CN113230248A - Application of licarin-B and composition thereof in preparation of medicines for treating or preventing toxoplasmosis - Google Patents
Application of licarin-B and composition thereof in preparation of medicines for treating or preventing toxoplasmosis Download PDFInfo
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- CN113230248A CN113230248A CN202110681144.8A CN202110681144A CN113230248A CN 113230248 A CN113230248 A CN 113230248A CN 202110681144 A CN202110681144 A CN 202110681144A CN 113230248 A CN113230248 A CN 113230248A
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- licarin
- toxoplasma
- toxoplasmosis
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- WKSAUQYGYAYLPV-UHFFFAOYSA-N pyrimethamine Chemical compound CCC1=NC(N)=NC(N)=C1C1=CC=C(Cl)C=C1 WKSAUQYGYAYLPV-UHFFFAOYSA-N 0.000 description 1
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Images
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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
- A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
- A61K31/36—Compounds containing methylenedioxyphenyl groups, e.g. sesamin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- 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)
- Veterinary Medicine (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention discloses application of licarin-B and a composition thereof in preparing a medicament for treating or preventing toxoplasmosis. The effective concentration of the licarin is 14-50 mug/mL. Also discloses application of the pharmaceutical composition containing the licarin-B in preparing a medicament for treating or preventing toxoplasmosis. According to the invention, the Toxoplasma gondii tachyzoites are treated by using different concentrations of licarin-B, and the results show that the Toxoplasma gondii tachyzoites treated by different concentrations of licarin-B become small and round, the surface is lost, the internal mitochondria of the tachyzoites swell, and the membrane structures of all organelles disappear with the time, and finally the insect body dies, which indicates that the licarin-B can play a role in treating or preventing the Toxoplasma gondii by inhibiting the invasion and intracellular replication of the Toxoplasma gondii tachyzoites. The licarin-B in vivo has good toxoplasma resisting effect, high safety, thorough treatment and difficult relapse, and is expected to become a first-line candidate drug for resisting the toxoplasma.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to application of licarin-B and a composition thereof in preparing medicines for treating or preventing toxoplasmosis.
Background
Toxoplasma is an obligate intracellular parasitic protozoa belonging to the phylum Toxoplasma, class Sporophytes, subclasses Coccidia, order Ceriporioles, order Eimeriales, family Toxoplasma, genus Toxoplasma. There is only one species under the genus Toxoplasma, Toxoplasma gondii (Toxoplasma gondii), which is commonly referred to as Toxoplasma. Toxoplasma gondii can infect all warm-blooded animals including humans, even some cold-blooded animals, and can parasitize all nucleated cells of the animal's body.
In individuals with a normal immune system, the clinical manifestations of toxoplasmosis are generally asymptomatic, and life can be compromised in immunodeficient patients. In these immunodeficient patients, toxoplasmosis is almost recurrent. The typical clinical manifestations of toxoplasmosis are: simple enlargement of the cervical or occipital lymph nodes. And retinochoroiditis caused by toxoplasma infection often presents a white focus of the overlay and a strong vitreous inflammatory response. The central nervous system is the most typical and most severe site of infection. Toxoplasma infections cause central nervous system damage, resulting in encephalitis, with clinical manifestations including symptoms of altered mental status, seizures, focal dyskinesias, cranial nerve disorders, paresthesia, cerebellar syndrome, movement disorders, and neuropsychiatric disorders. Patients with immunocompromised toxoplasmosis also develop chorioretinitis, pneumonia, often manifested as acute respiratory failure and hemodynamic abnormalities like septic shock. Toxoplasma pneumonia appears to be more common in bone marrow transplant patients and AIDS patients. The congenital toxoplasmosis of fetus is clinically manifested by hydrocephalus, malformation, intracranial calcification, chorioretinitis, strabismus, blindness, epilepsy, intellectual disturbance, hemorrhage and anemia caused by thrombocytopenia, etc.
In the prior art, the toxoplasma infection is generally treated by combined application of pyrimethamine, sulfadiazine and folic acid in clinic, but the treatment is often accompanied by side effects, incomplete treatment and easy relapse. licarin-B (C)20H20O4) Belongs to the lignanoid component of the traditional Chinese medicine, and is mainly derived from dried and mature nutmeg kernels in nature. Modern pharmacological studies show that nutmeg has antibacterial, anti-inflammatory, sedative, and anti-tumor effects. However, no studies have shown that licarin-B has an active inhibitory effect on Toxoplasma gondii.
Therefore, the medicines for treating toxoplasma infection in the market at present still cannot meet the clinical requirements, and a traditional Chinese medicine monomer licarin-B applied to the preparation of the medicines for treating or preventing toxoplasmosis is urgently needed to be found, so that the traditional Chinese medicine monomer licarin-B plays roles in inhibiting the invasion and increment of the toxoplasmosis in the preparation of the medicines for treating or preventing the toxoplasmosis, and the technical effects of remarkable effect and small toxic and side effects of the licarin-B in the aspect of treating or preventing the toxoplasmosis are realized.
Disclosure of Invention
The invention aims to solve the technical problem of providing the application of licarin-B in preparing the medicine for treating or preventing the toxoplasmosis, so that the licarin has obvious effect on treating or preventing the toxoplasmosis, has small toxic and side effects and is not easy to relapse.
In order to solve the technical problems, the invention provides an application of licarin-B in preparing a medicament for treating or preventing toxoplasmosis. The effective concentration of the licarin-B is 14-50 mu g/mL.
Further improved, the effective concentration of the licarin-B is 18-36 mu g/mL.
The invention also provides application of the pharmaceutical composition in preparing a medicament for treating or preventing toxoplasmosis, wherein the pharmaceutical composition comprises licarin-B and pharmaceutically acceptable auxiliary materials.
In a further improvement, the pharmaceutical composition also comprises an anti-insect drug which is combined with the licarin-B.
Further improved, the pharmaceutical composition is prepared into any one dosage form of powder, tablets, capsules, pills, dripping pills, injections, emulsions, suspensions or tinctures.
In a further improvement, the pharmaceutical composition is used as an animal feed additive.
After adopting such design, the invention has at least the following advantages:
according to the invention, the Toxoplasma gondii tachyzoites are treated by using different concentrations of licarin-B, and the results show that the Toxoplasma gondii tachyzoites treated by different concentrations of licarin-B become small and round, the surface is lost, the internal mitochondria of the tachyzoites swell, and the membrane structures of all organelles disappear with the time, and finally the insect body dies, which indicates that the licarin-B can play a role in treating or preventing the Toxoplasma gondii by inhibiting the invasion and intracellular replication of the Toxoplasma gondii tachyzoites. The licarin-B has good in-vivo insect-resistant effect, high safety, thorough treatment and difficult relapse, and is expected to become a first-line candidate drug for resisting toxoplasma gondii.
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 resistance of licarin-B of the present invention to intracellular invasion by Toxoplasma gondii.
FIG. 2 is a graph showing the intracellular proliferation of licarin-B of the present invention against Toxoplasma gondii.
FIG. 3 is a transmission electron microscope ultramicro structural diagram of the invention licarin-B affecting the structure of Toxoplasma gondii. Wherein: m: a mitochondrion; DG: a dense spot; r: a rod-shaped body; n: and (4) cell nucleus.
FIG. 4 is a confocal laser micrograph of the inventive licarin-B causing toxoplasma autophagy.
FIG. 5 is a confocal laser micrograph of the toxoplasma gondii nuclear alterations induced by licarin-B of the present invention.
FIG. 6 is a statistical plot of the survival rate of licarin-B of the present invention against a Toxoplasma gondii mouse model.
FIG. 7 is a statistical plot of the licarin-B of the present invention versus the insect load in various tissues, organs and blood of a Toxoplasma gondii mouse model.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Example 1: fluorescence quantitative PCR (polymerase chain reaction) determination of insect body load capacity after treatment of different concentrations of licarin-B
This test was performed to evaluate insect body loading for different drugs and at different concentrations.
1. Procedure of the test
Inoculating HFF to 6-well plate seeds one day before infection, adding licarin-B culture medium (4.5-22.5 mu g/mL) with different concentrations, selecting azithromycin (8.6 mu g/mL) as a positive drug, adding only infection culture medium to a negative control group, repeating three times for each group, inoculating toxoplasma gondii tachyzoite with the inoculation amount of 2 multiplied by 105Per hole, put in CO2Culturing in an incubator for 6h, discarding the culture medium, washing with PBS for 2 times to remove uninfected polypide, incubating for 8h, and extracting total DNA and measuring relative expression amount.
Similarly, HFF was inoculated in 6-well plates one day before infection at an inoculum size of 1X 105Inoculating toxoplasma tachyzoite when the next day cell grows up to 80%, wherein the inoculation amount is 2 × 105Per hole, put in CO2Culturing for 6h in an incubator, discarding the culture medium, washing for 2 times by PBS to remove uninfected polypide, adding different concentrations of licarin-B culture medium (4.5-45 mug/mL), selecting azithromycin as a positive drug (8.6 mug/mL), adding only the infection culture medium to a negative control group, repeating three times for each time, incubating for 24h, discarding the culture medium, washing for two times by PBS, extracting total DNA (deoxyribonucleic acid) and determining relative expression, and converting the polypide load capacity of each sample (T sample) and negative control (T negative). MeterAnd calculating the proliferation rate under each concentration, drawing a graph, and performing Probit regression analysis by using the inhibition rate to find the corresponding concentration when the inhibition rate is 50%. The proliferation rate was T-like/T-negative × 100%, and the inhibition rate was 1- (T-like/T-negative). The results are shown in FIGS. 1 and 2.
2. Test results
The results show that with increasing concentration of licarin-B, toxoplasma invasion into host cells and intracellular proliferation is inhibited and dose dependent. The half inhibition concentration IC of the licarin-B on the Toxoplasma gondii tachyzoite is obtained by calculation50The value was 14.05. mu.g/mL.
The results show that: licarin-B inhibits the invasion and intracellular replication of toxoplasma tachyzoites.
Example 2: transmission electron microscope observation of the effect of licarin-B on the inner ultrastructure of Toxoplasma gondii
1. Procedure of the test
One day before infection at T150HFF cells were inoculated in 2X 10 flasks6Inoculating tachyzoite in the amount of 1 × 10 when the cells grow up to 80% the next day6And (2) putting the cells/hole into an incubator for culturing for 12h, discarding the culture medium, washing the cells for 2 times by using PBS to remove uninfected worm bodies, adding 18 mu g/mL licarin-B culture medium, continuing culturing for 16h and 24h, taking the cells out after the incubation is finished, discarding the drug culture medium, washing the cells for two times by using PBS, digesting the cells for 3min by using TrypLE Express, stopping digestion by using the culture medium, washing the infected cells for two times by using PBS, finally collecting the infected cell blocks at each time point into a 1.5mL centrifuge tube (preventing the infected cell blocks from being attached to the wall of the centrifuge tube), adding 2.5% glutaraldehyde (0.01mol/L PBS, PH 7.4) for fixing at 4 ℃.
After 24h of pre-fixation (if the next step cannot be performed quickly, the cells can be stored in a sealed condition at 4 ℃ for about one month), the cells are washed repeatedly with PBS until the fixative is washed completely (more than 5 times). Postfixation with 1% osmic acid (0.01mol/L PBS, pH 7.4) over a period of 2h (protected from light). Washing with PBS for 3 times and 10min, dehydrating with gradient ethanol with concentration gradient of 30%, 50%, 70%, 80%, 90%, 95% and 100% (3 times) for 10min, and replacing with anhydrous acetone. The sample was soaked overnight in a 1:2 mixture of embedding medium Epon 812 and acetone at 35 ℃. And embedding the sample into the embedding plate, and embedding the sample into the embedding plate together with the mark. The embedded samples were placed in an incubator and polymerization hardened at progressively increasing temperatures (37 ℃, 12 h; 45 ℃, 12 h; 60 ℃, 48 h).
The tissue was trimmed to a tetrahedral pyramid, the sample was exposed, and the sample surface was trimmed to smooth. The preparation method comprises the steps of manufacturing a glass cutter by using a cutter manufacturing machine, carrying out ultrathin slicing on a sample by using a Leica UC6 type ultrathin slicer, fishing out the sample by using a 200-mesh copper net, dyeing the dried sample for 30min by using 2% uranyl acetate, taking out the sample, quickly cleaning the sample by using double distilled water, and dyeing the cleaned sample for 15min by using lead citrate. And finally observing by using a Hitachi H-7500 transmission electron microscope at a working voltage of 80 kv. The results are shown in FIG. 3.
2. Test results
The transmission electron microscopy results from fig. 3 show that: under the treatment of licarin-B, the inside of the tachyzoite has the phenomenon of mitochondrial swelling, and with the time prolongation, an autophagy-like double-layer membrane structure is generated, the membrane structure of each organelle disappears, and finally, the insect body dies.
The results show that: licarin-B causes mitochondrial swelling of toxoplasma, possibly causing autophagy of the insect.
Example 3: single dansyl cadaverine (MDC) staining detection of toxoplasma autophagy caused by licarin-B
1. Procedure of the test
Each sample contained 1X 106Toxoplasma tachyzoites incubated with different concentrations of either 18, 27 or 36. mu.g/mL licarin-B, control groups incubated without drug, after 8h, washed with PBS and centrifuged, suspended in 100. mu.M MDC staining solution at 37 ℃ for 1h and then washed twice with PBS. The Toxoplasma tachyzoites were resuspended in 500. mu.L of PBS and the fluorescence intensity of each group was observed with a confocal laser microscope. The results are shown in FIG. 4.
2. Test results
As can be seen from FIG. 4, the autophagy of Toxoplasma gondii after incubation with licarin-B was detected by MDC staining, and the intensity of green fluorescence indicates the degree of autophagy of Toxoplasma gondii, it can be seen that the degree of autophagy of Toxoplasma gondii is more and more severe as the incubation time of licarin-B is prolonged. And with the increase of the concentration of the licarin-B, the autophagy degree of the toxoplasma also becomes more and more serious.
The results show that: licarin-B causes autophagy of toxoplasma with time and dose dependence, also suggesting that toxoplasma autophagy is one of the pathways leading to death of the insect.
Example 4: DAPI staining detection of toxoplasma cell nuclear changes caused by licarin-B
1. Procedure of the test
Mixing extracellular parasites (1X 10)5Group) were incubated with different concentrations of licarin-B (18, 27 or 36. mu.g/mL) in DMEM at 37 ℃ for 16h, with no drug as a control group. After incubation, all samples were stained with 4', 6-diamino-2-phenylindole (DAPI) at 37 ℃ and fluorescence intensity was observed with confocal laser microscopy for determination of toxoplasma cell nuclear changes after incubation with licarin-B. The results are shown in FIG. 5.
2. Results of the experiment
As can be seen from FIG. 5, as the concentration of licarin-B increases, the nucleus of the toxoplasma gradually diffuses, the structure of the nucleus is destroyed, and the death of the toxoplasma body is finally caused.
Example 5: determination of the insect-resistant Effect of Ricarin-B on Toxoplasma gondii in vivo on a mouse model
1. Procedure of the test
In a BALB/c mouse, an in-vivo toxoplasma acute infection model is established. The in vivo insect-resistant effect of the licarin-B with different concentrations is evaluated by adopting an intraperitoneal injection mode. In the embodiment, the intraperitoneal injection concentration of the licarin-B is 25mg/kg.bw and 50mg/kg.bw, the intraperitoneal injection of physiological saline is used as a negative control, the intraperitoneal injection of azithromycin is used as a positive control group, the injection is continuously carried out for 20 days, the survival rate of a BALB/c mouse with acute toxoplasma infection is recorded, and the insect body load capacity in each tissue organ and blood of an infected mouse model after the injection is continuously carried out for 7 days is recorded. The results are shown in FIGS. 6 and 7.
2. Test results
As can be seen from FIG. 6, in the established Toxoplasma gondii acute infection mouse model, licarin-B was injected intraperitoneally at 50mg/kg. bw for 15 days, and 90% of BALB/c mice were protected from death. As can be seen from fig. 7: the intraperitoneal injection dose of the licarin-B is 50mg/kg.bw, the insect body load capacity in liver, brain, spleen and blood of the mouse is obviously reduced when the drug is continuously administered for 7 days, and the body temperature and the body weight of the mouse are not obviously changed.
The results show that the intraperitoneal injection of the licarin-B has better in-vivo activity against toxoplasma gondii.
According to the above embodiments, licarin-B 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 licarin-B has good effect of treating or preventing toxoplasmosis within the effective dosage range. 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.
The licarin-B can also be added into other existing anti-insect drugs to make them be used together, so as to enlarge the anti-spectrum range of parasites and further improve the prevention and treatment effect on parasitic diseases.
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 licarin-B in preparing medicine for treating or preventing toxoplasmosis is provided.
2. The use of licarin-B according to claim 1 in the preparation of a medicament for the treatment or prevention of toxoplasmosis, wherein the effective concentration of licarin-B is 14-50 μ g/mL.
3. The application of licarin-B in preparing a medicament for treating or preventing toxoplasmosis according to claim 2, wherein the effective concentration of licarin-B is 18-36 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 licarin-B and pharmaceutically acceptable auxiliary materials.
5. The use of a pharmaceutical composition according to 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 administered in combination with the licarin-B.
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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Citations (4)
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|---|---|---|---|---|
| US5641769A (en) * | 1993-05-05 | 1997-06-24 | Palo Alto Medical Foundation | treatment for toxoplasmoisis with a composition containing a hydroxynaphthoquinone and a spiropiperidyl derivative of rifamycin S. |
| KR20090093302A (en) * | 2008-02-29 | 2009-09-02 | 황재관 | Pharmaceutical composition for treating or preventing an inflammatory disease comprising licarin E |
| CN102391223A (en) * | 2011-10-10 | 2012-03-28 | 南京泽朗医药科技有限公司 | Method for separating and purifying licarin B |
| CN110876739A (en) * | 2019-08-09 | 2020-03-13 | 中国农业科学院兰州畜牧与兽药研究所 | Application of myristolignan in treating toxoplasmosis |
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2021
- 2021-06-18 CN CN202110681144.8A patent/CN113230248A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5641769A (en) * | 1993-05-05 | 1997-06-24 | Palo Alto Medical Foundation | treatment for toxoplasmoisis with a composition containing a hydroxynaphthoquinone and a spiropiperidyl derivative of rifamycin S. |
| KR20090093302A (en) * | 2008-02-29 | 2009-09-02 | 황재관 | Pharmaceutical composition for treating or preventing an inflammatory disease comprising licarin E |
| CN102391223A (en) * | 2011-10-10 | 2012-03-28 | 南京泽朗医药科技有限公司 | Method for separating and purifying licarin B |
| CN110876739A (en) * | 2019-08-09 | 2020-03-13 | 中国农业科学院兰州畜牧与兽药研究所 | Application of myristolignan in treating toxoplasmosis |
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Application publication date: 20210810 |