A powder of areca nut for treating chicken coccidiosis and / or anti-bacterial and anti-inflammatory and its preparation method and application

Abstract

The application discloses a areca powder for treating chicken coccidiosis and / or resisting bacteria and eliminating inflammation, and a preparation method and application thereof. The preparation method comprises the following steps: soaking areca nuts in water, heating and refluxing extraction, filtering, and obtaining areca nut extract; the areca nut extract is concentrated and spray-dried to obtain areca nut spray-dried powder; and the areca nut spray-dried powder is mixed with sucrose to obtain the areca powder for treating chicken coccidiosis and / or resisting bacteria and eliminating inflammation. The prepared areca powder can treat chicken coccidiosis, has the effects of inhibiting bacteria, eliminating inflammation, promoting digestion, protecting the intestinal tract and improving growth performance, has good treatment effect, and is non-toxic.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a areca nut powder for treating coccidiosis in chickens and / or for antibacterial and anti-inflammatory purposes, as well as its preparation method and application. Background Technology

[0002] The long-term, excessive, and abusive use of antibiotics in animal husbandry has led to increasingly serious bacterial resistance problems, posing a significant threat to human health. In response, the EU, Japan, and South Korea have completely banned the addition of antibiotics to feed. In 2018, my country's Ministry of Agriculture and Rural Affairs issued the "Pilot Program for Reducing the Use of Veterinary Antimicrobial Drugs (2018-2021)," deciding to launch an action to reduce the use of veterinary antimicrobial drugs. The program proposed to strive to implement pilot work on reducing the use of veterinary antimicrobial drugs in the breeding process within three years, promote the reduction model of veterinary antimicrobial drug use, reduce the use of antimicrobial drugs, achieve "zero growth" in the use of veterinary antimicrobial drugs, and effectively control veterinary drug residues and animal bacterial resistance. In 2019, the Ministry of Agriculture and Rural Affairs issued Announcement No. 194, stating that in order to safeguard the safety of animal-derived food and public health in my country, it decided to stop the production, import, sale, and use of certain drugs, and explicitly required that from 2020 onwards, all growth-promoting drugs, except for traditional Chinese medicine, be phased out. This indicates that feed containing antibiotics will be completely withdrawn from the market. Livestock and poultry production faces severe challenges. Research indicates that the oxidative stress-inflammation-immune imbalance chain is the root cause of non-communicable diseases in animals, and may also lead to stunted growth, metabolic disorders, and slow development in livestock and poultry. Therefore, finding alternatives to antibiotics to control livestock and poultry diseases is imperative. Natural plant-based herbal remedies, with their advantages of being green and environmentally friendly, having low toxicity and side effects, and leaving no residues, have attracted widespread attention from researchers and livestock industry practitioners.

[0003] Therefore, under the background of "reducing and limiting antibiotics" in animal husbandry, the research and development of safe, effective, and convenient natural products to replace antibiotics has become a major trend in the industry, among which traditional Chinese medicine for veterinary use is the most popular choice as an antibiotic substitute. The "Guiding Opinions of the Ministry of Agriculture on Promoting the Healthy Development of the Veterinary Drug Industry" (Agricultural Medicine Development

[2016] No. 15) pointed out that it is necessary to accelerate the development of the traditional Chinese medicine for veterinary use industry, integrate the advantageous resources of traditional Chinese medicine for veterinary use enterprises, increase the inheritance of traditional Chinese medicine for veterinary use and the innovative research of modern traditional Chinese medicine for veterinary use, strengthen the research and development of traditional Chinese medicine for veterinary use with definite curative effects, support the creation of substitutes for feed antibiotics, and support the construction of standardized planting bases for veterinary special medicinal materials.

[0004] Areca nut is a perennial evergreen palm tree belonging to the Areca palm family. Areca catechuThe dried, mature seeds of *L.* are light reddish-brown or pale yellowish-brown, oblate or conical in shape, and hard in texture. They are harvested from late spring to early autumn, boiled in water, dried, the pericarp removed, and the seeds extracted and dried. Areca nut, also known as Renpin, White Areca Nut, Olive Seed, Xizhangdan, Qingzai, Dafuzi, Langyu, Areca Seed, Binmen, and Binmen Yaojian, is one of my country's four famous southern medicinal herbs (areca nut, *Alpinia oxyphylla*, *Amomum villosum*, and *Morinda officinalis*). Areca nut cultivation in my country is mainly distributed in Yunnan, Hainan, and Taiwan. Global annual production is approximately 1.5 million tons. Food-grade areca nut is mainly processed into dried areca nuts, consumed as a chewing condiment, and sold in Pakistan, Indonesia, and Hunan and Hainan provinces of my country. The medicinal history of areca nut dates back over 1800 years, first recorded in *Li Dangzhi Yaolu*, written by Li Dangzhi during the Three Kingdoms period. Furthermore, the *Baoqing Materia Medica Compendium* records that "Li Dangzhi said it is also called Binmen. It grows in the South China Sea, namely Guangdi, as well as the East China Sea, Kunlun, Lingwai, and Jiaokuizhou," where Binmen refers to areca nut. The *Chinese Pharmacopoeia* has included areca nut since the 1953 edition and has undergone nine revisions to date, with increasingly mature clinical standards for medicinal areca nut. Areca nut is bitter, pungent, and warm in nature. It enters the stomach and large intestine meridians and has the effects of expelling parasites, eliminating food stagnation, promoting qi circulation, and promoting diuresis. It can be used to treat tapeworm disease, ascariasis, fascioliasis, abdominal pain due to intestinal parasites, indigestion, abdominal distension due to food stagnation, constipation, and edema.

[0005] Hainan Province has a history of cultivating and consuming areca nuts for over 2,000 years, making it the largest areca nut producing province in China, accounting for over 95% of the national output. It is Hainan's second largest tropical economic crop after rubber. Currently, the areca nut planting area is 1.56 million mu (approximately 104,000 hectares), with a harvest area of ​​about 1 million mu (approximately 66,667 hectares). The annual fresh fruit yield reaches 760,000 tons, and the dried fruit yield is about 180,000 tons, with a total output value exceeding 3 billion yuan. About 500,000 households and nearly 2.3 million farmers are involved in areca nut cultivation. Wanning City accounts for about 40% of the province's areca nut planting area and its annual fresh areca nut processing capacity accounts for two-thirds of Hainan's total, earning it the reputation of "Hainan's areca nuts half produced in Wanning." Currently, most of the areca nuts produced in Hainan are dried and sold to Hunan Province, where they undergo further processing such as soaking, slicing, adding brines, and drying to become edible areca nut blocks for chewing and sale.

[0006] Hainan possesses abundant areca nut resources with a monopolistic advantage, but due to insufficient research and development, it lacks sufficient competitiveness in the national areca nut industry market, particularly in the development of products utilizing the medicinal and edible value of areca nut. In 2015, the Hainan Provincial Government proposed further exploring the medicinal value of areca nut and strengthening the development of green biological pesticides and veterinary drugs. The proposal called for the prompt organization of pharmaceutical and related scientific and technological workers to establish standard extraction standards for areca nut extracts, develop standard extracts for total alkaloids and total polyphenols, and establish a standard substance library of major components; organize pharmacologists and related researchers to intensify pharmacological screening of standard extracts and substances; strengthen the comprehensive development and utilization of the medicinal value of the uniquely fragrant areca nut flower; research the formulation of areca nut with other herbal medicines; and develop new green biological pesticides and veterinary drugs, such as natural fishpond antibiotics, swine dewormers, goat veterinary drugs, and areca nut aquatic insecticides, to promote the development of areca nut medicinal products. Summary of the Invention

[0007] The purpose of this invention is to provide a areca nut powder for treating coccidiosis in chickens and / or for antibacterial and anti-inflammatory purposes, as well as its preparation method and application. The areca nut powder prepared by this invention can treat coccidiosis in chickens and exert antibacterial, anti-inflammatory, digestive, intestinal protective, and growth performance-enhancing effects. It has good therapeutic effects and is non-toxic.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0009] The first aspect of this invention provides a method for preparing areca nut powder for treating coccidiosis in chickens and / or for antibacterial and anti-inflammatory purposes, the preparation method comprising the following steps:

[0010] (a) Soak the areca nuts in water, then heat and reflux to extract and filter to obtain areca nut extract;

[0011] (b) The areca nut extract is concentrated and spray-dried to obtain areca nut spray-dried powder;

[0012] (c) The spray-dried areca nut powder is mixed with sucrose to obtain the areca nut powder used to treat coccidiosis in chickens and / or for antibacterial and anti-inflammatory purposes.

[0013] Preferably, the areca nut is selected from at least one of areca nut medicinal material, areca nut slices, fresh areca nut, dried areca nut, areca nut core, areca nut stem, and areca nut shell.

[0014] Preferably, in step (a), the soaking time is 0.8 to 2 hours.

[0015] Preferably, in step (a), the heating and reflux extraction is performed at least twice, and the mass ratio of areca nut to water is 1:(6~10) during each heating and reflux extraction, the extraction time is 1.5~2h, and the temperature is 95~100℃.

[0016] Preferably, in step (b), the concentration temperature is 65~75℃, and the relative density is 1.02~1.06 at 65~75℃.

[0017] Preferably, in step (b), the inlet air temperature of the spray dryer is 175~185℃, the outlet air temperature is 85~95℃, and the liquid inlet rate is 8~15ml / min.

[0018] Preferably, 1g of the areca nut powder used to treat coccidiosis in chickens and / or for antibacterial and anti-inflammatory purposes contains at least 4mg of arecoline.

[0019] The second aspect of the present invention provides a areca nut powder prepared by the above preparation method for treating coccidiosis in chickens and / or for antibacterial and anti-inflammatory purposes.

[0020] The third aspect of this invention provides the application of the areca nut powder prepared by the above-mentioned preparation method for treating coccidiosis in chickens and / or for antibacterial and anti-inflammatory purposes in the preparation of feed products for treating coccidiosis in chickens and / or for antibacterial and anti-inflammatory purposes.

[0021] The fourth aspect of the present invention provides a feed for treating coccidiosis in chickens and / or for antibacterial and anti-inflammatory purposes, wherein the feed for treating coccidiosis in chickens and / or for antibacterial and anti-inflammatory purposes contains areca nut powder prepared by the preparation method at a mass content of 0.4‰ to 1.0‰.

[0022] Compared with the prior art, the beneficial effects of the present invention include at least the following:

[0023] The areca nut powder prepared by the method of this invention can treat coccidiosis in chickens and exert antibacterial, anti-inflammatory, digestive aid, intestinal protection, and growth performance improvement effects. It has good therapeutic effect and is non-toxic.

[0024] The areca nut powder of this invention has no adverse effects on the mental state, movement, and drinking of experimental chickens; it has no significant effect on the feed intake of chicks; and it has no adverse effects on routine blood tests, serum biochemical indicators, and organ indices such as the heart, liver, spleen, lungs, and kidneys. The areca nut powder of this invention has high clinical safety for use in chicks.

[0025] This invention has high stability and is suitable for mass production. Attached Figure Description

[0026] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0027] Figure 1 This is a thin-layer chromatographic identification chromatogram of the areca nut powder in the experimental examples of this invention;

[0028] Figure 2 This is the liquid chromatogram of the blank sample in the experimental examples of this invention;

[0029] Figure 3 This is the liquid chromatogram of the reference solution in the experimental examples of this invention;

[0030] Figure 4 This is the liquid chromatogram of the test solution in the experimental examples of this invention;

[0031] Figure 5 The results of the mouse ear swelling test in the experimental examples of this invention;

[0032] Figure 6 The results of the mouse ear swelling rate test in the experimental examples of this invention;

[0033] Figure 7 These are anatomical images of the cecum from different groups in the experimental examples of this invention. Detailed Implementation

[0034] The embodiments of the technical solution of the present invention will be described in detail below with reference to the examples. The following embodiments are only used to illustrate the technical solution of the present invention more clearly, and are therefore only examples, and should not be used to limit the scope of protection of the present invention.

[0035] It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning as understood by those skilled in the art to which this invention pertains.

[0036] This invention provides a method for preparing areca nut powder for treating coccidiosis in chickens and / or for antibacterial and anti-inflammatory purposes. The preparation method includes the following steps:

[0037] (a) Soak the areca nuts in water, then heat and reflux to extract and filter to obtain areca nut extract;

[0038] (b) The areca nut extract is concentrated and spray-dried to obtain areca nut spray-dried powder;

[0039] (c) The spray-dried areca nut powder is mixed with sucrose to obtain the areca nut powder used to treat coccidiosis in chickens and / or for antibacterial and anti-inflammatory purposes.

[0040] In one embodiment, the areca nut is selected from at least one of areca nut medicinal material, areca nut slices, fresh areca nut, dried areca nut, areca nut core, areca nut stem, and areca nut shell.

[0041] In one embodiment, in step (a), the soaking time is 0.8 to 2 hours.

[0042] In one embodiment, in step (a), the heating and reflux extraction is performed at least twice, with the mass ratio of areca nut to water being 1:(6~10) during each heating and reflux extraction, the extraction time being 1.5~2 hours, and the temperature being 95~100℃.

[0043] In one embodiment, in step (b), the concentration temperature is 65~75°C, and the concentration is carried out to a relative density of 1.02~1.06 at 65~75°C.

[0044] In one embodiment, in step (b), the inlet air temperature of the spray dryer is 175~185℃, the outlet air temperature is 85~95℃, and the liquid inlet rate is 8~15ml / min.

[0045] In one embodiment, 1g of the areca nut powder used to treat coccidiosis in chickens and / or for antibacterial and anti-inflammatory purposes contains at least 4mg of arecoline.

[0046] Another embodiment of the present invention provides a areca nut powder prepared by the above preparation method for treating coccidiosis in chickens and / or for antibacterial and anti-inflammatory purposes.

[0047] In another embodiment of the present invention, the areca nut powder prepared by the above-mentioned preparation method for treating coccidiosis in chickens and / or for antibacterial and anti-inflammatory purposes is used in the preparation of feed products for treating coccidiosis in chickens and / or for antibacterial and anti-inflammatory purposes.

[0048] Another embodiment of the present invention provides a feed for treating coccidiosis in chickens and / or for antibacterial and anti-inflammatory purposes, wherein the feed for treating coccidiosis in chickens and / or for antibacterial and anti-inflammatory purposes contains areca nut powder prepared by the preparation method at a mass content of 0.4‰~1.0‰.

[0049] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0050] Example 1

[0051] This embodiment describes a method for preparing areca nut powder for treating coccidiosis in chickens and / or for antibacterial and anti-inflammatory purposes. The preparation method includes the following steps:

[0052] (a) Place the areca nut material in a container and soak it in room temperature water for 1 hour (material-to-liquid ratio 1:8). Heat it under reflux at 100°C for 1.5 hours. Filter the filtrate through gauze to obtain extract 1. Add 6 times the amount of water to the residue and heat it under reflux at 100°C for 1.5 hours. Filter the filtrate through gauze to obtain extract 2. Combine extract 1 and extract 2 to obtain areca nut extract.

[0053] (b) The areca nut extract was concentrated under reduced pressure at a temperature of 70°C until the relative concentration was 1.05 (70°C) to obtain a concentrated areca nut extract. Then, it was spray-dried (inlet air temperature was 180°C, outlet air temperature was 90°C, and liquid inlet rate was 10 mL / min) to obtain spray-dried areca nut powder.

[0054] (c) The spray-dried areca nut powder is mixed with sucrose to obtain the areca nut powder used to treat coccidiosis in chickens and / or for antibacterial and anti-inflammatory purposes.

[0055] Experimental Example

[0056] I. Determination of arecoline content in the areca nut powder prepared in the above examples:

[0057] 1. Solution preparation:

[0058] Carbonate buffer solution: Weigh 1.91g of sodium carbonate and 0.56g of sodium bicarbonate using an analytical balance, add 100ml of water to dissolve them, and the solution is ready.

[0059] 2. Preparation of the reference solution:

[0060] Accurately weigh 15 mg of arecoline hydrobromide reference standard, place it in a 10 ml volumetric flask, add methanol (chromatographic grade) to dissolve and dilute to the mark, mix well, and the solution is obtained.

[0061] 3. Preparation of the control herbal solution:

[0062] Take 1g of areca nut reference material into a round-bottom flask, add 50ml of ether, then add 5ml of carbonate buffer, let stand for 30 minutes, shake occasionally, heat under reflux for 30 minutes, then transfer to a separatory funnel to separate the ether solution into an evaporating dish, evaporate to dryness, add 1ml of methanol to dissolve the residue, let stand for 1 hour, centrifuge, and take the supernatant to obtain the final product.

[0063] 4. Preparation of the test solution:

[0064] Take 1g of areca nut powder, add 20ml of anhydrous ethanol, sonicate for 20 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1ml of anhydrous ethanol, and you have the product.

[0065] 5. Thin-layer chromatography conditions:

[0066] Take 5µl of each of the above three solutions and spot them separately on the same silica gel G thin-layer plate. Use cyclohexane-ethyl acetate-concentrated ammonia solution (7.5:7.5:0.2) as the developing solvent, place the plate in a developing tank pre-saturated with ammonia vapor, develop, remove, air dry, and fumigate in iodine vapor until the spots are clearly visible.

[0067] 6. Test Results:

[0068] Thin-layer chromatographic identification chromatograms as follows Figure 1 As shown, Figure 1 In the table, a represents arecoline hydrobromide; b represents areca nut reference material; c represents test sample 1; d represents test sample 2; and e represents test sample 3.

[0069] Depend on Figure 1 It can be seen that in the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatograms of the areca nut reference material and the reference standard.

[0070] II. Determination of arecoline content in areca nut powder by high performance liquid chromatography:

[0071] 1. Solution preparation:

[0072] Mobile phase phosphoric acid solution: Pipette 2 ml of phosphoric acid into 1000 ml of water, adjust the pH to 3.8 with concentrated ammonia solution, filter, and the solution is ready.

[0073] Treatment of phosphoric acid solution with test sample solution: Pipette 5 ml of phosphoric acid into 1000 ml of water to obtain the solution.

[0074] Carbonate buffer solution: Weigh 1.91g of sodium carbonate and 0.56g of sodium bicarbonate using an analytical balance, add 100ml of water to dissolve them, and the solution is ready.

[0075] 50% acetonitrile: Measure 50 ml of acetonitrile and add it to 50 ml of water, mix well, and you have the product.

[0076] 2. Preparation of the reference solution:

[0077] Take an appropriate amount of arecoline hydrobromide reference standard, accurately weigh it, and add it to the mobile phase to prepare a solution containing 0.1 mg per 1 ml, which is the result of (arecoline weight = arecoline hydrobromide weight / 1.5214).

[0078] 3. Preparation of the test solution:

[0079] Accurately weigh approximately 0.5g of areca nut powder and place it in a stoppered conical flask. Add 50ml of ether and 3ml of carbonate buffer (dissolve 1.91g of sodium carbonate and 0.56g of sodium bicarbonate in water to make 100ml). Let stand for 30 minutes, shaking occasionally. Heat under reflux for 30 minutes. Separate the ether solution and add it to an evaporating dish containing 1ml of phosphate solution (5→1000). Extract the residue twice with ether under reflux (30ml and 20ml each time) for 15 minutes each time. Combine the ether solutions in the same evaporating dish, evaporate the ether, dissolve the residue in 50% acetonitrile solution, transfer to a 25ml volumetric flask, and add 50% acetonitrile to the mark. Shake well, filter, and collect the filtrate.

[0080] 4. Chromatographic conditions and system suitability test:

[0081] The column was packed with strong cation exchange bonded silica gel (SCX-strong cation exchange resin column); the mobile phase was acetonitrile-phosphoric acid solution (2→1000, pH adjusted to 3.8 with concentrated ammonia solution) (55:45); the detection wavelength was 215 nm. The theoretical plate number, calculated based on the arecoline peak, should be no less than 3000.

[0082] 5. Measurement method:

[0083] Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0084] 6. Test Results:

[0085] The chromatogram of the sample solution is as follows Figures 2-4 As shown, Figure 2 This is the liquid chromatogram of a blank sample; Figure 3 Here is the liquid chromatogram of the reference solution; Figure 4 The liquid chromatogram of the test sample solution;

[0086] Depend on Figures 2-4 It can be seen that the liquid chromatogram of the test sample solution shows one characteristic peak (arecoline hydrobromide) corresponding to the liquid chromatogram of the reference solution, and this characteristic peak should be consistent with the retention time of the reference solution.

[0087] 7. Calculate the effective components and their content in areca nut extract powder:

[0088] Content ω = cr × Ax / AR × V / W, where,

[0089] ω — content of the test sample, mg / g;

[0090] cr—Concentration of reference standard, mg / ml;

[0091] Ax — Peak area of ​​the test sample;

[0092] AR—Peak area of ​​the control sample;

[0093] V—Dilution factor of the test sample, in ml;

[0094] W—Sample weight of the test sample, in grams;

[0095] The content of the active ingredient in the areca nut powder is as follows: each 1g contains areca catechin (C8H12H4 ...52H42H42H52H42H52H42H52H42H52H42H52H52H42H52H52H52H 13 The amount of NO2 was calculated to be 4.3 mg.

[0096] III. Study on the inhibitory effect of areca nut powder on xylene-induced ear swelling in mice:

[0097] 1. Test Procedure:

[0098] 1.1 Test Drug:

[0099] Betel powder, prepared by the examples.

[0100] 1.2. Control drug:

[0101] Flos Lonicerae and Scutellaria baicalensis Extract Powder, 20 kg / bag, batch number 2303081, Shengtai'er (Inner Mongolia) Technology Co., Ltd.

[0102] 1.3. Test animals:

[0103] SPF-grade Kunming mice, weighing 18 - 22 g, a total of 60 mice, half male and half female, provided by Beijing Weishang Lide Biotechnology Co., Ltd., animal production license number: SCXK (Beijing) 2021 - 0010. The mice were housed in the College of Veterinary Medicine of China Agricultural University. Laboratory environment: temperature: 20 - 26 °C, humidity: 40% - 70%, pressure difference 10 Pa, illuminance 15 - 20 lx.

[0104] 1.4. Animal grouping:

[0105] The mice were divided into six groups, namely blank control group (Control), model group (Model), Flos Lonicerae and Scutellaria baicalensis Extract Powder group (SHHQ), low-dose betel powder group (BLL), medium-dose betel powder group (BLM), and high-dose betel powder group (BLH). Each group was gavaged with different test drugs for 5 days. One hour after the last administration, modeling was performed using xylene. 20 μL of xylene was applied to both the front and back sides of the right auricle of the mice, and the left ear was used as the control.

[0106] Table 1 Grouping of anti-inflammatory test of betel powder

[0107] Control 10 water —— 0.2ml water Model 10 water —— xylene SHHQ 10 Scutellaria baicalensis extract powder 200mg / kg / d xylene BLL 10 Areca nut powder 62.5 mg / kg / d xylene BLM 10 Areca nut powder 125mg / kg / d xylene BLH 10 Areca nut powder 250mg / kg / d xylene

[0108] 1.5. Process of anti-inflammatory test of betel powder:

[0109] After 30 minutes of modeling, the mice were sacrificed by cervical dislocation. The auricles of the left and right ears of the mice at the same position were punched out with an 8 mm puncher, and the weight of the ear pieces was accurately weighed, the data was recorded, and the ear swelling degree and ear swelling inhibition rate were calculated.

[0110] Ear swelling degree = weight of the right ear piece - weight of the left ear piece;

[0111] Swelling rate = (weight of the right ear piece - weight of the left ear piece) / weight of the left ear piece × 100%;

[0112] Ear swelling inhibition rate (%) = [average ear swelling degree of the model group (mg) - average ear swelling degree of the drug administration group (mg)] / average ear swelling degree of the model group (mg) × 100%.

[0113] 1.6. Data processing:

[0114] Statistical analysis was performed using SPSS 20.0 software for significance analysis. Quantitative data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons between groups, and the least significant difference (LSD) method was used when the variances between groups were homogeneous. P <0.05 indicates a statistically significant difference.

[0115] 2. Results Analysis:

[0116] The results of the xylene-induced ear swelling test of areca nut powder are as follows: Figures 5-6 As shown, by Figures 5-6 It can be seen that, compared with the blank control group, the swelling degree in the model group was significantly increased ( P <0.01), indicating that the xylene-induced ear swelling test model was successfully established; the swelling degree of the low, medium and high dose groups of areca nut powder was significantly reduced compared with the model group (<0.01). P <0.01), the swelling degree in the Scutellaria baicalensis extract powder group was significantly reduced compared with the model group ( ). P <0.05), indicating that the low, medium and high doses of areca nut powder and the Scutellaria baicalensis extract powder all have anti-inflammatory effects, and the anti-inflammatory effect of areca nut extract powder is better than that of Scutellaria baicalensis extract powder.

[0117] Table 2 Results of anti-inflammatory tests in different groups

[0118] Control Equal volumes of physiological saline 0.22±1.48 99.15 Model Equal volumes of physiological saline 25.81±3.00## —— SHHQ 200 18.89±3.97* 26.81 BLL 60 12.99±3.53** 49.67 BLM 120 12.31±3.87** 52.31 BLH 240 11.50±3.75** 55.44

[0119] Note: ## indicates a highly significant difference compared to the blank control group. P <0.01); * indicates a significant difference compared to the model group ( P <0.05); ** indicates a significant difference compared to the model group ( P <0.01).

[0120] IV. Study on the antibacterial effect of areca nut powder:

[0121] 1. Test Procedure:

[0122] 1.1 Test Drug:

[0123] Areca nut powder, prepared according to the examples.

[0124] 1.2 Test strains:

[0125] Escherichia coli, ATCC 25922, was purchased from the China Industrial Microbial Culture Collection Center; Staphylococcus aureus, ATCC 29213, was purchased from the China Industrial Microbial Culture Collection Center.

[0126] 1.3 Preparation of bacterial suspension:

[0127] Activated *Escherichia coli* and *Staphylococcus aureus* were inoculated onto MHA plates using a sterile inoculation loop. The bacterial cultures were incubated at 37°C for 24 hours. Then, colonies showing typical growth on each culture medium were picked and inoculated into the broth. The total bacterial count was determined using the plate count method to a total bacterial count of 0.5 McFarland units (1 × 10⁻⁶). 8 Prepare a bacterial suspension (CFU / ml) for later use.

[0128] 1.4 Minimum Inhibitory Concentration and Minimum Bactericidal Concentration Tests:

[0129] The bacterial suspension was adjusted to 0.5 McFarland units with sterile broth medium. In a 96-well U-shaped plate, 100 μl of MHB was added to wells A1-H1. Then, 100 μl of 64 mg / ml areca nut powder solution was added to well A1. 100 μl of this solution was then added to well B1, and the mixture was stirred. Another 100 μl was added to well B3, and so on up to well 8. Finally, 100 μl of bacterial suspension was added to each well. A sterile solubilizer and 100 μl of bacterial suspension were added to well B2. A positive control (colistin) was added to well B3. Each experiment was repeated three times. The plates were incubated at 37°C for 24 hours. The MIC (minimum inhibitory concentration) was determined by visual inspection in clear wells. 20 μl of the liquid from the visually clear wells was transferred to agar plates and incubated at 37°C for 18 hours. Wells with fewer than 5 colonies counted on blood agar plates were considered the MBC (minimum inhibitory concentration) for this drug.

[0130] 2. Results Analysis:

[0131] The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of areca nut powder against Escherichia coli were both 16 mg / ml, while the MIC and MBC of areca nut extract powder against Staphylococcus aureus were both 1 mg / ml, indicating that areca nut extract powder has a strong antibacterial effect.

[0132] Table 3. MIC test results of areca nut extract powder against Escherichia coli and Staphylococcus aureus.

[0133] 16mg / ml — — 8mg / ml + — 4mg / ml + — 2mg / ml + — 1mg / ml + — 0.5mg / ml + + 0.25mg / ml + + 0.125mg / ml + +

[0134] Note: "—" indicates no bacterial growth; "+" indicates bacterial growth.

[0135] Table 4. Results of the areca nut extract powder against Escherichia coli and Staphylococcus aureus MBC test.

[0136] 16mg / ml — — 8mg / ml + — 4mg / ml + — 2mg / ml + — 1mg / ml + — 0.5mg / ml + + 0.25mg / ml + + 0.125mg / ml + +

[0137] Note: "—" indicates no bacterial growth; "+" indicates bacterial growth.

[0138] V. Study on the efficacy of areca nut powder in treating coccidiosis in experimental chickens:

[0139] 1. Test Procedure:

[0140] 1.1. Experimental subject: Hy-Line Grey laying hens;

[0141] 1.2 Test drug: Areca nut powder, prepared according to the example.

[0142] 1.3. Control drug:

[0143] When administering Tiefeng Anti-ball Powder, follow the instructions for use, i.e., mix it with feed. Add 10g of Tiefeng Anti-ball Powder per kilogram of feed, and administer it in the same manner as the instructions.

[0144] 1.4. Administration method:

[0145] Both the test drug and the control drug were administered via feed mixing. The daily feed consumption of each group of chickens was estimated based on their age and laying hen performance indicators. The feed consumption was measured, and the test drug was accurately weighed according to the prescribed dosage for each group. The drug-containing feed for chicks was then prepared by uniformly adding the drug to the feed troughs. Before the next day's administration, the feed troughs were thoroughly cleaned, and the newly prepared feed containing the test drug was added.

[0146] 1.5. Experimental Procedure:

[0147] A total of 1200 healthy 3-day-old chickens were selected from the experimental site for inclusion in the experiment. After acclimatization, the chickens were randomly divided into four groups of 300 each using body weight as the core indicator: a betel nut powder group (BLTQS), a TFKQS control group (Tiefeng Anti-ball Powder), a positive control group (Model), and a negative control group (Control). The 3-day-old chickens underwent 7 days of acclimatization. Subsequently, except for the negative and positive control groups, the chicks in the other groups began medication at 12 days of age until 14 days of age. Except for the negative control group, the chicks in the other groups received 0.2 ml of sporulated oocysts via gavage at 14 days of age, totaling 5.0 × 10⁶ ml. 3 The animals were fed the medication daily for 8 days, from 14 to 21 days old, and observed for 7 days after stopping the medication.

[0148] 1.6. Molding method:

[0149] Eimeria tenella var. truncatulae that have been rejuvenated in chicks ( E.tenellaOocysts were placed in a 2.5% K₂Cr₂O₇ solution and incubated at 28°C for 48–72 hours to induce sporulation, until 80%–90% of the oocysts were sporulated. The oocysts were then collected and stored at 4°C for later use. The concentration of sporulated oocysts was determined using a McMaster counting chamber. Next, the required volume of the egg-containing solution was centrifuged at 3600 rpm for 5 minutes. The supernatant was slowly aspirated, and the precipitate was resuspended in PBS. This washing process was repeated 2–3 times to remove K₂Cr₂O₇. Finally, PBS was added until the solution was nearly transparent, and the volume was adjusted to a concentration of approximately 2.5 × 10⁻⁶ oocysts. 4 Count / ml, seal and store at 4℃. At 14 days of age, administer 0.2ml of sporulated oocysts orally, 5.0×10⁶. 3 indivual.

[0150] 1.7 Relative weight gain rate:

[0151] Based on the weight of each group of experimental chickens (including the weight of chickens that died during the period) recorded before grouping (D0) and at the end of administration (D11), the weight gain of each group of experimental chickens during the period D1~D10 was calculated according to the following formula I. The weight gain rate of the experimental chickens in the drug group compared with the experimental chickens in the negative control group was further calculated according to the following formula II, and inter-group comparisons were performed.

[0152] Weight gain of experimental chickens (g / chicken) = (Total weight of surviving experimental chickens on day 11 + Total weight of experimental chickens that died during days 1 to 10) - Total weight of experimental chickens on day 0 (Formula I);

[0153] The relative weight gain rate (%) of experimental chickens = (weight gain in the drug-treated group / weight gain in the negative control group) × 100 (Formula II).

[0154] 1.8. Anticoccidial efficacy test:

[0155] ① Score for bloody stool:

[0156] On days 4-7 (D7-D10) of the parasite treatment, the bloody feces of the experimental chickens in each group were recorded daily. The scoring standard was based on the method of Morehouse and Baron (1970). The scores were 0, +1, +2, +3 and +4 points respectively, according to the proportion of bloody feces in the whole feces: 0%, 1-25%, 26-50%, 51-75%, and 76-100%.

[0157] ②Cecal lesion score:

[0158] On day 7 of the parasite treatment, six experimental chickens from each group were anesthetized and euthanized. The cecum was dissected, and its lesions were scored based on visual observation, following the criteria of Johnson and Reid (1970). No visible cecal lesions and normal cecal morphology: 0 points; Scattered hematomas visible, but no abnormal contents and unchanged cecal wall thickness: 1 point; Blood visible in the cecal contents, increased cecal wall thickness, and significantly increased hemorrhagic lesions: 2 points; Blood clots or large amounts of blood visible in the cecum, with extremely increased cecal wall thickness: 3 points; Significantly swollen cecum with large amounts of blood, including cases where the chicken died: 4 points. The cecum on the more severely affected side was used for scoring. The cecal lesion score was calculated by multiplying the average cecal score of each group by ten.

[0159] ③ Number of oocysts per gram of feces (OPG):

[0160] At the end of administration (D11), fecal samples were collected from each group. Fecal examination was performed using the saturated saline flotation method, and egg counts were conducted using the McMaster method. 2g samples were randomly placed in a beaker, and 58ml of saturated saline was added and stirred with a glass rod until well mixed (if the sample had too many eggs, it needed to be diluted to 60ml). After mixing, the liquid was filtered through a 60-mesh sieve, and immediately approximately 1.5ml of the supernatant was aspirated using a disposable Pasteur pipette and poured into both counting chambers of the counting chamber. After standing on the microscope stage for 5 minutes, the samples were observed under 100x magnification. Each counting chamber had 100 squares, and the volume of a single counting chamber was 0.15ml. The total number of oocysts in the two counting chambers was recorded as n1 and n2. The number of oocysts per gram of feces was then calculated. E.tenella The formula for calculating the number of ovarian cysts (OPG value) is: OPG=[(n1+n2) / (2×0.15)]×60÷2.

[0161] ④ Oocyte ratio:

[0162] According to the Kiyoshi Kakuda method, when the ovum-to-cyst ratio is 0~1, 1~25, 26~50, 51~75, and 76~100, the corresponding ovum-to-cyst values ​​are 0, 5, 10, 20, and 40, respectively. The formula for calculating the ovum-to-cyst ratio is: Ovum-to-cyst ratio (%) = OPG of negative control group or experimental group / OPG of positive control group × 100%.

[0163] ⑤Anticoccidial Index (ACI):

[0164] After measuring the above indicators, the anticoccidial index (ACI) is calculated according to ACI = (relative weight gain rate + survival rate) − (lesion value + oocyst value). Above 180: excellent efficacy (highly effective); 160~180: good efficacy (moderately effective); 120~160: moderate efficacy (lowly effective); below 120: poor efficacy (ineffective).

[0165] 1.9 Detection of inflammatory factor markers:

[0166] In the efficacy evaluation test, blood samples were collected from 10 chickens in each group before administration (D0) and at the end of administration (D11). The serum levels of TNF-α, IL-1β, IL-6, and IL-10 were detected using an ELISA kit.

[0167] 1.10 Data Processing:

[0168] The significance of data between groups was tested using SPSS software. Measurement data such as weight gain and feed intake of experimental animals were compared between groups and expressed as "mean ± standard deviation". For comparison of differences in data at the same time point between groups, if the data conformed to a normal distribution and the variances were homogeneous, the Duncan method of one-way ANOVA was used for testing. If the data did not conform to a normal distribution or the variances were unequal, the nonparametric Kruskal-Wallis method was used for testing.

[0169] 2. Test Results:

[0170] 2.1 Relative weight gain rate:

[0171] Table 5. Relative weight gain rate of the experimental chickens

[0172] Control 34.25 - Model 25.95 75.77 TFKQS 28.75 83.95 BLTQS 31.85 92.99

[0173] As shown in Table 5, the relative weight gain rate of chickens in the Model group was the lowest (75.77%). Compared with the Model group, the relative weight gain rate of chickens in each drug administration group was higher, and the relative weight gain rate of the BLTQS group was the highest (92.99%).

[0174] 2.2 Anticoccidial efficacy test:

[0175] ① Score for bloody stool:

[0176] Table 6. Statistics of bloody stools in chickens of each experimental group during the drug administration period.

[0177] Control <![CDATA[0.00±0.00 c ]]> <![CDATA[0.00±0.00 c ]]> <![CDATA[0.00±0.00 c ]]> <![CDATA[0.00±0.00 c ]]> Model <![CDATA[1.73±0.46 a ]]> <![CDATA[2.33±0.49 a ]]> <![CDATA[3.20±0.41 a ]]> <![CDATA[3.13±0.64 a ]]> TFKQS <![CDATA[0.67±0.49 b ]]> <![CDATA[1.13±0.35 b ]]> <![CDATA[2.13±0.35 ab ]]> <![CDATA[1.47±0.52 b ]]> BLTQS <![CDATA[0.47±0.52 bc ]]> <![CDATA[1.27±0.46 b ]]> <![CDATA[1.07±0.46 bc ]]> <![CDATA[0.60±0.51 bc <!-- 10 -->]]>

[0178] Note: In the same column, different letter subscripts indicate significant differences. P <0.05), the same letter subscript or no letter subscript indicates no statistically significant difference ( P >0.05).

[0179] As shown in Table 6, when chicks were inoculated with coccidiosis at 14 days of age, the amount of bloody stool excretion was significantly higher in the Model group compared to the Control group on days 4-7 post-infection. P<0.05); Compared with the Model group, on days 4-7 post-infection (D7-D10), the stool blood score decreased in all treatment groups, showing a trend of first increasing and then decreasing. Compared with the Control group, stool blood was reduced in all treatment groups, but there was no significant difference in stool blood score. P >0.05).

[0180] ②Cecal lesion scoring: Cecal necropsy image as shown Figure 7 As shown;

[0181] Table 7. Cecal lesion scores and values ​​for each group during the drug administration period.

[0182] Control <![CDATA[0.00±0.00 c ]]> 0 Model <![CDATA[2.70±0.67 a ]]> 27 TFKQS <![CDATA[1.50±0.97 ab ]]> 15 BLTQS <![CDATA[1.20±0.63 bc ]]> 12

[0183] Note: In the same column, different letter subscripts indicate significant differences. P <0.05), the same letter subscript or no letter subscript indicates no statistically significant difference ( P >0.05).

[0184] As shown in Table 7, compared with the Control group, the cecal lesion score of the Model group was significantly higher. P <0.05); compared with the Model group, the cecal lesion score of the treatment group was reduced.

[0185] ③ Oocyst count and anticoccidial index:

[0186] Table 8. Scores of oocyst counts and anticoccidial index for each group.

[0187]

[0188] Note: In the same column, different letter subscripts indicate significant differences. P <0.05), the same letter subscript or no letter subscript indicates no statistically significant difference ( P >0.05).

[0189] As shown in Table 8, at the end of administration (D11), the number of ovarian follicles (OPG) excreted in the feces of each treatment group was significantly lower than that of the Model group. P <0.05), with no significant difference between the treatment groups ( P >0.05). Compared with the Model group, the oocyst ratio and oocyst value decreased in the BLTQS group. The anticoccidial index results showed that all groups in the BLTQS group achieved a moderate anticoccidial effect.

[0190] 2.3 Detection of inflammatory factor markers:

[0191] Table 9 Results of serum inflammatory factor-related indicators

[0192]

[0193] Note: In the same column, different letter subscripts indicate significant differences. P <0.05), the same letter subscript or no letter subscript indicates no statistically significant difference ( P >0.05).

[0194] As shown in Table 9, before drug administration (D0): there were no significant differences in serum TNF-α, IL-1β, IL-6, and IL-10 levels among the groups before drug administration (D0). P >0.05), indicating that the chickens in each group were relatively stable in the early stages of the experiment. At the end of administration (D11): compared with the Control group, the Model group showed significantly increased levels of IL-1β, IL-6, and TNF-α ( P <0.05); compared with the Model group, the TFKQS and BLTQS groups showed significantly lower levels of IL-1β, IL-6, and TNF-α ( P <0.05). There was no significant difference in IL-10 levels between the Model group and the Control group. P >0.05); compared with the Model group, the IL-10 levels in the BLM and BLH groups were significantly increased ( P <0.05).

[0195] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A method for preparing areca nut powder for treating coccidiosis in chickens and / or for antibacterial and anti-inflammatory purposes, characterized in that, The preparation method includes the following steps: (a) Soak the areca nuts in water, then heat and reflux to extract and filter to obtain areca nut extract; (b) The areca nut extract is concentrated and spray-dried to obtain areca nut spray-dried powder; (c) The spray-dried areca nut powder is mixed with sucrose to obtain the areca nut powder used to treat coccidiosis in chickens and / or for antibacterial and anti-inflammatory purposes.

2. The preparation method according to claim 1, characterized in that, The areca nut is selected from at least one of the following: areca nut medicinal material, areca nut slices, fresh areca nut, dried areca nut, areca nut core, areca nut stem, and areca nut shell.

3. The preparation method according to claim 1, characterized in that, In step (a), the soaking time is 0.8 to 2 hours.

4. The preparation method according to claim 1, characterized in that, In step (a), the heating and reflux extraction is performed at least twice. During each heating and reflux extraction, the mass ratio of areca nut to water is 1:(6~10), the extraction time is 1.5~2 hours, and the temperature is 95~100℃.

5. The preparation method according to claim 1, characterized in that, In step (b), the concentration temperature is 65~75℃, and the relative density is 1.02~1.06 at 65~75℃.

6. The preparation method according to claim 1, characterized in that, In step (b), the inlet air temperature of the spray dryer is 175~185℃, the outlet air temperature is 85~95℃, and the liquid inlet rate is 8~15ml / min.

7. The preparation method according to claim 1, characterized in that, The areca nut powder described in 1g for treating coccidiosis in chickens and / or for antibacterial and anti-inflammatory purposes contains at least 4mg of arecoline.

8. The preparation method according to any one of claims 1 to 7 yields a areca nut powder for treating coccidiosis in chickens and / or for antibacterial and anti-inflammatory purposes.

9. The areca nut powder prepared by any one of claims 1 to 7 for treating coccidiosis in chickens and / or for antibacterial and anti-inflammatory purposes is used in the preparation of feed products for treating coccidiosis in chickens and / or for antibacterial and anti-inflammatory purposes.

10. A feed for treating coccidiosis in chickens and / or for antibacterial and anti-inflammatory purposes, characterized in that, The feed for treating coccidiosis in chickens and / or for antibacterial and anti-inflammatory purposes contains areca nut powder prepared by any one of claims 1 to 7 with a mass content of 0.4‰ to 1.0‰.

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