A compound plant composition for inhibiting intestinal inflammatory response of pigs, and a preparation method and application thereof

Abstract

This invention proposes a compound plant composition for inhibiting intestinal inflammatory response in pigs, its preparation method, and its application, relating to the field of veterinary technology. The compound plant composition contains the following active ingredients in parts by weight: 50-70 parts carvacrol, 10-20 parts thymol, 30-50 parts cinnamaldehyde, 5-10 parts baicalein, 5-10 parts apigenin, and 3-8 parts chlorogenic acid. The preparation method involves mixing the active ingredients in proportion, adding water and Tween-80, and stirring until homogeneous. This invention can significantly reduce the levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in the serum and small intestinal tissue of pigs infected with enterotoxigenic Escherichia coli, and increase the level of the anti-inflammatory cytokine IL-10. It also exerts its anti-inflammatory effect by inhibiting the activation of the TLR4-MyD88-NF-κB signaling pathway. This invention uses a compound of natural plant extracts, eliminating the risk of antibiotic resistance and veterinary drug residues, and is simple to prepare.

Description

Technical Field

[0001] This invention relates to the field of veterinary technology, and in particular to a compound plant composition for inhibiting intestinal inflammatory response in pigs, its preparation method, and its application. Background Technology

[0002] The intestines are a vital site for the digestion and absorption of nutrients in living organisms and also an important immune organ. After weaning, piglets are prone to intestinal inflammation due to changes in the feeding environment, management practices, feed sources, and pathogenic microbial infections. Intestinal inflammation caused by viruses and bacteria (such as Salmonella and Escherichia coli) has become a prominent problem hindering the healthy development of the pig industry. These pathogens, after invading the piglets' bodies, can activate their immune system, leading to an increase in the expression of pro-inflammatory factors such as tumor necrosis factor-alpha (TNF-α) and interleukin-1β (IL-1β) in the intestines, thus triggering an inflammatory response. Intestinal inflammation results in the excessive release of inflammatory factors, damaging intestinal tissues. Clinical manifestations include vomiting, diarrhea, lethargy, and loss of appetite, severely impacting piglet production performance, increasing mortality, and causing significant economic losses to the pig industry.

[0003] Enterotoxigenic Escherichia coli (ETEC) is one of the important intestinal pathogens that endanger the health of pigs. Its released endotoxin, LPS, binds to the leukocyte differentiation antigen 14 receptor on the surface of intestinal epithelial cells, thereby activating downstream responses mediated by the Toll-like receptor 4 (TLR4) myeloid differentiation factor MyD88 signaling pathway. This process rapidly upregulates the expression levels of interleukin-type inflammatory cytokines (such as IL-1β, IL-6, and IL-8), triggering an inflammatory response at the site of infection. The nuclear transcription factor NF-κB plays a crucial regulatory role in these inflammatory responses during this process.

[0004] Antibiotics are currently the most widely used and fastest-acting treatment for diarrhea caused by ETEC infection. However, the problem of bacterial resistance caused by antibiotic overuse poses a significant potential threat to global public health. As more and more countries and regions begin to strictly restrict the use of antibiotics, seeking safe and effective antibiotic alternatives has become a key research focus. Since July 2020, when my country banned the use of antibiotics in animal feed, probiotics, acidifiers, natural plant extracts, and enzyme preparations have all been found to be able to replace antibiotics in feed to some extent. Plant extracts have received widespread attention due to their wide availability, low cost, environmental friendliness, and significant effects.

[0005] Studies have shown that a considerable number of compound plant compositions possess anti-inflammatory effects, but their mechanisms of action are not entirely the same. For example, lemon essential oil exerts its anti-inflammatory properties by inhibiting 5-LOX; thymol essential oil significantly inhibits the synthesis of pro-inflammatory factors TNF-α, IL-6, and IL-1β induced by low-density lipoprotein oxide in human THP-1 monocytes, while simultaneously enhancing the gene and protein expression of the anti-inflammatory cytokine IL-10; researchers have conducted a 14-day gastric injury experiment using a claw edema model and found that carvacrol in oregano oil has anti-inflammatory effects. Studies suggest that carvacrol may exert its pharmacological effects through a unique target different from prostaglandins, promoting gastric ulcer repair while reducing inflammatory responses. Furthermore, researchers have also confirmed the anti-inflammatory effects of rosemary essential oil through studies using mouse models focusing on antioxidant pathways. Therefore, this invention proposes a compound plant composition for inhibiting intestinal inflammation in pigs, along with its preparation method and applications, to address the problems existing in the prior art. Summary of the Invention

[0006] To address the aforementioned problems, the present invention aims to provide a compound plant composition for inhibiting intestinal inflammatory response in pigs, its preparation method, and its application, thereby solving current problems such as antibiotic resistance and veterinary drug residues. Furthermore, there is no need to consider the withdrawal period when adding the compound, and it is safe and risk-free for long-term use.

[0007] To achieve the objective of this invention, the invention is achieved through the following technical solution: a compound plant composition for inhibiting intestinal inflammatory response in pigs, comprising the following active ingredients in parts by weight: 50-70 parts of carvacrol, 10-20 parts of thymol, 30-50 parts of cinnamaldehyde, 5-10 parts of baicalein, 5-10 parts of apigenin, and 3-8 parts of chlorogenic acid.

[0008] A further improvement is that the compound plant composition also contains a pharmaceutically acceptable carrier or a pharmaceutically acceptable excipient.

[0009] A further improvement is that the pharmaceutically acceptable excipients include Tween-80 and water.

[0010] A further improvement is that the compound plant composition is used to be added to pig diets at a dosage of 100-200 mg / kg.

[0011] A method for preparing a compound herbal composition for inhibiting intestinal inflammatory response in pigs includes the following steps: Step 1: Mix carvacrol, thymol, cinnamaldehyde, baicalin, apigenin and chlorogenic acid according to the following weight parts; Step 2: Add water and Tween-80, stir well, and you will get the compound plant composition.

[0012] A further improvement is that, by weight, the amount of water added is 120-180 parts, and the amount of Tween-80 added is 10-20 parts.

[0013] The application of a compound herbal composition for inhibiting intestinal inflammatory response in pigs in the preparation of feed additives or drugs for inhibiting intestinal inflammatory response in pigs.

[0014] A further improvement is that the inhibition of intestinal inflammatory response in pigs refers to the inhibition of intestinal inflammatory response in pigs caused by enterotoxigenic Escherichia coli infection.

[0015] A further improvement is that the feed additive or drug is used to achieve at least one of the following effects: One effect is to reduce the levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in pigs; The second effect is to increase the level of the anti-inflammatory cytokine IL-10; The third effect is the inhibition of activation of signaling pathways mediated by TLR4, MyD88, and NF-κB.

[0016] The beneficial effects of this invention are as follows: The compound plant composition of this invention can significantly reduce the levels of pro-inflammatory cytokines in the serum and small intestinal tissue of ETEC-infected pigs, while increasing the levels of anti-inflammatory cytokines, effectively inhibiting intestinal inflammatory responses. Furthermore, by inhibiting the activation of the TLR4-MyD88-NF-κB inflammatory signaling pathway, it regulates the expression of inflammatory factors upstream, thereby exerting an anti-inflammatory effect. Finally, the use of natural plant extracts in the formulation eliminates the risk of antibiotic resistance and veterinary drug residues, requires no withdrawal period, and is simple to prepare, making it suitable for industrial production. Attached Figure Description

[0017] Figure 1 This is a schematic diagram illustrating the effect of the compound plant composition of the present invention on the levels of inflammation-related cytokines in the serum of pigs infected with enterotoxin-producing Escherichia coli.

[0018] Figure 2 This is a schematic diagram illustrating the effect of the compound plant composition of the present invention on the expression level of inflammation-related cytokine mRNA in the duodenal tissue of pigs infected with enterotoxin-producing Escherichia coli.

[0019] Figure 3 This is a schematic diagram illustrating the effect of the compound plant composition of the present invention on the expression level of inflammation-related cytokine mRNA in the jejunal tissue of pigs infected with enterotoxin-producing Escherichia coli.

[0020] Figure 4 This is a schematic diagram illustrating the effect of the compound plant composition of the present invention on the expression level of inflammation-related cytokine mRNA in the ileal tissue of pigs infected with enterotoxin-producing Escherichia coli.

[0021] Figure 5This is an immunoblot diagram illustrating the effect of the compound plant composition of the present invention on the expression levels of TLR4, MyD88, IκBα, p-IκBα, p65, and p-p65 proteins in the small intestinal tissue of pigs infected with enterotoxigenic Escherichia coli. Detailed Implementation

[0022] To enhance understanding of the present invention, the present invention will be further described in detail below with reference to embodiments. These embodiments are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0023] Example 1 This embodiment provides a compound plant composition for inhibiting intestinal inflammatory response in pigs. The raw materials for its preparation are, by weight, 60 parts carvacrol, 15 parts thymol, 40 parts cinnamaldehyde, 8 parts baicalin, 8 parts apigenin, and 5 parts chlorogenic acid.

[0024] The preparation method is as follows: Mix the above-mentioned parts by weight of carvacrol, thymol, cinnamaldehyde, baicalin, apigenin and chlorogenic acid, add 149 parts by weight of water and 15 parts by weight of Tween-80, and stir quickly until homogeneous to obtain the compound plant composition.

[0025] Accordingly, the compound plant composition was added to the pig diet at a dosage of 150 mg / kg.

[0026] Example 2 This embodiment provides a compound plant composition for inhibiting intestinal inflammatory response in pigs. The raw materials for its preparation are, by weight, 50 parts carvacrol, 10 parts thymol, 30 parts cinnamaldehyde, 5 parts baicalein, 5 parts apigenin, and 3 parts chlorogenic acid.

[0027] The preparation method is as follows: Mix the above-mentioned parts by weight of carvacrol, thymol, cinnamaldehyde, baicalin, apigenin and chlorogenic acid, add 120 parts by weight of water and 10 parts by weight of Tween-80, and stir quickly until homogeneous to obtain the compound plant composition.

[0028] Accordingly, the compound plant composition was added to the pig diet at a dosage of 100 mg / kg.

[0029] Example 3 This embodiment provides a compound plant composition for inhibiting intestinal inflammatory response in pigs. The raw materials for its preparation are, by weight, 70 parts carvacrol, 20 parts thymol, 50 parts cinnamaldehyde, 10 parts baicalein, 10 parts apigenin, and 8 parts chlorogenic acid.

[0030] The preparation method is as follows: Mix the above-mentioned parts by weight of carvacrol, thymol, cinnamaldehyde, baicalin, apigenin and chlorogenic acid, add 180 parts by weight of water and 20 parts by weight of Tween-80, and stir quickly until homogeneous to obtain the compound plant composition.

[0031] Accordingly, the compound plant composition was added to the pig diet at a dosage of 200 mg / kg.

[0032] Application Example 1 according to Figures 1-5 As shown, this application example 1 provides the application of a compound plant composition that inhibits intestinal inflammatory response in pigs in the preparation of feed additives or drugs that inhibit intestinal inflammatory response in pigs.

[0033] Taking the compound plant composition prepared in Example 1 as an example, an animal experiment was conducted to prevent and treat inflammatory responses caused by enterotoxigenic Escherichia coli infection in pigs.

[0034] 1. Materials and Methods 1.1 Test strains ETEC K88 (K88 type ETEC or K88 type enterotoxin-producing Escherichia coli) was provided by Henan Agricultural University and published in the literature "The effect of overexpression of porcine β-defensin 2 (pBD2) on anti-Escherichia coli infection in IPEC-J2 cells", with a commitment to distribute it to the public for 20 years.

[0035] 1.2 Experimental Diets The composition and nutritional components of the basal diet are shown in Table 2. Table 2. Composition and nutrient levels of basal diet for piglets 1.3 Experimental Animals and Design Twenty 25-day-old weaned piglets were randomly divided into four groups: blank control group (CON), compound plant composition group (CEO), challenge control group (ETEC), and compound plant composition prevention and control group (CEO+ETEC), with five piglets in each group.

[0036] Blank control group: Weaned piglets fed a normal basal diet without any treatment; Compound plant composition group: Normal feeding of the basal diet, followed by 3 days of pre-feeding, with 150 mg / kg of the compound plant composition prepared in Example 1 added to the basal diet; Control group (ETEC group): fed a normal basal diet, and simultaneously fed 10 mg / day of the compound plant-based combination treatment group per pig. 10 CFU K88 ETEC, cesarean section 3 days later.

[0037] The compound plant-based diet control group: Normal basal diet was fed, followed by the addition of 150 mg / kg of the compound plant-based diet 3 days after pre-feeding; after 30 days of feeding the compound plant-based diet, each pig was fed 10 mg / kg of the compound plant-based diet. 10 CFU K88 ETEC, cesarean section 3 days later.

[0038] 1.4 Determination of the content of inflammation-related factors in pig serum by enzyme-linked immunosorbent assay (ELISA) After the experiment, blood was collected from the anterior vena cava of the pigs. 10 mL was collected from each pig and the serum was separated after standing at 37°C for 2 hours.

[0039] The levels of IL-1β, IL-6, IL-10, and TNF-α in porcine serum were detected using a porcine interleukin-1β (IL-1β) (H002-1-1, Nanjing Jiancheng), porcine interleukin-6 (IL-6) (H007-1-1, Nanjing Jiancheng), porcine interleukin-10 (IL-10) (H009-1-1, Nanjing Jiancheng), and tumor necrosis factor-α (TNF-α) (H052-1-1, Nanjing Jiancheng). The optical density was measured at 450 nm.

[0040] 1.5 Real-time quantitative PCR detection of expression levels of inflammation-related factors in porcine small intestinal tissue After the experiment, 0.3g of duodenum, jejunum, and ileum were collected from each group of pigs. After grinding, total RNA was extracted using the TRIzol method, and the total RNA was reverse transcribed into cDNA according to the instructions of Hifair® III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNAdigester plus) (11141ES, Yeasen). Real-time quantitative PCR (qPCR) was performed using Hieff UNICON® Advanced qPCR SYBR Master Mix (11185ES, Yeasen). The qPCR reaction system consisted of 10 μL of 2×SYBR qPCR Master Mix, 7 μL of ddH2O, 0.5 μL each of forward and reverse primers (10 μmol / L), and 2 μL of cDNA. The RT-qPCR reaction program was: 95 ℃ pre-denaturation for 3 min; 95 ℃ denaturation for 5 s, 60 ℃ annealing for 20 s, 72 ℃ extension for 15 s, for 45 cycles. Set up a negative control to confirm the primer melting curve and amplification curve. Use β-actin as an internal control and employ 2... -ΔΔCt The relative levels of gene expression were calculated. All primers were synthesized by Henan Shangya Biotechnology Co., Ltd. Primer information is shown in Table 1.

[0041] Table 1 qPCR primer sequences 1.6 Immunoblot analysis of TLR4, MyD88, IκBα, p-IκBα, p65, and p-p65 protein expression levels in porcine small intestinal tissue After the experiment, 0.3g of duodenum, jejunum, and ileum were collected from each group of pigs. The samples were ground and lysed with RIPA buffer to obtain the supernatant. The protein concentration of each sample was determined using the BCA method (a standard technique in this field). Equal volumes of protein samples were separated by SDS-PAGE electrophoresis and transferred to nitrocellulose membranes (NC membranes). These NC membranes were then blocked with 5% skim milk at room temperature for 3 hours. The samples were incubated overnight at 4°C with the following primary antibodies: anti-TLR4 (#66350-1-Ig, proteintech), anti-MyD88 (#67969-1-Ig, proteintech), anti-IκBα (#4812, CST), anti-p-IκBα (#2859, CST), anti-p65 (#6956, CST), and anti-p-p65 (#3033, CST). Wash five times with TBST for five minutes each time, then incubate with peroxidase-conjugated goat anti-rabbit IgG (#SA00001-1, proteintech) or goat anti-mouse IgG (#SA00001-1, proteintech) at room temperature for one hour. Finally, detect the protein using enhanced chemiluminescence.

[0042] 2. Experimental Results To assess the effect of CEO on inhibiting intestinal inflammation in pigs, this study first fed piglets with CEO mixed into their diet in a certain proportion, and then infected the piglets with ETEC. The secretion levels of inflammation-related factors in the serum of the piglets, the expression levels of inflammation-related factors in the intestinal tissue, and the expression levels of proteins related to the inflammatory response signaling pathway were comprehensively analyzed.

[0043] 2.1 Adding CEO to the diet significantly affected the secretion of inflammatory factors in the serum of piglets infected with ETEC. like Figure 1 As shown, compared with the control group, the levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in the serum of the CEO group were significantly reduced. p <0.01), the level of the anti-inflammatory cytokine IL-10 was significantly increased ( p <0.01); while the levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in the serum of the ETEC group were significantly increased ( p <0.01), the level of the anti-inflammatory cytokine IL-10 was significantly reduced ( p<0.01). Compared with the ETEC group, the levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in the serum of the CEO+ETEC group were significantly reduced ( p <0.01), the level of the anti-inflammatory cytokine IL-10 was significantly increased ( p <0.01). The above experimental results indicate that adding CEO to the diet can effectively inhibit the inflammatory response induced by ETEC infection.

[0044] 2.2 Dietary supplementation with CEO significantly affected the secretion of inflammatory factors in the intestinal tissue of piglets infected with ETEC. like Figures 2-4 As shown, compared with the control group, the ETEC group piglets had higher levels of pro-inflammatory cytokines in the duodenum, jejunum, and ileum. IL-1β , IL-6 and TNF-α The mRNA transcription level was significantly increased ( p <0.0001), while anti-inflammatory cytokines IL-10 The transcription level of genes was significantly reduced. p <0.0001). Compared with the ETEC-infected group alone, the CEO+ETEC group showed higher levels of [unclear text - likely referring to a specific metric or percentage] in each intestinal segment. IL-1 β , IL-6 and TNF-α The transcription level decreased significantly ( p <0.0001), the transcriptional level of IL-10 significantly rebounded ( p <0.0001). The above results indicate that dietary supplementation with CEO can effectively inhibit the expression of pro-inflammatory factors in the intestine of piglets induced by ETEC infection and promote the expression of anti-inflammatory factors, thereby alleviating the intestinal inflammatory response induced by ETEC in vivo.

[0045] 2.3 Dietary supplementation with CEO significantly affected the expression of proteins (TLR4, MyD88, IκBα, p-IκBα, p65, p-p65) regulating inflammatory response signaling pathways in the intestinal tissue of piglets infected with ETEC. like Figure 5 As shown, compared with the control group, the protein expression levels of TLR4, MyD88, and p-p65 in the duodenum, jejunum, and ileum of pigs in the ETEC group were significantly increased, while the expression level of IκBα protein was significantly decreased. Compared with the ETEC group, the protein expression levels of TLR4, MyD88, and p-p65 in all intestinal segments of pigs in the CEO+ETEC group were significantly decreased, while the expression level of IκBα protein was significantly increased. In conclusion, dietary supplementation with CEO can alleviate the ETEC infection-induced intestinal inflammatory response in piglets in vivo by inhibiting the overactivation of the TLR4 / MyD88 / NF-κB signaling pathway.

[0046] The above results indicate that adding CEO to the diet can significantly inhibit the occurrence of intestinal inflammation in pigs.

[0047] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the present invention without departing from its framework and scope of application, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A compound herbal composition for inhibiting intestinal inflammatory response in pigs, characterized in that: It contains the following active ingredients in parts by weight: 50-70 parts carvacrol, 10-20 parts thymol, 30-50 parts cinnamaldehyde, 5-10 parts baicalein, 5-10 parts apigenin, and 3-8 parts chlorogenic acid.

2. The compound herbal composition for inhibiting intestinal inflammatory response in pigs according to claim 1, characterized in that: The compound plant composition also contains a pharmaceutically acceptable carrier or a pharmaceutically acceptable excipient.

3. The compound herbal composition for inhibiting intestinal inflammatory response in pigs according to claim 2, characterized in that: The pharmaceutically acceptable excipients include Tween-80 and water.

4. The compound herbal composition for inhibiting intestinal inflammatory response in pigs according to claim 1, characterized in that: The compound plant composition is used to be added to pig diets at a dosage of 100-200 mg / kg.

5. A method for preparing a compound plant composition for inhibiting intestinal inflammatory response in pigs as described in any one of claims 1-4, characterized in that: Includes the following steps: Step 1: Mix carvacrol, thymol, cinnamaldehyde, baicalin, apigenin and chlorogenic acid according to the following weight parts; Step 2: Add water and Tween-80, stir well, and you will get the compound plant composition.

6. The method for preparing the compound plant composition for inhibiting intestinal inflammatory response in pigs according to claim 5, characterized in that: The amount of water added is 120-180 parts by weight, and the amount of Tween-80 added is 10-20 parts.

7. The use of a compound plant composition for inhibiting intestinal inflammatory response in pigs in the preparation of feed additives or drugs for inhibiting intestinal inflammatory response in pigs.

8. The application of the compound plant composition for inhibiting intestinal inflammation in pigs according to claim 7 in the preparation of feed additives or drugs for inhibiting intestinal inflammation in pigs, characterized in that: The inhibition of intestinal inflammatory response in pigs refers to the inhibition of intestinal inflammatory response in pigs caused by enterotoxigenic Escherichia coli infection.

9. The application of the compound plant composition for inhibiting intestinal inflammation in pigs according to claim 7 in the preparation of feed additives or drugs for inhibiting intestinal inflammation in pigs, characterized in that: The feed additive or drug is used to achieve at least one of the following effects: One effect is to reduce the levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in pigs; The second effect is to increase the level of the anti-inflammatory cytokine IL-10; The third effect is the inhibition of activation of signaling pathways mediated by TLR4, MyD88, and NF-κB.

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