Formulation drugs for blocking Toxoplasma gondii infection and preparation method thereof
Preparation of a combination of prescription drugs by the composition of kalein and azadirin has solved the problem of lack of effective and small side effects in the prior art to block the infection of Toxoplasma gondii, and achieved effective blocking of Toxoplasma gondii infection and low toxic side effects.
Patent Information
- Application Number
- CN202411148464.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-21
- Publication Date
- 2025-07-08
- Estimated Expiration
- 2044-08-21
AI Technical Summary
There is a lack of effective and minor side effects in the prior art that blocks Toxoplasma infection, especially for immunodeficient or immunosuppressed patients and pregnant women, Toxoplasma infection can be fatal, and existing drugs such as pyrimidine, sulfadiazine and spiromycin have toxic side effects.
The composition of kaleenol and azadirtin is prepared in a mass ratio of 2: (3 to 8), and is prepared into tablets, powders, oral liquids, capsules or granules. A group of prescription drugs are prepared through pharmaceutically acceptable auxiliary materials to block Toxoplasma infection.
It significantly improves the therapeutic effect on Toxoplasma infection, has few toxic and side effects, and has important practical application value.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biological medicine technology, and particularly to a compound drug for blocking Toxoplasma gondii infection and a preparation method thereof. Background Art
[0002] Toxoplasmosis is a natural zoonotic parasitic disease caused by Toxoplasma gondii. Toxoplasma gondii, also known as Toxoplasma, belongs to the phylum Protozoa and the class Sporozoasida, and is an opportunistic pathogenic protozoan that widely parasitizes all nucleated cells except red blood cells in humans and various animals.
[0003] Under normal circumstances of the body, Toxoplasma gondii infection is mostly latent. However, for immunodeficient or immunosuppressed individuals, such as patients with AIDS, organ transplantation, and malignant tumors, Toxoplasma gondii infection may be one of the lethal causes. Pregnant women have a relatively high probability of being infected with Toxoplasma gondii, and Toxoplasma gondii can be transmitted to the fetus through the placental barrier, resulting in miscarriage of pregnant women, developmental malformations, intellectual disabilities, encephalitis, and meningitis in infants. For animals, acute toxoplasmosis can cause fever, dyspnea, and exhaustion in piglets; stillbirth, miscarriage, or weak offspring in sows.
[0004] In recent years, with the continuous increase in clinical patients and the objective needs of related problems, the treatment of toxoplasmosis has attracted more and more attention from medical workers. At present, there is no specific drug for toxoplasmosis, and the existing pyrimethamine, sulfadiazine, and spiramycin all have varying degrees of toxic and side effects. The present invention intends to develop a new compound drug that can effectively block Toxoplasma gondii infection using plant-derived natural extracts. Summary of the Invention
[0005] The purpose of the present invention is to provide a compound drug for blocking Toxoplasma gondii infection and a preparation method thereof to solve the problems existing in the above-mentioned prior art. This compound drug can effectively block Toxoplasma gondii infection and has the advantage of small toxic and side effects, with important practical application value.
[0006] To achieve the above purpose, the present invention provides the following solutions:
[0007] The present invention provides a composition for blocking Toxoplasma gondii infection, including polydatin and azadirachtin, and the mass ratio of polydatin to azadirachtin is 2:(3 - 8).
[0008] Preferably, the mass ratio of polydatin to azadirachtin is 3:(7 - 12).
[0009] More preferably, the mass ratio of polydatin to azadirachtin is 3:7.
[0010] The present invention also provides the use of the above composition in the preparation of a formulated drug for blocking Toxoplasma gondii infection.
[0011] The present invention also provides a formulated drug for blocking Toxoplasma gondii infection, the active ingredient of which comprises the above composition.
[0012] Further, the formulated drug further comprises a pharmaceutically acceptable excipient.
[0013] Further, the excipient comprises a solubilizer, an emulsifier, a filler, a disintegrant, a flavoring agent, and / or a preservative.
[0014] Further, the dosage form of the formulated drug is a tablet, a powder, an oral liquid, an emulsion, a capsule, or a granule.
[0015] The present invention also provides a preparation method of the above formulated drug, which comprises the steps of uniformly mixing polydatin and azadirachtin, and then adding a pharmaceutical excipient to prepare the formulated drug.
[0016] The present invention discloses the following technical effects:
[0017] The present invention develops a formulated drug for blocking Toxoplasma gondii infection, and the active ingredient comprises polydatin and azadirachtin. The present invention finds through research that when polydatin and azadirachtin are compounded in a mass ratio within the range of 2:(3-8), they have a synergistic effect in blocking Toxoplasma gondii infection.
[0018] The formulated drug provided by the present invention can significantly improve the therapeutic effect on Toxoplasma gondii infection compared with a single agent. The formulated drug developed from plant-derived natural extracts by the present invention can effectively block Toxoplasma gondii infection, and has the advantages of low toxicity and side effects, and has important practical application value. Detailed Embodiments
[0019] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.
[0020] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.
[0021] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.
[0022] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the specification of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of this invention are obvious to those skilled in the art. The specification and examples of this invention are merely exemplary.
[0023] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.
[0024] The molecular formula of azadirachtin is C 35 H 44 O 16 , with the CAS number 11141-17-6, and the structural formula is as follows:
[0025]
[0026] The molecular formula of polydatin is C 20 H 22 O8, with the CAS number 65914-17-2, and the structural formula is as follows:
[0027]
[0028] The Toxoplasma gondii used in the following examples is the transgenic Toxoplasma gondii RH strain (RH-GFP) expressing green fluorescent protein, provided by the laboratory of Hu Xiaoyu at Hebei University.
[0029] Example 1
[0030] The MTT method was used to detect the cytotoxicity of azadirachtin and polydatin. The experimental groups and control groups of each drug were set up, and the specific method is as follows:
[0031] Vero cells in the logarithmic growth phase were pipetted into a single-cell suspension, and the cell suspension was added to a 96-well culture plate at an addition rate of 100 μL / well. After culturing for 24 h at 37 °C and 5% CO2, the supernatant was aspirated and discarded. Agents with final concentrations of 160, 80, 40, 20, 10, 5, 2.5, and 1.25 mg / L were added to different wells; the control group was replaced with an equal amount of solvent. After culturing at 37 °C and 5% CO2 for 20 h, 20 μL of 5 mg / mL MTT working solution was added to each well. After continued culturing for 4 h, the culture plate was taken out, and the supernatant was carefully aspirated and discarded. 200 μL of dimethyl sulfoxide was added to each well, and after shaking and mixing for 0.5 h, the OD value of each well was measured at a wavelength of 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and the average value of the duplicate wells was calculated.
[0032] Cell proliferation rate (%) = (OD value of experimental group - OD value of control group) / OD value of control group × 100%.
[0033] The results of the cytotoxicity experiment are shown in Table 1. The maximum dilution concentration with a cell proliferation rate greater than 90% is the drug safety concentration, and the in vitro anti-parasitic experiment is carried out within the drug safety concentration.
[0034] Table 1 Proliferation rate (%) of Vero cells at different drug concentrations (mg / L)
[0035]
[0036]
[0037] Example 2
[0038] 1. Experimental method
[0039] 1.1 Collection of Toxoplasma gondii tachyzoites
[0040] After resuscitating the RH strain in a 37 °C water bath and continuously passaging it 3 times, the parasites were collected by centrifugation. The parasites were resuspended in physiological saline to obtain a Toxoplasma gondii tachyzoite ascites suspension for the experiment.
[0041] 1.2 Virulence detection experiment
[0042] The Toxoplasma gondii tachyzoite ascites suspension was quantitatively dispensed into a cell culture plate, approximately 5×10 4 tachyzoites / well, and the virulence detection experiment was carried out using the direct contact killing method, and the corrected mortality of each treatment group was calculated.
[0043] The method for detecting the efficacy using the direct contact killing method is as follows:
[0044] The medicament solutions with different concentrations shown in Table 2 and the tachyzoite ascites suspension of Toxoplasma gondii were added to a multi-well culture plate at a volume ratio of 1:1, and then placed in an incubator at 37°C. After 20 h, 10 μL of 2% MTT working solution was added to each well, and the culture was continued for 4 h. The culture plate was taken out, centrifuged, and the supernatant was aspirated. 0.2 mL of dimethyl sulfoxide was added to each well to dissolve the MTT reduction product. The main wavelength was set at 490 nm and the secondary wavelength was set at 630 nm with an enzyme-labeled instrument to zero the blank control wells, and the absorbance OD values of each well in the experimental group were measured. The mortality rate and corrected mortality rate were calculated according to the following formulas:
[0045] Mortality rate = (OD value of control well - OD value of medicament-added well / OD value of control well) × 100%.
[0046] Corrected mortality rate = (mortality rate of treatment group - mortality rate of control group) / (1 - mortality rate of control group) × 100%.
[0047] 2.2 Statistical analysis
[0048] Analysis was performed using the SAS statistical analysis system to calculate the LC 50 , 95% confidence limit of single medicament and mixed medicaments with various ratios, and the CTC value of the mixed medicaments to judge the synergistic effect.
[0049] The CTC value of the mixed medicaments was calculated according to the following formula:
[0050] ATI = S / M × 100;
[0051] TTI = TI A ×P A +TI B ×P B ;
[0052] CTC = ATI / TTI × 100;
[0053] where, ATI——actual toxicity index of the mixed medicaments; S——LC 50 of the standard medicament, unit: mg / L; M——LC 50 of the mixed medicaments, unit: mg / L; TTI——theoretical toxicity index of the mixed medicaments; TI A ——toxicity index of medicament A; P A ——percentage content of medicament A in the mixed medicaments, unit: %; TI B ——toxicity index of medicament B; P B ——percentage content of medicament B in the mixed medicaments, unit: %.
[0054] Classification criteria for joint action: When CTC of the mixed medicaments ≥ 120, it shows a synergistic effect; when CTC ≤ 80, it shows an antagonistic effect; when 80 < CTC < 120, it shows an additive effect.
[0055] 2. Results
[0056] The inhibitory effects of azadirachtin, polydatin, and their mixture on Toxoplasma gondii were detected. The results are shown in Table 2. The results indicate that when polydatin and azadirachtin are used in combination and the mass ratio of the two is in the range of 2:(3 - 8), the CTC values are all greater than 120, showing a synergistic effect. Among them, when the mass ratio of polydatin to azadirachtin is in the range of 3:7, the CTC value reaches more than 170, and the synergistic effect presents the optimal result.
[0057] Table 2 Toxicity test results of each agent against Toxoplasma gondii
[0058]
[0059] Example 3
[0060] 1. Experimental method
[0061] 1.1 Construction of a mouse model of toxoplasmosis
[0062] Mice were infected with Toxoplasma gondii by intraperitoneal injection to construct a mouse model of toxoplasmosis. The injection volume was 1×10 3 tachyzoites of Toxoplasma gondii per mouse. The preparation method of the ascites of Toxoplasma gondii tachyzoites was the same as that in Example 2, and it was diluted with physiological saline to 5×10 3 tachyzoites of Toxoplasma gondii per mL. Each mouse was inoculated with 0.2 mL and mixed well before injection.
[0063] 1.2 Experimental grouping and drug administration method
[0064] The experiment was divided into an experimental group and a control group. Starting from 2 hours after infection, the mice in each group were administered drugs according to the drug administration method and dosage shown in Table 3 for 5 consecutive days, twice a day.
[0065] Table 3 Experimental grouping and drug administration method
[0066]
[0067]
[0068] 1.3 Index detection
[0069] The survival time, survival rate, and the number of ascites worms of the mice in each group were counted.
[0070] 2. Results
[0071] 2.1 Survival time and survival rate of mice
[0072] The survival time and survival rate of each group of mice are shown in Table 4. The results show that all the mice in the control group died within 8 days, and Experimental Group 3 had a better effect in protecting the survival of mice, with the survival rate of mice reaching 100%.
[0073] Table 4 Survival time and survival rate of each group of mice
[0074] Group Average survival time (d) Survival rate (%) Experimental group 1 12 60 Experimental group 2 18 85 Experimental group 3 20 100 Control group 7.8 0
[0075] 2.2 Results of counting the number of worms in the ascites of mice
[0076] The results of counting the number of worms in the ascites of each group of mice are shown in Table 5. The results show that the number of worms in the ascites of the control group was the largest on the 7th day after infection; the compound medicine of Experimental Group 3 could effectively inhibit the increase in the number of worms, and no worms were detected by microscopic examination of the ascites starting from the 10th day after infection.
[0077] Table 5 Results of counting the number of worms in the ascites of each group of mice
[0078] Group 3d 5d 7d 10d Experimental group 1 3864±704 6875±761 5987±734 1647±594 Experimental group 2 3627±651 5873±891 5437±857 979±138 Experimental group 3 1687±283 4581±521 2158±672 0 Control group 8517±861 654782±25243 350000±38347 -
[0079] The embodiments described above are only used to describe the preferred mode of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solution of the present invention shall fall within the protection scope determined by the claims of the present invention.
Claims
1. A composition for blocking Toxoplasma gondii infection, characterized in that, It includes polydatin and azadirachtin, and the mass ratio of the polydatin to the azadirachtin is 2:(3-8).
2. The composition according to claim 1, characterized in that, The mass ratio of the polydatin to the azadirachtin is 3:(7-12).
3. The composition according to claim 2, wherein The mass ratio of the polydatin to the azadirachtin is 3:
7.
4. Use of a composition according to any one of claims 1-3 in the preparation of a formulated drug for blocking Toxoplasma gondii infection.
5. A compound drug for blocking Toxoplasma gondii infection, characterized in that, The active ingredient includes the composition according to any one of claims 1-3.
6. The compound medicine according to claim 5, wherein The formulated drug further includes pharmaceutically acceptable excipients.
7. The compound medicine according to claim 6, wherein The excipients include solubilizers, emulsifiers, fillers, disintegrants, flavoring agents and / or preservatives.
8. The compound medicine according to claim 7, characterized in that, The dosage form of the formulated drug is tablets, powders, oral liquids, emulsions, capsules or granules.
9. A preparation method of the medicinal composition according to any one of claims 6-8, characterized in that, It includes the step of mixing polydatin and azadirachtin evenly and then adding pharmaceutical excipients to prepare the formulated drug.