Chinese herbal compound for resisting white spot syndrome virus and application thereof
Through the traditional Chinese medicine compound of rhubarb extract, sophora alcohol extract, quercetin and emodin, the environmental pollution and economic losses caused by the long-term use of chemical drugs have been solved, and the anti-WSSV capability of shrimps has been significantly improved, and effective Virus blockade and survival rates have increased.
Patent Information
- Application Number
- CN202411261967.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-10
- Publication Date
- 2024-12-17
AI Technical Summary
In the prevention and treatment of WSSV, the long-term use of chemical synthetic drugs leads to drug residues and environmental pollution, and the lack of effective natural alternatives, which leads to huge economic losses in the aquaculture industry.
Use the mixture of rhubarb extract, sophora alcohol extract, quercetin and emoflavin at a ratio of 1:1:0.05:0.02, as a compound prescription for anti-white spot syndrome virus, extracted through anhydrous ethanol and added to shrimp feed or In the aquaculture water, to block the outbreak of WSSV.
This Chinese medicine compound prescription significantly improves the survival rate and antiviral ability of shrimps, reduces the outbreak frequency and economic losses of WSSV, and is simple in preparation and use, is natural and pollution-free, and is not easy to induce drug resistance.
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Figure CN119139383A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to an antiviral Chinese medicinal compound, in particular to an anti-leukoplakia syndrome virus Chinese medicinal compound and application thereof. Background Art
[0002] White spot syndrome (WSD) caused by white spot syndrome virus (WSSV) is an acute viral disease of aquatic animals, with multiple transmission vectors, rapid onset and high mortality. Its outbreak has caused huge economic losses to the aquaculture industries of Penaeus vannamei, Procambarus clarkii, Chinese mitten crab and mud crab, and is one of the most severe challenges facing the global aquaculture industry. After being infected with WSSV, crustaceans have reddish color with jejunum, slow reaction to the outside world and often lie quietly at the bottom of the water. In the late stage of infection, there are white spots on the shell, and the mortality rate can generally reach 100% within 3 to 14 days after the onset of the disease. Due to the lack of acquired immunity in crustaceans, chemical drugs are currently mainly used to prevent and control WSD at home and abroad. Although the use of most chemical synthetic drugs can effectively block the outbreak of WSSV in the short term, the long-term irrational use of drugs induces a series of negative effects such as drug residues and environmental pollution, which endangers human health. Chinese herbal medicines are natural and safe, with little toxicity and side effects, low residue, and are not prone to drug resistance. They have attracted much attention in the prevention and control of aquatic animal diseases and are considered to be potential targets for replacing chemical synthetic drugs in the future. Studies have shown that the ethanol extracts of Chinese herbal medicines rhubarb (Rheumpalmatum L.) and Sophora japonica (Flossophora eimaturus) and their active ingredients rhubarb and quercetin have significant effects in anti-apoptosis, oxidative stress response, bactericidal, antiviral, improving immunity and enhancing appetite. At present, there is no research on the anti-WSSV activity of the above-mentioned Chinese herbal compound. Exploring the anti-WSSV activity of the above-mentioned Chinese herbal compound has important practical significance for blocking the outbreak of WSSV. Summary of the invention
[0003] The technical problem to be solved by the present invention is to provide an anti-WSSV Chinese medicinal compound capable of improving the anti-WSSV activity and survival rate of aquatic animals and application thereof.
[0004] The technical solution adopted by the present invention to solve the above technical problems is: an anti-white spot syndrome virus Chinese medicine compound composed of the following components: rhubarb ethanol extract, sophora japonica seed ethanol extract, quercetin and rhein.
[0005] Furthermore, the mass ratio of the rhubarb alcohol extract, the sophora japonica bud alcohol extract, quercetin and rhein is 1:1:0.05:0.02.
[0006] Furthermore, the preparation method of the rhubarb alcohol extract and the sophora japonica flower alcohol extract is as follows: take rhubarb and sophora japonica flower, dry and grind them, sieve them through a 100-mesh sieve, weigh 50g of each, add 300mL of anhydrous ethanol, place them in an ultrasonic cleaner for ultrasonic dissolution for 10 minutes, then place them in a 60°C water bath for heating for 30 minutes, repeat the above steps 3 times, add 150mL of anhydrous ethanol each time, concentrate the anhydrous ethanol extract by rotary evaporation, and finally place them in an oven for drying to obtain the rhubarb alcohol extract and the sophora japonica flower alcohol extract.
[0007] The present invention also provides application of the anti-white spot syndrome virus Chinese medicinal compound in preparing an anti-white spot syndrome virus feed additive for shrimps.
[0008] Furthermore, the amount of the Chinese medicinal compound added to the shrimp breeding water is 200 mg / L breeding water.
[0009] Furthermore, the amount of the Chinese medicinal compound added to commercial feed is 4wt%.
[0010] Compared with the prior art, the invention has the advantages that: the invention discloses a Chinese medicine compound for resisting white spot syndrome virus and its application. After rhubarb and sophora japonica seed are dried and crushed, effective components are extracted by anhydrous ethanol, rhubarb alcohol extract, sophora japonica seed alcohol extract, quercetin and rhein are weighed in proportion and mixed to obtain the Chinese medicine compound for resisting WSSV, which can effectively block the outbreak of WSSV. The anti-WSSV effect of the Chinese medicine compound is more significant than that of the single use of rhubarb alcohol extract, sophora japonica seed alcohol extract, quercetin and rhein. The Chinese medicine compound is simple in preparation and use, is natural and pollution-free, is not easy to cause drug resistance in shrimp, can effectively reduce the economic loss caused by the outbreak of WSSV to shrimp farming, has low cost, is simple in use, and is conducive to popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Figure 1 The effects of Chinese herbal extracts and their compound on the survival rate of shrimp larvae, where A is the alcohol extract of rhubarb; B is rhein; C is quercetin; D is the alcohol extract of Sophora japonica; E is the Chinese herbal compound A; F: Chinese herbal compound B;
[0012] Figure 2 The anti-WSSV activity of Chinese herbal medicine alcohol extracts and their compound preparations is analyzed, where AF are the viral replication levels in shrimp larvae treated with quercetin, emodin, rhubarb alcohol extract, sophora japonica extract, compound A and compound B for 72 hours; G is the survival curve of shrimp larvae under different treatment conditions within 72 hours;
[0013] Figure 3Figure 4 is the effect of Chinese herbal extracts and their compound on the disease resistance of shrimp pre-incubated with larvae. AB is the virus replication level and survival curve of shrimp infected with WSSV after different time of quercetin pretreatment; CD is the virus replication level and survival curve of shrimp infected with WSSV after different time of Sophora japonica ethanol extract pretreatment; EF is the virus replication level and survival curve of shrimp infected with WSSV after different time of emodin pretreatment; GH is the virus replication level and survival curve of shrimp infected with WSSV after different time of rhubarb ethanol extract pretreatment; IJ is the virus replication level and survival curve of shrimp infected with WSSV after different time of Chinese herbal compound A pretreatment; KL is the virus replication level and survival curve of shrimp infected with WSSV after different time of Chinese herbal compound B pretreatment;
[0014] Figure 4 The therapeutic effects of Chinese herbal medicine extracts and their compound on diseased shrimps, where AF are the survival curves of diseased shrimps treated with rhubarb alcohol extract, sophora japonica bud alcohol extract, quercetin, emodin, compound Chinese medicine A and compound Chinese medicine B, respectively;
[0015] Figure 5 The effects of Chinese herbal extracts and their compound recipes on the horizontal transmission of WSSV, where AF are the effects of rhubarb alcohol extract, sophora japonica bud alcohol extract, quercetin, rhein, Chinese herbal compound recipe A and Chinese herbal compound recipe B on the replication of WSSV genome, respectively;
[0016] Figure 6 The figure shows the effect of dietary supplementation of Chinese herbal compound A on the resistance of juvenile shrimp to WSSV, where A is the level of virus replication in diseased shrimp; B is the survival curve of shrimp 72 hours after WSSV infection. DETAILED DESCRIPTION
[0017] The present invention is further described in detail below with reference to the accompanying drawings.
[0018] To illustrate the essence of the present invention, the anti-WSSV activity of rhubarb, sophora japonica seed ethanol extract and its effective components, emodin and quercetin, in a Chinese medicine compound was analyzed with white shrimp larvae as experimental subjects to verify its role in blocking the outbreak of WSSV. Rhubarb, sophora japonica seed, emodin, quercetin, dimethyl sulfoxide (DMSO) and anhydrous ethanol were all provided by Shanghai Aladdin Biochemical Technology Co., Ltd., and WSSV and white shrimp were taken from Zhejiang Marine Aquaculture Research Institute. 1. Specific embodiments
[0020] 1. Preparation of Chinese herbal compound
[0021] Take rhubarb and sophora japonica seeds, dry and grind them respectively, weigh 50g of each, add 300mL of anhydrous ethanol, place in an ultrasonic cleaner for ultrasonic dissolution for 10min, then heat in a water bath at 60℃ for 30min, repeat the above steps 3 times, and add 150mL of anhydrous ethanol each time. Take the above anhydrous ethanol extracts and perform rotary evaporation concentration, and finally place in an oven to dry to obtain rhubarb ethanol extract and sophora japonica seeds ethanol extract. Weigh the Chinese medicine compound A (rhubarb ethanol extract: rhubarb ethanol extract: sophora japonica seeds ethanol extract: quercetin = 1: 0.05: 1: 0.02) and the Chinese medicine compound B (rhubarb ethanol extract: rhubarb ethanol extract: sophora japonica seeds ethanol extract: quercetin = 1: 0.02: 1: 0.05) according to the mass ratio, mix well, use DMSO to fully dissolve, and prepare a mother solution with a final concentration of 50000mg / L, and store at 4℃ for use.
[0022] 2. Determination of WSSV infection concentration
[0023] Healthy, vigorous, and uniform shrimps were placed in a 6-well plate, with 6 mL of water per well and 10 shrimps. The concentration of WSSV was set to: 1.6 × 10 4 , 1.6×10 5 , 1.6×10 6 , 1.6×10 7 and 1.6×10 8 The experiment was repeated three times, and the survival rate of shrimp larvae was counted every 24 hours. The virus concentration of 1.6×10 6 copies / μL for subsequent infection experiments.
[0024] 3. Acute toxicity test of rhubarb alcohol extract, sophora japonica extract, quercetin, rhein and their compound
[0025] Healthy, vigorous and uniform shrimps were placed in a 6-well plate, with 6 mL of water per well and 10 shrimps. Different concentrations of rhubarb alcohol extract, sophora japonica alcohol extract, quercetin, rhein and their compound (0-300 mg / L) were set to incubate the shrimps for 72 hours. The experiment was repeated 3 times, and the survival rate of the shrimps was counted every 24 hours. The safe concentration of the above-mentioned Chinese medicine extracts and their compound was determined based on the survival rate of the shrimps.
[0026] The cumulative survival rate of shrimp larvae was Figure 1 As shown. When the alcohol extract of rhubarb in aquaculture water ( Figure 1 A) Rhein Figure 1 B) Quercetin ( Figure 1 C), Sophora japonica bud ethanol extract ( Figure 1 D) Compound A( Figure 1 E) and compound B( Figure 1F) When the concentrations were 100 mg / L, 2 mg / L, 40 mg / L, 200 mg / L, 200 mg / L and below 200 mg / L, the cumulative survival rate of shrimp larvae was not significantly different from that of the control group and no precipitation was observed. Therefore, it is believed that the ethanol extract of rhubarb (100 mg / L), the ethanol extract of sophora japonica (200 mg / L), quercetin (40 mg / L), rhein (2 mg / L), compound A (200 mg / L) and compound B (200 mg / L) are safe for shrimp larvae and can be used in subsequent experiments.
[0027] 4. Analysis of the anti-WSSV activity of rhubarb alcohol extract, sophora japonica bud alcohol extract, quercetin, rhein and their compound
[0028] WSSV copy number detection: Use a marine animal tissue DNA extraction kit to extract shrimp DNA, detect its concentration with a spectrophotometer and dilute it to a final concentration of 50ng / μL before performing qPCR. Primer information is VP28 141 (VP28-F: 5'-AAACCTCCGCATTCCTGTGA-3', VP28-R: 5'-TCCGCATCTTCTTCCTTCAT-3'). The total qPCR reaction system was 25 μL, including: 2×Es Taq MasterMix 12.5 μL, VP28-F (10 μM) 0.5 μL, VP28-R (10 μM) 0.5 μL, ddH2O 10.5 μL, DNA 1 μL. The reaction procedure is shown in Table 1. pMD19T-VP28 was used. 141 The standard curve made from the standard product was used to convert the qPCR results to obtain the WSSV copy number.
[0029] Table 1 qPCR reaction procedure
[0030]
[0031]
[0032] (1) Evaluation of the anti-WSSV activity of rhubarb ethanol extract, sophora japonica bud ethanol extract, quercetin, rhein and their combination
[0033] Rheum officinale ethanol extract (100 mg / L), Sophora japonica bud ethanol extract (200 mg / L), quercetin (40 mg / L), rhein (2 mg / L), compound A (200 mg / L), compound B (200 mg / L) and WSSV (1.6×10 6 After mixing, the shrimps were incubated in a 6-well plate for 72 h. DMSO (V DMSO / V 水=0.4‰) and WSSV were incubated for 72 hours as a control. The mortality of the shrimp was counted every 24 hours during the experiment, and the shrimp were collected at 72 hours to detect the number of virus copies.
[0034] The results are as follows Figure 2 As shown in the results, the above Chinese herbal extracts and their compound formulas can effectively inhibit the virus replication in the diseased shrimp and improve the survival rate of the diseased shrimp. Among them, all the shrimps in the virus group died at 72 hours. Rhubarb ethanol extract, Sophora japonica ethanol extract, quercetin, emodin, compound A, and compound B can increase the cumulative survival rate of the diseased shrimp within 72 hours to 36%, 26%, 25%, 35%, 58%, and 46%, respectively. Figure 2 AG). The data also showed that the above-mentioned Chinese herbal extracts and their compound formulas had an inhibitory effect on the replication of the WSSV genome in shrimp larvae, and the inhibition rates at 72 hours were 63% (rhubarb ethanol extract), 50% (sophora japonica bud ethanol extract), 87% (quercetin), 90% (rhubarb), 91% (Chinese herbal compound formula A), and 84% (Chinese herbal compound formula B). In summary, the above-mentioned Chinese herbal extracts and their compound formulas all had good anti-WSSV activity, among which compound Chinese herbal formula A had the best antiviral activity and had the greatest potential to be used to prevent and control WSSV outbreaks.
[0035] (2) Experiment on pre-incubation of shrimp larvae with alcohol extract of rhubarb, alcohol extract of sophora japonica, quercetin, rhein and their combination
[0036] To further evaluate the anti-WSSV activity of rhubarb ethanol extract, sophora japonica extract, quercetin, rhein and their compound, a pre-incubation experiment was conducted. Rhubarb ethanol extract (100 mg / L), sophora japonica extract (200 mg / L), quercetin (40 mg / L), rhein (2 mg / L), compound A (200 mg / L) and compound B (200 mg / L) were pre-incubated with shrimp in 6-well plates for 24, 48 and 72 h, respectively. The original culture water was then discarded and washed three times with new culture water and replaced with WSSV-containing culture water (1.6 × 10 6 The shrimp were incubated with water containing 100 copies / μL for 72 hours. The experiment was repeated three times, and the survival of the shrimp was recorded every 24 hours. The shrimp were collected at 72 hours to detect the number of virus copies.
[0037] The results are as follows Figure 3 As shown in the figure, after shrimps were pretreated with Chinese herbal extracts and their compound for different time periods and then infected with WSSV, their cumulative mortality and viral genome copy levels within 72 hours were reduced. Among them, although quercetin pre-incubation of shrimps can reduce the level of viral replication in their bodies (maximum inhibition rate of 18%) and increase their cumulative survival rate (maximum 12%), the difference was not significant compared with the control group ( Figure 3 AB). In addition, the ethanol extract of Sophora japonica seed ( Figure 3 CD), rhein ( Figure 3EF), Rheum officinale ethanol extract ( Figure 3 Pre-incubation of shrimp with herbal compound A (Figure GH) and herbal compound B (Figure KL) for 72 hours can significantly inhibit the virus replication in the shrimp, with inhibition rates of 73%, 48%, 69%, and 61%, respectively. The corresponding cumulative survival rates of diseased shrimp were increased by 34%, 55%, 21%, and 36%, respectively. In addition, we also found that herbal compound A had the strongest anti-WSSV ability when pre-treated shrimp for 72 hours, with the cumulative survival rate of diseased shrimp increased by 44%, and the inhibition rate of viral genome replication in the body was 87% ( Figure 3 IJ). Therefore, the above-mentioned Chinese medicine compound A has the best effect in preventing WSSV outbreak.
[0038] (3) The therapeutic effects of rhubarb ethanol extract, sophora japonica bud ethanol extract, quercetin, rhein and their compound on diseased shrimp
[0039] The experiment was conducted in a 6-well plate, and the sick shrimp were obtained as described above. The sick shrimp were treated in a 6-well plate with aquaculture water containing only rhubarb ethanol extract (100 mg / L), sophora japonica ethanol extract (200 mg / L), quercetin (40 mg / L), rhein (2 mg / L) and its compound A (200 mg / L), and compound B (200 mg / L). The experiment set up a continuous dressing group and a discontinuous dressing group. The continuous dressing group replaced new aquaculture water and added new Chinese medicine compound every 24 hours, and the discontinuous dressing group did not do any treatment during the treatment period. The experiment was designed with 3 parallels, and the survival rate of sick shrimp was recorded every 24 hours, and the surviving sick shrimp were collected to detect the number of virus copies.
[0040] The results are as follows Figure 4 As shown in Figure 2, all shrimp infected with WSSV died on the third day. Figure 4 A), Sophora japonica bud ethanol extract ( Figure 4 B) Quercetin ( Figure 4 C) Rhein Figure 4 D) and its compound A( Figure 4 E), Compound B ( Figure 4 F) The diseased shrimps after treatment were still alive on the 3rd day, and their cumulative survival rates were 43%, 33%, 35%, 14%, 43%, and 48%, respectively, and all died on the 5th day after infection. The therapeutic effect of the above-mentioned Chinese herbal extracts and their compound prescriptions on the diseased shrimps was further enhanced by continuously changing the above-mentioned Chinese herbal extracts and their compound prescriptions, because on the 6th day, the cumulative survival rates of the diseased shrimps treated with rhubarb ethanol extract, sophora japonica bud ethanol extract, compound Chinese herbal medicine A and B were 3%, 5%, 15%, and 9%, respectively. In summary, the above-mentioned Chinese herbal extracts and their compound prescriptions all have good therapeutic effects on diseased shrimps, among which compound Chinese herbal medicine A has the best therapeutic effect.
[0041] (4) Effects of rhubarb extract, sophora japonica extract, quercetin, rhein and their combination on the horizontal transmission of WSSV
[0042] Horizontal transmission is the main cause of large-scale WSSV outbreaks. The following is an evaluation of the effects of Chinese herbal extracts and their compound on the horizontal transmission of WSSV. 6 The donor diseased shrimps were obtained by incubating healthy and uniform shrimp larvae with 100 copies / μL) for 24 hours, and healthy shrimp larvae were used as recipient shrimps. Experimental group: The donor and recipient shrimps were co-cultured in containers containing rhubarb ethanol extract (100 mg / L), sophora japonica ethanol extract (200 mg / L), quercetin (40 mg / L), rhein (2 mg / L) and its compound A (200 mg / L) and compound B (200 mg / L) for 72 hours. During the culture period, the donor and recipient shrimps were separated by gauze to ensure no direct contact. Control group: The donor and recipient shrimps were cultured in a container containing only DMSO (V DMSO / V 养殖水 =0.4‰) in a container and cultured for 72 hours. The culture method was the same as that of the experimental group. After the experiment, the recipient shrimp was taken to detect the number of virus copies.
[0043] The results are as follows Figure 5 As shown in Figure 2, the above-mentioned Chinese herbal extracts and their compound formulas can effectively inhibit the horizontal transmission of WSSV but cannot completely block it ( Figure 5 Among them, rhein ( Figure 5 D), Sophora japonica bud ethanol extract ( Figure 5 B) Chinese medicine compound A( Figure 5 E) and B( Figure 5 F) can significantly reduce the horizontal transmission of WSSV, and their inhibition rates on WSSV replication at 72h are 36%, 80%, 84%, and 59%, respectively. In addition, the maximum inhibition rate of rhubarb ethanol extract on WSSV replication is 49%, which is not significantly different from the control group ( Figure 5 A), quercetin can significantly inhibit the horizontal spread of WSSV in the early stage of WSSV infection, with an inhibition rate of 54% ( Figure 5 C). The above results show that the above-mentioned Chinese medicine compound A can most effectively inhibit the horizontal transmission of WSSV and is more suitable for preventing and controlling the outbreak of WSSV.
[0044] 2. Application Examples
[0045] Pharmacodynamics experiment of compound Chinese medicine A against white spot syndrome virus
[0046] The sources of Chinese herbal compound A (rhubarb ethanol extract, rhubarb extract, sophora japonica ethanol extract, quercetin in a mass ratio of 1:0.05:1:0.02), white shrimp and white spot syndrome virus (WSSV) are the same as the above specific embodiments. Healthy and uniform juvenile shrimps were randomly placed in a 500L breeding barrel, each barrel containing 150 juvenile shrimps, 400L of breeding water, and covered with gauze. A 15-day breeding experiment was carried out by adding 0%, 1%, 2% and 4% of Chinese herbal compound A to the commercial feed in mass percentage. During the experiment, the juvenile shrimps were fed 3 times a day at 5% of their body weight, and the experiment was repeated 3 times. At the end of the experiment, the juvenile shrimps in each group were taken and WSSV (1.6×10 6 The viral copy number was detected and the survival rate was recorded 72 hours after infection.
[0047] Effects of dietary supplementation with compound Chinese medicine A on WSSV infection Figure 6 Compared with the control group, dietary supplementation with compound Chinese medicine can effectively reduce the level of viral replication in juvenile shrimp infected with WSSV and increase their survival rate. Among them, dietary supplementation with 4% compound Chinese medicine inhibited WSSV replication in juvenile shrimp by 60%, and the survival rate of juvenile shrimp increased to more than 30%.
[0048] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.
Claims
1. A Chinese medicinal compound for resisting white spot syndrome virus, characterized in that It is composed of the following ingredients: rhubarb alcohol extract, rhubarb emodin, sophora japonica seed alcohol extract and quercetin.
2. The anti-white spot syndrome virus Chinese medicinal compound according to claim 1, characterized in that: The mixing mass ratio of the rhubarb alcohol extract, rhein, sophora japonica bud alcohol extract and quercetin is 1:0.05:1:0.
02.
3. An anti-white spot syndrome virus Chinese medicinal compound according to claim 1 or 2, characterized in that The preparation method of the rhubarb alcohol extract and the sophora japonica seed alcohol extract is as follows: take rhubarb and sophora japonica seed, dry and grind them, sieve them through a 100-mesh sieve, weigh 50g of each, add 300ml of anhydrous ethanol, place them in an ultrasonic cleaner for ultrasonic dissolution for 10min, then place them in a 60℃ water bath for heating for 30min, repeat the above steps 3 times, add 150ml of anhydrous ethanol each time, concentrate the anhydrous ethanol extract by rotary evaporation, and finally place them in an oven for drying to obtain the rhubarb alcohol extract and the sophora japonica seed alcohol extract.
4. Use of the anti-white spot syndrome virus Chinese medicinal compound according to claim 1 in the preparation of white spot syndrome virus inhibitors for shrimp.
5. Use of the anti-white spot syndrome virus Chinese medicinal compound according to claim 4 in the preparation of white spot syndrome virus inhibitors for shrimps, characterized in that: The amount of the Chinese medicinal compound added to the shrimp breeding water is 200 mg / L breeding water.
6. Use of the anti-white spot syndrome virus Chinese medicinal compound according to claim 4 in the preparation of white spot syndrome virus inhibitors for shrimps, characterized in that: The amount of the Chinese medicinal compound added to commercial feed is 4wt%.