Application of raspberry ketone glucoside in preparation of preparation for preventing and treating monocolonic insect

Abstract

The invention relates to the technical field of biological medicine, in particular to application of raspberry ketone glucoside in preparation of a preparation for preventing and treating monocolonic insect. Experiments and tests show that the raspberry ketone glucoside shows remarkable killing activity on fish monozoiasis, and meanwhile, the raspberry ketone glucoside also keeps good safety on fishes. After short-time immersion bath administration, the raspberry ketone glucoside can weaken the movement of the monoagonism and influence the generation of ATP (adenosine triphosphate) of the monoagonism. The raspberry ketone glucoside disclosed by the invention has a relatively good prevention and control effect on fish mononimus, and more solutions can be provided for disease prevention and control of aquaculture by developing a preparation based on the raspberry ketone glucoside.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to application of rubusoside in preparation of monogenea prevention and treatment preparation. BACKGROUND

[0002] Monogenea is a kind of ectoparasite that causes the most serious damage in aquaculture, and more than 5000 species have been recorded worldwide. Among them, Gyrodactylus and Dactylogyrus are of great concern due to their wide host spectrum and high pathogenicity. This kind of monogenea causes mechanical damage to the host epidermis, gills and fins through the chitin hook-like attachment organ, and causes anemia, inflammation and immunosuppression by feeding on host tissue fluid, blood and mucus. Under high-density parasitic conditions, it can cause fish growth retardation, secondary bacterial or fungal infection, and even large-scale death. For example, Gyrodactylus kobayashii, as a typical species of Gyrodactylus, mainly invades Cyprinidae fish (such as goldfish, silver carp, etc.), and the gill ulceration and abnormal secretion of body mucus caused by Gyrodactylus disease are characterized by an infection rate of more than 90%, which causes the extension of the breeding cycle and significant economic losses. The Norwegian Atlantic salmon fry population has decreased by 50% due to Gyrodactylus infection, and similar epidemics have also frequently occurred in silver carp and grass carp in freshwater aquaculture in China.

[0003] Rubusoside has a CAS number of 38963-94-9, a molecular formula of C 16 H 22 O7, and a chemical formula as shown below:

[0004] Rubusoside is a glycoside natural product extracted from Rubus idaeus in Rosaceae, and has excellent biodegradability and low ecological toxicity. Existing researches mainly focus on its antioxidant, anti-inflammatory and hypoglycemic activities, and no report has been found on its killing effect on monogenea. SUMMARY

[0005] The present application aims to provide an environmentally friendly natural product capable of preparing a prevention and treatment preparation for the prevention and control of fish monogenea disease in aquaculture, and provides an innovative solution to the ecological pollution and drug residue problems caused by chemical insecticides. In view of this, the present application provides the application of rubusoside in the preparation of monogenea prevention and treatment preparation, which can kill pathogenic parasites such as Gyrodactylus kobayashii, and meet the urgent needs of the aquaculture industry for high-efficiency and environmentally friendly prevention and treatment technology.

[0006] In one aspect, the present application relates to the application of rubusoside in the preparation of monogenea prevention and treatment preparation.

[0007] Further, in the application provided by the present application, the raspberry glycoside acts on the epidermis of the monogenean.

[0008] Further, in the application provided by the present application, the raspberry glycoside inhibits the generation of ATP in the epidermis of the monogenean.

[0009] Further, in the application provided by the present application, the monogenean includes Clonorchis sinensis and Prosthogonimus.

[0010] Further, in the application provided by the present application, the raspberry glycoside inhibits the pathogenicity of the monogenean.

[0011] Further, in the application provided by the present application, the pathogenicity includes the ability of the monogenean to infect fish.

[0012] In another aspect, the present application relates to the use of raspberry glycoside in the preparation of a drug for preventing and treating fish monogenean disease.

[0013] Further, in the application provided by the present application, the raspberry glycoside inhibits the generation of ATP in the epidermis of the monogenean, and the raspberry glycoside inhibits the ability of the monogenean to infect fish.

[0014] Further, in the application provided by the present application, the fish monogenean disease includes clonorchiasis and prosthogonimus disease.

[0015] Further, in the application provided by the present application, the use method of the drug for preventing and treating fish monogenean disease is external use.

[0016] Compared with the prior art, the technical scheme provided by the present application at least has the following beneficial effects or advantages:

[0017] The application provides a natural product-based raspberry ketone for preparing a preparation for preventing and treating fish monogenea, in particular tridacnids infection. The finding provides more choices for the field of aquaculture disease prevention. Experimental data show that the raspberry ketone has a significant killing effect on monogenea, especially on small Lin tridacnids and intermediate ringworms, and a significant reduction in the head activity frequency of the worms can be observed after 0.5 hours of contact with the drug, which shows the characteristics of rapid effect. In addition, the application first applies the raspberry ketone to the field of water production monogenea prevention, which fills the gap in the technical field. Compared with conventional chemical insecticides, the raspberry ketone as a natural product has excellent biodegradability and does not produce persistent residues in the water environment, and can significantly reduce the impact on non-target organisms, which shows good environmental friendly characteristics. This new prevention strategy based on plant secondary metabolites effectively solves the problem of damage to the water ecosystem caused by traditional chemical agents. More importantly, in terms of application value, the application has significant technical advantages: on the one hand, the raspberry ketone raw material is derived from natural plants, and the preparation process is simple and low in cost, which is conducive to large-scale production and application; on the other hand, by specifically killing monogenea, the survival rate of farmed fish can be significantly improved, which can create considerable economic benefits for the aquaculture industry. It is particularly worth emphasizing that the application first reveals the specific mechanism of action of raspberry ketone on the neuromuscular system of small Lin tridacnids through systematic research, which provides an important theoretical basis for the development of environmentally friendly water production insecticides. Therefore, as an active ingredient that can effectively act on monogenea, especially small Lin tridacnids and intermediate ringworms, the raspberry ketone has great potential to be developed into a drug for preventing and treating monogenea. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description only show some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor.

[0019] Figure 1 It is a line graph of in vitro deworming activity data of raspberry ketone on small Lin tridacnids.

[0020] Figure 2 It is a columnar data schematic diagram of in vivo deworming activity of raspberry ketone on small Lin tridacnids.

[0021] Figure 3 It is a data schematic diagram of the influence of raspberry ketone and other anti-worm drugs on the head stretching frequency of small Lin tridacnids. The dotted line represents the average number; *** represents that p<0.001 compared with the control group.

[0022] Figure 4Fig. 1 is a diagram of the data of the ATP content changes in the in vivo and in vitro of Trichuris trichiura treated by raspberry ketone. Wherein, Fig. 1A is the effect of the in vivo treatment of raspberry ketone on the ATP content of Trichuris trichiura; Fig. 1B is the effect of the in vitro treatment of raspberry ketone on the ATP content of Trichuris trichiura; the dotted line represents the average; *** represents p < 0.001 compared with the control group.

[0023] Figure 5 Fig. 2 is a diagram of the data of the in vitro anthelmintic activity of raspberry ketone on Trichuris trichiura.

[0024] Figure 6 Fig. 3 is a diagram of the data of the in vivo anthelmintic activity of raspberry ketone on Trichuris trichiura.

[0025] Figure 7 Fig. 4 is a diagram of the data of the ATP content changes in the in vivo and in vitro of Trichuris trichiura treated by raspberry ketone. Wherein, Fig. 4A is the effect of the in vivo treatment of raspberry ketone on the ATP content of Trichuris trichiura; Fig. 4B is the effect of the in vitro treatment of raspberry ketone on the ATP content of Trichuris trichiura; the dotted line represents the average; *** represents p < 0.001 compared with the control group.

[0026] Figure 8 Fig. 5 is a diagram of the anti-parasitic efficiency of raspberry ketone in the horizontal transmission of Trichuris trichiura. Wherein, Fig. 5A is a workflow diagram of the experimental design; Fig. 5B is a diagram of the anti-parasitic efficiency of the donor and recipient goldfish, the numerical value is represented by the average value ± standard deviation, p < 0.05. DETAILED DESCRIPTION

[0027] Hereinafter, the technical solutions of the present application will be described in conjunction with the examples, but the present application is not limited to the following examples. The experimental methods and detection methods described in each example are all conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified. The % in the following examples is the mass percentage unless otherwise specified. The ratio in the following examples is the mass ratio unless otherwise specified.

[0028] The compound raspberry ketone used in the examples was purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd., with a purity of greater than or equal to 98%.

[0029] The preparation method of raspberry ketone solution in the examples is as follows: 10 mg of raspberry ketone is weighed and placed in a 1.5 mL centrifuge tube, then 1 mL of DMSO is added for dissolution, to obtain a raspberry ketone solution with a concentration of 10 mg / mL.

[0030] Goldfish used in the examples were 5.7±0.3 cm in length and purchased from a local fish farm in Xi'an, China. After purchase, all fish were kept in an aerated recirculating tank (water temperature 21.0±0.3℃, pH 6.5-7.0) and fed with commercial feed at 3% of body weight once a day. Two weeks later, all monogenean worms were removed by a 12-hour bath in 2.5 mg / L piperazine. Subsequently, the monogenean-free goldfish were acclimated to laboratory conditions for one week to obtain test fish.

[0031] The test fish were introduced into donor goldfish with T. aculeatum to obtain test fish containing T. aculeatum;

[0032] The test fish were introduced into donor goldfish with T. aculeatum to obtain test fish containing T. aculeatum;

[0033] In Examples 1-4, a static water test method was used with tap water supplemented with aeration, pH 7.2, and the water temperature was controlled at 21.0±0.3℃, and the dissolved oxygen in the water was maintained at more than 5 mg / L throughout the test.

[0034] Example 1

[0035] This example was intended to demonstrate the safe concentration range of raspberry ketone for fish, as follows in detail.

[0036] Five fish tanks were each filled with 10 L of tap water supplemented with aeration, and then different volumes of raspberry ketone solution were added to prepare raspberry ketone solutions with mass concentrations of 7 mg / L, 7.5 mg / L, 8 mg / L, 8.5 mg / L, and 9 mg / L. Ten randomly healthy fish were placed in each fish tank, and each treatment was repeated three times, for 96 hours. During this period, no feed was given, and the condition of the goldfish was observed and recorded for poisoning or death, and dead fish were removed in time to avoid pollution of the water quality. The criteria for poisoning were head floating, bottom sinking, lateral swimming, or delayed response to the striking reaction. The criteria for death were cessation of gill cover activity or no stress response to a glass rod tapping the caudal peduncle. The time of death of the goldfish was recorded in detail, and the mortality rate of each treatment group was calculated. The median lethal concentration (LC 50 ) and 95% confidence interval of raspberry ketone were determined by Probit analysis, and the results are shown in Table 1.

[0037] Table 1: Death of goldfish in each concentration of raspberry ketone within 12 h, 24 h, 48 h, and 96 h

[0038]

[0039] As shown in Table 1, all fish that died died within 24 hours of the start of the experiment. Treatment with 7 mg / L raspberry glycoside had no significant effect on the parasite-free fish; however, when the raspberry glycoside concentration was increased to 9 mg / L, all parasite-free fish died within 24 hours. The median lethal concentrations (LC50) at 24, 48, 72, and 96 hours are also shown. 50 The value was the same, at 8.03 mg / L (95% confidence interval: 7.96–8.10 mg / L).

[0040] Example 2

[0041] This embodiment aims to illustrate the anthelmintic effect of raspberry glycosides on Gyrodactylus kobayii. The specific experiment is as follows:

[0042] 1. In vitro anthelmintic activity test of raspberry glycosides.

[0043] Fish severely parasitized with *Gyrodactylus kobayashi* were selected for testing and anesthetized with an overdose of MS-222. The caudal fins containing *Gyrodactylus kobayashi* were then cut into small pieces. Each piece containing at least 50 *Gyrodactylus kobayashi* was then transferred using forceps to glass petri dishes containing rhizoside solutions at concentrations of 3 mg / L, 4 mg / L, and 5 mg / L. Additionally, 0.2 mg / L mebendazole was used as a positive control, and 0.2% DMSO was used as a negative control to further evaluate the anthelmintic activity of rhizoside. The number of surviving monogenean trematodes in all groups was recorded using a stereomicroscope at 20-minute intervals from 0 to 120 minutes. The anthelmintic effect of each compound was calculated as follows: AE = (B - T) / B × 100%, where B is the number of *Gyrodactylus kobayashi* on the caudal fin before treatment, and T is the number of surviving monogenean trematodes after treatment.

[0044] The results are as follows Figure 1 As shown, the 4 mg / L and 5 mg / L raspberry glycoside treatment groups achieved 100% anthelmintic efficacy after 100 minutes and 60 minutes of bath treatment, respectively. When the raspberry glycoside solution concentration was reduced to 3 mg / L, the anthelmintic efficacy decreased to 82.17% after 100 minutes of bath treatment. In contrast, the mebendazole positive control killed only 4.84% of monogenean trematodes after 120 minutes of bath treatment, while the 0.2% dimethyl sulfoxide negative control and blank control groups showed negligible worm mortality.

[0045] 2. In vivo anthelmintic activity test of raspberry glycoside.

[0046] Seven 3L plastic tanks were selected, and 2L of fully aerated tap water was added to each of the plastic tanks. Twenty-one test fish containing T. aculeatum (T. aculeatum density of 40-200 per fish) in the infection tank were randomly allocated to the seven plastic tanks (three test fish containing T. aculeatum in each plastic tank). Thereafter, different volumes of raspberry solution were added according to the results of the pre-test to maintain a final concentration of 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, and 6 mg / L, and 0.2% DMSO was designed as a negative control group to eliminate the effect of the solvent on T. aculeatum. Each concentration was repeated three times, and the number of surviving T. aculeatum on the caudal fin of goldfish before and after treatment at 0, 24 hours, and 48 hours was counted under a stereomicroscope, and then the anthelmintic effect of the raspberry solution was calculated. In short, the anthelmintic effect of each solution was calculated according to the following formula: E = (N-NS) / N x 100% (for N>NS), E = 0 (for N≤NS). E is the anthelmintic effect, N is the number of T. aculeatum on the caudal fin of goldfish before treatment, and NS is the number of surviving T. aculeatum after treatment.

[0047] The results, as shown in Figure 2 , 0.2% DMSO did not kill T. aculeatum after 48 hours. In the selected concentration range, the inhibition rate of T. aculeatum was positively correlated with the concentration of raspberry. The half-inhibition concentration (EC 50 ) and 95% confidence interval (CI) values of raspberry were 2.67 (2.48-2.85) and 2.11 (1.91-2.30) mg / L, respectively, after 24 hours and 48 hours of medicated bath, and the 90% inhibition concentration (EC 90 ) and 95% confidence interval (CI) values were 4.48 (4.22-4.80) and 3.83 (3.58-4.14) mg / L, respectively, and the mortality rates of T. aculeatum treated with 5 mg / L and 6 mg / L of raspberry for 24 hours were 94.41% and 100%, respectively.

[0048] This example further illustrates the therapeutic index of the raspberry solution, and the specific data are shown in Table 2:

[0049] Table 2: Therapeutic index of the raspberry solution

[0050]

[0051] The therapeutic index (TI) is an important indicator for evaluating the efficacy and safety of a solution. In this study, the therapeutic index (TI) of the raspberry solution for goldfish was 3.01 and 3.81, respectively, after 24 hours and 48 hours of treatment. These results indicate that the safety of the raspberry solution for goldfish increases with time.

[0052] Example 3

[0053] This example is intended to illustrate the effect of raspberry solution on the activity of C. sinensis, in detail as follows:

[0054] The fish infected with C. sinensis at moderate intensity were selected for this experiment and treated in the water tank containing 5 mg / L raspberry solution. Commonly used anti-monogene solution was selected as positive control, including 0.2 mg / L of mebendazole and 0.5 mg / L of rotenone, and a negative control group containing 0.2% DMSO was also set up. All groups were repeated three times. After 0.5 hours of treatment with raspberry solution, the symptoms of C. sinensis before and after treatment with the solution were observed, and 30 C. sinensis were randomly observed in each group, and the head contraction frequency of each group of monogene was counted within 30 seconds.

[0055] The results are shown in Figure 3 Table 1. The head movement frequency of C. sinensis significantly decreased after 0.5 hours of contact with raspberry solution. The head contraction frequency of monogene in the blank control group, i.e. 0.2% DMSO negative control group and 0.2 mg / L mebendazole positive control group, was 22 times, 21.53 times and 20.87 times within 30 seconds, respectively, and the difference was not significant (p>0.05), indicating that 0.2% DMSO and 0.2 mg / L mebendazole had no significant effect on the activity of monogene. However, after 0.5 hours of treatment with 5 mg / L raspberry, it was observed that most of the monogene stopped moving and contracting, and the head movement frequency was only 0.4 times (every 30 seconds), which had a significant difference compared with the control group (p<0.001). The effect of rotenone on the activity of monogene was similar to that of raspberry, and the head movement frequency was only 0.67 times (every 30 seconds).

[0056] Example 4

[0057] This example is intended to illustrate the effect of raspberry solution on the ATP level of C. sinensis, in detail as follows:

[0058] The ATP level of monogene after treatment with raspberry was detected by using a commercial kit, and the results are shown in Figure 4 Table 2. The in vivo bath test results showed that, similar to the rotenone positive control group, after 0.5 hours of bath treatment with raspberry solution, the ATP content of monogene decreased sharply from 1.91 nmol / mg protein in the control group to 0.5 nmol / mg protein (2.67 mg / L raspberry treatment group) and 0.32 nmol / mg protein (2.67 mg / L raspberry treatment group) (p<0.001). The ATP content of monogene decreased with the increase of solution concentration. Similar phenomenon also occurred in the in vitro bath test group. The above results showed that raspberry can inhibit the production of ATP of C. sinensis.

[0059] Example 5

[0060] This example is intended to illustrate the anthelmintic effect of raspberry ketone on the intermediate host of D. intermedius. The specific test is as follows:

[0061] 1. In vitro anthelmintic activity test of raspberry ketone

[0062] The gills with D. intermedius from the test fish containing D. intermedius were taken and placed in a 12-well plate containing 2.0 mL water (from the filtered water tank). Two different concentrations of raspberry ketone, 1.29 mg / L and 2.58 mg / L, were added to the wells, while a negative control group without raspberry ketone and a positive control group containing 0.5 mg / L rotenone were set up, with each treatment repeated three times. After 0.5 hours of drug bath, the parasites were collected, lysed with 200 μL of pre-cooled lysis buffer, and then centrifuged for 6 minutes. Subsequently, 20 μL of each supernatant and 100 μL of ATP diluent were added to a 96-well plate, which was detected using an enzyme label instrument (Synergy2, BioTek, USA), an ATP standard curve was drawn, and the ATP concentration was calculated. The protein concentration of each group was also determined and used to calibrate the ATP level in the parasites.

[0063] The results are shown in Figure 5 mg / L and 4 mg / L raspberry ketone treatment groups reached 100% anthelmintic effect after 80 minutes and 40 minutes of drug bath, respectively. When the concentration of raspberry ketone was reduced to 1 mg / L, the anthelmintic effect was 91.09% after 100 minutes of drug bath. The positive control group of mebendazole killed only 6.24% of the parasites after 120 minutes of drug bath, while the 0.2% dimethyl sulfoxide negative control group and the blank control group showed negligible mortality of the parasites.

[0064] 2. In vivo anthelmintic activity test of raspberry ketone

[0065] The test fish containing D. intermedius were divided into five water tanks (3L, three test fish containing D. intermedius in each water tank), and 2L of fully aerated tap water was added. Two different concentrations of raspberry ketone, 1.29 mg / L and 2.58 mg / L, were used for immersion for 0.5 hours, while a negative control group without raspberry ketone and a positive control group containing 0.5 mg / L rotenone were set up. The subsequent treatment method was the same as above (in vitro drug bath treatment method), with each treatment repeated three times.

[0066] The in vivo anthelmintic activity of raspberry ketone on D. intermedius is shown in Figure 6 The 0.2% DMSO did not kill D. intermedius after 48 hours. Within the selected concentration range, the inhibition rate of D. intermedius was positively correlated with the concentration of raspberry ketone. The half-inhibitory concentration (EC 50The 95% confidence interval (CI) values ​​were 1.29 (1.01–1.53) and 1.02 (0.90–1.12) mg / L, respectively, and the 90% inhibitory concentration (EC50) was... 90 The 95% confidence interval (CI) values ​​were 2.48 (2.14–3.09) and 1.99 (1.84–2.20) mg / L, respectively, and the mortality rates of the insects after 24 h of treatment with 3 mg / L and 4 mg / L raspberry glycoside were 96.17% and 99.24%, respectively.

[0067] This embodiment further illustrates the therapeutic index of raspberry glycosides against Dactylogyrus intermedia, as detailed in Table 3.

[0068] Table 3: Therapeutic index of raspberry glycosides against Dactylogyrus intermedia

[0069]

[0070] Example 6

[0071] This example aims to illustrate the effect of raspberry glycosides on the ATP levels of Dactylogyrus intermedia.

[0072] The ATP levels of Gyrodactylus worms after treatment with raspberry glycosides were detected using a commercial kit, and the results are as follows: Figure 7 As shown in Figure A, the in vivo bath test results indicate that, similar to the rotenone positive control group, after 0.5 hours of in vivo bath treatment with raspberry glycoside, the ATP content of *Dactylogyrus intermedia* decreased sharply, from 1.02 nmol / mg protein in the control group to 0.42 nmol / mg protein (1.29 mg / L raspberry glycoside treatment group) and 0.18 nmol / mg protein (2.58 mg / L raspberry glycoside treatment group). The ATP content of *Dactylogyrus intermedia* decreased with increasing drug concentration. A similar phenomenon occurred in the in vitro bath test group, as shown in Figure B. These results indicate that raspberry glycoside can inhibit ATP production in *Dactylogyrus intermedia*.

[0073] Figure 8 Figure A illustrates the experimental procedure for the effect of raspberry glycosides on the horizontal transmission of Dactylogyrus intermedia. Figure 8 B in the study showed that rhizoside significantly inhibited the infection of Dactylogyrus intermedia in recipient fish after contact with infected donor goldfish, indicating that rhizoside can effectively reduce the horizontal transmission of Dactylogyrus intermedia.

[0074] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. Use of raspberry ketone in the preparation of a preparation for preventing and treating monogenea.

2. Use according to claim 1, characterized in that, Raspberry ketone acts on the epidermis of monogenea.

3. Use according to claim 2, characterized in that, Raspberry ketone inhibits the generation of ATP in the epidermis of monogenea.

4. Use according to claim 1, characterized in that, The monogenea includes Tridacna sp. and Intermediary ring worm.

5. The use according to claim 1, characterized in that, Raspberry ketone inhibits the pathogenicity of monogenea.

6. Use according to claim 5, characterized in that, The pathogenicity includes the ability of monogenea to infect fish.

7. Use of raspberry ketone in the preparation of a drug for preventing and treating fish monogenea disease.

8. Use according to claim 7, characterized in that, The raspberry ketone inhibits the generation of ATP in the epidermis of monogenea, and the raspberry ketone inhibits the ability of monogenea to infect fish.

9. Use according to claim 7, characterized in that, The fish monogenea disease includes Tridacna sp. disease and Intermediary ring worm disease.

10. Use according to claim 7, characterized in that, The method for using the drug for preventing and treating fish monogenea disease is external use.

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