Use of mangiferin in preparation of anti-toxoplasma drugs

Abstract

This invention relates to the field of biopharmaceutical technology, specifically to the application of mangiferin in the preparation of drugs against Toxoplasma gondii. In vitro plaque assays, adhesion assays, invasion assays, and proliferation assays have demonstrated that mangiferin significantly inhibits the adhesion, invasion, and intracellular proliferation of Toxoplasma gondii, with effects comparable to or better than sulfadiazine. Animal experiments show that mangiferin significantly prolongs the survival time of mice infected with Toxoplasma gondii, reduces the spleen index and tissue worm load, and alleviates pathological damage to the spleen. This invention provides a new drug option for the treatment of Toxoplasma gondii-related diseases.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to the application of mangiferin in the preparation of anti-Toxoplasma gondii drugs. Background Technology

[0002] Toxoplasma gondii can remain latent in the central nervous system for extended periods and can induce fatal encephalitis when the host's immune function is compromised. Currently, the disease burden of opportunistic infections related to Toxoplasma gondii is increasing. Among HIV / AIDS patients with co-infection with Toxoplasma gondii, approximately 25% to 60% may further develop toxoplasmic encephalitis. In organ transplant recipients, toxoplasmosis resulting from either primary infection or reactivation of latent infection has a high mortality rate.

[0003] Currently, the first-line clinical treatment for toxoplasmosis infection still primarily involves pyrimethamine combined with sulfonamides. However, this regimen has significant toxic side effects, including hematologic toxicities such as bone marrow suppression, which have even become a major factor limiting treatment continuity. Studies have shown that over 50% of research reports indicate treatment discontinuation or regimen adjustments due to adverse reactions. Common toxicities include bone marrow suppression, gastrointestinal reactions, and skin damage, and can further lead to serious adverse events such as aplastic anemia, Stevens-Johnson syndrome, and liver damage, thus significantly limiting its clinical application and patient compliance.

[0004] Therefore, developing novel anti-toxoplasmosis drugs with novel mechanisms of action, definite efficacy, and good safety is of great significance for improving the clinical treatment of toxoplasmosis and reducing the disease burden. Summary of the Invention

[0005] To address the above problems, this invention provides the application of mangiferin in the preparation of drugs against Toxoplasma gondii.

[0006] This invention is achieved through the following technical solution: Application of mangiferin in the preparation of drugs against Toxoplasma gondii.

[0007] Preferably, the drug has mangiferin as its sole active ingredient.

[0008] Preferably, the concentration of mangiferin in the drug is 160 μM to 320 μM.

[0009] Preferably, the drug further includes pharmaceutically acceptable pharmaceutical excipients.

[0010] Preferably, the pharmaceutically acceptable excipient is one or more of the following: diluent, disintegrant, precipitation inhibitor, flow aid, binder, dispersant, suspending agent, isotonic agent, thickener, emulsifier, preservative, and stabilizer.

[0011] Preferably, the pharmaceutically acceptable excipient is sodium carboxymethyl cellulose.

[0012] Preferably, the dosage form of the drug includes an oral preparation, an infusion, or an injection.

[0013] Preferably, the oral preparation is a suspension.

[0014] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention discloses for the first time the application of mangiferin in the preparation of anti-toxoplasmosis drugs, which broadens the pharmaceutical field of mangiferin and provides new active lead compounds and drug candidates for the treatment of toxoplasmosis.

[0015] (2) In vitro experiments showed that mangiferin could significantly inhibit plaque formation in Toxoplasma gondii. The number of plaques was significantly reduced at both 160 μM and 320 μM concentrations. The 320 μM concentration completely blocked plaque formation, and the relative plaque area was significantly lower than that of the sulfadiazine positive control group. P <0.0001).

[0016] (3) In vitro mechanism studies have shown that mangiferin can significantly inhibit the adhesion of Toxoplasma gondii to host cells. P <0.0001) and intrusion ( P <0.0001, and continuously inhibited the intracellular proliferation of Toxoplasma gondii within a time range of 12h to 48h. P The value <0.0001 indicates that mangiferin can exert its anti-Toxoplasma gondii effect through multiple processes such as adhesion, invasion, and proliferation.

[0017] (4) Animal experiments showed that mangiferin 200 mg / kg administered orally could significantly prolong the survival time of mice acutely infected with Toxoplasma gondii. P <0.01), reducing the spleen index ( P <0.01, alleviated pathological damage to spleen tissue, and significantly reduced the Toxoplasma gondii load in the blood, spleen, and brain tissue of mice. P <0.0001), the in vivo anti-Toxoplasma gondii effect was clear, and the high-dose group showed more significant anti-parasitic and tissue protection effects.

[0018] In summary, this invention provides new drug options and experimental evidence for the clinical treatment of toxoplasmosis. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a diagram illustrating the inhibition of Toxoplasma gondii plaque formation by mangiferin, as described in this invention. Figure 1 In the diagram, A is a representative image from the plaque experiment; B is a statistical graph of the number of plaques; C is a statistical graph of the relative plaque area; where ns indicates no statistically significant difference; **** indicates... P <0.0001.

[0021] Figure 2 This diagram illustrates the inhibition of Toxoplasma gondii adhesion and invasion by mangiferin, as described in this invention. Figure 2 In the figure, A is the statistical graph of mangiferin inhibiting Toxoplasma gondii adhesion; B is the statistical graph of mangiferin inhibiting Toxoplasma gondii invasion; where ns indicates no statistically significant difference; ** indicates P <0.01; **** indicates P <0.0001.

[0022] Figure 3 This diagram illustrates the inhibitory effect of mangiferin at different treatment times on Toxoplasma gondii proliferation. Figure 3 In the diagram, A represents the inhibitory effect of mangiferin treatment on Toxoplasma gondii proliferation after 12 hours; B represents the inhibitory effect of mangiferin treatment on Toxoplasma gondii proliferation after 24 hours; and C represents the inhibitory effect of mangiferin treatment on Toxoplasma gondii proliferation after 48 hours. Here, ns indicates no statistically significant difference; * indicates... P <0.05; *** indicates P <0.001; **** indicates P <0.0001.

[0023] Figure 4 The invention provides an illustration of how mangiferin prolongs the survival time of mice and alleviates splenic lesions. Figure 4 In the diagram, A represents the survival statistics of mice after mangiferin treatment; B represents the spleen index of mice after mangiferin treatment; and C represents the pathological analysis of paraffin-embedded spleen sections stained with hematoxylin and eosin (HE) after mangiferin treatment. Here, ns indicates no statistically significant difference; ** indicates... P <0.01.

[0024] Figure 5 The present invention provides a diagram showing how mangiferin reduces the Toxoplasma gondii load in blood, spleen, and brain tissue. Figure 5In the diagram, A shows the relative Toxoplasma gondii load in the erythrocyte-depleted blood of mice after mangiferin treatment; B shows the relative Toxoplasma gondii load in the spleen of mice after mangiferin treatment; C shows the relative Toxoplasma gondii load in the brain tissue of mice after mangiferin treatment; **** indicates... P <0.0001. Detailed Implementation

[0025] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0026] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention and in its specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0027] The beneficial effects of the present invention will be illustrated below through specific embodiments.

[0028] Mangiferin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0029] The RH strain of Toxoplasma gondii was preserved and provided by the Department of Pathogenic Biology, Henan University of Medical Sciences.

[0030] Sodium carboxymethyl cellulose (CMC-Na) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0031] Example 1: Effect of mangiferin on plaque formation in Toxoplasma gondii This embodiment aims to preliminarily evaluate the inhibitory effect of mangiferin on plaque formation by Toxoplasma gondii on host cell monolayers.

[0032] Human foreskin fibroblasts (HFF) were cultured at 4 × 10⁻⁶ cells per well. 3Cells were seeded at a density of 100 μL of complete DMEM medium in each well of a 96-well plate and allowed to adhere for 12 h. Cells were then treated with mangiferin at concentrations ranging from 10 μM to 2560 μM (concentration gradient: 10 μM, 20 μM, 40 μM, 80 μM, 160 μM, 320 μM, 640 μM, 1280 μM, 2560 μM). A DMSO solvent control group, a culture medium control group, and a cell-free blank control group were included. After 48 h of drug treatment, 10 μL of CCK-8 solution was added to each well, and incubation continued for 2 h. Absorbance at 450 nm was measured using a microplate reader (Thermo Fisher Scientific, USA), and cell viability was calculated. Results: The cytotoxicity of different concentrations of mangiferin to HFF cells was assessed using the CCK-8 assay. Cell viability remained above 90% when mangiferin concentration was ≤320 μM; cell viability dropped below 90% when concentration exceeded 320 μM. Based on the above results, two concentrations, 160 μM and 320 μM, were selected for subsequent in vitro experiments.

[0033] HFF cells were seeded in 6-well plates and cultured until complete confluence. After purifying RH strain Toxoplasma gondii tachyzoites, HFF cells were infected at a dose of 200 cells / well, with 3 replicates per group. The experiment was set up with 4 treatment groups: solvent control group (DMSO final dilution ratio of 1:1000 in the culture medium), 160 μM mangiferin group, 320 μM mangiferin group, and 100 μg / mL sulfadiazine (SD) positive drug group. The drug solution of each group was prepared in DMEM medium supplemented with 2% fetal bovine serum (FBS). Each group had 3 replicates, and the drug volume of each group was the same. The cells were incubated at 37℃ and 5% CO2 for 7 days. After observing the formation of obvious plaques, the plates were gently washed three times with PBS; fixed with 4% paraformaldehyde at room temperature for 15 min, and then washed three times with PBS; 2 mL of 0.5% crystal violet staining solution was added, and the plates were stained at room temperature for 60 min. The staining solution was then discarded and the plates were washed with running water. After the 6-well plates were allowed to dry naturally, images were taken with a scanner, and the number and area of ​​plaques in each well were analyzed using ImageJ software. Three biological replicates were performed.

[0034] In vitro plaque assays showed that, compared with the DMSO control group, the number of plaques formed by Toxoplasma gondii was significantly reduced after treatment with 160 μM and 320 μM mangiferin. P <0.0001), with no plaque formation observed in the 320 μM mangiferin treatment group. Compared to the SD positive control group, there was no significant difference (ns) in the number of plaques between the 160 μM and 320 μM mangiferin treatment groups. Figure 1 As shown in A and B in the diagram.

[0035] Further analysis of the relative plaque area revealed that, compared with the DMSO control group, the relative plaque area formed by Toxoplasma gondii was significantly reduced after treatment with 160 μM and 320 μM mangiferin. P <0.0001). Among them, the relative plaque area of ​​the 320 μM mangiferin treatment group was significantly lower than that of the SD positive control group ( P <0.0001), such as Figure 1 As shown in C.

[0036] Example 2: Effects of mangiferin on Toxoplasma gondii adhesion to host cells To investigate the possible mechanisms by which mangiferin inhibits plaque formation, this example examines its effect on the process of parasite adhesion to host cells. The experimental groupings in this example are the same as in Example 1.

[0037] HFF cells were seeded in 24-well culture plates and cultured until the cells completely fused to form a dense monolayer. The tachyzoites of the RH strain of *Toxoplasma gondii* were purified and cultured at 5 × 10⁻⁶ cells / well. 5 Cell monolayers were infected with a dose of 10 cells / well, and mangiferin or control culture medium of corresponding concentration was added simultaneously for grouping treatment, with 3 replicates per group; incubated at 4°C for 30 min. After incubation, pre-cooled PBS was slowly added along the well wall for gentle washing 3 times to remove free parasites that were not bound to the cells; then observed under a 40x inverted microscope, 10 fields of view were randomly selected from each coverslip, and the number of Toxoplasma gondii adhering to the cell membrane surface in each field of view was counted, and the biological replicates were performed three times.

[0038] Adhesion assay results showed that, compared with the solvent control group, the number of Toxoplasma gondii adhering to the SD treatment group did not change significantly (ns), while treatment with 160 μM and 320 μM mangiferin significantly reduced the number of Toxoplasma gondii adhering to the SD treatment group. P <0.0001), such as Figure 2 As shown in A in the diagram.

[0039] Example 3: Effects of mangiferin on Toxoplasma gondii invasion of host cells Based on the confirmed inhibition of adhesion, this embodiment further investigates whether mangiferin affects the parasite invasion process. The experimental groups in this embodiment are the same as in Embodiment 1.

[0040] HFF cells were seeded in 24-well plates and cultured until complete confluence. After purifying RH tachyzoites, they were labeled with 5 μmol / mL CFDA-SE. After incubation at 37°C for 30 min, the cells were washed three times with DMEM containing 2% FBS. The labeled cells were then counted at a density of 5 × 10⁻⁶ cells / well. 5HFF cells were infected with Toxoplasma gondii tachyzoites in three replicates per group. Simultaneously, appropriate concentrations of mangiferin or control medium were added, and the cells were incubated at 37°C with 5% CO2 for 2 hours to allow for complete parasite invasion. Cells were then gently washed three times with PBS to remove uninvaded parasites, and cultured in DMEM containing 2% FBS for 12 hours at 37°C with 5% CO2. Cells were then trypsinized and collected into 2 mL EP tubes, incubated at 2000 rpm for 5 min, the supernatant was discarded, and the pellet was resuspended in 500 μL of DMEM containing 2% FBS. The proportion of HFF cells invading by tachyzoites was analyzed by flow cytometry. A blank control (HFF cells only) was also included, and three biological replicates were performed.

[0041] Invasion experiment results showed that, compared with the solvent control group, the proportion of Toxoplasma gondii invasion in the SD treatment group did not change significantly (ns), while treatment with 160 μM or 320 μM mangiferin significantly reduced the proportion of Toxoplasma gondii invasion. P <0.01), such as Figure 2 As shown in B in the diagram.

[0042] Example 4: Effects of mangiferin on intracellular proliferation of Toxoplasma gondii To determine whether mangiferin can still inhibit intracellular proliferation of the invaded parasites, kinetic monitoring was conducted in this embodiment. The experimental groups in this embodiment are the same as in Example 1.

[0043] HFF cells were seeded into 12-well culture plates and cultured until 80% confluence was achieved. Rh-positive Toxoplasma gondii tachyzoites were purified and cultured at a rate of 1 × 10⁻⁶ cells per well. 6 Cells were infected with individual parasites. Simultaneously, appropriate concentrations of mangiferin or control culture medium were added, with three replicates per group. Samples were collected after 12h, 24h, and 48h of continuous drug treatment and analyzed by qPCR (target gene: Toxoplasma gondii). SAG1 Internal reference gene is human Actin (H-) Actin The intracellular Toxoplasma gondii proliferation level was detected and three biological replicates were performed.

[0044] Intracellular proliferation assays showed that mangiferin treatment for 12 h significantly inhibited the intracellular proliferation of *Toxoplasma gondii* after infection of HFF cells. Compared with the DMSO control group, the relative proliferation levels of *Toxoplasma gondii* in the 160 μM and 320 μM mangiferin treatment groups were significantly reduced. P <0.0001); among them, the Toxoplasma gondii proliferation level in the 320 μM mangiferin group was significantly lower than that in the SD positive control group ( P <0.0001), such as Figure 3 As shown in A in the diagram.

[0045] Even with treatment time extended to 24 h, the 160 μM and 320 μM mangiferin treatment groups still showed significant inhibitory effects on proliferation (both...). P <0.0001). Among them, the Toxoplasma gondii proliferation level in the 320 μM mangiferin group was further reduced, showing a significant difference compared to the SD positive control group, such as... Figure 3 As stated in B.

[0046] Even after 48 hours of treatment, mangiferin still significantly inhibited the intracellular proliferation of Toxoplasma gondii. P <0.0001), but there was no statistically significant difference (ns) between the 320 μM mangiferin group and the SD positive control group, suggesting that the proliferation inhibition effect of 320 μM mangiferin was still comparable to that of SD treatment at this time. Figure 3 As shown in C.

[0047] Example 5: Protective effect of mangiferin on mice acutely infected with Toxoplasma gondii and its effect on tissue load. After confirming the multi-faceted inhibitory effect of mangiferin on parasites in vitro, this embodiment further evaluates its in vivo efficacy in an animal infection model.

[0048] Fifty-six 8-week-old female BALB / c mice were purchased from Beijing SPAF Biotechnology Co., Ltd. After acclimatization, they were used to establish a Toxoplasma gondii RH strain intraperitoneal infection model. All mice were infected by intraperitoneal injection of 100 RH strain Toxoplasma gondii tachyzoites. Four hours after infection, the mice were administered the drug via gavage, and the intervention was continued for 7 days.

[0049] Mice were randomly divided into four groups: a sodium carboxymethyl cellulose (CMC-Na) solvent control group, a 50 mg / kg mangiferin group, a 100 mg / kg mangiferin group, and a 200 mg / kg mangiferin group. 0.5 g of CMCNa was dissolved in 100 mL of physiological saline to prepare a 0.5% CMCNa saline solution as the CMCNa solvent control solution. Simultaneously, mangiferin suspensions with concentrations of 5 mg / mL, 10 mg / mL, and 20 mg / mL were prepared using this solution as the solvent. The dosage for each group was calculated as 10 μL / g of mouse body weight.

[0050] The experimental detection indicators were set as follows: The survival status of mice was observed and recorded throughout the entire process, survival curves were plotted, and daily changes in mouse body weight were monitored (n=10); on day 7 post-infection, mouse body weight and spleen mass were measured, and the spleen index was calculated using the formula: Spleen Index = (Spleen weight / Mouse body weight) × 100%; peripheral blood samples treated with schizoaffected red blood cells were collected, and brain tissue was isolated. The relative Toxoplasma gondii load in blood, spleen, and brain tissue was detected using qPCR (n=4, target gene: Toxoplasma gondii). SAG1 The internal control is a gene-modified mouse. Actin (M- ActinThe spleen and brain tissue specimens were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) for histopathological observation and analysis (n=4).

[0051] Survival analysis showed that all mice in the CMC-Na solvent control group died on day 7 post-Toxoplasma gondii infection. Mice in the mangiferin 50 mg / kg and 100 mg / kg treatment groups all died on day 8 post-infection, with no significant prolongation of survival compared to the CMC-Na control group; however, all mice in the mangiferin 200 mg / kg treatment group died on day 15 post-infection, with a significantly longer survival time than the CMC-Na control group. P <0.01). The above results indicate that mangiferin has a dose-dependent protective effect against Toxoplasma gondii infection in mice, with a more significant effect at a dose of 200 mg / kg. Figure 4 As shown in A in the diagram.

[0052] Analysis of the spleen index in mice revealed that the spleen index of the 50 mg / kg and 100 mg / kg mangiferin treatment groups was not significantly different from that of the CMC-Na control group, while the spleen index of the 200 mg / kg mangiferin treatment group was significantly lower than that of the CMC-Na control group. P <0.01), such as Figure 4 As shown in B in the diagram.

[0053] Histopathological analysis of the spleen tissue showed significant structural abnormalities in the spleen of mice infected with Toxoplasma gondii, mainly manifested as dense lymphocyte aggregation, indistinct boundary between white and red pulp, and congestion of the red pulp. Compared with the CMC-Na control group, the spleen tissue structure of mice treated with mangiferin was improved, with reduced abnormal lymphocyte aggregation and red pulp congestion. The spleen tissue structure of mice treated with 200 mg / kg mangiferin was relatively intact, suggesting that mangiferin can alleviate spleen tissue damage caused by Toxoplasma gondii infection to some extent. Figure 4 As shown in C.

[0054] Further relative quantification of Toxoplasma gondii load in mouse blood, spleen, and brain tissue revealed that, compared with the CMC-Na solvent control group, the relative Toxoplasma gondii load in the blood, spleen, and brain tissue of mice treated with mangiferin was significantly reduced on day 7 post-infection. P <0.0001). The above results indicate that mangiferin treatment can significantly reduce the Toxoplasma gondii load in mice infected with Toxoplasma gondii, such as Figure 5 As shown.

[0055] The above results indicate that mangiferin has a significant inhibitory effect on the growth and development of Toxoplasma gondii, and plays a positive role in controlling the occurrence of toxoplasmosis.

[0056] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0057] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this invention should be determined by the appended claims.

Claims

1. Application of mangiferin in the preparation of drugs against Toxoplasma gondii.

2. The application as described in claim 1, characterized in that, The drug has mangiferin as its sole active ingredient.

3. The application as described in claim 2, characterized in that, The concentration of mangiferin in the drug is 160 μM to 320 μM.

4. The application as described in claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

5. The application as described in claim 4, characterized in that, The pharmaceutically acceptable excipients are one or more of the following: diluents, disintegrants, precipitation inhibitors, flow aids, binders, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, and stabilizers.

6. The application as described in claim 4, characterized in that, The pharmaceutically acceptable excipient is sodium carboxymethyl cellulose.

7. The application as described in claim 1, characterized in that, The dosage forms of the drug include oral preparations, infusions, or injections.

8. The application as described in claim 1, characterized in that, The oral preparation is a suspension.

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