Brevibacterium breviscapus extract and preparation method and application thereof

Abstract

The invention relates to a scutellaria breviscapus extract and a preparation method and application thereof, belonging to the technical field of animal medicine. The invention obtains an extract with high scutellaria breviscapus aglycone content by fermenting the whole plant of scutellaria breviscapus under anaerobic conditions; extracts the fermented product to obtain an extract with high scutellaria breviscapus aglycone content; the invention significantly increases the scutellaria breviscapus aglycone content by drying and crushing the whole plant of scutellaria breviscapus and then performing anaerobic fermentation. The obtained extract can significantly increase the effects of inhibiting virus replication and directly killing PRV, and is safe to use.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal medical disease prevention and control, and specifically relates to a breviscapus breviscapus extract and a preparation method and application thereof. Background Art

[0002] Chinese herbal medicines not only have antibacterial and antiviral effects, but also have the characteristics of wide sources, low prices, low toxicity and side effects, little drug residue, and no drug resistance. Breviscapine is the dried whole herb of the Asteraceae plant Erigeron breviscapine, which is cold, slightly bitter, sweet, warm and spicy. It is currently mainly used in the treatment of cardiovascular and cerebrovascular diseases.

[0003] The chemical components of Breviscapus scutellariae mainly include flavonoids, caffeoyl, aromatic acids, coumarins, pyrones, volatile oils, etc. Flavonoids mainly contain scutellarin, scutellarin and a very small amount of scutellarin aglycone. Scutellarin is a representative component of flavonoids in Breviscapus scutellariae, accounting for more than 90%. It is the main effective ingredient of Breviscapus scutellariae for the treatment of cerebrovascular diseases and the main active substance of Breviscapus scutellariae. According to Chinese national standards, scutellarin accounts for more than 90% in oral preparations of Breviscapus scutellariae and more than 98% in injection preparations. Scutellarin can be metabolized by enzymes, acids and intestinal flora in the body into scutellarin aglycone, the latter of which has higher biological activity and is easier to absorb, so scutellarin aglycone has better bioavailability. However, scutellarin aglycone is not easy to obtain commercially.

[0004] Pseudorabies (PR) in pigs is an acute infectious disease caused by pseudorabies virus (PRV). PRV can infect a variety of mammals, including carnivores, ruminants, rodents, and humans. After infection, it can cause central nervous system disorders accompanied by extreme itching, and cause severe clinical symptoms and acute death, resulting in significant economic losses to the breeding industry. Although scientists have been committed to the research and development of prevention and control methods and vaccines in the past few years, PR is still a serious infectious disease that is widely prevalent in China's pig industry, and its potential threat to human health has also attracted worldwide attention. Summary of the invention

[0005] In view of the problems existing in the background technology, the present invention provides a Breviscapus scutellariae extract and a preparation method and application thereof. The whole plant of Breviscapus scutellariae is dried and crushed and then subjected to anaerobic fermentation to significantly increase the content of scutellariae aglycone. The anaerobic fermentation product is extracted to obtain a Breviscapus scutellariae extract with a high scutellariae aglycone content. The extract can significantly improve the effects of inhibiting viral replication and directly killing PRV, and is safe to use.

[0006] In view of this, the first purpose of the present invention is to provide a method for fermenting Erigeron breviscapus, which can significantly increase the yield of scutellaria baicalensis aglycone in Erigeron breviscapus; the second purpose is to provide a Erigeron breviscapus extract, which is extracted from the anaerobic fermentation products of Erigeron breviscapus; the third purpose is to provide a medicine containing the Erigeron breviscapus extract, which can significantly improve the effect of inhibiting viral replication and directly killing PRV, and is safe to use.

[0007] The Breviscapus breviscapus fermentation method comprises the following steps:

[0008] (1) drying and crushing the whole plant of Erigeron breviscapus;

[0009] (2) Add sterile water for anaerobic fermentation without sterilization.

[0010] Furthermore, the solid-liquid ratio of Erigeron breviscapus pollen to sterile water is 1:8-10, the fermentation time is 4-8 days, the fermentation temperature is 34-40°C; and the fermentation process is left to stand.

[0011] Furthermore, the nitrogen content in the fermentation broth is 0.1-0.3 wt%, and the carbon content is 0.2-0.3 wt%.

[0012] A fermented product of Erigeron breviscapus is obtained by fermenting using the above-mentioned fermentation method.

[0013] A Brevibac breviscapus extract is obtained from the anaerobic fermentation product of Brevibac breviscapus.

[0014] Furthermore, the Breviscapus breviscapus extract is obtained by alcohol extraction.

[0015] Furthermore, the Breviscapus breviscapus extract is obtained by extraction with methanol.

[0016] The use of the above-mentioned Erigeron breviscapus extract in preventing or treating neurotropic porcine pseudorabies virus.

[0017] A medicine for preventing or treating neurotropic porcine pseudorabies virus, comprising the above-mentioned Erigeron breviscapus extract.

[0018] Beneficial effects of the present invention:

[0019] The present invention adopts an anaerobic fermentation process to ferment the whole plant of Breviscapus breviscapus, and does not perform sterilization treatment before fermentation, and does not add any fermentation bacteria during the fermentation process, so that the Breviscapus breviscapus can self-induce anaerobic fermentation, and the content of scutellaria baicalensis aglycone is significantly improved after fermentation, so as to obtain a fermentation product with a high scutellaria baicalensis aglycone content.

[0020] The fermentation product obtained after anaerobic fermentation of the present invention is extracted to obtain a scutellaria baicalensis aglycone extract. The extract has significantly improved light content compared with the original medicinal material and plays an anti-PRV effect by inhibiting viral replication and directly killing the virus in a dose-dependent manner, providing a material basis for further research and improvement of the active monomer scutellaria baicalensis aglycone component.

[0021] The Erigeron breviscapus extract obtained by the present invention has the characteristics of wide raw material sources, significant anti-neurotropic virus effect, safe use, etc. By using the extract to prepare veterinary drugs, veterinary drug residues in food can be reduced, veterinary public health safety can be improved, and the disease prevention and control capabilities of pig farms can be enhanced. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The content graphs of scutellarin and scutellarin aglycone and HPLC chromatograms of each treatment group in Example 2 of the present invention;

[0023] Figure 2 is a comparison chart of the single factor experimental results of Example 2 of the present invention;

[0024] Figure 3 Schematic diagram of the test steps for the in vitro inhibitory activity of the drug of Example 3 of the present invention on PRV;

[0025] Figure 4 This is a graph showing the effect of crude flavonoids from Example 3 of the present invention on the proliferation rate of trigeminal ganglion (TG) cells in mice;

[0026] Figure 5 is the protection rate of the drug in Example 3 of the present invention on TG cells;

[0027] Figure 6 The protection rate of the drugs on TG cells and the effect on the intracellular proliferation of PRV under different modes in Example 4 of the present invention;

[0028] Figure 7 This is the TG cell culture result of Example 4 of the present invention (100X);

[0029] Figure 8 is the immunofluorescence result of Example 4 of the present invention (100X);

[0030] Fig. 9 This is the 48h dynamic changes of TRL4TLR4 / MyD88 / NF-κB p65 protein in TG cells of Example 4 of the present invention;

[0031] Remark: Figure 1 In the table, ** indicates VS original drug group P < 0.01; * indicates VS original drug group P < 0.05; ++ indicates VS anaerobic self-fermentation group P < 0.01; 1 indicates scutellarin absorption peak; 2 indicates SCU absorption peak; 3, 4, 5, 6, 7, 8 indicate new absorption peaks;

[0032] Figure 2 In the figure, A is the material-liquid ratio; B is the time; C is the temperature; D is the nitrogen content; E is the carbon content; the same symbols between groups indicate no significant difference, P>0.05; adjacent symbols indicate significant difference, P<0.05; symbols are neither the same nor adjacent, P<0.01;

[0033] Figure 4 In the table, ns means P>0.05VS blank control; * means P<0.05VS blank control; ** means P<0.01VS blank control;

[0034] Figure 5 Figure A shows the protection rate of crude flavonoids from the original drug on cells; Figure B shows the protection rate of crude flavonoids from fermented Erigeron breviscapus on cells;

[0035] Figure 6 In the figure, A and B are blocking modes of action, C and D are inhibitory modes of action, and E and F are direct killing modes of action;

[0036] Figure 7 In the figure, A is the anatomical diagram of the mouse skull base; B is the TG cells cultured for 1 day; C is the TG cells cultured for 3 days; D is the TG cells cultured for 5 days; E is the TG cells inoculated with PRV for 24 hours; F is the TG cells inoculated with PRV for 48 hours;

[0037] Fig. 9 Figure A shows the expression of TRL4TLR4 / MyD88 / NF-κB; Figure B shows the comparison of TRL4 expression; Figure C shows the comparison of MyD88 expression; and Figure D shows the comparison of NF-κB p65 expression. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0039] In order to more clearly illustrate the present invention, it is described in detail through the following examples.

[0040] Example 1

[0041] Fermentation method of Erigeron breviscapus and extraction of fermentation products

[0042] The fermentation method of Erigeron breviscapus includes the following two steps:

[0043] (1) The whole plant of Erigeron breviscapus was collected from Yao'an County, Chuxiong Prefecture, Yunnan Province in July and August, and dried at 60°C and crushed;

[0044] (2) Without sterilization, sterile water was directly added at a material-liquid ratio of 1:8.4 for anaerobic fermentation at a fermentation temperature of 36°C-37°C; the fermentation process was kept constant temperature and allowed to stand, and the nitrogen content in the fermentation liquid was controlled to be 0.19wt% and the carbon content was controlled to be 0.22wt%. The fermentation lasted for 6 days to obtain a fermented product of Erigeron breviscapus. The fermentation nitrogen came from peptone and the carbon came from glucose.

[0045] Three parallel experiments were conducted under the above fermentation conditions, and the obtained scutellaria breviscapus fermentation product was analyzed, and the content of scutellaria breviscapus aglycone was (27.88±0.60) mg / g, with a deviation rate of 0.79%.

[0046] The fermentation product was added to 70% methanol solution and ultrasonically extracted for 50 minutes. The extraction was repeated twice and the residue was discarded and the extract was combined. The volume was rotary evaporated in a 45°C water bath until constant, and DMEM was added to make the volume to 100 mL and then packaged to obtain the Erigeron breviscapus extract (crude flavonoids liquid).

[0047] Example 2

[0048] Add different bacterial agents to compare the fermentation of Erigeron breviscapus

[0049] The collected Erigeron breviscapus is the same as that in Example 1, and is dried and crushed in the same manner as in Example 1.

[0050] (1) Selection of fermentation agents

[0051] Sources of the bacterial agents used: Bacillus subtilis (BNCC 188080) and Paecilomyces cicadae (BNCC 114807) were purchased from Beina Chuanglian Biotechnology Co., Ltd.; Lactobacillus plantarum (BMZ 133562), Bacillus coagulans (BMZ 132868) and Bacillus licheniformis (BMZ 132570) were purchased from Ningbo Mingzhou Biotechnology Co., Ltd.; Clostridium butyricum (CCTCC CB 20081667) and Aspergillus niger (CCTCC AF 2000036) were purchased from China Center for Type Culture Collection; three strains of distiller's yeast were purchased from Jinning, Kunming, and named JA, JB, and JC respectively.

[0052] Thawing and recovery of strains: Lactobacillus plantarum, Bacillus coagulans, and Clostridium butyricum were cultured in MRS liquid medium; three types of Yunnan local wine koji, Bacillus subtilis, Paecilomyces cicadae, and Bacillus licheniformis were activated in LB broth liquid medium at 37°C, 120r / min, and activated for 24h; Aspergillus niger was activated in potato dextrose liquid medium at 37°C, 120r / min for 36h. All strains were inoculated to the fourth generation every 24h at a rate of 10%.

[0053] (2) Fermentation

[0054] The experiment was divided into 7 probiotic fermentation groups, 3 koji fermentation groups, 2 self-induced fermentation groups (fermented by the method of Example 1), 1 sterile control group and 1 original drug group, and fermented under the same conditions. Each group was repeated 3 times, and the fermentation was terminated on the 5th day.

[0055] Probiotics and koji fermentation group: weigh 10g of Erigeron breviscapus into a sterile fermentation tank, sterilize with high-pressure steam at 121℃ for 30min, cool to room temperature, add 30mL of sterile water, and then add 10mL of activated bacterial solution respectively. The fermentation conditions are 37℃ and stirring at 120r / min.

[0056] Self-induced fermentation group: weigh 10g of Erigeron breviscapus into a sterile fermentation tank, add 40mL of sterile water without high pressure sterilization, and the self-induced aerobic fermentation conditions are 37℃, 120r / min shaking culture, and the self-induced anaerobic fermentation conditions are sufficient paraffin liquid sealing and constant temperature standing at 37℃.

[0057] Sterile control group: weigh 10g of Erigeron breviscapus into a sterile fermentation tank, sterilize with high-pressure steam at 121℃ for 30min, cool to room temperature, add 40mL of sterile water, and the treatment conditions are 37℃ and stirring at 120r / min.

[0058] Technical drug group: unfermented Erigeron breviscapus technical drug (Erigeron breviscapus whole plant dry powder).

[0059] (3) Extraction

[0060] The same method as in Example 1 was used to prepare different experimental groups to obtain Erigeron breviscapus extract.

[0061] (4) Results comparison

[0062] HPLC analysis was performed on the extracts of Breviscapus scutellariae in different experimental groups, and the contents of scutellarin and scutellarin aglycone were calculated.

[0063] HPLC chromatogram Figure 1 As shown in (A): Compared with the original drug group, the peak areas of peak 1 (scutellarin) and peak 2 (scutellarin aglycone) of all fermentation groups changed to varying degrees; the new absorption peaks 3, 4, 5, 6, and 7 (unidentified) produced by the 7 probiotic groups and 3 koji groups all appeared in the sterile control group, indicating that the selected bacteria under the experimental conditions failed to significantly change the composition of Scutellaria breviscapus; the peak 2 (scutellarin aglycone) of the two auto-induced fermentation groups became the main peak, and two obvious new absorption peaks 7 and 8 (unidentified) were also produced, indicating that the auto-induced fermentation had a significant effect on the composition of Scutellaria breviscapus.

[0064] The contents of scutellaria breviscapus extracts obtained from different experimental groups are shown in the attached figure. Figure 1(B) shows that compared with the original drug group, the content of scutellaria baicalensis in the sterile control group was significantly reduced, indicating that high temperature and high pressure treatment would destroy scutellaria baicalensis in Scutellaria breviscapus; the content of scutellaria baicalensis in the 7 probiotic groups and 3 koji groups was lower than that in the sterile control group, indicating that the fermentation treatment further consumed scutellaria baicalensis. The content of scutellaria baicalensis in all fermentation treatment groups was extremely significantly lower than that in the original drug group (P < 0.01), indicating that the fermentation treatment could not increase the content of scutellaria baicalensis.

[0065] The contents of scutellaria breviscapus aglycone in the extracts of different experimental groups are shown in the attached figure. Figure 1 (C) shows that compared with the original drug group, the content of scutellarin aglycone in the sterile control group was not significantly different (P>0.05), indicating that high pressure treatment had little effect on its content; the content of scutellarin aglycone in the Lactobacillus plantarum, Clostridium butyricum, Bacillus subtilis, Aspergillus niger and Paecilomyces cicadae groups was not significantly different (P>0.05), and the content of scutellarin aglycone in the fermentation groups of Bacillus coagulans, Bacillus licheniformis, koji A and koji B was significantly or extremely significantly reduced (P<0.05 or P<0.01). This shows that the selected 7 strains of probiotics and koji A and B are not suitable for fermentation of scutellarin aglycone; the content of scutellarin aglycone in koji C, self-induced anaerobic and aerobic fermentation groups was extremely significantly increased (P<0.01), which was 2.06, 13.04 and 11.39 times higher than that of the original drug, respectively, indicating that koji C and induced fermentation have the effect of enriching scutellarin aglycone. The content of scutellarin aglycone in the self-induced anaerobic fermentation group was significantly higher than that in all other fermentation groups (P<0.01). Therefore, the content of scutellarin aglycone was selected as the evaluation standard for the optimization of fermentation conditions, and the fermentation condition optimization experiment was carried out based on the self-induced anaerobic fermentation.

[0066] Single factor experiment:

[0067] The key factors affecting the fermentation of Erigeron breviscapus, namely, solid-liquid ratio, time, temperature, nitrogen source addition and carbon source addition, were selected as single factor variables, and other factors remained unchanged. The samples were divided into 5 groups and single factor experiments were carried out in sequence under self-induced anaerobic fermentation conditions.

[0068] (1) Water single factor experiment: fermentation time 5 days, temperature 37 °C, fermentation liquid is sterile water, water ratio is 1:2, 1:4, 1:6, 1:8, 1:10, 1:12, 1:14;

[0069] (2) Fermentation time single factor experiment: temperature 37°C, fermentation liquid was sterile water, material-liquid ratio was 1:8, and the time was 2, 4, 6, 8, 10, 12, and 14 days respectively;

[0070] (3) Temperature single factor experiment: temperature 37°C, fermentation time 6 days, fermentation liquid is sterile water, solid-liquid ratio is 1:8, temperatures are 28°C, 31°C, 34°C, 37°C, 40°C, 43°C, 46°C respectively;

[0071] (4) Single factor experiment of nitrogen source (peptone) addition: temperature 37°C, fermentation time 6 days, solid-liquid ratio 1:8, different concentrations of nitrogen source added to sterile water, respectively 0.4%, 0.6%, 0.8%, 1%, 2%, 3%, 4%;

[0072] (5) Single factor experiment on the amount of carbon source (glucose) added: temperature 37 °C, fermentation time 6 days, solid-liquid ratio 1:8, different concentrations of carbon source added to sterile water, respectively 0.4%, 0.6%, 0.8%, 1%, 2%, 3%, 4%.

[0073] The contents of scutellaria breviscapus aglycone in extracts under different conditions were obtained as follows Figure 2 shown.

[0074] Example 3

[0075] In vitro test on the anti-PRV effect of Erigeron breviscapus extract

[0076] 1. Pseudorabies virus (PRV) amplification:

[0077] Resuscitate ST cells in a T25 cell culture flask. When the cells grow to 80% to 90% confluence at the bottom of the culture flask, aspirate the culture medium, add 1mL PBS to wash, and repeat twice. Add 1mL of virus stock solution for virus infection, shake gently and place in a 37℃ cell culture incubator for adsorption for 1h. Aspirate the virus solution, add 1mL PBS to wash, repeat twice, and add 4-5mL maintenance culture medium to continue culturing. When the cell lesion reaches 80% to 90%, freeze and thaw repeatedly at -80℃ for 3 times. After thawing, centrifuge horizontally at 12000rpm, 4℃, centrifuge for 15min, take the supernatant as the virus solution, divide it into 1.5mL centrifuge tubes, store at -80℃ for a long time, and place on ice to thaw when using.

[0078] 2. PRV TCID50 determination

[0079] The cells were passaged to the second or third generation and inoculated into a 96-well cell culture plate, with 104 cells per well. When the cells grew into a monolayer, the medium was aspirated and 1 mL of PBS was added for washing. The plate was repeated twice. Different dilutions of virus solution were added (diluted by 10-fold gradient method with cell maintenance solution, with concentrations of 10-1 to 10-8, respectively). Eight replicates were performed for each dilution. A cell control was set up. The cytopathic effect (CPE) was observed daily, the number of cytopathic wells was recorded, and the TCID50 of the virus was calculated according to the Reed-Muench method.

[0080] 3. Determination of safe drug concentration

[0081] TG cells were seeded into 96-well plates at a density of 3×104 cells / well. After the cells grew to confluence, they were gently washed once with PBS, and then the test drugs diluted with maintenance medium were added. The working concentrations were 0.05, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 50, 60 mg / mL respectively. A cell blank control without drugs was also set up. There were 8 replicates in each group. After culturing for 48 h, the OD450 was measured according to the instructions of the CCK-8 kit, and the cell proliferation rate was calculated according to formula (2-1).

[0082]

[0083] 4. Determination of the minimum effective concentration of the drug against PRV

[0084] The experiment was divided into a raw drug group, a fermentation group (the Erigeron breviscapus extract obtained by the method of Example 1, the same below), a PRV group and a blank group. The experimental steps were as Figure 2 shown. Based on the maximum safe concentration of the drug, the drug was diluted with maintenance medium, and the working concentrations were 0.12, 0.23, 0.47, 0.94, 1.88, 3.75, 7.50, 15.00 mg / mL respectively; 100 μL / well. After treatment at 37 °C with 5% CO2 for 2 h, PRV was inoculated into the cells at a final concentration of 100-fold TCID50 dose. At the same time, a PRV group (only adding the virus solution) and a blank control (only adding the maintenance medium) were set. After 48 h of infection, the OD450 was measured according to the instructions of the CCK-8 kit. The protection rate of each concentration of the drug solution against PRV-infected cells was calculated according to formula (2-2), and the therapeutic index (TI) of the drug was calculated according to formula (2-3) to evaluate the inhibitory activity and safety index of Erigeron breviscapus and fermented Erigeron breviscapus crude flavonoids against PRV. The larger the therapeutic index, the safer the drug. Among them, TI>2 is effective and low-toxic, 1<TI<2 is low-effective and toxic, and TI<1 has no clinical use value.

[0085]

[0086] 5. Verification of the antiviral mode of the drug

[0087] It was divided into a raw drug group, a fermentation group, a PRV group and a blank group, and the experiment was carried out under three action modes of blocking, inhibition and direct killing. Based on the maximum safe concentration and the minimum effective concentration of the drug, four concentrations of 0.5, 1, 2, 4 mg / mL were selected. The raw drug group was abbreviated as Y0.5, Y1, Y2 and Y4, and the fermentation group was abbreviated as F0.5, F1, F2 and F4. (96-well plate: 3×104 cells / well, 0.1 mL / well of the drug solution or maintenance medium, 8 replicates; 24-well plate: 9×104 cells / well, 0.3 mL / well of the drug solution or maintenance medium, 3 replicates).

[0088] Blocking effect: The cells were treated with different concentrations of drug solution for 2 hours, then taken out, inoculated with PRV and incubated for 2 hours, washed once with PBS, and continued to be cultured with maintenance solution.

[0089] Inhibitory effect: The cells were inoculated with PRV and incubated for 2 hours, then taken out, washed once with PBS, and then different concentrations of drug solution were added. After culturing for 2 hours, the maintenance medium was changed and the culture was continued.

[0090] Direct killing effect: Mix different concentrations of drug solution with PRV, incubate at 37℃ for 2 hours, then add the mixture to the cells, incubate for 2 hours, take out, wash once with PBS, and continue culturing with maintenance medium.

[0091] PRV was inoculated into cells at a dose of 100 times TCID50, and a blank group (maintenance solution only) and a PRV group (PRV only) were set up. The cells were tested after 48 hours of culture.

[0092] Detection method: The 96-well plate was operated according to the CCK-8 kit, and the cell protection rate was calculated according to formula (2-2). The PRV DNA in TG cells was extracted using a DNA extraction kit in a 24-well plate. The virus copy number was analyzed by an absolute qPCR method based on SYBR Green and primers designed for the PRV gE gene (upstream primer: 5'-CTACAGCGAGAGCGACAACGA-3'; downstream primer: 5'-CGACAGCGAGCAGATGACCA-3'). The target gene amplification fragment was 139 bp. The reaction procedure was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 59.2°C for 30 s, extension at 72°C for 30 s, 40 cycles; complete extension at 65°C for 5 s, 96°C for 5 s. According to the qPCR method established in the laboratory in the early stage, the PRV standard curve is shown in formula (2-4), R2=0.999, x is the copy number, and y is the Ct value. The copy number of PRV in TG cells was calculated.

[0093] y=-3.679x+35.72#(2-4)

[0094] 6. In vitro anti-PRV results

[0095] 6.1 TCID50 determination results (Table 4)

[0096] According to the Reed-Muench method, the median infectious dose of PRV on BHK cells was 10-9.71 / 0.1mL.

[0097] Table 4 TCID50 determination results

[0098]

[0099] 6.2 Determination of safe drug concentration

[0100] Effects of the original drug group and the fermentation group on the proliferation rate of TG cells Figure 4 As shown in the figure, when the concentration of the original drug group is ≤15mg / mL, the proliferation rate of TG cells is higher than 70%, which is a non-toxic concentration. Compared with the blank group, the original drug group concentration between 0.25 and 10mg / mL can significantly increase the proliferation rate of TG cells (P < 0.01) (A); when the concentration of the fermentation group is ≤35mg / mL, the proliferation rate of TG cells is higher than 70%, which is a safe concentration. Compared with the blank group, the fermentation group concentration between 6 and 30mg / mL can significantly increase the proliferation rate of TG cells (P < 0.01) (B). The maximum safe concentrations of the original drug group and the fermentation group for TG cells were determined to be 15mg / mL and 35mg / mL.

[0101] 6.3 Results of determination of the minimum inhibitory concentration of drugs for PRV

[0102] The results of the determination of the protection rate of TG cells infected with PRV by different concentrations of drugs are as follows Figure 5 The results showed that the minimum inhibitory concentration of crude flavonoids from the original drug on PRV was 0.47 mg / mL (A), SI = 15 / 0.47 = 31.91; the minimum inhibitory concentration of crude flavonoids from fermented Erigeron breviscapus on PRV was 0.23 mg / mL (B), SI = 35 / 0.23 = 152.17. This shows that both the original drug of Erigeron breviscapus and the crude flavonoids from fermented Erigeron breviscapus are low-toxic and effective drugs, and the fermented Erigeron breviscapus is superior to the original drug.

[0103] 6.4 Results of in vitro drug action test on PRV

[0104] 6.4.1 Blocking mode of action

[0105] The blocking effect of the original drug group and the fermentation group was not good. In the blocking effect, the OD values ​​of the original drug and fermentation groups were significantly lower than those of the blank group (P < 0.05) (Table 5), and the cell protection rates were all negative (A). Except for F2 and F4, the virus copy numbers were higher than those of the PRV group (P < 0.01) (B), indicating that only the F2 and F4 groups had a certain blocking effect on PRV entry into the cells, and the drugs in the other groups could promote PRV to enter TG cells and aggravate the TG cell pathology caused by the virus.

[0106] 6.4.2 Mode of Inhibition

[0107] The original drug group and the fermentation group had a certain inhibitory effect on PRV replication and inhibited PRV proliferation in a concentration-dependent manner. In the inhibition mode, the OD values ​​of Y0.5 and Y1 groups were lower than those of PRV group, with no significant difference (P>0.05) (Table 5), the protection rate was negative (C), and the virus copy number was lower than that of PRV group, but the difference was not significant (P>0.05) (D); the OD values ​​of Y2 and Y4 groups were significantly or extremely significantly higher than those of PRV group (P<0.05 or P<0.01) (Table 5), the cell protection rate was between 10.07% and 35.09% (C), and the virus copy number was extremely significantly or significantly lower than that of PRV group (P<0.05 or P<0.01); the OD values ​​of fermentation groups at all concentrations were extremely significantly higher than those of PRV group (P<0.01) (Table 5), the OD values ​​increased with increasing concentration, and the cell protection rate was between 57.6% and 74.17% (C); the virus copy number was extremely significantly lower than that of PRV group (P<0.01) (D), and decreased with increasing concentration.

[0108] 6.5. Direct poisoning mode

[0109] The toxicity of the original drug group and the fermentation group was obvious. Under the direct toxicity mode, the OD values ​​of the original drug group and the fermentation group were significantly or extremely significantly greater than those of the PRV positive group (P < 0.05 or P < 0.01) (Table 5), and the OD value increased with the increase of concentration. The protection rate of the original drug group was between 70.80% and 102.94%, and the protection rate of the fermentation group was between 91.40% and 108.42% (E), and the protection rate increased with the increase of concentration; the virus copy number of the original drug group and the fermentation group was significantly or extremely significantly lower than that of the PRV group (P < 0.05 or P < 0.01), and the virus copy number decreased with the increase of concentration.

[0110] In the inhibition and direct killing mode, the OD value of the fermentation group was greater than that of the original drug group (P < 0.05), and the virus copy number was less than that of the original drug group (at 0.5, 1, and 2 mg / mL, the difference was significant (P < 0.05), and at 4 mg / mL, the difference was not significant (P > 0.05)), indicating that at the same concentration, the fermented Erigeron breviscapus had better inhibition and direct killing effects on PRV than the original drug.

[0111] In summary, the original crude flavonoids and fermented breviscapus crude flavonoids had poor blocking effects on PRV, and the inhibition and direct toxicity were dose-dependent, with the best toxicity effect. The fermentation group was better than the original group ( Figure 6 ). The direct toxic effect mainly occurs outside the cell, so 4 mg / mL of fermented Erigeron breviscapus was selected for subsequent research under the inhibitory mode.

[0112] Table 5 Cell OD values

[0113] Group Blocking inhibition Direct Kill Y0.5 <![CDATA[1.36±0.16 b ]]> <![CDATA[0.72±0.06 f ]]> <![CDATA[2.17±0.17 c ]]> Y1.0 <![CDATA[1.33±0.17 bc ]]> <![CDATA[0.74±0.10 f ]]> <![CDATA[2.24±0.12 c ]]> Y2.0 <![CDATA[1.32±0.17 bc ]]> <![CDATA[0.88±0.04 e ]]> <![CDATA[2.42±0.10 b ]]> Y4.0 <![CDATA[1.31±0.17 bcd ]]> <![CDATA[1.21±0.08 d ]]> <![CDATA[2.56±0.06 ab ]]> F0.5 <![CDATA[1.13±0.19 de ]]> <![CDATA[1.50±0.07 c ]]> <![CDATA[2.44±0.14 b ]]> F1.0 <![CDATA[1.15±0.15 cd ]]> <![CDATA[1.52±0.08 c ]]> <![CDATA[2.45±0.08 b ]]> F2.0 <![CDATA[1.16±0.15 cd ]]> <![CDATA[1.60±0.07 c ]]> <![CDATA[2.65±0.14 a ]]> F4.0 <![CDATA[0.95±0.2 e ]]> <![CDATA[1.72±0.09 b ]]> <![CDATA[2.66±0.15 a ]]> PRV <![CDATA[1.38±0.13 b ]]> <![CDATA[0.75±0.04 f ]]> <![CDATA[1.30±0.13 d ]]> blank <![CDATA[2.54±0.09 a ]]> <![CDATA[2.07±0.15 a ]]> <![CDATA[2.53±0.10 ab ]]>

[0114] The same symbol between groups indicates no significant difference, P>0.05; adjacent symbols indicate significant difference, P<0.05; symbols that are neither the same nor adjacent, P<0.01

[0115] Example 4

[0116] Effects of fermented Erigeron breviscapus on the expression of TLR4, MyD88 and NF-κB p65 proteins

[0117] The experiment was divided into blank group, control group, PRV group, fermented Erigeron breviscapus group (FEF), and PRV+fermented Erigeron breviscapus group (PRV+FEF). The contrl group was not incubated with virus but only with maintenance solution; the PRV group was inoculated with 100 times TCID 50 After incubation for 2 h, the virus solution was removed and replaced with maintenance solution. In the FEF group, 4 mg / mL of fermented Erigeron breviscapus was added and the maintenance solution was replaced after 2 h. In the PRV+FEF group, cells were inoculated with 100 times TCID 50 After incubation for 2 hours, the PRV was taken out, washed once with PBS, and then 4 mg / mL of fermented Erigeron breviscapus crude flavonoids was added. After culturing for 2 hours, the maintenance medium was changed. The time after the maintenance medium was changed in each experimental group was counted as 0 hours. The cells were collected at 0, 12, 24, 36, and 48 hours of the experiment, and the expression of TLR4, MyD88, and NF-κB proteins was semi-quantitatively analyzed by Western blot.

[0118] Western blot test steps: extract total protein with RIPA strong cell lysis buffer (containing 1% protease inhibitor) according to the instructions; add 1 / 5 volume of protein loading buffer after quantification by BAC method, and denature in boiling water bath; load 20μg of total protein, separate by 8% SDS-PAGE, and transfer to PVDF membrane; block with 3% BSA (prepared with PBST) for 1h, incubate in primary antibodies β-actin (1:6000), MyD88 (1:2000), TRL4 (1:4000), NF-κB p65 (1:3000) at 4℃ overnight; wash with PBS three times, incubate with goat anti-rabbit secondary antibody (1:5500) for 2h; detect and take pictures with ECL chemiluminescence method on Tianneng imaging system; use Image J software to quantify the gray density of the bands, and the protein expression level is normalized to the β-actin level. The results are expressed as the ratio relative to the blank group cells.

[0119] (1) TG cell culture and inoculation results

[0120] Mouse trigeminal ganglion dissection Figure 7 (A) The milky white tissue is the trigeminal ganglion (A1), and the transparent connective tissue is the vascular membrane (A2).

[0121] Mouse TG cell growth Figure 7As shown in (BD), on the first day, the cells completed attachment to the wall and were oval or thin and narrow spindle-shaped, and the residual tissue or dead cells were dark black grape-shaped; on the second day, the cells gradually became larger and increased in number; on the fifth day, the cells reached 90% fusion and were regular multipolar structures or spindle-shaped, and the axon directions of the spindle-shaped cells were uniformly distributed.

[0122] The pathological changes of mice inoculated with PRV are shown in Figure 7 As shown in (E&F), TG cells were inoculated with PRV for 24 hours, and a large number of cells shrank and became rounded, with a small number of cavities. At 48 hours, a large number of cells aggregated and stacked to form giant cells, showing typical CPE.

[0123] (2) Immunofluorescence identification results

[0124] GFAP immunofluorescence detection of mouse TG primary cells Figure 8 As shown in the figure, the P2X7 positive rate is about 65%, and the cells are spindle-shaped (A); the GFAP positive rate is about 3%, the cells are star-shaped, with large cell bodies and many axons (B); the NueN positive rate is about 8%, the cell nucleus is large, and the DAPI staining is light (C). Based on the above observations, it is judged that the cultured cells are composed of 65% satellite glial cells, 3% astrocytes, 8% neurons and 24% unknown cells. The PRV positive rate is 100%, most of the virus particles are in the cell membrane (D1), and a small amount of virus particles are expressed on the cell nucleus (D2), indicating that PRV can invade TG cells.

[0125] (3)TCID 50 Measurement results

[0126] TCID 50 The half-infectious dose of PRV on BHK cells was calculated according to the Reed-Muench method to be 10 -9.71 / 0.1mL; the infection rate on TG cells is 10 -5.12 / 0.1mL, indicating that TG cells are less sensitive to PRV.

[0127] (4) Effects of fermented Erigeron breviscapus and PRV on the expression of TLR4, MyD88, and NF-κB p65 proteins in TG cells

[0128] The expression levels of TLR4, MyD88, and NF-κB proteins were Fig. 9Compared with the blank group, both PRV and FEF significantly upregulated TRL4 at 0h (P<0.05), and significantly downregulated it at 12h and 24h (P<0.05). The expression of TRL4 in the PRV+FEF group was extremely significantly higher than that in the blank group at 0h (P<0.01) and significantly higher than that in the FEF group, and extremely significantly lower than that in the blank group at 12h and 24h (P<0.01) and lower than that in the FEF group, showing the regulatory effect of superimposing PRV and FEF; at 36h, the PRV group significantly upregulated TRL4 (P<0.05), and FEF significantly downregulated TRL4 (P<0.05). The expression of TRL4 protein in the PRV+FEF group was significantly lower than that in the blank group (P<0.05) but higher than that in the FEF group, showing the neutralization of the regulatory effects of the two; at 48h, there was no significant difference between each group and the control.

[0129] The regulatory effects of PRV and FEF on MyD88 and NF-κB p65 proteins were basically the same as the change trend of TRL4 at 0, 12, 24, and 36 hours. At 48 hours, MyD88 and NF-κB p65 proteins were significantly upregulated in the PRV group (P < 0.05), and extremely significantly downregulated in FEF (P < 0.01). The PRV+FEF group was extremely significantly lower than the blank group (P < 0.01) but significantly higher than the EFE group (P < 0.05), showing a trend of neutralizing the regulation of the two.

[0130] PRV and fermented Erigeron breviscapus crude flavonoids can rapidly up-regulate the expression of TLR4, MyD88, and NF-κB p65 proteins in TG cells at 0h, and down-regulate protein expression at 12h and 24h; the drugs begin to play a therapeutic regulatory role at 36h, gradually weakening the protein up-regulation caused by PRV. This shows that fermented Erigeron breviscapus crude flavonoids have a certain regulatory effect on the expression of TLR4, MyD88, and NF-κB p65 in TG primary cells infected with PRV.

[0131] Example 5

[0132] Results of HPLC Methodological Study on Scutellarin and Scutellarin Aglycone

[0133] (1) Establishment of HPLC detection method for scutellarin and scutellarin aglycone

[0134] Chromatographic conditions: chromatographic column: Inert Sustain C18 (4.6×150mm, 5μm) chromatographic column; mobile phase: 0.5% formic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, elution was performed according to the elution order in Table 2-4, flow rate 0.8mL / min, detection wavelength 335nm, column oven 30°C, injection volume 10μL.

[0135] Preparation of standard stock solutions: Accurately weigh appropriate amounts of scutellarin and scutellarin aglycone standards, respectively, and add 70% methanol to prepare standard stock solutions with concentrations of 9.088 mg / mL and 12.640 mg / mL, respectively.

[0136] Preparation of test drug solution: Accurately weigh 0.1000 g of dry drug, add 2 mL of 70% methanol solution, ultrasonically extract for 40 min, and filter through a 0.22 μm organic filter membrane to obtain the solution.

[0137] HPLC methodology investigation: Accurately pipette 1-10mL of the standard mother solution prepared by the above method into a 10mL volumetric flask, dilute to the scale with methanol and shake well, determine the peak area, use the mass concentration of the standard as the horizontal axis (x) and the peak area as the vertical axis (y), draw a standard curve, and examine the linear relationship; take the standard mother solution, repeat the injection for 6 times in a row, record the peak area of ​​the standard, and calculate the RSD to examine the precision; take the standard mother solution and the drug solution, inject them at 0, 12, 24, 36, and 48h, record the peak area, calculate the RSD to examine the stability; take 6 portions of standard and original flavonoid solution, sample and analyze to calculate the content and RSD value, and examine the repeatability; accurately measure 6 portions of drug solution with known content, 1.0mL each, accurately add appropriate amounts of 2 kinds of standard mother solutions, sample and analyze, and calculate the sample recovery rate.

[0138] Table 6 Mobile phase elution order

[0139] Time (min) Mobile phase A (%) Mobile phase B (%) 0-5 80 20 5-30 70 30 30-55 45 55 55-65 45 55 65-68 80 20 68-70 80 20

[0140] (2) Results of HPLC Methodological Study on Scutellarin and Scutellarin Aglycone

[0141] Linear relationship:

[0142] Chromatograms of scutellarin and scutellarin aglycone standards are shown in Figure 1 (C), it can be seen that there are few impurity peaks, and the peak area is ≥99%, indicating that the standard is of high purity; the linear relationship results of the standard are shown in Table 7, R 2 ≥0.997 indicated that the concentrations of scutellarin and scutellarin aglycone were in the ranges of 0.988-9.088ug and 1.264-12.640ug, respectively, and there was a good linear relationship between the injection volume and the peak area.

[0143] Table 7 Linear relationship between scutellarin and scutellarin aglycone

[0144] Compound Peak time / min Linear range / mg Linear equations <![CDATA[R 2 ]]> Scutellarin 27.873 0.988～9.088 y=2E-06x-0.5253 0.997 Scutellarin aglycone 53.535 1.264～12.64 y=2E-07x+0.3392 0.998

[0145] Results of precision, stability and repeatability inspection:

[0146] The results of precision, stability and repeatability are shown in Table 8. The RDS were all less than 3%, indicating that the instrument had good precision, the sample solution was stable within 48 h, and the method had good repeatability.

[0147] Table 8 Results of precision, stability and repeatability

[0148]

[0149] (3) Results of sample recovery rate investigation

[0150] The results of the sample recovery of the original flavonoid solution are shown in Table 9. The mean sample recoveries of scutellarin and SCU were 98.56% and 98.00%, respectively, and the sample recoveries were ≥98%; the RSD values ​​were 2.46% and 2.85%, respectively, and RSD < 3%, indicating that this method has good accuracy.

[0151] In summary, this method can be used for the qualitative and quantitative detection of scutellarin and scutellarin aglycone in Brevib.

[0152] Table 9 Sample recovery test results

[0153]

[0154]

[0155] Note: In the manual, unless otherwise specified, the maintenance solution used is composed of 98 mL DMEM medium, 2 mL fetal bovine serum, 1 mL double antibody, and 0.1 mL amphotericin B (2.5 mg / L.

[0156] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than 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 in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A method for fermenting Erigeron breviscapus, characterized in that: The following steps are included: (1) Dry and crush the whole plant of Erigeron breviscapus; (2) Adding sterile water for anaerobic fermentation without sterilization; the solid-liquid ratio of Erigeron breviscapus pollen to sterile water is 1:8-10, the fermentation time is 4-8 days, and the fermentation temperature is 34-40°C; the nitrogen content in the fermentation liquid is 0.10-0.30wt%, and the carbon content is 0.20-0.30wt%.

2. A fermented product of Erigeron breviscapus, characterized in that: Obtained by the fermentation method according to claim 1.

3. A Breviscapus breviscapus extract, characterized in that The method is obtained by extracting the fermentation product of Breviscapus breviscapus according to claim 2 with 70% methanol.

4. Use of the Erigeron breviscapus extract according to claim 3 in the preparation of a drug for preventing or treating neurotropic porcine pseudorabies virus.

5. A drug for preventing or treating neurotropic porcine pseudorabies virus, characterized in that: The invention comprises the Breviscapus breviscapus extract as claimed in claim 3.

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