阅读说明：本技术 桧烯在制备杀灭或预防寄生虫病药物中的应用 (Application of sabinene in preparation of medicine for killing or preventing parasite ) 是由 尚小飞 邱燕华 张继瑜 李冰 王玮玮 周绪正 苗小楼 翟斌涛 于 2020-12-18 设计创作，主要内容包括：本发明属于药物技术领域,具体涉及植物挥发油中单萜类化合物桧烯在制备杀灭或预防寄生虫病药物中的应用,还公开了包括桧烯的药物组合物作为动物饲料添加剂的应用,实验结果显示,①桧烯对弓形虫I型虫株(RH-2F)速殖子的半数抑制浓度为90.76μg/ml,能够显著降低弓形虫的存活率；②桧烯有较强的抗弓形虫增殖活性,可以防止弓形虫在细胞内的增殖,从而预防或治疗弓形虫病；③桧烯对弓形虫具有较强的抗入侵活性,可以防止细胞外的弓形虫入侵宿主细胞；且本发明所述的桧烯在治疗弓形虫病方面具有安全、有效、毒性小等特点,制备工艺简单,成本低,可制备成各种制剂,且容易降解,不会对环境造成任何污染。(The invention belongs to the technical field of medicines, in particular relates to application of a monoterpene compound sabinene in plant volatile oil in preparation of medicines for killing or preventing parasitic diseases, and also discloses application of a medicine composition comprising the sabinene as an animal feed additive, wherein experimental results show that firstly, the half inhibitory concentration of the sabinene to tachyzoites toxoplasma gondii I (RH-2F) is 90.76 mu g/ml, and the survival rate of the toxoplasma gondii can be obviously reduced; the sabinene has stronger anti-toxoplasmosis activity, can prevent the proliferation of toxoplasmosis in cells, thereby preventing or treating the toxoplasmosis; the sabinene has strong anti-invasion activity to the toxoplasma, and can prevent the toxoplasma outside the cell from invading the host cell; the sabinene has the characteristics of safety, effectiveness, low toxicity and the like in the aspect of treating toxoplasmosis, has simple preparation process and low cost, can be prepared into various preparations, is easy to degrade, and does not cause any pollution to the environment.)

1. Application of sabinene and its pharmaceutically acceptable salt in preparing medicine for killing or preventing parasite is provided.

2. Application of sabinene and its pharmaceutically acceptable salt in preparing medicine for killing or preventing intracellular parasite is provided.

3. The use according to claim 1 or 2, wherein the parasitic disease comprises toxoplasmosis.

4. The pharmaceutical composition for killing or preventing the parasitic diseases is characterized by comprising sabinene and other pharmaceutically acceptable auxiliary materials.

5. The pharmaceutical composition of claim 4, wherein the effective concentration of sabinene in the pharmaceutical composition is 90.76 μ g/ml.

6. The pharmaceutical composition of claim 5, wherein the pharmaceutical composition can be formulated into any one of powder, tablet, capsule, pill, drop pill, injection, emulsion, suspension or tincture.

7. A pharmaceutical composition for inhibiting the invasion of a parasite into a host cell, comprising sabinene and other pharmaceutically acceptable excipients.

8. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition is formulated for external use.

9. Use of sabinene and other insect-resistant drugs in combination for treating parasitic diseases.

10. Use of a pharmaceutical composition according to any one of claims 4 to 8 as an animal feed additive.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an application of a monoterpene compound sabinene in plant volatile oil in preparation of medicines for killing or preventing parasitic diseases.

Background

Toxoplasmosis is a global zoonosis which is a zoonosis caused by the parasitism of Toxoplasma gondii (Toxoplasma gondii) of Toxoplasma genus, Toxoplasma family, Toxoplasma in the nucleated cells of the host. Research shows that the feline is a terminal host, more than 100 animals such as human, livestock, poultry and the like belong to intermediate hosts, the feline has the greatest harm to pigs and sheep in livestock, and most of the feline is recessive infection. Feline infection leads to death, while infection of pregnant women leads to symptoms of premature delivery, abortion, etc. Toxoplasmosis seriously affects the healthy development of animal husbandry and human health, and brings serious economic loss. At present, the medicines for treating toxoplasmosis are mainly chemosynthetic medicines, such as pyrimethamine, sulfadiazine and folic acid which are jointly applied and the like, can control the development of toxoplasmosis, but the treatment is often accompanied by side effects, and the prognosis is poor and the toxoplasmosis is easy to relapse.

Sabinene (bicyclo [3.1.0] hexane, 4-methyl-1- (1-methyl) -) is a monoterpene compound which is widely distributed in various plant volatile oils such as Salvia officinalis, Zingiber striolatum, Dracocephalum integrifolium and the like, and the plant volatile oils can be extracted by methods such as supercritical extraction or steam distillation and then separated and extracted by methods such as solvent extraction, preparation of thin layer chromatography and the like, and can also be obtained by biological fermentation or chemical synthesis. The sabinene is convenient to obtain and low in cost. Sabinene is an important high-density fuel precursor, can be used as an additive of aviation fuel, has important application prospects in the fields of medicines, foods, energy sources and the like, and is attracted more and more attention in recent years. The research shows that sabinene has the activity of resisting bacteria, weeding, killing arthropod, etc., for example, the invention patent (CN201910847090.0) discloses the application of a natural monoterpene compound sabinene in the preparation of herbicide, the compound is applied under the lower concentration of 5 mu l/mL, can kill monocotyledonous and dicotyledonous plants such as green bristlegrass, and has obvious inhibiting effect on dicotyledonous plants such as amaranthus retroflexus and alfalfa, the compound is prepared into different concentrations, and can be used alone or mixed with other pesticides before or in the seedling stage, so that the compound can be naturally degraded, has no pollution to the environment, and can be used as herbicide. The invention patent (CN201710293058.3) discloses a perfume composition for reproducing the fragrance of white lotus flower, and particularly relates to a perfume composition for reproducing the fragrance of white lotus flower with excellent preference by adding 2-phenylethyl alcohol into pentadecane, p-xylylene ether, heptadecane, sabinene, limonene, myrcene, alpha-terpineol and the like which are analyzed by the SPME method to obtain the fragrance components of white lotus flower. The invention patent (CN201811348112.0) discloses an antibacterial, acarid-removing and peculiar smell-removing aqueous solution, which at least comprises tea tree essential oil, cedar essential oil, aromatic holly leaf essential oil, rosemary essential oil, lavender essential oil, geraniol, alpha pinene, beta pinene, myrcene, sabinene, cedar wood oil, borneol, menthol, thymol and other components. Has good antibacterial, deodorizing and astringing effects, and has effects of removing odor, controlling peculiar smell, and resisting virus. The invention discloses an attractant and a lure for trapping dichocrocis punctiferalis, the attractant is formed by mixing compounds of alpha-pinene, camphene, sabinene and beta-pinene, and each compound is a volatile active compound released by dichocrocis punctiferalis main host plants (Chinese chestnuts) in the field. The attractant is prepared by mixing volatile active compounds with the function of attracting dichocrocis punctiferalis, can attract and collect a large amount of dichocrocis punctiferalis female moths and a small amount of male moths, and can be applied to the dynamic monitoring and control of the population occurrence of dichocrocis punctiferalis in chestnut orchards. However, there is no technical proposal in the art to disclose the effect of sabinene on parasites.

The inventor unexpectedly discovers in the research process that sabinene can obviously reduce the survival rate of Toxoplasma gondii type I (RH-2F) tachyzoites, has good toxoplasma gondii-resisting proliferation activity, obviously reduces the number of intracellular polypides and cysts in vivo; inhibiting toxoplasma invasion activity, and enlarging the application of sabinene.

Disclosure of Invention

Aiming at the technical problems, the invention discloses a new application of sabinene, and the first purpose of the sabinene is to provide an application of sabinene and pharmaceutically acceptable salts thereof in preparation of medicines for killing or preventing parasitic diseases.

The second purpose of the invention is to provide the application of sabinene and the pharmaceutically acceptable salts thereof in preparing the medicines for killing or preventing intracellular parasitic diseases.

Preferably, the parasitic disease comprises toxoplasmosis.

The third purpose of the invention is to provide a pharmaceutical composition for killing or preventing parasitic diseases, which comprises sabinene and other pharmaceutically acceptable auxiliary materials.

Preferably, the effective concentration of sabinene in the pharmaceutical composition is 90.76 μ g/ml.

Preferably, the pharmaceutical composition can be prepared into any one dosage form of powder, tablets, capsules, pills, dripping pills, injections, emulsions, suspensions or tinctures.

The fourth purpose of the invention is to provide a pharmaceutical composition for inhibiting parasite host cells, which comprises sabinene and other pharmaceutically acceptable auxiliary materials, wherein the effective concentration of the sabinene in the composition is 221.5 mug/mL.

Preferably, the pharmaceutical composition can be prepared into an external preparation.

The fifth purpose of the invention is to provide the application of the combination of sabinene and other insect-resistant medicaments for treating parasitic diseases

The sixth purpose of the invention is to provide the application of the pharmaceutical composition in preparing animal feed.

The invention has the beneficial effects that: the invention provides a new application of sabinene, in particular to an application of sabinene in preparation of a medicament for killing or preventing parasite diseases, and experimental results show that firstly, the half inhibitory concentration of sabinene to Toxoplasma gondii I type insect strain (RH-2F) tachyzoite is 90.76 mu g/ml, and the survival rate of Toxoplasma gondii can be obviously reduced; the sabinene has stronger anti-toxoplasmosis activity, can prevent the proliferation of toxoplasmosis in cells, thereby preventing or treating the toxoplasmosis; the sabinene has strong anti-invasion activity to the toxoplasma, and can prevent the toxoplasma outside the cell from invading the host cell; the sabinene has the characteristics of safety, effectiveness, low toxicity and the like in the aspect of treating toxoplasmosis, has simple preparation process and low cost, can be prepared into various preparations, is easy to degrade, and does not cause any pollution to the environment.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1: inhibition of Toosendanin against Toxoplasma gondii type I insect (RH-2F)

FIG. 2: anti-proliferation effect of sabinene on toxoplasma gondii in immunofluorescence experiment

FIG. 3: antiproliferative effect of sabinene on toxoplasma II type insect strain (Pru) in cell

FIG. 4: anti-invasion effect of sabinene on extracellular toxoplasma

FIG. 5: cytotoxicity of sabinene against Vero cells

Detailed Description

Reference will now be made in detail to embodiments of the present invention, the results of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention. The experimental procedures in the following examples are conventional unless otherwise specified.

The Vero cell described in the examples below is a Hetian Green monkey kidney cell, an isoploid cell, a commonly used cell line derived from the culture of macaque kidney cells.

The cell slide described in the following examples is a slide which is immersed in a cell culture medium and on which cells are grown, and is mainly used for histology, immunohistochemistry, frozen sections, cell smears, in situ hybridization, and the like.

MGG consists of May-Grunwald dye and Giemsa dye according to the Giemsa staining method described in the following examples. The former is chemically known as eosin methylene blue II, and has good cytoplasma coloration effect; the latter stain nuclei better. Therefore, MGG staining can combine the advantages of both, and is commonly used for staining cell smears.

DAPI described in the following examples, 4', 6-diamidino-2-phenylindole, is a fluorescent dye that binds strongly to DNA and is commonly used for fluorescence microscopy. Because DAPI is permeable to intact cell membranes, it can be used for staining of both living and fixed cells.

The CCK-8 reagent described in the following example contains WST-8, the chemical name of which is 2- (2-Methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfonic acid benzene) -2H-tetrazole monosodium salt, which is reduced to a highly water-soluble yellow Formazan product (Formazan) by dehydrogenase in the mitochondria of cells under the action of an electron carrier, namely 1-Methoxy-5-methylphenazinium dimethylsulfate (1-Methoxy PMS). The amount of formazan product generated is directly proportional to the number of viable cells.

The light absorption value of the enzyme linked immunosorbent assay device is measured at the wavelength of 450nm, and the quantity of living cells can be indirectly reflected. The method is widely used for activity detection of some bioactive factors, large-scale screening of anti-tumor drugs, cell proliferation tests, cytotoxicity tests, drug sensitivity tests and the like.

The DPBS, fully known as Dulbecco's phosphate-buffered saline (Du's phosphate buffer), described in the examples below, is a balanced salt solution used for short-term maintenance of mammalian cell viability. It can maintain the structural and physiological integrity of cells ex vivo for a limited time. Wherein the presence of calcium and magnesium ions reduces trypsin activity, therefore EDTA is typically added to digest cells to sequester calcium and magnesium ions that may be present. However, EDTA cannot be neutralized by serum, so that DPBS containing no calcium and magnesium ions must be used after treating cells with EDTA. DPBS is generally used for cleaning before cell dissociation, cell or tissue transport, cell counting dilution, solution preparation, cleaning of containers and cell supplies, and the like.

The cysts described in the following examples refer to the membranes that secrete a protein around the body to withstand harsh conditions when lower plants and protozoa are in a resting state or when external environmental conditions change. Protozoa generally have a simple body structure and a high reproductive capacity. Among parasitic protozoa, some particular phenomenon of cyst formation can be seen. The cyst is covered by a very firm membrane that, even when expelled from the body, can survive for a long period of time. This state is called resistant type or infection type. The invention characterizes the cyst of toxoplasma.

Example I inhibitory Effect of sabinene on Toxoplasma gondii type I Strain (RH-2F)

1. Sources of test cells and reagents

vero cells: purchased from the cell bank of the Chinese academy of sciences;

toxoplasma RH-2F: purchased from ATCC;

toxoplasma RH and Pru: is a gift from the Lanzhou veterinary research institute of Chinese agricultural academy of sciences, Zhuxing, Henghui;

sabinene: purchased from Shanghai leaf Biotech, Inc.;

detection reagent: purchased from promo-maige, beijing biotechnology limited;

primary and secondary antibodies: purchased from Abcam;

2. procedure of experiment

Harvesting fresh and active Toxoplasma gondii type I strain (RH-2F) tachyzoite from vero cells, counting by using a blood cell counting plate, and adjusting the concentration of Toxoplasma gondii RH-2F to be 1 × 10⁵One per ml. Different concentrations of sabinene were prepared and added to 96-well plates, with 0.25% DMSO as control and medium alone as blank. Adding after culturing for 12hAnd (5) detecting the reagent, and detecting the luminescence value in a photometer after 30min of action. Calculating the survival rate of Toxoplasma gondii RH-2F, drawing a curve and calculating IC₅₀。

3. Results of the experiment

As shown in FIG. 1, the survival rate of Toxoplasma gondii type I strain (RH-2F) decreased with the increase of the concentration of sabinene drug, and the IC of Toxoplasma gondii type I strain (RH-2F) was obtained₅₀90.76. mu.g/ml. The sabinene has effect in inhibiting Toxoplasma gondii I strain (RH-2F).

EXAMPLE II antiproliferative effect of sabinene on Toxoplasma cells (immunofluorescence)

1. Procedure of experiment

The sources of cells and reagents used in the experimental procedure of this example are the same as those disclosed in the first example.

1×10⁵Vero cells per ml were seeded in cell slides in 12-well plates and 1X 10 cells were plated after 12h⁵A toxoplasma I type worm strain (RH-2F) tachyzoite is inoculated in a single-layer vero cell, after 8 hours of invasion, sabinene is added for 48 hours, 0.25% DMSO is used as a control group, and acetylpyrimidine (10 mu g/ml) is used as a positive control. Fixing with 4% paraformaldehyde, repairing antigen, sealing with 10% goat serum, penetrating with 0.2% Triton X-100, selecting rabbit anti-toxoplasma polyclonal antibody to mark toxoplasma, and using goat anti-rabbit lgG H as secondary antibody&L, DAPI stain nuclei. After dyeing and sealing, the film is observed and photographed under a confocal microscope.

2. Results of the experiment

The experimental result is shown in figure 2, at 48h, sabinene has obvious antiproliferative effect on toxoplasma I strain (RH-2F), and only a few insect bodies exist in the visual field. Although the positive control acetylpyrimidine can also reduce endosymptosporium, the activity is lower than sabinene, and the anti-proliferation effect is lower than sabinene. In conclusion, sabinene has a strong anti-toxoplasma proliferation activity.

EXAMPLE III antiproliferative Effect of sabinene on the intracellular Toxoplasma II Strain (Pru)

1. Procedure of experiment

The sources of cells and reagents used in the experimental procedure of this example are the same as those disclosed in the first example.

The inoculation concentration of the 6-well plate is 2 multiplied by 10⁵After culturing the vero cells of each/ml for 12h, adding 3X 10 of the cells into the monolayer⁵Individual/ml Toxoplasma gondii type II strain (Pru) tachyzoite. After 24h of invasion, uninvaded tachyzoites were washed away with DPBS and sabinene was added at a concentration of 80. mu.g/ml, with 0.25% DMSO as a control. And observing the result by Giemsa staining when the culture is carried out for 48 hours.

2. Results of the experiment

The experimental results are shown in FIG. 3, at 48h, the control group had been occupied by cysts in the visual field, and no cypress group had seen the polypide. Research results show that sabinene has strong toxoplasma gondii proliferation resisting activity.

Example IV anti-invasive Effect of sabinene on extracellular Toxoplasma

1. Procedure of experiment

The sources of cells and reagents used in the experimental procedure of this example are the same as those disclosed in the first example.

1×10⁵Vero cells/ml were seeded in cell slide in 12-well plate, and 1X 10 sabinene-containing concentrations were added after 12h⁵One/ml of Toxoplasma gondii type I strain (RH-2F) tachyzoite was inoculated in a monolayer of vero cells with 0.25% DMSO as a control. After 2h of invasion, the non-invading extracellular tachyzoites were washed out with PBS as much as possible. After fixing, antigen repairing and sealing, using mouse anti-toxoplasma monoclonal antibody to mark toxoplasma outside cell, and using goat anti-mouse lgG H as secondary antibody&And L. After permeation, selecting rabbit anti-toxoplasma polyclonal antibody to mark toxoplasma in cell, and using goat anti-rabbit lgG H as second antibody&L, DAPI stain nuclei. After staining and mounting, ten fields are randomly selected under a confocal microscope for observation, photographing and counting.

2. Results of the experiment

As shown in FIG. 4, sabinene showed dose dependence on the invasion-resistant activity of Toxoplasma gondii type I strain (RH-2F). The invasion rate was 80.70% at 40. mu.g/ml and 19.30% at 120. mu.g/ml. In conclusion, sabinene has a strong anti-invasive activity against toxoplasma.

EXAMPLE cytotoxicity of sabinene on vero cells

1. Procedure of experiment

Toxicity of sabinene to vero cells was determined by the CCK-8 method. Vero cell adjusted cell concentration to 1X 10⁵Each 100. mu.l of the suspension was inoculated into a 96-well plate per ml, and cultured for 12 hours, and then sabinene was added thereto at each concentration. Control group was 0.25% DMSO, and blank group was medium alone. After 24h of culture, CCK-8 reagent is added, and after 1h of action, the OD value of each hole is measured in a 450nm enzyme-labeling instrument. Cell viability was calculated according to the following formula, and the curve was plotted and IC was calculated₅₀。

2. The experimental results are as follows:

the survival rate of the vero cell is 93.63% when the sabinene is 128 mu g/mL, the proliferation promoting effect is realized when the sabinene is 64 mu g/mL, the cell survival rate reaches 106.90%, and the IC of the sabinene on the vero cell₅₀221.5 μ g/ml, which indicates that sabinene has low toxicity to host cells, and can be used for treating toxoplasmosis.

In conclusion, the invention discloses a new application of sabinene, and particularly discloses that the half inhibitory concentration of sabinene to the tachyzoite of toxoplasma gondii I (RH-2F) is 90.76 mug/ml, and the survival rate of toxoplasma gondii can be remarkably reduced; meanwhile, the sabinene has stronger anti-toxoplasma proliferation activity and can inhibit the proliferation of toxoplasma; meanwhile, sabinene also has strong anti-invasion activity to toxoplasma; the sabinene has the characteristics of safety, effectiveness, low toxicity and the like in the aspect of treating toxoplasmosis, has simple preparation process and low cost, can be prepared into various preparations, is easy to degrade, and does not cause any pollution to the environment.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.