| Berberine(BBR) is an isoquinoline alkaloid, and the main active ingredient lies in the rhizoma coptidis used for treatment of the gastrointestinal disease for long time in the aspect of Traditional Chinese Medicine. A plenty of modern researches have discovered that the BBR features of anti-hypertension,hypoglycemia,lipid-lowering, antiarrhythmia,anti-tumor,antianxiety,antibacterial,antivirus and anti-oxidation. 8-cetylberberine(8-BBR-C16)is a long chain alkylate derivative through the lipophilic modification of BBR to increase its bioavailability and pharmacological activity,and expand its scope of the treatment and application. In the study,the comparative pharmacokinetic research between 8-BBR-C16,8-BBR-C8 and BBR was conducted,and the metabolite and their metabolic pathway of BBR-C16 was analysed.Firstly, the Synthesis of 8-BBR-C16The 8-BBR-C16 was synthesized through the lipophilic substitution reaction on the C8 site of BBR with a long 18 alkyl chain through Grignard reagent. Then the product was structurally confirmed with infrared spectroscopy (IR),nuclear magnetic resonance(NMR)and liquid chromatographic tandem mass spectrometry(LC/MS/MS).Secondly,the establishment of HPLC and SPE of BBR,8-BBR-C8 and 8-BBR-C16 in the biological specimensThe HPLC conditions were as followed, mobile phase consists of acetonitrile and 20 mmol/L potassium dihydrogen phosphate solution (80:20,pH= 4),column:phenomenex Luna C8(5 μm,250 mm*4.6 mm),column temperature:30℃, flowrate:1.0 mL/min,wave length:345 nm,injection volume:100μL.The study determined the concentration of BBR,8-BBR-C8,8-BBR-C16 in plasma, and the results showed that the standard curve,LLOQ, precision,recovery and stability can reach the demand of assay for the biological specimens.The study developed a higher output and stable SPE pretreatment method to extract the BBR,8-BBR-C8 and 8-BBR-C16 in the tissues samples.The conditions were as followed:tissues homogenate were extracted by dichloromethane and then purified with the SPE column(Waters company, silica catridges),and the purfied solution was injected on the HPLC instrument. The verification results showed that this analytical method can also reach the assay demand for biological specimensThirdly, pharmacokinetic comparative research of 8-BBR-C16 in rat plasmaAfter oral administration of BBR,8-BBR-C8 and 8-BBR-C16 at the dosage of 80mg·kg-1 for rats respectively,the blood was collected at 0.5h,1h,1.5h,2h,3h,4h,6h, 8h,12h,24h,36h,48h and 72h.The drug concentrations in plasmas were determined with the HPLC and the pharmacokinetic parameters were evaluated by the non-compartment model.The results indicated that 8-BBR-C16 had the following characteristics compared with BBR and 8-BBR-C8:(1)Cmax became larger obviously from 45.92 μg·L-1 to 129.41 μg·L-1 referred to BBR.(2)The relative bioavailability was significantly elevated.Compared with 8-BBR-C16,BBR and 8-BBR had the relative bioavailability of 7.7% and 13.6% respectively.(3)The elimination of 8-BBR-C16 slowed down along with the longer residence time and cycle time in the body, and t1/2 became longer. t1/2 of 8-BBR-C16 was 11.90 h but BBR and 8-BBR-C8 were 3.61h,5.10 h respectivley.(4)The average plasma concentration-time curve of 8-BBR-C16 had the double peaks obviously.It could be deduced from the prominent shift of pharmacokinetic behavior and enhanced bioavailability that 8-BBR-C16 prone to access the celluar membrane as a result of long alkyl group addition to BBR.Fourthly, comparative research of 8-BBR-C16 in rat tissues distributionAfter oral administration of BBR,8-BBR-C8 and 8-BBR-C16 at the dosage of 80mg·kg-1 with rat respectively,the heart,liver,spleen,lung,kidney,brain,uterus, testis.stomach and small intestine was collected at lh,3h,8h and 12h.Drugs concentrations in tissues were determined with the HPLC after being pretreated by SPE.The results indicated that 8-BBR-C16 had the following characteristics compared with BBR and 8-BBR-C8:(1) The distribution concentrationsof 8-BBR-C16 in all tissues increased dras-tically.(2) The distribution concentrations ratio of 8-BBR-C16 in the rat tissues shifted and the lung and spleen had the higher concentration. Specially,the concentration distributed in lung was highest from that 8-BBR-C16 had the lung target could be deduced.It may attribute to the good lipotropism of 8-BBR-C16.(3) The distribution scope of 8-BBR-C 16 became more wider and the concentra -tions in brain and testis arised from nothing.It could be deduced from the prominent shift that 8-BBR-C16 prone to access the inner membrane of blood capillary and tissues to reached the inner of tissues,as a result of excellent lipid solubility of 8-BBR-C16.Fifthly, comparative research of 8-BBR-C16 excretion in rat urine and fecesThe rats were oral administration f BBR,8-BBR-C8 and 8-BBR-C16 at the dosag of 80mg’kg-’respectively and put into the metabolism cage.Then the urine and feces were collected in the intervals of 0-3 h,3-6 h,6-12 h,12-18 h,18-24 h,24-30 h,3O-36 h,36-42 h,42-48 h,48-60 h,60-72 h and 72-96h separately and purified with SPE pretreatment.The concentration of the drugs were determined by HPLC.The experiment results showed that the excretion of the 8-BBR-C16 obviously changed compared to BBR and 8-BBR-C8:(1)The accumulative excretion ratio of BBR,8-BBR-C8 and 8-BBR-C16 in the urine was 0.0291%,0.0148% and 0.0014% respectively.The 8-BBR-C16 excretion amount lowered an order of magnitude compared to the two others,only 4.1% of the excretion amount of BBR.(2)The accumulative excretion ratio of BBR,8-BBR-C8 and 8-BBR-C16 in the feces was 76.9%,57.1% and 20.5% respectively.It could be deduced that the main excretion way for BBR and its alkylation derivatives wree not urine,but feces.It were thought that the reasons of the evident decrease of the excretion amounts in the urine and feces was widely metabolism and drugs accumulation in some organs.Sixthly, research on the metabolic product and route of 8-BBR-C16 in rat urineRats were treated with single oral administration of 8-BBR-C16 at the dosage of 40mg-kg-1 by every three days and put into the metabolic cages for the collection of urine.The collected urine was purified with the macroporous resin and then was screened with thin-layer chromatography and HPLC.Meanwhile the purified urine was analysed with LC/MS/MS.The results showed:(1) 8-BBR-C16 had three metabolic products:product of demethylation,product conjuncted with glucuronic acid group,and product conjuncted with sulfat.(2) There was a wide metabolic way for 8-BBR-C16 in rat urine, first way was demethylation at C9 or C10 site through metabolism phase I,second was conjunction with sulfat after eliminating methylene at C2,C3-OCH2O through metabolism phase II,and third was conjunction with glucuronic acid group after demethylation at C9 or C10 site through metabolism phase Ⅱ. |
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