Application of anti-glycolysis medicine in preparation of medicine and / or preparation for improving anti-infection effect of anti-stress product

Abstract

The invention provides application of an anti-glycolysis drug in preparation of a drug and / or a preparation for improving the anti-infection effect of an anti-stress product, and belongs to the technical field of veterinary drugs. Experiments show that the anti-glycolysis drug can improve the anti-infection effect of the anti-stress product. The anti-glycolysis drug and the anti-stress product are used in cooperation according to a specific proportion, the anti-infection ability of animals in the stress state can be remarkably enhanced, the effect is not lower than the effect of cooperation of the anti-stress product and antibiotics, and the anti-glycolysis drug and the anti-stress product can be used instead of the antibiotics. The method for improving the anti-infection effect of the anti-stress product, provided by the invention, is simple, convenient, green and environment-friendly, bacteria are not easy to generate drug resistance, and the method has a relatively good application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of veterinary drugs, and in particular to the application of anti-glycolytic drugs in the preparation of drugs and / or preparations for enhancing the anti-infection effect of anti-stress products. Background Art

[0002] Stress is a series of neuroendocrine responses mainly characterized by sympathetic nerve excitement and increased secretion of pituitary-adrenal cortex caused by various strong stimulating factors (i.e., stressors) in the animal body, as well as various functional and metabolic changes caused thereby. Any stimulus, as long as it reaches a certain intensity, will become a stressor. Multiple links in the breeding process can cause stress, such as drastic weather changes, cold, stuffiness, long-distance transportation, weaning, castration, fright, and vaccination.

[0003] The stress response can enhance the blood supply, oxygen supply, and energy supply of the internal organs of the body, and enhance the body's resistance to adverse stimuli. However, continuous stress responses can cause various diseases such as transportation tetany in ruminants, porcine stress syndrome, and sudden death syndrome in broiler chickens. In addition to causing self-harm, stress responses can also induce various infectious diseases including bacterial infections. Endogenous infection is a common form of pathogenic bacteria infecting livestock and poultry at present. Pathogenic bacteria are often carried on the respiratory or digestive tract mucosa of healthy livestock and poultry, and it is easy to become infected and diseased when adverse stress factors occur.

[0004] Once the stress response occurs, its impact on the animal body is multi-faceted, one of which is the impact on animal metabolism. Stress will enhance catabolism, weaken anabolism, increase blood sugar, and induce infection. The current main methods for preventing and controlling livestock and poultry stress are to supplement nutrition, such as supplementing vitamin C to enhance the body's metabolism and anti-stress; or to give traditional Chinese medicines for calming the nerves to weaken the stress response. However, these methods have very limited effects on bacterial infections caused by stress. In the past, some anti-stress programs added antibiotics to enhance the anti-infection effect of anti-stress products. However, in the context of the country's implementation of the "Action to Reduce the Use of Veterinary Antibacterial Drugs", there is an urgent need to find products that can replace antibiotics and improve the efficacy of anti-stress drugs. Summary of the Invention

[0005] In view of this, the present invention provides the application of anti-glycolytic drugs in enhancing the anti-infection effect of anti-stress products, and solves the problem of the abuse of antibiotics in anti-stress programs.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides the use of anti-glycolytic drugs in the preparation of drugs and / or preparations for enhancing the anti-infection effect of anti-stress products, wherein the anti-stress products include traditional Chinese veterinary drugs or nutritional preparations. The anti-glycolytic drugs have the effects of inhibiting the glycolytic metabolic activity of pathogenic bacteria and inhibiting the growth of Pasteurella in vitro.

[0008] Preferably, the anti-glycolytic drugs include rhein, celastrol or shikonin.

[0009] Preferably, the traditional Chinese veterinary drug includes the following components in mass percentage: Acorus tatarinowii 20-30%, Fossilia Ossis Mastodi 20-30%, Semen Ziziphi Spinosae 20-30%, Eucommia ulmoides 8-16% and Cuscuta chinensis 8-16%.

[0010] Preferably, the nutritional preparation includes vitamin C.

[0011] The present invention also provides a method for improving the anti-infection ability of stressed animals, by feeding the traditional Chinese veterinary drug and the anti-glycolytic drug to the stressed animals, wherein the feeding amount of the traditional Chinese veterinary drug is 1.8-2.2‰ of the basal diet, and the feeding amount of the anti-glycolytic drug is 0.08-0.12‰ of the basal diet.

[0012] The present invention also provides a method for improving the anti-infection ability of stressed animals, by feeding vitamin C and the anti-glycolytic drug to the stressed animals, and adding the vitamin C and the anti-glycolytic drug to the drinking water of the stressed animals. The concentration of vitamin C in the drinking water is 40-50 mg / L, and the final concentration of the anti-glycolytic drug is 80-100 mg / L.

[0013] The present invention also provides a composite preparation for improving the anti-stress ability of animals, including an anti-stress product and an anti-glycolytic drug, wherein the anti-glycolytic drug includes rhein, celastrol or shikonin, and the mass ratio of the anti-stress product to the traditional Chinese veterinary drug is 0.4-20:1.

[0014] Preferably, the anti-stress product is a traditional Chinese veterinary drug, and the mass ratio of the traditional Chinese veterinary drug to the anti-glycolytic drug is 18-20:1; the traditional Chinese veterinary drug includes the following components in mass percentage: Acorus tatarinowii 20-30%, Fossilia Ossis Mastodi 20-30%, Semen Ziziphi Spinosae 20-30%, Eucommia ulmoides 8-16% and Cuscuta chinensis 8-16%.

[0015] Preferably, the anti-stress product is vitamin C, and the mass ratio of vitamin C to the anti-glycolytic drug is 0.4-0.5:1.

[0016] By adopting the above technical solutions, the present invention has the following beneficial effects: Through experiments, the present invention discovers that anti-glycolytic drugs can enhance the anti-infection effect of anti-stress products. The present invention uses anti-glycolytic drugs and anti-stress products in a specific ratio, or formulates an anti-stress compound preparation for administration to animals, which can significantly enhance the anti-infection ability of animals under stress conditions, and the effect is better than the combined application of antibiotics and anti-stress products, and can replace the use of antibiotics. The preparation method of the anti-stress compound preparation of the present invention is simple, environmentally friendly, and bacteria are not easily resistant, which is suitable for large-scale popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It shows the change of blood glucose level of animals under the cold stress model.

[0018] Figure 2 It shows the change of blood glucose level of animals under the adverse stimulus stress model.

[0019] Figure 3 It shows the growth of Pasteurella at different blood glucose concentrations; N, normal blood glucose; H, high blood glucose.

[0020] Figure 4 It shows the comparison of the glycolytic enzyme activities of Pasteurella at different blood glucose concentrations; N, normal blood glucose; H, high blood glucose.

[0021] Figure 5 It shows the comparison of the expression levels of glycolytic enzymes of Pasteurella at different blood glucose concentrations; N, normal blood glucose; H, high blood glucose.

[0022] Figure 6 It shows the effect of different drugs on the production of pyruvate, the end product of glycolytic metabolism of Pasteurella. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0024] Example 1. Determination of hyperglycemia caused by stress

[0025] (1) Cold stress model

[0026] Ten 1-month-old laying hens (purchased from Dazhuang Qingmu Farm, Baoding, Hebei) were transferred from a normal temperature (20 °C) breeding environment to a low temperature (10 °C) breeding environment, and a commercially available blood glucose meter was used to measure the fasting blood glucose levels of the laying hens at room temperature and low temperature. The measurement results ( Figure 1 ) showed that the blood glucose of the laying hens increased significantly at low temperature (P < 0.05).

[0027] (2) Adverse stimulus stress model

[0028] Twenty rabbits (purchased from Dazhuang Qingmu Farm, Baoding, Hebei) weighing 1 kg each were randomly divided into two groups of 10 rabbits each. One group served as the experimental group, and each rabbit was injected with 2000 μg of LPS (lipopolysaccharide) to establish an infection stress model. The other group was the control group. After injecting an equal amount of normal saline, the blood glucose differences in the rabbits were measured using a commercially available blood glucose meter 24 hours later. The measurement results ( Figure 2 ) showed that the blood glucose levels of rabbits under adverse stimulus stress were significantly increased (P<0.05).

[0029] Example 2. Effects of hyperglycemia on bacterial glycolysis and related enzymes

[0030] (1) Hyperglycemia promotes the growth of Pasteurella

[0031] Different concentrations of glucose were added to newborn bovine serum (purchased from Pingrui Biotechnology (Beijing) Co., Ltd., complement inactivated) to obtain two sera with different blood glucose concentrations, namely normal blood glucose concentration (56 mg / 100 mL) and hyperglycemia concentration (112 mg / 100 mL). The Pasteurella X7 strain (preserved and provided by the College of Veterinary Medicine, Hebei Agricultural University) was cultured until the logarithmic phase, counted, and cryopreserved for subsequent experiments.

[0032] The diluted bacterial solutions were respectively added to the above two sera at a final concentration of approximately 100 CFU. The mixtures were incubated in a water bath at 37°C for 2.5 h, and then the bacterial growth was analyzed by plate counting. The experimental results ( Figure 3 ) showed that the average number of Pasteurella in the hyperglycemic growth environment was 263 CFU, and the average number of Pasteurella in the normal blood glucose growth environment was 127 CFU, with a significant difference between the two (P = 0.0004).

[0033] (2) Effects of hyperglycemia on the glycolytic metabolism of Pasteurella

[0034] A pyruvate detection kit (purchased from Beijing Solarbio Science & Technology Co., Ltd.) was used for the experiment. The frozen bacterial solution in (1) was thawed, and the bacteria were collected into a centrifuge tube. After centrifugation, the supernatant was discarded; the bacteria were resuspended with the extraction solution in the kit to prepare a resuspended solution with a bacterial density of 500×10 4 CFU / mL. The resuspended solution was divided into two groups and sonicated under ice bath conditions to release pyruvate in the bacterial cells. The ultrasonic power was 200 W, sonicated for 3 sec, with an interval of 10 sec, repeated 30 times. After standing for 30 min, centrifugation was carried out at 8000 g and room temperature for 10 min. Different concentrations of glucose were added to the Pasteurella that had released pyruvate to obtain two reaction solutions with different blood glucose concentrations: one was a normal blood glucose reaction solution of 56 mg / 100 mL, and the other was a hyperglycemic reaction solution of 112 mg / 100 mL. The mixtures were incubated in a water bath at 37°C for 0.5 h, and the glycolytic enzyme activities were measured. The results are shown in Figure 4The results showed that in a hyperglycemic environment, the end product of glycolytic metabolism, pyruvate, in Pasteurella multocida increased significantly, indicating that hyperglycemia can significantly enhance the glycolytic metabolism level of Pasteurella multocida.

[0035] (3) Effect of hyperglycemia on the expression levels of glycolytic enzymes in Pasteurella multocida

[0036] The Pasteurella multocida in step (1) was divided into two parts and incubated in a normal blood glucose concentration (56 mg / 100 mL) and a hyperglycemic concentration (112 mg / 100 mL) for 2.5 h. Bacterial RNA was extracted by the Trizol method, reverse transcribed into cDNA, and then fluorescence quantitative PCR was performed using the primers in Table 1 to detect the expression levels of Pasteurella multocida glycolytic enzyme genes.

[0037] Table 1 Primers for real-time fluorescence quantitative PCR

[0038]

[0039]

[0040] The detection results are as Figure 5 shown. The results showed that the gene expression levels of all 9 members of the glycolytic enzymes in Pasteurella multocida in the hyperglycemic growth environment were significantly higher than those in the normal blood glucose growth environment. That is, the hyperglycemic growth environment promotes the up-regulated expression of Pasteurella multocida glycolytic enzyme genes.

[0041] Example 3. Bacteriostatic effect of glycolysis inhibitor drugs on Pasteurella multocida

[0042] Some drugs with glycolysis inhibitory effects (abbreviated as anti-glycolysis drugs) were selected from antitumor drugs for testing. The pyruvate determination test in Example 2 (2) was carried out. Different anti-glycolysis drugs were added to the hyperglycemic test group before incubation at 37 °C, and then the pyruvate content was measured. The measurement results are shown in Figure 6 . The results showed that the pyruvate production in the rhein, celastrol, and shikonin treatment groups decreased significantly (P < 0.05), indicating that these three anti-glycolysis drugs have an inhibitory effect on the glycolytic metabolism of pathogenic bacteria.

[0043] Determination of the minimum inhibitory concentration (MIC): Glucose solution was added to newborn bovine serum [purchased from Pingrui Biotechnology (Beijing) Co., Ltd.] to make the serum blood glucose concentration 56 mg / 100 mL, which was used as the normal blood glucose concentration; in another group of serum, the blood glucose concentration was 112 mg / 100 mL, which was used as the hyperglycemic growth environment. Bacterial suspension of Pasteurella multocida strain X7 with a turbidity of 0.5 McFarland was added to both sera, and the volume ratio of the bacterial suspension to the serum was 1:9. Different concentrations of the drug inhibiting glycolytic enzymes (the drugs shown in Table 1) were added to each EP tube, and the final concentrations of the drugs in the EP tubes were 0 μM (positive control), 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, and 1 mM, respectively. The EP tubes were placed in a constant temperature water bath at 37 °C for 2.5 h. After the water bath, plate counting was performed using TSA medium, and three parallel experiments were set up for each group of experiments. The lowest drug concentration with a plate counting result less than or equal to that of the positive control was the MIC value of the drug against Pasteurella multocida, and the minimum inhibitory concentrations of each drug are shown in Table 2.

[0044] Table 2 Minimum inhibitory concentrations of drugs inhibiting glycolytic enzymes against Pasteurella multocida

[0045]

[0046]

[0047] The results showed that rhein had the best antibacterial effect, and the in vitro antibacterial activities of celastrol, shikonin, resveratrol, pterostilbene, and coumaric acid decreased in turn. For clotrimazole, methyl jasmonate, ferulic acid, ginsenoside Rh2, metformin hydrochloride, dihydroartemisinin, chitosan, quercetin, ursolic acid, oleanolic acid, curcumin, 2-deoxy-D-glucose, genistein, sodium fluoride, aspirin, and coumaric acid, no antibacterial effect was observed when the drug concentration reached 1 mM, so the present invention did not continue the determination.

[0048] Example 4. Glycolysis-inhibiting drugs can enhance the anti-infection ability of hyperglycemic mice

[0049] KM mice [purchased from SPF Biotechnology (Beijing) Co., Ltd.] were selected and streptozotocin was intraperitoneally injected at a dose of 40 mg / kg (drug / mouse body weight) once to obtain hyperglycemic model mice. Forty KM hyperglycemic model mice were selected and evenly divided into 4 groups, and 100 CFU of Pasteurella multocida strain X7 was subcutaneously injected into each mouse. Each group of mice was treated by gavage with rhein (100 mM), celastrol (100 mM), or shikonin (100 mM) every day, and the control group was gavaged with 0.5 mL of water every day. The experimental results are shown in Table 3.

[0050] Table 3 Effects of anti-glycolysis drugs on the anti-infection ability of mice

[0051]

[0052]

[0053] The results show that the above glycolysis inhibitor drugs can all enhance the ability of hyperglycemic mice to resist Pasteurella infection.

[0054] Example 5. Anti-infection experiment of glycolysis inhibitor synergistic anti-stress products

[0055] (1) Anti-infection experiment of cold stress model

[0056] The anti-stress traditional Chinese medicine in this experiment is the Chinese veterinary medicine in the patent "ZL201511027082.X", which is composed of 20% Acorus tatarinowii, 20% fossil fragments, 28% Semen Ziziphi Spinosae, 16% Eucommia ulmoides, and 16% Cuscuta chinensis (the above percentage contents are mass percentage contents). The Chinese veterinary medicine is mixed in the basal diet at a ratio of 2‰ of the mass of the Chinese veterinary medicine to the mass of the basal diet for feeding.

[0057] Take 120 one-month-old laying hens, randomly divide them into 12 groups, with 10 hens in each group, and raise them all in a low-temperature (10°C) environment to prepare a cold stress model. The chicks in each group are allowed to freely eat the basal diet and drink water.

[0058] In order to determine the effect of the drug on the production performance of chicks, 6 groups of experiments were set. Among them, while adding the anti-stress traditional Chinese medicine to the feed in experimental groups 1-3, rhein, celastrol, and shikonin were added respectively. The addition amount of the anti-stress traditional Chinese medicine is 2‰ of the mass of the basal diet, and the addition amounts of rhein, celastrol, and shikonin are 0.1‰ of the basal diet, and the chicks were fed. In experimental group 4, the anti-stress traditional Chinese medicine and amoxicillin were fed. The addition amount of the anti-stress traditional Chinese medicine is 2‰ of the mass of the basal diet, and the addition amount of amoxicillin is 0.1‰ of the mass of the basal diet. In experimental group 5, only the anti-stress traditional Chinese medicine was added, and the addition amount of the anti-stress traditional Chinese medicine is 2‰ of the mass of the basal diet. Experimental group 6 is the control group, only fed the basal diet without adding any drugs. Observe the average daily weight gain and feed-to-meat ratio of the chicks within 7 days, and the results are shown in Table 4.

[0059] Table 4 Determination of production performance of chicks in each group

[0060]

[0061]

[0062] To determine the effect of drugs on the survival rate of infected chicks, 6 groups of experiments were set up. In experiments 7 - 12, 10,000 CFU of Pasteurella multocida strain X7 was subcutaneously injected, and then the chicks were fed with basal diet + drugs. Among them, in experimental groups 7 - 9, while adding anti - stress traditional Chinese medicine to the feed, rhein, celastrol, and shikonin were added respectively. The addition amount of anti - stress traditional Chinese medicine was 2‰ of the quality of the basal diet, and the addition amounts of rhein, celastrol, and shikonin were 0.1‰ of the basal diet, and the chicks were fed. In experimental group 10, the chicks were fed with anti - stress traditional Chinese medicine and amoxicillin. The addition amount of anti - stress traditional Chinese medicine was 2‰ of the quality of the basal diet, and the addition amount of amoxicillin was 0.1‰ of the basal diet. In experimental group 11, only anti - stress traditional Chinese medicine was added, and the addition amount of anti - stress traditional Chinese medicine was 2‰ of the quality of the basal diet. Experimental group 12 was the control group, only fed with the basal diet without adding any drugs. The survival rate of the chicks within 7 days was observed, and the results are shown in Table 5.

[0063] Table 5 Determination of the anti - infectious performance of chicks in each group

[0064] Group Drug administration Survival rate 7 Traditional Chinese medicine + Rhein 10 / 10 8 Traditional Chinese medicine + Celastrol 9 / 10 9 Traditional Chinese medicine + Shikonin 10 / 10 10 Traditional Chinese medicine + Amoxicillin 10 / 10 11 Traditional Chinese medicine 3 / 10 12 None (control) 0 / 10

[0065] In the prior art, antibiotics are usually combined with anti - stress drugs to reduce the bacterial infections accompanied by the stress process. The experimental results of Example 5 show that after adding glycolysis - inhibiting drugs to the anti - stress traditional Chinese medicine, the effect of the traditional Chinese medicine is not affected, and the average daily gain and feed - to - weight ratio of the chicks are similar to those of the group using only traditional Chinese medicine. The survival rate of the laying hens in the experimental group adding glycolysis - inhibiting drugs is close to that of the group adding antibiotics, that is, it can significantly enhance the anti - infectious ability of animals and replace antibiotics. It shows that glycolysis inhibitors can be used to replace antibiotics, providing a new idea and method for reducing the use of antibiotics.

[0066] (2) Anti - infectious experiment of the adverse - stimulus stress model

[0067] Sixty rabbits with a body weight of 1 kg were randomly divided into 6 groups, with 10 rabbits in each group. All were injected with 2000 μg of LPS (lipopolysaccharide) to establish an adverse - stimulus stress model. The rabbits in each group were allowed to freely eat the basal diet and drink water. In experimental groups 1 - 3, in addition to adding vitamin C (final concentration 50 mg / L) to the drinking water, rhein, celastrol, and shikonin were additionally added, and the final concentrations were all 100 mg / L. In experimental group 4, vitamin C (final concentration 50 mg / L) + amoxicillin (final concentration 100 mg / L) was added to the drinking water. In experimental group 5, vitamin C (final concentration 50 mg / L) was added to the drinking water. Experimental group 6 was the control group without adding any substances. All rabbits were subcutaneously injected with 10 CFU of Pasteurella multocida strain X7, and the morbidity and mortality were continuously observed and recorded for seven days. The results are shown in Table 6.

[0068] Table 6 Determination of the anti - infectious performance of stressed rabbits

[0069] Group Drug administration Survival rate 1 Vitamin C + Rhein 10 / 10 2 Vitamin C + Celastrol 10 / 10 3 Vitamin C + Shikonin 10 / 10 4 Vitamin C + Amoxicillin 10 / 10 5 Vitamin C 5 / 10 6 None (control) 0 / 10

[0070] The results show that adding glycolysis inhibitor drugs to drinking water can effectively improve the anti-infection ability of rabbits and replace the use of antibiotics.

[0071] As can be seen from the above embodiments, the application of anti-glycolysis drugs in the preparation of drugs and / or preparations for enhancing the anti-infection effect of anti-stress products. The anti-glycolysis drugs can enhance the anti-infection effect of anti-stress products, and are superior to the effect of combining anti-stress products with antibiotics.

[0072] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of anti-glycolytic drugs in the preparation of drugs and / or preparations for improving the anti-infection effect of anti-stress products, wherein the anti-stress products include traditional Chinese veterinary drugs or nutritional preparations.

2. The use according to claim 1, characterized in that: The antiglycolytic drugs include rhein, tripterygium wilfordii or shikonin.

3. The use according to claim 1, characterized in that: The Chinese veterinary medicine comprises the following components in percentage by mass: 20-30% of calamus calamus, 20-30% of dragon bone, 20-30% of spiny jujube seed, 8-16% of eucommia bark and 8-16% of dodder seed.

4. The use according to claim 1, characterized in that: The nutritional formulation includes vitamin C.

5. A method for improving the resistance of stressed animals to infection, characterized in that: The corresponding animal is fed with the Chinese veterinary medicine and the anti-glycolytic drug, and the feeding amount of the Chinese veterinary medicine is 1.8 to 2.2 of the basic feed. ‰ The feeding amount of the anti-glycolytic drug is 0.08 to 0.12 of the basic feed. ‰ .

6. A method for improving the resistance of stressed animals to infection, characterized in that: The stressed animals are fed vitamin C and antiglycolytic drugs, and the vitamin C and antiglycolytic drugs are added to the drinking water of the stressed animals. The concentration of vitamin C in the drinking water is 40-50 mg / L, and the final concentration of the antiglycolytic drugs is 80-100 mg / L.

7. A composite preparation for improving the anti-stress ability of animals, characterized in that: It comprises an anti-stress product and an anti-glycolytic drug, wherein the anti-glycolytic drug comprises rhein, celastrol or shikonin, and the mass ratio of the anti-stress product to the anti-glycolytic drug is 0.4-20:

1.

8. The composite formulation according to claim 7, characterized in that The anti-stress product is a traditional Chinese veterinary medicine, and the mass ratio of the traditional Chinese veterinary medicine to the anti-glycolysis drug is 18-20:1; the traditional Chinese veterinary medicine comprises the following components in mass percentage: 20-30% of Acorus calamus, 20-30% of Dragon Bone, 20-30% of Ziziphus jujuba seeds, 8-16% of Eucommia ulmoides and 8-16% of Cuscuta australis.

9. The composite formulation according to claim 7, characterized in that The anti-stress product is vitamin C, and the mass ratio of vitamin C to anti-glycolysis drugs is 0.4-0.5:1.

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