CN111671744B - Application of myricetin in preparation of medicine for treating schistosomiasis - Google Patents

Application of myricetin in preparation of medicine for treating schistosomiasis Download PDF

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CN111671744B
CN111671744B CN202010130233.9A CN202010130233A CN111671744B CN 111671744 B CN111671744 B CN 111671744B CN 202010130233 A CN202010130233 A CN 202010130233A CN 111671744 B CN111671744 B CN 111671744B
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myricetin
schistosoma japonicum
liver
schistosomiasis
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CN111671744A (en
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吕志跃
黄萍
成韶芸
胡玥
周旻昱
周洪利
马玉斌
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Sun Yat Sen University
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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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Abstract

The invention discloses an application of myricetin in preparing a medicament for treating schistosomiasis. The invention discovers that the myricetin has good schistosome disinsection effect no matter in vivo or in vitro and improves the hepatic fibrosis of the mouse infected by the schistosoma japonicum through in vitro observation of the schistosoma japonicum anti-schistosoma japonicum effect of the myricetin and establishment of a mouse model infected by the schistosoma japonicum for in vivo experiments. In addition, the insecticidal effect of myricetin is equivalent to the positive drug praziquantel, and the myricetin can replace the application of myricetin in schistosomiasis japonica, provides possibility for avoiding clinical drug resistance, and has a wide application prospect.

Description

Application of myricetin in preparation of medicine for treating schistosomiasis
Technical Field
The invention relates to the technical field of biology, in particular to the technical field of prevention and treatment of schistosomiasis japonica, and more particularly relates to a new application of myricetin in preparation of drugs for treating schistosomiasis japonica.
Background
Schistosomiasis is prevalent in 78 countries and regions worldwide as a neglected tropical disease, with 7.79 million people threatened, with a conservative estimate of about 2.30 million people infected, with 1.2 million people symptomatic, resulting in nearly 30 million deaths annually in africa in the south of sahara alone from schistosomiasis. Schistosomiasis is a parasitic disease caused by a parasite of the genus schistosoma, and there are mainly six human-related species of schistosomiasis organisms, of which schistosoma mansoni, schistosoma japonicum and schistosoma japonicum are the most prevalent. The average life span of schistosoma in the host is 3-10 years, and the inhabitation time can even be as long as 40 years in some cases. Although schistosoma japonicum cannot proliferate in the final host, it can produce large numbers of eggs deposited in the liver or other organs, the mature female worms will lay hundreds and thousands of eggs every day, long term parasitism and large numbers of eggs cause infection and disease transmission.
In the absence of an effective vaccine, chemotherapy is an important means of preventing and treating schistosomiasis today. The first choice of drug currently used in the clinic is praziquantel, which is effective against all species of schistosomes infecting humans and has low toxic side effects. However, the application of praziquantel has many limitations, because the praziquantel is only effective to early childhood insects (3-6 h) and adults just penetrating into the skin, but has weak effect on the childhood insects, has no preventive effect, and is a big problem in blood prevention work due to repeated infection of a treated person after treatment. Meanwhile, because the long-term large-scale repeated use of the praziquantel in the circulation area easily causes the drug resistance of schistosoma, british scholars adopt sub-dose praziquantel to treat infected mice in a laboratory to induce schistosoma mansoni praziquantel resistant strains, so that the search for an effective drug capable of replacing the praziquantel is very urgent.
Myricetin of formula C 15 H 10 O 8 The compound is a natural flavonol compound widely existing in a plurality of natural plants, fruits and vegetables, has wide pharmacological activities including oxidation resistance, tumor resistance, anti-inflammation, antimicrobial, antiallergic, cardiovascular and neuron protection and the like, has small toxic and side effects, and does not have any toxic effect or death after being injected into the abdominal cavity of a mouse by 1000mg/kg of myricetin. In addition, it has been shown that dihydromyricetin can reduce CCl 4 Protective effect of induced mouse liver fibrosis lesion (37021manyuan, luoming, jialei, et al. dihydromyricetin on lipid peroxidation injury of liver fibrosis rat [ J]Chinese medicine, 2009, 6 (18): 26-28.); dihydromyricetin has obvious therapeutic action on schistosomiasis hepatic fibrosis (Zhuilong, wangjunjie, chenmeizi, et al. Experimental research of dihydromyricetin for treating schistosomiasis hepatic fibrosis [ J]Chinese clinical pharmacology and therapeutics, 2010,15 (4): 381-384.). At present, only dihydromyricetin is found to have a treatment effect on organ pathological injury of schistosomiasis-induced hepatic fibrosis after schistosomiasis infection, but no report is found on the effect of myricetin in treating schistosomiasis and the effect of myricetin in schistosomiasis-induced hepatic fibrosis.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a new application of myricetin in preparing a medicament for treating schistosomiasis.
The above object of the present invention is achieved by the following technical solutions:
according to the invention, firstly, the anti-schistosoma japonicum effect of the myricetin is observed in vitro, the myricetin is found to have a good in vitro anti-schistosoma japonicum effect, a schistosoma japonicum infected mouse model is further established to carry out in vivo experiments, the myricetin has a good schistosoma japonicum insecticidal effect no matter inside and outside a body, the myricetin can effectively inhibit the egg laying amount of the schistosoma japonicum, the inhibition effect is not obviously different from that of a positive medicament praziquantel, and the myricetin and the praziquantel can effectively reduce liver worm egg deposition, so that the myricetin can reduce the infection of pathogenic worms from the source, and the effect of treating schistosomiasis is achieved. Meanwhile, the myricetin is found to have the improvement effect on the liver fibrosis of mice infected with schistosoma japonicum. The insecticidal effect of myricetin is equivalent to that of a positive drug praziquantel, and the myricetin can replace the application of myricetin in schistosomiasis japonica, provides possibility for avoiding clinical drug resistance and has a great application prospect.
Therefore, the following applications of myricetin are all within the scope of the present invention:
application of myricetin in preparing medicine for treating schistosomiasis is provided.
Application of myricetin in preparing medicine for inhibiting schistosomiasis is provided.
Application of myricetin in preparation of medicine for inhibiting oviposition of schistosome
The myricetin is used in preparing medicine for reducing the load of schistosome egg and liver egg in host infected by schistosome.
Application of myricetin in preparing medicine for improving liver disease injury caused by schistosome is provided.
Preferably, the pathological injury to the liver is liver fibrosis.
Preferably, the schistosoma japonicum is schistosoma japonicum.
The invention also provides a medicament for treating schistosomiasis, which contains myricetin.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides an application of myricetin in preparing a medicament for treating schistosomiasis. The research of the invention finds that the myricetin can have good anti-schistosoma japonicum effect, and the in-vivo experimental result proves that the myricetin obviously reduces the load of eggs and liver eggs in mice infected with schistosoma japonicum, and obviously improves the liver fibrosis lesion of the mice; the myricetin can be used for treating schistosomiasis japonica in the early stage, and has a treatment and improvement effect on liver pathological injury caused by the attack of schistosomiasis japonica in the later stage. Moreover, the myricetin has the effect equivalent to that of the positive drug praziquantel, can replace the application of the praziquantel in the diseases, and provides possibility for avoiding clinical drug resistance.
Drawings
FIG. 1 is the chemical structural formula of myricetin and its influence on Schistosoma japonicum; FIG. 1A is the structure of myricetin compound, and FIG. 1B is the survival curve of Schistosoma japonicum after the myricetin with different concentrations acts.
FIG. 2 is a scanning electron micrograph of Schistosoma japonicum after the action of myricetin (A, male worm; B, female worm; oral sucker; ventral sucker; tegument; gynecophoral canal).
FIG. 3 is H & E staining pattern of mouse liver infected with Schistosoma japonicum after myricetin action.
FIG. 4 is a Masson staining pattern of the liver of mice infected with Schistosoma japonicum after myricetin action.
FIG. 5 shows the expression of factors related to liver fibrosis of mice infected with Schistosoma japonicum after myricetin action.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. The reagents, methods and apparatus employed in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 in vitro insecticidal Effect assay of Myricetin
The preparation of the medicine comprises the following steps: myricetin was formulated with DMSO to a final concentration of 10 μ M.
Construction of a Schistosoma japonicum infected New Zealand rabbit model:
placing Schistosoma japonicum positive Oncomelania in 12-well plate at room temperature of 25 + -1 deg.C, adding dechlorinated water to 2/3 of the position, placing under incandescent lamp, irradiating for 2h to escape cercaria, removing animal abdominal skin, moistening skin with dechlorinated water, sticking the counted cercaria cover glass on the dehaired skin, and removing the glass after 20 min. New Zealand rabbits are infected with 1000-1200 cercaria/rabbit.
New Zealand rabbits were treated at 8 weeks after infection by heart blood sampling, killed by air embolism, dissected to collect adult Japanese blood fluke settled in inferior mesenteric vein and hepatic portal vein, washed with physiological saline and transferred to 24-well plate with 3 pairs of adult fluke per 1ml of complete medium (RP1640 MI medium containing 100U/ml penicillin, 100. Mu.g/ml streptomycin and 10% heat inactivated serum) at 37 ℃ and 5% CO 2 After the incubation in the incubator for 4 hours, adding myricetin (300-1000 mu M) with different concentrations, simultaneously taking 100 mu M praziquantel and 1% DMSO as a positive control and a negative control respectively, and observing the living state of the polypide under an inverted microscope during incubation for 24 hours, 48 hours and 72 hours; the results are shown in FIG. 1.
The results in FIG. 1 show that the negative control group survived 80% after 96h in vitro culture; all the insects die after being acted by positive control medicament praziquantel or myricetin (1000 mu M) for 8 hours. Myricetin acting on 24h IC of schistosoma japonicum 50 At 600 μ M, it was time and dose dependent on killing of adults in vitro. Collecting Schistosoma japonicum adults with a lethal dose of myricetin for 24h, and observing the damage condition of the schistosoma japonicum adults by a scanning electron microscope and a transmission electron microscope; the results are shown in FIG. 2.
FIG. 2 shows the result of normal worm sucking disc margin, male skin layer with spongy fold ridges and pits, and single hair ball shaped sensory papillae distributed on the body skin layer; the normal female body wall is in a regular strip shape, and depressions are distributed on the normal female body wall and are lined with sensory papillae. After the action of myricetin, the sucking discs of the male insect mouths and the abdomens are inwards sunken, the edges are swelled, the surface is irregularly contracted, the disc-shaped bulges are deformed, the folds on the surface of the cortex layer are arranged in a disordered way, the cilia on the mastoid are lost, and the mastoid is felt to be collapsed or disappear; the female insect mouth abdominal sucker edge is necrotic and burst, with the adhesion of a block, the cortical fold ridges in the middle section and the tail part of the insect body are fused into small pieces, and the mastoid is reduced, which shows that the myricetin can damage the integrity of the schistosoma japonicum body wall, and the myricetin has good function of resisting the schistosoma japonicum adult.
Example 2 Myricetin is effective in reducing the number of adult mice infected with Schistosoma japonicum and improving liver fibrosis lesions
1. Japanese blood fluke infected mouse model establishment
(1) Balb/c mice were randomly divided into four groups, namely a normal control group (ctrl), an infected group (infested), a praziquantel-treated group (pzq-treated) and a myricetin-treated group (me-treated), with 8 mice per group.
(2) Except for the normal control group, the other mice were infected with 30. + -. 2 pieces of Schistosoma japonicum cercaria as described above.
(3) At 36-42 days after infection, the praziquantel treatment group and the myricetin treatment group respectively irrigate the stomach of the mice with 500mg/kg of praziquantel and 250mg/kg of myricetin (prepared by 0.9 percent of normal saline) every day, and irrigate the stomach of the other two groups with normal saline with the same volume.
2. And (3) after 7 weeks of schistosoma japonicum infection, carrying out orbital bleeding after anesthetizing a mouse by intraperitoneal injection of pentobarbital sodium, perfusing the heart to collect adults of the hepatic portal vein and the mesenteric vein, rapidly collecting liver tissues, and preserving the tissues according to different experimental requirements after weighing. All collected worms were placed in a 60mm saline culture dish and the total number of worms and male worms were counted under a scope. Shearing central lobe of partial liver of each group of mice, weighing, placing in an EP tube, shearing, adding 4% potassium hydroxide solution, shaking in a constant temperature shaking table at 37 ℃ overnight until liver tissue is completely digested, centrifuging 1500g for 5min, removing upper layer liquid, and diluting lower layer precipitate with normal saline to 1ml. 10 μ l of the resuspension was tableted and counted under the microscope and repeated 5-7 times. Calculating the number of worm eggs contained in each gram of liver tissue; the results are shown in Table 1:
TABLE 1 adult insect and liver insect egg load of mice infected with Schistosoma japonicum after myricetin administration
Figure BDA0002395597020000051
The results in Table 1 show that myricetin can significantly reduce the adult load of mice, and little adult is observed in the praziquantel-treated group, which indicates that the in vivo insecticidal effect of praziquantel is more effective than myricetin. Interestingly, the result of the liver worm egg load of each group of mice shows that both praziquantel and myricetin can effectively inhibit the egg laying amount of schistosoma japonicum, and the inhibition effect has no obvious difference between the two drugs, which indicates that myricetin and praziquantel can effectively reduce the deposition of liver worm eggs.
3. Carrying out conventional paraffin section on the liver tissue, and observing pathological injury conditions of the liver tissue of the mouse by H & E and Masson staining; the results are shown in FIGS. 3 and 4.
FIG. 3 is a graph showing the H & E results of the livers of the mice in each group after myricetin action, and it can be seen from the graph that the lobules of the livers of the normal group under the microscope are complete in structure, the central veins and the radioactive liver cell cords can be seen, and the small bile ducts and fibrous tissue hyperplasia can not be seen in the areas of the junction and the periphery. After infection, the liver can be seen with worm egg deposition, a large amount of inflammatory cells infiltrate around the worm eggs, and collagen deposition is accompanied, typical worm egg granuloma is formed and is in diffuse distribution, and the granuloma area of the myricetin or praziquantel treatment group is obviously reduced compared with that of the infection group.
FIG. 4 is the Masson result chart of the livers of each group of mice after the myricetin acts, the livers of the mice in the infected group have a large amount of fresh collagen fibers deposited around granuloma and are dispersedly distributed in liver tissues, and the liver fibrosis of the myricetin or praziquantel treated group is obviously improved, which indicates that the myricetin can effectively improve the liver pathological injury caused by the schistosoma japonicum.
4. Liver tissues of each group of mice are respectively extracted with lysine buffer (Thermo Fisher) and TRIzol (Thermo Fisher) reagents to obtain total tissue protein and total RNA, and Western blot and RT-qPCR are used for detecting the expression of fibrosis-related molecules such as alpha-SMA, collagen I and Collagen IV.
The primers used were as follows:
α-SMA：
F：5’-CACAGCCCTGGTGTGCGACAAT-3’；
R：5’-TTGCTCTGGGCTTCATCCCCCA-3’；
Collagen I：
F：GGAGACAATGGAGACCGAAA-3’；
R：5’-CGACCAATTAGGGCTTGTGT-3’；
Collagen IV：
F：5’-ATGCCCTTTCTCTTCTGCAA-3’；
R：5’-GAAGGAATAGCCGATCCACA-3’；
the detection results are shown in FIG. 5, and FIG. 5 is a graph showing the influence of myricetin on the expression of factors related to liver fibrosis of mice infected with Schistosoma japonicum, and it can be seen from the graph that myricetin can effectively reverse the expression of alpha-SMA, collagen I and Collagen IV induced by Schistosoma japonicum infection (5A, RT-qPCR results; 5B,5C, western blot results).
Among the results of the detection, the quantitative results are expressed as mean ± Standard Deviation (SD). All data were statistically analyzed using SPSS 19.0 (SPSS Inc, new York, USA). Differences between groups were analyzed using one-way analysis of variance (ANOVA). All histograms were plotted by GraphPad Prism 7.0 (GraphPad Software, san Diego, USA). * P <0.05, P <0.01, P <0.001.
The above description is intended to describe in detail the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the claims of the present invention, and all equivalent changes and modifications made within the technical spirit of the present invention should fall within the scope of the claims of the present invention.

Claims (3)

1. The application of myricetin as a unique active ingredient in preparing a medicament for treating schistosoma japonicum is characterized in that the medicament has the effect of killing schistosoma japonicum, can inhibit the oviposition of schistosoma japonicum and/or reduce the load of eggs of insects and liver eggs in a host infected by schistosoma japonicum.
2. The use according to claim 1, wherein the medicament is capable of ameliorating pathological damage of the liver caused by schistosoma japonicum.
3. Use according to claim 2, wherein the pathological damage of the liver is liver fibrosis.
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