Application of garlic essential oil in the preparation of drugs against Trichomonas vaginalis

Abstract

This invention discloses the application of garlic essential oil in the preparation of an anti-Trichomonas vaginalis drug for pigeons. The garlic essential oil is prepared by steam distillation, and the drug is in the form of an emulsion for internal use. The invention first uses garlic essential oil in an in vitro inhibition test against Trichomonas vaginalis, determining the minimum inhibitory concentration (MIC) of the garlic essential oil. Trichomonas vaginalis was completely inhibited after 8 hours of treatment with a 50 μL / mL garlic essential oil emulsion. In vivo inhibition tests were also conducted using garlic essential oil. Each infected pigeon was administered 2.5 mL of a 20 μL / mL garlic essential oil emulsion (i.e., 50 μL of garlic essential oil). After 3 days of treatment, the oral trichomoniasis was completely cleared from the pigeons. During the treatment period, the pigeons did not experience any adverse reactions such as vomiting or diarrhea after oral administration. The garlic essential oil of this invention is naturally sourced, green and healthy, non-irritating to pigeons, and falls within the safe range for use, thus having significant application value in the preparation of an anti-Trichomonas vaginalis drug.

Description

Technical Field

[0001] This invention relates to the field of antiparasitic drug technology, specifically to the application of garlic essential oil in the preparation of anti-Trichomonas vaginalis drugs and the inhibition of Trichomonas vaginalis growth and reproduction. Background Technology

[0002] Trichomoniasis in pigeons is a common parasitic disease. The pathogen belongs to the phylum Protozoa, class Flagellates, subclass Phytoflagellates, order Polyflagellates, family Trichomonidae, genus Trichomonas. Depending on the site of infection, it can be classified into pharyngeal, umbilical, visceral, and cloacal types. The pharyngeal type is the most common and causes the most severe symptoms. The main characteristic is the presence of a yellowish-white pseudomembrane at the corner of the pigeon's beak, and a yellowish-white, well-defined, cheesy substance the size of a soybean or button on the mucous membrane of the throat, which can be easily peeled off with a cotton swab. The main symptoms include difficulty swallowing and breathing, often accompanied by a "gurgling" sound; ruffled and dull feathers; decreased appetite; empty crop; emaciation; lethargy; and yellowish-green feces adhering to the vent.

[0003] Trichomoniasis in pigeons is a global disease, with a carrier rate exceeding 60% in my country. Pigeons of all breeds, ages, and sexes can be infected, with young pigeons under 3 months old having the highest infection rate. Young pigeons aged 2-5 weeks show more severe clinical symptoms and have a significantly higher mortality rate compared to other age groups. This results in substantial economic losses for pigeon farms. The disease is typically transmitted through contact. When infected pigeons (both sick and infested) excrete the parasites, they can infect other pigeons by contaminating feed, water, nest mats, and the still-open umbilical cord. Adult pigeons can transmit the parasites through kissing. Suckling pigeons can transmit the parasites to their young through pigeon milk, making them lifelong carriers.

[0004] For a long time, metronidazole drugs have been the first-line treatment for trichomoniasis. However, these drugs have many adverse reactions, including genotoxicity, carcinogenicity, teratogenicity, and mutagenicity. Furthermore, in a 2002 document issued by the Ministry of Agriculture and Rural Affairs, metronidazole was explicitly prohibited from use in the production of all animal-derived foods. From January 1, 2020, my country officially banned the addition of all growth-promoting drugs (excluding traditional Chinese medicine) to veterinary feed additives; and from July 1, 2020, my country officially banned feed manufacturers from producing commercial feed containing growth-promoting drug feed additives (excluding traditional Chinese medicine). Therefore, adopting green and healthy prevention and control methods is necessary. Traditional Chinese medicine is naturally derived and has far fewer toxic side effects than Western medicine. At the same time, the use of traditional Chinese medicine can effectively solve problems such as drug residues and drug resistance.

[0005] Garlic essential oil is a natural substance extracted from garlic (Allium sativum Linnaeus), with its main components being allicin, allicin, and various sulfide compounds. Garlic essential oil has advantages such as being natural, multifunctional, non-toxic, and non-drug-resistant. It can promote animal feed intake and has antioxidant, antibacterial, anti-inflammatory, and immunomodulatory functions, and has been widely used in the prevention and treatment of livestock and poultry diseases. Studies have shown that garlic essential oil can treat diseases caused by various bacteria, viruses, and intestinal parasites, but there are no research reports on the use of garlic essential oil to inhibit the activity of Trichomonas vaginalis in pigeons. Summary of the Invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing an application of garlic essential oil in the preparation of drugs against Trichomonas vaginalis.

[0007] To address the aforementioned technical problems, this invention discloses the application of garlic essential oil in the preparation of an anti-trichomonal drug. The technical solution adopted by this invention is as follows:

[0008] The application of garlic essential oil in the preparation of an anti-trichomonal drug.

[0009] The garlic essential oil is obtained by steam distillation of fresh garlic to obtain an oil-gas mixture. After cooling, the mixture flows into an oil-water separator, where it is allowed to settle and separate into layers. The lower layer is the garlic essential oil. The steam distillation temperature is 110–125°C, the distillation time is 2.5–3.0 hours, and the steam pressure is 0.2 MPa. Preferably, after selection and cleaning, fresh garlic is fed into a distillation tower, and steam is introduced. The steam boiler pressurizes the steam to 0.2 MPa, and the steam temperature is maintained at 110–125°C. The distillation process lasts for approximately 2.5 hours. The distilled oil-gas mixture is cooled by a cooler and then flows into the oil-water separator. The separator utilizes the principle that garlic oil has a higher relative density than water and is insoluble in water to initially separate the layers. The lower layer is garlic oil containing a small amount of water, called crude oil, and the upper layer is an oil-water mixture. The initially separated crude oil is then allowed to settle and separate again in a storage tank, and the resulting lower layer is the garlic essential oil.

[0010] The garlic essential oil, when applied to Trichomonas vaginalis for 8 hours, caused significant morphological changes in the parasite. These changes included a roughened and concave cell membrane with gaps at the edges, and the presence of vacuoles of varying sizes and some highly refractive granular substances within the cell. With prolonged application of the garlic essential oil, the parasite's cell membrane ruptured, leading to a large leakage of intracellular contents and ultimately, lysis and death of the parasite.

[0011] The in vitro culture conditions for Trichomonas vaginalis were as follows: cultured in TYM medium (containing fetal bovine serum and antibiotics), at a temperature of 37°C, and at a rotation speed of 200–250 rpm. Each 3.5 mL of TYM medium contained 0.3 mL of fetal bovine serum and 0.3 mL of antibiotics (10 kU / mL penicillin and 10 mg / mL streptomycin).

[0012] The TYM powder culture medium was purchased from Jiangxi Yisenyuan Plant Fragrance Co., Ltd., and the TYM culture medium formula is as follows: 20.0 g / L casein peptone, 10.0 g / L yeast extract, 5.0 g / L maltose, 1.0 g / L L-cysteine ​​hydrochloride, 0.2 g / L vitamin C, 0.8 g / L dipotassium hydrogen phosphate, 0.8 g / L potassium dihydrogen phosphate, and 0.5 g / L agar.

[0013] The drug is described in the form of an emulsion.

[0014] The drug is prepared by mixing garlic essential oil with polysorbate-20, then adding physiological saline and stirring until emulsified. Preferably, a high-speed homogenizing emulsifier is used to stir until emulsified.

[0015] The volume ratio of garlic essential oil to polysorbate-20 solvent is 10:1.

[0016] The drug mentioned is an in vivo medication.

[0017] Each pigeon was administered 50 μL of garlic essential oil daily for 1–7 days. The garlic essential oil used was pure garlic essential oil without any other excipients. Preferably, each pigeon was administered 2.5 mL of a 20 μL / mL garlic essential oil emulsion daily. After 3 days of administration, the oral trichomoniasis in the pigeons was completely eradicated.

[0018] The garlic essential oil mentioned above includes allicin, allicin, and sulfide compounds.

[0019] Beneficial Effects: This invention provides a novel application of garlic essential oil in the preparation of drugs against Trichomonas vaginalis in pigeons. Firstly, an in vitro inhibition test was conducted using garlic essential oil to inhibit Trichomonas vaginalis, determining the minimum inhibitory concentration (MIC) of the garlic essential oil. Trichomonas vaginalis was completely inhibited after 8 hours of treatment with a garlic essential oil emulsion at a concentration of 50 μL / mL. In vivo inhibition tests were also conducted using garlic essential oil. Each infected pigeon was administered 2.5 mL of a 20 μL / mL garlic essential oil emulsion (i.e., 50 μL of garlic essential oil). After 3 days of treatment, the oral trichomoniasis in the pigeons was completely eradicated. During the treatment period, no adverse reactions such as vomiting or diarrhea occurred in the pigeons. The garlic essential oil used in this invention is naturally sourced, green, and healthy. The preparation method is simple and low-cost, non-irritating to pigeons, and falls within the safe usage range. It has significant application value in the preparation of drugs against Trichomonas vaginalis in pigeons. Attached Figure Description

[0020] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0021] Figure 1 The images show the morphology of Trichomonas vaginalis after treatment with a garlic oil emulsion at a concentration of 50 μL / mL. Images A, B, and D are 1000× oil immersion images, and image C is a Gymsa stain image. Detailed Implementation

[0022] In the following embodiments, the method for preparing garlic essential oil is as follows: Fresh garlic, after being selected and cleaned, is fed into a distillation tower, steam is introduced, the steam boiler is pressurized to 0.2 MPa, the steam temperature is maintained at 110–125°C, and the distillation process is maintained for approximately 2.5 hours. The distilled oil-gas mixture is cooled by a cooler and then flows into an oil-water separator. The separator utilizes the principle that garlic oil has a higher relative density than water and is insoluble in water, and initially allows the layers to separate. The lower layer is garlic oil containing a small amount of water, called crude oil, and the upper layer is an oil-water mixture. The initially separated crude oil is then allowed to stand again in a storage tank for further separation, and the resulting lower solution is garlic essential oil.

[0023] Example 1: In vitro inhibitory concentration of garlic essential oil against Trichomonas vaginalis.

[0024] (1) Isolation and culture of Trichomonas vaginalis: Weigh 38.3g of TYM medium powder (purchased from Jiangxi Yisenyuan Plant Fragrance Co., Ltd.) and place it in 900mL of distilled water. Adjust the pH to 6.0 with 1M HCl, heat to boiling until completely dissolved, pour into an Erlenmeyer flask, and autoclave at 121℃ for 15min. Shake well after sterilization. After cooling, store at 4℃ for later use. Before use, add 0.3mL of fetal bovine serum and 0.3mL of double antibiotics (10kU / mL penicillin and 10mg / mL streptomycin) to every 3.5mL of medium to provide sufficient nutrition and inhibit bacterial growth. Shake well before use for Trichomonas vaginalis passage and incubation. The TYM culture medium formula is as follows: 20.0 g / L casein peptone, 10.0 g / L yeast extract, 5.0 g / L maltose, 1.0 g / L L-cysteine ​​hydrochloride, 0.2 g / L vitamin C, 0.8 g / L dipotassium hydrogen phosphate, 0.8 g / L potassium dihydrogen phosphate, and 0.5 g / L agar.

[0025] Trichomonas vaginalis was inoculated into TYM medium (containing fetal bovine serum and antibiotics) and cultured in a 37°C shaker at 220 rpm for 48 h. The growth status of Trichomonas vaginalis was assessed using the trypan blue exclusion method. When the activity of Trichomonas vaginalis was greater than 95%, the cultured parasites were collected, centrifuged at 2000 rpm for 10 minutes, the supernatant was discarded, and 1 mL of TYM medium was added for resuspending. Subculture was performed every 48 h. After stable subculturing, the parasites were ready for experimentation. The parasites used in the experiment needed to be washed 2-3 times with phosphate-buffered saline (PBS) (pH = 7.0) and then resuspended in 1 mL of preheated 37°C TYM medium to prepare a concentration of 1×10⁻⁶. 6 Prepare insect solution at 37°C for later use.

[0026] (2) Preparation of drug solution: Mix 20 mL of garlic essential oil with 2 mL of polysorbate-20 (v:v = 10:1), then add 178 mL of physiological saline, and emulsify using a high-speed homogenizer to prepare a 100 μL / mL garlic essential oil emulsion stock solution (of which the polysorbate-20 content accounts for 1% v / v of the total stock solution), and store it in a refrigerator at 4℃ for later use. Weigh 6 mg of metronidazole powder using an electronic balance, add it to 10 mL of sterile distilled water, and prepare a stock solution with a final concentration of 600 μg / mL, and store it in a refrigerator at 4℃ for later use.

[0027] (3) Determination of the minimum inhibitory concentration (MIC) of Trichomonas vaginalis: The sensitivity of Trichomonas vaginalis to garlic essential oil was studied by incubating it with garlic essential oil emulsion, so as to determine the minimum inhibitory concentration of garlic essential oil against Trichomonas vaginalis. 500 μL of TYM medium (containing fetal bovine serum and antibiotics) was added to a 96-well autoclaved deep-well plate. Then, 500 μL of the drug solution to be tested was added to the first well, and the solution was diluted twofold to the sixth well. Then, 100 μL of Trichomonas vaginalis suspension was inoculated at a concentration of 1 × 10⁻⁶. 6 The concentration of each sample was 600 μL, with a final volume of 600 μL per tube. The experiment consisted of eight groups: 600 μg / mL metronidazole, solvent control (10 μL / mL polysorbate-20), 1.5625 μL / mL garlic oil, 3.125 μL / mL garlic oil, 6.25 μL / mL garlic oil, 12.5 μL / mL garlic oil, 25 μL / mL garlic oil, and 50 μL / mL garlic oil. After completion, the 96-well deep plate was sealed with sealing film and incubated on a 37°C shaker at 160 rpm. 10 μL of liquid was taken from each well of a 96-well deep-well plate after incubation for 4 h, 8 h, 12 h, and 24 h. *Trichomonas pigeonis* were counted under an optical microscope based on morphology and movement, and compared with a growth control group. The relative inhibition rate was calculated (relative inhibition rate = (number of parasites in the growth control group - number of parasites in the experimental group) / number of parasites in the growth control group × 100%). A relative inhibition rate greater than 0 indicates that garlic oil can inhibit the growth and reproduction of *Trichomonas pigeonis*. The lowest concentration that inhibits the growth of *Trichomonas pigeonis* is the minimum inhibitory concentration (MIC) of garlic oil. If the number of *Trichomonas pigeonis* in the field of view is large, it needs to be diluted with PBS (pH = 7.0), thoroughly mixed with a vortex mixer, and then the live parasites are counted using a hemocytometer. The minimum inhibitory concentration (MIC) is defined as the lowest drug concentration at which no active parasites are observed. The experiment was performed in quadruplicate.

[0028] Statistical analysis: Differences between different groups were analyzed using GraphPad Prism 9 ANOVA. A p-value less than 0.05 was considered statistically significant.

[0029] Results: The relative inhibition rates of garlic essential oil at all concentrations against Trichomonas vaginalis were above 75% at different time points. At the same drug concentration, the relative inhibition rate of Trichomonas vaginalis increased with time. The fastest increase in relative inhibition rate was observed within 4-8 hours, while the relative inhibition rate showed no significant difference among groups within 8-24 hours. After 8 hours of drug treatment, the relative inhibition rate of garlic essential oil emulsion at a concentration of 50 μL / mL against Trichomonas vaginalis was 100%. (See Table 1).

[0030] Table 1. Observation of the in vitro anti-Trichomonas vaginalis effect of garlic essential oil.

[0031]

[0032] Note: Different lowercase letters in the superscript of data in the same column indicate significant differences (P < 0.05).

[0033] Example 2: Effects of garlic essential oil on morphological changes of Trichomonas vaginalis in pigeons

[0034] Morphological observation of Trichomonas vaginalis: 10 μL of liquid was taken from a 96-well deep-well plate that had been incubated for 24 hours and dropped into the center of a glass slide. After covering with a coverslip, the slide was observed and photographed under an oil immersion microscope. After observation, smears were prepared by dropping 10 μL of liquid into the center of a glass slide and spreading it evenly to an appropriate size. The dried smear was then held with the smear side facing up, and its reverse side was passed back and forth three times in the outer flame of an alcohol lamp at an appropriate speed. The ideal temperature was when the reverse side of the slide felt warm but not hot to the touch. The prepared smears were then stained with Wright's Giemsa stain.

[0035] The procedure for staining with Giemsa Reischademay is as follows: First, add approximately 0.5-0.8 mL of Giemsa Reischademay solution A to the smear, ensuring the stain evenly covers the specimen, and stain for 0.5-1 minute. Then, add solution B on top of solution A (2-3 times the volume of solution A), and gently blow with a bulb syringe to ensure thorough mixing of solutions A and B, and stain for 5-10 minutes. Afterward, rinse the stain thoroughly with distilled water, dry the slide, and observe the morphological changes of the insects under an optical microscope.

[0036] result: Figure 1 A showed that after 4 hours of treatment with a 50 μL / mL garlic oil emulsion, *Trichomonas vaginalis* exhibited significant changes in its internal structure, with varying numbers of refractive granules observed intracellularly. However, the cell membrane remained intact at this stage. After 8 hours of treatment with 50 μL / mL garlic oil, the morphology of the parasite underwent significant changes. The cell membrane became rough and concave, with gaps appearing at the edges. Vacuoles of varying sizes and some highly refractive granular material were visible intracellularly. At this point, the cell nucleus was difficult to observe, suggesting possible nuclear dissolution. Figure 1 D. Figure 1 B shows that as the contact time with garlic essential oil was extended to 12 hours, the cell membranes of the parasites ruptured, resulting in a large leakage of intracellular contents and lysis and death of the parasites. After 12 hours of contact with garlic essential oil, numerous dead fragments of Trichomonas vaginalis were observed under a light microscope after staining with Gymnezae. Figure 1 C).

[0037] In conclusion, *Trichomonas vaginalis* was completely inhibited after 8 hours of treatment with a garlic oil emulsion at a concentration of 50 μL / mL. After treatment with garlic oil, numerous highly refractive particles formed inside the parasite, the cell nucleus disappeared, and finally, lysis occurred.

[0038] Example 3: In vivo inhibition test of garlic essential oil against Trichomonas vaginalis in pigeons

[0039] Twenty pigeons infected with Trichomonas vaginalis (all were examined and found to be free of other diseases) were selected and randomly divided into two groups of 10 pigeons each. Five swabs were used to clean the inside of the pigeons' mouths with sterile cotton swabs. The swabs were then placed in centrifuge tubes containing 1 mL of physiological saline, broken off at the point where the swabs were placed, and the tubes were capped. The tubes were inverted 20 times to mix the swabs. 10 μL of the solution was then drawn and dropped onto a hemocytometer. A coverslip was placed on the tubes, and the swabs were observed and counted under an optical microscope. The average number of Trichomonas vaginalis worms in each group was calculated.

[0040] The 100 μL / mL garlic essential oil emulsion stock solution prepared in Example 1 was diluted 5 times with physiological saline to prepare a 20 μL / mL garlic essential oil emulsion solution for in vivo testing. Treatment group: Each sick pigeon was administered 2.5 mL of the garlic essential oil emulsion solution (20 μL / mL) daily for 7 consecutive days. Blank control group: Sick pigeons were administered an equal volume of physiological saline without any additional medication.

[0041] The health status of each group of pigeons was observed and recorded before and after medication, as shown in Table 2. Samples were taken and counted before and after medication, and the average number of trichomoniasis in each group of pigeons was calculated. The results are shown in Table 3.

[0042] Table 2. Effects of garlic essential oil on the health status of pigeons suffering from trichomoniasis.

[0043]

[0044] As shown in Tables 2 and 3, the garlic essential oil used in this invention for preparing the anti-Trichomonas vaginalis drug, when applied to pigeons according to the provided dosage and administration method, completely eradicated Trichomonas vaginalis from the pigeons' mouths within 3 days, achieving a 100% eradication rate. During the 7-day oral administration of garlic essential oil, the pigeons did not exhibit any adverse reactions such as vomiting or diarrhea, indicating that the dosage of garlic essential oil used in this invention is non-irritating to pigeons and falls within the safe range for use.

[0045] This invention provides a concept and method for the application of garlic essential oil in the preparation of drugs against Trichomonas vaginalis in pigeons. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. The application of garlic essential oil as the sole active ingredient in the preparation of a drug for treating trichomoniasis in pigeons, wherein, The garlic essential oil is prepared as follows: fresh garlic is selected and cleaned, then fed into a distillation tower. Steam is introduced, the steam boiler is pressurized to 0.2 MPa, and the steam temperature is maintained at 110–125°C. The distillation process is maintained for 2.5 hours. The distilled oil-gas mixture is cooled by a cooler and flows into an oil-water separator. The separator utilizes the principle that garlic oil has a higher relative density than water and is insoluble in water to initially separate the layers. The lower layer is garlic oil containing a small amount of water, called crude oil, and the upper layer is an oil-water mixture. The initially separated crude oil is then separated again by settling in a storage tank. The resulting lower solution is the garlic essential oil. The drug is in the form of an emulsion; the drug is prepared by mixing garlic essential oil with polysorbate-20, and then adding physiological saline and stirring until emulsified; The medication is for internal use. For internal use, each pigeon is given 50 μL of garlic oil by gavage for 3 days. The volume ratio of garlic essential oil to polysorbate-20 solvent is 10:1.

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