| Flavonoids compounds are widely seen in nature world, with performances of preventing and treating of respiratory, cardiovascular and cerebrovascular diseases, anti-cancer, anti-tumor, reducing radiation damage, anti-inflammatory and antibacterial efficacy, which has attracted widespread attention. In the present study, in vivo experiment was conducted to research the effects of different flavonoids compounds on Newcastle disease vaccine efficacy, intestinal enzymes and intestinal homeostasis balance, so as to explore the best prescription of flavonoids compounds. Then the best prescription of flavonoids compounds was prepared as the microcapsules. The optimum conditions of microcapsule preparation were filtered based on an orthogonal test. The test is divided into the following four parts:Test Ⅰ Influence of traditional Chinese medicine flavonoids compounds on immuno effect of Newcastle Disease vaccine in chicken To study the effect of traditional Chinese medicine flavonoids compounds on Newcastle disease vaccine,300chickens were randomly divided into6groups(each two repeats, each repeat of25chickens):group Ⅰ(blank control group), group Ⅱ (vaccine control group), group Ⅲ (astragalus polysaccharide control group) and group IV, V, VI(treated with3compounds of traditional Chinese medicine flavonoids). At14-day-old, all the chickens in group Ⅱ～Ⅵ were vaccinated with Newcastle Disease Vaccine, re-vaccinated at28days of age. Before2days of the vaccination, the chickens in three flavone prescriptions groups were treated by aqueous flavonoids compounds solution at the dosage of15mg per feather, once a day for five days respectively. Astragalus polysaccharide group was administrated with astragalus polysaccharide soluble powder for5consecutive days. On day49after the first vaccination, the chickens except for blank control group were challenged with0.1mL of NDV at LD50by intramuscular injection. On days7,14,21,28and49after the first vaccination, and day8after the challenge (D57), blood was sampled for determination of serum hemagglutination inhibition (HI) antibody titer and T lymphocyte proliferation in peripheral blood. The pathogenic and dead statuses of chickens were clinically examined daily for8successive days after the challenge (D57). The results showed that on days21-57after the first vaccination, the antibody titers in the3flavonoid compounds groups were significantly higher than that in vaccine control group (p<0.05). Tlymphocyte proliferations were higher(p>0.05) or significantly higher(p<0.05), the mortality and morbidity after challenge in the3flavonoid compounds groups, especially in the epimedium flavone plus sanchi flavone plus wild dendranthema flower flavone plus baical skullcap root flavone group(ESWB), were lower or significantly lower than that of vaccine control group. The antibody titers and mortality in ESWB group were near to the astragalus polysaccharide control group, however, the aprotective rate was higher. In conclusion, flavonoid compounds can increase the immuno effect of Disease Vaccine. The prescription of ESWB has the best function than the others.Test Ⅱ Effect of traditional Chinese medicine flavonoids compounds on intestinal enzyme activities and microbiology flora in chickens The method of animals grouping and treating was same to that of Test Ⅰ. In2days of administration completed,6chickens with similar weight were selected from each group to be humanely sacrificed so as to aseptically collect the contents in their duodenum, jejunum, ileum, cecum and the junction of cecum and rectum. The protease and amylase activities of the contents in their duodenum, jejunum, ileum, also the counts of enteric bacilli and lactobacillus of the contents in their cecum and junction of cecum and rectum were determined. The results showed that the protease activities in the intestinal tracts of Ⅳ, Ⅴ, VI groups were significantly higher than those in Ⅰ, Ⅱ, Ⅲ control groups (except that of Group Ⅲ in jejunum was similar to that of Group VI), and group V (ESWB) was the highest. The amylase activities in the jejunum of groups IV, V, VI were significantly higher than those in groups Ⅰ, Ⅱ, Ⅲ, and in the duodenum group V was significantly higher those in groups Ⅳ and VI. The lactic acid bacteria counts in the cecum of groups V and VI were significantly higher than those of the other groups, and in the junction of cecum and rectum of groups IV, V and VI were higher than those in the other three groups, group V were significantly higher than groups Ⅰ, Ⅱ, Ⅲ. The enteric bacilli counts in the junction of cecum and rectum of groups Ⅲ, Ⅳ and V were significantly higher than those in groups I and VI. It indicated that the traditional Chinese medicine flavonoids compounds could selectively influence on the intestinal digestive enzymes and microbiology flora to enhance the activities of digestive enzymes and increase the number of beneficial flora to improve intestinal microflora balance. A comprehensive analysis suggested that the ESWB compounds (group V) has best efficacy, while the vaccine has nothing benefit in enhancing the intestinal enzymes activities or improving the microbial flora.Test III Preparation and technology optimization of traditional Chinese medicine flavonoids compound enteric microcapsules In order to explore the optimal preparation conditions of traditional Chinese medicine flavonoid compound microcapsules, hydroxypropyl methyl cellulose phthalate (HPMCP) was used as the capsule material, acetone as the organic solvent, liquid paraffin as continuous phase, Span80as the emulsifier and magnesium stearate as the dispersant for the preparation of microcapsules. Taking the entrapment efficiency, drug loading capacity and microcapsules morphology as the quality evaluating indicators, the individual impacts of HPMCP/acetone (M/V), dosage of dispersant, stirring time and other factors on the quality evaluating indicators were inspected. Based on the results of the single factor test, the orthogonal test was conducted by using the ratios of flavoniods compounds/HPMCP (A), Span80/liquid paraffin (B), and stirring speed (C) as the factor levels, and taking the entrapment efficiency, drug loading capacity and morphology of microcapsules as the quality evaluating indicators to optimize the test conditions. The results showed that the optimum conditions for the preparation of traditional Chinese medicine flavonoids compound microcapsules are as follows: flavonoids:HPMCP is1:2.5, HPMCP:acetone (M/V) is1.0:15, acetone:liquid paraffin (V/V) is1:7, the dosage of magnesium stearate is4%of that of liquid paraffin, the dosage of Span80is9%of that of liquid paraffin, and the stirring speed was300r/min. The microcapsules prepared with the best conditions has uniform appearance, with an average particle size of35.77μm within11-50μm, and with an average encapsulation efficiency of91.72%and average drug loading of26.84%.Test IV Influence of traditional Chinese medicine flavonoids compounds enteric microcapsules on chicken humoral immune,anti-infectious bronchitis virus and intestinal digestive enzyme activities To study the effect of traditional Chinese medicine flavonoids compounds microcapsules on chicken humoral immune and anti-infectious bronchitis virus,350chickens were randomly divided into7groups:group Ⅰ was the blank control group, group Ⅱ was immune control group, group Ⅲ was astragalus polysaccharide control group, group Ⅳ was flavonoids compound medicine control group, group V,VI,VII groups were respectively low, medium, high dose groups of Chinese medicine flavonoids compounds microcapsules. At14-day-old, all the chickens in group Ⅱ-Ⅵ were vaccinated with Newcastle Disease Vaccine, re-vaccinated at28days of age. Before2days of the vaccination, the chickens were treated medicines for five days respectively; Astragalus polysaccharide group was administrated with astragalus polysaccharide soluble powder for5consecutive days. In2days of administration completed,6chickens with similar weight were selected from each group to be humanely sacrificed so as to aseptically collect the contents in their duodenum, jejunum, ileum for determining the protease and amylase activities On days7,14,21and28after the first vaccination, six birds in each group were randomly selected to sample vein blood to determine serum hemagglutination inhibition antibody titer. On day29after the1st vaccination, the chickens except for immune control group were challenged with avian infectious bronchitis virus10-3(EID50)/0.1mL,0.5mL tracheal injection. Pathogenic and dead statuses of chickens were clinically examined daily after the challenge. The results showed that traditional Chinese medicine flavonoids compounds microcapsules medium, high dose can significantly increase serum antibody titers, reduce chicken morbidity and mortality after challenging and improve the protection rate, and significantly improve the digestive enzyme. Traditional Chinese medicine flavonoids compound microcapsules can increase the bioavailability of drug. The high dose group was the best based on the comprehensive evaluation and traditional Chinese medicine flavonoids compound showed better effect than astragalus polysaccharide. |
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