阅读说明：本技术 皱皮木瓜多糖在制备抗病毒的药物上的应用 (Application of chaenomeles speciosa polysaccharide in preparation of antiviral drugs ) 是由 宋桥明 汪鋆植 于 2021-09-15 设计创作，主要内容包括：本发明提供皱皮木瓜多糖在制备抗流感病毒的药物上的应用。所述的皱皮木瓜多糖在制备抗禽流感病毒的药物上的应用。所述的皱皮木瓜多糖与H7N9禽流感灭活疫苗的组合物在制备抗禽流感病毒的药物上的应用。本发明的木瓜多糖具有抗病毒作用,降低病毒感染鸡的死亡。与疫苗联用效果更显著。可与疫苗联用用于禽流感的预防与治疗。木瓜多糖具有促生长作用,可促进鸡生长。特别是鸡感染病毒后,既能抗病毒,又能促进鸡生长。与疫苗联用效果更显著。(The invention provides application of chaenomeles speciosa polysaccharide in preparation of a medicine for resisting influenza viruses. The application of the chaenomeles speciosa polysaccharide in preparing the medicine for resisting avian influenza virus is provided. The application of the composition of the chaenomeles speciosa polysaccharide and the H7N9 avian influenza inactivated vaccine in preparing the medicine for resisting avian influenza virus is provided. The pawpaw polysaccharide has an antiviral effect, and can reduce death of virus infected chickens. The effect is more obvious when the composition is used together with a vaccine. Can be used for preventing and treating avian influenza in combination with vaccine. The fructus Chaenomelis polysaccharide has growth promoting effect, and can promote chicken growth. Especially, after the chicken is infected with virus, the virus can be resisted, and the growth of the chicken can be promoted. The effect is more obvious when the composition is used together with a vaccine.)

1. Application of fructus Chaenomelis polysaccharide in preparing medicine for resisting influenza virus is provided.

2. Use of the chaenomeles speciosa polysaccharide of claim 1 in the preparation of a medicament against avian influenza virus.

3. The use of the composition of chaenomeles speciosa polysaccharide and H7N9 inactivated vaccine for avian influenza virus of claim 2 in the preparation of a medicament for treating avian influenza virus.

4. Use according to claim 2 or 3, wherein the chaenomeles speciosa is mixed with chicken feed and fed.

5. The use of claim 4, wherein the amount of the chaenomeles speciosa is 0.001-0.05% by weight of the chicken feed.

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 (Chaenmeles speciosa Nakai) is a dried nearly mature fruit of Chaenmeles speciosa, and the Yangtze yang in Hubei is the main production area of the Chaenomeles speciosa. Pawpaw is used as a good medicine for treating diseases since ancient times, and has the effects of calming the liver, relaxing the muscles and tendons, harmonizing the stomach, eliminating dampness, reducing blood pressure, reducing blood sugar, softening blood vessels, protecting the liver, reducing enzyme, beautifying, resisting aging, preventing senile dementia, promoting blood circulation, prolonging life, improving human immunity and the like. The pawpaw is medicinal and edible, and contains various nutrient components and medicinal components which are beneficial to a human body, such as polysaccharide, organic acid, flavone, triterpene, amino acid, protease, vitamin and the like. Pharmacological researches on organic acids, flavones and triterpenes of pawpaw are reported earlier, and the researches on pawpaw polysaccharide are mainly in the aspects of oxidation resistance and tumor resistance. In recent years, the polysaccharide has good immunoregulation effect, so that the polysaccharide becomes a research hotspot, and the polysaccharide is widely applied to the fields of food, medical treatment, health care and the like. The polysaccharide is one of the important components of the wrinkled pawpaw. The research finds that the pawpaw 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 the chaenomeles speciosa polysaccharide in preparation of a medicine for resisting influenza viruses.

Furthermore, the chaenomeles speciosa polysaccharide is applied to preparation of drugs for resisting avian influenza viruses.

The application of the composition of the chaenomeles speciosa polysaccharide and the H7N9 avian influenza inactivated vaccine in preparing the medicine for resisting avian influenza virus is provided.

The cockle pawpaw polysaccharide is mixed with the 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 pawpaw polysaccharide has an antiviral effect, and can reduce death of virus infected chickens. The effect is more obvious when the composition is used together with a vaccine. Can be used for preventing and treating avian influenza in combination with vaccine.

The fructus Chaenomelis polysaccharide has growth promoting effect, and can promote chicken growth. Especially, after the chicken is infected with virus, the virus can be resisted, and the growth of the chicken can be promoted. The effect is more obvious when the composition is used together with a vaccine.

The papaya polysaccharide can improve the chicken immunity. The pawpaw polysaccharide can improve the content of an antibody (IgG) in serum and immune organ indexes after the chicken is infected with virus, is beneficial to resisting virus and ensures the health of the chicken.

Detailed Description

Example 1

Experimental materials and instruments

Experimental materials: chaenomeles speciosa Nakai is collected from the gateway and puerto of the town of the aretan of the autonomous county of the Changyang Tujia of Hubei province and identified by professor of the royal jade soldier of the university of the three gorges.

The main reagents are as follows: 95% ethanol, trichloroacetic acid, NaOH, 3500Da semipermeable membrane, Shanghai-sourced leaf biology company.

Main experimental instrument

Freezone vacuum freeze dryer, LABCONCO, Inc., USA

5415R Small bench high speed refrigerated centrifuge German Albende Pvt Ltd

The NanoQuant microplate reader Tecan company, Switzerland

Model N-1300 rotary evaporator, EYELA Japan

GZX-9240MBE digital display air blast drying oven Shanghai Bochen industry Co., Ltd medical equipment factory

SB-2000 model Water bath Shanghai Ailang Instrument Co Ltd

Extraction of papaya polysaccharide

10kg of dried pawpaw fruits are added with 95% ethanol for refluxing twice, 100L each time and 2h each time, and degreased. Volatilizing ethanol from the pawpaw water bath pot after ethanol reflux extraction, and drying at 80 ℃. Extracting with boiling water for three times (150L each time, 3 hr each time), 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 the ethanol in a water bath kettle, adding water for dissolving, adding trichloroacetic acid at 40g/L in ice bath, reacting for 20min, taking out, adding NaOH solution, adjusting pH to neutral, centrifuging at 4000RPM for 10min, dialyzing the supernatant in a dialysis bag with flowing water for two days, concentrating the dialysate to 2L, adding 6L of 95% ethanol, precipitating with ethanol overnight, centrifuging at 4000RPM for 10min, volatilizing the ethanol in the water bath kettle, and freeze-drying to obtain crude Chaenomeles speciosa polysaccharide. 420g of brown powdery papaya polysaccharide was obtained, the yield being 4.2%.

Example 2

Experimental study on avian influenza resistance and chicken immunity growth promotion effect of pawpaw polysaccharide

Experimental materials and instruments

Experimental materials:

a vaccine; H7N9 inactivated vaccine for avian influenza, Qingdao Yibang bioengineering Co. H7N9 avian influenza virus, provided by the college of animal science and technology of the university of agriculture in Huazhong.

MDCK cells, Shanghai Zhongyao cell bank

Chicken immunoglobulin g (igg) ELISA kit knodi bio ltd;

papaya polysaccharides, obtained from example 1.

Main experimental instrument

Clean bench, Suzhou clean plant Co., Ltd

Carbon dioxide incubator, Shanghai Sagitar instruments Ltd

Amplification of viruses in chick embryos

And (3) taking the primary H7N9 avian influenza virus infected experimental chicken, and taking the nasal swab supernatant and the lung tissue grinding fluid of the experimental chicken on the death day to amplify in 9-10 day old chick embryos. The amplification method is as follows:

(1) inspecting chick embryos and marking chick embryo air chambers: and (5) checking the chick embryos by using an egg lighting lamp, removing the unqualified chick embryos which are dead, and marking the qualified chick embryos at the air chamber.

(2) Disinfecting chicken embryos and opening the chicken embryos: the chicken embryos which are marked and disinfected by alcohol are lightly placed in a biological safety cabinet, and the tip of a sterile forceps is used for punching a hole 5mm above a marking air chamber.

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

(4) Sealing and incubating chicken embryos: the chicken embryo hole is sealed by using sterile wax sheets and then transferred into a carbon dioxide incubator to be continuously cultured for 48 hours at 37 ℃.

(5) Chick embryo allantoic fluid harvest: after the culture, the embryos were stored in a refrigerator at 4 ℃ and cooled overnight. After the overnight cooling, 75% alcohol is sprayed on the chick embryos to sterilize, the chick embryos are placed in a biological safety cabinet, the shells of the chick embryo air chambers are knocked apart by a blunt instrument, and the chick embryo air chambers are opened by sterile forceps. The air chamber was opened and allantoic fluid was aspirated using a sterile pipette and harvested in a 15ml centrifuge tube.

(6) Subpackaging and storing chick embryo allantoic fluid: allantoic fluid harvested in a 15ml centrifuge tube is centrifuged to take supernatant, and the supernatant is subpackaged in a freezing tube and sealed. And storing in a refrigerator at 4 ℃ for later use.

Virus TCID₅₀Measurement of/ml

Microtiter plate preparation of MDCK cells

1) The single layer of MDCK cells in the cell culture flasks was rinsed with sterile PBS solution.

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

3) Cells were resuspended to 10⁵One/ml, added to a 96-well plate at 100. mu.l per well. Culturing in an incubator at 37 deg.C and 5% carbon dioxide for 20 hr.

Gradient dilution of virus

1) Prepare 8 tubes of 15ml centrifuge. The labels are-1, -2, -3, -4, -5, -6, -7 and-8 in the order of the dilution gradient from high to low, respectively. Each branch was filled with 9ml of virus growth medium.

2) Adding 1ml of allantoic fluid to be titrated into a "-1" tube, blowing and stirring uniformly, sucking 1ml from the "-1" tube, and adding into a "-2" tube. And the virus stock solution is diluted by 10 times by the same analogy.

3) Virus growth medium was used to rinse and stand overnight for MDCK cell growth medium to discard.

4) After rinsing, the virus solution is added in a gradient way in each row. The same analogy is repeated for 8 columns, each column of the sample adding and replying holes is 6, and each hole is 100 mu l. After the sample addition was completed, the cell plate was left to stand in an incubator at 37 ℃ and a carbon dioxide concentration of 5% for 1 hour to adsorb viruses.

5) After adsorption, the solution in the plate was discarded, 200. mu.l of the virus growth medium was added to each well, and the mixture was allowed to stand in an incubator at 37 ℃ and a carbon dioxide concentration of 5% for 72 hours. And counting according to the cytopathic condition. And rechecking by using a 1% concentration turkey blood suspension after statistics. According to the number of cytopathic holes of different dilutions, the titer of the sample was calculated according to the Reed-Muench formula. Determining TCID₅₀Ml (half of the tissue cell infectious dose).

Example 3

Papaya polysaccharide for resisting avian influenza and promoting chicken immunity

140 chickens of 90 days old are selected and randomly divided into 7 groups of 20 chickens.

Verify the correct or incorrect blank set of the following descriptions: healthy chickens not infected with H7N9 avian influenza disease are not inoculated with vaccine and fed with pawpaw polysaccharide, and only fed with basal feed.

Model group: chickens infected with H7N9 avian influenza disease were fed with neither vaccine nor papaya polysaccharide, but only basal feed.

Vaccine groups: the chicken infected with H7N9 avian influenza disease is inoculated with inactivated H7N9 avian influenza vaccine.

Group 1 of papaya polysaccharides: the chicken infected with H7N9 avian influenza disease is added with 0.01 percent of pawpaw polysaccharide in the basal feed.

And 2 groups of pawpaw polysaccharide: the chicken infected with H7N9 avian influenza disease is added with 0.05% of pawpaw polysaccharide in the basal feed.

Pawpaw polysaccharide group 3 (vaccine): the chicken infected with the H7N9 avian influenza disease is inoculated with the H7N9 avian influenza inactivated vaccine through muscles, and the pawpaw polysaccharide with the mass fraction of 0.01 percent is added into the basal feed.

Pawpaw polysaccharide group 4 (vaccine): the chicken infected with the H7N9 avian influenza disease is inoculated with the H7N9 avian influenza inactivated vaccine through muscles, and the pawpaw polysaccharide with the mass fraction of 0.05 percent is added into the basic feed.

One week later, each chicken was inoculated nasally with 10 diluted with 0.1ml of physiological saline, except for the blank group⁷TCID₅₀Allantoic fluid/ml virus from chick embryos. The blank group of chickens were inoculated nasally with 0.1ml of physiological saline. And (5) carrying out isolated feeding in groups. The chicken house is cleaned in time. Washing the water tank and the trough every day, ventilating every day at regular time, and spraying and sterilizing the henhouse and the surrounding environment every week.

The pawpaw polysaccharide group is respectively given to the feed added with the pawpaw polysaccharide from the day of vaccination, wherein 1 group and 3 group of pawpaw polysaccharide are added with the pawpaw polysaccharide with the mass fraction of 0.01 percent in the basal feed, and 2 group and 4 group of pawpaw polysaccharide are added with the pawpaw polysaccharide with the mass fraction of 0.05 percent in the basal feed; feeding continuously for 21 days. The blood was collected by the infrawinged vein 2 and 4 weeks after the vaccination, and the serum was isolated and the immune antibody level (IGg) was measured. And feeding the other groups and the pawpaw polysaccharide group with basic feed in the non-additive period. The basic feed comprises corn flour, bean pulp and fish meal in a mass ratio of 10: 2: 1.

Effect of papaya polysaccharide on the survival rate of chickens

The results are shown in Table 1. At 10⁷TCID₅₀The chicken infected by the dose of per ml obviously shows clinical symptoms such as listlessness, anorexia and the like, and the infected chicken 5-10 is the death peak. With the time, the combination of the pawpaw polysaccharide and the vaccine recovers firstly and gradually becomes normal. The pawpaw polysaccharide can reduce the death of virus infected chickens, and has more obvious effect when being combined with the vaccine.

TABLE 1 Effect of papaya polysaccharide on the survival of chickens

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

P < 0.05P < 0.01 in comparison with model group

Effect of pawpaw polysaccharide on chicken weight gain.

The results are shown in Table 2. At 10⁷The chicken infected by the dose of the EID50 has obviously slowed growth and obvious difference (P is less than 0.05) compared with a blank group, the weight gaining effect of the pawpaw polysaccharide group, the pawpaw polysaccharide group combined vaccine group and a model group is obvious compared with the chicken, the pawpaw polysaccharide can promote the growth of the virus-infected live chicken, and the combined effect of the pawpaw polysaccharide and the vaccine is more obvious.

TABLE 2 Effect of papaya polysaccharide on chicken weight gain

P < 0.05P < 0.01 in comparison with model group

And (3) toxin attacking detection: inoculating virus on day 3, collecting chicken blood, detecting virus hemagglutination titer (HA) on 96-hole micro hemagglutination plate with 1% chicken red blood cell suspension prepared at present by conventional method, wherein HA is not less than 2⁴Is positive, HA < 2⁴It was negative. As a result: performing hemagglutination potency detection on chicken blood sample by hemagglutination inhibition test, wherein the hemagglutination potency is more than or equal to 2 after 3 days of virus attack⁴Blank control hemagglutination titer < 2⁴Indicating successful counteracting toxic substance.

Effect of pawpaw polysaccharide on IgG in chicken serum.

The determination of (1): and (3) detecting the concentration of IgG in the preserved serum sample by adopting ELISA, and specifically operating according to the instruction of the IgG kit. The concentrations of the chicken IgG standard substances are as follows in sequence: 2400. 1200, 600, 300, 150, 75 μ g/μ L. Setting standard holes and sample holes according to the instruction, then incubating for 60min in a constant temperature box at 37 ℃, washing for 5 times, incubating for 15min in a light-proof constant temperature box at 37 ℃ after adding a substrate, finally adding a stop solution, and measuring the OD value of each hole at the wavelength of 450nm in 15 min. Drawing a standard curve according to the OD value of the standard as the abscissa and the concentration value as the ordinate, and calculating the content of IgG in the sample according to the standard curve (y is 2465.1X-201.3R)²0.999). The results are shown in Table 3. The IgG content in serum is increased compared with that in serum of a model group and a blank group 2 and 4 weeks after the immunization of the vaccine, but the difference is not obvious, the IgG content in serum is increased compared with that in serum of the vaccine group, the pawpaw polysaccharide group and the pawpaw polysaccharide group, the difference is significant (P is less than 0.05 or P is less than 0.01), the pawpaw polysaccharide can be used alone to increase the IgG content in serum of a virus-infected live chicken, and the effect of combining the pawpaw polysaccharide and the vaccine is more significant.

TABLE 3 Effect of papaya polysaccharide on IgG in Chicken serum (μ g/ml)

P < 0.05P < 0.01 in comparison with the blank

The effect of papaya polysaccharide on chicken immune organ index.

The results are shown in Table 4. 4 weeks after the vaccine immunization, the immune organ indexes of the vaccine group, the pawpaw polysaccharide group and the pawpaw polysaccharide group are increased compared with the immune organ indexes of the model group and the blank group, but the difference is not obvious, the vaccine group, the pawpaw polysaccharide group and the pawpaw polysaccharide group are combined, the immune organ indexes of the vaccine group and the blank group are increased compared with the immune organ indexes of the blank group, the difference is significant (P is less than 0.05 or P is less than 0.01), the pawpaw polysaccharide can increase the immune organ indexes of the virus-infected live chickens when being used alone, and the effect of the pawpaw polysaccharide and the vaccine combination is more significant.

TABLE 4 Effect of papaya polysaccharide on chicken immune organ index

Group of	Quantity (only)	Spleen index%	Thymus index%	Bursa index%
					Blank group	20	0.35±0.05	0.48±0.10	0.41±0.05
Model set	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**
Pawpaw polysaccharide 3 group (vaccine)	16	0.57±0.06**	0.74±0.12**	0.56±0.08**
					Pawpaw polysaccharide 4 group (vaccine)	18	0.59±0.07**	0.76±0.13**	0.59±0.11**

P < 0.05P < 0.01 in comparison with the blank