| BackgroundThe Xinmeng granule are mainly constituted of five Chinese medicines: Danshen,semen ziziphi spinosae,Schisandra chinensis,Mulberry,and so on.The drug is designed to treat or relieve insomnia caused by nephropathy.The Xinmeng granule were jointly created by Huasen pharmaceutical industry of Chongqing and Traditional Chinese Medicine College of Southwest university,and now is normatively studyed as the 6th class new drug.Actually,the preparation of Xinmeng granule has already gained the national technical invention patent.Meanwhile,the GLP safety evaluation as well as pesticide effect study have been finished.Now the Xinmeng granule is being studyed clinically,and its preclinical pharmaceutics study is subsidized by the national science and technology key project for special drug discovery（2014ZX09304-306-04）.ObjectiveDetect the content of several major active components in Xinmeng granule,which aimes to enhance its quality control.Meanwhile,study the preclinical pharmaceutics of Danshensu in plasma and brain from rats,which designs to provide basic informations for clinical study and use,such as clinically set the dose,the interval of the treatment and the course of the treatment.Methods and Results1.The content detection of major active components in Xinmeng granuleEstablished a RP-HPLC method to detect the content of Danshensu,protocatechualdehyde,spinosin,ferulic acid and schizandrin,which are five major active components of Xinmeng granule.The method was as follow: Chromatographic column: Xterra C18 chromatographic column（250 mm × 4.6 mm ×3.5 μm）,Mobile phase: methanol-0.1% ice acetic acid solution,gradient elution;Detection wavelength: 235 nm and 335 nm;Flow rate: 0.5 m L·min-1;Column temperature: 30 ℃;Sample quantity: 10 μL.Accorded the "9101 drug quality standard analytical method validation guidelines",which is in the fourth part of the Chinese pharmacopoeia（revised in 2015）,to verify the methodology.Under the chromatographic conditions above,the retention time of Danshensu,protocatechualdehyde,spinosin,ferulic acid and schizandrin successively were 8.5 min,15.7 min,21.4 min,22.3 min and 30.7 min.The system applicability,precision,recovery rate and stability of the investigation results about each components conformed to the stipulations of the Chinese pharmacopoeia（revised in 2015）.The average contents of each components in Xinmeng granule from 3 batches successively were 18.24±0.77 mg·g-1,7.57±0.18 mg·g-1,5.61±0.08 mg·g-1,1.86±0.05 mg·g-1 and 17.95±0.25 mg·g-1.2.The preclinical pharmaceutics of Danshensu in plasma from ratsUsed the ethyl acetate liquid-liquid extraction method to pretreat the rat plasma sample.And used the RP-HPLC gradient elution method,combined with internal standard method,to detect the contents of Danshensu in rat plasma.The internal standard was p-hydroxy benzoic acid.Accorded the "9012 biological samples quantitative analytical method validation guidelines",which is in the fourth part of the Chinese pharmacopoeia（revised in 2015）,to verify the methodology.Under the established method above,the retention time of Danshensu and internal standard successively were 9.6 min and 15.7 min.The specificity,precision,recovery rate and stability of the investigation results conformed to the stipulations of the Chinese pharmacopoeia（revised in 2015）.After the rats were irrigated with Xinmeng granule at a single dose of 1.25 g·kg-1、2.50 g·kg-1 and 5.00 g·kg-1,the blood were collected at different time.After the contents of Danshensu in rats plasma were detected,the pharmacokinetic parameters were calculated by the DAS3.2.1 software.The results are as follows: the AUC（0-t）respectively were 5.88±1.02 μg·mL-1·h,19.44±2.22 μg·m L-1·h and 64.14±14.28 μg·m L-1·h;the peak Cmax respectively were 2.931±0.479 μg·m L-1,5.413±0.429 μg·mL-1 and 11.509±2.684 μg·mL-1;the Tmax respectively were 30.0±5.4 min,60.0±9.7 min and 115.0±31.3 min;the t1/2 respectively were 123.8±17.8 min,264.9±79.1 min and 280.8±71.9 min;and the CL respectively were 199±28 m L·h-1·kg-1,128±16 m L·h-1·kg-1 and 80±18 m L·h-1·kg-1.With the increase of the intragastric doses,both the AUC（0-t）and the Cmax had increased,but non-linearly.Meanwhile,the Tmax and the t1/2 also increased,but the CL decreased with the dose.Then six rats have been intragastricly administrated of XinMeng granules at the doses of 2.50 g·kg-1 everyday at 9:30 a.m.,and continued for 7 days.The plasma of the rats were collected everyday before the gavage,and were collected at different times after the gavage of the 7th day.Then the concentrations of Danshensu in the plasma were detected and the pharmacokinetic parameters of Danshensu were analyzed by the DAS3.2.1 software.The results showed that the steady concentration state of Danshensu reached at the 5th day,and the AUCss were 45.12±0.71 ug·m L-1·h,meanwhile the Css,av were 1.880±0.030 ug·mL-1.Compared with the single-dose group,all of the AUC（0-t）,the MRT and the t1/2 increased from the repeated-dose group.Meanwhile the CL oppositely decreased,but the Cmax and the Tmax changed nothing.The result of the accumulation coefficient（R）was 1.16.3.The preclinical pharmaceutics of Danshensu in brain from ratsUsed the RP-HPLC method,which original desighned to detect the contents of Danshensu in rats plasma,to detect the content of Danshensu in rats brain.According to the "9012 biological samples quantitative analytical method validation guidelines",which is in the fourth part of the Chinese pharmacopoeia（revised in 2015）,to verify the methodology.Under the established method,the retention time of Danshensu and the internal standard successively were 9.6 min and 15.7 min.The specificity,precision,recovery rate and stability of the investigation results conformed to the stipulations of the pharmacopoeia（revised in 2015）.After the rats have been intragastricly administrated of Xinmeng granules at a single dose of 2.50 g·kg-1,the brain were collected at different times.After the contents of Danshensu in rats brain were detected,the pharmacokinetic parameters were calculated by the DAS3.2.1 software.The results showed that the Danshensu distributed quickly to the brain,and the Tmax was 30 min.A second absorbtion peak arised at 90 min.Compared with plasma,all of the MRT、the t1/2 and the Tmax decreased notablely in brain.The result of brain distribution ratio（Kp）was 20%.Conclusion1.A RP-HPLC gradient elution method was established to detect the contents of Danshensu,protocatechualdehyde,spinosin,ferulic acid and schizandrin,which are five major active components of Xinmeng granule.The average contents of each components in Xinmeng granule from 3 batches successively were 18.24±0.77 mg·g-1,7.57±0.18 mg·g-1,5.61±0.08 mg·g-1,1.86±0.05 mg·g-1 and 17.95±0.25 mg·g-1.2.The pharmacokinetics of Danshensu in rats plasma were significantly influenced by the Xinmeng granule doses,which ranged from 1.25 g·kg-1 to 5.00 g·kg-1,with a non-linear feature.When the rats were intragastricly administrated with XinMeng granules for 7 days repeatedly at the dose of 2.50 g·kg-1,and the drug intervation is 24 h,the elimination rate of Danshensu in rats decreased slightly,but showed no effect of accumulation toxicity.3.When the rats were intragastricly administrated with XinMeng granules at a single dose of 2.50 g·kg-1,the Danshensu distributed quickly to the brain,and showed a quickly absorbtion-quickly elimilation characteristic.The result of the brain distribution ratio was 20%. |
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