CN105012288A - Composition and application thereof in treatment of liver cancer - Google Patents

Abstract

The present invention provides a composition, including vitamin A or its prodrug, and aplysia; the molar ratio of the two components is: vitamin A or its prodrug: aplysia=1:0.1～3; Vitamin A or a prodrug thereof is preferably β-carotene. The present invention also provides the application of the composition and the composition comprising vitamin A or its prodrug and aplysia in the preparation of medicament for treating liver cancer. The composition of the invention can effectively inhibit the proliferation of liver cancer cells through the synergistic effect of the active components.

Description

A composition and its application for treating liver cancer

technical field

The invention belongs to the field of medicine, and in particular relates to a composition comprising aplysia and vitamin A or its prodrug and its medical application in treating liver cancer.

Background technique

Hepatocellular carcinoma is one of the most common malignant tumors in China, which has the characteristics of insidious onset, high metastasis rate, and poor prognosis. Although surgery is the first choice and the most effective method for the treatment of liver cancer, chemotherapy is one of the main methods of treatment for inoperable patients. Some patients can also get the chance of surgery because of chemotherapy, which prolongs their lives and improves their quality of life. However, while chemotherapy drugs kill tumor cells, they also cause serious damage to normal cells in the body. Therefore, searching for a highly effective and low-toxic anticancer drug has become the focus of attention in recent years.

Aplysia is a brominated sesquiterpene fat-soluble compound isolated and purified from the extract of S. triheliensis, with a molecular weight of 295 and a structural formula as shown in 1. Studies have found that Aplysia has biological activities such as anti-inflammation, antibacterial, anti-oxidation, and anti-tumor, and has inhibitory effects on breast cancer, gastric cancer, and S180 sarcoma (1. Aplysia on human breast cancer SK-BR-3 Research on cell inhibition and mechanism[J]; Ma Wenlong, Liang Hui, et al.; Natural Product Research and Development, 2012, 24(9): 120l-1205. 2. Aplysia on the proliferation and apoptosis of human gastric cancer cell line SGC 7901 Liu Ying, Liang Hui, et al.; Chinese Pharmacology Bulletin, 2010, 26(3): 333-337. 3. The effect of Aplysia on tumor-inhibitory activity and immune effect of S180 tumor-bearing mice Experimental observation [J]; Liu Ying, Liang Hui, etc.; Chinese Pharmacology Bulletin, 2006, 22(11): 1403-1405.)

β-Carotene is one of the carotenoids with a molecular weight of 537 and a structural formula as shown in 2. It has the functions of anti-oxidation, protecting eyesight, and promoting bone growth and development. Beta-carotene is a prodrug of vitamin A; however, the body converts beta-carotene into vitamin A only when needed. This feature makes β-carotene a safe source of vitamin A, without the accumulation of vitamin A poisoning caused by excessive intake.

So far, there is no research report on the synergistic effect of aplysia and β-carotene on liver cancer.

Contents of the invention

In view of the above problems, an object of the present invention is to provide a composition comprising aplysatin and vitamin A or its prodrug, especially aplysatin and β-carotene. The two active components in the composition work synergistically, have significant anti-liver cancer activity, and are expected to develop new anti-liver cancer drugs, providing doctors and patients with a new treatment option.

In order to realize the foregoing invention object, the present invention adopts following technical scheme:

A composition, comprising vitamin A or its prodrug, and Aplysatin; The molar ratio of the two components is:

Vitamin A or its prodrug: Aplysia = 1:0.1~3.

Preferably, in the composition, the molar ratio of the two components is:

Vitamin A or its prodrug: Aplysia = 1:0.15-2.6.

More preferably, in the composition, the molar ratio of the two components is:

Vitamin A or its prodrug: Aplysia = 1:0.2~2.

In the above composition, the vitamin A or its prodrug is preferably β-carotene.

Another object of the present invention is the application of the above composition in the preparation of medicaments for treating liver cancer.

The present invention also provides an application of a composition comprising vitamin A or its prodrug and aplysia in the preparation of a drug for treating liver cancer.

The vitamin A or its prodrug is preferably β-carotene.

In the above application, the liver cancer refers to primary liver cancer or secondary liver cancer.

Another object of the present invention is to provide a drug for treating liver cancer, including the above composition.

Preferably, the medicine may also include pharmaceutically acceptable auxiliary materials.

Preferably, the medicine is an oral preparation, selected from one of tablets, capsules, dropping pills, granules and oral liquids.

Another object of the present invention is to provide a preparation method of the above-mentioned medicine for treating liver cancer, comprising mixing the composition with pharmaceutically acceptable adjuvants, and preparing a clinically acceptable preparation according to conventional methods in the art.

The pharmaceutically acceptable adjuvant of the present invention includes (1) diluent, such as starch, powdered sugar, dextrin, lactose, pregelatinized starch, microcrystalline fiber, inorganic calcium salt (such as calcium sulfate, calcium hydrogen phosphate, pharmaceutical calcium carbonate, etc.), mannitol, etc., vegetable oil, polyethylene glycol, etc.; (2) binders, such as distilled water, ethanol, starch slurry, sodium carboxymethylcellulose, hydroxypropylcellulose, methylcellulose (3) Disintegrants, such as dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, cross-linked carboxymethyl Sodium cellulose, etc.; (4) lubricants, such as magnesium stearate, micronized silica gel, talcum powder, hydrogenated vegetable oil, polyethylene glycols, magnesium lauryl sulfate, etc. (5) Flavoring agents, such as sucrose, stevia, etc.

When used for treating liver cancer, the composition of the present invention is administered to humans or mammals. For this purpose, the quality of ingestion or the quality of administration of the composition of the present invention varies depending on the purity of the components in the composition, the age, weight, etc. of the subject to be administered. For the treatment of liver cancer, based on the adult body weight of 70kg, usually 150-200 mg per person per day, more preferably 160-180 mg per person per day.

β-carotene is a prodrug of vitamin A, and its physiological activity is basically the same as that of vitamin A. Therefore, in the research on the activity of the composition in the present invention, β-carotene is used as the representative of the "vitamin A or its prodrug".

The study found that when β-carotene and Aplysia were used in combination, both in vitro and in vivo, they showed a synergistic effect on inhibiting the proliferation of liver cancer cells.

When the effects of β-carotene and aplysia on the proliferation of human liver cancer HepG2 cell lines were detected by CCK-8 method in vitro, the IC ₅₀ values of β-carotene or aplysia in HepG2 cells alone for 24 hours were (86.50± 6.98) μmol/L and (100.05±8.24) mg/L. When Aplysia was combined with β-carotene 20, 40, 60 μmol/L, the IC ₅₀ were A(80.97±7.25)mg/L, B(21.46±1.88)mg/L and C(18.91±1.42)mg/L respectively . With the increase of the dose of β-carotene, the IC ₅₀ of Aplysia decreased gradually. In the diagram of the relationship between β-carotene and Aplysia in vitro as shown in Figure 1, the 95% confidence interval of point A intersects with the additive line, indicating that the two drugs produce additive effects, while points B and C, 95 The % credible interval does not overlap with the addition line and is on the left side, indicating that the two drugs produce a synergistic effect, and point B (40 μmol/L β-carotene combined with 21.5 mg/L aplysia) has the strongest synergistic effect. It shows that the composition of the present invention has a synergistic effect of inhibiting the proliferation of liver cancer cells in vitro.

The composition of β-carotene and aplysia in the present invention also shows good anti-tumor synergistic effect in vivo. β-carotene or Aplysia acted alone on tumor-bearing mice (hepatoma tumor strain H ₂₂ ), the IC ₅₀ values were (312.2±38.3) mg/kg and (146.5±16.7) mg/kg, respectively. When aplysia was combined with β-carotene 100, 150, and 200 mg/kg, the IC ₅₀ were A (108.1±12.0) mg/kg, B (35.0±3.6) mg/kg and C (28.7±1.8) mg/kg, respectively . With the increase of the dose of β-carotene, the IC ₅₀ of Aplysia decreased gradually. In the diagram of the relationship between β-carotene and Aplysia in vivo as shown in Figure 2, there are three points in the combined drug group, and the 95% confidence interval of point A intersects with the addition line, indicating that the two drugs produce additive effects, while B Points and C, the 95% confidence interval does not overlap with the summation line and is on the left, indicating that the two drugs produce a synergistic effect, and point B (150mg/kg β-carotene combined with 35.0mg/kg aplysia) The synergistic effect is the strongest. It shows that the composition of the present invention has significant synergistic effect against liver cancer in vivo.

The above in vitro and in vivo experiments have proved that aplysia and vitamin A represented by β-carotene or its prodrug has a clear anti-hepatic effect, and there is a synergistic effect between them, which can exert a significant anti-cancer effect at a relatively small dose. tumor effect. Preliminary mechanism studies also show that the composition of the present invention can significantly down-regulate the expression of MMP-9, VEGF and PCNA in tumor tissue, and significantly increase the levels of IL-6 and TNF-α in the serum of tumor-bearing mice, suggesting that β-carrot Aplysia and aplysia may play a synergistic anti-hepatocarcinoma effect by inhibiting angiogenesis and metastasis, promoting the secretion of immune inflammatory factors, and enhancing the immune function of the body. Therefore, the composition of the present invention is expected to be developed into an effective and low-toxic anti-liver cancer drug, thereby benefiting patients.

Description of drawings

Below, describe embodiment of the present invention in detail in conjunction with accompanying drawing, wherein:

Figure 1 shows the interaction relationship between β-carotene and Aplysia in Test Example 1, where the solid oblique line is the sum of the two effects, and the part between the dotted lines is the 95% confidence interval of point A . When point A is 20 μmol/L of β-carotene, the ED ₅₀ of aplysatin; when point B is 40 μmol/L of β-carotene, the ED ₅₀ of aplysatin; when point C is 60 μmol/L of β-carotene, ED ₅₀ of Aplyssatin.

What Fig. 2 shows is the influence of each treatment group on the apoptosis of HepG2 cells in Test Example 2, wherein,

A: control group; B: 21.5mg/L aplysia; C: 40μmol/L β-carotene; D: 40μmol/L β-carotene combined with 21.5mg/L aplysia.

Figure 3 shows the interaction relationship between β-carotene and Aplysia in Test Example 3, where the solid oblique line is the sum of the two effects, and the part between the dotted lines is the 95% confidence interval of point A . Point A is when β-carotene is 100mg/kg, the ED ₅₀ of aplysia; point B is when β-carotene is 150mg/kg, the ED ₅₀ of aplysia; point C is when β-carotene is 200mg/kg, ED ₅₀ of Aplyssatin.

The photograph of Fig. 4 is the tumor tissue section through optical microscope magnification 200 times, what show is the influence of the medicine in test example 4 on the expression of MMP-9 in mouse _H22 tumor tissue, wherein,

A: model group; B: 150mg/kg β-carotene intervention group; C: 35.0mg/kg aplysiathin intervention group; D: 150mg/kg β-carotene and 35.0mg/kg aplysatin intervention group.

The photograph of Fig. 5 is the tumor tissue section through optical microscope magnification 200 times, what show is the influence of the medicine in the test example 4 on mouse _H22 tumor tissue VEGF expression, wherein,

A: model group; B: 150mg/kg β-carotene intervention group; C: 35.0mg/kg aplysiathin intervention group; D: 150mg/kg β-carotene and 35.0mg/kg aplysatin intervention group.

The photograph of Fig. 6 is the tumor tissue section through optical microscope magnification 200 times, what show is the influence of the medicine in test example 4 on mouse _H22 tumor tissue PCNA expression, wherein,

A: model group; B: 150mg/kg β-carotene intervention group; C: 35.0mg/kg aplysiathin intervention group; D: 150mg/kg β-carotene and 35.0mg/kg aplysatin intervention group.

Detailed ways

The present invention will be described below with reference to specific examples. Those skilled in the art can understand that these examples are only used to illustrate the present invention and do not limit the scope of the present invention in any way.

The experimental methods in the following examples are conventional methods unless otherwise specified. The medicinal raw materials, reagent materials, etc. used in the following examples are all commercially available products unless otherwise specified.

Among them, Aplysia was extracted and purified from S. triretiformis by the Institute of Medical Nutrition, Qingdao University School of Medicine, and identified by the Institute of Oceanology, Chinese Academy of Sciences. Its molecular formula is C ₁₅ H ₁₉ OBr, and its molecular weight is 295.

Embodiment 1 a kind of composition

Raw materials (molar ratio):

β-carotene: Aplysia = 1:3

Embodiment 2 a kind of composition

Raw materials (molar ratio):

β-carotene: Aplysia = 1:2.6

Embodiment 3 a kind of composition

Raw materials (molar ratio):

β-carotene: Aplysia = 1:2

Embodiment 4 a kind of composition

Raw materials (molar ratio):

β-carotene: Aplysia = 1:1.69

Embodiment 5 a kind of composition

Raw materials (molar ratio):

β-carotene: Aplysia = 1:0.43

Embodiment 6 A kind of composition

Raw materials (molar ratio):

β-carotene: Aplysia = 1:0.15

Embodiment 7 a kind of composition

Raw materials (molar ratio):

β-carotene: Aplysia = 1:0.1

Embodiment 8 A kind of tablet

Get the composition described in Example 4 and mix it with an appropriate amount of starch evenly, and compress it into tablets.

A kind of capsule of embodiment 9

Take the composition described in Example 7 and mix it with an appropriate amount of starch evenly, wet granulate, granulate, and pack into capsules to obtain

Test Example 1 CCK-8 method to detect the effect of β-carotene and aplysia on the proliferation activity of liver cancer cell lines in vitro

1. Experimental method

1.1 Cell culture

Human liver cancer HepG2 cells were cultured in DMEM high-glucose full medium (containing 10% fetal bovine serum) at 37°C in a 5% CO ₂ incubator, the medium was changed every other day, and passaged every three days, and the cells in the logarithmic growth phase were collected conduct experiment. β-carotene and Aplysia are all dissolved with dimethyl sulfoxide (DMSO), and are diluted to final concentration with DMEM high-sugar full culture solution during use, and the final concentration of DMSO in all treatment groups is 0.1% (experiments confirm that this concentration has a significant impact on cells without obvious damage).

1.2 Determination of IC ₅₀

Liver cancer HepG2 cells in the logarithmic growth phase were seeded in 96-well plates at 5×10 ³ /well, and after 24 hours of culture, they were divided into the following experimental groups:

Group A: Add β-carotene culture solution with final concentrations of 20, 40, 60, 80, 100, and 120 μmol/L respectively;

Group B: Add Aplysia culture solution with final concentrations of 20, 40, 60, 80, 100, 120 mg/L respectively;

Group C: Add β-carotene with a final concentration of 20 μmol/L and Aplysia culture solution with a final concentration of 70, 80, and 90 mg/L respectively;

Group D: add β-carotene with a final concentration of 40 μmol/L and Aplysia culture solution with a final concentration of 10, 20, and 30 mg/L;

Group E: Add β-carotene with a final concentration of 60 μmol/L and Aplysia culture solution with a final concentration of 5, 10, and 20 mg/L respectively;

Group F: control group, culture solution containing equal volume of PBS and 0.1% DMSO was added.

Each group has 6 replicate wells. After adding the drug solution, continue to cultivate for 24 hours, discard the supernatant, add 100 μl of fresh culture medium and 10 μl of CCK-8 solution to each well, continue to cultivate for 2 hours, measure the OD value of each well at 450 nm in a microplate reader, and the size of the OD value and cell activity positively correlated. Calculation of _IC50 . Experiments were repeated three times.

2. Relevant standards

2.1 Establishment of Effective Standards for Tumor Inhibitory Effect

Compared with the control group, the tumor cell inhibition rate > 50% in the experimental group is used as the effective standard for the anti-tumor effect of the drug.

2.2 Criteria for judging the anti-tumor effect of combined application of β-carotene and aplysia

Calculate the 50% inhibition rate (IC ₅₀ ) of tumor cells with β-carotene or aplysiastatin alone. Taking the IC ₅₀ value of β-carotene on the abscissa and the IC ₅₀ value of Aplysia on the y-coordinate, draw a diagram of the interaction between β-carotene and Aplysia: the calculated IC of β-carotene alone ₅₀ is marked on the X-axis, IC ₅₀ of Aplysia alone is marked on the Y-axis, and the two points are connected to form the action summation line. Mark the IC ₅₀ of the combined application of β-carotene and Aplysia at different doses on the coordinate axis, and mark its 95% confidence interval at the same time. If the IC ₅₀ falls on the left side of the addition line, the two drugs have a synergistic effect , if it falls on the right side of the addition line, the two drugs are antagonistic, and if they fall on the addition line, they are additive.

3. Results and Discussion

3.1 The effect of drugs on the proliferation of HepG2 cells and the interaction of β-carotene and Aplysia in anti-tumor effect

The results of CCK-8 showed that after different doses of β-carotene or Aplysia acted on HepG2 cells alone for 24 hours, compared with the control group, the growth and proliferation of tumor cells in each group were significantly inhibited in a dose-dependent manner (P<0.05 ). The IC ₅₀ values of β-carotene or Aplysia in HepG2 cells for 24 hours were (86.50±6.98) μmol/L and (100.05±8.24) mg/L, respectively. When Aplysia was combined with β-carotene 20, 40, 60 μmol/L, the IC ₅₀ were A(80.97±7.25)mg/L, B(21.46±1.88)mg/L and C(18.91±1.42)mg/L respectively . With the increase of the dose of β-carotene, the IC ₅₀ of Aplysia decreased gradually. The specific results are shown in Table 1.

In the interaction diagram of β-carotene and Aplysia shown in Figure 2, there are three points in the combined drug group, and the 95% confidence interval of point A intersects with the addition line, indicating that the two drugs produce additive effects, while B Point and C, the 95% confidence interval does not overlap with the summation line and is on the left, indicating that the two drugs produce a synergistic effect, and point B (40μmol/L β-carotene combined with 21.5mg/L Aplysia) The synergistic effect is the strongest.

Table 1 Effects of β-carotene and Aplysia on the proliferation of HepG2 cells

*: Compared with the control group, P<0.05.

Test Example 2 Effect of β-Carotene and Aplysia on HepG2 Cell Apoptosis Synergistically

1. Experimental method

1.1 Cell culture

Same as "Cell Culture" under item 1.1 of Test Example 1.

1.2 Effect of drugs on HepG2 cell apoptosis

Liver cancer HepG2 cells in the exponential growth phase were respectively treated with 1) control group (culture solution containing equal volume of PBS and 0.1% DMSO), 2) 40 μmol/L β-carotene, 3) 21.5 mg/L aplysia, 4) 40 μmol /L β-carotene combined with 21.5 mg/L Aplysia, after 24 hours of treatment, the cells were digested with trypsin and collected, washed with PBS, and the cell density was adjusted to 1×10 ⁶ /ml. Pipette 100 μl of cell suspension from each treatment group into a special analysis tube for flow cytometry, add 5 μl FITC Annexin V and 5 μl PI solution, gently pipette evenly, and incubate at room temperature in the dark for 15 minutes. Add 400 μl 1× buffer, and analyze cell apoptosis by flow cytometry. Normal unstained cells were used to debug the instrument, and FITC Annexin V and PI single-stained cells were used to adjust the fluorescence compensation and define the quadrants of normal and apoptotic cells. The data was analyzed by WinMDI2.9 software, and the percentage of total apoptotic cells was equal to that in the middle and late stage The sum of the percentages of apoptotic cells (FITC Annexin V ⁺ /PI ⁺ ) and early apoptotic cells (FITC Annexin V ⁺ /PI ^- ). Experiments were repeated three times.

2. Results and Discussion

Figure 2 shows the effect of each treatment group on the apoptosis of HepG2 cells.

The results showed that after HepG2 cells were treated with 40μmol/L β-carotene, 21.5mg/L Aplysia, and 40μmol/L β-carotene and 21.5mg/L Aplysia for 24 hours, 40μmol/L β-carotene and 21.5mg/L Aplysatin group HepG2 cell apoptosis rate were (13.8±1.6)% and (3.0±0.1)% respectively, while 40μmol/L β-carotene and 21.5mg/kg Aplysatin combined group, The apoptosis rate of HepG2 cells was (58.2 ± 1.6)%, which was significantly higher than the apoptosis rate of the control group (0 ± 0.2)% and the single use of 40μmol/L β-carotene or 21.5mg/kg Aplysia treatment, there was a significant difference (P<0.05).

Test Example 3 Effects of β-carotene and Aplysia on _H22 Transplanted Tumor Mice

1. Experimental method

1. Preparation of 1H ₂₂ tumor cell suspension

The _H22 ascites-type liver cancer breed mice were killed by neck dislocation, the ascites was extracted, and the density of the cell suspension was adjusted to 2×10 ⁶ /mL, and the viable cell rate >95% was used for the experiment when checked with trypan blue.

1.2 Grouping and handling of animals

After Kunming mice were fed for one week, the _H22 tumor cell suspension was inoculated subcutaneously in the axilla of the left forelimb of the mice, with a dose of 0.2 mL, and the whole operation was required to be completed within half an hour. After 24 hours, all mice were randomly divided into 6 groups according to body weight, which were:

1) Model group: There is 1 subgroup, and soybean oil is administered into the stomach;

2) β-carotene intervention group: 3 subgroups were given intragastric administration of 200, 300, and 400 mg·kg ^-1 ·d ^-1 β-carotene respectively;

3) Aplysia intervention group: 3 subgroups were given 50, 100, and 150 mg·kg ^-1 ·d ^-1 aplysia by intragastric administration;

4) Drug combination intervention group 1: 3 subgroups were given β-carotene 100 mg·kg ^-1 ·d ^-1 + aplysia 90, 110, 130 mg·kg ^-1 ·d ^-1 by intragastric administration;

5) Drug combination intervention group 2: There are 3 subgroups, which are given β-carotene 150 mg·kg ^-1 ·d ^-1 + aplysia 20, 40, 60 mg·kg ^-1 ·d ^-1 by intragastric administration;

6) Drug combination intervention group 3: There are 3 subgroups, which are given 200 mg/kg·bw of β-carotene+10, 20, and 30 mg·kg ^-1 ·d ^-1 of aplysia by intragastric administration respectively.

There were 10 animals in each group. The mice in each group were gavaged continuously for 14 days from the 24h of the transplanted tumor. After the last gavage, they were fasted for 24h and their body weight was measured. Mice were sacrificed by cervical dislocation, dissected under sterile conditions, the tumor tissue was completely stripped, and weighed accurately.

2. Establishment of tumor models and effective standards for drug anti-tumor effects

The subcutaneous growth of tumor tissue in the left forelimb armpit of the mice in the model group was used as the establishment standard of the tumor model. Compared with the model group, the tumor inhibition rate > 50% (IC ₅₀ ) in the drug group was used as the effective standard for the anti-tumor effect of the drug.

3. Criteria for judging the anti-tumor effect of combined application of β-carotene and aplysia

Calculate the 50% inhibition rate (IC ₅₀ ) of tumor cells with β-carotene or aplysiastatin alone. Taking the IC ₅₀ value of β-carotene on the abscissa and the IC ₅₀ value of Aplysia on the ordinate, draw the interaction diagram between β-carotene and Aplysia: mark the IC ₅₀ of β-carotene alone on the On the X-axis, the IC ₅₀ of Aplysia alone is marked on the Y-axis, and the connection of two points is the action summation line. Mark the IC ₅₀ of the combined application of β-carotene and Aplysia at different doses on the coordinate axis, and mark its 95% confidence interval at the same time. If the IC ₅₀ falls on the left side of the addition line, the two drugs have a synergistic effect , if it falls on the right side of the addition line, the two drugs are antagonistic, and if they fall on the addition line, they are additive.

4. Results and Discussion

4.1 The inhibitory effect of drugs on _H22 liver cancer xenografts and the interaction of β-carotene and aplysia in anti-tumor effect

The tumor mass of the tumor-bearing mice in the model group increased significantly, while the tumor growth of the mice in each drug intervention group was inhibited, and with the increase of the drug dose, the tumor mass gradually decreased, showing a dose-effect relationship. Compared with the model group, the differences were significant. Significant (P<0.05). β-carotene or Aplysia acted alone on tumor-bearing mice, the IC ₅₀ values were (312.2±38.3) mg/kg and (146.5±16.7) mg/kg, respectively. When aplysia was combined with β-carotene 100, 150, and 200 mg/kg, the IC ₅₀ were A (108.1±12.0) mg/kg, B (35.0±3.6) mg/kg and C (28.7±1.8) mg/kg, respectively . With the increase of the dose of β-carotene, the IC ₅₀ of Aplysia decreased gradually. See Table 2 for specific data.

Table 2 Antitumor effects of different doses of β-carotene and Aplysia on _H22 tumors ( n=10)

*: Compared with the model group, P<0.05.

In the interaction diagram of β-carotene and Aplysia shown in Figure 3, there are three points in the combined drug group, and the 95% confidence interval of point A intersects with the addition line, indicating that the two drugs produce additive effects, while B Points and C, the 95% confidence interval does not overlap with the summation line and is on the left, indicating that the two drugs produce a synergistic effect, and point B (150mg/kg β-carotene combined with 35.0mg/kg aplysia) The synergistic effect is the strongest.

Test Example 4 Effect of β-carotene and Aplysia synergistically on mice with _H22 transplanted tumor

1. Experimental method

1. Preparation of 1H ₂₂ tumor cell suspension

According to the method described under "1.1" of Test Example 3, the _H22 tumor cell suspension was prepared.

1.2 Grouping and handling of animals

After Kunming mice were fed for one week, the _H22 tumor cell suspension was inoculated subcutaneously in the axilla of the left forelimb of the mice, with a dose of 0.2 mL, and the whole operation was required to be completed within half an hour. After 24 hours, all mice were randomly divided into 4 groups according to body weight, which were:

1) Model group: intragastric administration of soybean oil for 14 days;

2) β-carotene intervention group: β-carotene 150mg/kg, orally;

3) Aplysia intervention group: Aplysia 35.0 mg/kg, orally administered;

4) Cooperative intervention group: β-carotene 150mg/kg and aplysia 35.0mg/kg, orally administered.

There were 10 animals in each group, and the mice in each group were gavaged continuously for 14 days. After the last gavage, they were fasted for 24 hours, weighed their body weight, collected whole blood from the eyeballs, and separated the serum for cytokine detection. Mice were sacrificed by cervical dislocation, dissected under sterile conditions, the tumor tissue was completely stripped, and weighed accurately. Tumor tissue blocks were fixed with 10% neutral formaldehyde buffer for tissue sectioning.

1.3 Immunohistochemical method to detect the expression of MMP-9, VEGF and PCNA in tumor tissue

The tumor tissues were fixed and dehydrated in 10% neutral formaldehyde; transparent in xylene, embedded in paraffin, and serially sectioned with a thickness of 4 nm. Sections were dewaxed with xylene, rinsed with gradient ethanol, and washed twice with distilled water for 5 min each time. After soaking in 3% hydrogen peroxide solution for 10 minutes, wash with distilled water 3 times, 2 minutes each time. The slices were placed in EDTA antigen retrieval solution (0.01M, PH=8.0), heated to 95°C by microwave for 20min, and then cooled to room temperature. Wash 3 times with 0.01M PBS, 2min each time. The corresponding primary antibodies were added dropwise on the slices, incubated in a 37°C incubator for 70 minutes, and then washed with PBS containing 0.05% Tween-20 for 3 times, 2 minutes each time. PV6000 secondary antibody was added dropwise, incubated at 37°C for 20 min, and washed 3 times with PBS containing 0.05% Tween-20, 2 min each time. Use freshly prepared DAB solution to develop color for 5 minutes, and observe the color development result under a microscope. After rinsing with distilled water, counterstain with hematoxylin, then dehydrate and dry the counterstained sections with ethanol, make them transparent with xylene, and seal with neutral gum. 5 slices were randomly selected in each group, and 10 fields of view were randomly selected for each slice, and observed under an optical microscope (×400). Positive judging criteria: MMP-9 and VEGF are positive cells when the cytoplasmic staining is brownish yellow; while for PCNA, brownish yellow granular staining in the nucleus is the positive judging standard.

1.4 ELISA test to detect the concentration of IL-6 and TNF-α in serum of tumor-bearing mice

The mouse serum was separated and stored at -80°C for future use. The kit is stored at 2-8°C and equilibrated at room temperature for 20 minutes before use. Set the standard hole, sample hole and blank hole respectively, add different concentrations of standard substance to the standard hole, add the sample to be tested and sample diluent successively to the sample hole, and not add to the blank hole. Add 100 μl of horseradish peroxidase (HRP)-labeled antibody to each well except the blank well, and incubate in a constant temperature water bath at 37°C for 60 min. Discard the liquid, fill each well with washing solution, let stand at room temperature for 1 min, discard the washing solution, pat dry on absorbent paper, and wash the plate 5 times repeatedly. Add substrates A and B to each well, and incubate at 37°C for 15 minutes in the dark. Add stop solution to each well to terminate the reaction, measure the absorbance (OD value) of each well at a wavelength of 450 nm within 15 minutes, and calculate the concentration of each sample.

2. Results and Discussion

2.1 Effects of β-carotene and Aplysia on the expression of MMP-9, VEGF and PCNA in mouse _H22 tumor tissue

From the microphotographs of the stained tumor tissue sections shown in Figure 4-Figure 6, it can be seen that compared with the model group, the β-carotene intervention group and the aplysia intervention group, the β-carotene and aplysia In the synergistic intervention group, the expression of MMP-9, VEGF and PCNA in tumor tissue were all significantly reduced. It is suggested that β-carotene and Aplysia can act synergistically, which may inhibit the growth of tumor tissue by inhibiting angiogenesis and metastasis.

2.2 Effects of β-carotene and Aplysia on serum levels of IL-6 and TNF-α in _H22 tumor-bearing mice

The results are shown in Table 3. Compared with the model group, β-carotene intervention group and aplysia in the intervention group, the levels of IL-6 and TNF-α in the serum of tumor-bearing mice were significantly increased in the β-carotene and aplysia in the intervention group, suggesting that The synergistic effect of β-carotene and Aplysia may enhance the immune function of the body by promoting the secretion of immune inflammatory factors, thereby exerting an anti-tumor effect.

The influence of table 3 medicine on IL-6, TNF-α level in _H22 mice serum ( n=10)

^* : Compared with the model group, P<0.05;^# : Compared with the β-carotene intervention group, P<0.05; ^▲ : Compared with the Aplysia intervenin group, P<0.05.

The above-mentioned in vivo and in vitro tests prove that the composition provided by the present invention comprising β-carotene and Aplysia has a definite activity of inhibiting the proliferation of liver cancer cells; The active components can produce significant antitumor activity at a relatively small dose. Beta-carotene is a safe source of food coloring and vitamin A, and aplysia is less toxic. The composition formed by them is a liver cancer cell proliferation inhibitor. The above research results also suggest that a safe, low-toxic and effective anti-liver cancer drug can be developed using the composition of the present invention as an active ingredient.

The above description of the specific embodiments of the present invention does not limit the present invention, and those skilled in the art can make various changes or deformations according to the present invention, as long as they do not depart from the spirit of the present invention, all should belong to the scope of the appended claims of the present invention.

Claims (10)

1. A composition, comprising vitamin A or its prodrug, and Aplysatin; The molar ratio of the two components is: Vitamin A or its prodrug: Aplysia = 1:0.1~3. 2. composition according to claim 1, is characterized in that, in described composition, two component mol ratios are: Vitamin A or its prodrug: Aplysia = 1:0.15-2.6. 3. composition according to claim 2, is characterized in that, in described composition, two component mol ratios are: Vitamin A or its prodrug: Aplysia = 1:0.2~2. 4. The composition according to any one of claims 1 to 3, wherein the vitamin A or its prodrug is preferably β-carotene. 5. The use of the composition according to any one of claims 1 to 4 in the preparation of a medicament for treating liver cancer. 6. Application of a composition comprising vitamin A or its prodrug and aplysia in the preparation of a drug for treating liver cancer; The vitamin A or its prodrug is preferably β-carotene. 7. The application according to claim 5 or 6, characterized in that said liver cancer refers to primary liver cancer or secondary liver cancer. 8. A medicine for treating liver cancer, comprising the composition according to any one of claims 1 to 4; Preferably, the medicine may also include pharmaceutically acceptable auxiliary materials. 9. The medicine according to claim 8, characterized in that the medicine is an oral preparation selected from one of tablets, capsules, dropping pills, granules and oral liquids. 10. The preparation method of the medicine for treating liver cancer according to claim 8 or 9, comprising mixing the composition according to any one of claims 1 to 4 with pharmaceutically acceptable adjuvants, and preparing according to conventional methods in the art into clinically acceptable formulations.