Evaluation Of Phylogenetic Effect Of Fructus Aurantii Immaturus And Citrus Aurantium On Dry Knot Constipation Mice

ObjectiveTo research the differences of chemical composition between Fructus Aurantii Immaturus and Fructus Aurantii, and the efficacy differences between Fructus Aurantii Immaturus and Fructus Aurantii on the treatment of the constipated mice. To analysis the chemical Composition which may result in the efficacy differences between Fructus Aurantii Immaturus and Fructus Aurantii. To research the material foundation that cause the homologous plant becomes different traditional Chinese medicine.Methods1. To develop the fingerprint spectrum of Fructus Aurantii Immaturus and Fructus Aurantii byhigh-performance liquid chromatography,10 batches of Fructus Aurantii Immaturus and Fructus Aurantii were chosen from different producing areas, by using the statistical methods of similarity;, comparing, cluster analysis and principal component analysis, to find the main related components resulting to the different of chemical compositions in Fructus Aurantii Immaturus and Fructus Aurantii.2. the central composite design-response surface methodology was used to selecting the optimum purification process of purifying all flavonoids from Fructus Aurantii Immaturus with macroporous resin, and the volatile oil of the Fructus Aurantii Immaturus and Fructus Aurantii was extracted by steam distillation.3. Water deprivation for seventy-two hours and dry rice-feed were performed to built the model of constipated mice, at the same time, the water decoction of Fructus Aurantii Immaturus, the water decoction of Fructus Aurantii,the flavonoids of Fructus Aurantii Immaturus, the flavonoids of Fructus Aurantii, the essential oil of Fructus Aurantii Immaturus、the essential oil of Fructus Aurantii were given by gavage for 8 days. To evaluate the effect of the compositions from the Fructus Aurantii Immaturus and Fructus Aurantii, by the determination of different pharmacodynamics indexes.4. By using the statistical methods of person correlation and Grey Correlation, combined with the fingerprint spectrum and pharmacodynamics indexes, the main related components resulting to the different of efficacy in Fructus Aurantii Immaturus and Fructus Aurantii were found out.Results1. The HPLC fingerprint spectrum of Fructus Aurantii Immaturus and Fructus Aurantii was established to Choose 17 characteristic peak in the fingerprint spectrum of Fructus Aurantii Immaturus,while 18 in that of Fructus Aurantii, as thus, the same and different points between Fructus Aurantii Immaturus and Fructus Aurantii on the chemical composition could be seen intuitively. The HPLC fingerprint spectrum of the 10 batches of Fructus Aurantii Immaturus from different producing areas showed a high similarity(> 0.9), on the contrary, it showed large differences in that of Fructus Aurantii. By using cluster analysis to campare the fingerprint spectrum of 20 batches of Fructus Aurantii Immaturus and Fructus Aurantii, it seems that Fructus Aurantii Immaturus and Fructus Aurantii did not have overlap region on the chemical composition though they grew from the homologous plant. The method of principal component analysis was applied to show the result that the primary compositions which cause the differences between Fructus Aurantii Immaturus and Fructus Aurantii are as follows: P1 (Synephrine),P2(unknown, RT 43.484 min), P4(unknown, RT 47.897 min), P7(Neohesperidin), P8(unknown, RT 67.379 min), P10(unknown, RT 95.185 min).2. The optimum purification process of all flavonoids from Fructus Aurantii Immaturus was developed by macroporous resin:the ratio of diameter to height was1:8,the concentration of water-extraction was3.5 mg · ml-1, the pH of water-extraction was 6.00, the volume of water-extraction was 18 ml, the adsorption time was 77 min, the volume of water for removing impurity was 30 ml,the ethanol concentration was 88%, the volume of ethanol was 72 ml. To establish the optimum purification method of purifying all flavonoids from Fructus Aurantii by macroporous resin:the ratio of diameter to height was 1:23, the concentration of water-extraction was 5.3 mg · ml-1, the pH of water-extraction was 6.00,the volume of water-extraction was 40 ml, the adsorption time was 53 min, the volume of water for removing impurity was 100 ml,the ethanol concentration was 74%, the volume of ethanol was 124 ml.3. It seems that the efficacy of the decoction of Fructus Aurantii Immaturus was higher than the decoction of Fructus Aurantii in The treatment of the constipated mice and was dose-dependent by comparing with the normal group and the model group, at the same time, it is also better than the others components in the Fructus Aurantii Immaturus, and the effect of components in the Fructus Aurantii Immaturus was better than that of in the Fructus Aurantii.4. Through the analysis of person correlation, it seems that the main related components resulting to the effect of Fructus Aurantii Immaturus were, P3(unknown, RT 43.195 min), P5(Naringin), P8(unknown, RT 53.879 min), P9(Neohesperidin), P11 (unknown, RT 67.388 min), P13(unknown, RT 95.189 min); the main related components resulting to the effect of Fructus Aurantii were, P3 (unknown, RT 46.597 min), P4(Narirutin), P6(Hesperidin), P7(Naringin), P13(unknown,RT 95.188 min), P14(Limonin). Through the analysis of Grey Correlation, it is found that the main related components resulting to the effect of Fructus Aurantii Immaturus were, P3(unknown, RT 43.195 min), P5(Hesperidin), P6(Naringin), P8(unknown, RT 53.879 min) P9 (Neohesperidin), P11 (unknown, RT 67.388 min), P13 (unknown, RT 95.186 min) 和 P14(Limonin); the main related components resulting to the effect of Fructus Aurantii were, P1(Synephrine), P2(unknown, RT 43.434 min), P4(Narirutin), P5(Naringin), P6(Hesperidin), P7(Neohesperidin), P8(unkno wn, RT 59.314 min), P9(unknown, RT 67.37 min), P10(unknown, RT 68.626 min), P11 (unknown, RT 79.991 min), P12(unknown, RT 89.35 min),P13 (unknown, RT 95.188 min), P14 (Limonin), P15 (unknown, RT 105.869 min), P17 (unknown, RT 109.274 min), P18(unknown, RT 111.572 min).ConclusionThere are many differences between Fructus Aurantii Immaturus and Fructus Aurantii in chemical composition and content, and these differences might be due to combined effect by several components. In the matter of medicinal efficacy, the effect of the decoction of Fructus Aurantii Immaturuswas is better than that of the decoction of Fructus Aurantii, at the same time, it also better than the other components in the Fructus Aurantii Immaturus. By comprehensive analysis of the fingerprint and the efficacy of purgation, the efficacy foundation of Fructus Aurantii Immaturus and Fructus Aurantii were not identical. To conjecture the differences of them were induced by the interaction of many compositions, therefore, we should not only detecting the single substance while evaluating them by quality.