Application of angelica sinensis extract in medicine preparation

Abstract

The invention relates to the technical field of biological medicines, in particular to application of an angelica sinensis extract in medicine preparation. The medicine is used for resisting the white spot syndrome virus, can be used for preventing and / or treating the white spot syndrome of the aquatic animals, particularly has an excellent antiviral effect on crustaceans in the aquatic animals, can reduce the loss of the crustaceans in the aquatic animals due to the virus, and provides a powerful guarantee for the development of the aquaculture industry.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to application of angelica extract in preparing medicines. Background Art

[0002] Angelica extract is a kind of traditional Chinese medicine widely found in nature. It has many unique biological effects, such as promoting hematopoiesis, anticoagulation, lowering blood pressure, antiviral, anti-inflammatory and analgesic, anti-oxidant, anti-tumor, protecting liver and kidney, etc. Moreover, it is distributed in both north and south my country, with sufficient raw materials and low extraction technology cost. At present, the research on Angelica extract has not involved the antiviral activity of aquatic animals.

[0003] White Spot Syndrome is a major infectious disease of aquatic animals caused by White Spot Syndrome Virus (WSSV). It has been clearly classified as a Category II animal disease in my country and is also an epidemic disease affecting crustaceans worldwide. In view of the wide host range and high lethality of WSSV, the World Organization for Animal Health has included it in the list of diseases since 1997.

[0004] In recent years, in order to effectively respond to the challenges of aquatic biological viruses, a series of environmentally friendly preparations and drugs have emerged and have been widely used in practice, including immunopotentiators, Chinese herbal extracts, probiotics, and aquatic vaccines. As a country with abundant Chinese herbal medicine resources, my country has occupied an important position in the development of medicine since ancient times. Many Chinese herbal medicines are rich in natural active ingredients such as organic acids, flavonoids, terpenes, saponins, and alkaloids. These ingredients not only have significant effects of antiviral, enhancing the body's immunity and anti-stress ability, but also can synergistically act on pathogens through multiple pathways and targets, thereby achieving the effect of synergistic inhibition and even elimination of pathogens. These new preparations and drugs have shown unique advantages with their high safety, low residue, and low resistance to drug resistance.

[0005] Therefore, in-depth innovative research on natural antiviral active ingredients is not only a positive response to the current urgent needs of the aquaculture industry, but also an important way to ensure the safety of aquatic products and achieve an environmentally friendly farming model. It has far-reaching and significant significance. Summary of the invention

[0006] The inventors of the present application have demonstrated the effectiveness of Angelica sinensis extract in resisting white spot syndrome virus through animal experiments.

[0007] Therefore, the present invention provides the use of the angelica extract in preparing a medicine, wherein the medicine is used for resisting white spot syndrome virus.

[0008] In certain embodiments, the medicament comprises an angelica extract and a diluent.

[0009] In certain embodiments, the diluent is DMSO.

[0010] In certain embodiments, the mass concentration of the Angelica extract in the medicament is 60 mg / kg to 120 mg / kg.

[0011] In certain embodiments, the angelica extract is obtained by extracting angelica with an alcohol extraction agent;

[0012] The method for extracting the angelica extract comprises: providing dried angelica, crushing it and then extracting it with an alcohol extraction reagent; optionally, filtering the obtained extract and concentrating it to obtain the angelica extract.

[0013] In certain embodiments, the alcoholic extraction agent is methanol or ethanol.

[0014] In certain embodiments, the ratio between the mass of Angelica sinensis and the volume of the solvent is 10 g:500 ml to 50 g:500 ml.

[0015] In certain embodiments, the medicament further comprises a pharmaceutically acceptable carrier and / or excipient;

[0016] In certain embodiments, the drug is in a form selected from a pill, a powder, a capsule, a tablet (eg, an effervescent tablet), a film-coated tablet, a granule, a liquid, or a suppository.

[0017] In certain embodiments, the medicament is used for preventing and / or treating white spot syndrome in aquatic animals.

[0018] In certain embodiments, the aquatic animal is a crustacean.

[0019] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0020] (1) The present invention has proved that the angelica extract has excellent antiviral efficacy through animal virus determination, and has excellent therapeutic and preventive effects on white spot syndrome virus. This will effectively reduce the losses suffered by crustaceans in aquaculture due to the virus and provide a strong guarantee for the stable development of the aquaculture industry.

[0021] (2) The angelica extract of the present invention is a natural active ingredient with the characteristics of low toxicity, low residue, low cost, environmental protection and safety. With its biological activity, it can effectively fight against the white spot syndrome virus that harms crustacean aquaculture, which provides a new way to develop and research green aquaculture biological agents, and also expands the application field of angelica extract. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 It is a schematic diagram of the process of preparing the angelica extract provided by the present invention;

[0024] Figure 2 It is the absolute quantitative standard curve diagram of the present invention;

[0025] Figure 3 This is a graph showing the inhibitory effect of different concentrations of the Angelica extract of the present invention on WSSV;

[0026] Figure 4 This is a graph of the cumulative survival rate data of Procambarus clarkii under different treatments using the Kaplan-Meier method. DETAILED DESCRIPTION

[0027] The technical scheme of the present invention is described below in conjunction with the embodiments, but the present invention is not limited to the following embodiments. The experimental methods and detection methods described in each embodiment are conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0028] White spot syndrome virus strain: provided by the Aquatic Disease Laboratory of Northwest Agriculture and Forestry University;

[0029] Angelica: provided by Shaanxi Traditional Chinese Medicine Store;

[0030] The experimental animals used Procambarus clarkii were purchased from the Xianyang Aquarium Market in Shaanxi Province.

[0031] Preparation of the drug to be tested: Accurately weigh the angelica extract, prepare it into a standard solution with a concentration of 200 mg / mL with chromatography-grade DMSO, then dilute it with DMSO to prepare the drug solution of different concentrations to be tested, and transfer it into a brown chromatography bottle for later use.

[0032] Example 1

[0033] This embodiment provides a method for preparing an angelica extract;

[0034] 50 g of angelica sinensis was washed and dried in the shade. The dried angelica sinensis was fully crushed with a high-speed crusher, and the crushed angelica sinensis was filtered through a 60-mesh screen to obtain angelica sinensis powder. The angelica sinensis powder was placed in an electric constant temperature blast drying oven at 56°C to dry until the weight was stable, and then placed in a distillation flask. 500 ml of analytical grade pure methanol was added to the distillation flask, and then ultrasonic reflux extraction was performed at 68°C for 4 hours. This step was repeated three times to obtain a liquid to be filtered; the liquid to be filtered was filtered with filter paper to obtain a clear liquid, and the clear liquid was placed in a rotary vacuum evaporator at 68°C for evaporation and concentration. The concentrated liquid was placed in an electric constant temperature blast drying oven at 60°C to dry until the weight no longer changed, to obtain an angelica sinensis extract.

[0035] The safe concentration of the Angelica sinensis extract provided by the present invention and the determination of the anti-white spot syndrome virus activity of the Angelica sinensis extract are described below through Examples 2 to 4.

[0036] Example 2

[0037] This example provides a safety evaluation of angelica extract, and the specific scheme is as follows:

[0038] Healthy Procambarus clarkii were randomly selected and divided into groups, with 12 in each group, and placed in a plastic box with a water temperature of 24-27℃ for experimental treatment. The experiment set up a blank control group and a drug treatment group. The blank control group was only injected with TM (100mMTris-HCl, 10mMMgCl2, pH7.5) buffer, while the drug treatment group was injected with different concentrations of Angelica extract. The test concentrations of Angelica extract were: 200mg / kg, 180mg / kg, 160mg / kg, 140mg / kg, 120mg / kg, 100mg / kg, 80mg / kg, and 60mg / kg. The drug injection volume was 100μL / claw. After drug injection, the survival status of the shrimp was observed and recorded for 72 consecutive hours.

[0039] The safety evaluation results showed that within the tested time and concentration range (60-120 mg / kg), the angelica extract was safe for Procambarus clarkii, and the crayfish did not show any discomfort symptoms; within the tested time and concentration range (140-200 mg / kg), 2-9 crayfish died in each group. Therefore, it was determined that the safe concentration of angelica extract was higher than 120 mg / kg and should not be higher than 140 mg / kg. In subsequent experiments, angelica extract concentrations lower than 120 mg / kg were used to test Procambarus clarkii.

[0040] Example 3

[0041] This example tests the antiviral activity of the angelica extract of the present invention, and the specific scheme is as follows:

[0042] Healthy crayfish were randomly selected and divided into three groups (WSSV treatment group, Angelica extract and WSSV co-treatment group, blank control group), 25 in each group, and three parallel experiments were set up. The experiment was carried out in 25℃ aquaculture water. The test concentrations of Angelica extract were 100 and 50mg / kg. The volume of the treatment solution injected into each crayfish was 100μL. The drug treatment method was to premix the WSSV virus and the monomer compound and then inject it into the abdomen immediately (the virus dosage for each shrimp was 6.9×107copies, premixing: the virus solution was mixed with the drug solution at 25℃ and then injected immediately). After 24h, 5 crayfish were randomly selected from each treatment group, and the gill tissue of the shrimp was collected and stored at -80℃ for use.

[0043] The collected samples were used for absolute quantification of WSSV genomic DNA copy number. Absolute fluorescence quantitative PCR was used to detect the viral genome copy number in the gill tissue of the treated samples. The DNA of the collected tissue samples was extracted using a marine animal tissue genomic DNA extraction kit. The concentration and purity of the extracted DNA were detected using an ultra-micro spectrophotometer, and then the DNA concentration was uniformly adjusted to 50 ng / μL. RT-qPCR and the constructed absolute quantitative standard curve ( Figure 2 ) Quantification of viral copy number in tissues.

[0044] DNA extraction from gill tissue of Procambarus clarkii:

[0045] 1) Weigh about 15 mg of tissue material, grind it thoroughly, put it into a centrifuge tube containing 200 μL GA buffer, and vortex for 15 seconds;

[0046] 2) Add 20 μL of Proteinase K (20 mg / mL) solution, vortex to mix, briefly centrifuge to remove water droplets on the inner wall of the tube cap, place at 56°C until the tissue is completely dissolved, and briefly centrifuge to remove water droplets on the inner wall of the tube cap;

[0047] 3) Add 200 μL of buffer GB, mix thoroughly by inversion, and place at 70°C for 10 min. The solution should become clear and centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0048] 4) Add 200 μL of anhydrous ethanol and mix thoroughly by inversion. Flocculent precipitation may appear at this time. Centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0049] 5) Add the solution and flocculent precipitate obtained in the previous step to the adsorption column CB3 (the adsorption column is placed in the collection tube), centrifuge at 12000 rpm for 30 seconds, discard the waste liquid, and put the adsorption column CB3 back into the collection tube;

[0050] 6) Add 500 μL of buffer GD to the adsorption column CB3, centrifuge at 12000 rpm for 30 seconds, discard the waste liquid, and place the adsorption column CB3 in a collection tube;

[0051] 7) Add 600 μL of rinse solution PW to the adsorption column CB3, centrifuge at 12000 rpm for 30 seconds, discard the waste liquid, and place the adsorption column CB3 in a collection tube;

[0052] 8) Repeat step 7);

[0053] 9) Place the adsorption column CB3 back into the collection tube, centrifuge at 12000 rpm for 2 min, discard the waste liquid, and place the adsorption column CB3 at room temperature for several minutes to completely dry the residual rinse liquid in the adsorption material;

[0054] 10) Transfer the adsorption column CB3 into a clean centrifuge tube, add 100 μL of elution buffer ddH2O to the middle part of the adsorption membrane, leave it at room temperature for 2-5 minutes, centrifuge it at 12000 rpm for 2 minutes, and collect the solution into the centrifuge tube;

[0055] 11) Repeat step 10)

[0056] 12) The solution obtained by centrifugation contains DNA products, and the DNA products are stored at -20°C for future use.

[0057] CFX-96 was used for real-time PCR quantitative detection. VP28-141 was used as the quantitative primer, and the primer sequence is shown in Table 1. The quantitative kit was Vazyme GreenMasterMix. The quantitative reaction system is shown in Table 2, and the reaction procedure is shown in Table 3.

[0058] Table 1

[0059]

[0060] Table 2

[0061] Reagents Volume (μL) 2×SYBR Green Master Mix 7.5 Forward Primer (10μM) 0.3 Reverse Primer (10μM) 0.3 template 1 <![CDATA[ddH2O]]> Up to 15

[0062] Table 3

[0063]

[0064] Example 4

[0065] This example shows the protective effect of the angelica extract on Procambarus clarkii; the specific scheme is as follows:

[0066] Healthy crayfish were randomly divided into 3 groups, 100 in each group, and injected with TM buffer (control group), TM-WSSV mixture (positive control group) and drug-WSSV mixture (treatment group, 100 mg / kg and 50 mg / kg). Each group of crayfish was observed for 8 days and the mortality was recorded. The survival rate was calculated as follows: survival rate (%) = number of survivors in the treatment group / total number in each group × 100%.

[0067] Depend on Figure 3 It can be seen that different concentrations of Angelica extract (100 mg / kg and 50 mg / kg) significantly reduced the copy number of WSSV in Procambarus clarkii, and had good anti-WSSV biological activity, with inhibition rates reaching 69.84% and 88.56%.

[0068] Depend on Figure 4 It can be seen that the angelica extract can significantly reduce the mortality of WSSV-infected crayfish. During the test, no death or only a small number of crayfish were found in the TM buffer-treated crayfish. A large number of crayfish treated with WSSV died, and the survival rate was 5% at 8 days. The survival rate of diseased crayfish treated with different concentrations of angelica extract (50mg / kg and 100mg / kg) was improved to 21% and 35%.

[0069] Depend on Figure 3 , Figure 4 It can be seen that Angelica sinensis extract can significantly inhibit the replication of WSSV in Procambarus clarkii and at the same time improve the survival rate of Procambarus clarkii infected with WSSV.

[0070] In summary, the results of the examples of the present invention fully demonstrate that the angelica extract can inhibit the replication of WSSV in Procambarus clarkii by significantly reducing the expression of WSSV replication-related genes, thereby improving the survival rate of the host infected with the virus. This study provides a theoretical basis for the development of angelica extract as an efficient and practical anti-WSSV drug, and also provides an important reference for the study of the antiviral effect of angelica extract on other aquatic animal viruses.

[0071] As described above, the basic principle, main features and advantages of the present invention are well described. The above embodiments and descriptions are only descriptions of the preferred implementation modes of the present invention, and the present invention is not limited by the above embodiments. Without departing from the spirit and scope of the present invention, various changes and improvements made by ordinary technicians in this field to the technical solution of the present invention should fall within the protection scope determined by the present invention.

Claims

1. The use of angelica extract in the preparation of medicines, characterized in that: The drug is used to fight against white spot syndrome virus.

2. The use of the angelica extract in the preparation of medicines according to claim 1, characterized in that: The medicine comprises angelica extract and diluent.

3. The use of the angelica extract in the preparation of medicines according to claim 2, characterized in that: The diluent is DMSO.

4. The use of the angelica extract in preparing medicine according to claim 3, characterized in that: The mass concentration of the angelica extract in the medicine is 60 mg / kg to 120 mg / kg.

5. The use of the angelica extract according to any one of claims 1 to 4 in the preparation of medicines, characterized in that: The angelica extract is obtained by extracting angelica with an alcohol extraction reagent; The method for extracting the angelica extract comprises: providing dried angelica, crushing it and then extracting it with an alcohol extraction reagent; optionally, filtering the obtained extract and concentrating it to obtain the angelica extract.

6. The use of the angelica extract in preparing medicine according to claim 5, characterized in that: The alcohol extraction reagent is methanol or ethanol.

7. Use of the angelica extract according to claim 5 or 6 in preparing medicines, characterized in that: The ratio between the mass of the angelica sinensis and the volume of the solvent is 10g:500ml to 50g:500ml.

8. The use of the angelica extract according to any one of claims 1 to 7 in the preparation of medicines, characterized in that: It has one or more characteristics selected from the following: (1) The drug further comprises a pharmaceutically acceptable carrier and / or excipient; (2) The drug is in the form of pills, powders, capsules, tablets (e.g., effervescent tablets), film-coated tablets, granules, liquids or suppositories.

9. The use of the angelica extract in preparing medicine according to claim 1, characterized in that: The medicine is used for preventing and / or treating white spot syndrome in aquatic animals.

10. The use of the angelica extract in preparing medicine according to claim 9, characterized in that: The aquatic animals are crustaceans.

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