Application of brucea javanica bitter alcohol in preparing medicine for treating malaria

Abstract

The invention discloses an application of brucea javanica bitter alcohol (BRU) in preparation of a medicine for treating malaria. The invention belongs to the technical field of biological medicine. According to the invention, a model that mice are infected with a Plasmodium berghei (Plasmodium berghei, pb) ANKA insect strain and a Plasmodium johnsonii (Plasmodium yoelii, py) 17XNL insect strain is established, and BRU in-vivo insecticidal effect observation is carried out. Observation indexes mainly comprise insect blood rate calculation, anemia condition, liver and kidney function influence, spleen form and insect body ultrastructure detection. Results show that the BRU (as shown in the formula I) has a remarkable effect of killing the plasmodium, and can directly damage the ultrastructure of the plasmodium body, block the development of the plasmodium body and remarkably improve the degree of mouse spleen damage caused by plasmodium infection so as to improve the anemia condition of the mouse. And the influence on liver and kidney functions is slight and reversible. Therefore, a new technical means is provided for malaria treatment, and the brucea javanica bitter alcohol has a wide application prospect in the aspect of treating parasitic infection. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a new use of brusatol (picras-3-en-21-oic acid, 13,20-epoxy-3,11,12-trihydroxy-15-[(3-methyl-1-oxo-2-buten-1-yl)oxy]), and in particular to the use of brusatol in the preparation of a drug for treating malaria. The present invention belongs to the field of biomedicine technology. Background Art

[0002] Malaria is a global acute parasitic infectious disease caused by Plasmodium and transmitted through mosquito bites. It has high morbidity and mortality rates among people around the world.

[0003] In 1820, Pelletier and Caventou of France isolated quinine and cinchonine from the bark of quia. In 1854, botanist Hasskarl transplanted cinchona seeds from Peru and Bolivia to Indonesia and planted them on a large scale. Quinine was mass-produced and became the world's best medicine for treating malaria. In 1944, Robert Burns Woodward and William Doering of the United States synthesized chloroquine and later developed primaquine. These synthetic drugs soon played an important role in treating malaria because they were more effective than quinine, had a long-lasting effect and few side effects. ICI (Imperial Chemical Industries) of the United Kingdom also developed drugs such as proguanil and pyrimethamine for the treatment and prevention of malaria, which are still the basic antimalarial drugs listed by the World Health Organization. However, with the intensification of drug use, malarial parasites have developed resistance to the most commonly used chloroquine, and chloroquine-resistant malignant malaria is facing a situation where there is no cure. In 1969, Tu Youyou joined the 523 antimalarial drug research project and isolated an effective antimalarial monomer, artemisinin. Later, Chinese scientists developed a series of antimalarial drugs such as artesunate, artemether, and dihydroartemisinin based on artemisinin. Artemisinin drugs have attracted the attention of the international community for their high efficiency, low toxicity, and outstanding efficacy against chloroquine malarial parasites. With the widespread use of artemisinin, malarial parasites have become less sensitive to artemisinin, and even in Africa, a high-incidence area for malaria, artemisinin resistance has emerged. The World Health Organization has completely banned the use of single artemisinin and adopted artemisinin-based combination therapies (ACTs) as the first-line treatment method.

[0004] Brusatol (picras-3-en-21-oic acid, 13,20-epoxy-3,11,12-trihydroxy-15-[(3-methyl-1-oxo-2-buten-1-yl)oxy]) belongs to the quassin lactone compounds and is an important active ingredient extracted from Brucea brucea. Brucea brucea was first recorded in "Supplement to Materia Medica" and is also known as Sophora flavescens, Old Crow Gallbladder, Bitter Stick, Crow Egg, Duck Egg, Duck Gallbladder, and Small Melia. It is bitter and cold in nature and has a slight toxicity. It has the functions of clearing heat, stopping dysentery, stopping malaria, drying dampness, and killing insects. It is mainly used for dysentery, bleeding, malaria, cold and heat. As a traditional Chinese herbal medicine, Brucea brucea is widely used among the people and has been an anti-malarial medicine since ancient times. With the continuous in-depth study of the chemical components of Brucea brucea, researchers have gradually determined that the main active ingredients in Brucea brucea are brucein A, brucein alcohol, and brucein lactone. Among them, brucein alcohol is composed of a C8-CH2-OC 13 A tetracyclic triterpenoid compound composed of a five-membered oxygen ring, three six-membered rings A, B, and C, and a lactone ring (D ring). Studies have shown that the C-2 enolate oxygen and unsaturated carbonyl oxygen of brucea brucea and the β-hydroxyl group at C-11 are important active sites, and the ester alkoxy side chain at C-21 has a slight effect on the activity. Modern pharmacological studies believe that brucea brucea has antibacterial, antiviral, anticancer, anti-peptic ulcer, and lipid-lowering effects. However, there are no reports on whether brucea brucea has a therapeutic effect on mice infected with Plasmodium and its safety.

[0005] The present invention studies the effect of brucein on mice infected with malarial parasites, and finds that brucein can directly damage the ultrastructure of the parasite, block the development of malarial parasites, significantly improve the degree of spleen damage in mice caused by malarial parasite infection, and then improve the anemia of mice, thereby achieving the purpose of killing malarial parasites and treating malaria. Therefore, it can be used as a new type of antimalarial drug for the prevention and treatment of malaria. Summary of the invention

[0006] The purpose of the present invention is to provide the use of brusatol (Picras-3-en-21-oic acid, 13,20-epoxy-3,11,12-trihydroxy-15-[(3-methyl-1-oxo-2-buten-1-yl)oxy]) in treating malaria.

[0007] In order to achieve the above purpose, the present invention adopts the following technical means:

[0008] The present invention constructs a malaria animal model by establishing mice infected with Plasmodium yoelii and Plasmodium berghei. Four hours after infection with Plasmodium, each group of mice is intraperitoneally injected with brucea brucei (4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg), artesunate (140 mg / kg) and normal saline for four consecutive days. The results show that brucea brucei can effectively inhibit Plasmodium infection, which is equivalent to the effect of artesunate, and can control the parasite blood rate below 5%. The optimal effective dose is 2 mg / kg, and significantly improves anemia and spleen damage in infected mice. That is, the present invention has found through research that brusatol (BRU) can treat Plasmodium-infected mice and has an antimalarial effect.

[0009] Therefore, based on the above research, the present invention proposes the use of brusatol (BRU) in the preparation of drugs for treating malaria. The chemical structure of brusatol (Picras-3-en-21-oic acid, 13,20-epoxy-3,11,12-trihydroxy-15-[(3-methyl-1-oxo-2-buten-1-yl)oxy]) is shown in Formula I:

[0010]

[0011] Preferably, the malaria is caused by infection with Plasmodium berghei (pb) or Plasmodium yoelii (py).

[0012] Among them, preferably, the brucea javanica alcohol blocks the development of Plasmodium by directly damaging the ultrastructure of the parasite, significantly improves spleen damage caused by Plasmodium infection, and further improves the anemia of the patient, thereby achieving the purpose of killing Plasmodium and treating malaria.

[0013] Preferably, the drug is an injectable drug.

[0014] In addition, the present invention also proposes the use of brusatol (BRU) in the preparation of a drug or reagent for inhibiting the growth of Plasmodium. The chemical structure of the brusatol (Picras-3-en-21-oic acid, 13,20-epoxy-3,11,12-trihydroxy-15-[(3-methyl-1-oxo-2-buten-1-yl)oxy]) is shown in Formula I:

[0015]

[0016] Preferably, the malarial parasite is Plasmodium berghei (pb) or Plasmodium yoelii (py).

[0017] Preferably, the brucea javanica directly damages the ultrastructure of the parasite and blocks the development of the parasite to achieve the purpose of inhibiting the growth of the parasite.

[0018] Preferably, the drug or reagent is a drug or reagent that inhibits the growth of Plasmodium in vitro.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention proposes a new drug for treating malaria, brucein, which can be used for the treatment of malaria. As a unique Nrf2 pathway inhibitor, brucein is widely used in the field of tumor research. A single intraperitoneal injection of 2 mg / kg can significantly inhibit tumor growth. By screening the data related to effective insecticidal dose and liver and kidney toxicity, the present invention found that brucein at a dose of 2 mg / kg / d, intraperitoneal injection, and continuous use for 4 days can achieve a similar effect of killing malarial parasites as artesunate, thereby achieving the purpose of treating malaria. Therefore, the proposal of the present invention provides a new treatment direction for the treatment of malaria, and will also give brucein a broad application prospect in the field of anti-parasitic infection diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The insecticidal effect of different concentrations of bruceiol on mice infected with Plasmodium;

[0022] Among them, A: The insecticidal effect of different doses of brucei alcohol on mice infected with Plasmodium berghei; B: Brucei alcohol on thin blood smears of mice infected with Plasmodium berghei (a: normal control group (Control); b: model control group (Model); c: ART 140mg / kg; d: BRU 2mg / kg; magnification: 1000×); C: The effect of brucei alcohol on the parasite blood rate of mice infected with Plasmodium berghei; D: Brucei alcohol on thin blood smears of mice infected with Plasmodium yoelii (a: normal control group; b: model control group; c: ART 140mg / kg; d: BRU 2mg / kg; magnification: 1000×); E: The effect of brucei alcohol on the parasite blood rate of mice infected with Plasmodium yoelii, indicating that brucei alcohol has a killing effect on different types of Plasmodium;

[0023] Figure 2 The effect of brucein on liver and kidney function of experimental mice;

[0024] Among them, AST: aspartate aminotransferase; ALT: alanine aminotransferase; TP: total protein; ALB: albumin; GLOB: globulin; CREA: creatinine; UREA: urea, indicating that brucein has a mild toxicity to liver and kidney damage, and all indicators returned to normal after drug withdrawal; brustol: brucein; artesunate: artesunate;

[0025] Figure 3 The effect of brucein on the ultrastructure of Plasmodium falciparum;

[0026] Among them, A: Model control group (Model, 60000×); B: Artesunate treatment group (ART, 20000×); C: Brucea bruceiol treatment group (BRU, 25000×); Note: The red arrow is the nucleus; the yellow arrow is the surface membrane; the blue arrow is the ribosome; the green arrow is the apex;

[0027] Figure 4 To investigate the effects of bruceiol on red blood cells (RBC) and hemoglobin (HBG) in mice infected with Plasmodium falciparum.

[0028] Among them, A: The effect of brucei alcohol on RBC and HGB of mice infected with Plasmodium berghei; B: The changes of RBC and HGB of mice infected with Plasmodium yoelii after brucei alcohol treatment;

[0029] Figure 5 Effects of bruceiol on spleen morphology in mice infected with Plasmodium berghei;

[0030] Among them, A: percentage change of spleen coefficient in BRU, ART, Control, and Model groups; B: gross morphology of spleen in BRU, ART, Control, and Model groups; C: pathological changes of spleen under HE staining in BRU, ART, Control, and Model groups; red arrows: malarial pigment deposition;

[0031] Figure 6 Effects of bruceiol on spleen morphology in mice infected with Plasmodium yoelii;

[0032] Among them, A: percentage change of spleen coefficient in BRU, ART, Control, and Model groups; B: gross morphology of spleen in BRU, ART, Control, and Model groups; C: pathological changes of spleen under HE staining in BRU, ART, Control, and Model groups; red arrows: malarial pigment deposition;

[0033] In the above figures, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with specific examples, and the advantages and features of the present invention will become clearer as the description proceeds. However, these examples are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0035] Example 1 Application of Brucea bruceiol in treating mice infected with Plasmodium

[0036] 1. Animals and experimental reagents:

[0037] Mice (strains: C57BL6 / J, BALB / c, purchased from Liaoning Changsheng Biotechnology Co., Ltd.);

[0038] Experimental parasite strains: (P.berghei ANKA strain, P.yoelii 17XNL).

[0039] 2. Establishment of Plasmodium infection mouse animal model:

[0040] Mouse model of Plasmodium berghei infection: 56 healthy BALB / c male mice (weight 22-25 g) were intraperitoneally injected with 0.2 ml of infected blood (containing approximately 1×10 7 The mice were randomly divided into 7 groups, 8 in each group. 4 hours after infection with Plasmodium berghei, brucei alcohol (BRU, 4mg / kg, 3mg / kg, 2mg / kg, 1mg / kg, 0.5mg / kg) and artesunate (ART, 140mg / kg) were injected intraperitoneally for 4 consecutive days (from 0th to 3rd day), and the model control group (Model) was injected with normal saline intraperitoneally. At the same time, healthy mice that were not given any drugs were used as the normal control group (Control). On the 4th day, the tail tip blood of the mice was collected, and the blood rate of the parasites was observed and counted by oil microscopy after Giemsa staining; the whole blood was collected and centrifuged, the serum was subjected to biochemical detection, and the red blood cell mass was observed by electron microscopy; the spleen was sterilely taken for morphological observation.

[0041] Plasmodium yoelii infection mouse model: 56 healthy C57BL / 6J mice (weight 22-25 g) were intraperitoneally injected with 0.2 ml of infected blood (containing approximately 1×10 7 yoelii infected red blood cells), and the rest were infected with Plasmodium berghei as mentioned above.

[0042] 3. Results

[0043] 3.1 Brucea brucei can reduce the parasite blood rate in mice infected with Plasmodium

[0044] After mice were infected with Plasmodium, they were intraperitoneally injected with different concentrations of brucei alcohol (4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, and 0.5 mg / kg), and compared with the model control group (Model) and the artesunate treatment group (ART, 140 mg / kg). The results of the four-day continuous inhibition experiment showed that compared with the model control group, brucei alcohol could significantly reduce the parasite blood rate, and 2 mg / kg was the most ideal dose for treating mice infected with Plasmodium berghei. When the parasite blood rate was lower than 5%, the insecticidal effect of brucei alcohol at a dose of 2 mg / kg on Plasmodium berghei was similar to that of 140 mg / kg of artesunate. The results are as follows: Figure 1 shown.

[0045] 3.2 Brucea bruceiol has drug safety

[0046] After mice were infected with Plasmodium, they were given bruceiol (BRU, 2 mg / kg), artesunate (ART, 140 mg / kg) or normal saline by intraperitoneal injection for 4 consecutive days. Serum was collected on the 4th and 28th days of the experiment to detect changes in AST, ALT, TP, ALB, GLOB, CREA and UREA in the serum. The experimental results showed that on the 4th day of the experiment, compared with the model control group (Model) and the artesunate group (ART), the AST, ALT, CREA and UREA in the serum of the bruceiol group were significantly increased, and TP, ALB and GLOB were significantly decreased. On the 28th day of the experiment, there was no statistical difference in the above indicators among the bruceiol group, artesunate group and control group. The results are as follows Figure 2 shown.

[0047] 3.3 Effects of bruceiol on the ultrastructure of Plasmodium berghei

[0048] After mice were infected with Plasmodium, they were intraperitoneally injected with brucea rutin (BRU, 2 mg / kg), artesunate (ART, 140 mg / kg) or saline for 4 consecutive days. The whole blood of the mice was collected, and the red blood cells were harvested, washed, fixed, embedded and stained, and then observed under a transmission electron microscope. Figure 3 As shown in the figure, at the ultrastructural level, the model control group (Model), i.e., normal Plasmodium berghei without drug intervention, has a complete oval body in the schizont stage, with a bulge visible on the surface as the apex of the body, a clear surface membrane consisting of two layers of inner and outer membranes, a large number of ribosomes in the cytoplasm, ribosomes around a single nucleus, coarse and dense heterochromatin and scattered granular and dense euchromatin in the nucleus, and the nucleolus is not obvious ( Figure 3 A); After intervention with brucein, the membrane integrity of the worm body was lost, the endoplasmic reticulum was not obvious, the number of ribosomes was significantly reduced, and the nuclear condensation and dissolution were obvious ( Figure 3C), which has the same effect as artesunate (ART) ( Figure 3 B). This indicates that bruceiol blocks the development of Plasmodium berghei and has a significant killing effect on Plasmodium berghei.

[0049] 3.4 Brucea brucei can improve anemia in mice infected with Plasmodium berghei and Plasmodium yoelii

[0050] After mice were infected with Plasmodium, they were intraperitoneally injected with brucea rutin (BRU, 2 mg / kg), artesunate (ART, 140 mg / kg) or saline for 4 consecutive days. The anticoagulated blood was collected from the mice and the changes of RBC (red blood cell count) and HGB (hemoglobin) were detected by blood routine instrument. Figure 4 As shown, compared with the model control group (Model), brucein (BRU) can significantly increase the RBC and HGB of infected mice, and its effect is basically the same as that of artesunate (ART). It further shows that brucein can significantly improve the anemia caused by malaria.

[0051] 3.5 Brucea brucei can alleviate spleen damage in mice infected with Plasmodium berghei and Plasmodium yoelii

[0052] After mice were infected with Plasmodium, bruceiol (BRU, 2 mg / kg), artesunate (ART, 140 mg / kg) or saline was intraperitoneally injected into the experimental animals for 4 consecutive days, and the spleens of the mice were collected. Figure 5 , 6 As shown, the spleen of the brucea brucei alcohol group (BRU) was significantly smaller than that of the model control group (Model), with a significant difference ( Figure 5 A. Figure 6 A); The gross morphology of the spleen ( Figure 5 B. Figure 6 B), the spleen of the model control group was significantly enlarged and congested with pigmentation, while the spleen size of the brucea rutin group (BRU) was similar to that of the normal spleen, and the pigmentation and spleen congestion were significantly reduced; the pathological HE staining results showed that the spleen of normal mice was Figure 5 As shown in the Control group in C and 6C, a large number of red pulps can be seen, the white pulp is wrapped by the marginal zone, the boundary between the red pulp and the white pulp is clearly visible, the lymphocytes in the white pulp are dense, and the red pulp is mainly composed of red blood cells and a small number of lymphocytes, and the cell morphology is relatively regular. Figure 5 C, 6C shows that the boundary between the red pulp and the white pulp disappeared, the red pulp was infiltrated by lymphocytes, the cell morphology was changed and irregular, and brown malarial pigment was deposited in the red pulp. Figure 5As shown in the ART group and BRU group in C and 6C, the boundary between the red pulp and the white pulp is clearly visible, the lymphocytes in the white pulp are dense, and the cell morphology in the red pulp is relatively regular, similar to the normal group. The above results show that brucein can significantly improve the spleen damage of mice caused by Plasmodium infection.

Claims

1. Use of brusatol (BRU) in the preparation of a drug for treating malaria, wherein the brusatol (Picras-3-en-21-oic acid, 13,20-epoxy-3,11, The chemical structure of 12-trihydroxy-15-[(3-methyl-1-oxo-2-buten-1-yl)oxy]) is shown in Formula I:

2. The use according to claim 1, characterized in that: The malaria is caused by infection with Plasmodium berghei (pb) or Plasmodium yoelii (py).

3. The use according to claim 1 or 2, characterized in that: The brucea javanica alcohol blocks the development of malarial parasites by directly damaging the ultrastructure of the parasite, significantly improves spleen damage caused by malarial parasite infection, and further improves the anemia of the patient, thereby achieving the purpose of killing malarial parasites and treating malaria.

4. The use according to claim 1 or 2, characterized in that: The medicine is an injectable medicine.

5. Use of brusatol (BRU) in the preparation of a drug or reagent for inhibiting the growth of Plasmodium, wherein the brusatol (Picras-3-en-21-oic acid, 13,20-epoxy-3,11, The chemical structure of 12-trihydroxy-15-[(3-methyl-1-oxo-2-buten-1-yl)oxy]) is shown in Formula I:

6. The use according to claim 5, characterized in that: The malarial parasite is Plasmodium berghei (pb) or Plasmodium yoelii (py).

7. The use according to claim 5, characterized in that: The brucea brucea alcohol directly damages the ultrastructure of the parasite and blocks the development of the parasite to achieve the purpose of inhibiting the growth of the parasite.

8. The use according to claim 5, characterized in that: The medicine or reagent is a medicine or reagent for inhibiting the growth of malarial parasites in vitro.

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