CN115737641A - Application of pure penicilliol in preparation of medicine for treating inflammatory bowel disease - Google Patents

Abstract

The invention relates to application of pure penicillium chlororaphe alcohol in preparation of a medicament for treating inflammatory bowel diseases, belongs to the technical field of new medical application, and particularly relates to application of pure penicillium chlororaphe alcohol in preparation of a medicament for treating inflammatory bowel diseases.

Description

Application of pure green penicillol in preparation of medicine for treating inflammatory bowel disease

technical field

The invention belongs to the technical field of new uses of medicine, and in particular relates to the application of pure green penicillol in the preparation of medicines for treating inflammatory bowel disease.

Background technique

Inflammatory bowel disease (IBD) is a general term for gastrointestinal diseases that exhibit chronic or recurrent immune response and inflammatory symptoms. With the changing of people's lifestyles and the increasing pressure of life, the global incidence of IBD is increasing year by year, and the incidence of IBD in my country ranks first in Asia. The clinical manifestations of IBD patients mainly include: intestinal manifestations such as abdominal pain, diarrhea, mucus and bloody stools, extraintestinal manifestations such as fistula formation, perianal abscess, bloody stools, abdominal cramps, fever, fatigue, severe internal cramps in the pelvic region, muscle spasms, and appetite reduction and weight loss etc. At present, there is a shortage of drugs for the treatment of IBD, and there is no effective treatment method. Although conventional treatment can improve symptoms, the recurrence rate is high and the curative effect is not good for severe patients. The overall clinical remission rate does not exceed 50%. The development of innovative drugs for the treatment of IBD has become an urgent clinical need.

Copper sulfate (CuSO ₄ ) is widely used to establish a model of acute neuroinflammation in zebrafish. Copper sulfate damages the peripheral organ neuromast of the zebrafish lateral line, causing zebrafish inflammatory cells to migrate around the neuromast (lateral line); a systemic infectious inflammation model caused by lipopolysaccharide (LPS); the above two inflammatory models and 2, Different models of inflammatory bowel disease induced by 4,6-trinitrobenzenesulfonic acid (TNBS).

2,4,6-trinitrobenzenesulfonic acid (2,4,6-trinitrobenzenesulfonic acid, TNBS), dextran sodium sulfate (dextran sodium sulfate, DSS), oxazolone, etc. Model mutagens. Among them, TNBS induction has the advantages of short modeling time, high reproducibility, and easy induction, and is a classic IBD chemical mutagen. TNBS can cause the disappearance of intestinal peristalsis, intestinal dilatation, and intestinal obstruction in zebrafish, as well as shorten the length of intestinal villi and increase the number of goblet cells, which is very similar to the pathological manifestations of IBD patients.

Fleming et al. used TNBS used in experimental mouse models to induce intestinal inflammation in zebrafish, soaked zebrafish in TNBS, zebrafish had enlarged intestinal lumen, disappeared intestinal folds, increased the number of goblet cells, and increased the number of epithelial cells. Tumor necrosis factor-α (TNF-α) staining was positive. Oscar et al. used zebrafish as a model to study intestinal inflammation, exposed zebrafish juveniles to TNBS from 72hpf to 120hpf, observed that TNBS led to the recruitment of neutrophils to the intestine, and proved that TNBS can induce zebrafish intestinal inflammation. He et al. used TNBS to induce IBD-like enterocolitis in zebrafish juveniles and collected zebrafish larvae for detection. The study found that the expression of toll-like receptor 3 (TLR3) and TLRs signaling pathway molecules MyD88 and TRIF in TNBS-treated zebrafish , activation of NF-κB, and production of the inflammatory cytokine tumor necrosis factor-α were all stimulated.

Pure green penicillol is a quinolinone alkaloid isolated from the secondary metabolites of the marine fungus Aspergillus austroafricanus (Aspergillus austroafricanus) Y32-2, with a molecular formula of C ₁₅ H ₁₁ NO ₃ .

It has been reported that pure viridinol has strong antibacterial activity against Staphylococcus aureus, but there is no report on the effect of pure viridinol on inflammatory bowel disease.

Contents of the invention

Aiming at the deficiencies of the prior art, the present invention provides the application of pure viridinol in the preparation of medicines for treating inflammatory bowel disease.

Technical scheme of the present invention is as follows:

Application of pure virididillol in the preparation of medicines for treating inflammatory bowel disease, wherein the structural formula of pure virididillol is as follows:

Preferably according to the present invention, in the application, the drug can regulate MAPK and NF-κB signaling pathways by activating the expression of pparγ, so as to treat inflammatory bowel disease.

A medicine for treating inflammatory bowel disease, which contains pure green penicillol.

Preferably according to the present invention, the medicine contains pharmaceutically acceptable carriers and or auxiliary materials.

Preferably according to the present invention, the dosage form of the drug is tablet, capsule, granule, pellet, drop pill, oral liquid, aqueous injection, powder injection, infusion, ointment, gel or microemulsion.

Beneficial effect

The invention discovers for the first time that pure green penicillol can treat inflammatory bowel disease, increases the new use of pure green penicillol, provides a new treatment method for the treatment of inflammatory bowel disease, and also develops the application and development of pure green penicillol for reference.

Description of drawings

Figure 1 is a schematic diagram of the effect of pure green penicillol on the migration of zebrafish intestinal inflammatory cells induced by TNBS;

In the figure: TNBS represents 2,4,6-trinitrobenzenesulfonic acid; the red box is the part where the number of intestinal inflammatory cells in zebrafish is counted, and the right side is the enlarged picture of the intestinal statistical area.

Figure 2 is a statistical histogram of the effect of pure green penicillol on the migration of zebrafish intestinal inflammatory cells induced by TNBS;

In the figure: compared with blank control group, ^## P<0.01; compared with TNBS modeling group, ^* P<0.05, ^** P<0.01.

Figure 3 is a schematic diagram of the influence of pure green penicillol on the intestinal tissue structure of inflammatory bowel disease zebrafish;

In the figure: the black solid arrow indicates necrolysis of the mucosal epithelium, the black dotted arrow indicates the disappearance of intestinal folds, and the scale bar of the intestinal pictures is 50 μm.

Figure 4 is a schematic diagram of the influence of pure green penicillol on the intestinal ultrastructure of inflammatory bowel disease zebrafish;

In the figure: the black arrow indicates the state of intestinal microvilli; Mv indicates microvilli, and Ec indicates epithelial cells; the scale bar of the intestinal pictures is 20 μm.

Fig. 5 is a schematic diagram of the influence of pure green penicillol on the gene expression level of inflammatory bowel disease zebrafish;

In the figure: pparγ indicates peroxisome proliferator-activated receptor γ, zak indicates a protein kinase; hsp70.1 indicates heat shock protein, ap-1 indicates activator protein-1, map3k8 indicates a serine/threonine Kinase, ikbαa indicates nuclear transcription factor inhibitory gene, nf-κb indicates nuclear transcription factor, il8a indicates an inflammatory factor; compared with blank control group, ^# P<0.05, ^## P<0.01; compared with TNBS modeling group , ^* P<0.05, ^** P<0.01.

Fig. 6 is a schematic diagram of the influence of pure green penicillol on the systemic infectious inflammation caused by LPS;

In the figure: LPS means lipopolysaccharide; the picture shows the overall distribution of inflammatory cells in zebrafish.

Figure 7 is a schematic diagram of the effect of Epimedin B on _CuSO4- induced acute neuroinflammation in zebrafish;

In the figure: CuSO ₄ represents copper sulfate; the picture shows the migration of zebrafish inflammatory cells around the neuromast (lateral line) and the enlarged picture of its area.

Figure 8 is a histogram of the anti-inflammatory effect of Epimedin B on _CuSO4- induced acute neuroinflammation in zebrafish;

In the figure: CuSO ₄ represents copper sulfate; compared with blank control, ^### P<0.001; compared with CuSO ₄ model group, ***P<0.001.

Fig. 9 is a schematic diagram of the effect of Epimedin B on the local inflammation induced by tail docking in zebrafish;

In the figure: The picture shows the migration of zebrafish inflammatory cells to the site of tail docking and the enlarged picture of the area.

Figure 10 is a histogram of the anti-inflammatory effect of Epimedin B on local inflammation in zebrafish induced by tail docking;

In the figure: compared with the blank control group, ^### P<0.001; compared with the docked model group, ***P<0.001.

Figure 11 is a schematic diagram of the effect of Epimedin B on the aggregation and migration of zebrafish intestinal inflammatory cells;

In the figure: TNBS represents 2,4,6-trinitrobenzenesulfonic acid; the red box is the part where the number of inflammatory cells in the intestinal tract of zebrafish is counted, and the right side is the enlarged picture of the statistical area of the intestinal tract.

Figure 12 is a histogram of the aggregation and migration of zebrafish intestinal inflammatory cells induced by Epimedin B on TNBS; in the figure: compared with the blank control group, ^### P<0.001; compared with the TNBS model group, ***P< 0.001.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific examples, but the embodiments of the present invention are not limited thereto.

The medicines and reagents used in the examples, unless otherwise specified, are common products on the market, and the contents not described in detail in the examples are all according to the prior art in the art.

main source of material

2,4,6-Trinitrobenzenesulfonic acid (TNBS) was purchased from Sigma Company, electron microscope fixative was purchased from Wuhan Google Biotechnology Co., Ltd., 812 embedding agent was purchased from SPI Company, and zebrafish juvenile culture water was purchased at a concentration of 0.4 mM CaCl ₂ , 5 mM NaCl, 0.17 mM KCl, 0.16 mM MgSO ₄ .

Pure green penicillol: pure green penicillol was purchased from TargetMol Company (production batch number: 155521; purity 98%).

Epimedin B: purchased from Shanghai Yeyuan Biotechnology Co., Ltd. (production batch number: J12HB184812; purity ≥ 98%).

Lipopolysaccharide LPS: purchased from Sigma Company of the United States (production batch number: 0000155411).

Example 1

Effect of pure green penicillol on the aggregation degree of intestinal inflammatory cells

Using transgenic zebrafish with 3dpf healthy inflammatory cells marked with green fluorescence as experimental animals, they were randomly divided into blank control group (pisciculture water), model group (TNBS) and different concentration sample groups (TNBS+30μg/mL pure green Penicillol, TNBS+60 μg/mL pure chloridol, TNBS+90 μg/mL pure chloridol). There are 10 larvae in each group, and 2 replicate holes are set at the same time. 2,4,6-trinitrobenzenesulfonic acid (TNBS) with a final concentration of 50 μg/mL was added simultaneously to the model group and the sample group with different concentrations (without adding pure green penicillol). The above-mentioned groups of zebrafish were placed in a constant temperature incubator at 28.5°C and incubated continuously for 2 days under dark conditions. After the end, the larvae of each group were cleaned, and the blank control group and the model group zebrafish were placed in fresh culture water, respectively, and the zebrafish of different concentration sample groups were placed in culture water containing different concentrations of the samples to be tested. The above-mentioned groups of zebrafish were placed in a constant temperature incubator at 28.5°C, and continued to act for 1 day. After the treatment, the zebrafish were anesthetized with 0.2% ethyl m-aminobenzoate methanesulfonate, observed and photographed under a fluorescent microscope. The number of inflammatory cells showing green fluorescence in the zebrafish gut was counted.

Hematoxylin-eosin (H&E) staining of zebrafish intestinal sections: Zebrafish treated with compounds were collected and fixed with 4% paraformaldehyde. The zebrafish samples were trimmed, dehydrated, embedded, sliced, stained, and mounted in strict accordance with the pathological test procedures. Observe the section of intestinal tissue under a microscope and image the typical lesion.

Transmission electron microscope detection of zebrafish intestine: collect the zebrafish treated with the compound, fix with electron microscope fixative solution at 4°C for 2-4 hours, and then rinse with 0.1M phosphate buffer PB (PH7.4) for 3 times, each time for 15min . The target slices were obtained through fixation, dehydration, infiltration, embedding, sectioning, and staining. Intestinal tissue was observed under a transmission electron microscope, and images were collected for analysis.

The expression of key genes related to inflammatory bowel disease was detected by PCR: Zebrafish tissues treated with pure chloridol (blank control group, TNBS group, TNBS+90μg/mL pure chloridol group) were collected, and FastPure Cell/ Tissue TotalRNA Isolation Kit V2 for RNA extraction. The RNA of each sample was reverse-transcribed to obtain cDNA, and the expression levels of genes related to inflammation were measured using the BIO-RAD CFX96 real-time system, which was divided into three replicates. QRT-PCR amplification reaction conditions are 95°C pre-denaturation for 30s for 1 cycle, denaturation at 95°C for 10s in each cycle, annealing at 60°C for 10s, a total of 40 cycles, and finally 95°C for 15s, 60°C for 60s, and 95°C for 15s. cycles. When analyzing the results, β-actin was used as an internal reference, and the relative quantitative analysis was performed on the results. The PCR primers of the target gene and internal reference gene were synthesized and purified by Shanghai Jierui Bioengineering Co., Ltd., and passed the quality inspection. The gene sequences of relevant primers are listed in Table 1.

Table 1 Gene amplification primer sequences for real-time fluorescent quantitative PCR

Experimental results:

(1) Effect of pure green penicillol on aggregation and migration of intestinal inflammatory cells in zebrafish

The TNBS-induced inflammatory bowel disease model of zebrafish was established by using TNBS to induce the accumulation of inflammatory cells in the intestine of zebrafish. The anti-inflammatory activity of compounds on zebrafish intestinal inflammation was evaluated by counting the number of inflammatory cells in the zebrafish intestinal site. As shown in Figure 1 and Figure 2, compared with the blank group, the number of inflammatory cells migrating to the intestinal tract of zebrafish in the TNBS group was significantly increased. When the concentration of pure green penicillol was 60 and 90 μg/mL, the number of intestinal inflammatory cells in zebrafish decreased significantly compared with the model group.

(2) Effect of pure green penicillol on intestinal tissue of zebrafish with inflammatory bowel disease

Hematoxylin-eosin (H&E) staining of intestinal sections of zebrafish in blank group, TNBS group, and pure viridin (90 μg/mL) group. The experimental results showed that the mucosal layer, muscular layer and serosal layer of the zebrafish intestinal tissue in the blank group had a clear structure, and a single layer of columnar epithelium was lined on the mucosal surface, which was tall columnar and neatly arranged, and no obvious pathological changes were seen in the intestinal tissue; The cells of fish intestinal tissue are sparsely arranged, the mucosal epithelium is necrotic and dissolved (black solid arrow), and intestinal folds disappear (black arrow). Compared with the TNBS group, the zebrafish intestinal tissue folds were restored after the administration of pure green penicillol, and the cells were arranged more tightly, as shown in Figure 3.

(3) Effects of pure green penicillol on the intestinal ultrastructure of zebrafish with inflammatory bowel disease

Using transmission electron microscopy, the ultrastructure of zebrafish intestines in each group was observed and analyzed. In the blank group, intestinal cell membranes were complete, organelles were abundant, and microvilli (Mv) were arranged neatly and uniform in thickness (arrows). In the TNBS group, the distribution of intestinal microvilli was uneven, and the local area was slightly sparse and disordered (indicated by the arrow). As shown in the pictures of the pure viridin group, the intestinal microvilli returned to a rich state (shown by the arrow), see Figure 4.

(4) Effect of pure green penicillol on key genes of inflammatory bowel disease

The PCR results showed that compared with the blank control group, the mRNA expressions of pparγ and zak in the zebrafish of the TNBS group decreased significantly (P<0.01), and the mRNA expressions of hsp70.1, ap-1, map3k8, il8a, ikbαa, and nf-κb raised. Compared with the TNBS group, the mRNA expressions of pparγ and zak in the zebrafish treated with pure green penicillol were significantly increased, and the mRNA expressions of hsp70.1, ap-1, map3k8, il8a, ikbaαa, and nf-κb were significantly decreased (P <0.01), see Figure 5.

The results showed that TNBS was used to cause intestinal inflammation in zebrafish. Compared with the blank group, the number of intestinal inflammatory cells in the TNBS group increased significantly, the arrangement of intestinal tissue cells was sparse, intestinal folds disappeared, and the distribution of intestinal microvilli was uneven. Slightly sparse and disordered. The compound pure green penicillol can significantly reduce the accumulation of inflammatory cells in the zebrafish intestine caused by TNBS, restore intestinal folds and intestinal microvilli, up-regulate the expression of pparγ, and regulate the MAPK and NF-κB signaling pathways, thereby effectively alleviating the zebrafish intestinal inflammation. tract inflammation.

Experimental example 1

Pure green penicillol has no anti-inflammatory effect on systemic infectious inflammation caused by endotoxicity (LPS)

Using transgenic zebrafish with 3dpf healthy inflammatory cells marked with green fluorescence as experimental animals, they were randomly divided into blank control group (pisciculture water), LPS inflammation model group (LPS, 25ug/mL), pure green penicillol with different concentrations Treatment groups (LPS+30 μg/mL pure chloridol, LPS+60 μg/mL pure chloridol, LPS+90 μg/mL pure viride). Endotoxicity (LPS) with a final concentration of 25 μg/mL was added to the model group and different concentrations of pure virididol treatment groups (without adding pure viridicillol), respectively, with 10 larvae in each group, and 2 replicate wells were set at the same time . The above-mentioned groups of zebrafish were placed in a constant temperature incubator at 28.5°C and incubated continuously for 2 days under dark conditions. After the end, the larvae of each group were cleaned, and the larvae of the blank control group and the model group were placed in fresh culture water respectively, and the larvae of the sample groups with different concentrations were placed in the culture water containing the samples to be tested at different concentrations. The above-mentioned groups of zebrafish were placed in a constant temperature incubator at 28.5°C, and continued to act for 1 day. After the treatment, the zebrafish were anesthetized with 0.2% ethyl m-aminobenzoate methanesulfonate, and the changes of inflammatory cell aggregation in each group of zebrafish were observed under a fluorescence microscope. Statistical analysis of the number of inflammatory cells in zebrafish body.

As shown in Figure 6, compared with the control group, the number of inflammatory cells in the zebrafish body of the LPS model group increased significantly. Compared with the model group, there was no significant change in the number of inflammatory cells in the zebrafish body in each concentration group of pure green penicillol. It shows that pure green penicillol has no anti-inflammatory effect on systemic infectious inflammation caused by LPS.

It can be seen from the above experimental results that the systemic infectious inflammation model caused by endotoxicity (LPS) is different from the inflammatory bowel disease model caused by 2,4,6-trinitrobenzenesulfonic acid (TNBS), pure green green There was no correlation between the anti-inflammatory effects of the two mycodols.

Experimental example 2

Epimedin B has anti-inflammatory effects on the acute neuroinflammation model induced by CuSO ₄ and local inflammation induced by tail docking in zebrafish, but has no effect on inflammatory bowel disease induced by TNBS

(1) Anti-inflammatory effect of Epimedin B on _CuSO4 -induced acute inflammation in zebrafish

A transgenic zebrafish whose 72hpf healthy inflammatory cells were marked with green fluorescence was selected as the experimental animal. Set blank control group (pisciculture water), inflammation model group (CuSO ₄ ), positive control group (CuSO ₄ + ibuprofen 10 μM) and epimedin B group (CuSO ₄ + epimedin B 80, 160, 320 μM) . There were 10 zebrafish in each group, and 2 replicate wells were set up at the same time. Zebrafish were exposed to Epimedin B solution for 24 hours, and after 24 hours, except the blank control group, the other groups were exposed to CuSO ₄ (40 μM) for 1 hour under dark conditions. The inflammatory cells migrating to the lateral line were observed and photographed under a Zeiss fluorescence microscope, and the number of inflammatory cells migrating to the lateral line was counted.

(2) Anti-inflammatory effect of Epimedin B on local inflammation induced by tail docking in zebrafish

A transgenic zebrafish whose 72hpf healthy inflammatory cells were marked with green fluorescence was selected as the experimental animal. Set blank control group (undocked zebrafish + fish water), docked model group (docked zebrafish + fish water) and epimedin B group (docked zebrafish + epimedin B 80, 160 , 320 μM). The blank control is the zebrafish with normal development, and the tails of the other groups are cut off with the same length of tail at a fixed position with a scalpel under the microscope. Move the zebrafish into a 24-well plate, with 10 zebrafish per well, and set up two replicate wells. After the tail-docked zebrafish were exposed to Epimedin B liquid for 6 hours, the accumulation of inflammatory cells at the docked tail was observed under a Zeiss fluorescence microscope.

(3) Effect of Epimedin B on the degree of inflammatory cell aggregation in zebrafish-induced intestinal tract induced by TNBS

Using transgenic zebrafish with 3dpf healthy inflammatory cells marked with green fluorescence as experimental animals, they were randomly divided into blank control group (pisciculture water), model group (TNBS) and sample groups with different concentrations (TNBS+80μM Epimedin B , TNBS+160 μM Epimedin B, TNBS+320 μM Epimedin B). 2,4,6-trinitrobenzenesulfonic acid (TNBS) with a final concentration of 50 μg/mL was added to the model group and the sample group with different concentrations (without adding Epimedin B), respectively, with 10 larvae in each group. 2 duplicate wells. The above-mentioned groups of zebrafish were placed in a constant temperature incubator at 28.5°C and incubated continuously for 2 days under dark conditions. After the end, the larvae of each group were cleaned, and the larvae of the blank control group and the model group were placed in fresh culture water respectively, and the larvae of the sample groups with different concentrations were placed in the culture water containing the samples to be tested at different concentrations. The above-mentioned groups of zebrafish were placed in a constant temperature incubator at 28.5°C, and continued to act for 1 day. After the treatment, the zebrafish were anesthetized with 0.2% ethyl m-aminobenzoate methanesulfonate, observed and photographed under a fluorescent microscope. The number of inflammatory cells showing green fluorescence in the zebrafish gut was counted.

Experimental results:

1.1 Anti-inflammatory effect of Epimedin B on _CuSO4 -induced acute neuroinflammation in zebrafish

As shown in Figure 7 and Figure 8, compared with the blank control group, the number of inflammatory cells migrating to the lateral line in the CuSO ₄ model group zebrafish was significantly increased, and copper sulfate caused an inflammatory response in the zebrafish. Compared with the copper sulfate model group, the number of inflammatory cells migrating to the lateral line in the zebrafish body of the positive drug (ibuprofen) group and the epihudine B administration group was significantly reduced. With the increase of the dose concentration of Epimedin B, the number of inflammatory cells migrating to the lateral line also decreased. 73.5%. It shows that Epimedin B has obvious anti-inflammatory effect on CuSO ₄ -induced acute neuroinflammation in zebrafish.

1.2 Anti-inflammatory effect of Epimedin B on local inflammation induced by tail docking in zebrafish

As shown in Figure 9 and Figure 10, compared with the blank control group, the number of inflammatory cells in the docked tail of zebrafish in the docked model group increased. Compared with the docked tail model group, the number of inflammatory cells in the tail docked part of zebrafish decreased with the increase of the dose of epimedin B. It shows that Epimedin B has anti-inflammatory effect on the local inflammation induced by tail docking in zebrafish.

1.3 Epimedin B has no anti-inflammatory effect on inflammatory bowel disease caused by TNBS

As shown in Figure 11 and Figure 12, compared with the control group, the number of intestinal inflammatory cells in the TNBS model group increased significantly. Compared with the model group, there was no significant change in the number of inflammatory cells in the intestinal tract of zebrafish in each concentration group of Epimedin B. It shows that Epimedin B has no anti-inflammatory effect on inflammatory bowel disease caused by TNBS.

From the above experimental results, it can be seen that chaohouding B has anti-inflammatory effect on the inflammatory model induced by CuSO ₄ , and has anti-inflammatory effect on the local inflammation of zebrafish induced by tail docking, but has no effect on the inflammatory bowel disease induced by TNBS. It can be proved that the anti-inflammatory effect of the same drug on different inflammatory models is not related; it further proves that the neuroinflammatory model caused by CuSO ₄ and the inflammatory bowel disease caused by TNBS are different in mechanism and inflammatory effect. No relevance.

The invention discovers for the first time that pure green penicillol can treat inflammatory bowel disease, increases the new use of pure green penicillol, provides a new treatment method for the treatment of inflammatory bowel disease, and also develops the application and development of pure green penicillol for reference.

Claims (5)

1. the application of pure viridillol in the preparation of medicine for the treatment of inflammatory bowel disease, wherein the structural formula of pure viridillol is as follows:

2 . The application according to claim 1 , characterized in that the drug can treat inflammatory bowel disease by activating the expression of pparγ and regulating MAPK and NF-κB signaling pathways. 3. A medicine for treating inflammatory bowel disease, characterized in that the medicine contains pure green penicillol. 4. The medicine according to claim 3, characterized in that the medicine contains pharmaceutically acceptable carriers and or auxiliary materials. 5. The medicine according to claim 3, wherein the dosage form of the medicine is tablet, capsule, granule, micropill, drop pill, oral liquid, water injection, powder injection, transfusion, ointment formulations, gels or microemulsions.

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