A rock extract, its preparation method and application

Abstract

This invention relates to a *Pseudorabies rubra* extract, its preparation method, and its application, belonging to the field of natural extract technology. The invention uses an ethanol extract of *Pseudorabies rubra* as raw material, further extracting the ethanol extract with ethyl acetate to obtain a *Pseudorabies rubra* extract with antiviral activity against porcine pseudorabies, and also enhances the effect of ST cells in preventing PRV infection. The obtained *Pseudorabies rubra* extract has no toxicity to ST cells at concentrations of 25–0.78 µg / mL and can be used as a natural medicine for the treatment or prevention of porcine pseudorabies. This invention can effectively solve the problem that the current treatment of porcine pseudorabies mainly relies on antibiotics, reducing antibiotic use, reducing veterinary drug residues, and improving pork quality.

Description

Technical Field

[0001] This invention belongs to the field of natural extract technology, specifically, it relates to a rock extract, its preparation method and application. Background Technology

[0002] Traditional Chinese medicinal herbs not only possess antibacterial and antiviral effects, but also have the advantages of wide availability, low price, minimal toxicity and side effects, low drug residues, and no drug resistance. With the implementation of China's policy of completely banning the addition of antibiotics to animal feed starting in 2020, finding effective antibacterial and antiviral drugs from traditional natural medicines, especially Chinese herbal medicines, has become a major research direction. *Rodgersia pinnata* Franch. or *Rodgersia sambuccifolia* Hemsl., both belonging to the genus *Rodgersia* in the family Saxifragaceae, are dried rhizomes. *Rodgersia pinnata* and other plants in the *Rodgersia* genus prefer shady or semi-shady environments and are suitable for growth in forests, rock crevices, thickets, damp rock faces, or along valley streams in areas with high relative humidity. They are mainly distributed in Yunnan, Sichuan, and Guizhou provinces.

[0003] In traditional Chinese medicine, *Gnaphalium affine* has astringent, anti-inflammatory, and wind-dispelling properties. Its main chemical components are found in its roots. Studies have shown that *Gnaphalium affine* is rich in polyphenolic compounds, with bergenin content second only to the traditional Chinese medicine *Gnaphalium affine*. Currently, its development and application in veterinary medicine are relatively limited.

[0004] Porcine pseudorabies is an acute infectious disease of pigs caused by the pseudorabies virus (PRV). It is primarily transmitted from infected pigs to healthy pigs, causing abortion and stillbirth in pregnant sows, infertility in boars, high mortality rates in newborn piglets, and respiratory distress and growth retardation in finishing pigs. It is one of the major infectious diseases threatening the global pig industry. Currently, vaccination and boosting immunity are the main measures for preventing and controlling pseudorabies. Once infected, antibiotic treatment is still necessary.

[0005] The overuse and abuse of antibiotics have led to serious veterinary drug residues, directly endangering human health. As people's living standards improve, the demand for meat quality is increasing. In conjunction with the "Regulations on the Administration of Veterinary Drugs" and the "Regulations on the Administration of Feed and Feed Additives," the state has introduced corresponding policies to ban or restrict antibiotics. Developing alternative antibiotic veterinary products by searching for active ingredients in traditional Chinese medicine has become a current research hotspot. Summary of the Invention

[0006] To overcome the problems existing in the background art, the present invention provides a *Pseudorabies rubracoides* extract, its preparation method and application, obtained from the ethyl acetate extraction phase of *Pseudorabies rubracoides* ethanol extract. The obtained *Pseudorabies rubracoides* extract has antiviral activity against PRV and is not toxic to ST cells at 25–0.78 μg / mL, and can be used as a natural medicine for the treatment or prevention of porcine pseudorabies.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0008] The aforementioned rock extract and its preparation method include the following steps:

[0009] (1) Crush the rock powder and sieve it;

[0010] (2) Extract the rock jujube with ethanol to obtain an ethanol extract of the rock jujube;

[0011] (3) Extract the ethanol extract with ethyl acetate to obtain the ethyl acetate extract phase.

[0012] Furthermore, the obtained ethyl acetate extract was concentrated by rotary evaporation, and the concentrate was freeze-dried at low temperature to obtain a powdered rock extract.

[0013] Furthermore, the ethanol used in step (2) is 70% ethanol.

[0014] A rock extract, obtained by the above preparation method.

[0015] Furthermore, the aforementioned rock extract exhibits antiviral activity against porcine pseudorabies.

[0016] Furthermore, the aforementioned rock extract has a significant protective effect against ST cells from porcine pseudorabies virus infection in prophylactic mode.

[0017] The application of the aforementioned rock extract in the prevention or treatment of pseudorabies in pigs.

[0018] A drug for the prevention or treatment of pseudorabies in pigs contains the aforementioned rock extract.

[0019] The beneficial effects of this invention are:

[0020] The ethyl acetate phase of the crude ethanol extract from Yantuo exhibits significant anti-pseudorabies virus activity and enhances the effect of ST cells in preventing PRV infection, providing a material basis for further research and discovery of active monomeric components.

[0021] The ethyl acetate phase of the crude ethanol extract of this invention has the characteristics of wide availability of raw materials, significant antiviral effects, and safe use. By using this extract to prepare veterinary drugs, the use of antibiotics can be reduced, veterinary drug residues can be reduced, and the quality of pork can be improved. Attached Figure Description

[0022] Figure 1 This is a comparison chart of the yields of different extraction phases in Example 1 of the present invention;

[0023] Figure 2 This is a comparative graph showing the effects of different petroglyph extracts of this invention on the survival rate of ST cells;

[0024] Figure 3 This invention compares the effects of different extracts of *Gnaphalium affine* on the survival rate of ST cells under preventive mode.

[0025] Figure 4 This invention compares the effects of different extracts of *Gnaphalium affine* on ST cell survival rate under different treatment modes.

[0026] Figure 5 This invention compares the effects of different extracts of *Gnaphalium affine* on the survival rate of ST cells under toxicity treatment modes.

[0027] Figure 6 It is the total ion spectrum (TIC) collected in positive ion mode;

[0028] Figure 7 This is the total ion spectrum (TIC) collected in negative ion mode;

[0029] Remark: Figure 2 In the mean squares, ***P<0.001; **P<0.01; *P<0.05;

[0030] Figures 3 to 5 In the study, ***P<0.001; **P<0.01; *P<0.05; compared with the viral group, ###P<0.001; ##P<0.01; #P<0.05;

[0031] Figures 2 to 5 In the diagram, A represents the ethanol extract, B represents the ethyl acetate extract phase of the ethanol extract, C represents the n-butanol extract phase of the ethanol extract, and D represents the aqueous phase of the ethanol extract. Detailed Implementation

[0032] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are all within the scope of protection of this invention.

[0033] Example 1: Crude Ethanol Extract from Rock-like Materials and its Preparation Method

[0034] The rock powder was passed through a 60-mesh sieve, and 50g was weighed out. 3000mL of 70% ethanol was added, resulting in a material-to-liquid mass ratio of 1:60. The mixture was sonicated for 15 minutes and then magnetically rotated overnight at room temperature. After filtration and rotary evaporation, the ethanol was replaced with distilled water, and the mixture was concentrated to a certain volume. Finally, it was freeze-dried using a freeze dryer and weighed (W).

[0035] The same method was used to extract the crude extract with methanol and acetone respectively, with an extraction yield of (W / 50)×100%.

[0036] Table 1 Extraction yields using different solvents

[0037] name Extraction rate (%) 70% ethanol extract 33.2 methanol extract 29.80 Acetone extract 14.02

[0038] As shown in Table 1, in terms of the extraction yield from rock extract, the 70% ethanol extract > methanol extract > acetone extract.

[0039] The crude extract of *Lycopodium clavatum* was dissolved in purified water to a certain volume, and then extracted three times with the same volume of ethyl acetate for 30 min each time. The liquids from the three extractions were combined to obtain the ethyl acetate extract solution. Extraction was then performed with n-butanol and purified water using the same method to obtain the n-butanol extract solution and the aqueous phase solution. The three extract solutions were concentrated to a certain volume by rotary evaporation, then freeze-dried and weighed. The extraction yield was calculated. The ethyl acetate, n-butanol, and purified water extracts of the crude *Lycopodium clavatum* extract were then used for further processing.

[0040] See attached table for yields of different extraction phases. Figure 1 The yield of the n-butanol extract phase was higher than that of the ethyl acetate extract phase, which was higher than that of the aqueous phase. The yield of the 70% ethanol extract in the aqueous phase was greater than that in the ethyl acetate phase.

[0041] Based on the overall analysis and comparison of the extraction yields of different solvents and different extraction phases of *Lycopodium clavatum*, the crude ethanol extract of *Lycopodium clavatum* was finally selected as the effective part of the candidate raw material for veterinary drugs, and related experiments on anti-swine pseudorabies virus were carried out in sequence.

[0042] Example 2: In vitro anti-PRV test of ethanol extract of *Lycopodium clavatum*

[0043] Main solution preparation:

[0044] Fetal bovine serum, trypsin, and antibiotics were filtered, aliquoted, and stored at -20°C for later use. Complete cell growth medium: 90 mL DMEM high-glucose medium was mixed with 10 mL fetal bovine serum and 1 mL antibiotics, and stored at 4°C. Cell maintenance medium: 98 mL DMEM high-glucose medium was mixed with 2 mL fetal bovine serum, and stored at 4°C. Cell cryopreservation solution: prepared according to a ratio of DMEM high-glucose medium: fetal bovine serum: DMSO = 6:3:1.

[0045] Drug solution preparation:

[0046] Concentration of crude ethanol extract: Weigh 1g of crude ethanol extract and dilute to 10mL with DMSO, achieving a concentration of 100mg / mL. Filter sterilize using a 0.22μm microporous membrane and store at 4℃ for later use. Dilute to the required concentration before use. The preparation methods for each extract phase of the crude ethanol extract are the same as above.

[0047] Cell resuscitation, culture, and cryopreservation:

[0048] Remove ST cells and thaw them rapidly in a 37°C water bath. Add 4 mL of culture medium and mix well. Centrifuge at 1000 rpm for 4 min to collect the cells. Aspirate the supernatant, add 2 mL of complete culture medium, and mix well. Add the cell suspension to a T25 culture flask and add 3 mL of complete culture medium. Incubate at 37°C with 5% CO2. When the cells reach approximately 90% confluence, aspirate the culture medium, wash with 1 mL of PBS, and repeat twice. Digest with 0.25% trypsin, gently pipette to form a cell suspension, and aliquot into 2–3 T25 culture flasks. Incubate at 37°C with 5% CO2. Digest and collect cells as before, calculate cell concentration and viability, add cryopreservation solution, mix well, aliquot into sterile cryovials, label, place in a programmed freezing box, and freeze overnight at -80°C. Transfer to a liquid nitrogen tank for storage.

[0049] Porcine pseudorabies virus (PRV) amplification:

[0050] Resuscitate ST cells into T25 cell culture flasks. When cells reach 80%–90% confluence at the bottom of the flask, aspirate the culture medium, wash with 1 mL of PBS, and repeat twice. Add 1 mL of viral stock solution for viral infection, gently shake, and incubate at 37°C for 1 hour for adsorption. Aspirate the viral solution, wash with 1 mL of PBS, and repeat twice. Add 4–5 mL of maintenance medium and continue culturing. When cytopathic effects reach 80%–90%, perform a freeze-thaw cycle at -80°C three times. After thawing, centrifuge horizontally at 12000 rpm at 4°C for 15 minutes. Collect the supernatant as the viral solution, aliquot it into 1.5 mL centrifuge tubes, and store at -80°C for long-term storage. Thaw on ice before use.

[0051] PRV TCID50 determination:

[0052] Cells were passaged to the second and third generations and seeded into 96-well cell culture plates at 10⁴ cells per well. Once a confluent monolayer was formed, the culture medium was aspirated, and the cells were washed with 1 mL of PBS twice. Different dilutions of virus solution (diluted 10-fold serially with cell maintenance medium to concentrations of 10⁻⁶) were then added. -1 -10 -8 Eight replicates were performed for each dilution, with an additional cell control. Cytopathic effect (CPE) was observed daily, and the number of wells with CPE was recorded. The viral TCID50 was calculated using the Reed-Muench method.

[0053] Example 3: Effect of rock extract on ST cell activity

[0054] Cells were passaged to the second or third generation and seeded into 96-well cell culture plates at 104 cells per well. Once the cells had grown into a confluent monolayer, the culture medium was aspirated, and the cells were washed with 1 mL of PBS. This process was repeated twice. The extract of *Gnaphalium affine* obtained in Example 1 was added to prepare drug solutions (diluted with serum-free culture medium at concentrations of 200, 100, 50, 25, 12.5…0.78 μg / mL), with six replicates for each concentration. Cells were cultured for 24 h, and cell viability was measured according to the CCK-8 kit instructions. The safe concentrations of each drug were then calculated.

[0055] Table 2. PRV in ST cells with TCID 50 Measurement

[0056]

[0057]

[0058] The TCID50 of the virus, calculated using the Reed-Muench method, is 105.39 / 0.1 ml. This means that diluting the virus solution to 105.39 and inoculating 100 μL of the solution can induce cytopathic effects in 50% of the cells, as shown in Table 2.

[0059] The extract of *Lycopodium clavatum* obtained in Example 1 was diluted to different concentrations, and cell lethality experiments were performed accordingly. The experimental results are shown in the appendix. Figure 2 .

[0060] From the appendix Figure 2 It was found that the stock solution of the ethanol extract of *Rhododendron molle* was toxic to ST cells at concentrations of 200–6.25 μg / mL, but not at 3.13–0.78 μg / mL. The ethyl acetate extract of the ethanol extract was toxic to cells at concentrations of 100 and 50 μg / mL, but not at 25–0.78 μg / mL. The n-butanol extract of the ethanol extract was toxic to ST cells at concentrations of 100–12.5 μg / mL, but not at 6.25–0.78 μg / mL. The aqueous phase of the ethanol extract was toxic to ST cells at 100 μg / mL, but not at other concentrations.

[0061] Example 4: Anti-PRV Infection Test of Rock Extract

[0062] (1) Administering medication first, followed by antiviral testing (prevention mode)

[0063] Cells were passaged to the second and third generations and seeded into 96-well cell culture plates at 10⁴ cells per well. Once a confluent monolayer was formed, the culture medium was aspirated, and the cells were washed twice with 1 mL of PBS. Drug solution was added, with six replicates per concentration. Cell and virus controls were also included. Cells were incubated at 37°C and 5% CO₂ for 4 hours. After incubation, the culture medium was aspirated, and the cells were washed twice with 1 mL of PBS. 100 μL of 100 TCID₅₀ virus solution was added per well, and the cells were incubated at 37°C and 5% CO₂ for 2 hours. After incubation, the virus solution was aspirated, and the cells were washed twice with 1 mL of PBS. Maintenance medium was added, and culture was stopped when 80% cytopathic effect was observed in the virus control. Cell viability was measured according to the CCK-8 assay kit instructions. The survival rates of ST cells in prophylactic mode with different extracts of *Gnaphalium affine* are shown in the attached figure. Figure 3 As shown.

[0064] From the appendix Figure 3 Compared with the virus group, treatment of ST cells with 0.78 and 1.56 μg / mL *Isochrysis galbana* ethanol extract increased PRV cell survival by 19.99% (P<0.01) and 20.47% (P<0.01), respectively. Treatment of ST cells with 0.78 μg / mL *Isochrysis galbana* ethanol extract in ethyl acetate phase increased PRV cell survival by 13.7% (P<0.01). Treatment of ST cells with 12.5 μg / mL *Isochrysis galbana* ethanol extract in n-butanol phase increased PRV cell survival by 30.54% (P<0.001). Treatment of ST cells with 0.78, 1.56, and 6.25 μg / mL *Isochrysis galbana* ethanol extract in n-butanol phase increased PRV cell survival by 15.46% (P<0.05), 15.31% (P<0.05), and 18.42% (P<0.01), respectively. This indicates that the ethanol extract and various extract phase substances of the rock ginseng have a significant protective effect against viral infection of ST cells in the preventive mode.

[0065] (2) Antiviral assay after adding virus and then adding drug (treatment mode)

[0066] Cells were passaged to the second and third generations and seeded into 96-well cell culture plates at 10⁴ cells per well. Once a confluent monolayer was formed, the culture medium was aspirated, and the cells were washed twice with 1 mL of PBS. Then, 100 μL of 100 TCID₅₀ virus solution was added to each well, and the cells were incubated at 37°C with 5% CO₂ for 2 hours. After incubation, the virus solution was aspirated, and the cells were washed twice with 1 mL of PBS. Then, drug solution at a safe concentration was added, with six replicates for each concentration. Cell and virus controls were also included. Cells were incubated at 37°C with 5% CO₂ until 80% cytopathic effect was observed in the virus control. Cell viability was then measured according to the CCK-8 assay kit instructions. The survival rates of ST cells with different extracts of *Gnaphalium affine* under different treatment modes are shown in the attached figure. Figure 4 As shown.

[0067] Depend on Figure 4 It was found that, compared with the virus group, the cell survival rate was increased by 12.26% when the concentration of the ethanol extract of *Isodon japonicus* was 1.56 μg / mL (P<0.01). The cell survival rates of the ethyl acetate, n-butanol, and aqueous phases of the ethanol extract of *Isodon japonicus* were not significantly different from those of the virus group, indicating that the extracts of the ethanol extract of *Isodon japonicus* had no significant therapeutic effect on virus-infected ST cells under the treatment modality.

[0068] (3) Antiviral assay of infected cells after drug-virus interaction (virus-killing mode)

[0069] Cells were passaged to the second and third generations and seeded into 96-well cell culture plates at 10⁴ cells per well. Once a confluent monolayer was formed, the culture medium was aspirated, and the cells were washed with 1 mL of PBS, repeated twice. Virus solution was prepared and mixed with a safe concentration of the drug solution to achieve a final viral concentration of 100 TCID₅₀, and incubated at 37°C with 5% CO₂ for 4 hours. The above virus solution was added to 96-well cell culture plates, with 6 replicates per concentration (100 μL per well), and incubated at 37°C with 5% CO₂ for 2 hours. The cells were then removed, the virus solution was aspirated, and the cells were washed with 1 mL of PBS, repeated twice. Maintenance medium was added for continued culture. Cell and virus controls were also included. Culture was stopped when 80% cytopathic effect was observed in the virus control. Cell viability was measured according to the CCK-8 assay kit instructions. The survival rates of ST cells under different cytotoxicity modes are shown in the attached figure. Figure 5 As shown.

[0070] From the appendix Figure 5 Compared with the virus group, treatment of ST cells with 0.78 and 1.56 μg / mL *Isochrysis galbana* ethanol extract increased PRV cell survival by 19.99% (P<0.01) and 20.47% (P<0.01), respectively. Treatment of ST cells with 0.78 μg / mL *Isochrysis galbana* ethanol extract in ethyl acetate phase increased PRV cell survival by 13.7% (P<0.01). Treatment of ST cells with 12.5 μg / mL *Isochrysis galbana* ethanol extract in n-butanol phase increased PRV cell survival by 30.54% (P<0.001). Treatment of ST cells with 0.78, 1.56, and 6.25 μg / mL *Isochrysis galbana* ethanol extract in n-butanol phase increased PRV cell survival by 15.46% (P<0.05), 15.31% (P<0.05), and 18.42% (P<0.01), respectively. This indicates that the ethanol extract and various extract phase substances of the rock ginseng have a significant protective effect against viral infection of ST cells in the preventive mode.

[0071] Analysis of the effective components of rock extract

[0072] Sample pretreatment:

[0073] The ethyl acetate phase of the crude ethanol extract of *Rhododendron molle* was screened for optimal activity through in vitro antiviral assays and then analyzed for composition. 50 mg of powder sample was weighed, added to 500 μL of 75% methanol-water mixture, vortexed for 60 s, sonicated for 30 min, centrifuged at 17000 g (relative centrifugal force) for 20 min, and the supernatant was collected for analysis.

[0074] Parameter settings:

[0075] The column temperature was 40℃, the sample loading volume was 2μL, the positive ion mode was: A: 0.1% formic acid water; B: 0.1% formic acid acetonitrile, the negative ion mode was: A: water (2mM ammonium acetate); B: acetonitrile, and the flow rate elution gradient is shown in Table 3.

[0076] Table 3 Elution gradient of mobile phase

[0077]

[0078] Mass spectrometry conditions:

[0079] The AB 5600 Triple TOF mass spectrometer can perform primary and secondary mass spectrometry data acquisition based on IDA function under the control of the control software (Analyst TF 1.7, AB Sciex). In each data acquisition cycle, the molecular ions with the strongest intensity greater than 100 are selected for acquisition of the corresponding secondary mass spectrometry data. The primary acquisition range is 50–1200, and the bombardment energy is 30 eV. The ESI ion source parameters are set as follows: nebulizer pressure (GS1): 60 Psi, auxiliary gas pressure: 60 Psi, curtain gas pressure: 35 Psi, temperature: 650℃, spray voltage: 5000V (positive ion mode) or -4000V (negative ion mode).

[0080] Data processing:

[0081] Format conversion: The data was first converted to .abf format using Analysis Base File Converter. Then, MSDIAL ver 4.24 software was used to perform peak finding, peak alignment, and other data processing on the converted .abf files. At the same time, based on the primary and secondary spectrum searches, the databases of Metlin, MassBank, MoNA, and HMDB were independently integrated (version V6.0). Blank samples were removed to obtain the identification results.

[0082] Data Analysis:

[0083] Excel 2016 was used for data organization and statistics, and Graphpad Prism 8 was used for data processing and analysis. Data are expressed as mean ± standard error. Statistical analysis used univariate analysis for comparison; P < 0.05 was considered statistically significant, P < 0.01 was considered statistically significant, and P < 0.001 was considered highly statistically significant.

[0084] Table 4. Mass Spectrometric Structure Analysis of Ethyl Acetate Phase Bioactive Components from Ethanol Extract

[0085]

[0086]

[0087] Note: "√" indicates that the substance was present under the conditions of this test, and "-" indicates that the substance was not detected under the conditions of this test.

[0088] The optimal active site was analyzed by LC-MS, and the results are shown in Table 5. Figure 5 As shown, the total ion chromatogram of the ethyl acetate phase of the ethanol extract was collected in both positive and negative ion modes (see attached image). Figure 6 and 7 Through literature and database retrieval and analysis, 19 compounds were identified, including 8 flavonoids, 4 polyphenols, 3 fatty acids, 1 sugar, 1 quinone, 1 vitamin, and 1 phenolic acid.

[0089] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. The application of *Rhizoctonia solani* extract in drugs for the prevention or treatment of pseudorabies in pigs; wherein the *Rhizoctonia solani* extract is the ethyl acetate phase of crude ethanol extract of *Rhizoctonia solani*.

2. The application as described in claim 1, characterized in that: The method for obtaining the rock extract includes the following steps: (1) Crush the rock powder and sieve it; (2) Extract the rock jujube with ethanol to obtain an ethanol extract of the rock jujube; (3) Extract the ethanol extract with ethyl acetate to obtain the ethyl acetate extract phase.

3. The application as described in claim 2, characterized in that, The ethanol mentioned is 70% ethanol.

4. The application as described in claim 2, characterized in that, The ethyl acetate extract from step (3) was concentrated by rotary evaporation, and the resulting concentrate was freeze-dried at low temperature to obtain a powdered rock extract.

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