| Objective:According to our previous research, combination drugs were conducted, colon-specific preparation was prepared to improve cancer chemopreventive effect.1. Establish an inflammation associated colorectal cancer model, the active ingredients in Radix Angelicae Sinensis and Radix sophora flavescens were combined to improve the effect of colorectal cancer chemoprevention;2. Preparation colon-specific pellets of the supercritical fluid extraction of Radix Angelicae Sinensis, to increase drug concentration in parts of the colon, to improve cancer prevention effect.Methods:1. The BALB/C mice was administrated by intraperitoneal injection mutagen 10mg· kg-1 azoxymethane of (AOM) in the beginning, and the 2% dextran sulfate sodium (DSS) in the drinking water of mice 1,3,5 weeks after injected the mutagen mouse model to establish inflammation of colorectal cancer; mice were fed with 30mg · mL-1, matrine, oxymatrine and 60mg · mL-1 the supercritical fluid extraction (SFE) of Radix Angelicae Sinensis and 30mg · mL-1 matrine, and oxymatrine were combined with 60mg · mL-1 SFE of Radix Angelicae Sinensis, respectively, until the end of the experiment. During the experiment, the mice were weighed weekly, observed living conditions in mice. At 12th week after modeling, the mice were sacrificed, mice were weighed, colon and rectum were harvested, and colorectal length, thickness and width of the tumor site were measured, then pictures of issues were taken using a camera, colorectal macro index in each group was analysized by factor analysis to evaluate the effect of combination therapy. Colorectal tissue were evaluated by HE staining and immunohistochemical analysis, and the results of immunohistochemical were conducted by semi-quantitative analysis. The protein expression of iNOS, COX-2, P53, levels of NF-kB P65 in tumor tissue were determinated, the results were analysed to evaluated the effect of drug combination.2. The preparation process of SFE of Radix Angelicae Sinensis. pill core:low methoxyl pectin was used as matrix materials, the pills were prepared by ionic crosslinking method. The drug loading and encapsulation efficiency of pill cores were used as indicators to select the main factors crosslinking agents, the concentration of crosslinking agent, crosslinking temperature and crosslinking time, mixing temperature, concentration of pectin, the rate of lecithin and pectin, the rate of pectin and drug concentrations by single factor test. Using BOX-Behnken experimental design, to optimize the drug formulation and verify the best prescription.3. Studying coating preparation process of colon targeted pellets of SFE of Radix Angelicae Sinensis:In vitro cumulative release was used as the indicators to select the coating technology, select the different coating materials:Eudragit S100, Eudragit S100-L100 (4:1), Eudragit FS30D; after select the optimum coating material, the impact of different coating weight gaining of 5%,10%,15% on the colon targeting; select the different amounts of triethyl citrate, Tween 80, monostearate in coating formulation affecting the colon targeting and release. The optimal formulation was verified by three batchs, the similarity was evaluated by f2 similarity factor.4. In vivo evaluation of colon targeted pellets of SFE of Radix Angelicae Sinensis:SD rats were given oral colon-specific pellets, the rats were sacrificed at different time points, the stomach, the upper part of small intestine, the lower part of small intestine, cecum and colon contents of rat were harvest to determine the contents of the main ingredient ligustilide in the SFE of Radix Angelicae Sinensis to observe release behavior in the rat gastrointestinal tract.Results:1. The chemopreventive effect of the active ingredients in Radix Angelicae Sinensis and Radix sophora flavescens against colorectal inflammation:the drug can delay the progress and development of colorectal cancer, and its potential mechanism was associated with inhibition of cell proliferation, the expression of inflammation-related proteins COX-2 and i-NOS were reduced, and the expression of P53 was decreased and NF-κB pathway was also down regulated; compared with administered alone, drugs combination could reduce the incidence of colorectal tumors in mice, could also inhibit tumor cell proliferation, significantle, and reduce inflammation-related proteins COX-2 and i-NOS, decrease the protein expression of P53 and down-regulated NF-κB pathway, with a better effective, but the difference was not significant in statistically.2. The of SFE of Radix Angelicae Sinensis pills core preparation process:According to single factor and Box-Behnken optimization results, the best formulation and preparing conditions of SFE of Radix Angelicae Sinensis pills was 3% pectin, the rate of pectin and lecithin was 4:1, fully mixed, the drug was added with a rate of the pectin and drug 4:5, mixed for 10 min at 35 ℃, the dropped in 4% zinc acetate solution with a syringe, crosslinking at 35 ℃ for 30 min after dropped, the pill were washed with deionized water sufficiently, and drying at 35 ℃. Drug loading and encapsulation efficiency of the optimum preparation of pills core were 50.29%,89.31% respectively.3. Colon targeted pellets of SFE of Radix Angelicae Sinensis colon-preparation process: according to the results of in vitro release, the good colon targeting pellets can be prepared by the coating pan coating method; the optimum coating process were as follows:Eudragit FS30D was used as colon targeted materials, coupled with 1.4% Triethyl citrate, Tween-80,1.2% Monostearate, coating weight were gained 15%. Evaluation in vivo and in vitro had some differences, but the results still showed that the prepared colon targeting pellets had a colon targeting.Conclusion:The SFE of Radix Angelicae Sinensis, matrine and oxymatrine had a chemopreventive effect in inflammation associated with colorectal cancer, and drugs combination had raised the trend of chemopreventive. Colon targeted pellets of SFE of Radix Angelicae Sinensis with high drug loading, encapsulation efficiency and good colon targeting was got, and the preparation process was simple, stabile, repeatable good and. The results basically reached the expected goals. |
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