Inhibitory Effect Of ASP On Hepcidin In IDA Rats And The Study Of APIC Oral Liquid

According to the listing iron supplement Niferex, Angelica sinensis polysaccharide iron complex (APIC) was synthesized. The pharmacological experiments confirmed that APIC had better hematonic effects than Niferex. Therefore, we speculated APIC to participate in iron metabolism, increasing body iron absorption and utilization. The purpose of this paper was to study the inhibitory effect of ASP on hepcidin, IDA rats given the solution of Angelica sinensis polysaccharide (ASP), ELISA and Western blot were respectively used to measure hepcidin levels in serum and C/EBPα, SMAD4, STAT3/5, P-STAT3/5 of hepcidin gene upstream in liver, to find out the effect of ASP on iron metabolism and its mechanism, laying the basis for further research on new pharmacological effects of Angelica sinensis polysaccharides acting in iron homeostasis; in addition, to study APIC oral liquid and its pharmacokinetics, providing new drugs for clinical treatment of IDA and scientific data for iron pharmacokinetic studies.Part One The inhibitory effect of ASP on hepcidinThe IDA model was established by feeding iron-deficiency diet and periodic bleeding. Rats were divided into three groups: rhEPO group(i.v rhEPO 2000 IU·kg^-1); ASP group(ig ASP solution 1g·kg^-1); Control group(ig Equal volume NS). EPO and hepcidin levels in serum were detected by ELISA, the expression of C/EBPα, SMAD4, STAT3/5 and P-STAT3/5 proteins were detected by Western blot. The statistical test was carried out by SPSS 13.0.ELISA showed that ASP could significantly increase EPO concentration. EPO levers rised and then fell that showed a trend of changes in peak shape. It reached the maximum 12h after administration, later than the rhEPO group. As with the rhEPO, ASP could markedly inhibit hepcidin expression. Hepcidin concentration reduced to the minimum at 24h after ASP administration and 12h after rhEPO administration. The descent rate were respectively 13.4% and 31.8% compared to that before administration. the decrese in ASP group was more pronounced than it in rhEPO group.Western blot showed that ASP and rhEPO could significantly inhibit the expression of C/EBPα, P-STAT3/5 proteins in IDA rats compared to that in the control group. In addition, ASP could inhibit the expression of SMAD4 protein. Nevertheless, the expression of total-STAT3/5 was not affected by ASP and rhEPO. It indicated that the inhibitory effect on hepcidin of ASP was mediated by downregulating SMAD4 and stimulating endogennous EPO secretion, which further decreased the signal proteins of C/EBPαand P-STAT3/5.Part Two The study of APIC Oral LiquidAPIC was stable, near neutral in aqueous solution and not easy to oxidation or hydrolysis. According to the modern pharmaceutical theory, the prescription should be as single as possible. In this experiment,as the index to taste, appearance and stability, the best additional materials and its amount were detemrined as follows: CMC-Na which could increase the stability of the effctive element 0.1% and potassium sorbate as the perserative part was 0.1%, stevioside and aspartame as the flavoring agent was respectively 0.05%, the oral liquid had appropriate taste and good stability.This paper researhed the preparation in quality standard and tentative stability systemically. According to the oral liquid stipulation from chinese pharmacopoeia (Edition 2005), each target confirmed to the requirement.The iron content was determined with iodometry and the polysaccharide-iron with Phenol-Sulphate Acid Method; the stability of samples were studied with thermostatic accelerated test and permanent stability test. The results showed that the recovery and RSD of iodometry are respectively 99.63%, 0.64%, the recovery and RSD of Phenol-Sulphate Acid Method are respectively 99.71%, 2.4%; there was no obivious change with all indicates of the samples of the experiments monitored through the three-month period of aceelerating and long-term tests. The quality standard of the preparation was feasible and controllable and its stability was sound.The study was to investigate the relationship between the pharmacokinetic parameters and dose as well as the pharmacokinetics differences in IDA rats and normal rats after oral administration of the preparation. The IDA model rats were established by feeding iron-deficiency diet and periodic bleeding. Serum iron concentration were assayed by atomic absorption spectrometry. The pharmacokinetic parameters were analyzed by DAS 2.0 program and the statistical test was carried out by SPSS 13.0. It was investigated that the concentration-time curves in normal and IDA rats were fitted to two-compartment open model. In normal rats, the t_1/2α, t_1/2β, t_1/2Ka, T_max, C_max had no obvious difference, but CL/F increased as dose increased. AUC was not linear correlation with dose, when the dose was greater than 5.83 mg·kg^-1, AUC had no significant difference as dose increased. The serum iron concentration in IDA rats were significantly (p<0.05)higher than those in normal rats 4h after administration. t_1/2β of the IDA rats were significantly longer than those of the normal groups and CL/F significantly lower than those of the normal groups. The results indecated that the preparation did not show the linear absorption, there were significant differences in APIC pharmacokinetics between IDA rats and normal rats, and the differences had relationship on the dosage.