Study On The Cathartic Effect Of Anthraquinones By Colon-targeted Drug Delivery And Pharmacokinetics Of Anthraquinone Extractions Of Rhubarb

ObjectivesThis study focuses on the cathartic effection and mechanism of different anthraquinone extracts (the free anthraquinone, combined anthraquinone and the total anthraquinone), whatever administed in colon targeting-capsule or through the tube. And also cloud-point extraction (CPE) was successfully applied for determination of aloe-emodin, emodin and chrysophanol in rat plasma.Methods1. On one hand, Rhubarb powder, which passed a 24-mesh sieve, were extracted with sulfuric acid and chloroform. Followed filter and concentrate, crude extracts were recrystallized with ethyl acetate. And the free anthraquinone were got. On the other hand, the powder were extracted with 95% ethanol. Followed filter and concentrate, the tannin was removed by MgO, and the total anthraquinone gained. Anthraquinone aglycones were extracted from the total anthraquinones using chloroform, and the combined anthraquinone gained. The total anthraquinones and the combined anthraquinone were hydrolyzed in sulfuric acid and chloroform for 1h and twice. Chloroform phase was separateed from aqueous phase. After being concentrated, the extracts was dissolved with methanol into constant volume and injected into the HPLC. The free anthraquinone was dissolved with methanol into constant volume and determined by the HPLC directly.2. Female Wistar rats (180-220 g) fasted for 16 h with free access to water, prior to the experiments. One end of the PVC catheter was inserted into the cecum while the animal was under anesthesia with ether, and the other end was fixed on the neck. After 3 days, this rats were randomly divided into 4 groups:the free anthraquinone group, the combined anthraquinone group, the total anthraquinone group and the control (NS). Carmine red, as a marker, was administered togerther with drugs and NS through the PVC catheter. Faeces samples were collected at 2、4、6、10、14、20、24 h after dosing. And LIT (large intestine transit time) and moister content(%) of faeces were detected. On the other hand, fasted female Wistar rats (180-220 g) were randomly divided into 4 groups:the free anthraquinone group, the combined anthraquinone group, the total anthraquinone group and the control (NS) group, the combined anthraquinone group, the total anthraquinones group and the control (NS). Large intestine was washed by NS with peristaltic pump, both ends of the colon were ligated while the animal was under anesthesia with ether.2 mL Tyrode solution(g/L, NaCl,8.0; KCl,0.2; CaCl2·6H2O,0.2; MgCl2·6H2O,0.1; NaHCO3,1.0; NaH2PO4,0.05; Sugar,2.7) with drugs were injected into the colon. After 1 h, rats were killed and the colon was resected. Sodium, chlorine, calcium, magnesium and potassium concentration and residual fluid in colon segment were detected.3. Female Wistar rats (180-220 g) fasted for 16 h with free access to water, prior to the experiments. Rats were randomly divided into 4 groups:low-dose group(12 mg/kg), medium-dose group(24 mg/kg), high-dose group(48 mg/kg) and the control. Carmine red, as a marker, was administered togerther with the free anthraquinone and NS in a colon targeting capsules by PCcaps-kit. Faeces samples were collected at 2、4、6、10、14、20、24 h after dosing. And LIT (large intestine transit time) and moister content(%) of faeces were detected. In order to detect the stability of colon targeting capsule in the gastrointestinal tract, colon targeting capsules with carmine red were administered by PCcaps-kit. Rats were killed at different time, and the location and condition of the capsules were observated.4. The non-ionic surfactant Triton X-114 was chosen as the extract solvent. A Kromasil C18 (4.6 mm×150 mm,5.0μm) column was used for elution separation at 40℃with detection wave length at 254 nm and gradient elution was used during the analysis. 1. The extraction yield and the purity of the free anthraquinone, combined anthraquinone and the total anthraquinone were 0.57%、2.03%、2.49% and 59.5%、14.8%、18.6% respectively.2. Compared with the comtrol, the free anthraquinone group and the total anthraquinone group could significantly reduce the LIT(p<0.01), increase the moister content(%) of the feces (p<0.01) and the concentration of K+、Na+、Cl- (p<0.05). But the combined anthraquinone group showed no significant difference.3. Before reached the cecum colon, the colon targeting capsule does not dissolve. Compared with the comtrol, high dose-group could significantly reduce the LIT (p<0.05), and increase the moister content(%)of the faeces, but low-dose group showed no significant difference during 9 h to 23 h.4. The concentration-response relationship was linear over a concentration range of 0.12-15μgmL-1,0.506-16.5μgmL-1 and 0.13-16.2μgmL-1 for aloe-emodin, emodin and chrysophanol, respectively. The limit of detection (LOD) was less than 24 ng mL-1 (S/N=3) and the limit of quantification (LOQ) was less than 38 ngmL-1 (S/N=10). All calibration curves showed good linearity (r2> 0.9997) within the test ranges. The intra-and inter-day variations for 3 analytes were less than 9.83% and 8.05%, respectively. The recovery was more than 93.6%. Their Cmax were 3.44μg·mL-1、6.04μg·mL-1 and 1.95μg·mL-1at 0.17 h. Their pharmacokinetic features are compatible with two-compartment open model. Conclusions1. Due to the successful method for the different anthraquinone extraction, high purity productions were got.2. The results showed that the total rhubarb anthraquinones and the free anthraquinones had cathartic effect, but the combined anthraquinone had no difference with the control. Therefore, we believe that only anthraquinone aglycones had cathartic effect, and it was obvious dose-dependent. The main mechanisms of the cathartic effect of anthraquinone aglycones:accelerating colonic transit, reducing the absorption of water and increasing the concentration of Na+, K+, Cl- in colon.3. Rhubarb colon targeted capsules were stabile in the stomach and small intestine, but started to decompose in the colon, with colon targeting. Anthraquinone aglycones colon targeted capsules could accelerate colonic transit and increase the water in the colon, and the effect is dose-dependent.4. The method was successfully applied for the evaluation of pharmacokinetic profiles of aloe-emodin, emodin and chrysophanol in rat after administrating the extracts of Rhubarb.