Crude extract of fermented purslane liquid for anti-feline parvovirus and its application

Abstract

The present invention provides a crude extract of fermented purslane liquid against feline parvovirus and its application. The present invention uses Bacillus licheniformis to ferment purslane. The crude extract of the fermented purslane liquid prepared can not only resist feline parvovirus infection and inhibit the invasion of feline parvovirus into host cells, but also has the effect of improving the taste of cat food. When used to make traditional Chinese medicine, it can improve the taste of traditional Chinese medicine, has a feeding-inducing effect, and has little side effect on cats. The present invention has important theoretical and practical significance for the research of drugs against feline parvovirus.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a crude extract of purslane fermentation liquid for resisting feline parvovirus and application thereof. Background Art

[0002] Feline parvovirus (FPV), belonging to the Parvoviridae family and the Parvovirus genus, mainly causes panleukopenia in felines, with a high infection rate and high mortality rate, among which the mortality rate of kittens is even higher. Currently, feline parvovirus is prevalent worldwide, causing serious damage to domestic pets, wild animals, and economic animal breeding industries.

[0003] At present, there is no specific medicine or effective treatment for feline parvovirus. Clinically, the main treatment is to regulate the body's immunity, provide nutritional support to sick animals through intravenous injection, and provide symptomatic treatment such as fluid replacement. At the same time, treatment is carried out in combination with traditional Chinese and Western medicine. Acute deterioration is prone to occur in the late stage of treatment, and Chinese medicine tastes bitter, so animals do not like to eat it. Therefore, how to provide a drug with good taste and no toxic side effects against feline parvovirus is a technical problem that technicians in this field need to solve urgently.

[0004] Purslane is a common wild vegetable, and it is also recorded as a medicine in various medicines. It has the characteristics of fast reproduction, wide distribution, and abundant resources. It is cold in nature and sour in taste. It enters the liver and large intestine meridians and has obvious effects of clearing away heat and detoxifying, cooling blood and stopping bleeding, and stopping dysentery. In the current existing technology, there are few methods of using traditional Chinese medicine to fight against feline parvovirus, and there is no relevant record of Purslane fighting against feline parvovirus. Summary of the invention

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A crude extract of purslane fermentation liquid is prepared by fermenting purslane with probiotics, wherein the probiotics are Bacillus licheniformis.

[0007] Furthermore, the crude extract of purslane fermented liquid includes one of a water extract of purslane fermented liquid and an alcohol precipitate of a water extract of purslane fermented liquid.

[0008] A method for preparing a crude extract of purslane fermentation broth comprises the following steps:

[0009] (1) Grind purslane, pass through an 80-mesh sieve, weigh 7.5 g of purslane into a conical flask, and sterilize under high pressure at 121°C for 20 min;

[0010] (2) Mixing the sterilized purslane with LB medium, inoculating with Bacillus licheniformis culture liquid, and placing in a shaking incubator for fermentation;

[0011] (3) Boil the fermented purslane in a 100 °C water bath, and gently aspirate and collect the supernatant after centrifugation;

[0012] (5) Place the supernatant collected in step (3) in a 60 °C oven until completely dry, then add PBS buffer for dilution to obtain the water extract of the purslane fermentation broth;

[0013] (6) Add absolute ethanol to the supernatant collected in step (3), seal and refrigerate, let stand for 24 h, centrifuge to discard the supernatant, and retain the precipitate at the same time. The rotation speed is 5000 r, the time is 5 min, and place it in a 60 °C oven to volatilize the ethanol to concentrate it. The water extract and alcohol precipitate are finally diluted with PBS buffer to obtain the water extract and alcohol precipitate of the purslane fermentation broth.

[0014] Furthermore, the 50 mL system of the LB medium described in step (2) is: 0.5 g of tryptone, 0.25 g of yeast extract, 0.5 g of sodium chloride, and made up to 50 mL with ultrapure water.

[0015] Furthermore, the volume ratio of the Bacillus licheniformis bacterial liquid inoculated in step (2) is 3%, the fermentation temperature is 37 °C, the fermentation rotation speed is 180 r / min, and the fermentation time is 72 h.

[0016] Furthermore, the boiling time in step (3) is 30 min, the temperature is 100 °C, the centrifugation rotation speed is 5000 r, and the time is 15 min.

[0017] Furthermore, after adding absolute ethanol in step (6), the volume ratio of absolute ethanol in the supernatant reaches 80%.

[0018] An antiviral application of the crude extract of the purslane fermentation broth, wherein the crude extract of the purslane fermentation broth is used to resist feline parvovirus infection and inhibit the invasion of feline parvovirus into host cells.

[0019] An application of the crude extract of the purslane fermentation broth in improving the taste of cat food, by adding the crude extract of the purslane fermentation broth to cat food.

[0020] The use of the above-mentioned crude extract of the purslane fermentation broth in the preparation of drugs and / or foods for relieving, adjuvantly treating or treating animal diseases caused by feline parvovirus infection.

[0021] Advantages of the present invention:

[0022] The present invention ferments purslane with Bacillus licheniformis. The crude extract of the purslane fermentation broth prepared can not only resist feline parvovirus infection and inhibit the invasion of feline parvovirus into host cells, but also has the effect of improving the taste of cat food. When used in making traditional Chinese medicine, it can improve the taste of traditional Chinese medicine, has a palatability effect, and has little side effect on cats. The present invention has important theoretical and practical significance for the research of drugs against feline parvovirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 These are the experimental results of the crude extract of Purslane fermentation broth inhibiting virus invasion after infection, where A: FPV-infected cell pathological changes; B: normal cell control; C: positive drug ribavirin control; D: anti-FPV effect of ethanol precipitate extracted from water extract of Purslane fermentation broth, with a concentration of 2.5 mg / mL; E: anti-FPV effect of ethanol precipitate extracted from water extract of Purslane fermentation broth, with a concentration of 1.25 mg / mL.

[0024] Figure 2 These are the experimental results of the crude extract of Purslane fermentation broth inhibiting the early invasion of viruses into host cells, including: A: FPV-infected cell pathological changes; B: normal cell control; C: positive drug ribavirin control; D: the inhibitory effect of the alcohol precipitate extracted from the water extract of Purslane fermentation broth on FPV, with a concentration of 2.5 mg / mL; E: the inhibitory effect of the alcohol precipitate extracted from the water extract of Purslane fermentation broth on FPV, with a concentration of 1.25 mg / mL; F: the therapeutic effect of the alcohol precipitate extracted from the water extract of Purslane fermentation broth on FPV, with a concentration of 2.5 mg / mL; G: the therapeutic effect of the alcohol precipitate extracted from the water extract of Purslane fermentation broth on FPV, with a concentration of 1.25 mg / mL.

[0025] Figure 3 These are the experimental results of using crude extract of purslane fermented liquid to improve the taste of cat food. DETAILED DESCRIPTION

[0026] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0027] Example 1 A method for preparing a crude extract of purslane fermentation broth

[0028] (1) Use a grinder to grind purslane, pass it through an 80-mesh sieve, and weigh 7.5 g of purslane. Put it into a conical flask and sterilize it at 121°C for 20 min.

[0029] (2) Prepare 50 mL of LB medium: 0.5 g of tryptone, 0.25 g of yeast extract, and 0.5 g of NaCl, dilute to 50 mL with ultrapure water, and sterilize with high-pressure steam at 121°C for 20 min;

[0030] (3) Sterilized purslane was mixed with LB medium, and 3% of the volume ratio of Bacillus licheniformis solution was inoculated, and the mixture was placed in a shaking incubator at 37°C for 72 hours at a rotation speed of 180 r / min. At the same time, a control group without inoculation was prepared;

[0031] (4) Boil the fermented purslane in a 100 °C water bath for 30 min. After centrifugation, gently aspirate and collect the supernatant at a rotation speed of 5000 r for 15 min;

[0032] (5) Take half of the supernatant and place it in a 60 °C oven to evaporate the water and concentrate it. After complete drying, weigh it. The water extract is finally diluted with PBS buffer to obtain the water extract of the purslane fermentation broth, and store it in a -20 °C refrigerator;

[0033] (6) Add absolute ethanol to the other half of the supernatant to make the volume ratio of absolute ethanol in the supernatant reach 80%. Seal and refrigerate, let it stand for 24 h, centrifuge to discard the supernatant, and retain the precipitate at a rotation speed of 5000 r for 5 min;

[0034] (7) Place it in a 60 °C oven to evaporate the ethanol and concentrate it. After complete drying, weigh it. The water extract and alcohol precipitate are finally diluted with PBS buffer to obtain the water extract and alcohol precipitate of the purslane fermentation broth, and store it in a -20 °C refrigerator.

[0035] Example 2 Antiviral Application of a Crude Extract of Purslane Fermentation Broth

[0036] (1) Prepare a positive control drug, and the drug used is ribavirin;

[0037] (2) Cell culture: Place F81 cells (feline kidney cells) in a 1640 medium (1640 complete medium) containing 10% fetal bovine serum and 1% double antibody, and culture them in an incubator at 37 °C and 5% CO2. When the cells grow to 90%, digest and passage them;

[0038] (3) Virus activation: Take 1 mL of FPV virus seed and inoculate it on the monolayer F81 cells that have grown. Add 4 mL of cell maintenance medium and culture it in an incubator at 37 °C and 5% CO2. Observe the cytopathic effect of the cells under a microscope every 12 h. When the cytopathic effect reaches 90%, collect the virus. After collecting the cell flask, freeze-thaw it 3 times repeatedly to harvest the virus. After pipetting, transfer it to a centrifuge tube, centrifuge at 10000 r / min for 10 min, collect the supernatant for aliquoting, and store it in a -80 °C refrigerator for standby. The virus titer is determined by the Reed-Muench method and expressed as the 50% tissue culture infective dose (TCID50);

[0039] (4) Determination of the drug safety concentration of the water extract and the water extract and ethanol precipitation of Portulaca oleracea fermentation broth: When subculturing cells, prepare a cell suspension with 1640 complete medium, inoculate the cell suspension at 100 μL per well into a 96-well plate, and culture it in an incubator at 37 °C and 5% CO2 until the cells grow to a monolayer. Then add the crude extract of Portulaca oleracea fermentation broth at different concentrations, with 3 replicates for each concentration, and set a blank control at the same time. After 72 h, use the CPE (cytopathic effect) observation method to observe cell lesions and determine the drug safety concentration of the water extract and the water extract and ethanol precipitation of Portulaca oleracea fermentation broth;

[0040] (5) Mix the water extract and the water extract and ethanol precipitation of Portulaca oleracea fermentation broth at concentrations within the cell safety concentration range with an equal volume of 100 TCID50 virus solution for 2 h, and add 100 μL per well to a 96-well plate of F81 that has grown to a monolayer. Culture it in an incubator at 37 °C and 5% CO2 for 72 h, observe cell lesions under a microscope, and record them using the CPE method;

[0041] (6) Anti-invasion effect of the water extract and the water extract and ethanol precipitation of Portulaca oleracea fermentation broth after FPV infection: Add 2 mL of 100 TCID50 FPV to a 6-well plate with 80% cell growth. After incubating at 37 °C for 2 h, discard the supernatant, wash twice with PBS buffer, add the crude extract of Portulaca oleracea fermentation broth, discard the supernatant after 2 h, and add 1640 cell maintenance medium containing 2% fetal bovine serum and 1% double antibody. Set a virus control and a cell control at the same time, and culture in an incubator at 37 °C and 5% CO2 for 72 h ( Figure 1 );

[0042] (7) Early inhibitory effect of the water extract and the water extract and ethanol precipitation of Portulaca oleracea fermentation broth on FPV: Mix 100 TCID50 FPV and the drug solution in equal proportion and add them to a 6-well plate with 80% cell growth. After incubating at 37 °C for 2 h, discard the supernatant, wash twice with PBS buffer, and then add 1640 cell maintenance medium containing 2% fetal bovine serum and 1% double antibody. Set a virus control and a cell control at the same time, and culture in an incubator at 37 °C and 5% CO2 for 72 h ( Figure 2 ).

[0043] Table 1 Determination of the drug safety concentration of the water extract of Portulaca oleracea

[0044]

[0045] Note: The more "+", the better the cell state. The influence of the cell state at high concentrations shown in the experimental results may be that the insoluble fine particles in the crude extract have environmental hindrance effects such as attachment on the growth of pure cells in vitro culture.

[0046] As shown in Table 1, the drug safety concentration of the unfermented aqueous extract of purslane on cells is 2.5 mg / mL or lower, and after fermentation, the drug safety concentration is 5 mg / mL or lower.

[0047] Table 2 Determination of the drug safety concentration of the aqueous extract of purslane precipitated with ethanol

[0048]

[0049] Note: The more "+", the better the cell state. The influence of the cell state at high concentrations shown in the experimental results may be that the insoluble fine particles in the crude extract have an obstructive effect on the growth of pure cells in vitro culture, such as attachment.

[0050] As shown in Table 2, the drug safety concentrations of the aqueous extract of purslane precipitated with ethanol before and after fermentation are both 2.5 mg / mL or lower.

[0051] Table 3 Inhibitory effect of the aqueous extract of purslane on FPV

[0052]

[0053] Note: The more "+", the better the cell state

[0054] As shown in Table 3, the aqueous extract of fermented purslane shows an obvious antiviral effect on FPV at 5 mg / mL.

[0055] Table 4 Inhibitory effect of the aqueous extract of purslane precipitated with ethanol on FPV

[0056]

[0057] Note: The more "+", the better the cell state

[0058] As shown in Table 4, the aqueous extract of fermented purslane precipitated with ethanol has an obvious antiviral effect on FPV at 2.5 mg / mL and 1.25 mg / mL.

[0059] As Figure 1 shown, compared with the virus-infected control, the virus-infected cells treated with the aqueous extract of fermented purslane precipitated with ethanol grow better and there is no obvious cytopathic effect. It can be seen that the crude extract of the fermented purslane has a good effect of inhibiting virus invasion.

[0060] As Figure 2 shown, compared with the virus control, the cells treated with the crude extract of the fermented purslane grow better and there is no obvious cytopathic effect. It can be seen that the crude extract of the fermented purslane has a good early inhibitory effect on feline parvovirus.

[0061] Example 3 Application of a crude extract of fermented purslane liquid in improving the taste of cat food

[0062] The water-extracted and alcohol-precipitated extract of the purslane fermentation broth prepared in Example 1 was mashed and evenly added to the cat food. At the same time, cat food without the water-extracted and alcohol-precipitated purslane fermentation broth was used as a control to observe the feeding situation of the cats. The results are shown in Figure 3 .

[0063] As Figure 3 shown, compared with the non-added one, the cat food added with the water-extracted and alcohol-precipitated purslane fermentation broth is more favored by cats and has a feeding-inducing effect. Therefore, the extract of the purslane fermentation broth is made into traditional Chinese medicine, which improves the taste compared with traditional Chinese medicine, and cats do not reject eating traditional Chinese medicine.

[0064] In the present invention, specific embodiments are applied to elaborate the principle and implementation manner of the present invention. The description of the above embodiments is only used to help understand the method and its core idea of the present invention; at the same time, for those of ordinary skill in the art, according to the idea of the present invention, there will be changes in the scope of the specific implementation manner. In summary, the content of this specification should not be construed as a limitation to the present invention.

Claims

1. Use of a crude extract of fermented purslane liquid in the preparation of a drug against feline parvovirus, characterized in that, The crude extract is obtained by fermenting single purslane with Bacillus licheniformis; The preparation method of the crude extract of Purslane fermentation broth comprises the following steps: (1) Grind purslane, pass through an 80-mesh sieve, weigh 7.5 g of purslane into a conical flask, and sterilize under high pressure at 121°C for 20 min; (2) Mix the sterilized purslane with LB medium, inoculate with Bacillus licheniformis culture medium, place in a shaker, and ferment; (3) Boil the fermented purslane in a 100°C water bath, centrifuge and gently collect the supernatant; (5) placing the supernatant collected in step (3) in a 60° C. oven until completely dry, and then adding PBS buffer to dilute it to obtain a water extract of purslane fermentation liquid; (6) Add anhydrous ethanol to the supernatant collected in step (3), seal and refrigerate, let stand for 24 hours, centrifuge and discard the supernatant, while retaining the precipitate, at a speed of 5000r for 5 minutes, place in a 60°C oven to evaporate the ethanol and concentrate it, and finally dilute the water-extracted alcohol precipitate with PBS buffer to obtain the water-extracted alcohol precipitate of Portulaca oleracea fermentation liquid.

2. According to the preparation method described in claim 1, characterized in that, The 50 mL LB medium system described in step (2) is: 0.5 g tryptone, 0.25 g yeast extract, 0.5 g sodium chloride, and the volume is adjusted to 50 mL with ultrapure water.

3. According to the preparation method described in claim 1, characterized in that, The volume ratio of the Bacillus licheniformis culture solution introduced in step (2) was 3%, the fermentation temperature was 37° C., the fermentation speed was 180 r / min, and the fermentation time was 72 h.

4. According to the preparation method described in claim 1, characterized in that, In step (3), the boiling time is 30 minutes, the temperature is 100°C, and the centrifugal speed is 5000r for 15 minutes.

5. According to the preparation method described in claim 1, it is characterized in that, After anhydrous ethanol is added in step (6), the anhydrous ethanol volume ratio of the supernatant reaches 80%.

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