| Rhubarb is commonly used as laxatives in Asian countries, of which anthraquinones(AQs) are the major active ingredients, but there are an increased number of concerns regarding the nephrotoxicity of AQs. To solve this problem, in our previous study, rhubarb total free AQs containing no less than 50% free AQs had been extracted, which was used to prepare rhubarb total free AQs oral colon-specific drug delivery granules(RTFA-OCDD-GN) through oral colon-specific drug delivery technology. And we have proved that RTFA-OCDD-GN could not only produce considerable purgative efficacy but also reduce nephrotoxicity of AQs compared with rhubarb. In this study, a reliable high performance liquid chromatography(HPLC) method was established and validated for simultaneous determination of rhein, aloe-emodin, emodin, physcion and chrysophanol in rat plasma, urine and faece using danthron as the internal standard(IS). Simultaneously, the method was successfully applied to investigate and compare the remarkable differences in pharmacokinetic and excretion kinetic study of rhubarb AQs in rats after orally administered with rhubarb and RTFA-OCDD-GN, and then explained why these granules could reduce the nephrotoxicity of rhubarb AQs when they produced purgative efficacy.Objective:1. A reliable HPLC method was established, validated and optimized for simultaneous determination of five rhubarb AQs in rat plasma, faece and urine, which provided the methodology based on comparing the pharmacokinetic and excretion kinetic characteristics of rhubarb AQs in rats after orally administered with RTFA-OCDD-GN and rhubarb.2. To determine the content of five rhubarb AQs in rat plasma, faece and urine after orally administered with RTFA-OCDD-GN and rhubarb using HPLC. The purgative efficacy of rhubarb was considerable with the RTFA-OCDD-GN under the dosages. Then we compared the remarkable differences in pharmacokinetic and excretion kinetic study of rhubarb AQs in rats after orally administered with RTFA-OCDD-GN and rhubarb, and explained why the granules could reduce the nephrotoxicity of rhubarb AQs when they produced purgative efficacy.Methods:1. An HPLC analysis was performed using an Agilent 1260 Infinity liquid chromatograph, and chromatography was carried out on an Agilent Eclipse Plus-C18 column(250 mm×4.6 mm, 5 μm) using danthron as IS. For the determination of plasma and urine, the mobile phase consisted of methanol-1% formic acid aqueous(78:22, v/v) at a flow rate of 1 m L·min-1. The detection wavelength was set at 254 nm. The final injection volume was 10 μL. The column oven temperature was maintained at 30°C.2. An HPLC analysis was performed using an Agilent 1260 Infinity liquid chromatograph, and chromatography was carried out on an Agilent Eclipse Plus-C18 column(250 mm×4.6 mm, 5 μm) using danthron as IS. To determine the faece, the mobile phase consisted of methanol-1% formic acid aqueous(75:25, v/v) at a flow rate of 0.8 m L·min-1. The detection wavelength was set at 254 nm. The final injection volume was 10 μL. The column oven temperature was maintained at 30°C.3. The medication groups were orally administered at a single intragastric gavage dose of RTFA-OCDD-GN and rhubarb, respectively. The purgative efficacy of rhubarb was considerable with the RTFA-OCDD-GN under this dosage. All animals were held in metabolic cages for the collection of the plasma, faece and urine samples within 72 h after oral administration. All biological samples analysis were performed by HPLC. Non-compartmental pharmacokinetic parameters and excretion kinetic parameters were calculated by drug and statistics(DAS) pharmacokinetic program.Results:1. Using the established HPLC method, all five ingredients exhibited good linearity with r greater than 0.9991 over the concentration range of 0.2000~15.20 μg·m L-1 for rhein, 0.2000~12.40 μg·m L-1 for aloe-emodin, 0.7000 ~ 22.40 μg·m L-1 for chrysophanol, 0.7000 ~ 16.30 μg·m L-1 for physcion and 0.6000~18.80 μg·m L-1 for emodin, respectively. For the five analytes, intra- and inter-day precision were less than 1.0% and 3.5%(n=6), respectively. And the extraction recoveries were all above 83.72% with all RSD values less than 7.3%. Results of stability studies were in line with requirement with all RSD values less than 4.8%.2. Using this HPLC method, all five ingredients in rat urine exhibited good linearity with r greater than 0.9994 over the concentration range of 0.3040~608.0 μg·m L-1 for rhein, 0.3040~608.0 μg·m L-1 for aloe-emodin, 0.1206 ~ 241.2 μg·m L-1 for chrysophanol, 0.07024 ~ 70.24 μg·m L-1 for physcion and 0.09840~196.8 μg·m L-1 for emodin, respectively. For the five analytes, intra- and inter-day precision were less than 2.7% and 2.3%(n=6), respectively. The extraction recoveries were all above 77.50% with all RSD values less than 12.6%. Results of stability studies were in line with requirement with all RSD values less than 6.8%.3. Using this HPLC method, all five ingredients in rat faece exhibited good linearity with r greater than 0.9994 over the concentration range of 0.3040~608.0 μg·m L-1 for rhein, 0.1590~318.4 μg·m L-1 for aloe-emodin, 0.1206 ~ 241.2 μg·m L-1 for chrysophanol, 0.07024 ~ 70.24 μg·m L-1 for physcion and 0.09840~196.8 μg·m L-1 for emodin, respectively. For the five analytes, intra- and inter-day precision were less than 2.7% and 3.5%(n=6), respectively. The extraction recoveries were all above 74.30% with all RSD values less than 8.1%. Results of stability studies were in line with requirement with all RSD values less than 6.2%.4. Only rhein, aloe-emodin, emodin and chrysophanol could be detected in rat plasma after orally administered with rhubarb and RTFA-OCDD-GN, and physcion was undetectable in some time points using HPLC. The results showed that, compared with the rhubarb group, the AUC, Cmax, t1/2z and Vz/F of rhein, aloe-emodin, emodin and chrysophanol in rats receiving the RTFA-OCDD-GN were significantly decreased, and the Tmax values were prolonged. Moreover, the Tmax of rhein, the Cmax values of chrysophanol and emodin all have significant differences(P<0.05).5. The five anthraquinone ingredients could be detected in rat urine and faeces by HPLC. By comparing the excretion rates of anthraquinone prototype after oral administration we found that the total excretion in 72 h through urine and faece of five components was 48.97%, of which 28.92% was excreted in the urine and fecal excretion rate was 19.95%. While the corresponding datas of rhubarb group were 23.93%, 14.42% and 9.51%, respectively. Moreover, the prototype excretion rates of the single component in RTFA-OCDD-GN group were all higher than the rhubarb group, and the excretion rates of emodin and physcion had significant differences between the two groups(P<0.05).Conclusion:1. An accurate and reliable HPLC method was developed and validated for the simultaneous determination of rhein, aloe-emodin, chrysophanol, physcion and emodin, and was successfully applied to investigate and compare the remarkable differences in pharmacokinetic and excretion kinetic study of rhubarb AQs in rats after orally administered with RTFA-OCDD-GN and rhubarb. The result is a new breakthrough in rhubarb toxicity attenuated research at home and abroad.2. The achieved comparative pharmacokinetic and excretion kinetic results gave a preliminary analysis and explanation to the mechanism of toxicity attenuation of RTFA-OCDD-GN coupled to the purgative mechanism. The results suggested that OCDDS reduced nephrotoxicity of AQs not merely due to reduced absorption of AQs, but also reduced the t1/2z and Vz/F of rhubarb AQs. Simultaneously, OCDDS improved the excretion rate of AQs prototype through the urine and feces, such that the AQs absorbed into the blood stream was rapidly excreted most in prototype form and then reduced accumulation. The result is a new breakthrough in rhubarb toxicity attenuated research at home and abroad. The thorough investigation of the mechanism of toxicity attenuation of RTFA-OCDD-GN will provide theoretical support for the further development of rhubarb and traditional Chinese medicine preparations containing rhubarb. |
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