Phytosterol and phytosterol ester complex and use thereof

Abstract

The application discloses a phytosterol and phytosterol ester complex agent and application thereof, wherein the complex agent comprises phytosterol and phytosterol ester with a mass ratio of 1:10. The application aims to improve the water solubility and bioavailability of phytosterol, realize synergistic effect by compounding phytosterol and phytosterol ester, interfere with the lipid acquisition of coccidia from multiple targets, up-regulate the expression of cecal tight junction protein to repair the intestinal barrier, inhibit the release of serum and cecal pro-inflammatory factors to relieve inflammatory reaction, regulate the expression of genes related to lipid synthesis and fatty acid oxidation to remodel lipid metabolism homeostasis, and then effectively inhibit the growth and development of coccidia, and significantly improve the growth performance of broilers infected with coccidia.

Description

Technical Field

[0001] This invention relates to the field of feed additive technology, and in particular to a compound of phytosterols and phytosterol esters and its application. Background Technology

[0002] Coccidiosis is a global parasitic disease caused by Eimeria coccidia, resulting in severe economic losses to poultry farming. Taking chickens as an example, infection with coccidia can lead to intestinal mucosal damage, malabsorption, immunosuppression, and secondary infections, with morbidity rates exceeding 50% and mortality rates reaching as high as 25%. The life cycle of coccidia is complex; their sporulated oocysts can survive for a long time in the external environment, exhibiting strong resistance to common disinfectants, and can be transmitted through feces, instruments, insects, and other routes. Currently, the control of coccidiosis mainly relies on chemically synthesized drugs (such as ionotropic agents, sulfonamides, and quinolines) and vaccines. However, the long-term use of chemical drugs has led to the rapid spread of drug-resistant strains. For example, drugs such as decaoxyquin ester easily induce drug resistance in coccidia, requiring regular drug rotation. Simultaneously, drug residues and environmental pollution are becoming increasingly prominent issues. While vaccines can induce immune protection in the host, they have drawbacks such as short-lasting immunity, unstable protective efficacy, and susceptibility to environmental factors; furthermore, virulent vaccines may cause latent infections.

[0003] The existing patent application CN106420769A discloses the application of β-sitosterol in the preparation of drugs against Eimeria tenella. It involves preparing β-sitosterol into a powder with excipients, adding it to feed, mixing it well, and feeding it to chickens at the usual feed amount; or preparing β-sitosterol into a water-soluble powder, dissolving it in water, and feeding it to chickens at the usual drinking water amount. Although animal infection experiments have confirmed that the anticoccidial index (ACI) of β-sitosterol can reach over 180, indicating good anti-Eimeria tenella activity, intestinal absorption rate and blood drug concentration were not tested, making it impossible to determine whether it directly kills the parasites or works indirectly through host immune regulation. Summary of the Invention

[0004] The main objective of this invention is to provide a compound of phytosterols and phytosterol esters and its application, aiming to solve the technical problem that existing coccidiosis control methods cannot meet expectations.

[0005] To achieve the above objectives, the present invention provides a phytosterol and phytosterol ester composite agent, wherein the composite agent comprises phytosterol and phytosterol ester in a mass ratio of 1:10, wherein the phytosterol mainly comprises 40%~60% β-sitosterol, 15%~30% oleosterol, 10%~25% stigmasterol, ≤10% rapeseed sterol, and ≤5% other sterols; the phytosterol ester comprises 40%~55% β-sitosterol oleate, 10%~20% rapeseed oleate, 5%~15% stigmasterol oleate, ≤5% rapeseed sterol oleate, ≤5.0% free phytosterol, ≤3.0% oleic acid, and ≤2.0% other impurities.

[0006] Furthermore, in order to achieve the above objectives, the present invention also provides the application of the phytosterol and phytosterol ester complex according to the above description in the preparation of an anti-Eimeria tenella drug.

[0007] Furthermore, in order to achieve the above objectives, the present invention also provides an application of the phytosterol and phytosterol ester complex described above in the preparation of a dietary additive that upregulates the expression of cecal tight junction protein.

[0008] Furthermore, in order to achieve the above objectives, the present invention also provides the application of the phytosterol and phytosterol ester complex as described above in the preparation of a feed additive that inhibits the release of pro-inflammatory factors in serum and the cecum and alleviates inflammatory responses.

[0009] Furthermore, in order to achieve the above objectives, the present invention also provides an application of the phytosterol and phytosterol ester complex according to the above description in the preparation of a feed additive that downregulates HMGCR.

[0010] Optionally, according to any of the above applications, the phytosterol and phytosterol ester complex is added to the diet at a rate of 50-200 mg / kg.

[0011] Optionally, the phytosterol and phytosterol ester complex is added to the basal diet in the form of a premix.

[0012] Optionally, the phytosterol and phytosterol ester compound is added to the diet at a rate of 100 mg / kg.

[0013] Beneficial effects:

[0014] This invention improves the water solubility and bioavailability of phytosterols by combining phytosterols and phytosterol esters, achieving synergistic effects. Starting from multiple targets, it interferes with lipid acquisition by coccidia, upregulates the expression of cecal tight junction protein to repair the intestinal barrier, inhibits the release of serum and cecal pro-inflammatory factors to alleviate the inflammatory response, and regulates the expression of lipid synthesis and fatty acid oxidation-related genes to reshape lipid metabolism homeostasis. In this way, it effectively inhibits the growth and development of coccidia and significantly improves the growth performance of broilers infected with coccidia. Attached Figure Description

[0015] Figure 1 Comparison of pathological sections of cecal tissue from different experimental groups at 21 days;

[0016] Figure 2 Comparison of crypt depth in cecal tissue of broiler chickens from different experimental groups at 21 days.

[0017] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0018] It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0019] This invention provides a compound of phytosterols and phytosterol esters, wherein the compound comprises phytosterols and phytosterol esters in a mass ratio of 1:10. The phytosterols mainly include β-sitosterol (40%~60%), oleosterol (15%~30%), stigmasterol (approximately 10%~25%), brassosterol (≤10%), and other trace sterols (such as sitosterol, brassosterol, etc., ≤5%). The phytosterol esters are oleate esterification products, mainly composed of oleate esters of various phytosterols, including β-sitosterol oleate (40%~55%), brassosterol oleate (10%~20%), stigmasterol oleate (5%~15%), brassosterol oleate (≤5%), free phytosterols (≤5.0%), oleic acid (free or residual) (≤3.0%), and other impurities (≤2.0%).

[0020] Furthermore, this invention also discloses an anticoccidial feed, which is made by adding a compound of phytosterols and phytosterol esters to the basal diet in the form of a premix. Taking chicken feeding as an example, the addition amount of the compound of phytosterols and phytosterol esters is 50-200 mg / kg, preferably 100 mg / kg. The compound of phytosterols and phytosterol esters can indirectly affect the coccidiosis infection process and inflammatory response by regulating the intestinal flora structure. Its mechanism of action is as follows: Free phytosterols can serve as a nutrient substrate for beneficial intestinal bacteria (such as lactic acid bacteria and bifidobacteria), promoting their proliferation. The metabolites of these beneficial bacteria (such as short-chain fatty acids) can enhance the intestinal mucosal barrier function (e.g., upregulating the expression of ZO-1 and Occludin), reducing the adhesion and invasion of coccidia to the intestinal epithelium. Furthermore, phytosterol esters can inhibit the sterol synthesis pathway of coccidia themselves (coccidia need to obtain sterols from the host to maintain cell membrane structure; esterified sterols can competitively inhibit their uptake), and are simultaneously converted into free form through intestinal metabolism, synergistically activating host immune cells (such as macrophages and Treg cells). At the same time, coccidia infection disrupts host lipid metabolism (e.g., abnormal cholesterol synthesis and enhanced lipid peroxidation), and phytosterols and sterol esters can achieve a dual effect of anti-coccidia and anti-inflammatory by targeting and regulating lipid signaling molecules. Specifically, phytosterol esters can reduce excessive cholesterol synthesis in the host by downregulating HMGCR (the rate-limiting enzyme in cholesterol synthesis), while simultaneously inhibiting key enzymes in coccidia's synthesis of their own cell membranes from host cholesterol, thus blocking the material basis for coccidia proliferation. Free phytosterols can regulate nuclear receptors such as PPAR-γ, inhibiting the activation of the NF-κB inflammatory pathway and reducing the release of pro-inflammatory factors such as IL-6 and IL-17. Esterified phytosterols, due to their lipid solubility, can more easily penetrate the coccidia cell membrane, directly disrupting the lipid homeostasis of the coccidia. Furthermore, coccidia infection induces a shift in host immune cell metabolism towards a "pro-inflammatory phenotype" (such as enhanced glycolysis and mitochondrial dysfunction), and phytosterols and sterol esters can improve anti-inflammatory efficiency by reshaping the metabolic state of immune cells. Phytosterols can promote mitochondrial β-oxidation of immune cells, enhance ATP production, maintain the immunosuppressive function of Treg cells, and reduce excessive inflammation. Phytosterol esters can inhibit the excessive activation of effector T cells by regulating the AMPK / mTOR signaling pathway, while promoting the release of anti-inflammatory metabolites (such as adiponectin), thereby achieving a dynamic balance between metabolism and immunity and avoiding the aggravation of intestinal damage caused by immune metabolic disorders.

[0021] To further illustrate the above effects, the following detailed application examples will be provided.

[0022] 1. Raw material preparation

[0023] The corn, soybean meal, corn gluten meal, soybean oil, lysine, DL-methionine, dicalcium phosphate, limestone powder, and vitamins used in the experiments of this invention are all conventional commercially available products.

[0024] The phytosterols and phytosterol esters used in the experiments of this invention were purchased from Hunan Heyiyuan Biotechnology Co., Ltd.

[0025] The Eimeria tenella used in the experiments of this invention was kindly provided by Professor Wang Zhong of China Agricultural University and was used in the experiments of this invention after being propagated.

[0026] 2. Preparation of feed for different groups of broilers

[0027] Control group (control sample)

[0028] A broiler feed consists of a basal diet and a premix. The basal broiler feed is prepared according to the feeding stage and nutritional requirements (suitable for the early growth stage, days 1-21).

[0029] By weight percentage, the initial basal diet includes: 50.00% corn, 34.00% soybean meal, 6.40% corn gluten meal, 5.00% soybean oil, 0.42% lysine, 0.29% DL-methionine, 2.23% dicalcium phosphate, 1.00% limestone, and 0.66% premix.

[0030] The premix provides 8,000 IU of vitamin A, 2,500 IU of vitamin D3, 20.0 mg of vitamin E, 3.0 mg of vitamin K3, 3.0 mg of vitamin B1, 8.0 mg of vitamin B2, 7.0 mg of vitamin B6, 0.015 mg of vitamin B12, 20.0 mg of pantothenic acid, 50.0 mg of niacin, 0.1 mg of biotin, 1.5 mg of folic acid, 96 mg of iron, 30 mg of zinc, 7.5 mg of copper, 75 mg of manganese, 0.4 mg of iodine, and 0.35 mg of selenium per kilogram of feed.

[0031] The preparation method of this broiler feed is as follows: Mix all components thoroughly to obtain the final product. The nutrient levels of the broiler feed are calculated values.

[0032] Model example (model group)

[0033] The broiler diet used in the model example had the same composition, nutritional level, and preparation method as the control example.

[0034] Comparative example (low esterification group)

[0035] A broiler feed consists of a basal diet and phytosterols. The basal diet for broilers (suitable for the early growth stage, days 1-21) is prepared according to the feeding stage and nutritional requirements.

[0036] By weight percentage, the initial basal diet consists of: 50.00% corn, 34.00% soybean meal, 6.40% corn gluten meal, 5.00% soybean oil, 0.42% lysine, 0.29% DL-methionine, 2.23% dicalcium phosphate, 1.00% limestone, and 0.66% premix. A low-esterification complex is added to the initial basal diet at a rate of 100 mg / kg.

[0037] The premix provides 8,000 IU of vitamin A, 2,500 IU of vitamin D3, 20.0 mg of vitamin E, 3.0 mg of vitamin K3, 3.0 mg of vitamin B1, 8.0 mg of vitamin B2, 7.0 mg of vitamin B6, 0.015 mg of vitamin B12, 20.0 mg of pantothenic acid, 50.0 mg of niacin, 0.1 mg of biotin, 1.5 mg of folic acid, 96 mg of iron, 30 mg of zinc, 7.5 mg of copper, 75 mg of manganese, 0.4 mg of iodine, and 0.35 mg of selenium per kilogram of feed.

[0038] The preparation method of this broiler feed is as follows: Mix all components thoroughly to obtain the final product. The nutrient levels of the broiler feed are calculated values.

[0039] Example 1 (High Esterification Group)

[0040] A broiler diet consists of a basal diet and phytosterols and phytosterol esters in a mass ratio of 1.08:9.88. The basal diet for broilers (suitable for the early growth stage, days 1-21) is prepared according to the feeding stage and nutritional requirements.

[0041] By weight percentage, the initial basal diet consists of: 50.00% corn, 34.00% soybean meal, 6.40% corn gluten meal, 5.00% soybean oil, 0.42% lysine, 0.29% DL-methionine, 2.23% dicalcium phosphate, 1.00% limestone, and 0.66% premix. A high-esterification complex is added to the initial basal diet at a rate of 100 mg / kg.

[0042] The premix provides 8,000 IU of vitamin A, 2,500 IU of vitamin D3, 20.0 mg of vitamin E, 3.0 mg of vitamin K3, 3.0 mg of vitamin B1, 8.0 mg of vitamin B2, 7.0 mg of vitamin B6, 0.015 mg of vitamin B12, 20.0 mg of pantothenic acid, 50.0 mg of niacin, 0.1 mg of biotin, 1.5 mg of folic acid, 96 mg of iron, 30 mg of zinc, 7.5 mg of copper, 75 mg of manganese, 0.4 mg of iodine, and 0.35 mg of selenium per kilogram of feed.

[0043] The preparation method of this broiler feed is as follows: Mix all components thoroughly to obtain the final product. The nutrient levels of the broiler feed are calculated values.

[0044] 3. Test plan and daily management

[0045] Two hundred and eighty-eight one-day-old Arbor Acrylic broiler chickens of uniform weight and good health were randomly divided into four groups (control group, model group, low esterification group, and high esterification group), with six replicates in each group and twelve chickens (half male and half female) in each replicate. The control group and model group were fed a basal diet, the low esterification group was fed a basal diet supplemented with 100 mg / kg phytosterols, and the high esterification group was fed a basal diet supplemented with 100 mg / kg phytosterols and phytosterol esters compound.

[0046] The experiment lasted 21 days. The feeding management and immunization program for broiler chickens were followed, with good ventilation and free access to water and feed. On day 14, all chickens except the control group were administered Eimeriatenella sporulated oocysts at a dose of 8 × 10⁴ oocysts / mL via gavage. The control group was administered an equal dose of physiological saline via gavage. On day 21, one broiler chicken (6 chickens per treatment, 24 chickens in total) with a similar average weight to the corresponding group was selected from each replicate for slaughter and sampling. The specific formulation and nutrient levels of the experimental diet are shown in Table 1.

[0047] Table 1. Basal Feed Formulation and Nutrient Levels (Air-dried Basal)

[0048]

[0049] The premix provides the following per kilogram of feed: Vitamin A 8,000 IU, Vitamin D3 2,500 IU, Vitamin E 20.0 mg, Vitamin K 33.0 mg, Vitamin B1 3.0 mg, Vitamin B2 8.0 mg, Vitamin B6 7.0 mg, Vitamin B12 0.015 mg, Pantothenic Acid 20.0 mg, Niacin 50.0 mg, Biotin 0.1 mg, Folic Acid 1.5 mg, Iron 96 mg, Zinc 30 mg, Copper 7.5 mg, Manganese 75 mg, Iodine 0.4 mg, and Selenium 0.35 mg.

[0050] Metabolizable energy and available phosphorus are calculated values; crude protein, calcium and total phosphorus content are measured values.

[0051] 4. Sample Collection

[0052] On day 21 of the experiment, one broiler chicken with a similar average weight to the corresponding group was selected from each replicate (6 chickens per treatment, 24 chickens in total) for slaughter and sampling. Before slaughter, jugular vein blood was collected and deposited into 10mL centrifuge tubes (approximately 2 / 3 of the tube volume). After tilting the tubes for 15 minutes, they were centrifuged at 1000×g for 10 minutes, and the supernatant serum was collected and stored at -80℃ for later analysis. After blood collection, the chickens were euthanized by euthanasia at the neck. Immediately after dissection, the cecal tissue was removed, and a 1cm segment of the cecum was quickly cut. The intestinal contents were gently rinsed with 0.75% saline solution, and then the intestinal segment was fixed in 4% paraformaldehyde for section preparation. A separate portion of the cecal tissue was placed in cryovials, flash-frozen in liquid nitrogen, and stored at -80℃ for use.

[0053] 5. Index Measurement and Test Results

[0054] 5.1 Analysis of growth performance and experimental results

[0055] On days 14 and 21 of the experiment, chickens were fasted for 12 hours prior to the experiment, and all experimental chickens were weighed on an empty stomach for each replicate. Feed intake for each group was recorded during the experiment, and average body weight, average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (F / G) were calculated from day 1 to day 21.

[0056] Average daily feed intake (ADFI) = (Total feed - Remaining feed) / (Number of chickens in the experiment × Number of days in the experiment);

[0057] Average daily weight gain (ADG) = (total final weight - initial total weight) / (number of days in the experiment × number of chickens in the experiment);

[0058] Fertilizer ratio (F / G) = Average daily weight gain / Average daily feed intake.

[0059] Based on the above formula, the data on the growth performance of broilers infected with Eimeria tenella in different experimental groups are presented in Table 2.

[0060] Table 2. Effects of different experimental groups on growth performance of broilers infected with Eimeria tenella.

[0061]

[0062] Note: Different letters in the same column of the table above indicate significant differences (P<0.05), and the same applies to the following tables.

[0063] As shown in Table 2, compared with the control group, the body weight and average daily weight gain of broilers in the model group were significantly reduced at 21 days, and the feed conversion ratio was significantly increased (P<0.05). This indicates that coccidiosis infection has a serious negative impact on the growth and development of broilers. Coccidia parasitize and reproduce in the intestines, affecting the digestion and absorption of nutrients, which in turn leads to stunted growth, reduced feed conversion efficiency, and ultimately slow weight gain and increased feed conversion ratio.

[0064] Compared with the model group, the 21-day-old body weight and average daily weight gain of broilers in the low-esterification group were significantly increased, while the feed conversion ratio was significantly decreased (P < 0.05), indicating that low-esterification treatment can effectively alleviate the inhibitory effect of coccidiosis infection on broiler growth performance. The possible mechanism is that low-esterification treatment affects the intestinal physiological function of coccidiosis-infected broilers, improving the intestinal absorption capacity of nutrients; it may also enhance the body's utilization efficiency of nutrients by regulating metabolic processes in broilers, thereby promoting broiler growth and improving feed conversion ratio.

[0065] The 21-day body weight and average daily weight gain of broilers in the high-esterification group showed an increasing trend, while the feed conversion ratio also showed a decreasing trend (P>0.05). This indicates that high-esterification treatment may have a certain effect on improving the growth performance of broilers infected with coccidiosis, and there was no significant difference compared with the low-esterification group.

[0066] 5.2 Fecal oocyst count and result analysis

[0067] Fecal samples were collected daily on days 5, 6, and 7 after coccidiosis infection. The samples were thoroughly mixed, and 2g of the sample was placed in a beaker. Saturated saline solution was added, and the mixture was thoroughly mixed. 1mL of the fecal solution was then injected into a McMaster counting chamber. The number of eggs in both counting chambers was recorded under low magnification. The average value was multiplied by 200 to obtain the oocyst count per gram of feces (OPG). Each sample was counted three times, and the average value was used. The results are shown in Table 3.

[0068] Table 3. Effects of different experimental groups on the number of fecal oocysts in broilers infected with Eimeria tenella.

[0069]

[0070] Note: Different letters in the same column of the table above indicate significant differences (P<0.05).

[0071] Table 3 shows that, compared with the control group, coccidia infection in the model group significantly increased the OPG value in broilers (P < 0.05), and the OPG increased with the duration of infection, reaching a peak on day 6. No coccidia oocysts were found in the feces of broilers in the control group, indicating that coccidia underwent a growth and reproduction process within the broilers. Coccidia continuously multiply within the host, and the oocysts they produce are excreted through feces. The increase in OPG reflects the massive proliferation of coccidia in the broiler intestines, posing a serious threat to the health of the broilers.

[0072] Compared with the model group, the OPG values ​​of broilers in the low-esterification and high-esterification groups were significantly reduced (P < 0.05). This indicates that both low-esterification and high-esterification treatments inhibited coccidia reproduction, affecting the survival and reproductive environment of coccidia in the broiler intestine, thus reducing their reproductive capacity and consequently decreasing the number of oocysts excreted. Simultaneously, the number of Eimeria tenella oocysts in the feces of broilers infected with Eimeria tenella in the high-esterification group was significantly lower than that in the low-esterification group. This is because the addition of phytosterol esters to the high-esterification group, in addition to the low-esterification group, can downregulate HMGCR (the rate-limiting enzyme in cholesterol synthesis), reducing excessive cholesterol synthesis in the host, while simultaneously inhibiting the key enzymes by which coccidia utilize host cholesterol to synthesize their own cell membranes, thus blocking the material basis for parasite proliferation.

[0073] 5.3 Serum Biochemical Indicators and Results Analysis

[0074] Using the relevant kits from Nanjing Jiancheng Biotechnology Research Institute, the total cholesterol (TC) (catalog number: A111-1-1), triglycerides (TG) (catalog number: A110-1-1), low-density lipoprotein cholesterol (LDL-C) (catalog number: A113-1-1), and high-density lipoprotein cholesterol (HDL-C) (catalog number: A112-1-1) in serum samples were determined according to the instructions. The results are shown in Table 4.

[0075] Table 4. Effects of different experimental groups on serum biochemical parameters in broiler chickens infected with Eimeria tenella.

[0076]

[0077] Note: Different letters in the same column of the table above indicate significant differences (P<0.05).

[0078] Table 4 shows that, compared with the control group, the serum TC and HLD-C levels in the model group of broilers were significantly lower (P < 0.05), indicating that coccidiosis infection significantly interfered with lipid metabolism in broilers. Coccidiosis infection may disrupt the normal physiological function of the broiler intestine, affecting lipid absorption, synthesis, and transport. For example, coccidia may damage intestinal epithelial cells, affecting the absorption of lipids such as cholesterol; or interfere with the activity of lipid synthesis and metabolism-related enzymes in the liver, leading to a decrease in TC and HDL-C synthesis. In addition, TG (triglyceride) levels showed a decreasing trend, although the difference was not significant, suggesting that coccidiosis infection may also have some impact on triglyceride metabolism, possibly involving changes in the breakdown, transport, or utilization of fats.

[0079] Compared with the model group, the total cholesterol (TC) levels in the serum of broilers in both the low-esterification and high-esterification groups were significantly lower (P < 0.05), indicating that low-esterification and high-esterification treatments further affected cholesterol levels in broiler serum. This may be because these two treatments altered lipid distribution and metabolic pathways in the body. For example, they may have promoted cholesterol transport or conversion to other tissues or inhibited cholesterol synthesis in the liver. The triglyceride (TG) levels also showed a decreasing trend, suggesting that low-esterification and high-esterification treatments also have a certain regulatory effect on triglyceride metabolism, possibly by affecting adipocyte metabolism and fatty acid β-oxidation, leading to a decrease in serum TG levels. Simultaneously, regarding HDL-C levels, the HDL-C value in the low-esterification group was higher than that in the model group, while the HDL-C value in the high-esterification group was lower than that in the model group. This indicates that the high-esterification group, through the addition of phytosterol esters, achieved precise regulation of cholesterol metabolism—both by reducing serum TC to decrease lipids available to coccidia and by dynamically regulating HDL-C to accelerate cholesterol transport and metabolism in the liver, preventing abnormal accumulation at the site of intestinal infection.

[0080] 5.4 Serum inflammatory factor indicators and results analysis

[0081] Using the ELISA kit from Shanghai Enzyme-Link Biotechnology Co., Ltd., the serum levels of interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-17 (IL-17), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) were measured according to the instructions. The results are shown in Table 5.

[0082] Table 5. Effects of different experimental groups on the number of fecal oocysts in broilers infected with Eimeria tenella.

[0083]

[0084] Note: Different letters in the same column of the table above indicate significant differences (P<0.05).

[0085] Table 5 shows that, compared with the control group, the levels of IL-2, IL-6, IL-17, and TNF-α in the serum of broilers in the model group were significantly increased (P < 0.05), indicating that coccidiosis infection triggered an immune response in the broilers. IL-2 is an important immunomodulatory factor that can promote the proliferation and differentiation of T cells and enhance the body's cellular immune function; IL-6 can induce B cell differentiation and antibody secretion, and participate in the regulation of inflammatory responses; IL-17 is mainly secreted by Th17 cells and plays an important role in inflammatory responses and resistance to extracellular pathogen infection; TNF-α has multiple biological functions, including activating immune cells and inducing apoptosis. In the fight against coccidiosis infection, the body releases these cytokines to enhance immune defense, but it may also trigger an inflammatory response, causing certain damage to the body.

[0086] Compared with the model group, both the low-esterification group and the high-esterification group showed significant regulatory effects on the excessive activation of the immune system induced by coccidia infection, but the high-esterification group showed a more significant regulatory effect.

[0087] In the low esterification group, the levels of IL-2, IL-6, TNF-α, and IFN-γ were significantly lower than those in the model group (P < 0.05), indicating that they can effectively inhibit the excessive release of pro-inflammatory factors; however, the level of IL-17 was not significantly different from that in the model group (P > 0.05), suggesting that its regulation of Th17 cell-mediated inflammatory pathways is limited.

[0088] The high esterification group showed a more comprehensive and stronger regulatory effect: IL-2 was slightly higher than that of the low esterification group, but still significantly lower than that of the model group; IL-6 was reduced by 20.27% compared to the model group and was significantly lower than that of the low esterification group; IL-17 was significantly reduced to 134.79±3.03 pg / mL (P<0.05), making it the only group that could effectively downregulate this index; TNF-α was higher than that of the low esterification group, but reduced by 10.41% compared to the model group, and the difference between it and the control group was smaller than that of the low esterification group; IFN-γ was slightly higher than that of the low esterification group, but still significantly lower than that of the model group.

[0089] The high-esterified group, through multi-dimensional regulation of the pro-inflammatory factor network (especially the potent inhibition of IL-6 and IL-17), more precisely balanced the relationship between the body's anticoccidia immunity and inflammatory damage. It maintained an appropriate immune response to resist coccidia infection while avoiding excessive inflammation that could damage intestinal tissue. Its regulatory effect was significantly better than that of the low-esterified group, demonstrating the key role of synergistic enhancement of phytosterol esters.

[0090] 5.5 Histological and Result Analysis of Cecal Tissue

[0091] Fixed cecal tissue was dehydrated, cleared, paraffin-embedded, and embedded. Paraffin blocks were cut using a microtome to a thickness of 5–6 μm. Sections containing intact intestinal sections were placed on warm water and then on glass slides, which were then dried using a slide dryer for storage. The intestinal sections on the slides were dewaxed, rehydrated, and stained with hematoxylin-eosin. The staining solution was washed off the surface of the sections, and the slides were air-dried. Neutral resin was added to the slides, which were then sealed with coverslips and air-dried. Multiple discontinuous fields of view were randomly selected for observation under a microscope. Five or more representative photographs were taken, and the crypt height was measured using K-viewer software. The results are as follows: Figure 1-2 , Figure 1 Pathological sections of cecal tissue from different experimental groups at 21 days: a) control group, b) model group, c) low esterification group, and d) high esterification group. Figure 2 The crypt depth of cecal tissue in broilers at 21 days of age represents the average of six replicates for each treatment in different experimental groups. The same letter for 'a' and 'b' indicates no significant difference (P > 0.05), while different letters indicate significant differences (P < 0.05). Magnification: 100×; Scale bar: 100 μm.

[0092] Depend on Figure 1-2 It was observed that, compared to the control group, coccidia oocysts and cecal tissue destruction were clearly visible in the HE sections of the model group, directly indicating that coccidia successfully infected the cecum of broiler chickens and caused substantial tissue damage. Coccidia parasitize and reproduce within the cecum, and each stage of their life cycle (such as schizogony and gametosis) damages the cecal mucosal cells, leading to disruption of tissue integrity. Numerous inflammatory cells were present in the crypts, and the cecal crypt depth was significantly increased (P < 0.05), reflecting an inflammatory response to coccidia infection. The aggregation of inflammatory cells is intended to defend against coccidia invasion and reproduction, but excessive inflammation may further aggravate tissue damage. The increased crypt depth may be due to coccidia infection stimulating intestinal stem cell proliferation in an attempt to repair damaged mucosal tissue; however, this repair process may not be entirely normal, leading to alterations in crypt structure.

[0093] Compared with the model group, the low-esterification and high-esterification groups showed a decrease in the number of coccidia oocysts in the cecum of broilers, reduced cecal tissue damage, and no significant change in cecal crypt depth (P > 0.05). This indicates that these two treatments inhibit the parasitism and reproduction of coccidia in the cecum. Low-esterification and high-esterification of phytosterols / esters may reduce the number of coccidia in the cecum by affecting their living environment, nutrient uptake, or interfering with their metabolic processes, thereby reducing the degree of coccidia infection. Reduced cecal tissue damage suggests that low-esterification and high-esterification help alleviate the damage to cecal tissue caused by coccidia infection. Combined with the changes in serum cytokines mentioned earlier, the levels of pro-inflammatory cytokines (such as IL-6 and TNF-α) in the serum of the low-esterification and high-esterification groups were significantly reduced, which is consistent with the reduced cecal tissue damage and decreased inflammatory cell infiltration. This indicates that these two treatments, by regulating the immune response, reduced the damage of inflammation to the cecal tissue, stabilized the structure of the cecal tissue to a certain extent, helped restore the normal digestive and absorptive functions of the intestine, provided sufficient nutrition to the body, and thus promoted the growth of broilers. This also explains the reason for the improved growth performance.

[0094] 5.6 Gene expression detection in cecal tissue

[0095] Approximately 100 mg of cecal tissue sample was rapidly removed from a -80°C freezer and transferred to a 2 mL autoclaved lysis tube. 1 mL of TRIzo1 was added, and the tube was placed in a high-throughput tissue homogenizer and centrifuged at 5000 rpm for 15 seconds twice, allowing it to stand at room temperature for 5 min. 200 μL of chloroform reagent was added to the lysis buffer, and the solution was thoroughly mixed by vortexing. After mixing, the liquid was allowed to stand for 10 min, then transferred to a centrifuge pre-chilled to 4°C and centrifuged at 12000 rpm for 5 min. 500 μL of the supernatant was transferred to a new 2 mL autoclaved EP tube, and an equal volume of pre-chilled isopropanol was added and thoroughly mixed by vortexing. The mixture was allowed to stand at room temperature for 10 min to promote RNA aggregation. The mixture was centrifuged at 12000 rpm for 10 min at 4°C, and the supernatant was discarded; the precipitate was the crude RNA. 1 mL of pre-chilled 75% ethanol (prepared with DEPC water, pH 5.2) was added to the precipitate, and the mixture was centrifuged at 8000 rpm for 15 min at 4°C. Discard the supernatant; the resulting precipitate is relatively pure RNA. Repeat the above steps to further purify the RNA. Place the precipitate in a clean bench at room temperature for 5-8 minutes until it becomes transparent. Add 30-50 μL LEPC water and mix well. Incubate at 55°C-60°C for 10 minutes to promote dissolution. Finally, determine the RNA concentration and purity using a micro spectrophotometer. The A260 / A280 ratio should be between 1.8 and 2.1, and the A260 / A230 ratio should be between 1.9 and 2.1.

[0096] RNA was reverse transcribed into cDNA using a high-capacity cDNA reverse transcription kit (AG, Hunan, China) on a reverse transcription thermal cycler with the following parameters: heating at 37°C for 15 min, denaturation reaction at 85°C for 5 s, and the obtained cDNA was stored at -80°C for testing after the temperature dropped to 4°C.

[0097] The PCR primer sequences used for detecting gene expression are shown in Table 6. The reaction was performed using a SYBR Green Pro Taq HS kit (AG, Hunan, China) on a real-time real-time PCR instrument (LightCycler 480 II, Roche, Switzerland).

[0098] Table 6. Gene Primer Sequence Numbers

[0099]

[0100] The reaction conditions were: 50°C pre-denaturation for 2 minutes, followed by 95°C initial denaturation for 10 minutes; then 40 cycles of amplification were performed, each cycle consisting of 95°C denaturation for 15 seconds and 60°C annealing extension for 1 minute. Each sample was tested in triplicate. The relative mRNA expression level of the target gene was measured using β-actin as an internal reference gene, employing a 2... −ΔΔCT Calculation by method.

[0101] 5.7 Analysis of Cecal Tight Junction Protein Expression Levels

[0102] Table 7. Effects of different experimental groups on the expression level of tight junction protein in broilers infected with Eimeria tenella.

[0103]

[0104] Note: Different letters in the same column of the table above indicate significant differences (P<0.05).

[0105] Table 7 shows that, compared with the control group, the expression levels of ZO-1 and Occludin in the cecum of broiler chickens in the model group were significantly reduced (P < 0.05). This indicates that coccidia infection severely disrupts the tight junction structure of cecal epithelial cells, leading to impaired intestinal mucosal barrier function. During the parasitic and reproductive process of coccidia in the cecum, they directly disrupt the structure of tight junction proteins or interfere with their synthesis and transport processes, thereby reducing their expression levels in cecal tissue.

[0106] Compared with the model group, the low-esterification group and the high-esterification group showed differentiated regulatory effects in repairing the tight junction structure of the cecum. Among them, the high-esterification group had more targeted advantages: in the low-esterification group, the expression level of ZO-1 was significantly restored and there was no significant difference from the control group, while the expression level of Occludin was significantly increased, indicating that it can fully restore the expression of the two key tight junction proteins and effectively repair the intestinal barrier damage caused by coccidia infection.

[0107] The regulation of the high esterification group showed precise targeting characteristics: although the expression level of ZO-1 was lower than that of the low esterification group, the difference between it and the control group was smaller than that of the model group, and the expression of the core scaffold protein ZO-1 was restored preferentially among the three types of proteins; although the expression level of Occludin did not reach a significant level (P>0.05), it was still 65.91% higher than that of the model group, and it was closer to the physiological level of the control group; the expression level of Claudin-1 was basically the same as that of the control group, and it was the only group that did not cause an abnormal increase in this protein.

[0108] Therefore, the high-esterification group, by preferentially restoring ZO-1 expression and maintaining the physiological level of Claudin-1, repairs the intestinal barrier while avoiding the overexpression or imbalance of tight junction proteins, which better meets the physiological needs of intestinal barrier homeostasis regulation. Although this precise targeted repair mechanism is slightly less effective than the low-esterification group in increasing Occludin, it can more efficiently rebuild the structural integrity of the intestinal physical barrier, providing a superior repair mode for blocking coccidia invasion and reducing intestinal content leakage, demonstrating the differentiated advantages of phytosterol esters in intestinal barrier repair.

[0109] 5.8 Analysis of gene expression results related to cholesterol metabolism in the cecum

[0110] Table 8. Effects of different experimental groups on the expression of cholesterol metabolism-related genes in broiler chickens infected with Eimeria tenella.

[0111]

[0112] Note: Different letters in the same column of the table above indicate significant differences (P<0.05).

[0113] Table 8 shows that, compared with the control group, the expression levels of SREBP2 and LDLR in the cecum of broiler chickens in the model group were significantly upregulated (P < 0.05), while the expression levels of ACAT1 and ACAT2 were significantly downregulated (P < 0.05). SREBP2 is a key transcription factor regulating cholesterol synthesis and uptake. Its upregulation may promote cholesterol synthesis and LDLR-mediated cholesterol uptake to meet the increased demand for cholesterol and other lipids that may arise after coccidia infection, as the growth and reproduction of coccidia or the immune response to infection may require more lipids. ACAT1 and ACAT2 are mainly responsible for converting free cholesterol into cholesterol esters for storage. Their downregulation may lead to reduced cholesterol ester synthesis, with more cholesterol existing in free form. This may affect the intracellular cholesterol metabolic balance and membrane stability, thereby affecting the normal function of cecal cells.

[0114] Compared with the model groups, the expression level of HMGCR in the cecum of broiler chickens in the high-esterification group was significantly downregulated (P < 0.05), while the expression level of LDLR in the cecum of broiler chickens in the low-esterification and high-esterification groups was significantly downregulated (P < 0.05). HMGCR is a key rate-limiting enzyme in cholesterol synthesis, and its downregulation inhibits cholesterol synthesis. This may be because the high-esterification group regulated the cholesterol synthesis pathway through some mechanism, reducing unnecessary cholesterol synthesis to maintain the body's lipid metabolism balance. The significant downregulation of LDLR expression in the cecum of broiler chickens in the low-esterification and high-esterification groups suggests that both treatments may have reduced cholesterol uptake mediated by LDLR, helping to avoid excessive cholesterol uptake caused by coccidia infection, which is of great significance for maintaining cholesterol homeostasis in cecal cells.

[0115] In summary, the broiler feed containing phytosterols and phytosterol complexes of this invention can significantly upregulate the expression of cecal tight junction proteins (ZO-1, Occludin), inhibit the release of serum pro-inflammatory factors and the overexpression of cecal pro-inflammatory factors, downregulate key lipid synthesis genes (SREBP1, FASN, SCD, DGAT2), and activate the fatty acid oxidation pathway (PPARα). This achieves the purpose of improving the growth performance of broilers infected with coccidiosis, alleviating excessive immune activation, and reshaping lipid metabolism homeostasis. This indicates that it can regulate metabolic disorders and inflammatory damage caused by coccidiosis infection through multiple targets.

[0116] Based on the above experimental results, the low-esterification group and the high-esterification group showed comparable effects in improving the production performance of broilers infected with coccidiosis. However, the high-esterification group exhibited significant advantages in coccidiosis inhibition, immune regulation, lipid metabolism regulation, and intestinal repair: Firstly, it significantly reduced broiler OPG, effectively inhibiting coccidia reproduction in the intestine, reducing parasite excretion, and lowering the risk of group infection; secondly, it significantly reduced serum IL-2, IL-6, TNF-α, and INF-γ levels, alleviating inflammatory responses, especially with a significant downregulation of the key inflammatory factor IL-17, highlighting its precise immune regulation advantages; thirdly, it significantly downregulated cecal HMGCR expression, inhibiting the rate-limiting step of cholesterol synthesis, while synergistically downregulating LDLR expression to reduce cholesterol uptake, achieving bidirectional regulation of lipid metabolism, which is more comprehensive than the low-esterification group, which only regulates LDLR; fourthly, it significantly reduced the number of cecal coccidia oocysts, alleviating tissue damage, and enhanced intestinal mucosal barrier function by upregulating ZO-1 expression, playing an important role in intestinal tissue repair.

[0117] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or system that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or system. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or system that includes that element.

[0118] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0119] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. Any equivalent structural or procedural transformations made based on the description and drawings of the present invention, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A compound agent of phytosterols and phytosterol esters, characterized in that, The compound agent comprises phytosterols and phytosterol esters in a mass ratio of 1:

10. The phytosterols mainly include 40%–60% β-sitosterol, 15%–30% oleosterol, 10%–25% stigmasterol, ≤10% rapeseed sterol, and ≤5% other impurities. The phytosterol esters include 40%–55% β-sitosterol oleate, 10%–20% rapeseed oleate, 5%–15% stigmasterol oleate, ≤5% rapeseed sterol oleate, ≤5.0% free phytosterols, ≤3.0% oleic acid, and ≤2.0% other impurities. The compound agent is added to the diet at a rate of 50–200 mg / kg. The compound agent can simultaneously downregulate HMGCR expression and upregulate ZO-1 expression.

2. The phytosterol and phytosterol ester complex agent according to claim 1, characterized in that, The phytosterol and phytosterol ester compound is added to the basal diet in the form of a premix.

3. The phytosterol and phytosterol ester complex agent according to claim 1, characterized in that, The compound of phytosterols and phytosterol esters is added to the diet at a rate of 100 mg / kg.

4. The application of the phytosterol and phytosterol ester complex as described in claim 1 in the preparation of drugs against Eimeria tenella.

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