Pharmaceutical Study On The Further Development Of Xianglian Pill Into Colon-targeted Preparation

Objective: Ulcerative Colitis （UC） is a kind of nonspecific inflammatory bowel disease which etiology is unclear. But it is thought to be due to infection, hereditary, autoimmune and environment factors in modern medical science. These make UC very difficult to cure, and it strongly affects the patients’ daily living and work. The western medicine to treat UC mainly includes amino salicylic acid, adrenocortical hormones, antibacterial drug, immunosuppressant, ect. These medicines are effective in controlling the symptoms of UC, but bringing severe side effects, also with very high cost. Traditional Chinese Medicine （TCM） has great advantage in the therapy of UC, because its therapeutic effects are remarkable and side effects are minimal, and the cost is much lower. The Xianglian pill we have studied on in this subject has been included by Pharmacopoea Chinensis. It is composed by rhizoma coptidis, processed by evodia rutaecarpa, namely Yuhuanglian, and aucklandia lappa. This preparation is clinic used for UC treatment and proved to have obvious curative effects by practice. But the existing Xianglian preparation technology is rough, and the preparations are low in effective ingredient contents, large in dose. They are also high in viscosity, readily absorbing moisture and agglomerating, unqualified disintegration and unfavorable to storage because of lots of impurities. In bioavailability, the effective ingredient concentrations are low in focus of colon and the residence time is limited in diseased region. All these do not meet the needs of mordern TCM. This study was designed to further develop Xianglian pill. First, effective fractions were extracted and refined; secondary, the total alkaloid of Yuhuanglian and volatile oil from aucklandia lappa were prepared into colon-targeted and gastro-release micropellet respectively; at last, the two types of micropellets were filled into capsules in fixed proportion. Thus, a new generation of Xianglian preparation was developed with colon-targeted property, in order to enhance therapeutic effects.Methods: （1） The total alkaloid of Yuhuanglian was extracted by hot water extraction combined with ultrasonic treatment, and refined by salting out. The optimum extraction conditions were investigated by the orthogonal design with temperature of the water, duration of extraction, and times of extracting as the factors and the extraction rate of the total alkaloid of rhizoma coptidis as the index. Thin layer chromatography （TLC） was used for the identification of evodia rutaecarpa and rhizoma coptidis. The contents of the total alkaloid of rhizoma coptidis in Yuhuanglian extract were determined by UV spectrophometry and the detection wavelength was 349nm. （2） Supercritical carbon dioxide fluid extraction （SFE-CO₂） was adopted to extract the volatile oil from Aucklandia lappa. The optimum extraction conditions were investigated by the orthogonal design with the total contents of costuslactone and ehydrocostuslacton as the index. Three factors including extraction pressure, temperature and time were abserved. TLC was used for the identification of costuslactone and ehydrocostuslacton in the volatile oil extract. The total contents of costuslactone and ehydrocostuslacton in the extract were determined by HPLC. （3） The total alkaloid extract of Yuhuanglian was developed into pH-dependent colon-targeted micropellet coated by acrylic resin, and evaluate its release in vitro. The optimum coating prescription was also investigated by the orthogonal design with proportion of Eudragitl L100-55 and Eudragitl S100, the ratio of plasticizer to Eudragitl, and the ratio of the weight of the membrane to micropellet as the factors; the comprehensive grade of coating effect as the index. The same methods were adopted in the identification and content determination of the micropellet as the total alkaloid extract. （4） The aucklandia lappa’s volatile oil was included byβ-cyclodextrin（β-CD）. The experiment was carried out with orthogonal design, with the proportion of the oil andβ-CD, inclusion temperature, and inclusion time as factors and utilization ratio of volatile oil as the index. The inclusion compound was developed to gastro-release micromicropellet, uncoated. The methods of identification and content determination of the micromicropellet were as the same as the volatile oil extract. （5） Filled the two types of micropellet into capsules in a fixed proportion. （6） Variance analysis of all orthogonal designs were done by SPSS 10.0; the difference was statistically significant if P＜0.05, or else, no significant difference was observed. Intiutive analysis was also done to fix the optimum conditions.Results: （1） The optimum extraction condition of total alkaloid of Yuhuanglian obtained was: adding 8 fold of water, with the temperature at 60℃, extracting 3 times, 1.0 hours every turn. The extracting temperature of the water and extracting times had significant effects on the extraction rate of the total alkaloid （P＜0.05）, but the duration of extraction had no significant effect on the index （P＞0.05）. TLC was used for the identification of evodia rutaecarpa with a mixed solvent of cyclohexane-ethyl acetate-methanol-triethylamine （19:5:1:1） as the developer and spraying with 10% sulfuric acid ethanol solution as colorimetric method, while rhizoma coptidis with a mixed solvent of n-butyl alcohol-acetic acid-water （7:1:2） as the developer. The contents of the total alkaloid of rhizoma coptidis were determined by UV spectrophometry in Yuhuanglian extract; the linear range of berberine hydrochloric was 1.04～11.44μg/ml, R²=0.9999; the recovery rate and RSD were 99.2% and 1.0%; the average contents of the total alkaloid determined in the extract were 65.9%. （2） The optimum extraction condition of volatile oil obtained was: temperature of 30℃, pressure of 25 MPa and time of 2.5 hours. The effects of temperature and pressure on the contents of extract were significant（P＜0.05）, but the extraction time had no significant effect on the index. The costuslactone and dehydrocostuslactone in the supercritical CO₂ extract were identified by silica gel G-CMC plate and the developing reagents consisted of petroleum ether（30～60℃）-benzene-ethyl acetate （14:1:3） with 5% vanillic aldehyde dissovled in sulfuric acid as the colour developing reagent. The contents of costuslactone and dehydrocostuslactone were determined by HPLC on a Hewlett Packard ODS column （125mm×4mm, 5μm） using a mobile phase of methyl alcohol-water （65:35） at the flow rate of 0.5ml/min, column temperature of 25℃, detected by VWD detector, and the detection and reference wavelength was 225nm and 360nm respectively. The linear concentrations of costuslactone and dehydrocostuslactone were 11.48～114.80μg/ml and 10.78～107.80μg/ml respectively. Their recovery rates and RSD were 99.9%, 99.8% and 0.93%, 0.77% respectively. It was determined that the average contents of costuslactone and dehydrocostuslactone were 27.59% and 36.34% respectively, and the average total contents were 63.92% in the volatile oil extract. （3） The preparation technology of Yuhuanglian’s total alkaloid micropellet （Yuhuanglian micropellet for short） was using microcrystalline cellulose （MCC） as the excipient adding 5% starch to it as disintegrating agent and using 25% alcoholic solution as binding agent. The proportion of extract and mixed excipients was 3:7. All the materials were homogenized to prepare granule and sphere in coating pan. The optimum coating prescription was as: the proportion of Eudragitl L100-55 and Eudragitl S100 was 1:3, the ratio of plasticizer to Eudragitl was 30%, and the ratio of the membrane weight to micropellet was 30%; the proportion of the two acrylic resin and the ratio of the membrane weight to micropellet had significant affect on the index （P＜0.05）, while the ratio of plasticizer to Eudratitl had no significant affect on it （P＞0.05）. In vitro release experiment, the linear range of berberine hydrochloric in pH1.2 medium was 1.096/μg/ml～9.864μg/ml, R²=0.9999, and the cumulative release of coated micropellet was less than 5% within 2h. In pH7.5 medium, the linear range of berberine hydrochloric was 1.096μg/ml～9.864μg/ml, R²=0.9998. The cumulative release of coated micropellet was more than 50% within 5h, and approximate 100% within 10h. （4） The optimum preparation conditions for inclusion were established as: oil:β-CD was 1: 9, the inclusion temperature was at 50℃and the inclusion time was for 3h. The utilization ratio of oil is 75.5%. All factors have significant affect on utilization ratio of oil （P＜0.05）. The preparation technology of the aucklandia lappa’s volatile oil micropellet （aucklandia lappa micropellet for short） obtained was: MCC adding the 5% starch as excipients and 25% alcoholic solution as binding agent. The proportion of inclusion compound and mixed excipients was 3:7. All the materials were homogenized to prepare granule and sphere in coating pan. In time limit experiment of collapse, the micropellet disintegrate in 16 min in the pH1.2 medium. （5） The two types of micropellet above were filled into capsules in fixed proportion of 3:1.Conclusion: （1） It is short in production cycle, high in yield and reliable adopting hot water extraction combined with ultrasonic treatment to extract total alkaloid of Yuhuanglian. The contents of the total alkaloid of rhizoma coptidis in the extract were more than 50%, according with the criterion of effective fractions or ingredients preparation. Using TLC to identify the evodia rutaecarpa and rhizoma coptidis is simple, feasible and fast. The method to determine the contents of the total alkaloid of rhizoma coptidis by UV spectrophometry is accurate, reliable and rapid. It can be used for quality control of Yuhuanglian extract. （2） Using supercritical CO₂ to extract volatile oil from aucklandia lappa is short in production cycle and harmless to the workers. Also no organic solvent is residual in the extract and the total contents of costuslactone and dehydrocostuslactone were more than 50%. It accords with the criterion of effective fractions or ingredients preparation. It is simple, feasible and fast to identify costuslactone and dehydrocostuslactone by TLC. It is accurate, sensitive and no disturbance to determine the contents of costuslactone and dehydrocostuslactone by HPLC. It is suitable for quality control of the extract. （3） The technology of Yuhuanglian micropellet is simple, no special excipient required at all. The release experiment in vitro shows that the coated micropellet has a certain degreee of acid resistane and property of colon-targeted release. （4） The optimized inclusion process of volatile oil is efficient and high in utilization ratio of volatile oil. The method is suitable for practice. The technology of aucklandia lappa micropellet is simple, with the same excipients as Yuhuanglian micropellet. The time limit experiment of collapse shows that the uncoated micropellet can disintegrate in stamoach in a short time. （5） The capsules with separate gastric and enteric solubility and colon-targeted release property are formed after the two types of micropellet wre enclosed.