Application of anti-glycolytic drugs in the preparation of drugs and / or formulations that enhance the anti-infective effects of anti-stress products

Abstract

This invention provides the application of anti-glycolytic drugs in the preparation of drugs and / or formulations that enhance the anti-infective efficacy of anti-stress products, belonging to the field of veterinary drug technology. Experiments have shown that anti-glycolytic drugs can enhance the anti-infective efficacy of anti-stress products. This invention combines anti-glycolytic drugs with anti-stress products in a specific ratio, significantly enhancing the anti-infective ability of animals under stress, with an effect no less than that of anti-stress products combined with antibiotics, and can replace antibiotics. The method for enhancing the anti-infective efficacy of anti-stress products provided by this invention is simple, environmentally friendly, and bacteria are less likely to develop drug resistance, showing good application prospects.

Description

Technical Field

[0001] This invention relates to the field of veterinary drug technology, and in particular to the application of anti-glycolytic drugs in the preparation of drugs and / or formulations that enhance the anti-infective effects of anti-stress products. Background Technology

[0002] Stress is a series of neuroendocrine responses in animals caused by various strong stimuli (stressors), primarily involving sympathetic nerve excitation and increased secretion from the pituitary-adrenal cortex, resulting in various functional and metabolic changes. Any stimulus, if it reaches a certain intensity, can become a stressor. Stress can occur in multiple stages of animal husbandry, such as drastic weather changes, cold, heat, long-distance transportation, weaning, castration, fright, and vaccination.

[0003] Stress responses can enhance blood supply, oxygen supply, and energy supply to internal organs, and strengthen the body's resistance to adverse stimuli. However, prolonged stress responses can cause various diseases such as transport tetany in ruminants, swine stress syndrome, and sudden death syndrome in broilers. Besides causing self-damage, stress responses can also induce various infectious diseases, including bacterial infections. Endogenous infection is currently a common form of pathogenic bacterial infection in livestock and poultry. Healthy livestock and poultry often carry pathogens on their respiratory or digestive tract mucosa, making them susceptible to infection and disease when adverse stress factors occur.

[0004] Once a stress response occurs, its effects on the animal's body are multifaceted, one of which is its impact on metabolism. Stress increases catabolism and decreases anabolism, leading to elevated blood sugar and inducing infection. Current methods for controlling stress in livestock and poultry mainly involve nutritional supplementation, such as vitamin C supplementation, to enhance the body's metabolic resistance to stress; or administering sedative and tranquilizing traditional Chinese medicine to reduce the stress response. However, these methods have very limited effectiveness against bacterial infections caused by stress. In the past, some anti-stress programs included antibiotics to enhance the anti-infective effects of anti-stress products. However, given the national "Action Plan for Reducing the Use of Veterinary Antibiotics," there is an urgent need to find alternatives to antibiotics that can 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 antibiotic overuse in anti-stress regimens.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of an anti-glycolytic drug in the preparation of drugs and / or formulations that enhance the anti-infective effects of anti-stress products, wherein the anti-stress products include traditional Chinese veterinary medicines or nutritional preparations. The anti-glycolytic drug inhibits the glycolytic metabolic activity of pathogenic bacteria and inhibits the growth of Pasteurella multocida in vitro.

[0008] Preferably, the anti-glycolytic drug includes rhein, tripterygium wilfordii, or shikonin.

[0009] Preferably, the traditional Chinese veterinary medicine comprises the following components in the following mass percentages: 20-30% Acorus tatarinowii, 20-30% dragon bone, 20-30% Ziziphus jujuba var. spinosa, 8-16% Eucommia ulmoides, and 8-16% Cuscuta chinensis.

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

[0011] The present invention also provides a method for improving the infection resistance of stressed animals, wherein the stressed animals are fed the traditional Chinese veterinary medicine and the anti-glycolytic drug, wherein the amount of the traditional Chinese veterinary medicine is 1.8 to 2.2‰ of the basic feed and the amount of the anti-glycolytic drug is 0.08 to 0.12‰ of the basic feed.

[0012] The present invention also provides a method for improving the resistance of stress animals to infection, wherein the stress animal is fed vitamin C and an anti-glycolytic drug, and the vitamin C and the anti-glycolytic drug are added to the stress animal's drinking water, wherein 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 compound preparation for improving the stress resistance of animals, comprising an anti-stress product and an anti-glycolytic drug, wherein the anti-glycolytic drug comprises rhein, tripterygium oleracea or shikonin, and the mass ratio of the anti-stress product to the traditional Chinese veterinary medicine is 0.4 to 20:1.

[0014] Preferably, 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% Acorus tatarinowii, 20-30% dragon bone, 20-30% Ziziphus jujuba var. spinosa, 8-16% Eucommia ulmoides, and 8-16% Cuscuta chinensis.

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

[0016] By adopting the above technical solution, the present invention has the following beneficial effects: Experiments have shown that anti-glycolytic drugs can enhance the anti-infective effect of anti-stress products. The present invention combines anti-glycolytic drugs with anti-stress products in a specific ratio, or prepares an anti-stress compound preparation for administration to animals, which significantly enhances the animals' anti-infective ability under stress, and the effect is superior to the combined use of antibiotics and anti-stress products, and can replace antibiotics. The preparation method of the anti-stress compound preparation of the present invention is simple, environmentally friendly, and bacteria are less likely to develop drug resistance, making it suitable for widespread application. Attached Figure Description

[0017] Figure 1 This represents the changes in blood glucose levels in animals under a cold stress model.

[0018] Figure 2 This represents the changes in blood glucose levels in animals under adverse stress models.

[0019] Figure 3 The growth of Pasteurella bacteria under different blood glucose concentrations; N, normal blood glucose; H, hyperglycemia.

[0020] Figure 4 Comparison of Pasteurella glycolytic enzyme activity under different blood glucose concentrations; N, normal blood glucose; H, hyperglycemia.

[0021] Figure 5 Comparison of glycolytic enzyme expression levels in Pasteurella multocida under different blood glucose concentrations; N, normal blood glucose; H, hyperglycemia.

[0022] Figure 6 The effects of different drugs on the production of pyruvate, the end product of Pasteurella multocida glycolysis. Detailed Implementation

[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 scope of protection of the present invention.

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

[0025] (1) Cold stress model

[0026] Ten one-month-old layer chicks (purchased from Dazhuang Qingmu Farm, Baoding, Hebei Province) were transferred from a normal temperature (20℃) rearing environment to a low temperature (10℃) rearing environment. Fasting blood glucose levels in the chicks under both room temperature and low temperature conditions were measured using a commercially available blood glucose meter. Results ( Figure 1 The results showed that blood glucose levels in broiler hens increased significantly at low temperatures (P<0.05).

[0027] (2) Adverse Stimulus Stress Model

[0028] Twenty rabbits (1 kg each, purchased from Qingmu Farm, Dazhuang Town, Baoding, Hebei Province) were randomly divided into two groups of ten each. One group served as the experimental group, where each rabbit was injected with 2000 μg of LPS (lipopolysaccharide) to create an infection stress model. The other group served as the control group, receiving an equal volume of physiological saline. Blood glucose levels in the rabbits were measured using a commercially available glucometer 24 hours later. Results were presented (…). Figure 2 The results showed that blood glucose levels were significantly elevated in rabbits under adverse stress (P<0.05).

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

[0030] (1) High blood sugar promotes the growth of Pasteurella multocida.

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

[0032] The diluted bacterial suspension was added to both types of serum, with a final concentration of approximately 100 CFU. The solution was incubated at 37°C for 2.5 hours, followed by plate counting to analyze bacterial growth. Experimental results ( Figure 3 The results showed that the average number of Pasteurella bacteria in the hyperglycemic environment was 263 CFU, while the average number of Pasteurella bacteria in the normalglycemic environment was 127 CFU, and the difference between the two was significant (P = 0.0004).

[0033] (2) Effects of hyperglycemia on Pasteurella multocida glycolysis

[0034] The experiment was conducted using a pyruvate detection kit (purchased from Beijing Solarbio Science & Technology Co., Ltd.). The frozen bacterial culture in (1) was thawed, and the bacteria were collected into centrifuge tubes. After centrifugation, the supernatant was discarded. The bacteria were resuspended using the extraction buffer from the kit to prepare a resuspension with a bacterial density of 500 × 10⁻⁶. 4 CFU / mL. Divided into two groups, the bacteria were ultrasonically treated under ice bath conditions to release intracellular pyruvate. The ultrasonic power was 200W, and the treatment lasted 3 seconds with a 10-second interval, repeated 30 times. After standing for 30 minutes, the bacteria were centrifuged at 8000g at room temperature for 10 minutes. Different concentrations of glucose were added to the pyruvate-releasing Pasteurella multocida to obtain two reaction solutions with different blood glucose concentrations: one group was a normal blood glucose reaction solution (56 mg / 100 mL), and the other group was a hyperglycemic reaction solution (112 mg / 100 mL). The solutions were incubated at 37℃ for 0.5 hours, and the glycolytic enzyme activity was measured. The results are shown below. Figure 4The results showed that pyruvate, the end product of glycolysis, was significantly increased in Pasteurella in a hyperglycemic environment, indicating that hyperglycemia can significantly improve the glycolytic metabolism level of Pasteurella.

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

[0036] The Pasteurella multocida from step (1) was divided into two portions and incubated for 2.5 h at normal blood glucose concentration (56 mg / 100 mL) and high blood glucose concentration (112 mg / 100 mL), respectively. Bacterial RNA was extracted using the Trizol method, and cDNA was generated by reverse transcription. Then, the expression level of Pasteurella multocida glycolytic enzyme gene was detected by real-time PCR using the primers in Table 1.

[0037] Table 1 Primers for Real-Time Quantitative PCR

[0038]

[0039]

[0040] Test results as follows Figure 5 As shown in the figure, the results indicated that the gene expression levels of all nine members of Pasteurella glycolytic enzymes were significantly higher in the hyperglycemic environment than in the normal glycemic environment. This means that the hyperglycemic environment promotes the upregulation of Pasteurella glycolytic enzyme gene expression.

[0041] Example 3. Antibacterial effect of glycolysis inhibitors on Pasteurella multocida.

[0042] A selection of anti-glycolytic drugs (hereinafter referred to as anti-glycolytic drugs) were selected from anti-tumor drugs for testing. The pyruvate assay in Example 2(2) was performed. Different anti-glycolytic drugs were added to the hyperglycemic test group before incubation at 37°C, and then the pyruvate content was measured. The results are shown in […]. Figure 6 The results showed that the pyruvate production in the rhein, tripterygium nitrate, and shikonin treatment groups was significantly reduced (P < 0.05), indicating that these three anti-glycolytic drugs have the effect of inhibiting the glycolytic metabolism of pathogens.

[0043] Minimum inhibitory concentration (MIC) determination: Glucose solution was added to newborn calf serum [purchased from Pingrui Biotechnology (Beijing) Co., Ltd.] to achieve a serum blood glucose concentration of 56 mg / 100 mL, representing a normal blood glucose concentration; another group of serum had a blood glucose concentration of 112 mg / 100 mL, representing a hyperglycemic environment. Pasteurella X7 bacterial suspension with a turbidity of 0.5 McFarland turbidity was added to both sera, with a bacterial suspension to serum volume ratio of 1:9. Different concentrations of glycolytic enzyme inhibitors (drugs shown in Table 1) were added to each EP tube, with final drug concentrations of 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. The EP tubes were incubated at 37℃ for 2.5 h. After the incubation, plate counting was performed using TSA medium. Each experiment was conducted in triplicate. The minimum inhibitory concentration of a drug against Pasteurella multocida is defined as the lowest concentration of the drug whose plate count is less than or equal to that of the positive control. The minimum inhibitory concentrations of each drug are shown in Table 2.

[0044] Table 2. Minimum inhibitory concentrations of drugs that inhibit glycolytic enzymes against Pasteurella multocida.

[0045]

[0046]

[0047] The results showed that rhein had the best antibacterial effect, followed by triptolide, shikonin, resveratrol, pterostilbene, and coumaric acid, whose in vitro antibacterial activity decreased in that order. 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 did not show antibacterial effect at a drug concentration of 1 mM, therefore, this invention did not further test them.

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

[0049] KM mice [purchased from Spifort (Beijing) Co., Ltd.] were selected and hyperglycemic model mice were obtained by intraperitoneal injection of streptozotocin at a single dose of 40 mg / kg (drug / mouse body weight). Forty KM hyperglycemic model mice were selected and randomly divided into 4 groups. Each mouse was subcutaneously injected with 100 CFU of Pasteurella X7 strain. The mice in each group were treated daily by gavage with rhein (100 mM), tripterygium wilfordii (100 mM), and shikonin (100 mM), respectively. The control group was gavaged with 0.5 mL of water daily. The experimental results are shown in Table 3.

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

[0051]

[0052]

[0053] The results showed that all of the above-mentioned glycolysis inhibitors could enhance the resistance of hyperglycemic mice to Pasteurella infection.

[0054] Example 5. Anti-infection experiment of glycolysis inhibitors synergistically acting as anti-stress products

[0055] (1) Cold stress model anti-infection experiment

[0056] The anti-stress traditional Chinese medicine used in this experiment was a traditional Chinese veterinary medicine according to patent "ZL201511027082.X", which consisted of 20% Acorus tatarinowii, 20% dragon bone, 28% Ziziphus jujuba var. spinosa, 16% Eucommia ulmoides, and 16% Cuscuta chinensis (all percentages are by weight). The traditional Chinese veterinary medicine was mixed into the basic feed at a ratio of 2‰ of the weight of the traditional Chinese veterinary medicine to the basic feed and fed to the patient.

[0057] 120 one-month-old chicks were randomly divided into 12 groups of 10 chicks each. All chicks were kept in a low-temperature (10℃) environment to create a cold stress model. Each group of chicks had free access to basic feed and water.

[0058] To determine the effects of drugs on the production performance of chicks, six experimental groups were set up. In experimental groups 1-3, anti-stress traditional Chinese medicine was added to the feed along with rhein, triptolide, and shikonin, respectively. The dosage of the anti-stress traditional Chinese medicine was 2‰ of the basal feed weight, and the dosages of rhein, triptolide, and shikonin were 0.1‰ of the basal feed weight. In experimental group 4, chicks were fed both the anti-stress traditional Chinese medicine and amoxicillin, with the dosage of the anti-stress traditional Chinese medicine being 2‰ of the basal feed weight and the dosage of amoxicillin being 0.1‰ of the basal feed weight. In experimental group 5, only the anti-stress traditional Chinese medicine was added, at a dosage of 2‰ of the basal feed weight. In experimental group 6, the control group, chicks were fed only the basal feed without any drugs. The average daily weight gain and feed conversion ratio of the chicks were observed over 7 days, and the results are shown in Table 4.

[0059] Table 4. Measurement of chick production performance in each group

[0060]

[0061]

[0062] To determine the effect of drugs on the survival rate of infected chicks, six experimental groups were set up. In experiments 7–12, chicks were subcutaneously injected with 10,000 CFU of Pasteurella X7 strain, followed by a basal diet plus drug administration. In experimental groups 7–9, the feed included an anti-stress traditional Chinese medicine (TCM) supplemented with rhein, triptolide, and shikonin, respectively. The amount of anti-stress TCM added was 2‰ of the basal diet weight, and the amounts of rhein, triptolide, and shikonin added were 0.1‰ of the basal diet weight. In experiment 10, chicks were fed both the anti-stress TCM and amoxicillin, with the anti-stress TCM added at 2‰ of the basal diet weight and amoxicillin added at 0.1‰ of the basal diet weight. In experiment 11, only the anti-stress TCM was added at 2‰ of the basal diet weight. Experiment 12 served as the control group, fed only the basal diet without any drugs. The survival rate of chicks was observed over 7 days, and the results are shown in Table 5.

[0063] Table 5. Determination of anti-infection performance of chicks in each group.

[0064] Group Dosage Survival rate 7 Traditional Chinese medicine + rhein 10 / 10 8 Traditional Chinese medicine + Tripterygium wilfordii 9 / 10 9 Traditional Chinese medicine + Shikonin 10 / 10 10 Traditional Chinese medicine + amoxicillin 10 / 10 11 traditional Chinese medicine 3 / 10 12 No (control) 0 / 10

[0065] In existing technologies, antibiotics are typically used in conjunction with anti-stress drugs to reduce bacterial infections associated with stress. The experimental results of Example 5 show that adding a glycolysis inhibitor to the anti-stress traditional Chinese medicine does not affect its efficacy; the average daily weight gain and feed conversion ratio of chicks are similar to those of the group using the traditional Chinese medicine alone. The survival rate of chicks in the group with the added glycolysis inhibitor was close to that in the group with added antibiotics, indicating that it can significantly enhance the animals' anti-infection ability and replace antibiotics. This demonstrates that using glycolysis inhibitors can replace antibiotics, providing a new approach and method for reducing antibiotic use.

[0066] (2) Anti-infection experiment using an adverse stimulus stress model

[0067] Sixty rabbits weighing 1 kg each were randomly divided into 6 groups of 10 each. All rabbits were injected with 2000 μg of LPS (lipopolysaccharide) to create an adverse stress model. The rabbits in each group had free access to basal feed and drinking water. In experiments 1-3, in addition to vitamin C (final concentration 50 mg / L), rhein, tripterygium wilfordii, and shikonin were added to the drinking water, all at a final concentration of 100 mg / L. In experiment 4, vitamin C (final concentration 50 mg / L) and amoxicillin (final concentration 100 mg / L) were added to the drinking water. In experiment 5, vitamin C (final concentration 50 mg / L) was added to the drinking water. Experiment 6 served as a control without any added substances. All rabbits were subcutaneously injected with 10 CFU of Pasteurella multocida strain X7. Disease and mortality were observed and recorded for seven consecutive days. The results are shown in Table 6.

[0068] Table 6. Determination of Anti-infection Performance in Stress-Induced Rabbits

[0069] Group Dosage Survival rate 1 Vitamin C + Rhein 10 / 10 2 Vitamin C + Tripterygium wilfordii 10 / 10 3 Vitamin C + Shikonin 10 / 10 4 Vitamin C + Amoxicillin 10 / 10 5 Vitamin C 5 / 10 6 No (control) 0 / 10

[0070] The results showed that adding glycolysis inhibitors to drinking water could effectively improve the anti-infection ability of rabbits and replace antibiotics.

[0071] As can be seen from the above examples, the application of anti-glycation drugs in the preparation of drugs and / or formulations that enhance the anti-infective effect of anti-stress products shows that anti-glycation drugs can enhance the anti-infective effect of anti-stress products, and are superior to the effect of anti-stress products used in combination with antibiotics.

[0072] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A compound preparation for enhancing the stress resistance of animals, characterized in that, The application relates to an anti-stress product and an anti-glycolysis drug, wherein the anti-glycolysis drug is celastrol, the anti-stress product is a Chinese veterinary medicine, the mass ratio of the Chinese veterinary medicine to the celastrol is 18-20:1; the Chinese veterinary medicine is composed of the following components with mass percentage: acorus gramineus 20-30%, dragon bone 20-30%, zizyphus jujuba mill 20-30%, eucommia ulmoides oliver 8-16% and cuscuta chinensis lam 8-16%; and the stress is caused by pasteurella multocida X7 strain infection.

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