阅读说明：本技术 荔枝草多糖的抗病毒用途 (Antiviral application of common sage herb polysaccharide ) 是由 张敦林 唐波 周业飞 常晨 张道华 华涛 于 2021-08-17 设计创作，主要内容包括：本发明公开了荔枝草多糖的抗病毒用途,所述的荔枝草多糖为从荔枝草(Salvia plebeiaR．Br．)的全草中提取的多糖。所述的病毒为抗猪伪狂犬病毒,荔枝草多糖通过减少猪伪狂犬病毒引起的细胞病变,抑制猪伪狂犬病毒的吸附、入胞和复制,进而杀死猪伪狂犬病毒。本发明还提供一种抗病毒制剂,是将荔枝草多糖溶解于0.05～0.20 mol/L的PBS缓冲液或者DMSO得到浓度为200～800μg/mL的溶液,该水剂对猪伪狂犬病毒(Pseudorabies virus,PrV)具有良好的防治作用,是发展高效、绿色、生态农业的理想生物兽药。(The invention discloses an antiviral application of common sage herb polysaccharide, wherein the common sage herb polysaccharide is prepared from common sage herb (common sage herb) Salvia plebeia R.br.) from the whole plant. The virus is anti-porcine pseudorabies virus, and the common sage herb polysaccharide inhibits the adsorption, cell entering and replication of the porcine pseudorabies virus by reducing cytopathic effect caused by the porcine pseudorabies virus, so that the porcine pseudorabies virus is killed. The invention also provides an antiviral preparation, wherein the common sage herb polysaccharide is dissolved in 0.05-0.20 mol/L PBS buffer solution or DMSO to obtain a solution with the concentration of 200-800 mu g/mL, and the aqueous solution has a good prevention and treatment effect on porcine Pseudorabies virus (PrV), and is an ideal biological veterinary drug for developing high-efficiency, green and ecological agriculture.)

1. The application of herba Salviae Plebeiae polysaccharide in preparing antiviral medicine is provided.

2. The use as claimed in claim 1, wherein the preparation process of the common sage herb polysaccharide solid is as follows:

(1) weighing 20g of whole herb of common sage herb, cutting into pieces, putting the cut herb into a beaker, adding 500mL of water, heating and boiling for 1-2 h, filtering, removing filter residue, distilling to remove part of water to obtain 100mL of common sage herb water extract solution, and cooling to room temperature;

(2) adding 400mL of absolute ethanol to precipitate the polysaccharide in the solution, refrigerating and standing overnight, filtering, and drying to obtain crude polysaccharide;

(3) dissolving the obtained crude polysaccharide in 50mL of hot water, adding Sevege reagent into the dissolved liquid according to the volume ratio of 3:1 for protein removal treatment, and repeating the process until no protein is separated out;

(4) and (3) fully and uniformly mixing the dissolved liquid with a Sevage reagent, performing suction filtration, and performing freeze drying to obtain 4.28g of salvia plebeian polysaccharide.

3. The use of claim 1, wherein the virus is anti-porcine pseudorabies virus, and the lychee polysaccharide inhibits the adsorption, encytosis and replication of the porcine pseudorabies virus by reducing cytopathic effect caused by the porcine pseudorabies virus, thereby killing the porcine pseudorabies virus.

4. The use as claimed in claim 1, wherein the amount of the sage polysaccharide is 200-800 μ g/mL.

5. The use as claimed in claim 1, wherein the amount of the sage polysaccharide is 400-600 μ g/mL.

6. The use as claimed in claim 1, wherein the amount of the sage polysaccharide is 600 μ g/mL.

7. The use as claimed in claim 1, wherein the common sage herb polysaccharide is a solid obtained by extracting common sage herb polysaccharide with water as a solvent by a hot-boiling method and precipitating with ethanol.

8. An antiviral agent characterized by being obtained by dissolving the litchi polysaccharide obtained in claim 2 in a buffer solution, wherein the solvent used is PBS buffer solution or DMSO.

9. The antiviral preparation according to claim 7, wherein the concentration of the PBS buffer is 0.05-0.20 mol/L, and the concentration of the sage polysaccharide is 200-800 μ g/mL.

Technical Field

The invention relates to a new application of common sage herb polysaccharide in preparing antiviral drugs.

Background

The porcine Pseudorabies virus (PrV) belongs to the herpesviridae family, is similar to other herpesviruses, has latent infection on various nervous tissues of pigs under normal conditions, can cause various livestock and wild animals to generate heat, can cause acute infectious diseases with extreme itch (except pigs) and encephalomyelitis as main symptoms, is fulminant epidemic, can cause abortion, stillbirth and boar sterility of pregnant sows, mass death of newborn piglets, dyspnea and growth arrest of fattening pigs and the like, is one of important infectious diseases which harm the global pig industry, and is particularly important for preventing and controlling the spread of the virus in livestock.

At present, the drugs applied to resisting the porcine pseudorabies virus are mainly antiviral biological agents and western medicines. However, there is no drug that interferes with the host cell metabolism and only interferes with virus replication or directly kills viruses, and some viruses have drug resistance to antiviral drugs, and even have drug-dependent variations, which makes antiviral drugs not effective. The Chinese herbal medicine has less toxic and side effects and less residue, and has the advantages which cannot be achieved by western medicines in the aspect of antivirus. Many experiments show that the Chinese herbal medicine can interfere virus replication, and can play an antiviral role by regulating the immunity of an organism and the nonspecific anti-inflammatory reaction.

Common sage herb is the whole herb of common sage herb Salvia plebeia R.Br. of Salvia of Labiatae, also called as evening primrose, Chinese forest frog grass, wrinkled gianthyssop herb, and the like, is mainly produced in Jiangsu, Zhejiang, Anhui, Henan, Shandong and other places, is rich in resources, is firstly recorded in compendia of materia Medica, mainly contains polysaccharides, terpenoids, flavonoids, phenylpropanoids and volatile oils, has pharmacological activities of antibacterium, anti-inflammation, antioxidation and the like, and is mainly used for treating human tonsillitis, bronchitis, metrorrhagia, hematochezia, nephritic edema and the like. In the aspect of application of antiviral veterinary drugs, only see the patent application of compound veterinary drugs of Liujiakui and the like, namely an antiviral veterinary drug liquid and a preparation method thereof (application number: 201811238488.6), the patent adopts a plurality of composite components such as decoquinate, radix isatidis, honeysuckle, liquorice, radix bupleuri, astragalus mongholicus, white hyacinth beans, acanthopanax senticosus, honeysuckle stem, common sage herb and the like to prepare the antiviral veterinary medicine liquid, but the active ingredients of the medicine liquid are not clear, the types of viruses inhibited by the veterinary medicine are not clear, it only shows that the added radix isatidis, honeysuckle, liquorice, radix bupleuri and the like have obvious antiviral effect, and also shows that the added decoquinate can play a synergistic effect with important antiviral active ingredients to improve the efficacy of the finished product liquid medicine, the antiviral effect of common sage herb is not clear, and the patent adopts lactobacillus fermentation liquor of common sage herb extract in the compound veterinary drug. In a method for treating herpes virus infection, with publication number CN102083415A, entitled as a method for treating herpes virus infection, there is provided topical application of a nanoemulsion composition having antiviral or virustatic properties against herpes virus, which includes an antiviral main drug and other pharmaceutical adjuvants, in the oil from which the nanoemulsion is prepared, oil of litchis leaves is used as an alternative oil, but its use is only as an oil phase.

So far, no report of the application of the salvia plebeian polysaccharide to the livestock-type transmitted diseases caused by the porcine pseudorabies virus exists.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a new antiviral application of common sage herb polysaccharide.

The virus is porcine pseudorabies virus resistant. The action mechanism is that the litchis polysaccharide can reduce cytopathic effect caused by the porcine pseudorabies virus, inhibit the adsorption, cell entry and replication of the porcine pseudorabies virus and directly kill the porcine pseudorabies virus.

The common sage herb is the whole herb of common sage herb Salvia plebeia R.Br.

The invention provides a preparation for preventing or treating porcine pseudorabies virus infection, which comprises solid substances obtained by extracting common sage herb polysaccharide by a hot boiling method with water as a solvent and precipitating with ethanol.

The content of the solid sage polysaccharide in the preparation is 200-800 mug/mL, preferably 400-600 mug/mL, more preferably 600 mug/mL.

The preparation is obtained by dissolving the obtained common sage herb polysaccharide in a buffer solution, and the adopted solvent is PBS buffer solution or DMSO;

the concentration of the PBS buffer solution is 0.05-0.20 mol/L, preferably 0.10-0.12 mol/L.

The invention has the beneficial effects that the litchi chinensis polysaccharide has an antiviral effect on the porcine pseudorabies virus, inhibits the adsorption, cell entrance and replication of the porcine pseudorabies virus by reducing cytopathic effect caused by the porcine pseudorabies virus, has small environmental pollution and has no toxic or side effect on animals.

Drawings

FIG. 1 shows the standard curve and results of the measurement of the content of common sage herb polysaccharide, as shown in FIG. 1, the concentration of the glucose standard solution is in a good linear relationship with the absorbance (A: absorbance, C: concentration of the glucose standard solution) within the range of 10-50 mg/L;

FIG. 2 is a linear relationship diagram of the virus inhibition effect of common sage herb polysaccharide.

Detailed Description

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Preparation of solid sage polysaccharide (references: Wanya, Tian \31054; \31310, Huangjun Lin, et al., common sage Total polysaccharide extraction Process research [ J ]. Guangzhou chemical engineering, 2018,046(010):75-77)

(1) Weighing 20g of common sage herb, cutting, putting into a 1000mL beaker, adding 500mL of water, heating, boiling for 1-2 h (in the reference, the heating temperature is not more than 90 ℃, the temperature is increased to facilitate complete extraction), filtering, removing filter residues, distilling to remove part of water to obtain 100mL of common sage herb water extract solution, and cooling to room temperature.

(2) Adding 400mL of absolute ethanol to precipitate the polysaccharide in the solution, then placing the solution in a refrigerator at 4 ℃ for standing overnight, filtering, and drying to obtain crude polysaccharide. (none of the references, this step helps to remove impurities from the polysaccharide)

(3) Dissolving the obtained crude polysaccharide in 50mL of hot water, adding Sevege reagent (chloroform: n-butanol is 4: 1) into the dissolved liquid according to the volume ratio of 3:1 for protein removal treatment, and repeating the process until no protein is separated out.

(4) And (3) fully and uniformly mixing the dissolved liquid with a Sevage reagent, performing suction filtration, and performing freeze drying to obtain 4.28g of salvia plebeian polysaccharide.

Example 2

Determination of polysaccharide content of Salvia plebeia (reference: Wanya, Tian \31054; \31310, Huangjun Lin, et al., Total polysaccharide extraction of Salvia plebeia [ J ]. Guangzhou chemical, 2018,046(010):75-77)

(1) Preparation of glucose standard solution: accurately weighing 1.0100g of a glucose analytical pure sample, adding a small amount of deionized water for dissolving, and metering the volume to a 100mL volumetric flask to obtain a glucose standard solution with the concentration of 0.1000 g/L.

(2) Preparation of a standard curve: respectively and precisely measuring 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL and 1.0mL of glucose standard solution, placing the glucose standard solution into test tubes, adding distilled water into each test tube to 2.0mL, adding 1mL of 5% phenol solution, fully and uniformly mixing the solution, dropwise adding 5mL of concentrated sulfuric acid, uniformly mixing, standing for 5min, placing the solution into a 55 ℃ water bath, heating for 15min, taking out, naturally cooling for 5min to terminate the reaction, rapidly measuring, measuring the absorbance (A) at 490nm by adopting a UV method, taking the distilled water as a blank control, finally drawing a standard curve by taking the glucose concentration as an abscissa and the absorbance as an ordinate to obtain a glucose linear equation, wherein the glucose linear equation is shown in figure 1.

(3) Preparation of a sample solution: taking 1.0g of crude polysaccharide sample, adding distilled water to dissolve the crude polysaccharide sample, and fixing the volume to a 100mL volumetric flask to obtain a crude polysaccharide sample solution with the concentration of 0.1 g/L. Adding 0.25mL of crude polysaccharide sample solution into a test tube, adding distilled water to 2.0mL, adding 1mL of 5% phenol solution, fully and uniformly mixing the solution, dropwise adding 5mL of concentrated sulfuric acid, uniformly mixing, standing for 5min, placing in a water bath at 55 ℃ for heating for 15min, taking out, naturally cooling for 5min to terminate the reaction, rapidly measuring, and measuring the absorbance (A) at 490nm by adopting a UV method. The absorbance of the crude polysaccharide sample solution was measured to be 0.288, by the linear equation a ═ 8.56c +0.1936 (R)²0.99497) calculating the polysaccharide content in the crude polysaccharide sample to be 81.09 percent and the polysaccharide content in the whole herb of common sage herb to be 5.51 percent.

Example 3 preparation of an anti-porcine pseudorabies virus preparation of Salvia plebeia polysaccharide

An anti-porcine pseudorabies virus preparation is obtained by dissolving the solid litchi polysaccharide obtained in example 1 in 0.1mol/L PBS buffer solution, and the concentrations of the litchi polysaccharide in the obtained solution are 200 mug/mL, 400 mug/mL and 600 mug/mL respectively.

Example 4 measurement of Activity of Salvia plebeian polysaccharide against porcine pseudorabies Virus

The common sage herb polysaccharide prepared in example 3 is treated with the preparation with the concentration of 200 mug/mL, 400 mug/mL and 600 mug/mL for 10^3.0TCID₅₀the/mL PRV virus solution was inoculated simultaneously with ST cells grown in advance as a dense monolayer, with drug treatment and virus inoculation controls and cell controls (24-well plates). Culture supernatants from (un) treated PRV infected cells were collected at 24 hours and 48 hours (freeze-thaw) and virus content was measured. The results are shown in table 1 and fig. 2, and about 50% of cytopathic effect of the PRV inoculated control cell wells can be seen in the litchi polysaccharide antiviral experiment ST cell inoculation 24 hours. No lesion appeared in the 400. mu.g/mL and 600. mu.g/mL dose groups, about 10-20% of the slight lesions appeared in the 200. mu.g/mL dose groups, and the lichen polysaccharide control wells showed no visible change compared to the cell control wells. In the litchi polysaccharide antiviral experiment, after ST cells are inoculated for 48 hours, all cells in PRV-inoculated cell control wells are found to float. No lesions appeared in the 600. mu.g/mL dose group, about 20% lesions were seen in the 400. mu.g/mL dose group, and about 80% lesions were seen in the 200. mu.g/mL dose group.

TABLE 1

Example 5 real-time fluorescent quantitative PCR of Pseudorabies Virus wild Virus SYBR-Green I

The common sage herb polysaccharide prepared in example 3 is treated with the preparation with the concentration of 200 mug/mL, 400 mug/mL and 600 mug/mL for 10^3.0TCID₅₀The PRV virus solution/ml was inoculated simultaneously with ST cells grown in advance as a dense monolayer, while drug treatment and virus inoculation controls and cell controls (24-well plates) were set up. Culture supernatants from (un) treated PRV infected cells were collected at 48 hours (freeze-thaw)) The viral genome is extracted, and the fluorescent quantitative PCR is determined, with three replicates. According to a 'internal standard curve' established by a real-time fluorescent quantitative PCR method of porcine pseudorabies virus wild virus SYBR-Green I in the literature, and a cp value of a quantitative sample is analyzed. The results are shown in Table 2, the concentration of the lychee polysaccharide can inhibit the replication of the pseudorabies virus, and the virus copy number of the cell pores is lower as the concentration of the lychee polysaccharide is increased.

TABLE 2

Example 6 protection of Lissapo polysaccharide against lethal challenge in mice

Virulent PRV strain 100LD at lethal dose₅₀(SQ-20181210) the mice were challenged intraperitoneally with the comfrey polysaccharides (40mg/kg body weight, 60mg/kg body weight, 80mg/kg body weight, 100mg/kg body weight, 120mg/kg body weight and 140mg/kg body weight) 24 and 48 hours before (after) challenge, respectively, and a challenge control group and a drug control group were established. Survival was observed daily. The results are shown in Table 3, wherein the weight protection rate of 40mg/kg is 1/5, the weight protection rate of 60mg/kg is 1/5, the weight protection rate of 80mg/kg is 2/5,100mg/kg is 3/5,120mg/kg is 4/5,140mg/kg is 4/5, and therefore the optimum dosage of the litchis polysaccharide is 120 mg/kg.

TABLE 3

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.