| Objective: Baicalin（BC）is flavonoid extracted from dried roots of Labiatae Scutellaria baicalensis（scutellaria baicalensis Georigi）.Baicalin is composed of baicalein and a molecular glucuronide glycoside compound formation,which exist in the scutellaria baicalensis georigi.Baicalin has a variety of pharmacological effects,such as antihypertensive,anti-cancer,anti-bacterial,free radical scavenging,antipyretic,inhibition of angiogenesis,anti-inflammatory,antitiviral,antioxidant effects and so on.Clinically used for infectious hepatitis,acutebiliary tract infection,lead poisoning,upper respiratory tract infection,Hyperactivity,jaundice,dysentery,enteritis,keratitis.Baicalin can be absorbed into the blood after scutellarein by enzymatic hydrolysis in the gut,quickly transformed into baicalin in vivo.However,poor solubility and stability（decomposition under alkaline conditions）,greatly limits its wide spread application in clinical practice.As one of the widely accpeteded sustained release drug deliverysystems,Microspheres have been deeply investigated.It exerts sustained-release effeets through the specific skeleton material,Poly（lactic-co-glycolic）acid（PLGA）,which is biodegradable with harmless produets H2O and CO2.PLGA was used asadjuvant which had been approved by the Food and Drug A dministration（FDA）,USA.The intra-artieular injectable sustained-release microspheres Preparation to heal OA can reduce administration frequency,extent therapy duration,reduce the Patient pain and enhance the Patient compliance.The aim of our study is to prepare biodegradable baicalin-loaded microspheres with polymers,in order to reduce its stimulation,maintain drug concentration for a long time,so as to reduce the frequency of administration,decrease its side effects,improve patients’ compliance,thereby,establish the basis for further development of new form of baicalin.Method: HPLC method was established in the determination of the content of baicalin in PLGA microspheres.Baicalin-loaded PLGA microspheres had been prepared using the solvent evaporation method based on O/W emulsion.The single factor tests were practiced for the identification of main factors affecting the preparation of baicalin-loaded PLGA microspheres,including the addition method of oil phase,the rotation speed,the reaction temperature,the kind of dispersing agent of aqueous phase,the overall volume of reaction system,the concentration of PLGA,the concentration of gelatin,the mass ratio（BC/PLGA）,the reaction time,the composition of organic solvent and the water/oil phase ratio,and then Orthogonal design was employed to select the optimal prescription and preparation technology,taking particle size,drug loading,entrapment efficiency and score of microspheres as indexes,and then the baicalin-loaded PLGA microspheres were prepared according to the optimal formulation to inspect its reproducibility.We adopt microscopic count to assay the mean diameter and particle size distribution,utilize HPLC to determine the drug loading and entrapment efficiency.The HPLC method was established in the determination of the content of baicalin in releasing media during in vitro release.The characteristics of in vitro release of baicalin solution and baicalin-loaded PLGA microspheres were investigated by dynamic dialysis method.The accumulative release percentage-time curve was obtained and the observed data were fitted to several release models（zero order,first order,Higuchi,Weibull Ritger-peppas）,to clarify the drug releasing mechanism.The HPLC method with ultraviolet detection was developed for the determination of baicalin in rat Plasma.The rats were injected the single dose baicalin and microspheres suspension（10.5mg·kg-1）though intravenous.the drug concentration in plasma versus time curve was obtained and PKSolver was used to calculate the relevant pharmacokinetic parameters.The baicalin-loaded PLGA microspheres were stored under hightemperature,huge humidity or highlight condition to perform the influential factor tests,and the permanent stability was investigated by determination of its appearance and drug loading at different times under environmental conditions: sealed up,room temperature,and protected from light.Result: The HPLC method was established in the determination of the content of baicalin in PLGA microspheres,and the method is reliable.The initiatory preparative parameters of the baicalin microspheres was determined by single factor tests: injecting the oil phase into the aqueous phase with glass syringe,gelatin as the dispersing agent for the aqueous phase,selecting dichloromethane as the organic solvent,the temperature of water bath was 20℃,the rotation speed was 1000 rpm,the rotation time was 2h.The orthogonal design was employed to refine the prescription and preparation technology,taking particle size,drug loading rate,the entrapment efficiency and score of microspheres as indexes.The optimal prescription and preparation technology was subsequently obtained : the concentration of PLGA wass 6%,the concentration of dispersing agent was 1.5%,the mass ratio（CAP/PLGA）is 1∶5,and,the water/oil phase ratio was 10∶1.According to the optimum formulation,the mean particle size,drug-loading rate,entrapment rate and score of 3 batches microspheres prepared from the formulations were: 1.89μm,12.79%,85.40% and 35.92.Using the microscope,we found that the microspheres resulted in spherical shapes and possessed a smooth surface.The result of the baicalin’s solubility in different release medium shows in a pH 7.4 PBS buffer containing 0.05% NaN3 media has the highest solubility as 22.61±0.70 μg·mL-1.The in vitro release of the BC-S and BC-MS results shows that baicalin-loaded PLGA microspheres represented a significant sustained release with a burst release of 32.20% during the first 24 h followed by a gradual release of 84.71% at 11 d compared to the BC-S’s fast release（91.58% in 3h）.By fitting the observed data to several release models（zero order,first order,Higuchi,Weibull,Ritger-peppas）,the release profiles of BC-MS can be better agreed with Ritger-Peppas equation.The releasingindexes（n）was 0.6525 which can be used to speculated that the release mechanism of BC-MS was non-Fick’s diffusion which was resulting from the combined effect of drug proliferation as well as lipid skeleton dissolution.Determination of baicalin in rats plasma concentration by HPLC method with ultraviolet detection,which was strong specificity and good stability.The Plasma concentration versus time curve was obtained after single dose tail vein injection of baicalin and baicalin-loaded PLGA microspheres.The dates was analyzd by PKSolver software,the results were that: the elimination half-life of baicalin and baicalin-loaded PLGA microspheres were respectively1.27 h and 258.98 h;mean residence time（MRT）as 0.88 h and 373.01 h;which showed baicalin-loaded PLGA microspheres has more significant slow-release effect.Influential factor test results showed that baicalin PLGA microspheres are sensitive to temperature,they should be storage under 4℃ or 25℃ to avoid high temperature,and they are also sensitive to humidity,the morphology of the microspheres would be changed and microspheres were going to aggregate,so they must be sealed up to preserve,they are stable under 4500 lx of the light condition.Long-term test suggested that microspheres could be storaged for at least 3 months under environmental conditions: sealed up,room temperature,and protected from light.Conclusion: Baicalin-loaded PLGA microspheres have been developed using the solvent evaporation method based on O/W emulsion and an optimization strategy,the preparation process is stable,this formulation showed good drug loading rate and encapsulation efficiency,and the microspheres resulted in spherical shapes,smooth surface,with good dispersion.The microspheres showed a prolonged duration of release with regard to suspension when administrated via single intravenous injection or in vitro release.The microspheres could be storaged for at least 3 months sealed up under room temperature condition,keeping away from light. |
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