source,and positive ion MRM scan were employed.Result The linearity correlation coefficients(r)was≥0.992 2.The limit of quantification (LOQ S/N=5) was 2.0 ng/ml for Aconitine,0.5 ng/ml for Hypaconitine,and 0.5 ng/ml for Mesaconitine.The recovery of appended contrast in blank blood sample ranged from 91.25% to 103.1% with coefficient of variation (CV,n=6) less than 10.93%.Conclusion The method of LC/MS/MS is sensitive and reliable,and the sample handling is quick and simple,which was suitable for determination of the Aconitine and Hypaconitine and Mesaconitine in blood sample.
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Objective To determine the contents of hypaconitine and mesaconitine in Shubining Granules. Methods The HPLC method was established for the determin ation of hypaconitine and mesaconitine in Shubining Granules. The HPLC was perfo rmed on Akasil C18(4.6 mm?250 mm,5 ?m) column,mobile phase consisted of meth anol-0.1 %triethylamine (70 ∶30),column temperature 30 ℃,and the flow rare was 1.0 mL/min.The detection wavelength was 230 nm.Results A good linearity w as obtained in the range of 0.126～2.520 ?g of Hypaconitine and 0.152～3.04 ?g of Mesaconitine.The average recovery of Hypaconitine was 101.12 %.The average recovery of Mesaconitine was 99.68 %. Conclusion This method is simple and qui ck,and can be used as quality control of Shubining Granules.
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AIM:To establish a method for detecting three aconitum alkaloids(aconitine,mesaconitine,hy-paconitine) in Zhentong Huoluo Tincture(Radix Aconiti,Radix Aconiti kusnezoffii,Radix et Rhizoma Rhei,Rhizoma Pinelliae,Rhizoma Arisaematis ect.).METHODS:A ZOBAX Extend-C_ 18(250 mm?4.6 mm,5 ?m) column was used to determine the aconitum alkaloids in Zhentong Huoluo Tincture.The mobile was gradient elution,0.1% die-thylamine(adjust pH 9.0 with phosphoric acid) as mobile A;0-20 min A∶B(63∶37),20-45 min A∶B(63∶55∶37→45),at the rate of 1 mL/min.The temperature of column was 30 ℃,and the wavelength was at 232 nm.RESULTS:The linearity of the aconitine in 9.74 ng-974 ng was 0.999 9;that of the mesaconitine in 23.86-2 386 ng was 0.999 9 and that of the hypaconitine in 29.94 ng-2 994 ng was 0.999 9.The RSD of precision for these three were 1.50%、0.68% and 0.49%,respectively.The average recoveries and its RSD were 100.2%(RSD 3.2 %)、102.9%(RSD 2.2%) and 98.2(RSD 2.0%),respectively for aconitin,mesaconitine and hypaconitine.The reproducibilities were all less than 2.0%,and the sample solution was stable in 24 h.CONCLUSION:The method is simple,accurate and has good stability,can be used for the quantity control in the product of Zhentong Huoluo Tincture.