Use of bayberry bark in the preparation of a medicament for preventing or treating a parasitic infection or a complication caused by a parasitic infection

Abstract

The application provides use of waxberry tree bark in preparation of a medicine for preventing or treating parasitic infection or complications caused by parasitic infection, and belongs to the field of veterinary drugs. In the application, the waxberry tree bark can be used alone for killing of balantidium coli or prevention and treatment of chicken coccidiosis or avian trichomoniasis. The application has the characteristics of simple preparation and obvious prevention and treatment effect, and can be used for prevention and treatment of parasitic diseases in veterinary clinics, and has a higher insecticidal effect compared with many traditional Chinese medicinal materials.

Description

Technical Field

[0001] This invention relates to the field of veterinary medicine technology, specifically to the use of bayberry bark in the preparation of drugs for the prevention or treatment of parasitic infections or complications caused by parasitic infections. Background Technology

[0002] *Scutociliformes* belongs to the phylum Ciliata, class Oligohymena, subclass Scutociliformes, order Scutociliformes, and suborder Scutociliformes. The worm's body is spindle-shaped, slightly pointed at the anterior end and blunt at the posterior end. *Scutociliformes* causes significant losses to large yellow croaker fry and primarily harms marine aquaculture fish. Studies have shown that turbot, flounder, redfin pufferfish, and sea bass can also be infected and cause damage.

[0003] Coccidiosis in chickens is a general term for diseases caused by one or more intracellular parasitic protozoa of the genus *Eimeria*. It is a protozoan disease that causes significant losses in poultry farming. Common chicken coccidia include *Eimeria tenella*, *Eimeria necatrix*, *Eimeria acervulina*, *Eimeria maxima*, *Eimeria brunetti*, *Eimeria mitis*, and *Eimeria praecox*. Clinically, it manifests as chicken mortality, bloody diarrhea, stunted growth, reduced egg production, and decreased feed conversion ratios, resulting in substantial economic losses. Currently, chicken coccidiosis is mainly controlled through chemically synthesized anticoccidial drugs, vaccines, and traditional Chinese medicine. However, with the long-term and extensive use of anticoccidial drugs, chicken coccidiosis has developed increasingly severe drug resistance. Furthermore, with increasing demands for meat safety and stricter drug regulations, the use of these drugs is becoming more restricted or even prohibited. The European Union completely banned the addition of anticoccidial drugs to feed in 2012, making the control of chicken coccidiosis increasingly difficult. Coccidiosis vaccines are used in coccidiosis control, but traditional vaccines are all live vaccines, requiring monitoring during use to avoid the appearance of obvious symptoms of coccidiosis. Overdose also necessitates the use of anticoccidial drugs for control.

[0004] Avian trichomoniasis is a parasitic disease caused by the parasite *Trichomonas vaginalis*, which infects the upper digestive tract of poultry. This disease primarily affects pigeons, causing oral mucosal ulcers and necrosis. In severe cases, it can lead to death due to respiratory distress and exhaustion, resulting in significant economic losses for the pigeon farming industry. *Trichomonas vaginalis* is mainly transmitted through contaminated feed and water. Pigeons, turkeys, chickens, ducks, geese, and hawks are all susceptible, with young birds being particularly vulnerable.

[0005] Traditional Chinese medicine is an important source for the research and development of innovative drugs, and it is also one of the important sources for the development of antiparasitic drugs. Searching for anticoccidial drugs from traditional Chinese medicine and natural plants is a hot topic in the research.

[0006] The bark of the Chinese bayberry tree (Myrica rubra) is the bark, root bark, or root of the plant *Myrica rubra* (family Myricaceae). According to literature, it possesses antibacterial and hemostatic properties. It tastes bitter, pungent, and slightly astringent, and is warm in nature. It enters the liver and stomach meridians. Its functions include promoting blood circulation, relieving pain, stopping bleeding, detoxifying, and reducing swelling. It is used to treat abdominal pain, hypochondriac pain, toothache, hernia, traumatic injuries, fractures, hematemesis, epistaxis, hemorrhoidal bleeding, metrorrhagia, external bleeding, sores and swelling, mumps, gingivitis, burns, leg ulcers, eczema, scabies, colds, diarrhea, and dysentery. However, there are currently no reports on the application of Chinese bayberry bark in killing scutellaria barbata, preventing coccidiosis in chickens, and preventing trichomoniasis in poultry. Summary of the Invention

[0007] Purpose of the invention

[0008] The purpose of this invention is to provide the application of *Myrica rubra* bark (or *Myrica rubra* bark as the sole active ingredient) in the preparation of medicines for the prevention or treatment of parasitic infections or complications caused by parasitic infections. In this invention, *Myrica rubra* bark can be used alone for the killing of scutellaria or the prevention and treatment of coccidiosis in chickens or trichomoniasis in birds. It features simple preparation and significant preventive and therapeutic effects, and can be used for the prevention and control of parasitic diseases in veterinary clinical practice.

[0009] Solution

[0010] To achieve the objectives of this invention, the technical solution adopted is as follows:

[0011] In a first aspect, the present invention provides the use of bayberry bark in the preparation of a medicine for the prevention or treatment of parasitic infections or complications caused by parasitic infections.

[0012] Secondly, the invention provides the application of bayberry bark as the sole active ingredient in the preparation of a drug for the prevention or treatment of parasitic infections or complications caused by parasitic infections.

[0013] In either the first or second aspect, the parasite is a parasite that infects birds, mammals and / or fish, optionally a scutellarinus and / or coccidia and / or trichomoniasis.

[0014] In either the first or second aspect, the parasite is a shield ciliate, and the drug can be a water extract of the bark of the Chinese bayberry tree.

[0015] In either the first or second aspect, the drug is a medicated bath preparation.

[0016] In either the first or second aspect, the parasite is chicken coccidia and / or avian trichomoniasis; optionally, the chicken coccidia is Eimeria tenella; optionally, the avian trichomoniasis is Trichomonas pigeonis. The drug can be pulverized bayberry bark (optionally, the pulverized bayberry bark is sieved through a 40-mesh sieve).

[0017] In either the first or second aspect, the drug is an oral drug or a nasal spray.

[0018] In either the first or second aspect, the dosage form of the drug is any one of powder, granules, capsules, tablets, or liquid. Optionally, the crude amount of bayberry bark in the liquid is 0.1–1 g / mL, or optionally, the crude amount is 0.4–1 g / mL. Optionally, the liquid includes oral liquid or medicated bath.

[0019] In either the first or second aspect, the drug is a myrica bark extract or myrica bark powder (optionally, the myrica bark powder passes through a 40-mesh sieve), optionally a water extract (optionally, the water extract can be used directly as a liquid preparation, or dried to obtain powder, granules, capsules or tablets), optionally an aqueous extract, optionally a hydrothermal extract.

[0020] Furthermore, the bayberry bark extract is obtained by decocting and concentrating bayberry bark, optionally by decocting with water.

[0021] Furthermore, the preparation method of the bayberry bark extract is as follows: add 5 to 15 times the amount of water to every 1000g of bayberry bark, decoct 1 to 3 times, combine the extracts, and concentrate under reduced pressure to 1 to 2.5L.

[0022] Furthermore, the bark of the bayberry tree can be replaced with Changshan or Artemisia annua.

[0023] Beneficial effects

[0024] In this invention, the bark of the Chinese bayberry tree can be used alone to kill scutellaria or prevent and treat coccidiosis or trichomoniasis in chickens. It is characterized by its simple preparation and significant preventative and therapeutic effects, and can be used for the prevention and control of parasitic diseases in veterinary clinical practice, exhibiting a higher insecticidal effect compared to many traditional Chinese medicinal materials. Attached Figure Description

[0025] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0026] Figure 1 The insecticidal effect of the extract of bayberry bark (crude drug amount 0.5g / mL) diluted 4096 times in Experiment 1 of this invention (microscopic observation), the arrow points to the shield ciliate.

[0027] Figure 2 The blank group of the present invention, Shield ciliates (observed under a microscope), is shown by the arrow pointing to Shield ciliates.

[0028] Figure 3 The insecticidal effect of the extract of bayberry bark (crude drug amount 0.5g / mL) diluted 2048 times in Experiment Example 1 of this invention (microscopic observation). The arrow points to the shield ciliate, and the morphology of the shield ciliate has changed.

[0029] Figure 4 The insecticidal effect of the extract of bayberry bark (crude drug amount 0.5g / mL) diluted 1024 times in Experiment Example 1 of this invention (microscopic observation). The arrow points to the shield ciliate, and the morphology of the shield ciliate has been destroyed.

[0030] Figure 5 The bloody stool conditions of diseased chickens in groups G1 to G8 of Experiment 2 of this invention; wherein, the bloody stool conditions of drug A group G1; drug B group G2; drug C group G3; drug D group G4; drug E group G5; drug F group G6; drug G7; and drug H group G8.

[0031] Figure 6 The bloody stool condition of diseased chickens in groups G9 to G15 of Experiment Example 2 of this invention; wherein, the bloody stool condition of drug I group G9; the bloody stool condition of drug J group G10; the bloody stool condition of drug K group G11; the bloody stool condition of drug L group G12; the bloody stool condition of sensitive drug M group G13; the bloody stool condition of red control group G14; and the bloody stool condition of blank group G15.

[0032] Figure 7 The cecal lesions of chickens in groups G1 to G8 of Experiment 2 of this invention; wherein, the cecal lesions were as follows: Group A: G1; Group B: G2; Group C: G3; Group D: G4; Group E: G5; Group F: G6; Group G: G7; Group H: G8.

[0033] Figure 8 The cecal lesions of diseased chickens in groups G9 to G15 of Experiment Example 2 of this invention; wherein, the cecal lesions were as follows: G9 in drug I group; G10 in drug G group; G11 in drug K group; G12 in drug L group; G13 in drug M control group; G14 in red N control group; and G15 in blank O group. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In some embodiments, materials, elements, methods, and means well known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0036] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0037] The following examples investigated the effects of bayberry bark on in vitro resistance to scutellaria barbata, oral resistance to coccidiosis in chickens, and oral resistance to trichomoniasis in birds:

[0038] Experimental Example 1

[0039] Test materials

[0040] Waxberry bark, purchased from Chengdu Yuhetang E-commerce Co., Ltd.; 96-well cell culture plates; 96-well deep-well plates; 3.5% seawater, homemade.

[0041] Test methods

[0042] Preparation of extract from bayberry bark Take 500g of bayberry bark, add 10 times the amount of water, decoct twice, one hour each time, combine the decoctions, concentrate to 1000mL, and prepare a bayberry bark extract containing 0.5g / mL of raw medicinal material.

[0043] In vitro test method for killing ciliates Take a 96-well plate and add 0.6 mL of seawater to each well, followed by 0.6 mL of *Myrica rubra* bark extract (0.5 g / mL). Serially dilute with seawater up to the 12th well, achieving drug concentrations of 1 / 2 to 1 / 4096 of the stock solution. Add 120 μL of the diluted *Myrica rubra* bark extract to each well of the 96-well cell culture plate, then add 120 μL of *Scutellaria baicalensis* spore solution to each well. At this point, the concentration in each well is 1 / 4 to 1 / 8192 of the stock solution. Gently tap the cell culture plate 10-20 times, then let it stand for 1 hour. Observe under an inverted microscope to see if the *Scutellaria baicalensis* have died, thus determining the drug's effectiveness. A blank control group was also set up, with the same *Scutellaria baicalensis* concentration as the experimental group, except that no drug solution was added; instead, an equal volume of seawater was added.

[0044] Test results

[0045] Microscopic observation revealed that the minimum insecticidal concentration of the extract from the bark of the Chinese bayberry tree (crude drug content 0.5 g / mL) was 1 mL: 4096 mL. At this concentration, the scutellaria ceased to move. Figure 1 As shown, with Figure 2 Compared with the blank control group, Figure 1 A small number of scutellariae undergo morphological changes, exhibiting excellent in vitro scutellariae-killing effects. At a 2048-fold dilution, the morphology of the scutellariae is as follows: Figure 3 ,and Figure 2 The changes were significant compared to the control group. At a 1:1024 dilution of the bayberry bark extract, the morphology of the shield ciliates was as follows: Figure 4 This concentration can completely destroy the structure of the shield ciliate, making its morphology and structure no longer visible. This indicates that the bark of the bayberry tree is an excellent traditional Chinese medicine for killing shield ciliates.

[0046] Experimental Example 2

[0047] To evaluate the efficacy of 12 drugs at recommended doses against Eimeria tenella infection.

[0048] Test materials

[0049] The experimental strain was *Eimeria tenella* GD strain, isolated and preserved by the Parasitic Biology Research Laboratory of the Animal Health Institute, Guangdong Academy of Agricultural Sciences. The following experiments were conducted by the Parasitic Biology Research Laboratory of the Animal Health Institute, Guangdong Academy of Agricultural Sciences. However, it should be understood that the implementation of this invention does not depend on the strains represented by the above names or numbers. Each strain can be replaced by other strains of the same type; for example, *Eimeria tenella* can be replaced by other *Eimeria tenella* strains.

[0050] test drug

[0051] Twelve drugs to be screened (denoted as G1 to G12) were provided by Beijing Shengtai Technology Co., Ltd.; the drugs include:

[0052] Group G1 consists of: Polyporus umbellatus;

[0053] Group G2 consists of Changshan.

[0054] Group G3 consists of: bark of the bayberry tree;

[0055] Group G4 consists of: Mulberry bark;

[0056] Group G5 is: Rosehip;

[0057] Group G6 consists of: Eclipta prostrata;

[0058] Group G7 is: Polygonum cuspidatum;

[0059] Group G8 consists of: Panax notoginseng;

[0060] Group G9 consists of: Poria cocos;

[0061] Group G10 consists of: Artemisia capillaris;

[0062] Group G11 consists of Artemisia annua.

[0063] Group G12 is: Sanguisorba officinalis.

[0064] Prepare traditional Chinese medicine powders G1 to G12 separately, and pass them through a 40-mesh sieve.

[0065] Positive test drug (dicrazine solution), provided by Adison (Beijing) Biotechnology Co., Ltd.

[0066] Experimental animals and feed

[0067] 1-day-old 818 fast-growing broiler chickens; 161Q complete broiler feed, free of any anticoccidial drugs and antibiotics, purchased from the Institute of Animal Science, Guangdong Academy of Agricultural Sciences.

[0068] Test methods

[0069] Experimental Design Grouping

[0070] Chicks were raised to 12 days of age. After weighing each chick, weak or overweight chicks were removed, and healthy chicks with an individual weight difference within 10g were selected and randomly divided into 15 groups of 21 chicks each. Groups G1-12 were treated with the worm-inducing drug, group G13 was treated with the sensitive drug, and there were also two control groups: G14 (model group) and G15 (blank control group). Drug administration began at 12 days of age for all drug test groups. Groups G1-12 were administered the drug via feed mixing, and sensitive drugs were administered according to the instructions. At 14 days of age, each chick in all drug test groups and the control group was orally inoculated with 1.0 × 10⁻⁶ ppm. 4 One sporulated oocyst of Eimeria tenella GD strain (ET). All experimental chickens had free access to feed and water until the end of the experiment on day 7, as detailed in Table 1.

[0071] Table 1 Experimental Design Grouping

[0072]

[0073] Anticoccidial Index Assessment Standard

[0074] Blood in stool scoring

[0075] Fecal scoring was performed according to the method of Suo Xun (1997). On the fifth day after infection, the proportion of bloody stools was observed, photographed, and scored. The fecal score reflects the proportion of individuals in a group infected with coccidia who exhibit abnormal fecal characteristics: For Eimeria tenella infection, within a given time range of 12-24 hours, 0 points indicate 100% of the stools are not bloody, +1 point indicates 25% of the stools are bloody, +2 points indicates 50% of the stools are bloody, +3 indicates 75% of the stools are bloody, and +4 points indicates 100% of the stools are bloody.

[0076] The results are as follows Figure 5 , 6 As shown, the results indicated that chickens in the challenge model group gradually exhibited reduced feed intake and decreased mental state after infection with sporulated oocysts. On the 4th day post-infection, all drug groups and the control group without medication showed varying degrees of bloody feces and reduced water intake, which became more severe on the 5th and 6th days. Necropsy revealed cecal lesions with varying degrees of hemorrhage or blood clots, but no lesions were observed in other organs. The control group without medication and without challenge showed no bloody feces, and their feed and water intake were normal. Bloody feces observation on the 5th day post-infection showed that G4, G5, G9, G12, and the G14 group (infected but not treated model group) all scored +1 point for bloody feces; the remaining drug groups and the blank control group G15 scored 0 points for bloody feces.

[0077] relative weight gain

[0078] At the beginning and end of the experiment, the weight of the chickens was measured, and the average weight gain and relative weight gain rate were calculated. Relative weight gain rate = (weight gain rate of the experimental group / weight gain rate of the experimental group without medication or parasite treatment) × 100%.

[0079] The results are shown in Table 1. The results in Table 1 show that, except for the G15-blank control group, the weight of each group decreased. Among them, the relative weight gain rate of G2 (Changshan), G3 (Yangmei bark), and G4 (Sangbai bark) was over 95%, which was significantly higher than that of the G13 positive drug group.

[0080] Survival rate

[0081] Record the number of dead chickens in each group, perform necropsy to determine the cause of death, and calculate the survival rate. Survival rate = (number of surviving chickens at the end of the experiment / number of chickens in the experimental group) × 100%.

[0082] The results are shown in Table 1, indicating that the survival rate of each group was 100%.

[0083] lesion value

[0084] Chickens were slaughtered on the 7th day after infection. The intestinal lesion score of each chicken was calculated according to the lesion scoring method designed by Johnson and Reid (1970), and the lesion score was converted into a lesion value.

[0085] Lesion scoring: (When lesions on both sides of the cecum are inconsistent, the more severe side shall be used as the score):

[0086] 0 points, no visible lesions;

[0087] 1 point, the cecal wall has a few scattered petechiae, the intestinal wall is not thickened, and the contents are normal;

[0088] 2 points, numerous lesions, obvious blood in the cecal contents, slightly thickened cecal wall, normal contents;

[0089] 3 points, the cecum contains a large amount of blood or a cecal core (blood clots or grayish-white, cheese-like banana-shaped masses), the cecal wall is significantly thickened, and the cecum contains little feces;

[0090] 4 points. Chickens that die from coccidiosis are also given 4 points, as their cecum is swollen due to being filled with a large amount of blood or intestinal contents, and the intestinal contents may or may not contain fecal matter.

[0091] Lesion score (0-40) = Average lesion score (0-4) of each experimental group × 10.

[0092] Reduction in Lesion Score (RLS) = (Mean lesion score in the non-medicated control group - Mean lesion score in the medication-treated group) / Mean lesion score in the non-medicated control group * 100%

[0093] The lesion score reduction rate (RLS) evaluation criteria are as follows: in the drug control group, an RLS of <= 30% indicates drug resistance, 31-49% indicates partial drug resistance, and >= 50% indicates sensitivity.

[0094] The results of cecal lesions are as follows Figure 7 , Figure 8 As shown in Table 1, the results indicate that the lesion values ​​in the G3-Myrica rubra bark group and the G13-positive drug group were less than 10, while the lesion values ​​in the other groups were 14 or higher.

[0095] Oocyte count and relative ovum production (ROP)

[0096] The fecal oocyst count was performed using the McMaster counting method to determine the number of oocysts per gram of feces (OPG), and the oocyst value was calculated based on Table 2.

[0097] Relative ovum yield (ROP) = (average ovum yield in the infected drug-treated group / average ovum yield in the infected non-drug-treated group) × 100%. ROP ≥ 15% indicates drug resistance; ROP < 15% indicates no drug resistance.

[0098] Table 2. Conversion between number of ovules and ovule value

[0099]

[0100] The results are shown in Table 1. The results indicate that the oocyst values of the G3 - Myrica rubra bark group and the G13 - positive drug group are ≤5, and the ROP values are <8. The oocyst values of the other groups are 10 or above.

[0101] Anticoccidial Index (ACI)

[0102] According to the calculation formula of Merck & Co., Inc. in the United States, that is:

[0103] ACI = (relative weight gain rate + survival rate) × 100 - (lesion value + oocyst value) is calculated.

[0104] Pharmacodynamic judgment criteria: ACI > 180 belongs to highly effective anticoccidial drugs; 160 < ACI < 180 belongs to moderately effective anticoccidial drugs; 120 < ACI < 160 belongs to low - effective anticoccidial drugs; ACI < 120 is ineffective against coccidia.

[0105] The results are shown in Table 1. The results indicate that the ACI values of the G3 - Myrica rubra bark group and the G13 - positive drug group are > 180, belonging to highly effective anticoccidial drugs.

[0106] The above drug sensitivity test results are shown in Table 1. The results suggest that among the test drugs, sample 3 (G3, Myrica rubra bark) has the lowest oocyst production (ROP) (7.48) and the highest reduction rate of lesion score (RLS) (67.43%) against Eimeria tenella GD strain under the action of 10000 ppm feed mixing, showing a high - potency anticoccidial effect; followed by sample 2 (G2, Dichroa febrifuga) group under the action of 10000 ppm feed mixing, and the ROP shows a certain improvement (but greater than 15%); sample 11 (G11, Artemisia annua) group under the action of 10000 ppm feed mixing, and the RLS shows a certain improvement (but less than 50%); the ROP and lesion values of other groups are not ideal and are judged as ineffective. The sensitive drug control group has obvious improvement in various indicators and has a high - potency anticoccidial effect. The results are shown in Table 3 for details.

[0107] Table 3 Drug Sensitivity Test Results

[0108]

[0109] in conclusion

[0110] 1) The infection - without - drug control and blank control in this experiment are established; the sensitive drug control can achieve a high - potency treatment effect on this strain.

[0111] 2) The drug of the present invention was subjected to anticoccidial animal drug sensitivity test at the recommended dosage concentration. The results showed that: Sample 3 (G3, Myrica rubra bark) had the lowest oocyst yield (ROP) and the highest lesion score reduction rate (RLS) against Eimeria tenella GD strain at 10,000 ppm feed, showing a high-potency anticoccidial effect; Sample 2 (G2, Dichroa febrifuga) and Sample 11 (G11, Artemisia annua) had a certain improvement effect at 10,000 ppm feed; the ROP and RLS of the other groups were not ideal and were judged to be ineffective.

[0112] Screening report analysis: Group G3 was a blind sample group of bayberry bark. According to the screening test report analysis, bayberry bark has excellent in vivo anti-coccidial effect and has a significant preventive and therapeutic effect on coccidiosis in chickens. The anticoccidial index is a recognized indicator for evaluating the effect of anticoccidial drugs. The anticoccidial index of bayberry bark can reach 181.13, which achieves a highly effective anticoccidial effect.

[0113] The above test report shows that the bark of the bayberry tree has a good anti-parasitic effect, and it is very effective against both scutellaria and coccidioidomyces. Anti-parasitic treatment is a new application direction for bayberry bark.

[0114] Experimental Example 3

[0115] The inventors also studied the effects of the extract from the bark of the Chinese bayberry tree on the in vivo and in vitro activity of Trichomonas vaginalis strain P421 at the recommended dosage.

[0116] The experimental strain was Trichomonas vaginalis strain P421, isolated and preserved by the Parasitic Biology Research Laboratory of the Animal Health Institute, Guangdong Academy of Agricultural Sciences. The following experiments were conducted by the Parasitic Biology Research Laboratory of the Animal Health Institute, Guangdong Academy of Agricultural Sciences. However, it should be understood that the implementation of this invention does not depend on the strains represented by the above names or numbers; each strain can be replaced by other strains of the same type. For example, Trichomonas vaginalis can be replaced by other Trichomonas vaginalis strains.

[0117] Preparation of extract from bayberry bark Take 1000g of bayberry bark, add 10 times the amount of water, decoct twice, one hour each time, combine the decoctions, concentrate to 1000mL, and prepare bayberry bark extract containing 1g / mL of raw medicinal material.

[0118] Evaluation of in vitro cell-free anti-trichomonal efficacy

[0119] This experiment included a group using bayberry peel extract, with three replicate wells for each drug concentration. A 96-well plate was used for detection.

[0120] (1) A 96-well plate was used for detection. The drug sensitivity test system was set to 100 μL, including 90 μL of Trichomonas vaginalis and 10 μL of drug.

[0121] (2) Take the pigeon trichomonas that has been cultured for 48 hours and is in good growth condition, dilute it with TYM medium to a solution of 125,000 insects / mL, and add 90 μL of the solution to a 96-well plate.

[0122] (3) Add 10 μL of the drug to be tested to the detection well containing the insects, and set different concentration gradients for 1 g / mL bayberry bark extract: dilute by 10, 20, 30, 40, 50...80 times with culture medium.

[0123] (4) Mix the above reaction system and place it in a 37°C incubator for anaerobic culture for 48 hours. Examine it under a microscope. The drug concentration corresponding to the well where no insect growth is found is the maximum tolerated concentration of the drug, i.e., the MLC value.

[0124] The results showed that 1 g / mL of bayberry bark extract could kill all trichomonads at a dilution of 1:80.

[0125] Animal testing evaluation

[0126] Experimental animals: 15-day-old squabs; feed, which did not contain any anti-trichomonal drugs or antibiotics, was purchased from Guangzhou Houde Pigeon Breeding Co., Ltd.

[0127] Test methods Experimental squabs were raised to 15 days of age. After being weighed and divided into groups, thin or overweight squabs were removed, and healthy squabs with an individual weight difference within 50g were selected and randomly divided into 4 groups of 12 squabs each. The groups were: a *Myrica rubra* bark group, a model group (treated with parasites but not given medication), and a control group (neither treated with medication nor treated with parasites). Parasite treatment and medication began at 18 days of age. The *Myrica rubra* bark group received oral administration of *Myrica rubra* bark extract; each squab in the *Myrica rubra* bark group and the model group was orally inoculated with 5.0 × 10⁻⁶ ppm. 5 Trichomonas vaginalis. All experimental pigeons had free access to food and water until the end of the experiment on the 7th day. The experimental groups are detailed in Table 4.

[0128] Table 4 Experimental Design Grouping

[0129]

[0130] Evaluation indicators of the efficacy of anti-pigeon trichomoniasis drugs

[0131] After being infected with trichomoniasis, the experimental pigeons in the model group gradually showed symptoms such as reduced feed intake and decreased mental state, and obvious lesions appeared in the oral cavity. On the 8th day, the pigeons were euthanized and the oral and pharyngeal lesions were observed. The model group had varying degrees of caseous contents, but no lesions were observed in other organs. The control group, which did not receive any medication or trichomoniasis treatment, had no lesions and normal feed and water intake.

[0132] Based on the microscopic examination and culture of throat swabs from each pigeon, the positive rate of each group was compared, and the relative weight gain and survival rate were assessed.

[0133] Positive rate

[0134] On the first day after the administration of medication, throat swabs were collected from each pigeon using cotton swabs. The swabs were then incubated in 1.5 ml of TYM medium at 37°C under anaerobic conditions for 48 hours before microscopic examination. A sample showing the presence of Trichomonas vaginalis was considered positive; the absence of Trichomonas vaginalis was considered negative. The microscopic results were recorded. Simultaneously, the positive rate was calculated using the following formula:

[0135]

[0136] The results showed that, based on the microscopic examination and culture of throat swabs from each pigeon, the positive rates of each group were compared (see Table 5). The infection rate in the control group (no medication, no parasite treatment) was 0%, while the infection rate in the model group (no medication, no parasite treatment) was 100%, indicating that the infection model was valid and had certain drug evaluation value. The results of the medication group showed that the positive rate in the *Myrica rubra* bark group decreased, and the severity of oral lesions also improved, indicating its protective efficacy. This suggests that *Myrica rubra* bark extract has an anti-Trichomonas vaginalis infection effect.

[0137] relative weight gain

[0138] The weight of each group of pigeons was weighed at the beginning and end of the experiment, and the average weight gain and relative weight gain rate were calculated. The formula is as follows:

[0139]

[0140]

[0141] The relative weight gain rate results are shown in Table 5. The results show that the relative weight gain rate of the bayberry bark group is close to that of the control group compared with the model group, indicating that the bayberry bark extract can improve the problem of weight loss in pigeons caused by Trichomonas vaginalis infection.

[0142] Survival rate

[0143] After artificially infecting squabs and administering medication, daily mortality rates of the experimental animals were recorded. At the end of the experiment, the survival rate was calculated using the following formula:

[0144]

[0145] The survival rate results are shown in Table 5. The results indicate that, compared with the control group, the extract of bayberry bark can ensure the survival of pigeon trichomoniasis infection.

[0146] The results indicate that the extract from the bark of the Chinese bayberry tree has a good effect on improving oral lesions caused by Trichomonas vaginalis. See Table 5 for details.

[0147] Table 5. Results of drug susceptibility testing

[0148]

[0149] Table 5 shows that the extract of bayberry bark has in vitro and in vivo anti-trichomonal infection effects, reduces oral lesions caused by trichomonal infection, reduces mortality, and reduces the positive rate.

[0150] To investigate which active ingredient in the bark of the Chinese bayberry tree plays an insecticidal role, the insecticidal effects of its main components, myricetin and myricetin glycoside, were studied (according to literature reports, the bark of the Chinese bayberry tree contains myricetin and myricetin glycoside). The results are as follows:

[0151] Test Example 4

[0152] Myricetin, purchased from Yuanye Biotechnology Co., Ltd., CAS No. 529-44-2, 98%, 500mg / bottle.

[0153] Myricetin, purchased from Aladdin Company, CAS No. 17912-87-7, 98%, 20mg / vial.

[0154] Test methods

[0155] Myricetin solution: Take 10mg of myricetin, add 1mL of purified water, and repeatedly blow with a pipette tip to dissolve it fully. If it cannot be completely dissolved, form a saturated solution and set aside for later use.

[0156] Myricetin solution: Take 10 mg of myricetin, add 1 mL of purified water, and repeatedly blow with a pipette tip to dissolve it fully. If it cannot be completely dissolved, form a saturated solution and set aside for later use.

[0157] In vitro insecticidal assay: Take a 96-well plate, add 0.6 mL of seawater to each well, then add 0.6 mL of myricetin solution, serially diluting until the 12th well, with drug concentrations ranging from 1 / 2 to 1 / 40.96 of the stock solution. Add 120 μL of the diluted myricetin bark extract to each well of the 96-well cell culture plate, then add 120 μL of *Scutellaria barbata* spore solution to each well, resulting in concentrations of 1 / 4 to 1 / 81.92. Gently tap the cell culture plate 10-20 times, then let it stand for 1 hour. Observe under an inverted microscope to determine if the *Scutellaria barbata* have died, thus assessing the drug's effectiveness. A blank control group was also set up, with the same *Scutellaria barbata* concentration as the experimental group.

[0158] The insecticidal effect of myricetin solution was tested using the same method.

[0159] Test results

[0160] In vitro insecticidal tests showed that neither myricetin solution nor myricetin solution, even in saturated form, could kill shield ciliates, indicating that neither myricetin nor myricetin had any effect on killing shield ciliates.

[0161] analyze

[0162] According to literature reports, the bark of the Chinese bayberry tree contains myricetin and myricetin, but experimental results show that neither myricetin nor myricetin can kill shield-shaped ciliates. Therefore, the active substances in the bark that kill shield-shaped ciliates need further investigation. The insect-resistant components in the bark of the Chinese bayberry tree are neither myricetin nor myricetin.

[0163] 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 of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of bayberry bark as the sole active ingredient in the preparation of drugs for the prevention or treatment of parasitic infections or complications caused by parasitic infections; in, The parasites are scutiformis, coccidioidomyces, and avian trichomoniasis; When the parasite is chicken coccidioidomycetes, the drug is a powdered product of bayberry bark; When the parasite is a shield-like ciliate or a avian trichomonad, the drug is an aqueous extract of bayberry bark.

2. The application according to claim 1, characterized in that, When the parasite is a shield-shaped ciliate, the drug is a medicated bath preparation.

3. The application according to claim 1, characterized in that, The chicken coccidia is Eimeria tenella.

4. The application according to claim 1, characterized in that, The avian trichomoniasis is *Trichomonas guinea*.

5. The application according to claim 1, characterized in that, When the parasite is chicken coccidioidomycetes and / or avian trichomoniasis, the drug is an oral or nasal spray.

6. The application according to claim 1, characterized in that, The dosage form of the drug is any one of powder, granules, capsules, tablets, or liquid.

7. The application according to claim 6, characterized in that, The crude drug content of bayberry bark in the liquid formulation is 0.1~1g / mL.

8. The application according to claim 6, characterized in that, The crude drug content of bayberry bark in the liquid formulation is 0.4~1g / mL.

9. The application according to claim 6, characterized in that, The liquid form is an oral solution.

10. The application according to any one of claims 1 to 9, characterized in that, The drug is an aqueous extract of bayberry bark.

11. The application according to any one of claims 1 to 9, characterized in that, The drug is a hydrothermal extract of bayberry bark.

12. The application according to any one of claims 1 to 9, characterized in that, The water extract of bayberry bark is obtained by boiling and concentrating bayberry bark with water.

13. The application according to any one of claims 1 to 9, characterized in that, The method for preparing the aqueous extract of bayberry bark is as follows: add 5 to 15 times the amount of water to every 1000g of bayberry bark, decoct 1 to 3 times, combine the extracts, and concentrate under reduced pressure to 1 to 2.5L.

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