| Cortex Periplocae, usually called Xiangjiapi, is the dried root bark of Asclepiadaceae and Periplocca sepium Bge.. It has been recorded in the edition 2005 of Pharmacopoeia of the PRC for treating rheumatic arthritis and it is toxic. The main toxicity are gastrointestinal tract and arrhythmia. In this paper, chemical constituents, quality control method,cardiac toxic effect, pharmacokinetics and metabonomics on the toxic component of Xiangjiapi were studied.The chemical constituents of Xiangjiapi were studied by various modern isolation and purification techniques. Eight constituents were isolated from the chloroform extract and n-butanol extract by silica gel, Sephadex LH-20, preparation HPLC techniques.Utilizing chemical and spectroscopic method, the structures were fully characterized.They were lupeol, isovanillin,periplocoside M, isovanillic acid,2-hydroxy-5-(2-hydroxy-4-methoxybenzyl)-4-methoxybenzaldehyde,periplocin, periplogenin and sucrose. The standards could guarantee the further study.The quality control methods of Xiangjiapi were studied.An HPLC-DAD method with gradient elution had been developed to determine isovanillin,isovanillic acid, periplocin,2-hydroxy-5-(2-hydroxy-4-methoxybenzyl)-4-methoxybenzaldehyde and 4-methoxy salicylaldehyde.The mobile phase was acetonirile-0.05% phosphate solution and UV detection wavelength was 220 nm.HPLC fingerprints from different origin was also developed.Periplocin was used as the refernce.Eighteen common peaks of the fingerprints were marked, and five of them were identified. The quality evaluation standard was established by determination the cosine between the standard chromatogram and samples.The effects of Xiangjiapi on cardiac function and electrocardiogram(ECG) were established to investigated the cardiac toxic effect. All the rat’s were divided into 4 groups at random:the low, middle, high dose of Xiangjiapi and NS group.Xiangjipi was given for 3 days by oral administration and one times each day. The results of the study show that the low, middle and high dose of Xiangjiapi could significantly effect the hemodyamic. The LVSP and+dp/dtmax were increased and the HR, LVEDP and-dp/dtmax were reduced, significantly. It means that Xiangjiapi could increased the contraction force and reduced the diastolic function of the cardiac muscle.The ECG results of the study show that the middle and high dose of Xiangjiapi could decrease P-R and Q-T interval, and the R-R interval could be increased by the three doses.Also, the positive inotropic effect and negative chronotropic effect. It means that the feature of ECG of Xiangjiapi accord with the trait of cardenolide.It is certified that the cardenolide may be the toxic component which caused the cardiac toxic effect.A UPLC-MS/MS method was developed to investigate the acute toxicity of Xiangjiapi.The plasma and urine samples were pretreated with protein precipitation, and the supernatants were separated on a reversed-phase C18 Column with gradient elution using a mobile phase of acetonitrile-0.1% formic acid in water at a flow-rate of 0.30 mL·min-1.The analytes were ionized by an ESI source in positive ion scan mode.The plasma and urine samples from before and after administration rats were analyzed with the established method. The analytical data were processed via multivariate analysis such as (Principle component analysis) PCA. PCA score plot of analytical data describes the general differences between before and after administration samples.The dominant metabolites obtained from loading plot of Masslynex were the differential metabolites between given and ungiven dose rats, and 8 of them have been confirmed.They are Hexadecasphinganine, Phytosphingosine, LPC(18:2), LPC(20:4), LPC(16:0), LPC(18:0), Hippuric acid and Phenylacetyglycine. The mechanism of pathological changes may be elucidated with the up-or down-regulated metabolic pathways.An accurate and sensitive LC-MS method was firstly developed for determination of periplocin in biological samples.Plasma and urine samples were extracted using ethyl acetate. Liquid chromatographic separations were achieved by Kromasil C18/ Column (150 mm×4.6 mm,5μm) at room temperature with the mobile phase of methanol-water (76:24, v/v) at a flow rate of 0.8 mL·min-1.The analytes were ionized by an ESI source in positive ion mode under the following source conditions:Nebulizing gas 1.5 L·min-1;Drying gas 2.0 L·min-1; CDL temperature 250℃;Heat block temperature 200℃;Detector voltage 1.60 kV; the other parameters were fixed as for the tuning file. Analysis was carried out by selected ion monitoring for periplocin [M+Na]+ at m/z 719.50 and IS [M+Na]+ at m/z 803.50.The validated method was applied to the pharmacokinetic and excretion study of them after different administration.In our study, we found that periplocin could not be detected under the oral administration of the pure periplocin in rat plasmer, but can be detected after intravenous and intramuscular administration.The pharmacokinetic parameters were as follows:Tmax(h):0.033 and 0.112±0.054, Cmax(ng·mL-1):313.6±122.3 and 254.4±108.3, T1/2(h):0.507±0.187 and 0.351±0.088, AUC0-t(μg·h·L-1):79.18±16.38 and 145.4±26.6, AUC1-∞(μg·h·L-1):84.6±21.0 and 147.8±26.0, CL/F(L·(h·kg)-1):4.081±1.022 and 2.281±0361,MRT(h):0.444±0.125 and 0.519±0.098.It was demonstrated that oral bioavailability of periplocin very low, which may be transferred into other metabolite in the stomach and intestines. So it is necessary to be further studied. A UPLC-MS/MS method was developed to investigate the metablites of urine, fece and bile samples.A gradient liquid chromatographic system composed of acetonitrile -0.1% formic acid in water at a flow-rate of 0.25 mL·min-1 was used for metabolite separation on a reversed-phase C18 Column. A total of 5 metabolites were detected.The mass charge ratio and retention time were as follows:m/z 719(3.12 min), m/z 535(3.07 min), m/z 391(3.01 min),m/z 551(3.08 min)and m/z 407(2.93 min).The corresponding product ion spectra were acquired and interpreted, and structures were proposed. |
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