An essential oil monoterpene composition for preventing and treating mite diseases and its application

Abstract

This invention discloses an essential oil monoterpene composition for preventing and treating mite infestations, comprising carvacrol and eugenol; and the application of this essential oil monoterpene composition in the treatment of animal mite infestations. The essential oil monoterpene composition of this invention for preventing and treating mite infestations possesses the characteristics of high efficiency, low toxicity, no residue, no pollution, and low likelihood of inducing drug resistance; this invention provides a new approach for the development of anti-mite drugs and offers effective drugs and technical means for the clinical control of ectoparasitic diseases in animals.

Description

Technical Field

[0001] This invention belongs to the field of parasitic disease prevention and control technology, and specifically relates to an essential oil monoterpene composition for preventing and controlling mite diseases and its application. Background Technology

[0002] Mites belong to the subclass Acari of the class Arachnida, comprising approximately 32,000 species. Most are tiny, and the majority parasitize livestock, humans, and plants, playing a vital role in human and animal health. Sarcoptes mites are obligate ectoparasites belonging to the genus *Sarcoptes* of the family Sarcoptidae. They cause scabies in humans and over 100 species of mammals. Characterized by intense itching, exudative inflammation, and highly contagious contact transmission, they are widespread globally. Itchy mites belong to the genus *Psoroptes* of the family Psoroptidae. They parasitize the skin surface of animals, causing contact dermatitis characterized by crusting, hair loss, intense itching, and weight loss. Their host range is broad, including livestock such as sheep, cattle, buffalo, and rabbits, as well as various wild animals. Mite infestations can occur in animals of all ages, leading to decreased productivity and product quality in affected animals, and in severe cases, even death. In my country, animal mange can occur year-round, but is particularly severe in winter and spring. It is most common in pigs, sheep, rabbits, and dogs, with a high morbidity rate. In large-scale pig farms, the infection rate of swine mange can reach 70-90%. This disease can cause a 4.5-12% decrease in growth rate and a 2% reduction in feed conversion rate. Furthermore, it can lead to reduced farrowing rates in sows and decreased immunity in pigs, and in severe cases, death. Studies have shown that the direct economic loss to breeding pig farms caused by mange can reach as high as 800 yuan per head. At the same time, mange is also a significant public health problem, seriously threatening human health, with over 200 million cases worldwide each year. In 2017, it was listed as a "neglected tropical disease" by the World Health Organization.

[0003] Chemical acaricides such as organophosphates, pyrethroids, macrolides, and formamidins are commonly used to treat scabies. In recent years, the widespread and prolonged use of synthetic acaricides has led to the "3R" problem (residual residue, resistance, and resurgence), seriously threatening public health and food safety. While the era of antibiotic reduction and replacement is rapidly approaching, there are currently no alternative antibiotics for scabies, and treatment remains primarily based on chemical drugs.

[0004] Plant essential oils possess numerous advantages, including being residue-free, pollution-free, less prone to inducing drug resistance, and exhibiting various biological activities such as anti-inflammatory and antibacterial properties. In recent years, they have become a research hotspot for alternatives to antibiotics. With more and more chemically synthesized acaricides being forced out of the market, there is an urgent need to develop safe and highly effective new acaricides.

[0005] Current articles have reported on the resistance of scabies mites to pyrethroids and ivermectin, as well as clinical cases of ivermectin treatment failure in humans and animals. Although resistance to acaricides in scabies mites is not yet widespread, the development of new acaricides and their rotation with existing ones could significantly delay the emergence of resistance problems.

[0006] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0007] In view of the above-mentioned technical problems, the present invention provides an essential oil monoterpene composition for preventing and treating mite diseases, aiming to obtain an essential oil monoterpene composition that can effectively prevent and treat animal mite diseases and has a fast effect.

[0008] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0009] An essential oil monoterpene composition for preventing and treating mite infestations, comprising carvacrol and eugenol.

[0010] Furthermore, the volume ratio of carvacrol to eugenol is 3:1.

[0011] Furthermore, the carvacrol volume concentration is 1%, and the eugenol volume concentration is 0.33%.

[0012] The application of the essential oil monoterpene composition for preventing and treating mite diseases as described above in the treatment of animal mite diseases.

[0013] Furthermore, the mites are one or more of the following: scabies mites, ear mites, itch mites, demodicus mites, rodent mites, or dust mites.

[0014] A medicament for preventing and treating mite infestations, the active ingredient comprising an essential oil monoterpene composition for preventing and treating mite infestations as described above, and further comprising other acceptable excipients or auxiliary ingredients for use in the medicament.

[0015] Furthermore, the medicine is a topical medicine, and is one of the following: ointment, solution, emulsion, film or spray.

[0016] The method of using the essential oil monoterpene composition for preventing and treating mite diseases as described above is as follows: On day 0, day 3, and day 7, spray or apply the essential oil monoterpene composition for preventing and treating mite diseases to the affected areas of the animal. The dosage varies depending on the severity of the disease. Generally, the agent should completely soak into the scab-covered areas.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The essential oil monoterpene composition for preventing and treating mite diseases of this invention has the characteristics of high efficiency, low toxicity, no residue, no pollution, and low tendency to induce drug resistance. This invention provides a new solution for the development of anti-mite drugs and provides effective drugs and technical means for the clinical control of ectoparasitic diseases in animals. Detailed Implementation

[0019] The following detailed description is provided in conjunction with specific embodiments, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Unless otherwise specified, the raw materials and reagents used in the examples are commercially available. The reagents carvacrol, eugenol, geraniol, and liquid paraffin oil used in the examples were all purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0020] Example 1

[0021] Screening of essential oil monoterpene synergistic combinations

[0022] 1.1 Collection of scabies mites

[0023] Collect scabs from the ear margins and toes of rabbits naturally infected with scabies mites, cut them into small pieces, place them in 9cm disposable petri dishes, and incubate them in an incubator at 35℃ and 80% humidity for a period of time. Select scabies mites that are in good condition and have intact bodies for subsequent experiments.

[0024] 1.2 Determination of the acaricidal activity of essential oil monoterpenes

[0025] To test the acaricidal activity of carvacrol, eugenol, and geraniol, based on the preliminary experimental results, five volume concentration gradients of the three essential oils were prepared using liquid paraffin oil, namely 10%, 5%, 2.5%, 1%, and 0.5%, to obtain essential oil solutions; the negative control was liquid paraffin.

[0026] The method for determining the susceptibility of acaricides is as follows: Ten scabies mites were placed in a 3.5 cm petri dish as a group. 600 μL of the aforementioned essential oil solution was added to each dish. The petri dishes containing the essential oil solution were placed in a constant temperature and humidity incubator at 25℃ and 70% humidity. The treatment time and the number of mites killed were observed and recorded every 15 minutes until all scabies mites died. If all scabies mites were observed dead under a stereomicroscope after 15 minutes of treatment, the same concentration of essential oil solution was added to the same group, and observation under a stereomicroscope was continued, recording the time and the number of mites killed, until all scabies mites died. Each concentration of essential oil and the control group were repeated 5 times. A mite was considered dead when it remained motionless under a stereomicroscope and did not move when gently touched with a dissecting needle. The test results are shown in Table 1.

[0027] 1.3 Determination of Synergistic Toxicity Index

[0028] Three essential oils were prepared individually to a certain concentration (concentrations are shown in the first column from the left in Table 2, all are volume concentrations). Then, the three essential oils were mixed in pairs to form blends (mixtures, concentrations are shown in the first column from the left in Table 2). The concentration of each individual oil in the blend was the same as the concentration of each essential oil used alone. The killing effect of each individual oil and the blend on scabies mites was then tested. The theoretical mortality rate of the blend (P0.05) was determined. m )=1-(1-P A )×(1-P B ), P A P B This refers to the mortality rate of each single agent tested on the test insects individually. Finally, the synergistic toxicity index (cf) was calculated using the following formula: cf = [(actual mortality rate - theoretical mortality rate) / theoretical mortality rate] × 100. A cf value ≥ 20 indicates a synergistic effect when the two drugs are used together, ≤ -20 indicates an antagonistic effect, and a value between -20 and 20 indicates an additive effect. Essential oil combinations with synergistic effects were screened using this method, and the test results are shown in Table 2.

[0029] 1.4 Determination of co-toxic factors

[0030] After screening in section 1.3, the combination with the best synergistic effect was selected. Different proportions of this combination were made using monoterpenes, the essential oil with the best acaricidal effect, as the main component, and another essential oil as a synergist. The co-toxicity factor method was used to calculate whether the mixture had a synergistic effect. The specific calculation method is as follows: By testing the toxicity regression curve of each drug, the mortality rate of the test insects at the corresponding concentration of the drug was calculated from the toxicity regression curve. The theoretical mortality rate of the mixture is equal to the sum of the theoretical mortality rates of each individual drug. The actual observed mortality rate of the mixture was substituted into the following formula to determine whether the two drugs have a synergistic effect: Co-toxicity factor = (Observed mortality rate of the mixture - Theoretical mortality rate of the mixture) / Theoretical mortality rate of the mixture × 100.

[0031] Among them, a co-toxicity factor >20 indicates a synergistic effect, <-20 indicates an antagonistic effect, and a value between -20 and 20 indicates an additive effect. The results are shown in Table 3.

[0032] The results of the above operations are as follows:

[0033] 1.5 Determination of the acaricidal activity of essential oil monoterpenes

[0034] The measured data were imported into statistical analysis software. The results showed that when scabies mites were exposed to carvacrol, eugenol and geraniol for 30 minutes, carvacrol had the highest toxicity, with an LC50 of 0.24%, followed by eugenol, with an LC50 of 0.80%, and lastly geraniol, with an LC50 of 0.91%.

[0035] Table 1. Toxicity determination of carvacrol, eugenol and geraniol

[0036]

[0037]

[0038] 1.6 Determination of Synergistic Toxicity Index

[0039] The acaricidal activity of carvacrol, eugenol and geraniol in combination was determined. The results showed that the combination of 0.2% carvacrol and 0.7% eugenol had a significant synergistic effect, with a cf value of 80.47.

[0040] Table 2. Determination of the synergistic toxicity index of carvacrol and eugenol.

[0041]

[0042] 1.7 Determination of co-toxic factors

[0043] According to section 1.6, carvacrol and eugenol exhibit a synergistic effect when combined in a binary blend. Furthermore, section 1.5 indicates that carvacrol has the best acaricidal effect. Therefore, different ratios of these two essential oils were mixed using carvacrol as the main ingredient and eugenol as the adjuvant. The co-toxicity factor method was used to screen for the blend ratio with the greatest synergistic effect between carvacrol and eugenol. Table 3 shows that when the carvacrol:eugenol ratio is 3:1, 1:2, and 1:3, the co-toxicity factors are 48.51, 44.54, and 24.58, respectively, all exceeding 20, indicating a synergistic effect. However, when the carvacrol:eugenol ratio is 1:1 and 2:1, the co-toxicity factors are -9.15 and -0.89, respectively, falling between 20 and -20, indicating an additive effect. The results indicate that a 3:1 ratio of carvacrol to eugenol results in the highest co-toxicity factor and a synergistic effect.

[0044] Table 3. Determination of toxic factors in the ratio of carvacrol and eugenol.

[0045]

[0046]

[0047] Based on the results in Table 3 above, and the maximum skin application concentrations of carvacrol and eugenol being 1% and 0.5% respectively, the optimal synergistic ratio of carvacrol and eugenol was determined to be a binary compound of carvacrol:eugenol = 3:1; wherein, the carvacrol concentration was configured as 1% by volume and the eugenol concentration was configured as 0.33% by volume (hereinafter referred to as the essential oil monoterpene composition for the prevention and control of mite diseases).

[0048] To determine the optimal in vitro acaricidal efficacy of this essential oil monoterpene composition for controlling mites, which is superior to single agents of the same concentration, acaricidal susceptibility testing was performed on 1% carvacrol + 0.33% eugenol, 1% carvacrol, and 0.33% eugenol according to the acaricidal susceptibility testing method in section 1.2. In this experiment, the mortality rate of scabies mites was 100% after 10 minutes of treatment with 1% carvacrol, 10% after 180 minutes of treatment with 0.33% eugenol, and 100% after 8 minutes of treatment with 1% carvacrol + 0.33% eugenol. These results indicate that this binary compound composition has the best acaricidal efficacy, superior to single agents, and can be further used for clinical efficacy evaluation.

[0049] Example 2

[0050] Clinical efficacy evaluation of essential oil monoterpenoid composition for the prevention and treatment of scabies in rabbits.

[0051] 2.1 Establishment of the scabies mite animal model

[0052] Twenty-four healthy New Zealand White rabbits were selected and purchased from TuBage Agricultural and Animal Husbandry Technology Co., Ltd. in Nanning City, Guangxi Zhuang Autonomous Region. Eighteen of these rabbits were housed in the same environment as rabbits naturally infected with rabbit scabies mites, and a rabbit scabies mite animal model was established through direct contact infection.

[0053] 2.2 Selection and grouping of clinical cases of scabies mites in rabbits

[0054] Eighteen New Zealand White rabbits meeting the clinical diagnostic criteria and exhibiting obvious clinical characteristics of scabies were selected from 2.1, along with six healthy New Zealand White rabbits. The specific clinical diagnostic criteria were: itching, localized hair loss on the toes, nose, testicles, etc., and the appearance of scabs; microscopic examination of scraped scabs revealed active scabies mites or their eggs; no other skin diseases; and no prior drug treatment.

[0055] The 24 rabbits that met the relevant clinical diagnostic criteria were divided into 4 groups of 6 rabbits each. Six healthy rabbits served as the healthy control group; six rabbits infected with scabies mites served as the infection control group and received no treatment; and six rabbits infected with scabies mites served as the essential oil treatment group, receiving topical application of the essential oil, which was a monoterpene composition for preventing and treating mites (a mixture of 1% carvacrol and 0.33% eugenol at a volume ratio of 3:1). The standard procedure for topical application was to soak cotton swabs or cotton balls in the essential oil and gently apply it to the infected areas such as the ears and toes, ensuring that all observed infected areas were covered with the essential oil. Approximately 3 ml was used per toe and approximately 4 ml per ear, with three applications administered on days 0, 3, and 7. Six rabbits infected with scabies served as the control group, receiving a 0.3% ivermectin solution applied according to the usual method for treating scabies, i.e., by applying the solution to the infected areas with a cotton swab, on days 0 and 7. Neither the infected nor healthy control groups received any medication. All rabbits were weighed on days 0, 7, and 14, with a trial period of 14 days.

[0056] 2.3 Evaluation of efficacy before and after medication

[0057] Clinical manifestations of the tested New Zealand white rabbits were recorded before the first treatment and on days 7 and 14. The scabs on the ear margin and auricle were collected, accurately weighed, and placed in 9cm petri dishes. The petri dishes containing the scabs were placed in a constant temperature incubator at 35°C and 80% humidity for about 30 to 60 minutes. The petri dishes were then removed and placed under a stereomicroscope to accurately count the number of scabies mites.

[0058] The reduction rate of scabies mites per test rabbit was calculated using the following formula: Reduction rate % = (Number of live scabies mites before treatment - Number of live scabies mites after treatment) / Number of live scabies mites before treatment × 100%

[0059] The infection status of the rabbit's ear margins and toes was scored and graded, as shown in Table 4.

[0060] Table 4. Clinical Symptoms and Recovery Description Scoring Grades of Rabbits Infected with Sarcoptes

[0061] rabbit ear margin infection degree rabbit toe infection severity Rating No infection symptoms No infection symptoms 0 Skin becomes red and dry Skin becomes red and dry 1 Hair loss along the ear margins, with a few scabs appearing. Hair loss on toes, with a few scabs appearing. 2 About half of the ear rim was covered with scabs Increased scabs on toes and severe hair loss 3 The scabs become thicker and more numerous The scab thickens and the affected area expands. 4 Extensive thick crusts on the outer ear margin Excessive keratinization of the scab, covering the entire toe 5

[0062] Rabbits were weighed on days 0, 7, and 14 after drug administration. The average weight gain of each group of rabbits before and after drug administration was calculated, and the differences in weight gain among the groups were compared.

[0063] The results of the above operations are as follows:

[0064] 2.4 Evaluation of the clinical therapeutic effect of blended essential oils on rabbit scabies mites

[0065] 1) Score for the recovery of rabbit ears and toes

[0066] Before medication, carefully examine the ears and toes of the infected rabbits. Score the clinical symptoms and recovery levels of the scabies mite infection in each group of rabbits according to Table 4, and record the scores. Calculate the average clinical scores for each experimental group. Record the number of scabies mites in the rabbits and the weight of each group of rabbits on days 0, 7, and 14 to comprehensively observe the rabbits' recovery.

[0067] Seven days after treatment, clinical manifestations were observed, and the clinical infection symptoms in the rabbit ears treated with the essential oil monoterpene composition for the prevention and treatment of mite infestation were significantly improved. The scabs were observed to fall off and become thinner, and the degree of recovery was comparable to that of ivermectin. By day 14 after treatment, the scabs in both the essential oil treatment group and the drug control group had largely fallen off, clearly exposing the normal skin before infection, and hair had begun to grow.

[0068] Seven days after treatment, compared to the infection control group, the essential oil treatment group and the drug control group showed significant shedding of scabs on the toes, revealing bare, pale red skin. On day 14, compared to the infection control group, the essential oil treatment group and the drug control group showed hair growth on the toes, and almost all scabs had fallen off. Fourteen days after medication, the clinical manifestations of the rabbits showed significant improvement; the severe itching and other symptoms present before medication disappeared, and behaviors such as head shaking, paw shaking, ear scratching, and nibbling on the affected area vanished. The rabbits were relatively calm, exhibiting behavior consistent with that of healthy control rabbits.

[0069] Based on the clinical results and the scoring criteria in Table 4, the following tables 5 and 6 are derived:

[0070] Table 5. Statistical results of the treatment and recovery status of rabbit ear infection with scabies mites (mean score ± SE)

[0071]

[0072] Table 6. Statistical results of treatment and recovery scores for scabies mite infection on rabbit toes (mean score ± SE)

[0073]

[0074]

[0075] 2) Scabies mite reduction rate

[0076] Microscopic examination of the samples showed a significant reduction in scabies mites after medication. On day 7, the mite reduction rates in the essential oil treatment group and the drug control group reached 99.29% and 99.25%, respectively. Moreover, the mite reduction rates in both the essential oil treatment group and the drug control group were significantly different from those in the infected control group (P < 0.01).

[0077] Table 7. Effect of compound essential oils on the reduction rate of scabies mites (mean score ± SE)

[0078] Group Scabies mite count before administration 7-day scabies mite reduction rate 14-day scabies mite reduction rate Essential oil therapy group 182.50±38.60 92.27±2.34 0.83±0.31 Drug control group 167.00±30.98 2.33±0.21 0.50±0.22 Infection control group 148.33±21.13 4.67±0.21 4.8±0.17 healthy control group 0 0 0

[0079] 3) Weight changes

[0080] As shown in Table 8, the weight gain of the essential oil treatment group and the drug control group was lower than that of the healthy control group on the 7th and 14th days after the first administration, but there was no statistically significant difference between them (P>0.05), indicating that the drug treatment not only relieved the scabies infection but also restored animal production to a certain extent.

[0081] Table 8 Comparison of weight gain among experimental groups (mean ± SE)

[0082] Group Average weight on day 0 Average weight gain on day 7 Average weight gain on day 14 Essential oil therapy group 2.09±0.09 0.07±0.04 0.17±0.04 Drug control group 2.11±0.04 0.05±0.04 0.15±0.01 Infection control group 2.17±0.06 0.06±0.04 -0.15±0.03 healthy control group 2.32±0.09 0.10±0.02 0.21±0.03

[0083] Based on the comparison of clinical symptom scores, scabies mite reduction rates, and weight gain among the experimental groups, the results showed that the topical application of an essential oil monoterpene composition for the prevention and treatment of mite diseases to rabbits with scabies, applied once on days 0, 3, and 7, had a significant therapeutic effect on rabbit scabies.

[0084] Example 3

[0085] Clinical efficacy evaluation of essential oil monoterpenoid composition for the prevention and treatment of mite infestations in cat ear mites

[0086] 3.1 Collection and clinical scoring of feline ear mite clinical cases

[0087] Eleven cases of cats naturally infected with ear mites were collected from a veterinary hospital, and each cat was isolated and managed individually. In the experimental group, after cleaning the outer ear with pet-specific ear cleaner on day 0, approximately 1 mL of an essential oil monoterpene composition for preventing and treating ear mites was instilled into each ear, and the base of the ear was gently massaged to ensure even distribution of the essential oil within the ear canal. Medication was administered according to the data shown in column 2 of Table 10 throughout the entire course of the disease.

[0088] During the experiment, microscopic examinations were performed on days 0 and 14. Based on the clinical scoring criteria in Table 9, the levels of ear mites and cerumen were assessed and clinical scores were recorded before and after drug treatment, including any adverse reactions.

[0089] Table 9 Otoscopic and Clinical Scores

[0090]

[0091] 3.2 Evaluation of Clinical Treatment Efficacy in Feline Ear Mite Cases

[0092] Before and after treatment of feline ear mite cases, clinical scores were calculated according to the scoring criteria in Table 9. The total score was calculated as: ear mite level score + cerumen score + pruritus score. Table 10 shows the experimental results. In all cases treated with the essential oil monoterpene composition for preventing and treating ear mites, microscopic examination revealed no ear mites, the external auditory canal was clean, there was no cerumen or secretions, and no itching was observed. The total clinical score for ear mites was 0, indicating good recovery. The blended essential oils showed significant efficacy in the clinical treatment of feline ear mites.

[0093] Table 10 Clinical Case Scoring Table for Feline Ear Mite

[0094]

[0095]

[0096] Table 10 lists essential oil treatment groups 1 to 11, all of which are essential oil monoterpene compositions for preventing and treating mite diseases, namely, a 3:1 volume ratio of caraway (1% by volume) and eugenol (0.33% by volume).

[0097] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A monoterpene essential oil composition for preventing and treating animal mite diseases, characterized in that: The monoterpene composition consists of carvacrol and eugenol; the volume ratio of carvacrol to eugenol is 3:

1.

2. The use of the essential oil monoterpene composition for preventing and treating animal mite diseases as described in claim 1 in the preparation of drugs for preventing and treating animal mite diseases, characterized in that: The mites are one or both of scabies mites or ear mites.

3. A medicine for preventing and treating animal mite diseases, characterized in that: The active ingredient is the essential oil monoterpene composition for preventing and treating animal mite diseases as described in claim 1, and further comprises other acceptable excipients for use in the pharmaceutical preparation.

4. The agent for preventing and treating animal mite diseases according to claim 3, characterized in that: The medicine is one of the following: ointment, solution, emulsion, film, or spray.

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