CN113786413B

CN113786413B - Application of papaya polysaccharide on preparing antiviral drugs

Info

Publication number: CN113786413B
Application number: CN202111082845.6A
Authority: CN
Inventors: 宋桥明; 汪鋆植
Original assignee: Hubei Shaner Agricultural Development Co ltd
Current assignee: Hubei Shaner Agricultural Development Co ltd
Priority date: 2021-09-15
Filing date: 2021-09-15
Publication date: 2023-09-29
Anticipated expiration: 2041-09-15
Also published as: CN113786413A

Abstract

The invention provides application of wrinkled papaya polysaccharide in preparation of anti-influenza virus medicines. The application of the wrinkled papaya polysaccharide in preparing medicaments for resisting avian influenza virus. The composition of the wrinkled papaya polysaccharide and the H7N9 avian influenza inactivated vaccine is applied to preparation of medicines for resisting avian influenza virus. The papaya polysaccharide has antiviral effect, and can reduce death of chicken infected by viruses. The effect of the combination with vaccine is more obvious. Can be used in combination with vaccine for preventing and treating avian influenza. The papaya polysaccharide has growth promoting effect, and can promote chicken growth. Especially after the chicken is infected with virus, the chicken can resist virus and promote chicken growth. The effect of the combination with vaccine is more obvious.

Description

Application of papaya polysaccharide on preparing antiviral drugs

Technical Field

The invention relates to an antiviral drug for poultry, in particular to a drug for H7N9 avian influenza virus.

Background

The chaenomeles speciosa is the dry near-mature fruit of chaenomeles speciosa (Chaenmeles speciosa Nakai), and the long yang in Hubei is the main production place of the chaenomeles speciosa. Papaya has been used as a good medicine for treating diseases since ancient times, and has the effects of calming liver, relaxing tendons, harmonizing stomach, resolving dampness, reducing blood pressure, reducing blood glucose, softening blood vessels, protecting liver, reducing enzymes, maintaining beauty, resisting aging, preventing senile dementia, promoting blood circulation, prolonging life, improving human immunity and the like. Papaya has homology of medicine and food, and contains various nutritional ingredients and medicinal ingredients beneficial to human body, such as polysaccharide, organic acid, flavone, triterpene, amino acid, protease, vitamins, etc. The pharmacological researches on papaya organic acid, flavone and triterpene are more advanced, and the researches on papaya polysaccharide are mainly on the aspects of antioxidation and anti-tumor. In recent years, the polysaccharide has good immunoregulatory efficacy, so that the polysaccharide becomes a research hot spot, and the polysaccharide has wide application in the fields of food, medical treatment, health care and the like. Polysaccharide is one of the important components of wrinkled papaya. The research shows that the papaya polysaccharide has good antiviral activity, can be used as a feed additive, and can also be used for developing functional beverages, medicines and health care products.

Disclosure of Invention

Aiming at the technical problems, the invention provides application of wrinkled papaya polysaccharide in preparing anti-influenza virus medicines.

Furthermore, the wrinkled papaya polysaccharide is applied to the preparation of medicines for resisting avian influenza virus.

The composition of the wrinkled papaya polysaccharide and the H7N9 avian influenza inactivated vaccine is applied to preparation of medicines for resisting avian influenza virus.

The wrinkled papaya polysaccharide is mixed with chicken feed and then fed.

The mixing amount of the wrinkled papaya polysaccharide is 0.001-0.05% of the mass of the chicken feed.

The papaya polysaccharide has antiviral effect, and can reduce death of chicken infected by viruses. The effect of the combination with vaccine is more obvious. Can be used in combination with vaccine for preventing and treating avian influenza.

The papaya polysaccharide has growth promoting effect, and can promote chicken growth. Especially after the chicken is infected with virus, the chicken can resist virus and promote chicken growth. The effect of the combination with vaccine is more obvious.

Papaya polysaccharide can improve chicken immunity. The papaya polysaccharide can improve the content of antibodies (IgG) in serum and immune organ index of chicken after virus infection, is beneficial to antivirus and ensures chicken health.

Detailed Description

Example 1

Experimental material and instrument

Experimental materials: chaenomeles speciosa (Chaenmeles speciosa Nakai) was collected from the county prefecture of the long yang earth family, hubei province, areca county, town gate village, identified by the university of three gorges Wang Yubing professor.

The main reagent comprises: 95% ethanol, trichloroacetic acid, naOH,3500Da semipermeable membrane, shanghai Source leaf Bio Inc.

Main experimental instrument

Freezone vacuum freeze dryer, LABCONCO Inc. in America

5415R Small desk-top high-speed refrigerated centrifuge, ai Ben Germany, inc

NanoQuant microplate reader Tecan, switzerland

N-1300 rotary evaporator EYELA Co., ltd

GZX-9240MBE digital display air blast drying oven Shanghai Bo Xie medical equipment factory

SB-2000 type water bath Shanghai Ailang instruments Co., ltd

Extraction of papaya polysaccharide

10kg of papaya dried fruits, adding 95% ethanol, refluxing twice, 100L each time, and degreasing for 2h each time. Volatilizing ethanol on a papaya water bath kettle after ethanol reflux extraction, and drying at 80 ℃. Extracting with boiling water three times each time of 150L for 3 hr, mixing the three filtrates, and concentrating under reduced pressure to 10L. Adding 30L of 95% ethanol into the concentrated solution, precipitating with ethanol for 48h, centrifuging at 4000RPM for 10min, volatilizing ethanol on a water bath kettle, adding water for dissolution, adding trichloroacetic acid into an ice bath according to 40g/L, taking out, adding NaOH solution to adjust pH to neutrality after reacting for 20min, centrifuging at 4000RPM for 10min, dialyzing supernatant in a dialysis bag with running water for two days, concentrating the dialysate to 2L, adding 95% ethanol for 6L for precipitating overnight, centrifuging at 4000RPM for 10min, volatilizing ethanol on the water bath kettle, and freeze-drying to obtain the crude polysaccharide of the wrinkled papaya. 420g of brown powdery papaya polysaccharide is obtained, and the yield is 4.2%.

Example 2

Experimental study on anti-avian influenza effect of papaya polysaccharide and chicken immune growth promotion effect

Experimental material and instrument

Experimental materials:

a vaccine; an H7N9 avian influenza inactivated vaccine, qingdao Yibang bioengineering Co. H7N9 avian influenza virus, provided by the university of agricultural science and technology university of China.

MDCK cells, shanghai department of science cell bank

Chicken immunoglobulin G (IgG) ELISA kit kenodi biology ltd;

papaya polysaccharide was obtained from example 1.

Main experimental instrument

Ultra clean bench, su zhou purification plant limited

Carbon dioxide incubator, shanghai Santeng instruments Co., ltd

Amplification of virus in chick embryo

Taking a primary H7N9 avian influenza virus vein infection experimental chicken, and taking a nasal swab supernatant and lung tissue grinding fluid of the experimental chicken on the death day to amplify in 9-10 days old chick embryos. The amplification method is as follows:

(1) Checking the chick embryo and marking the chick embryo air chamber: the chick embryos were inspected using an egg lamp, the dead, unacceptable chick embryos were removed, and acceptable chick embryos were marked at the air chamber.

(2) Sterilizing chick embryo and perforating chick embryo: the labeled and alcohol sterilized chick embryos were gently placed in a biosafety cabinet and perforated 5mm above the labeling air chamber using the tips of sterile forceps.

(3) Inoculating a sample: the diluted virus sample was aspirated using a 1ml sterile syringe and the sample (0.1 ml/piece) was needled vertically down the opening.

(4) Hole sealing and incubation of chick embryos: after closing the chick embryo well with sterile wax sheet, it was transferred to a carbon dioxide incubator and incubated continuously for 48h at 37 ℃.

(5) Harvesting chick embryo allantoic fluid: after the cultivation, the chick embryos were stored in a refrigerator at 4℃overnight. After cooling overnight, the chick embryo is sprayed with 75% alcohol for disinfection, then placed in a biosafety cabinet, the outer shell of the chick embryo air chamber is broken by a blunt instrument, and the chick embryo air chamber is opened by sterile forceps. After opening the air chamber, allantoic fluid was aspirated using a sterile pipette and harvested in a 15ml centrifuge tube.

(6) Packaging and preserving chick embryo allantoic fluid: allantoic fluid harvested in a 15ml centrifuge tube is centrifuged to obtain a supernatant, and the supernatant is packaged in a frozen tube for sealing. And (5) preserving the mixture in a refrigerator at the temperature of 4 ℃ for standby.

Virus TCID ₅₀ Measurement of/ml

Microtiter plate preparation of MDCK cells

1) The monolayer MDCK cells in the cell culture flasks were rinsed with sterile PBS solution.

2) Cells were digested with trypsin at 37℃in an incubator with a carbon dioxide concentration of 5% for about 10min. After cell dispersion, the cell suspension was centrifuged and the supernatant was discarded.

3) Cell resuspension to 10 ⁵ Mu.l/ml, added to 96-well plates, 100. Mu.l/well. Culturing in an incubator with carbon dioxide concentration of 5% at 37deg.C for 20 hr.

Gradient dilution of viruses

1) 8 15ml centrifuge tubes were prepared. The dilution gradient is marked from high to low by-1, -2, -3, -4, -5, -6, -7, -8, respectively. 9ml of virus growth solution was added to each branch.

2) 1ml of allantoic fluid to be titrated is taken and added into a "-1" tube, and is blown and evenly mixed, and then 1ml is sucked from the "-1" tube and added into a "-2" tube. And so on 10-fold dilution of the virus stock.

3) The virus growth solution was rinsed and allowed to stand overnight and the MDCK cell growth solution was discarded.

4) And after rinsing, adding gradient dilution virus liquid into each column. And the like, 8 columns are used, each column is provided with 6 sample adding compound holes, and each hole is 100 mu l. After the completion of the sample addition, the cell plates were placed in an incubator at 37℃and a carbon dioxide concentration of 5% for 1 hour to adsorb viruses.

5) After the completion of the adsorption, 200. Mu.l of the virus growth solution was added to each well, and the mixture was allowed to stand in an incubator at 37℃with a carbon dioxide concentration of 5% for 72 hours. Statistics were performed based on cytopathic conditions. After statistics, the samples were rechecked with 1% turkey blood suspension. The titer of the samples was calculated according to the Reed-Muench formula based on the number of cytopathic wells at different dilutions. Determination of TCID ₅₀ /ml (half of the tissue cell infection dose).

Example 3

Papaya polysaccharide has effects of resisting avian influenza and promoting chicken immunity

140 chickens were selected at 90 days of age and randomly divided into 7 groups of 20 chickens.

Verify the following description is correct or not blank: healthy chickens not infected with H7N9 avian influenza disease are not vaccinated nor fed papaya polysaccharide, only with basal feed.

Model group: chickens infected with H7N9 avian influenza disease are not vaccinated nor fed papaya polysaccharide, only with basal feed.

Vaccine group: chickens infected with H7N9 avian influenza disease are vaccinated with the H7N9 avian influenza inactivated vaccine.

Papaya polysaccharide 1 group: papaya polysaccharide with mass fraction of 0.01% is added into basic feed for chickens infected with H7N9 avian influenza disease.

Papaya polysaccharide 2 group: papaya polysaccharide with mass fraction of 0.05% is added into basic feed for chickens infected with H7N9 avian influenza disease.

Papaya polysaccharide 3 group (vaccine): chicken infected with H7N9 avian influenza disease are vaccinated with H7N9 avian influenza inactivated vaccine by muscle, and papaya polysaccharide with mass fraction of 0.01% is added into basic feed.

Papaya polysaccharide 4 group (vaccine): chicken infected with H7N9 avian influenza disease are vaccinated with H7N9 avian influenza inactivated vaccine by muscle, and papaya polysaccharide with mass fraction of 0.05% is added into basic feed.

After one week, each chicken was inoculated nasally with 10 diluted with 0.1ml of physiological saline, except for the blank group ⁷ TCID ₅₀ /ml virus chick embryo allantoic fluid. The blank group of chickens was inoculated nasally with 0.1ml of physiological saline. And (5) grouping and isolating and feeding. When the chicken house is cleaned, the chicken house is sanitary. The water tank and the trough are cleaned every day, ventilation and ventilation are carried out every day, and the henhouse and the surrounding environment are sprayed and disinfected every week.

On the day of vaccination, papaya polysaccharide groups are respectively given to feeds added with papaya polysaccharide, papaya polysaccharide 1 and 3 groups are added with papaya polysaccharide with mass fraction of 0.01% in basic feeds, and papaya polysaccharide 2 and 4 groups are added with papaya polysaccharide with mass fraction of 0.05% in basic feeds; feeding continuously for 21 days. The subpterygium vein was collected 2 and 4 weeks after vaccination, serum was separated, and immune antibody level (IGg) was detected. And feeding basal feed to other groups and papaya polysaccharide groups in a non-adding period. The basic feed is corn flour, soybean meal and fish meal in a mass ratio of 10:2: 1.

Effect of papaya polysaccharide on chicken survival

The results are shown in Table 1. At 10 ⁷ TCID ₅₀ The dosage of/ml infects chicken, and the chicken obviously has clinical symptoms such as listlessness, anorexia and the like, and 5-10 infected chicken is death peak period. Over time, the papaya polysaccharide and vaccine combination group is recovered and gradually returns to normal. Papaya polysaccharide can reduce death of chicken infected by virus, and has combined effect with vaccineMore remarkable.

TABLE 1 Effect of papaya polysaccharide on chicken survival

Group of	Survival number (only)	Death (only)	Survival (%)
				Blank group	20	0	100**
Model group	4	16	20
				Vaccine group	13	7	65**
Papaya polysaccharide 1 group	8	12	40
				Papaya polysaccharide 2 group	10	10	50*
Papaya polysaccharide 3 group (vaccine)	16	4	80**
				Papaya polysaccharide 4 group (vaccine)	18	2	90**

P < 0.05P < 0.01 compared to model group

Effect of papaya polysaccharide on chicken weight gain.

The results are shown in Table 2. At 10 ⁷ The EID50 dose is used for infecting chickens, the chicken growth is obviously slowed down, the difference is obvious (P is less than 0.05) compared with a blank group, the weight gain effect of the papaya polysaccharide group combined vaccine group and model group is obvious compared with that of chickens, and the papaya polysaccharide can promote the growth of virus infected survival chickens, and the combined effect of the papaya polysaccharide and the vaccine is more obvious.

TABLE 2 Effect of papaya polysaccharide on chicken weight gain

P < 0.05P < 0.01 compared to model group

And (3) toxin counteracting detection: on day 3 of virus inoculation, chicken blood is collected, virus hemagglutination titer (HA) detection is carried out on 96-well micro-hemagglutination plates by using the 1% chicken erythrocyte suspension prepared at present according to a conventional method, and the HA is more than or equal to 2 ⁴ Positive, HA < 2 ⁴ Negative. Results: the hemagglutination inhibition test is used for detecting the hemagglutination titer of the chicken blood sample, and after toxicity attackThe hemagglutination titer is equal to or more than 2 in 3 days ⁴ Blank control hemagglutination potency < 2 ⁴ Indicating successful attack of the toxin.

Effect of papaya polysaccharide on IgG in chicken serum.

Is determined by: the concentration of IgG in the preserved serum samples was measured by ELISA, and the specific procedure was performed according to the IgG kit instructions. The chicken IgG standard concentration is as follows: 2400. 1200, 600, 300, 150, 75 μg/μl. Setting standard holes and sample holes according to the instruction, then incubating for 60min at 37 ℃ with a constant temperature oven, washing for 5 times, adding a substrate, incubating for 15min at 37 ℃ with a light-proof constant temperature oven, and finally adding a stop solution, and measuring the OD value of each hole at 450nm wavelength within 15 min. Drawing a standard curve according to the OD value of the standard substance as an abscissa and the concentration value as an ordinate, and calculating the content of IgG in the sample according to the standard curve (y= 2465.1X-201.3R ² =0.999). The results are shown in Table 3. The IgG content in serum is increased compared with that in a model group and a blank group 2 and 4 weeks after vaccine immunization, but the difference is not obvious, the IgG content in serum is increased compared with that in a blank group by a vaccine group, a papaya polysaccharide group and a papaya polysaccharide group, and the difference is obvious (P is less than 0.05 or P is less than 0.01), and the IgG content in serum of a virus infected surviving chicken can be increased by using the papaya polysaccharide alone, so that the effect of combining with the vaccine is more obvious.

TABLE 3 Effect of papaya polysaccharide on IgG in chicken serum (μg/ml)

P < 0.05P < 0.01 compared to blank

Effect of papaya polysaccharide on chicken immune organ index.

The results are shown in Table 4. The immune organ index of the vaccine group, the papaya polysaccharide group and the blank group is increased compared with the immune organ index of the vaccine group, the papaya polysaccharide group and the blank group, but the difference is not obvious, the difference is obvious (P is less than 0.05 or P is less than 0.01), the immune organ index of the surviving chicken infected by the virus can be increased by using the papaya polysaccharide alone, and the combined effect of the papaya polysaccharide group, the papaya polysaccharide group and the vaccine is more obvious.

TABLE 4 Effect of papaya polysaccharide on chicken immune organ index

Group of	Quantity (only)	Spleen index%	Thymus index%	Bursa fabricius index%
					Blank group	20	0.35±0.05	0.48±0.10	0.41±0.05
Model group	4	0.38±0.07	0.52±0.08	0.44±0.09
					Vaccine group	13	0.45±0.07*	0.57±0.05*	0.46±0.08*
Papaya polysaccharide 1 group	8	0.50±0.12**	0.71±0.09**	0.53±0.07**
					Papaya polysaccharide 2 group	10	0.54±0.08**	0.73±0.11**	0.55±0.12**
Papaya polysaccharide 3 group (vaccine)	16	0.57±0.06**	0.74±0.12**	0.56±0.08**
					Papaya polysaccharide 4 group (vaccine)	18	0.59±0.07**	0.76±0.13**	0.59±0.11**

P < 0.05P < 0.01 compared to blank

Claims

1. Application of Chaenomeles speciosa polysaccharides in preparing medicine for resisting avian influenza virus H7N9 is provided.

2. Application of composition of Chaenomeles speciosa polysaccharides and H7N9 avian influenza inactivated vaccine in preparing medicine for resisting avian influenza virus H7N9 is provided.

3. Use according to claim 1 or 2, characterized in that the wrinkled papaya polysaccharide is fed after being mixed with chicken feed.

4. The use according to claim 3, wherein the amount of the wrinkled papaya polysaccharide is 0.001-0.05% of the chicken feed.