| OBJECTIVE To simultaneously quantify the eight major bioactive Anthraquinones in C. tora using the specific HPLC-DAD method. And to determine three anthraquinones (aloe-emodin, obtusifoline, chrysophanol) in rat plasma after oral administration of Cassia obtusifolia L. extract, and to evaluate its pharmacokinetics.METHODS A reversed phase high performance liquid chromatography (HPLC) method was established for the simultaneous determination of three anthraquinones, Chromatographic separation was achieved on a Zorbax Extend-C18(250mm×4.6mm,5μm)(Palo Alto, CA, USA) at28℃, with a linear gradient elution of acetonitrile and0.3%aqueous acetic acid (v/v), at the flow rate of1mL/min. The eluent was detected at258nm for the eight anthraquinones and internal standard.Randomly divide98SD rates into14groups. Administrate anthraquinones extract of cassia seed at a dosage of1.0g/kg via oral gavage. Separately, narcotize at0,0.0833,0.1667,0.3333,0.5,1,2,3,4,5,6,8,10,12and24h. Then Take0.3ml of blood sample from orbit. After pretreating the blood samples, determine the drug concentration by HPLC method. Calculate the pharmacokinetics parameters of three target compounds:aloe-emodin, obtusifoline and chrysophanol, based on above test results.Choose six SD rats, and one as the blank control. Administrate another five rats with anthraquinones extract of cassia seed at a dosage of1.0g/kg. Put them into metabolism cage, separately. Collect urine samples and accurately record their volume at following time periods:0～4h,4～8h,8～12h,12～20h,20～28h,28～40h,40～52h,52～60h, and60～72h. Pretreat the urine samples and determine the drug concentration by HPLC method. Calculate accumulative discharge rate based on the volumes collected. Draw a discharge curve, analyze and determine the excretion pattern of the three targets compounds in urine.Choose five rats, perform abdominal operation for bile collecting. Via gavage, administrate them with anthraquinones extract of cassia seed at a dosage of1.0g/kg. Then collect bile samples every four hours and accurately record their volume. At the same time, to maintain a normal body fluid ilevel in rats and control them under narcotism, appropriate amount of normal saline and urethane solution (10%) should be supplied. After pretreatment, determine the collected bile samples by HPLC method. Draw a discharge curve according to the test results. Analyze the excretion time-cuve of the three targets compounds in bile.RESULTS This assay was applied to the evaluation of15samples from different origins in China. The results indicated that the developed assay could be readily utilized for the quality control of C. obtusifolia.After oral administration, aloe-emodin, obtusifoline and chrysophanol were quickly absorbed into the blood from the gastrointestinal tract (the tmax is observed at20-60min). The cumulative excretion of aloe-emodin, obtusifoline and chrysophanol was4.87%,2.74%and3.65%in urine samples,1.71%,7.2%and4.9%in bile samples, respectively. The maximum urinary and biliary excretions in different intervals of aloe-emodin, Obtusifoline and chrysophanol were observed at12～20and4～8h after oral administration, respectively.CONCLUSION The HPLC-DAD method is useful for the quality control of Cassia obtusifoliaAfter one-time administration with anthraquinones extract of cassia seed via gavage, the Tmax of blood concentration of aloe-emodin, obtusifoline and chrysophanol in male SD rats, after non-compartmental model fitting, are0.667,1and0.333h separately. From the above results, we can draw the following conclusion: generally, anthraquinones extract is absorbed quickly in intestinal tract, and then eliminated rapidly.24h later, no residues of aloe-emodin, obtusifoline and chrysophanol are observed in blood plasma. Therefore, both renal excretion and biliary excretion are important routines for drug excretion. |
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