| Aeromonas hydrophila is an emerging aquatic pathogen which has caused a great economic losses in freshwater aquaculture. At present, the measure used mostly in preventing and controlling aquatic animal diseases resulted by A. hydrophila is antibiotics. Due to the abuse of antibiotics, the drug-resistance of A. hydrophila has become increasingly serious, it not only adds difficulty to the cure of fish diseases but also brings great damages to the environment and human. With the increasing demand for organic aquaculture, there has been a growing interest in using natural products in aquaculture to prevent diseases for their less side effects than antibiotics. In this study, the prescription that restrained A. hydrophila was screened by the antibacterial experiment, at the same time, the extraction was optimized and the effective component was analysed. Moreover, the mode of action of prescription was investigated against A. hydrophila. The present study can provided basis for the application of polypharmacy.1、Antimicrobial activity of plant extracts against A. hydrophila was expressed as the minimal inhibitory concentration (MIC). The MIC showed that extracts of Galla Chinensis (0.097-0.195 mg/mL), rhubarb (0.78-1.560 mg/mL), Scutellaria (1.560-3.125 mg/mL) Terminalia chebula (1.560-3.125 mg/mL), and tea polyphenol (0.195-0.390 mg/mL) had excellent antibacterial activity. The prescription was made up according to a orthogonal t value design using five plant extracts as factors.The prescription formulated from tea polyphenol, Galla chinensis and rhubarb at the rate of 5:2:1 (w/w/w). The minimal inhibitory concentration (MIC) of prescription against A. hydrophila was 0.078 mg/mL.2、In order to enhance the contents of active component, the ionic liquid was used as extraction solvents and applied to the extraction of tannins and anthraquinones from Galla chinensis and rhubarb by the simultaneous ultrasonic and microwave assisted extraction (UMAE) technique. The optimal UMAE conditions were as follows, for Galla chinensis: 2.5 mol/L 1-butyl-3-methylimidazolium bromide ([bmim]Br) as extraction solvent, microwave irradiation power of 400 W, extraction time of 1 min, solid-liquid ratio of 1:15 (g/mL). For rhubarb: the solvent was 2.0 mol/L [bmim]Br solution, the ration of solid/liquid (g/mL) was 1:15, time was 2 min, and microwave power was 500 W. Under these UMAE conditions, the yields of tannins and anthraquinones were compared with the conventional extraction, the proposed approach exhibited higher efficiency and shorter extraction time. The content of tannins in Galla chinensis and anthraquinones in rhubarb collected from different cultivated areas was analyzed by the Phosphomolybdium and UPLC. The data demonstrated that the content of tannins in Dubei (Hubei Prov.) and anthraquinone in R. palmatum (Gansu Prov.) was the highest.The analysis of commercial tea polyphenols was performed using industry standard (QB 2154-1995) and high performance charomatographic (HPLC). The result showed that the content of tea polyphenols (85.40%) was close to the value of product report (88.9%). HPLC analysis results showed that the content of catechin was 51.42%, and account for 60.20% in tea polyphenols. 3、Macroporous resin was used to enrich the effective component and remove ionic liquid. In static adsorption experiment, XDA-6 and HPD-100 were selected to separate and isolate the tannins and anthraquinones, the loading temperature was 28°C. Dynamic adsorption and desorption tests were carried, and the optimum conditions were as follows. The ratio of column height to diameter was 1:8, loading volume 2BV, flow rate 1.0 BV/h, 85% ethanol was used as eluant, and the elution volume were 2BV and 3BV, respectively. Under the optimized conditions, the dynamic adsorption and desoption rate of tannins in XDA-6 was 94.78% and 91.62%, respectively. The content of tannins increased from 70.24% to 85.12% with a recovery of 99.06%. The dynamic adsorption and desoption rate of anthraquinones in HPD-100 was 86.34% and 85.92%, respectively. The content of anthraquinones increased from 7.13% to 20.52% with a recovery of 98.73%. The result of UPLC-MS/MS analysis showed that the residual [bmim]Br could be removed from crude extract.The minimal inhibitory concentration (MIC) of optimal prescription against A. hydrophila was 0.060 mg/mL. In order to evaluate and control effectively the quality of prescription, a sensitive and accurate UPLC-MS/MS method has been developed for the simultaneous determination of ten active compound in prescription. The analysis was performed on a Diamonsil-C18 column at 45℃using acetonitrile / formic acid gradient system at a flow rate 0.3 mL/min and diode array detection (DAD) at wavelengths 280 nm. Source block temperature 100°C, desolvation temperature 250°C, capillary voltage 3.0 kV and cone voltage 20 V, collision energy 6 eV and nitrogen as desolvation gas (500L L/h), spectra were scanned over a mass range of m/z 100–1500. The method showed good linearity and satisfactory accuracy. The newly established UPLC-MS/MS method was subsequently utilized to determine the content of ten compound in prescription. The result showed that contents of gallic acid, (-)-epigallocatechin (EGC), (+)-catechin, (-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate (ECG), aloe-emodin, rhein, emodin, chrysophanol, and physcion, were 5.27%, 3.30%, 1.08%, 19.32%, 5.46%, 0.23%, 0.56%, 1.28%, 0.75% and 0.39%, respectively.4、In order to provide theoretical basis for the further application of prescription. Bacteriostasis mechanisms against A. hydrophila of the optimal prescription were investigated used transmission electron microscopy, atomic absorption spectrometer and flow cytometry. The results demonstrated that the antibacterial activity of prescription was due to the disruption of the cell membrane which caused the leakage of cytoplasmic contents.5、The influence of prescription on growth, nonspecific immunity, anti-oxidation, and disease resistance to A. hydrophila in crucian carp (Carassius auratus gibelio var. E’erqisi, Bloch) was assayed. The prescription was added in the basal diet for the crucian carp at 0, 0.5, 1.0 and 2.0 g per 100 g, feeding for eight weeks, the growth, nonspecific immune response and anti-oxidization enzymes activity of the fish were investigated. Then, the fish was infected with A. hydrophila and mortalities were recorded. The results showed that crucian carp supplemented with 1.0% and 2.0 % prescription had a significant increase in specific growth rate (SGR), serum total protein, lysozyme, acid phosphatase(ACP), alkaline phosphatase(ALP), catalase (CAT) and superoxide dismutase (SOD) activities (P < 0.05) of experimented fish feeding after 8 weeks. In addition, the feed conversion rate (FCR), hydroxyl free radical ( ?O H), malondialdehyde (MDA) contents and the mortality rate significantly decreased (P < 0.05) when crucian carp was fed with medium and high doses of prescription. It is concluded that prescription can act as natural antimicrobial and immunostimulants to prevent the pathogenic infection, enhance the immune response, and promote growth of the fish. |
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