CN112843046A - Application of naringenin and composition thereof in preparation of medicine for treating or preventing toxoplasmosis - Google Patents
Application of naringenin and composition thereof in preparation of medicine for treating or preventing toxoplasmosis Download PDFInfo
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- CN112843046A CN112843046A CN202110262137.4A CN202110262137A CN112843046A CN 112843046 A CN112843046 A CN 112843046A CN 202110262137 A CN202110262137 A CN 202110262137A CN 112843046 A CN112843046 A CN 112843046A
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- 229940117954 naringenin Drugs 0.000 title claims abstract description 89
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- DFPMSGMNTNDNHN-ZPHOTFPESA-N naringin Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](OC=2C=C3O[C@@H](CC(=O)C3=C(O)C=2)C=2C=CC(O)=CC=2)O[C@H](CO)[C@@H](O)[C@@H]1O DFPMSGMNTNDNHN-ZPHOTFPESA-N 0.000 description 1
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- SEEPANYCNGTZFQ-UHFFFAOYSA-N sulfadiazine Chemical compound C1=CC(N)=CC=C1S(=O)(=O)NC1=NC=CC=N1 SEEPANYCNGTZFQ-UHFFFAOYSA-N 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/116—Heterocyclic compounds
- A23K20/121—Heterocyclic compounds containing oxygen or sulfur as hetero atom
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
- A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Polymers & Plastics (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Animal Husbandry (AREA)
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- Engineering & Computer Science (AREA)
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- Tropical Medicine & Parasitology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses an application of naringenin and pharmaceutically acceptable salts thereof in preparing a medicament for treating or preventing toxoplasmosis. The effective concentration of naringenin is 50-120 mug/mL. Also discloses application of the pharmaceutical composition containing naringenin and pharmaceutically acceptable salts thereof in preparing medicaments for treating or preventing toxoplasmosis. According to the invention, the naringenin is obtained to have good effects of treating or preventing toxoplasmosis by utilizing the inhibition and anti-proliferation effects of the naringenin on toxoplasma gondii and the anti-invasion effect on extracellular toxoplasma gondii, and the naringenin is found to have small toxicity, safety and reliability by the toxicity research of the naringenin on Vero cells. Therefore, the naringenin and the composition thereof have obvious application effect and small toxic and side effect in preparing the medicine for treating or preventing toxoplasmosis.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to application of naringenin and a composition thereof in preparing medicines for treating or preventing toxoplasmosis.
Background
Toxoplasma belongs to the phylum Toxoplasma, Sporophyceae, Coccidia, Eucoccidia, Eimeriales, Toxoplasma. There is only one species under the genus Toxoplasma, Toxoplasma gondii (Toxoplasma gondii), which is commonly referred to as Toxoplasma. However, depending on different regions, different hosts, different virulence, life history and development time, they can be divided into three different genotypes, type I (RH and GT-1 strains), type II (ME49 and PRU strains) and type III (CEP and VEG strains). Toxoplasma is an intracellular parasitic protozoan that infects all warm-blooded animals including humans, even some cold-blooded animals, and that parasitizes all nucleated cells of the animal's body. Toxoplasmosis is divided into congenital toxoplasmosis and acquired toxoplasmosis, with ocular toxoplasmosis and cerebral toxoplasmosis being the most common clinical manifestations. Patients with toxoplasmosis who are immunocompromised typically do not require treatment, but can have fatal consequences for patients with immunocompromised or low immune function.
In recent years, although great progress has been made in diagnosis, epidemiology, etiology, and the like, few new progress has been made in treatment. Because of the complexity of the toxoplasma life cycle, diversity in pathogenesis, and variability in biological properties, there is no preventive or specific drug treatment available. Although the combined medication of pyrimethamine and sulfadiazine is the gold standard for clinically treating toxoplasmosis at present, the treatment method is often accompanied by serious side effects, the treatment is incomplete, and the defect of easy relapse exists.
Naringenin is aglycone of naringin, belongs to flavanone compounds, and is mainly derived from flower buds of oriental cherry (Prunus yedonsis Mate.) and fruit shells of stem tree (Amacardi-um occidentale L.) in Anacardiaceae in nature. Pharmacological studies at home and abroad show that the naringenin has various pharmacological activities of resisting bacteria, inflammation, oxidation, fibrosis, cancer, tumor, virus, arrhythmia, cough, atherosclerosis, immunity, fat metabolism, aging, liver function and estrogen-like substances. However, no studies have shown that naringenin has an active effect on protozoa.
In the research process of the toxoplasmosis, the inventor finds the application of naringenin in preparing the medicine for treating or preventing the toxoplasmosis, for example, the naringenin has obvious inhibition activity on intracellular parasitic protozoan toxoplasmosis, can obviously inhibit the formation of nauplius, can also obviously inhibit the invasion activity of toxoplasmosis, and expands the application range of the naringenin; furthermore, naringenin has the characteristics of safety, effectiveness, low toxicity and the like in the aspect of treating toxoplasmosis.
Disclosure of Invention
The invention aims to solve the technical problem of providing an application of naringenin and pharmaceutically acceptable salts thereof in preparing a medicine for treating or preventing toxoplasmosis, so that the medicine for treating or preventing toxoplasmosis has an obvious effect and small toxic and side effects.
Further improved, the effective concentration of naringenin is 50-120 mug/mL.
Preferably, the effective concentration of naringenin is 60 μ g/mL.
The invention also aims to solve the technical problem of providing an application of a pharmaceutical composition in preparing a medicament for treating or preventing toxoplasmosis, wherein the pharmaceutical composition comprises naringenin or pharmaceutically acceptable salts of naringenin and pharmaceutically acceptable auxiliary materials. The medicinal composition has the advantages of remarkable effect and small toxic and side effects.
Preferably, the pharmaceutical composition further comprises an insect-resistant drug which is used in combination with the naringenin or the pharmaceutically acceptable salt of the naringenin, so as to expand an insect-resistant spectrum and improve an insect-resistant effect.
Further improved, the pharmaceutical composition is prepared into any one dosage form of powder, tablets, capsules, pills, dripping pills, injections, emulsions, suspensions or tinctures.
Further improved, the pharmaceutical composition can also be used as an animal feed additive, and the treatment or prevention of the toxoplasmosis of animals can be simply and conveniently realized.
After adopting such design, the invention has at least the following advantages:
1. according to the invention, the naringenin is obtained to have a good effect of treating toxoplasmosis by utilizing the inhibition and anti-proliferation effects of the naringenin on the toxoplasma gondii of the parasite, the naringenin is obtained to have a good effect of preventing the toxoplasmosis by utilizing the anti-invasion effect of the naringenin on the toxoplasmosis outside cells, and the toxicity of the naringenin on Vero cells is researched, so that the naringenin is found to be small in toxicity, safe and reliable. Therefore, the naringenin and the pharmaceutically acceptable salt thereof have obvious application effect and small toxic and side effect in preparing the medicine for treating or preventing toxoplasmosis.
2. Naringenin and pharmaceutically acceptable salts thereof are used as effective components of the pharmaceutical composition or are used together with other insect-resistant medicaments, so that the pharmaceutical composition has a good effect of preventing toxoplasmosis, and has the advantages of remarkable effect, small toxic and side effects and wide insect-resistant spectrum. When the pharmaceutical composition is used as an animal feed additive, the treatment or prevention of the toxoplasmosis of animals can be realized more conveniently.
Drawings
The foregoing is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and the detailed description.
FIG. 1 is a graph showing the results of toxicity test of naringenin on Vero cells in accordance with the present invention.
FIG. 2 is a graph showing the inhibitory effect of naringenin on Toxoplasma gondii in accordance with the present invention.
FIG. 3 is a microscopic view of naringenin of the present invention and the antiproliferative effect of the negative and positive control groups on intracellular RH.
FIG. 4 is a microscopic view of the antiproliferative effect of intracellular Pru by naringenin and negative and positive control groups of the present invention.
FIG. 5 is a graph showing the results of the anti-invasion effect of naringenin, negative control group and positive control group on the toxoplasma gondii outside the cell.
Detailed Description
The following detailed description of embodiments of the invention is intended to be illustrative, but not limiting, of the invention. The experimental procedures in the following examples are conventional and commercially available reagents unless otherwise specified.
EXAMPLE A toxicity study of naringenin on Vero cells
1. Procedure of experiment
And (3) measuring the toxicity of the naringenin to the Vero cell by adopting a CCK-8 method.
Adjusting the concentration of Vero cells to 1X 105And (4) inoculating the cells/mL of the cells into a 96-well plate, culturing for 12h, adding naringenin solutions with different concentrations, taking 0.25% DMSO as a control group, and taking a pure culture medium as a blank group. After 24h of culture, CCK-8 reagent is added, and after 1h of action, the optical density OD value of each well is measured in a 450nm microplate reader. Calculating the survival rate of Vero cells according to the following formula, drawing a curve and calculating IC50The value is obtained.
Cell survival (%) ═ (OD)Test group－ODBlank group)/(ODControl group－ODBlank group)×100％
2. Results of the experiment
The survival rate of Vero cells in the naringenin solutions with different concentrations is calculated, and the results are shown in table 1 below, and a curve is drawn, as shown in fig. 1.
Table 1: toxicity test result of naringenin on Vero cells
As is clear from the numerical values in Table 1 and FIG. 1, the survival rate of Vero cells was 98.84% at a naringenin concentration of 50. mu.g/mL, and the survival rate of Vero cells was 84.20% at a naringenin concentration of 100. mu.g/mL. And obtaining the semi-inhibitory concentration IC of naringenin on Vero cells50Is 320 mu g/mAnd L. The naringenin is shown to have lower toxicity to Vero cells.
Example inhibition of Toxoplasma by Dichron
1. Procedure of experiment
Toxoplasma gondii RH-2F strains expressing beta-galactosidase were used for the determination of growth inhibition.
Harvesting fresh and active Toxoplasma gondii RH-2F tachyzoite from Vero cells, inoculating the Toxoplasma gondii RH-2F tachyzoite to a 96-well plate, counting by a blood cell counting plate, and adjusting the concentration of the Toxoplasma gondii RH-2F tachyzoite to be 1 × 105one/mL. Naringenin solutions with different concentrations were prepared and added to 96-well plates, respectively, with 0.25% DMSO as a control group and pure medium as a blank group. Adding after culturing for 12h
And (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 tachyzoite, drawing a curve and calculating IC50The value is obtained.
2. Results of the experiment
The survival rate of the Toxoplasma gondii RH-2F tachyzoite from the naringenin solution with different concentrations is calculated, the result is shown in the following table 2, and a curve is drawn, as shown in the attached figure 2.
Table 2: experimental result of inhibiting effect of naringenin on toxoplasma gondii
As is clear from the numerical values in Table 2 and FIG. 2, the survival rate of Toxoplasma gondii RH-2F tachyzoites was 57.28% at a naringenin concentration of 50. mu.g/mL, decreased with an increase in the naringenin concentration, and only 19.62% at a naringenin concentration of 200. mu.g/mL. It can also obtain the semi-inhibitory concentration IC of naringenin on Toxoplasma gondii50Is 63.57μ g/mL. The naringenin is shown to be capable of remarkably reducing the survival rate of Toxoplasma gondii type I strain RH-2F tachyzoite, the inhibition effect is obvious, and the inhibition effect is dose dependent.
According to the results of the above first and second examples, naringenin was selected at a concentration of 60. mu.g/mL as a concentration for the subsequent study of naringenin's antiproliferative effect on Toxoplasma gondii. And the toxicity test result of the first embodiment shows that the naringenin has the characteristics of safety, reliability and small toxic and side effect when the concentration of the naringenin is below 120 mu g/mL.
Example anti-proliferative Effect of Trinaringenin on Toxoplasma I Strain (RH) in cells
1. Procedure of experiment
Will be 1 × 105Inoculating toxoplasma I strain RH tachyzoite in a 12-well plate into a single-layer Vero cell slide, after invading for 4H, adding naringenin solution with the concentration of 60 mug/mL, taking 0.25% DMSO as a negative control group, taking 10 mug/mL pyrimethamine as a positive control group, acting for 24H and 48H, then fixing with 4% paraformaldehyde, performing antigen retrieval, sealing with 10% goat serum, penetrating with 0.2% Triton X-100, selecting a rabbit anti-toxoplasma polyclonal antibody to mark toxoplasma, and using a second antibody of the goat anti-rabbit lgG H&L, DAPI stain nuclei. After dyeing and sealing, the film is observed and photographed under a confocal microscope.
2. Results of the experiment
FIG. 3 is a graph showing the anti-proliferative effect of Toxoplasma gondii in cells after 24h and 48h of the negative control group, naringenin and positive control group, as shown in FIG. 3: at 24h, compared with a negative control group, the naringenin group can reduce the number of toxoplasma gondii, but a certain amount of naematococcus still exists, and the effect is not as good as that of the positive control group. However, when the naringenin group acts for 48 hours, the naringenin group has a remarkable effect of reducing the number of toxoplasma gondii, and the effect is even better than that of a positive control group, which shows that naringenin solution with the concentration of 60 mug/mL can generate an anti-proliferation effect on toxoplasma gondii type I (RH) strains in cells, and the effect is remarkable.
Example antiproliferative Effect of Tetranaringenin on the intracellular Toxoplasma II Strain (Pru)
1. Procedure of experiment
Will be 1 × 105Inoculating one/mL toxoplasma II strain Pru tachyzoite into a single-layer Vero cell slide in a 12-well plate, after invading for 6H, adding naringenin solution with the concentration of 60 mug/mL, taking 0.25% DMSO as a negative control group, taking 10 mug/mL pyrimethamine as a positive control group, acting for 24H, then fixing with 4% paraformaldehyde, repairing antigens, sealing with 10% goat serum, penetrating with 0.2% Triton X-100, selecting a mouse anti-toxoplasma monoclonal antibody to mark toxoplasma, wherein a second antibody is goat anti-mouse lgG H&L(
647) DAPI stained nuclei. After dyeing and sealing, the film is observed and photographed under a confocal microscope.
2. Results of the experiment
FIG. 4 is a picture of the antiproliferative effect on Toxoplasma gondii in cells after 24 hours of the negative control group, naringenin and positive control group, as can be seen from FIG. 4: naringenin has obvious antiproliferative effect on toxoplasma gondii II strain Pru in cells, and is superior to a positive control group of pyrimethamine with 10 mug/mL. The naringenin solution with the concentration of 60 mug/mL is shown to generate an antiproliferative effect on the Toxoplasma gondii II strain Pru in the cell, and has the effect of obviously inhibiting the formation of naxoplasma gondii in the cell and the effect is obvious.
Example anti-invasive Effect of Pentanaringenin on extracellular Toxoplasma
1. Procedure of experiment
Mixing 1 × 10 solutions containing naringenin with different concentrations5A toxoplasma I strain RH tachyzoite of each/mL is inoculated in a reptile in a 12-well plate full single-layer Vero cell, 0.25% DMSO is used as a negative control group, and 10 mug/mL pyrimethamine is used as a positive control group. After 2h of invasion, the non-invading extracellular tachyzoites were washed out with PBS as much as possible. Then after fixing, antigen repairing and sealing, using mouse anti-toxoplasma monoclonal antibody to mark toxoplasma outside cells, and using secondary antibody as goat anti-mouse lgG H&L(Alexa
647). Selection of the cells after 0.2% Triton X-100 permeabilization Using Rabbit anti-Toxoplasma polyclonal antibodyThe toxoplasma in the cell is marked, and the second antibody is goat anti-rabbit lgG H&L(Alexa
488) DAPI stained nuclei. After mounting, randomly selecting ten visual fields under a confocal microscope to observe, photograph and count. The invasion rate of the cells by the Toxoplasma gondii was calculated according to the following formula.
The invasion rate (%) — the number of cells invaded by toxoplasma per total number of cells × 100%.
2. Results of the experiment
The invasion rate of the naringenin solution with different concentrations on Toxoplasma gondii invading Vero cells is calculated, the result is shown in the following table 3, and a curve is drawn, as shown in the attached figure 5.
TABLE 3 anti-invasion effect experiment result of naringenin on extracellular Toxoplasma gondii
From the values in Table 3 and FIG. 5, it can be seen that naringenin has a significant anti-invasion effect (P < 0.05) on Toxoplasma gondii type I strain RH at a concentration of 60. mu.g/mL or more and is dose-dependent. When the naringenin concentration is more than or equal to 70 mu g/mL, the effect of resisting the RH invasion of the toxoplasma I type insect strain is more excellent than that of pyrimethamine (Pyr) in a positive control group (P is less than 0.01). When the concentration of naringenin is 120 mug/mL, the invasion rate is only 3.55% (P is less than 0.001). The naringenin is shown to have obvious function of resisting the invading cells of the toxoplasma outside the cell and has obvious effect.
In addition, the inventor selects the pharmaceutically acceptable salts of naringenin, such as sodium salt and the like, and performs the pharmacological tests to obtain similar pharmacological effects, which shows that both the naringenin and the pharmaceutically acceptable salts thereof have the effect of treating or preventing toxoplasmosis.
The naringenin or naringenin salt is added with corresponding existing auxiliary materials to prepare any one dosage form of powder, tablets, capsules, pills, dripping pills, injections, emulsions, suspensions or tinctures, and the same pharmacological test is carried out, and the test results show that the naringenin has good effect of treating or preventing toxoplasmosis within the effective dosage range of the naringenin. The medicinal composition prepared from the Chinese herbal medicines can be added into animal feed and can be directly used for treating or preventing toxoplasmosis infected by animals.
Naringenin or naringenin salt is added into other existing anti-insect drugs, so that the naringenin or naringenin salt is used in combination, the anti-spectrum range of parasites is expanded, and the prevention and treatment effect on the parasitic diseases is further improved.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the present invention in any way, and it will be apparent to those skilled in the art that the above description of the present invention can be applied to various modifications, equivalent variations or modifications without departing from the spirit and scope of the present invention.
Claims (7)
1. Application of naringenin and its pharmaceutically acceptable salt in preparing medicine for treating or preventing toxoplasmosis is provided.
2. The use of naringenin and its pharmaceutically acceptable salts as claimed in claim 1 in the preparation of a medicament for treating or preventing toxoplasmosis, wherein the effective concentration of naringenin is 50-120 μ g/mL.
3. The use of naringenin and its pharmaceutically acceptable salts for the manufacture of a medicament for the treatment or prevention of toxoplasmosis according to claim 2, wherein the effective concentration of naringenin is 60 μ g/mL.
4. The application of a pharmaceutical composition in preparing a medicament for treating or preventing toxoplasmosis is characterized in that the pharmaceutical composition comprises naringenin or pharmaceutically acceptable salts of naringenin and pharmaceutically acceptable auxiliary materials.
5. The use of the pharmaceutical composition of claim 4 for the preparation of a medicament for the treatment or prevention of toxoplasmosis, wherein the pharmaceutical composition further comprises an anti-insect agent in combination with the naringenin or a pharmaceutically acceptable salt of naringenin.
6. The use of the pharmaceutical composition according to claim 4 or 5, for the preparation of a medicament for the treatment or prevention of toxoplasmosis, wherein the pharmaceutical composition is formulated as any one of a powder, a tablet, a capsule, a pill, a drop pill, an injection, an emulsion, a suspension, or a tincture.
7. Use of a pharmaceutical composition according to claim 6 for the preparation of a medicament for the treatment or prevention of toxoplasmosis, wherein the pharmaceutical composition is used as an animal feed additive.
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Cited By (1)
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| CN117045641A (en) * | 2023-10-11 | 2023-11-14 | 广东医科大学附属医院 | Application of stone-like chlorophyllin in preparing toxoplasma resistant medicine |
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| WO2001003681A2 (en) * | 1999-07-08 | 2001-01-18 | Prendergast Patrick T | Use of flavones, coumarins and related compounds to treat infections |
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| WO2001003681A2 (en) * | 1999-07-08 | 2001-01-18 | Prendergast Patrick T | Use of flavones, coumarins and related compounds to treat infections |
| JP2003504327A (en) * | 1999-07-08 | 2003-02-04 | プレンダーガスト、パトリック、ティ． | Use of flavones, coumarins and related compounds for the treatment of infectious diseases |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117045641A (en) * | 2023-10-11 | 2023-11-14 | 广东医科大学附属医院 | Application of stone-like chlorophyllin in preparing toxoplasma resistant medicine |
| CN117045641B (en) * | 2023-10-11 | 2024-01-23 | 广东医科大学附属医院 | Application of stone-like chlorophyllin in preparing toxoplasma resistant medicine |
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