CN112569214A - Application of diallyl sulfide in preparation of medicines for preventing and/or treating hepatic fibrosis and liver cancer - Google Patents
Application of diallyl sulfide in preparation of medicines for preventing and/or treating hepatic fibrosis and liver cancer Download PDFInfo
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Abstract
The invention provides application of diallyl sulfide in preparation of a medicament for preventing and/or treating hepatic fibrosis and liver cancer, belonging to the technical field of medicaments. The invention firstly proves that DAS can obviously improve hepatic fibrosis and liver cancer, and the DAS can obviously reduce the collagen fiber hyperplasia of the mouse liver, reduce the liver tumor cell profile, obviously reduce the hepatocellular carcinoma area, lead the hepatic lobule structure to be regular, lead the arrangement of the hepatic cells to be more regular and orderly and reduce the number of nodules on the surface of the liver. From the biochemical indexes, DAS can obviously reduce ALT, AST, gamma-GT activity and AFP activity rising phenomenon caused by hepatic fibrosis and liver cancer, and presents a certain dosage dependence relationship. The invention lays a foundation for further development and utilization of DAS as a natural medicine raw material, in particular as a raw material of medicines for preventing and/or treating liver diseases such as hepatic fibrosis and liver cancer.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to application of diallyl sulfide in preparation of medicines for preventing and/or treating hepatic fibrosis and liver cancer.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Liver cancer accounts for about 63 million new cases worldwide each year, with morbidity and mortality rates at the fifth and third sites of malignancy, respectively. Wherein hepatocellular carcinoma (HCC) accounts for 83.9-92.3% of the total number of liver cancer in China, and 70-80% of HCC patients have liver cirrhosis, while liver fibrosis is a common way for abnormal proliferation of connective tissues in liver and development of liver cirrhosis caused by various chronic liver diseases and liver injury. Therefore, inhibiting liver fibrosis may become an important potential target for inhibiting liver cancer progression.
Diallyl sulfide (DAS) is an important sulfhydryl-containing compound in garlic oil, and studies show that DAS has various biological effects such as anti-inflammation, anti-oxidation, and inhibition of various metabolic enzymes in vivo. And compared with diallyl disulfide (DADS) and diallyl trisulfide (DATS) which are also extracted from garlic, the compound has lower toxicity and simpler structure, thereby having wider application prospect. Early studies found that DAS is a carbon tetrachloride (CCl)4) The induced fibrosis has obvious inhibition effect, however, the effect of DAS on the intervention of the occurrence and development of liver cancer is not reported.
Disclosure of Invention
The invention provides application of diallyl sulfide (DAS) in preparation of a medicine for preventing and/or treating liver fibrosis and liver cancer, and the DAS is used for diethyl nitrosamine (DEN) and carbon tetrachloride (CCl)4) CoupletThe induced hepatic fibrosis-liver cancer, the changes of body weight, liver weight and liver coefficient are evaluated, the content of Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), gamma-glutamyl transpeptidase (gamma-GT) and tumor marker alpha-fetoprotein (AFP) in serum is detected, and the pathological histology and hepatic fibrosis of the liver are examined. The results show that the DAS remarkably inhibits DEN and CCl4The induced liver fibrosis and liver cancer progress can obviously relieve and improve liver fibrosis and liver cancer related symptoms, so the derivative can be used as an effective component for preparing related medicines for preventing and/or treating liver fibrosis and liver cancer, and has good practical application value.
In a first aspect of the present invention, the present invention provides use of DAS as an active ingredient in the preparation of a medicament for preventing and/or treating liver fibrosis and liver cancer.
The liver cancer is chemical induced liver cancer, and further is DEN-CCl4Combined application of the above two drugs for treating liver cancer.
In the second aspect of the invention, the invention also provides the application of DAS as an effective component in preparing medicines for relieving and improving liver collagen fiber hyperplasia, liver cell arrangement disorder and liver tumor cell heterogeneity and/or reducing liver cancer variable area and liver surface nodule number.
In a third aspect of the invention, the invention also provides the use of DAS as an active ingredient in the manufacture of a medicament for improving hepatic lobular structure.
In a fourth aspect of the present invention, the present invention provides a pharmaceutical composition for preventing and/or treating liver fibrosis and liver cancer, which comprises DAS as an active ingredient.
Compared with the prior art, the method has the following advantages and progressions:
the invention firstly proves that DAS can obviously improve hepatic fibrosis and liver cancer, and the DAS can obviously reduce the collagen fiber hyperplasia of the mouse liver, obviously reduce the hepatocellular carcinoma zone, reduce the liver tumor cell profile, lead the liver lobule structure to be regular, lead the arrangement of the liver cells to be more regular and orderly, reduce the number of nodules on the surface of the liver and the like. From the biochemical indexes, DAS can obviously reduce ALT, AST, gamma-GT activity and AFP activity rising phenomenon caused by hepatic fibrosis and liver cancer, and presents a certain dosage dependence relationship.
The invention lays a foundation for further development and utilization of DAS as a natural medicine raw material, in particular as a raw material of medicines for preventing and/or treating liver diseases such as hepatic fibrosis and liver cancer.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a mouse liver image of a normal control group, a liver fibrosis liver cancer model group, a DAS low dose group, a DAS medium dose group and a DAS high dose group in example 1 of the present invention;
FIG. 2 is a graph of H & E staining results (x 20) of liver tissue sections of mice in a normal control group, a liver fibrosis and liver cancer model group, a DAS low dose group, a DAS medium dose group and a DAS high dose group according to example 1 of the present invention; wherein, T: tumor region NT: a non-tumor region.
FIG. 3 is a graph showing the results of sirius red staining of liver tissue sections of mice in the normal control group, liver fibrosis and liver cancer model group, DAS low dose group, DAS medium dose group and DAS high dose group in example 1 of the present invention (X10).
FIG. 4 is a graph showing AFP staining results (X20) of liver tissue sections of mice in a normal control group, a liver fibrosis and liver cancer model group, a DAS low dose group, a DAS medium dose group and a DAS high dose group in example 1 of the present invention.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
In one exemplary embodiment of the present invention, there is provided a use of DAS as an active ingredient in the preparation of a medicament for preventing and/or treating liver fibrosis and liver cancer.
In another embodiment of the present invention, the application is an application of DAS as the only active ingredient or one of the active ingredients in preparing a medicament for preventing and/or treating liver fibrosis and liver cancer.
In yet another embodiment of the present invention, the liver cancer is a chemical-induced liver cancer, preferably DEN-CCl4Liver cancer induced by combination; bw, 25mg/kg per dose of DEN; CCl4Bw of 1ml/kg.
In another embodiment of the present invention, in the above application, the DAS single effective dose is 10 to 50 mg/kg.bw; still further, a single effective dose of said DAS comprises 20mg/kg.bw, 40 mg/kg.bw. Experiments prove that DAS obviously improves hepatic fibrosis and liver cancer and shows a certain dose dependence relationship. The DAS has the most obvious curative effect on relieving and improving liver cancer when the single effective dose is 40mg/kg.
In another embodiment of the present invention, the present invention further provides an application of DAS as an active ingredient in the preparation of a medicament for alleviating and improving collagen fiber proliferation of liver, hepatocyte arrangement disorder, liver tumor cell heterogeneity and/or reducing liver cancer variable area and liver surface nodule number.
In another embodiment of the present invention, the application is an application of DAS as the only active ingredient or one of the active ingredients in the preparation of a medicament for alleviating and improving liver collagen fiber proliferation, hepatocyte arrangement disorder, liver tumor cell heterogeneity and/or reducing liver cancer lesion area and liver surface nodule number.
In another embodiment of the present invention, there is provided a use of DAS as an active ingredient in the preparation of a medicament for improving hepatic lobular structure.
In another embodiment of the present invention, the use is the use of DAS as the sole active ingredient or one of the active ingredients in the manufacture of a medicament for improving hepatic lobular structure.
The improvement of the hepatic lobule structure includes that the hepatic lobule structure tends to be regular, and the hepatic cords tend to be orderly arranged.
In another embodiment of the present invention, in any of the above applications, DAS is used as a pharmaceutically active ingredient, and can be formulated into a pharmaceutically acceptable pharmaceutical preparation.
In still another embodiment of the present invention, the pharmaceutical preparation is selected from the group consisting of a preparation for oral administration, a preparation suitable for mucosa, an injection preparation, an inhalation preparation, and an external preparation.
In yet another embodiment of the present invention, the formulation for oral administration is selected from the group consisting of tablets, pills, powders, granules, capsules.
In another embodiment of the present invention, the present invention provides a pharmaceutical composition for preventing and/or treating liver fibrosis and liver cancer, which comprises DAS as an active ingredient (which may be the only active ingredient or one of the active ingredients).
In yet another embodiment of the present invention, the liver cancer is a chemical-induced liver cancer, preferably DEN-CCl4Combined application of the above two drugs for treating liver cancer.
The pharmaceutical composition for preventing and/or treating liver cancer may further comprise pharmaceutically acceptable auxiliary materials, wherein the auxiliary materials are at least one of diluents, excipients, disintegrants, fillers, binders, lubricants, flavoring agents, surface active agents and stabilizers.
And the composition can also be prepared into pharmaceutically acceptable pharmaceutical preparations.
In still another embodiment of the present invention, the pharmaceutical preparation is selected from the group consisting of a preparation for oral administration, a preparation suitable for mucosa, an injection preparation, an inhalation preparation, and an external preparation.
In yet another embodiment of the present invention, the formulation for oral administration is selected from the group consisting of tablets, pills, powders, granules, capsules.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that the following examples are illustrative of the present invention only and are not intended to limit the scope of the present invention.
Example 1
1 materials and methods
1.1 materials SPF grade ICR mice 30 (male 10, female 20), weighing 18-22g, purchased from Beijing Wittingle laboratory animal technology Co., Ltd (license number: SYXK (Jing) 2012-0036). DAS (W204218), DEN (N0756), Anti-Alpha Fetoprotein (AFP) primary antibody (SAB3500533) supplied by Sigma-Aldrich; CCl4(10006480), available from national drug group chemical agents, Inc.; goat anti-rabbit horseradish peroxidase labeled secondary antibody purchased from sequoia Jinqiao biotechnology limited, beijing; alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), and gamma-glutamyl transferase (gamma-GT) kits, which are available from ningbo meikang biotechnology limited; mouse AFP enzyme-linked immunosorbent assay kit (MAFP00) purchased from R&D systems, Inc.; sirius red staining solution (G1471) was purchased from Beijing Solebao scientific Co.
1.2 animal treatment mice were acclimatized for 7d, and then treated in a male-female ratio of 1: 2, the obtained male mice were randomly divided into 4 groups (n ═ 15): normal control group, liver fibrosis liver cancer model group, DAS low dose intervention group and DAS high dose intervention group. Mice, except for the normal control group, were intraperitoneally injected once on postnatal day 14 or 15 with N-diethylnitrosamine (DEN, 25mg/kg. bw, in physiological saline); the DAS low and high dose intervention group started gavage DAS (20 or 40mg/kg.bw) at 5 weeks of age, 1 time/day, and continued until the end of the experiment; except for the normal control groupAll mice were injected intraperitoneally with CCl at 6 weeks of age4Bw (1ml/kg. in corn oil), 2 times per week for 14 weeks. The general condition of the mice was closely observed and weighed once a week. Stopping the administration of CCl4After 2 weeks, mice were anesthetized and the abdominal aorta bled and the liver removed.
1.3 liver appearance, liver coefficient and number of nodules on the surface of the liver after the mouse liver was removed relatively quickly, photographed, weighed, the liver coefficient (liver weight/body weight x 100) calculated, and the number of nodules on the surface of the liver counted.
1.4 detection of ALT, AST, gamma-GT activity and AFP content in serum after abdominal aorta blood taking, centrifuging (1000g,15min) to separate serum, Beckman AU480 full-automatic biochemical instrument to detect ALT, AST, gamma-GT activity in serum, and using mouse AFP enzyme linked immunosorbent assay kit to detect AFP content in serum.
1.5 pathological examination of each group of mice the livers of the mice were removed rapidly and the left lobes of the livers were fixed with 4% paraformaldehyde solution for 48h and used to prepare paraffin sections 4 μm thick. Hematoxylin and eosin (H & E) staining, observed under Olympus DP74 microscope.
1.6 liver tissue Tianlangsu red collagen staining liver tissue paraffin section after antigen retrieval, staining with Tianlangsu red staining solution for 1h, washing with running water, mounting, and observing with Olympus DP74 microscope.
1.7 immunohistochemical detection of AFP expression in mice of each group paraffin sections were repaired with antigen and then treated with 3% H2O2Blocking normal serum, adding anti-AFP primary antibody (1: 200) diluted with antibody diluent (S3022, Dako Co.), overnight at 4 deg.C, amplifying signal with SP (rabbit IgG) -POD immunohistochemical kit (SP0021, Beijing Soilebao Tech Co., Ltd.), and performing ImmPACTTMDAB peroxidase substrate kit (Vector Laboratories, Inc.) was developed, and nuclei were counterstained with hematoxylin and observed under an Olympus DP74 microscope.
1.8 statistical analysis of the data
Or
One-way ANOVA analysis was performed using SPSS20.0 software and a Least Significant Difference (LSD) pairwise comparison, P<A difference of 0.05 is statistically significant.
2 results
2.1 general status, liver appearance, liver coefficients and number of nodules on the surface of the liver in groups of mice
The diet and activity of each group of mice had no obvious abnormality. Compared with a normal control group, the liver weight and the liver coefficient of the mice in the liver fibrosis and liver cancer model group are obviously increased (P is less than 0.05), and the livers show that white nodules with different sizes are full of; compared with the liver fibrosis and liver cancer model group, the liver weight and liver coefficient of the mice in the DAS intervention group are obviously reduced, the number of nodules is obviously reduced by the liver (P <0.05), and the number of the nodules on the surface of the liver of the mice in the DAS high-dose intervention group is obviously less than that of the liver of the DAS low-dose intervention group (P < 0.05). Specific results are shown in table 1 and fig. 1.
TABLE 1 comparison of fasting body weight, liver weight and liver coefficient for each group of rats
Note: p compared with normal control group<0.05; compared with the DEN model group, the method has the advantages that,#P<0.05; compared with the DAS low-dose intervention group,&P<0.05。
2.2 comparison of ALT, AST, Gamma-GT activity and AFP content in the serum of mice in each group
Compared with a normal control group, the activity of serum liver injury markers ALT and AST, the activity of a precancerous injury marker gamma-GT and the content of a liver cancer marker AFP of a mouse in a liver fibrosis and liver cancer model group are all obviously increased (P is less than 0.05); compared with mice in a liver fibrosis and liver cancer model group, the ALT, AST, gamma-GT activity and AFP content in serum of the mice in a DAS low-dose intervention group and a DAS high-dose intervention group are remarkably reduced (P is less than 0.05). And the content of AFP in the serum of the mouse in the DAS high-dose intervention group is obviously lower than that in the DAS low-dose intervention group (P < 0.05). The specific results are shown in Table 2.
TABLE 2 comparison of ALT, AST, gamma-GT activities and AFP content in serum of mice of each group
Note: p compared with normal control group<0.05; compared with the DEN model group, the method has the advantages that,#P<0.05; compared with the DAS low-dose intervention group,&P<0.05。
2.3 pathological observation of liver in groups of mice
Under a microscope, the liver lobules of the mice of the normal control group are clear in structure, and the liver cells are arranged in a cord shape. The hepatic lobule structure of the mouse in the hepatic fibrosis and hepatic carcinoma model group is completely destroyed, the arrangement of hepatic cells is disordered and has different sizes, a large tumor area with slight cytoplasm staining can be seen, the cell nucleus is deeply stained, and the abnormal cell shape is obviously increased. The liver lobule structure of the mouse in the DAS low-dose intervention group is improved, but the mouse also has an obvious canceration area, and the cellular heterogeneity is reduced; the liver slices of the DAS high-dose pre-treated mice can see more non-tumor areas, the cells are relatively regularly arranged, and the cellular heterogeneity is obviously reduced. The specific results are shown in FIG. 2.
2.4 sirius red staining to detect the condition of hepatic fibrosis of each group of mice:
the results of sirius red staining show that the liver lobule structure of the mice in the normal control group is clear, and the collagen fibers are only limited around the blood vessel wall. The livers of the mice in the liver fibrosis and liver cancer model group have obvious collagen fiber hyperplasia and are mutually connected to form false lobule structures with different shapes; the liver collagen fiber hyperplasia of mice in DAS low and high dose intervention groups is obviously reduced, and a small amount of false leaflet structures are formed.
2.5 immunohistochemical method for detection of AFP expression in mouse liver
To further determine whether the hyperplastic nodules in the liver were HCC, liver cancer marker-AFP expression was examined in the liver by immunohistochemical methods. As can be seen in FIG. 4, there was no specific expression of AFP in the liver of normal control mice; large AFP strong positive staining areas can be seen in liver tissues of the liver fibrosis and liver cancer model group; the liver tissue of mice in DAS low-dose intervention group had significantly reduced AFP-positive staining areas, while DAS high-dose intervention group had only a few scattered AFP-positive staining areas.
It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.
Claims (10)
1. Application of diallyl sulfide as an active ingredient in preparing a medicament for preventing and/or treating hepatic fibrosis and liver cancer.
2. The use according to claim 1, wherein the liver cancer is a chemical-induced liver cancer, preferably DEN-CCl4Combined application of the above two drugs for treating liver cancer.
3. The use according to claim 1, wherein the use is the use of diallyl sulfide as the sole active ingredient or as one of the active ingredients in the manufacture of a medicament for the prevention and/or treatment of liver cancer.
4. The use of claim 1, wherein the single effective dose of diallyl sulfide is 10 to 50 mg/kg.bw; preferably, the single effective dose of diallyl sulfide comprises 20mg/kg.bw or 40 mg/kg.bw.
5. Application of diallyl sulfide as an effective component in preparing medicines for relieving and improving liver collagen fiber hyperplasia, liver cell arrangement disorder and liver tumor cell heterogeneity and/or reducing liver cancer variable area and liver surface nodule number.
6. The use according to claim 5, wherein diallyl sulfide is used as the sole active ingredient or one of the active ingredients in the preparation of a medicament for alleviating and improving liver collagen fibroplasia, liver cell arrangement disorder, liver tumor cell heterogeneity and/or reducing liver cancer lesion area and liver surface nodule number.
7. Application of diallyl sulfide as an active ingredient in preparing a medicament for improving a lobular structure of a liver.
8. Use according to claim 7, wherein diallyl sulfide is used as the sole active ingredient or as one of the active ingredients in the manufacture of a medicament for improving the lobular structure of the liver.
9. The use according to any one of claims 1 to 8, wherein diallyl sulfide is used as a pharmaceutically active ingredient to form a pharmaceutically acceptable pharmaceutical formulation;
preferably, the pharmaceutical preparation is selected from the group consisting of a preparation for oral administration, a preparation suitable for mucosa, an injection preparation, an inhalation preparation, and an external preparation;
further preferably, the formulation for oral administration is selected from the group consisting of tablets, pills, powders, granules, capsules.
10. A pharmaceutical composition for preventing and/or treating hepatic fibrosis and liver cancer, which is characterized in that diallyl sulfide is used as an effective component;
preferably, the pharmaceutical composition further comprises pharmaceutically acceptable auxiliary materials, wherein the auxiliary materials are at least one of diluents, excipients, disintegrants, fillers, binders, lubricants, flavoring agents, surface active agents and stabilizers;
and, the pharmaceutical composition is prepared into a pharmaceutically acceptable pharmaceutical formulation;
preferably, the pharmaceutical preparation is selected from the group consisting of a preparation for oral administration, a preparation suitable for mucosa, an injection preparation, an inhalation preparation, and an external preparation;
preferably, the formulation for oral administration is selected from the group consisting of tablets, pills, powders, granules, capsules.
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