Application of oxidized berberine and neohesperidin in improving intestinal health of broiler chickens

Abstract

The invention discloses application of oxidized berberine and neohesperidin in improving intestinal health of broiler chickens, and belongs to the technical field of feed additives. Research finds that oxidized berberine and neohesperidin can increase the intestinal villus height, crypt depth and goblet cell number of broiler chickens and improve intestinal health; the slaughter indexes such as daily weight gain and feed intake can be improved, the feed-weight ratio is reduced, and the slaughter indexes such as carcass weight and semi-eviscerating weight are increased; meanwhile, the serum immune factor and antioxidant index level is improved, the safety is high, and no obvious toxicity exists; and the two components are combined for use, so that a synergistic interaction effect is also achieved. The invention provides a new raw material resource for improving the intestinal health of the broiler chicken and improving the production performance and economic value of the broiler chicken.

Description

Technical Field

[0001] This invention relates to the field of feed additive technology, particularly the application of oxidized berberine and neohesperidin in improving the intestinal health of broilers. Background Technology

[0002] In modern broiler farming, gut health remains a key factor restricting broiler production performance and profitability. Broiler intestinal diseases are numerous, exhibiting complex and diverse pathogenesis and clinical symptoms. Intestinal flora imbalance is a common problem, clinically manifesting as foamy cecal feces, orange feces, and large clumps of cecal feces, accompanied by increased water intake. When flora imbalance occurs, broilers experience reduced feed intake, impaired growth, and foot diseases are also common. While antibiotics can sometimes temporarily restore normal feces, broilers remain susceptible throughout their growth period and cannot develop permanent immunity. Relapse is highly likely if the underlying cause is not addressed. Coccidiosis poses a significant threat to broiler farming. Although caged broilers have relatively less contact with feces and a lower infection rate than floor-raised and net-raised broilers, for broiler farms that have used cages for many consecutive years, inadequate cleaning and disinfection of cages, cage racks, and manure scrapers can still lead to serious coccidiosis problems. Coccidiosis infection not only damages the intestinal mucosa, affecting the absorption of feed nutrients and water, and causing loose stools containing undigested feed, but also changes the pH value of the intestinal environment, leading to an imbalance of intestinal flora and a decrease in the activity of digestive enzymes.

[0003] Gut health is crucial for broilers. A healthy gut is the foundation for broilers' normal digestion and absorption of nutrients, directly affecting their growth rate, feed conversion ratio, and immunity. When the intestinal barrier function is normal, it effectively prevents pathogen invasion and maintains the stability of the internal environment. Once gut health is impaired, broiler growth performance will decline severely, resulting not only in slow growth and failure to reach target weight, but also increased susceptibility to disease, higher mortality rates, and significantly increased farming costs, seriously impacting the economic benefits of broiler farming.

[0004] Currently, the industry has adopted various measures to improve the gut health of broilers. In terms of feeding management, this includes strengthening environmental control in chicken houses, such as regularly disinfecting chicken houses, cages, and floors; promptly cleaning and harmlessly treating feces and damp bedding; and maintaining appropriate stocking density, humidity, and good ventilation to reduce the growth and spread of pathogens. Regarding disease prevention, antibiotics are used to treat intestinal diseases in sick broilers, such as sulfadiazine sodium soluble powder, colistin sulfate soluble powder, and amoxicillin soluble powder. However, the long-term and excessive use of antibiotics has led to increasingly serious problems of bacterial resistance, and drug residues can affect chicken quality and endanger consumer health. Furthermore, antibiotic use cannot fundamentally solve gut health problems and cannot effectively improve the intestinal microecological environment and intestinal mucosal barrier function. Against this backdrop, the development of safe, efficient, and green products to improve the gut health of broilers is urgently needed. Summary of the Invention

[0005] The purpose of this invention is to provide the application of oxidized berberine and neohesperidin in improving the intestinal health of broilers, thereby addressing the problems existing in the prior art. This invention has found that oxidized berberine and neohesperidin can increase the height of intestinal villi, crypt depth, and the number of goblet cells in broilers, thus improving intestinal health; they can also increase daily weight gain and feed intake, reduce feed conversion ratio, and increase slaughter weight, semi-eviscerated weight, and other slaughter indicators; simultaneously, they improve serum immune factors and antioxidant levels, and are highly safe with no significant toxicity; the combined use of the two components also has a synergistic effect. This invention provides a new raw material resource for improving the intestinal health of broilers and enhancing their production performance and economic value.

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

[0007] This invention provides the application of oxidized berberine in the preparation of feed or feed additives that improve the intestinal health of broilers.

[0008] This invention also provides the application of novel hesperidin in the preparation of feed or feed additives that improve the gut health of broilers.

[0009] This invention also provides the application of oxidized berberine and neohesperidin in the preparation of feed or feed additives that improve the intestinal health of broilers.

[0010] Furthermore, improving the gut health of broilers refers to increasing the height of intestinal villi, the depth of crypts, and the number of goblet cells in the broiler gut.

[0011] This invention also provides the application of oxidized berberine in the preparation of feed or feed additives that improve the growth and slaughter performance of broilers.

[0012] This invention also provides the application of novel hesperidin in the preparation of feed or feed additives that improve the growth and slaughter performance of broilers.

[0013] This invention also provides the application of oxidized berberine and neohesperidin in the preparation of feed or feed additives that improve the growth and slaughter performance of broilers.

[0014] Furthermore, the growth performance includes daily weight gain, feed intake, and feed conversion ratio; the slaughter performance includes carcass weight, semi-eviscerated weight, fully eviscerated weight, breast muscle weight, leg muscle weight, and abdominal fat weight.

[0015] The present invention also provides a feed additive for improving the intestinal health of broilers, wherein the feed additive includes oxidized berberine and neohesperidin.

[0016] The present invention also provides a feed for improving the intestinal health of broilers, the feed comprising oxidized berberine, neohesperidin and a basal feed.

[0017] The present invention discloses the following technical effects:

[0018] This invention has found that oxidized berberine and neohesperidin can increase the height of intestinal villi, crypt depth, and number of goblet cells in broilers, thus improving intestinal health. They can also increase daily weight gain and feed intake, reduce feed conversion ratio, and increase slaughter weight, semi-eviscerated weight, and other slaughter indicators. Simultaneously, they improve serum immune factors and antioxidant levels, with high safety and no significant toxicity. The combined use of the two components also has a synergistic effect. This invention provides a new raw material resource for improving broiler intestinal health and enhancing broiler production performance and economic value. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Berberine oxide is an oxidized derivative of berberine, which is mainly derived from the dried rhizomes of plants in the Ranunculaceae family such as Coptis chinensis and Phellodendron chinense, and plants in the Berberidaceae family such as Meringue and Mahonia fortunei. Berberine is typically extracted from these plants first, and then oxidized berberine is obtained through chemical oxidation. Berberine extraction often employs methods such as ethanol percolation and acid-water extraction. After extraction, relatively pure berberine is obtained, which is then oxidized under the action of a suitable oxidizing agent to synthesize berberine oxide. Current research indicates that berberine oxide possesses anti-inflammatory effects and has a good therapeutic effect on ulcerative colitis.

[0025] Neohesperidin belongs to the flavonoid family and is mainly found in the peel, pulp, and juice of citrus fruits such as oranges, tangerines, and lemons. It is typically extracted using solvent extraction methods, employing organic solvents such as ethanol and methanol to extract the relevant parts of the citrus plants. Neohesperidin is then separated and purified using methods such as chromatography and crystallization. Current research indicates that neohesperidin possesses anti-inflammatory, antioxidant, antibacterial, anticancer, immunomodulatory, anti-radiation, and cardiovascular protective effects.

[0026] The research approach of this invention is as follows:

[0027] First, the effects of berberine oxide and neohesperidin on broiler gut health and growth performance were individually verified. The results showed that 25 mg / kg berberine oxide and 80 mg / kg neohesperidin were more effective in maintaining broiler gut health and improving broiler growth and slaughter performance. Based on the results of independent experiments, this invention combines the two components to verify their effects on maintaining broiler gut health and improving broiler growth and slaughter performance.

[0028] Berberine oxide: CAS No. 549-21-3, purity: HPLC ≥ 98%.

[0029] Neohesperidin: CAS No. 13241-33-3, purity: HPLC ≥ 98%.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0031] Example 1

[0032] First, toxicity tests were conducted on oxidized berberine and neohesperidin, mainly including long-term toxicity tests and acute toxicity tests.

[0033] Acute toxicity test: The median lethal dose (LD50) of oxidized berberine and neohesperidin was determined according to the People's Republic of China National Standard "Chemicals: Acute Oral Toxicity Test Methods (GB / T21603-2008)". 50 The specific method is as follows: 110 KM mice, half male and half female, were used. After fasting for 12 hours with free access to water, they were randomly divided into a control group, five groups of oxidized berberine (50.2 mg / kg, 162 mg / kg, 512.2 mg / kg, 1638 mg / kg, 5243.6 mg / kg), and five groups of neohesperidin (40.2 mg / kg, 129 mg / kg, 411.2 mg / kg, 1314 mg / kg, 4221 mg / kg), with 10 mice in each group, half male and half female. The oxidized berberine and neohesperidin groups were administered 0.1 mL / 10g body weight via gavage, while the control group received an equal volume of 0.5% Tween 80. After administration, the mice had free access to food and water and were observed continuously for 2 weeks. Daily changes in body weight, food intake, daily living conditions, and mortality were recorded. In addition, the toxic reactions and time of death of mice in each group were observed and recorded every 3 hours within 12 hours of administration, and the above indicators were observed and recorded every 6 hours after 12 hours of administration. The experimental results showed that no mice in the oxidized berberine and neohesperidin groups died within 14 days, and the mice had normal activity and smooth, shiny fur.

[0034] Long-term toxicity test: Forty SD rats were randomly divided into two groups, one group was fed oxidized berberine feed additive and the other group was fed neohesperidin feed additive (directly fed feed additive, 2 g / 100 g body weight, three times a day, morning, noon and evening) for 120 consecutive days. 25 hours after the last feeding, half of the animals in each group (half male and half female) were sacrificed, and the remaining half were observed for another 2 weeks before being sacrificed. During the experiment, the animals' appearance, general behavior, food intake, and weight changes were observed. Hematological (RBC, HB, PLT, CT, WBC and differential) and blood biochemistry (ALT, ALT, ALP, Glu, BUN, CRE, AST, ALB), urine biochemistry, organ coefficients, and histopathological examinations were performed after 120 days of feed additive feeding and 2 weeks after feed additive withdrawal. The experimental results showed that animals fed with oxidized berberine and neohesperidin were generally in good condition, with no abnormal changes in appearance, behavior, food intake, or weight gain; hematological examinations, blood biochemistry tests, and urine biochemistry tests were all within the normal range; and no obvious abnormalities were found in the histopathological examination of major organs. These indicators remained unchanged even after two weeks of discontinuation of the feed additive. Continuous feeding of oxidized berberine and neohesperidin for 120 days had no significant effect on rats, with no clearly defined toxic target organs or sensitive indicators. No delayed toxic reactions were observed during the recovery period, suggesting that these two components have a high safety profile for clinical application.

[0035] Example 2

[0036] After confirming that oxidized berberine and neohesperidin have no significant toxic effects on animals, the effects of the two components on broiler chickens were verified.

[0037] 1. Test materials

[0038] Forty-eighty healthy AA broilers of similar weight at one day old were randomly divided into eight groups, with six replicates per group and ten birds per replicate. The basal diet used in the experiment was a corn-soybean meal diet. The specific diet composition and nutrient levels are shown in Table 1. The control group was fed the basal diet. The groups with different doses of oxidized berberine (OBB) were fed a basal diet of 25 mg / kg, 50 mg / kg, and 75 mg / kg OBB, respectively. The groups with different doses of neohesperidin (NEO) were fed a basal diet of 40 mg / kg, 80 mg / kg, and 120 mg / kg NEO, respectively. The combined application group (OBB+NEO) was fed a basal diet of 25 mg / kg OBB and 80 mg / kg NEO, respectively.

[0039] The entire experiment lasted 42 days. During the experiment, all chickens had free access to feed and water. The mental state, feed intake, and health status of the experimental chickens were observed daily.

[0040] Table 1. Composition and nutrient levels of basal diet (air-dried basal diet)

[0041] Note: 1 The premix provides the following per kilogram of feed: Vitamin A 8000 IU; Vitamin D3 2000 IU; Vitamin E 20 IU; Vitamin K3 15 mg; Vitamin B1 3 mg; Vitamin B2 8 mg; Vitamin B6 3 mg; Vitamin B12 0.015 mg; Folic acid 0.95 mg; Niacin 35 mg; D-Pantothenic acid 15 mg; Biotin 0.1 mg; Copper (as copper sulfate) 7 mg; Iron (as ferrous sulfate) 130 mg; Zinc (as zinc sulfate) 70 mg; Manganese (as manganese sulfate) 84 mg; Iodine (as potassium iodide) 0.7 mg; Selenium (as sodium selenite) 0.24 mg. 2 Metabolizable energy was calculated based on the Chinese Feed Composition and Nutritional Value Table (34th Edition, 2023). Crude protein was based on GB / T6432-2018, amino acids on GB / T18246-2019, calcium on GB / T6436-2018, and available phosphorus on GB / T6437-2018.

[0042] 2. Feeding and Management

[0043] The feeding experiment was conducted at the experimental chicken farm of the Institute of Animal Husbandry and Veterinary Medicine, Jiangxi Academy of Agricultural Sciences, and was approved by the Ethics Committee of the Institute of Animal Husbandry and Veterinary Medicine, Jiangxi Academy of Agricultural Sciences, strictly following local laws and regulations and institutional requirements. Before the experimental chickens were placed in the coop, the entire coop and pens were thoroughly cleaned and disinfected. The experiment used a wire mesh cage system, with 5 chickens per cage (each cage measuring 75 cm * 70 cm * 40 cm), and free access to feed and water. During the brooding period, the temperature inside the coop was maintained at 30–35℃, and the relative humidity at 70%–80%; during the rearing period, the temperature was controlled at approximately 23℃, and the relative humidity at 65%–75%. Lighting was set to 24-hour continuous illumination (light intensity of 5 lkx).

[0044] 3. Measurement methods for each indicator

[0045] 1) Growth performance indicators

[0046] Feed and water were withheld for the first 12 hours on days 21 and 42 of the experiment. On the last day of the experiment, the weight of the broilers was measured in replicates to calculate the average daily gain (ADG) for each individual. At the same time, the total feed intake for each replicate during the experimental period was recorded to calculate the average daily feed intake (ADFI) for each individual and the feed conversion ratio (F / G) for each group.

[0047] 2) Serum markers

[0048] Before slaughter, the experimental chickens were fasted for 12 hours and provided with drinking water only. Two chickens with normal growth and development, good body condition and close to average weight were randomly selected from each replicate. Blood was collected from the wing vein and transferred to a 1.5 mL sterile centrifuge tube. The tube was centrifuged at 1000 g for 10 min. The separated serum was stored at -80℃ for the detection of serum indicators.

[0049] Serum immune markers: The levels of immunoglobulin A (IgA), immunoglobulin M (IgM), and immunoglobulin G (IgG) in serum were measured using an enzyme-linked immunosorbent assay (ELISA) kit manufactured by Shanghai Enzyme Linked Products Co., Ltd., and a Thermo Fisher Scientific Multiskan SkyHigh microplate reader.

[0050] Serum antioxidant indicators: The levels of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), and catalase (CAT) in serum were detected by colorimetric method. All kits were purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.

[0051] 3) Slaughtering performance

[0052] On day 42 of the experiment, two broilers were selected from each replicate and weighed. After recording the live weight, the broilers were bled to death by bleeding from the neck. Their feathers were plucked, the water was drained, and they were weighed and the following weights were recorded: broiler carcass weight, semi-eviscerated weight, fully eviscerated weight, breast muscle weight, leg muscle weight, and abdominal fat weight.

[0053] 4) Intestinal structure

[0054] After the fixed jejunal segments were trimmed, dehydrated, embedded, sectioned, and stained, the stained paraffin sections were dried. Multiple images of villi and crypts were selected under an Eclipse Ci-L upright white light microscope. Visiopharm software was used to identify intestinal villi, crypts, and intestinal walls, and to measure villi height (VH), crypt depth (CD), and villi-to-crypt ratio (V / C).

[0055] 4. Test Results

[0056] 1) Effects on growth performance

[0057] As shown in Table 2, compared with the control group, the addition of 25 mg / kg OBB and 80 mg / kg NEO to the diet significantly improved the average daily weight gain of broilers from days 21 to 42 and from 0 to 21, as well as the feed conversion ratio from day 21 to 42. In contrast, the combined application group had a more significant effect on reducing the feed conversion ratio of broilers. This indicates that the combined use of OBB and NEO can play a synergistic role in promoting the utilization and conversion efficiency of feed in broilers.

[0058] Table 2 Effects on broiler growth performance (0-42 days)

[0059] 2) Impact on slaughter performance

[0060] Table 3 shows that, compared with the control group, the addition of different doses of OBB and NEO to the feed had a certain impact on slaughter performance. Specifically, compared with the control group, the addition of 25 mg / kg OBB and 80 mg / kg NEO to the feed significantly increased breast muscle weight, eviscerated weight, and semi-eviscerated weight; the improvement effect of adding 25 mg / kg OBB and 80 mg / kg NEO to the feed on broiler slaughter performance was even more pronounced.

[0061] Table 3 Effects on broiler slaughter performance (0-42 days)

[0062] 3) Effects on serum immune markers

[0063] Table 4 shows that, compared with the control group, the addition of different doses of OBB and NEO to the feed had a certain regulatory effect on serum immune factors. Specifically, compared with the control group, the addition of 25-50 mg / kg OBB and 40-80 mg / kg NEO to the feed significantly increased the levels of IgA, IgM, and IgG in the serum; the effects were even more pronounced with the addition of 25 mg / kg OBB and 80 mg / kg NEO. These results indicate that the addition of OBB and NEO to the diet can significantly increase the levels of immune factors in the serum of broilers, thereby enhancing the body's disease resistance.

[0064] Table 4 Effects on serum immune factors in broilers

[0065] 4) Effects on serum antioxidant indicators

[0066] Table 5 shows that, compared with the control group, the addition of 25 mg / kg OBB, 80 mg / kg NEO, or both 25 mg / kg OBB and 80 mg / kg NEO to the diet significantly increased the levels of antioxidant markers (SOD, CAT, T-AOC, and GSH-Px) in broiler serum. These results indicate that the addition of OBB and NEO to the diet can improve the antioxidant capacity of broiler serum.

[0067] Table 5 Effects on antioxidant capacity of broilers

[0068] 5) Effects on the gut

[0069] Table 6 shows that, compared with the control group, the addition of 25 mg / kg OBB, 80 mg / kg NEO, or both 25 mg / kg OBB and 80 mg / kg NEO to the diet significantly increased the number of goblet cells in the broiler intestine. Furthermore, the addition of 25-50 mg / kg OBB, or both 25 mg / kg OBB and 80 mg / kg NEO to the diet, increased the villus height in the broiler intestine; the addition of 25 mg / kg OBB, or both 25 mg / kg OBB and 80 mg / kg NEO to the diet, increased the crypt depth in the broiler intestine. Compared with the control group, the addition of different doses of OBB and NEO to the diet had little effect on the villus height / crypt depth ratio and the villus epithelial length.

[0070] Table 6. Effects of villus height, crypt depth, villus epithelial length, goblet cells, and villus height / crypt depth on broiler jejunum.

[0071]

[0072] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of oxidized berberine in the preparation of feed or feed additives that improve the intestinal health of broilers.

2. Application of neohesperidin in the preparation of feed or feed additives that improve the intestinal health of broilers.

3. Application of oxidized berberine and neohesperidin in the preparation of feed or feed additives that improve the intestinal health of broilers.

4. The application according to any one of claims 1-3, characterized in that, Improving the gut health of broilers refers to increasing the height of intestinal villi, the depth of crypts, and the number of goblet cells.

5. Application of oxidized berberine in the preparation of feed or feed additives that improve the growth and slaughter performance of broilers.

6. Application of neohesperidin in the preparation of feed or feed additives that improve the growth and slaughter performance of broilers.

7. Application of oxidized berberine and neohesperidin in the preparation of feed or feed additives that improve the growth and slaughter performance of broilers.

8. The application according to any one of claims 5-7, characterized in that, The growth performance includes daily weight gain, feed intake, and feed conversion ratio; the slaughter performance includes carcass weight, semi-eviscerated weight, fully eviscerated weight, breast muscle weight, leg muscle weight, and abdominal fat weight.

9. A feed additive for improving the intestinal health of broilers, characterized in that, The feed additives include oxidized berberine and neohesperidin.

10. A feed for improving the intestinal health of broilers, characterized in that, The feed includes oxidized berberine, neohesperidin, and a basal diet.

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