| Luteolin is an active ingredient in traditional Chinese medic ine(TCM) which belongs to flavonoids. As one kind of topoisomerase inhibitors, the pharmacological activity of anticancer makes it promising. Studies also confirmed that it have other effects to our body, such as antioxidant, anti-inflammatory, antibacterial, et al. Its sparingly water-solubility limits its clinical application. Sulfobutyl ether-β- cyclodextrin(SBE-β-CD), which have lower toxicity, stronger solubilization and inclusion ability could make luteolin into luteolin /SBE-β-CD inc lusion complex to increase the solubility and stability, and enhance the bioavailability.This research used SBE-β-CD to prepare luteolin/SBE-β-CD inclusion complex, HPLC method to determine the concentration of luteolin, completed the preparation optimization and quality evaluation of luteolin/SBE-β-CD inclusion complex, and studied the pharmacokinetics of luteolin and its SBE-β-CD inclusion complex. Specific research content mainly inc ludes the following two parts:The first part: Preparation technology study of luteolin/SBE-β-CD inclus ion complex by orthogonal designObjective: To establish a method for the measurement of the concentration of luteolin in luteolin/SBE-β-CD inc lus ion complex,optimize the preparation technology and evaluate the quality of luteolin/SBE-β-CD inclus ion complex. Methods: A quantitative analysis method of luteolin by HPLC was achieved on an Agilent ZORBAX SB-C18 column(4.6×250mm,5μm) with a mobile phase consisted of 0.2%H3PO4-methanol(30:70)at a flow rate of 1 m L/min. Samples were monitored at 348 nm and the column temperature was 25℃. Luteolin/ SBE-β-CD inclus ion complex was prepared by grinding method. Through investigating the effects of ingredient proportion of SBE-β-CD to luteolin, water amount, grinding time on composite score which achieved by inclus ion rate and yield, we used orthogonal design to select the best preparation technology. Phase solubility diagram, infrared spectroscopy, differential scanning calorimetry were used for the validation of luteolin/SBE-β-CD inclusion complex, and the solubility and Log P of luteolin and luteolin/SBE-β-CD inc lus ion complex were measured. Results: It had a good linearity over the concentration range of 6.69~20.08μg/m L of luteolin. The regression equation was Y=23794 X﹣17234(r=0.999).The average recovery was 101.51%, and the RSD was 1.62%. All the performance met the design demand by methodological investigation. The optimal preparation conditions of luteolin/SBE-β-CD inclus ion complex were: ingredient proportion of SBE-β-CD to luteolin(2:1), water amount(100μL), grinding time(70min). The analysisshowed that the molecular ratio of luteolin to SBE-β-CD was 1:1. After being inc luded in SBE-β-CD, the solubility of luteolin had increased 62.90 times, and the Log P of luteolin increased from 0.7656 to 1.0084.Conclusion: The established HPLC method for the determination of the concentration of luteolin in luteolin/SBE-β-CD inclus ion complex has met the design demand by methodological investigation. The luteolin/SBE-β-CD inclus ion complex show good inclusion rate and yield after being optimized with orthogonal design. The solubility and the Log P of luteolin increased after being included in SBE-β-CD.The second part:Pharmacokinetic study of luteolin and its SBE-β-CD inclus ion complexObjective: To establish a method for the determination of the concentration of luteolin in plasma of rat, study the pharmacokinetics of luteolin and its SBE-β-CD inc lusion complex in rat. Methods: Dosed SD rats with luteolin and luteolin/SBE-β-CD inclusion complex in two ways of intragastric administration and intravenous injection, respectively. Plasma samples were pretreated by liquid-liquid extraction, detected by HPLC with a mobile phase consisted of 0.2%H3PO4-methanol( 48:52). Relevant pharmacokinetic parameters were obtained by DAS software. Results: The standard curve was recorded over the range of 0.06~13.00μg/m L. The RSD of intra- and inter-day precisions were within 5.43~13.33% and 7.89~14.29%, respectively; accuracy ranged from 89.85 to 113.85% and88.69 to 107.69%, respectively. The extraction recovery and the RSD of luteolin were within 72.19-86.62% and 5.60-12.77%, respectively; the extraction recovery and the RSD of IS were within 70.24-75.30% and 4.69-11.99%, respectively. The plasma samples could withstand three freeze-thaw cycles, and luteolin was stable in plasma after being kept at 4℃ for 24 h and stored at-20 ℃ for 1 month. The pharmacokinetic data indicated an advanced Tmax and an increased AUC in luteolin/SBE-β-CD inclus ion complex group compared with luteolin group, which resulted in an improved bioavailability in luteolin/SBE-β-CD inclusion complex group.Conclusion: The fully-validated HPLC method is successfully applied to the pharmacokinetic study of luteolin in rats. The pharmacokinetic data indicate the bioavailability of luteolin increased after being prepared into luteolin/SBE-β-CD inclus ion complex. The increased solubility of luteolin/SBE-β-CD inclus ion complex promotes luteolin cross the mucus layer and unstirred water layer. The transfer process of SBE-β-CD in the interface of water- biological phase make the penetration amounts of luteolin increased may be the reason why Log P increased and then bioavailability increased after luteolin being prepared into luteolin/SBE-β-CD inclus ion complex. |
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