EFFECT OF FIBROUS ROOT OF COPTIS CHINENSIS FRANCH AND BERBERINE ON THE NON-SPECIFIC IMMUNITY AND RESISTANCE AGAINST AEROMONAS HYDROPHILAINFECTION IN GRASS CARP (CTENOPHARYNGODON IDELLA)

MOU Shao-Xia, ZHOU Xia, PENG Yao-Zong and LI Xue-Gang



EFFECT OF FIBROUS ROOT OFFRANCH AND BERBERINE ON THE NON-SPECIFIC IMMUNITY AND RESISTANCE AGAINSTINFECTION IN GRASS CARP ()

MOU Shao-Xia, ZHOU Xia, PENG Yao-Zong and LI Xue-Gang

(Southwest University, School of Pharmaceutical Sciences, Chongqing 400715, China)

To determine the effects ofFibrous root ofFranch (FRC) and berberine (BBR) on non-specific immunity and disease resistance againstinfection in grass carp (), fish were fed with diets containing 1.00%, 0.50%, 0.25%, 0.10%, 0.00% of FRC powder and 0.05% of BBR for 28 days. Fish were challenged with28 days post feeding and survival rate () was recorded over 14 days post challenge. Nitroblue tetrazolium (NBT) activity, serum lysozyme activity, phagocytic activity and complement C3 level were studied to assess the immune response of fish. The results revealed that dietary FRC and BBR increase the NBT activity, serum lysozyme activity, phagocytic activity and complement C3 level in grass carp when compared to the model group (MD). Feed containing 0.50% FRC was the most effective with theof the fish significantly increased by 44% compared to MD. Theantibacterial activity of FRC and BBR were investigated with the minimum inhibitory concentration (MIC) method and showed strong inhibitory effects. The results indicated that FRC and BBR can be used as immunostimulants to enhance the non-specific immunity and disease resistance of grass carp against.

Fibrous root ofFranch(FRC); Berberine (BBR); Grass carp; Non-specific immunity;; Antibacterial activity; Survival rate

Fish culture is an important industry. The grass carp is one of the major varieties of freshwater fish cultured in China. Due to its intensive culture and the depravation of environmental, grass carp often encounters the high temperature, overcrowding and poor water quality[1], which increase the opportunity of bacterial infection for grass carp, consequently resul­ting in high mortality rate, especially during the summer months[2]. One of the most common and frequently encountered bacterial pathogen in grass carp is()[3], which ma­king grass carp suffering from haemorrhages in gills and vent, ulceration, abscesses, exophthalmia and abdominal distension. It is reported that infected fish pathogens decreased immunity, specifically for the non-specific immunity, including the decline in the activities of lysozyme, the ability of phagocytes and respiratory burst, and the level of immune factors[4]. Decreased immunity leads to death of fish. Therefore, the effective methods to control and prevent bacterial diseases are to strengthen antibacterial activity and the non-specific immunity of fish in aquaculture.

Although antibiotics are widely used to get rid of the infections, their constant application leads to bacterial resistance, consequently cause adverse impacts on human health[5]. Vaccination is an effective precaution for controlling fish diseases. However, vaccines are expensive for fish farmers and specific to particular pathogens[6]. Most important of all, there is no effective vaccine available for preventing pathogens includingdue to its complex antigenic structure[7]. Immunostimulant plants are recognized as ideal alternatives for preventing fish infectious diseases[8]. Many herbal immunostimulants as feed supplement have been reported to increase the survival rate of fish by enhancing the non-specific immune capability and disease resistance[9—11]. What is more, herbs are inexpensive, easy to prepare, no- toxic side effects, no drug residue and no drug resistance. Thus, there is an increasing interest in using herbs as immunostimulants in aquaculture.

s, the rhizome ofFranch, has been widely used in traditional Chinese medicine for treating diarrhea and gastrointestinal disorders via its obvious antibacterial activity. However, the application ofs in aquaculture will greatly increase the economical pressure of farmers due to its higher price. In the research for the comprehensive utilization ofFranch, our group found that the fibrous root (FRC), usually abandoned, contained about 1/3 of total alkaloids of, and harbored similar active ingredients, including berberine, coptisine, palmatine, jatrorrhizine and epiberberine[12]. Like, berberine (BBR) is the most abundant alkaloids in FRC which has been reported as antibacterial[13], hypolipidemmic[14], antidiabetic[15], cholesterol- lowering[16]and anti-inflammatory activities[17]. However, no research has been carried out concerning whether FRC and BBR can inhibitand increase the immunity of fish.

Thus, the purpose of the present study was carried out to evaluate the antibacterial activity againstand its possible immune mechanisms on improving survival rate of grass carp to explore cheap, effective and safe antibacterial agents.

1 Material and method

1.1 Chemical

FRC was collected from shizhu, Chongqing. BBR was purchased from Shanghai Sunny Biotech Co., Ltd (Shanghai, China).was obtained from the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). The standard of lysozyme was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Kits for analysis of the level of complement C3 was purchased from Shanghai Yuping Biotech Co., Ltd (Shanghai, China). Nitroblue tetrazolium (NBT) was purchased from Beijing Notlas Biotech Co., Ltd. (Beijing, China).

1.2 Experimental animal and preparation of herbal diet

Non-vaccinated and three-month old healthy grass carp with an average weight of (100.3±2.8) g were obtained from Sichuan aquatic school. The fish were acclimatized for 7 days in the recirculation system of the laboratory in glass tanks at (25±2)℃ under continuous aeration. The water in the tank was exchanged once in two days to maintain the water quality. The fish were fed basal diets at the rate of 3% of body weight twice a day.

Basal feed was purchased from Chongqing hope Feed Co., Ltd. (Chongqing, China). The fodder was grounded into a fine powder through a 100-mesh sieve and mixed with the experimental drugs contained either FRC (1.00%), or FRC (0.50%), or FRC (0.25%), or FRC (0.10%), or BBR (0.05%). The drug was added by method of step by step to the feed power, then both of them were thoroughly mixed and water was added to produce thick dough. The dough was pressed using a laboratory small feed machine and dried in a ventilated oven at 40℃. After drying, pellets were packed in polythene bags respectively according to the different concentrations of FRC and BBR, sealed and labeled.

1.3 Preparation for FRC extract and BBR stocking solution

FRC was dried naturally and ground into powder using an electric grinder. The extraction was done by following the method of Natarajan,.[18]with slight modifications. The FRC power was exhaustively extracted with sterile distilled water for 24h at room temperature. BBR was dissolved thoroughly in sterile distilled water to make stock solution. After centrifuged, the supernatant was filtered through a sterile 0.2 μm pore size filter for antibacterial experiments.

1.4 Antibacterial activity of FRC and BBR against

was cultured in broth at 28℃ for 24h. The cultures were centrifuged at 3000×for 15min. The supernatant fluid was removed and the bacterial pellet was suspended in phosphate buffered saline (PBS, pH 7.4). The final bacterial concentration was adjusted to 5×105cfu/mL.

The minimal inhibitory concentrations (MICs) of FRC extract and BBR were determined with a microdilution assay. The assay was performed by a modification that described by Guo,[19]. The MIC was determined as the lowest drug concentration that significantly inhibitedgrowth.

1.5 The effects of FRC and BBR on immunological parameters andexperiment on survival rate ()

Experimental design One hundred and sixty eight grass carps with an average weight of (100.3±2.8) g were equally and randomly allocated into two sections. One section was for therapeutic experiment, the other section was for prophylactic experiment. Then each section was randomly divided into seven groups including normal control group (NC), model group (MD), four FRC dose groups (1.00% FRC, 0.50% FRC, 0.25% FRC and 0.10% FRC) and one BBR dose group (0.05% BBR). During the period of all experiment, the fish in NC and MD were fed with basal diets. At the beginning of the therapeutic experiment, the fish of all groups except NC were injected with 0.2 mL of the suspension of. After that, fish were fed with corresponding diets at the rate of 3% of body weight twice a day for 14 days. The1were noted daily for 14 days. In prophylactic experiment, fish were fed with respectively diets twice a day for a period of 28 days. Ten fish from all groups except NC were sampled and blood was collected from the caudal vein on the 14th and 28th days for different immunological assays. After 28 days of feeding, twelve fish from all groups except NC were also injected withsuspension 0.2 mL. The2were recorded over 14 days post infection.

Calculation of survival rate(%) Survival rate (%)=(Number of surviving fishes after challenge) / (Number of fishes injected with bacteria)× 100

Blood collection and serum separation The blood samples were drawn from caudal vein of fish using 1.0 mL hypodermal syringe. Heparin was used as an anticoagulant. 800 μL of blood was collected into plastic Eppendorf tubes and shaked well in order to prevent clotting of blood. After collection, 200 μL whole blood was used for the analysis of respiratory burst activity and phagocytic activity. The remaining whole blood was centrifuged at 3000×for 20min to obtain serum for assaying lysozyme activity and complement C3 level.

NBT for respiratory burst assay NBT assay was detected using the method described by Secombes[20]. 100 μL of blood was placed into the wells of the 96-well plates and incubated at 37℃ for 1h to make adhesion of cells. Then the supernatant was removed and the wells were washed thrice with PBS. After washing, 100 μL of 0.2% NBT was added and the plate was then incubated for 1h. The cells were fixed with 100% methanol for 2—3min and washed three times with 70% methanol. The wells were air-dried and 120 μL of 2 N potassium hydroxide and 140 μL dimethyl sulphoxide were added to each well. The OD was recorded in an ELISA reader (Bio Tek Instruments, USA) at 630 nm.

Serum lysozyme activity Serum lysozyme activity was detected using the method described by Yeh,[21]. Briefly, 20 μL of serum was mixed well with 200 μL of 0.2 mg/mL(Bioengineering Institute, Nanjing, China) suspension in phosphate buffer sodium. The optical density was detected after 1min and 5min at 490 nm in an ELISA plate reader. A unit of lysozyme activity was defined as the amount of sample causing a reduction in absorbance of 0.001 per minute.

Phagocytic activity Phagocytic activity was modified from the methods described by Wang,.[22]. A sample (0.1 mL) of blood was placed in plastic Eppendorf tube, 0.1 mL of1×107cfu/mL (450= 0.5—0.6) suspended in phosphate buffered saline (pH 7.2) were added and mixed well. The bacteria-blood solution was incubated for 20min at room temperature. 2 μL of this solution was taken on to a clean glass slide and a smear was prepared. The smear was air-dried, then fixed with ethanol (95%) for 5min and air dried. The air-dried smear was stained with 7% Giemsa for 10min. Two smears were made from each fish. 100 phagocytes from each smear were observed under the light microscope and the numbers of phagocytizing cells were counted. Phagocytic activity equals the number of phagocytizing cells divided by the total number of phagocytes counted.

Complement C3 level The levels of complement C3 were measured in an ELISA plate reader using kit. The assay kit is specially designed for fish detection.

1.6 Statistical analysis

Results for each parameter measured were expressed as the mean±SE. The data were statistic­cally analyzed by statistical package SPSS version 16 in which data were subjected to one-way ANOVA and Duncan’s multiple range test (DMRT) was used to determine the significant differences between the means. Differences were considered statistically significant when<0.05.

2 Result

2.1 The effects of FRC extract and BBR on antibacterial activity against

The solution of FRC extract and BBR showed strong antagonistic activities against. The minimal inhibitory concentrations are 3.125 mg/mL and 39.062 mg/L, respectively.

2.2 The effects of FRC and BBR on SR of grass carp infected with

In therapeutic experiment, after infected with,feeding with the FRC and BBR supple­mented diet for 14 days led to an increase inof fish. In Fig. 1, all treated groups showed significantly (<0.05) higherwhen compared with MD. The highestwas recorded in the 0.50% FRC group followed by 1.00% FRC and 0.25% FRC groups. The NC group fish were no mortality.

In prophylactic experiment,of grass carp after challenge within different experimental groups is presented in Fig. 2. The grass carps fed with FRC and BBR supplemented diet showed significantly (<0.05) high resistance againstwhen compared to the MD. The 0.50% FRC treatment conferred the best protection againstinfection. The unchallenged fish registered no mortality.

2.3 The effects of FRC and BBR on NBT activity

The effect of FRC and BBR on NBT activity is shown in Fig. 3. On the 14th day, the NBT activity in all treated groups except 0.10% FRC and 0.05% BBR groups showed a statistically (<0.05) significant increase when compared to the MD. On the 28th day, the NBT activity in 0.50% FRC, 0.25% FRC and 0.05% BBR groups were significantly higher than that of the MD (<0.05). The highest NBT activity was found in 0.50% FRC group in all sampling days.

Data (mean ± SE; *<0.05) difference from the model group is indicated with asterisk

Data (mean ± SE; *<0.05) difference from the model group is indicated with asterisk

Data (mean ± SE; *<0.05) difference from the model group is indicated with asterisk

2.4 The effects of FRC and BBR on serum lysoz­yme activity

As shown in Fig. 4, the serum lysozyme activity was higher in all the treated groups than the MD on all the days tested. On the 14th day, the lysozyme activity in 0.25% FRC, 0.10% FRC and 0.05% BBR groups were almost similar to the MD (>0.05), but 1.00% FRC and 0.50% FRC groups showed a significantly (<0.05) higher lysozyme activity than the MD. On the 28th day, the 0.50% FRC, 0.25% FRC and 0.05% BBR groups showed a significantly (<0.05) higher serum lysozyme activity than the MD. The level of lysozyme activity in all administration groups except 1.00% FRC group showed an increasing trend from 14 to 28days. The highest serum lysozyme activity was found in 1.00% FRC and 0.50% FRC groups on the 14th and 28th days of the study respectively.

Data (mean ± SE; *<0.05) difference from the model group is indicated with asterisk

2.5 The effects of FRC and BBR on phagocytic activity

Fig. 5 indicates that the phagocytic activity (%) was elevated for the whole period of the experiment. The phagocytic activity showed an increasing trend in the treatment groups from 14 days to 28 days. However, significant differences (<0.05) in phagocytic activity were observed in 0.50% FRC and 0.05% BBR groups on the 14th day as compared to the MD. On the 28th day, all treated groups showed a significantly (<0.05) higher phagocytic activity than the MD. The highest phagocytic activity was observed in 0.50% FRC group in all the sampling periods.

2.6 The effects of FRC and BBR on complement C3 level

The complement C3 level was higher in all treated groups than the MD throughout the study period (Fig. 6). On the 14th day, only the 0.50% and 0.25% FRC groups showed a significantly (<0.05) higher com­plement C3 level than the MD. Though the complement C3 level in 0.10% FRC and 0.05% BBR groups were higher than the MD, the difference was not significant (>0.05) on the 28th day. The highest value of complement C3 level was found in 0.50% FRC group on the 14th and 28th days of the study and differed significantly (<0.05)compare to the MD. The complement C3 level showed an increasing trend in all the treatment groups during the whole experimental period.

Data (mean ± SE; *<0.05) difference from the model group is indicated with asterisk

Data (mean ± SE; *<0.05) difference from the model group is indicated with asterisk

3 Discussion

The bacterial infection disease give great threat to cultured fish and it may result in major losses in aquaculture industry. Antimicrobial treatment is the main method to control disease problem in aquaculture. However, in many countries, the use of antibiotics is facing tougher control in aquaculture industry. Herbs are receiving greater attention for the substitution of chemical antimicrobials. In the present study, the antibacterial activity of FRC extract and BBR were investigated against pathogenicand showed strong inhibitory activities. Their MICs were 3.125 mg/mL and 39.062 mg/L, respectively. The results indicated that FRC and BBR can be used as antimicrobial agents for aquaculture use. In this way, it will reduce the use of antibiotics in some extent.

In therapeutic experiment and prophylactic experiment, compared with the MD, the treated groups increased theof the fish infected with. The results of this study indicated that the FRC and BBR are beneficial for fish by conferring protection against infectious disease. The similar results of improvedwere reported in juvenile rockfish fed with diet containing aloe after infected with[23],fed with diet containing propolis and herba epimedii extracts after infected with[24], juvenile Wuchang bream fed with diet containing emodinafter infected with[25]andinjected with sodium alginate after challenged with[26].

Based only on theantibacterial activity, FRC and BBR are not supposed to show significantly higheragainst diseases caused by, so there should be some unknown mechanisms to support the successful resistance of bacterial diseases.It has been reported that the fish increase the resistance to bacterial pathogens through an increase in the level of lysozyme and complement as well as an enhancement of the phagocytic and respiratory burst activities[27, 28].

The respiratory burst activity of the neutrophils was measured by nitroblue tetrazolium (NBT) assay. As we all know that respiratory burst plays an important role in the immune system, fish neutrophils after activation are able to release reactive oxygen species (superoxide radical and hydrogen peroxide) during a period of intense oxygen consumption, called the respiratory burst. These reactive oxygen species are toxic for bacterial pathogens of fish[29]. In the present study, the NBT activities in all treated groups were higher than the MD. The similar effects on respiratory burst activity were previously reported in rainbow trout fed with cholesterol[30];fed with Kalopanax pictus[31];fed with ascorbic acid[32]andfed with levamisole[33].

Lysozyme activity has been considered to be a natural defensive factor against invasive pathogens in fish. Lysozyme also called muramidase or N-acetyl­muramide glycanohydrlase is a alkaline enzyme, arising from neutrophils and monocytes, and secreted into blood and mucus to play a bactericidal role[34]. It destroys the β-1, 4 glycosidic bond between N-acetyl­muramic acid and N-acetylglucosamine in cell wall, and results in the death of pathogenic bacteria. In this study, the serum lysozyme activities in all treated groups were much higher than the MD. There have been reports on the increase in serum lysozyme acti­vity when fed with Mentha piperita[35]in; Eclipta alba[36]inand Liquorice extract[37]in.

The basic function of phagocytic cells is to identify pathogens and limit the spread and growth of pathogens. Their phagocytic activity is considered as an important defense mechanism against pathogenic bacteria[38]. In the present study, the phagocytic acti­vity was increased in the treatment groups fed with BBR and FRC at various doses. This elevated activity was maintained during the whole experiment. Previous studies have reported that administration other immunostimulants induced an enhancement of the phagocytic activity in fish[7, 9, 24, 39].

The complement system is composed of more than 35 soluble plasma proteins that plays an essential role in clearing pathogens and its activated products can contribute significantly to the immune response[40]. Complement component C3 is the most important and abundant protein in the complement system. The present study indicates that certain dose of FRC and BBR could increase the level of complement component C3 after 14 or 28 days of administration. Oral application pachymaran has been reported to elevate the level of complement component C3 in[41].

4 Conclusion

In conclusion, FRC and BBR increase theof grass carp by improving non-specific immunity and antibacterial activity against.FRC can influence the non-specific immune response in grass carp that could be due to the chemical entity especially BBR. The effect of other alkaloids through oral administration should be investigated and determined their molecular mechanism in further work.

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黄连须和小檗碱对草鱼非特异性免疫系统的影响及对嗜水气单胞菌感染的抵抗作用

牟绍霞 周 霞 彭耀宗 李学刚

(西南大学药学院, 重庆 400715)

为了评估黄连须(FRC)和小檗碱(BBR)对草鱼()的非特异性免疫及对抵抗嗜水气单胞菌感染的影响, 鱼喂食含1.00%、0.50%、0.25%, 0.10%, 0.00%的FRC和0.05%BBR的饲料28d。喂养28d后, 鱼注射嗜水气单胞菌, 记录感染14d后的存活率。检测NBT活性、血清溶菌酶活性、吞噬活性和补体C3水平来评价药物对草鱼的免疫反应。结果表明, 与模型组相比, 喂养饲料含有FRC和BBR的各组提高NBT活性、血清溶菌酶活性、吞噬活性和草鱼补体C3水平。喂食0.50%FRC组感染细菌后的存活率最高, 与模型组比较增加44%。FRC和BBR的体外抗菌活性采用最小抑菌浓度的方法进行了研究, 并显示出较强的抑制作用。结果表明, FRC和BBR可以用作免疫刺激剂以提高草鱼非特异性免疫和对嗜水气单胞菌引起疾病的抵抗。

黄连须; 小檗碱; 草鱼; 非特异性免疫; 嗜水气单胞菌; 抗菌活性; 存活率

10.7541/2015.35

S968.1 Document code: A Article ID: 1000-3207(2015)02-0267-08

date:2014-10-21; Accepted date:2014-12-22

The National Key Technology R&D Program (2011BAI13B02-1); Specialized Research Fund for the Doctoral Program of Higher Education (20130182110023); The high-end engineering and technical personnel training plan of Chongqing (2013-2016); The County-University cooperation Innovation funds of Southwest University (Sz201401, Sz201302)

Li Xue-Gang, E-mail: xuegangli@swu.edu.cn

MouShao-Xia (1986—), female, born in Yantai, Shandong; Master student: major in basic Pharmacy. E-mail: yiqifei1236@163.com