| Puerarin and catalpol were found to have the ability of improving the neurologic symptoms of stroke and promoting the regeneration of neuron and blood vessel of brain when used simultaneously. The protective effect of puerarin and catalpol against cranial nerve and the promotive effect on the regeneration of neuron and blood vessel of brain were especially beneficial to the prognosis treatment after the aute period of cerebral stroke. So we decided to develop an new oral preparation of puerarin and catalpol. To develop this new preparation, two key problems must be solved. One is the poor oral absorption of puerarin owing to its low solubility and the other is the low penetration of drug into brain caused by blood-brain barrier(BBB). Borneol, a “messenger†drug in traditional Chinese medicine which could direct drugs upward to head targeting the brain, was added into the oral preparation. At the same time, some new techniques such as self-microemulsifying drug delivery system(SMEDDS)and inclusion complex were used to increase the absorption of puerarin after oral administration. The whole work were as follows:(1) The determination methods of puerarin, catalpol and borneol in different situation were established.(1)HPLC was used to determie puerarin and catalpol in Cell infiltration fluid;(2)HPLC-MS/MS was developed to determine puerarin and catalpol in plasma and brain tissue of rats simultaneously;(3)puerarin and catalpol in inclusion complex or SMEDDS were determined by HPLC;(4)borneol in inclusion complex or SMEDDS was determined by GC.(2) The promoting effect of borneol on the oral absorption and penetration into brain of puerarin and catalpol and the suitable dose was selected in vitro and in vivo.(1)in vitro: an BBB model,co-culturing the brain microvessel endothelial cell(BMEC)and astrocyte(As) from the same genus for 7 days,was used to investigate the effect of borneol concentration from 6.25 μg·m L-1 to 100 μg·m L-1 on the transportion of puerarin and catalpol. The permeability of puerarin and catalpol were increased significantly(P<0.01) and TEER values at 2 h were decreased significantly(P<0.05) when the concentration of borneol was over 12.5 μg·m L-1compared with control group.(2)in vivo: the pharmacokinetics of puerarin and catalpol in plasma and brain of rats were studied after oral administration of borneol solution(0 g·kg-1, 0.025 g·kg-1, 0.05 g·kg-1 and 0.1 g·kg-1) followed by puerarin(0.2 g·kg-1) and catalpol(0.045 g·kg-1) nanocrystals suspension immediately. Borneol with the dosage of 0.05 g·kg-1and 0.1 g·kg-1 could significantly increase the oral absorption of puerarin(P<0.05), but there was no obvious difference for catalpol(P>0.05). As far as AUCbrain was concerned, the dose of 0.1 g·kg-1was better than others for puerarin(P<0.05); but for catapol 0.05 g·kg-1 was better than 0.025 g·kg-1(P<0.05) and was not different obviously with 0.1 g·kg-1(P>0.05).So the optimized dosage of borneol used in the new oral preparation was determined to be 0.1 g·kg-1.(3) The SMEDDS and inclusion complex loaded puerarin, catalpol and borneol were prepared and characterized.(1)SMEDDS: Saturated solubilities of puerarin and borneol in different lipids and surfactants were determined. The materials with better solubility were choose to study the compatibility of materials. Subsequently, pseudo-ternary phase diagrams were built. The properties of SMEEDS, such as emulsifying time, particle size and zeta potential was used to select the optimized formulation. Two SMEDDS were prepared as follows:(A) 10.00 g of Capmul MCM, 16.00 g of tween-80, 4.00 g of Labrasol and 20.00 g of 1,2-propylene glycol were mixed by stirring at 37 ℃, and then 0.50 g of puerarin and 0.25 g of borneol were added and stirred. After puerarin and borneol were completely dissolved, 0.11 g of catalpol was added. The particle size of SMEEDS was(151.6±1.92) nm and zeta potential was(-4.73±0.38) m V;(B) 10.00 g of Capmul MCM, 13.40 g of RH-40, 13.40 g of Labrasol and 13.40 g of 1,2-propylene glycol were mixed by stirring at 37 ℃, and then 0.50 g of puerarin and 0.25 g of borneol were added and stirred. After puerarin and borneol were completely dissolved, 0.11 g of catalpol was added. The particle size of SMEDDS was(25.37±1.60) nm and zeta potential was(-7.18±0.42) m V.(2)Inclusion complex: with HP-β-CD as the inclusion material, the freeze-drying method was used to prepare the inclusion complex. The inclusion mechanism was tested by measuring the inclusion concentration, DSC and X-ray diffraction. There is a competition in the simultaneous inclusion between puerarin and borneol and puerarin was included preferentially. Catalpol could be included with HP-β-CD with no impact on the inclusion of puerarin or borneol. The final preparation technology of inclusion complex was as follows: 0.50 g of puerarin and 0.25 g of borneol were added into 50 m L of 40% HP-β-CD solution. The obtained mixture was then ultrasounded for 30 mins, and 0.11 g of catalpol was added. Ultrasound was continued until drugs were dissolved completely. Puerarin and borneol can be included into the HP-β-CD completely leading to an inclusion rate of 100%. Catalpol can be complexed with the hydroxyl of HP-β-CD and recombination rate was 100%. The concentration of puerarin, catalpol and borneol were 10 mg·m L-1, 2.2 mg·m L-1 and 5 mg·m L-1, respectively.(4) The pharmacokinetic parameters of SMEDDS and inclusion complex of puerarin and catalpol in plasma and brain tissue of rats after oral administration were compared. The SMEEDS(tween-80) could significantly increase the oral absorption of puerarin than others(P<0.05), and inclusion complex could remarkably increase the oral absorption of catalpol than nanocrystal(P<0.01). There were no obvious difference between other groups.The SMEEDS(tween-80) could increase the AUCbrain of puerarin compared with other groups(P<0.01); the inclusion complex had the highest AUCbrain for catalpol, but there was no significant difference compared with SMEEDS(0.05<P<0.1).An oral formulation of puerarin and catalpol was developed in the present study to enhance both oral bioavailability of puerarin and brain penetration of puerarin and catalpol using borneol as an oral brain-targeting enhancer. The brain microvascular endothelial cells and astrocytes from the same genus were co-cultured for 7 d to establish BBB model. The effect of borneol concentration from 6.25 μg·m L-1 to 100 μg·m L-1 on the transportion of puerarin and catalpol was investigated. Three oral formulations of nanocrystals, SMEDDS and inclusion compounds were prepared. The effects of borneol and formulations on the pharmacokinetic behaviors of puerarin and catalpol in mice blood and brain after oral administration were investigated. The SMDDES using Tween-80 as surfcant with 0.1 g·kg-1 of borneol could increase the amount of puerarin and catalpol in brain and was promising to be used in Zi Ge oral new preparation. |
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