| Objective: Capsaicin, the main natural active ingredient that a sort of vanillyl amide alkaloids which is extracted from solanaceae chili genus chili,and its chemical name is:N-[(4-hydroxy-3-methoxyphenyl)methyl]-8- methyl-(E)-6-nonenamide.Capsaicin has shown a wide variety of physiological pharmacology properties, including eliminating inflammation, analgesic effect,bacteriostasis,insecticidal action,relieving itching,prevention and cure of angiocardiopathy,promoting lipometabolism,regulating tumor cell apoptosis,resist radiation,fight mutagenesisand so on.Among them,the long- acting analgesic effect and new analgesic mechanism of capsaicin is the most unique and outstanding.Because capsaicin can relieve pain and itching mainly through acting on the release, synthesis and storage of neuropeptide substance P that reduces inflammatory mediators histamine,slow shock peptide and prostaglandin generation and release.Thus it has stronger and lasting analgesic effect than opioid analgesics,while without the side effects usually generated by non-steroid anti-inflammatory agents and addiction of opioid analgesics. Due to the definite effect and low side effects,it is the firstchoice to treat postherpetic neuralgia and diabetic neuralgia in DRUGEVALUASION that presided by America Medical Association.Besides currently,capsaicin in clinical also mainly used in the treatment of sciatica,severe rheumatoid arthritis,severe psoriasis,allergy or uremia itching,cluster headaches,bald hair, and peripheral neuralgia which are related to HIV infection.Capsaicin can also prolong analgesia time when combined with morphine and exhibit an outstanding analgesia effect on advanced stage cancer patient.However,since capsaicin possesses significant first pass effect of hepar,excessive short half life,low oral bioavailability,at present,capsaicin is mostly used in topical drug administration,such as cream,patch,ointments, membrane,cataplasm and so on.But all the routes of administration mentioned above have showed drug effect to be slow,absorption incompletely,and different extents of irritant effects on skin which limited its clinical application severely. At present, there has been some oral drug injections of clinical pharmacology and toxicology studies completed.In view of its disadvantages existed in the process of clinical application,the aim of our study is to prepare a type of intestines-dissolving biodegradable capsaicin-loaded microcapsules with natural polymers.Intestines delivering drug not only can reduce irritation, improve capsaicin bioavailability,and by controlling capsaicin release in the intestines and colon,can realize drug slow-release to maintain effective drug concentration in a fairly long period of time,thus to reduce the frequency of administration,decrease its side effects and improve patients'compliance, especially to exbit a long lasting analgesia effect for patients who have suffered from the advanced stage of small bowel and colon cancer,thereby,establish the basis for further development of new form of capsaicin.Method:HPLC spectroscopy was established in the determination of the content of capsaicin in Alginate calcium-Chitosan microcapsules. Capsaicin- loaded Alginate calcium-Chitosan microcapsules had been prepared using the two step method of O/W emulsification - ion exchange - coacervation method, and both drop method and spray method were applied respectively.The single factor tests were practiced for the identification of main factors affecting the preparation of capsaicin-loaded Alginate calcium-Chitosan microcapsules, including the drops of height,the balance time of CaCl2,the incubation time of chitosan, the diameter of injector,spraying height,spraying pressure,the concentration of sodium alginate as carrier material,the concentration of chitosan and CaCl2,CaCl2/ chitosan ratios,and the oil phase/water pahse ratio.After the main influencing factors were Preliminary determined,the drop method experiments were conducted using Orthogonal design with the factors of the concentration of sodium alginate,the diameter of injector,the concentration of CaCl2 and oil phase/water pahse ratio.Meanwhile,spray method experiments were conducted also using Orthogonal design with the factors of the concentration of sodium alginate,spraying pressure,the concentra -tion of CaCl2 and spraying height.Taking particle size,particle distribution and irregular degrees as indexes,comprehensive point–rating and Multivariate linear regression were applied to optimize the formulation and two optimal formulations were obtained finally.Then 6 batches of Capsaicin-loaded Alginate calcium-Chitosan microcapsules were prepared according to the optimal formulation to inspect its reproducibility.We adopt microscopic count to assay the mean diameter and particle size distribution, use the scanning electron microscope to observe the shape and surface of the microcapsules, utilize HPLC spectroscopy to determine the drug loading and entrapment efficiency.High performance liquid chromatography was established in the determination of in vitro release of capsaicin-loaded Alginate calcium- Chitosan microcapsules,the in vivo release profile of capsaicin-loaded Alginate calcium-Chitosan microcapsules was investigated at 55℃and 37℃,and the accumulative release percentage-time curve was obtained. And then the observed data were fitted to several release models(zero order,first order, Higuchi, Ritger-peppas),to clarify the drug releasing mechanism.The capsaicin-loaded Alginate calcium-Chitosan microcapsules were stored under high temperature,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:HPLC spectroscopy was established in the determination of the content of capsaicin in Alginate calcium-Chitosan microcapsules,and the method is reliable,simple and easy.The initiatory preparative parameters of the capsaicin microcapsules was determined by single factor tests: a)for drop method:Drops of height was 8 to 12 cm from the needle tip to liquid surface,CaCl2 balance time was 0.5 h, Chitosan incubation time was 1 h,chitosan concentration was 0.75 %,the oil /water ratio was 9:1,and the microcapsules was Natural dryed. b)for spray method CaCl2 balance time was 0.25 h,chitosan incubation time was 0.5 h,2# the nozzle,Chitosan concentration was 0.75 %,the capsaicin concentration in oil phase was 6 mg·mL-1),water/oil ratio was 9:1,and the microcapsules was natural dryed.The orthogonal design was employed to refine the prescription and preparation technology,drop method taking mean particle size,drug-loading rate,entrapment rate as indexes,while? spray method taking average particle size, particle distribution and Irregular degrees as indexes.The quantitative equation was obtained through multiple linear regression,the quantitative effect of the formulation parameters at different levels on the above indexes could be predicted using quantitative equation.The optimal prescription and preparation technology was subsequently obtained by intuitive analysis and Z -points comprehensive method evaluation. a)For drop method: Sodium alginate concentration was 2.5 %,CaCl2 concentration was 4 %,syringe diameter was 0.26 mm,water/oil ratio was 9:1(2.5 % sodium alginate:60 mg·mL-1) CAP cod liver oil solution).According to the optimum formulation,the mean particle size,drug-loading rate,entrapment rate and score of 6 batches microcapsules prepared from the formulations were:1435.30μm,2.09%,69.55% and 3.00 respectively.the microcapsules resulted in yellow-white particles and had spherical shapes,with good liquidity and dispersion and possessed a smooth surface. b)For spray method:Sodium alginate concentration was 2.5 %,CaCl2 concentration was 4%, spray height was 30cm,spray pressure was 0.02 MPa,water/oil ratio was 9:1(2.5 % sodium alginate:60 mg·mL-1) CAP Cod liver oil solution).According to the optimum formulation,the mean particle size,drug-loading rate and score of 3 batches microcapsules prepared from the formulations were:180.84μm,1.89 % and 7.92 respectively.The microcapsules resulted in white particles and had good liquidity and dispersion.Using the scanning electron microscope,we found that the microcapsules resulted in spherical shapes and possessed a smooth surface.The dissolubility of capsaicin in three different receiving liquid containing 0.1 % Tween 80 are 196.94±22.43μg·mL-1,227.90±23.69μg·mL-1, 215.17±8.83μg·mL-1 respectively,while in three different receiving liquid containing 0.05 % Tween 80,the dissolubility of capsaicin are 131.08±19.54μg·mL-1,145.62±6.59μg·mL-1,138.27±4.63μg·mL-1 respectively.The dissolu -bility of capsaicin in three different receiving liquid containing 10% PEG 400 are 103.07±5.36μg·mL-1,125.97±5.36μg·mL-1,120.53±9.59μg·mL-1 respecti -vely, while in three different receiving liquid containing 30 % PEG 400,The dissolubility of capsaicin are 366.96±20.35μg·mL-1,590.44±25.33μg·mL-1, 448.75±24.40μg·mL-1 respectively.For drop method and spray method,the in vitro release profiles of capsaicin from Alginate calcium-Chitosan microcapsules at the previously defined time at 55℃and 37℃demonstrated:At the condition of 55℃, the drug was released for 9.06 % and 5.92 % in 2 h inartificial gastric liquid respectively,then a 24.16% and 15.58 % drug release was observed in 3 hours inartificial intestinal liquid,50.08 % in all for 24 h in artificial colon liquid, whereas.At the condition of 37℃,after an initial burst 4.93 % and 3.07 % in 2 h artificial gastric liquid respectively,a continuous drug release of 13.98 % and 8.37 %was observed for 3h in artificial intestinal liquid.The accumulated amount was 50.08 % and 29.56 % in all after 24 h observation in artificial colon liquid respectively.By fitting the observed data to several release models(zero order,first order,Higuchi,Ritger-peppas),the drug release from capsaicin-loaded Alginate calcium-Chitosan microcapsules by drop method and spray method can be better agreed with Higuchi equation because of the higher R2 value,1.000 and 0.996at 55℃,0.995 and 0.998 at 37℃respective -ly.From the Ritger-peppas equation Q=ktn,the releasing indexes (n) were 0.803 and 0.781 at 55℃,0.941 and 0.912 at 37℃respectively.By two method indicated that drug release was non-Fick diffusion controlled at 55℃,the mechanism may be involved:the dissolution/diffusion of drug from the matrices,and the matrix erosion resulting from degradation/dissolution of Alginate calcium.However the matrix erosion resulting from degradation/ dissolution of Alginate calcium at 37℃mainly.Influential factor test results showed that Capsaicin-loaded Alginate calcium-Chitosan microcapsules are sensitive to temperature,they should be storage under 4℃or 25℃to avoid high temperature.Capsaicin-loaded Alginate calcium-Chitosan microcapsules are also sensitive to humidity. Although humidity has little influence on the drug loading rate of the microcapsules,the morphology of the microcapsules would be changed and microcapsules were going to aggregate under high humidity level,so they must be sealed up to preserve.Moreover,when the microcapsules were placed under 4500lx of the light condition, the drug loading rate reduced,so they must be protected from ligh.Long-term test suggested that microcapsules could be storaged for at least 3 months under environmental conditions:sealed up,room temperature, and protected from light.Conclusions: Capsaicin-loaded Alginate calcium-Chitosan microcapsules have been developed using the two step method of O/W Emulsification-ion exchange-coacervation method,drop method and spray method were applied respectively.Both of the preparation process is stable,and the microcapsules resulted in spherical shapes, smooth surface,with good dispersion.The optimal formulation showed good drug loading rate and encapsulation efficiency.The microcapsules could be storaged for at least 3 months sealed up under room temperature condition, keeping away from light.
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