阅读说明：本技术 原海葱苷a在制备治疗肝细胞肝癌药物中的用途 (Application of raw onionsoside A in preparation of medicine for treating liver cell and liver cancer ) 是由 谢幼华 罗萌君 刘晶 刘艳丰 潘少坤 于 2020-06-23 设计创作，主要内容包括：本发明属生物医药技术领域,涉及原海葱苷A新的制药用途,具体涉及原海葱苷A(Proscillaridin A)(CAS 466-06-8)在制备治疗肝癌尤其是肝细胞肝癌(HCC)药物中的用途。本发明经实验显示,原海葱苷A能显著抑制肝癌细胞的生长,促进肝癌细胞凋亡,所述的原海葱苷A可作为药物前体制成治疗肝癌尤其是肝细胞肝癌的药物以及制成包含原海葱苷A或者以原海葱苷A作为药物前体的药物和药学上可接受的载体的药物组合物用于治疗肝癌尤其是肝细胞肝癌,本发明为治疗肝癌尤其是肝细胞肝癌提供了新的药物和方法。(The invention belongs to the technical field of biological medicines, relates to a new pharmaceutical application of procheilin A, and particularly relates to an application of procheilin A (Proscillaridin A) (CAS466-06-8) in preparation of a medicine for treating liver cancer, particularly hepatocellular carcinoma (HCC). Experiments show that the alliin A can obviously inhibit the growth of liver cancer cells and promote the apoptosis of the liver cancer cells, and the alliin A can be used as a prodrug to prepare a medicament for treating liver cancer, particularly liver cell cancer, and a pharmaceutical composition containing the alliin A or the alliin A as the prodrug and a pharmaceutically acceptable carrier for treating the liver cancer, particularly the liver cell cancer.)

1. The application of the raw onionsoside A with the structure of the formula 1 in preparing the medicine for treating liver cancer,

the molecular weight of the procollagen A is 530.7, the molecular formula is C30H42O8, and CAS 466-06-8.

2. The use according to claim 1, wherein: the liver cancer is hepatocellular carcinoma.

3. Use according to claim 1 or 2, characterized in that: the final concentration of the dissolved medicine of the raw onionsoside A is 10mM, and the raw onionsoside A is stored at minus 80 ℃ for a long time and at minus 20 ℃ for a short time.

4. Use according to claim 1 or 2, characterized in that: the procollagen A can obviously inhibit cell growth after acting on a liver cancer cell line MHCC 97H.

5. Use according to claim 1 or 2, characterized in that: the procollagen A can obviously inhibit cell cycle and apoptosis after acting on a liver cancer cell line MHCC 97H.

6. A medicament for treating liver cancer, which is characterized in that: the drug comprises the raw onionsoside A as claimed in claim 1 as a prodrug.

7. A pharmaceutical composition for treating liver cancer, which comprises the procallicin a of claim 1 or a drug comprising the procallicin a as a prodrug and a pharmaceutically acceptable carrier.

Technical Field

The invention belongs to the technical field of biological medicines, relates to a new pharmaceutical application of prochloraz A, and particularly relates to an application of prochloraz A (CAS466-06-8) in preparation of a medicine for treating liver cancer, in particular to hepatocellular carcinoma (HCC).

Background

The prior art discloses hepatocellular carcinoma (HCC for short) as one of the most common malignant tumors in China and even all over the world, which seriously harms human health. According to the latest data of global cancer statistics, HCC has become one of ten gross death malignant tumors worldwide, male death patients are obviously more than female, more than 50% of new onset and death cases occur in China, and the mortality rate of HCC morbidity shows the trend that rural areas are higher than urban areas and male are higher than female.

Studies have shown that the development of HCC is associated with a number of factors, such as hepatitis virus infection, long-term alcohol intake, etc. At present, the treatment of HCC is mainly surgical treatment, sorafenib and regorafenib are the only existing drugs approved by FDA to treat HCC, however, sorafenib has less than 3 months of overall survival gain for patients with advanced liver cancer, and therefore, the search for other drugs targeting HCC is of great significance. Cardiac glycoside compounds are organic compounds mainly distributed in more than one hundred plants of more than ten families, such as oleaceae, liliaceae, cruciferae, Moraceae and the like, and are formed by combining an alcohol group or an alcohol-like group (ligand or aglycone) with different numbers of sugar molecules, and the structures of the compounds are shown in figure 1. Cardiac glycoside compounds are mainly classified into two classes, namely, dienolide (e.g., procymidone A) and digitalis (e.g., digoxin), and the two classes are mainly distinguished by the difference in R structure. Under normal conditions, Na⁺-K⁺ATP enzyme can maintain Na inside and outside cells⁺，K⁺Relatively stable, cardiac glycoside compounds capable of inhibiting Na⁺-K⁺-ATPase activity to reduce Na⁺Outflow sum K⁺Internal flow further causes intracellular Ca²⁺The concentration is increased, thereby improving the conductivity of myocardial cells, acting on the heart and strengthening myocardial contractility, and is mainly used for treating congestive heart failure and arrhythmia.

The cardiac glycoside compounds are used for treating cancers, and no report on the application of the raw onionside A in preparing the medicaments for treating the liver cancer exists so far. Based on the current situation of the prior art, the inventor of the application plans to provide a new pharmaceutical application of the protopanaxadiol A, and particularly relates to an application of the protopanaxadiol A (ProscillaridinA) (CAS466-06-8) in preparing medicines for treating liver cancer, especially liver cell cancer (HCC).

Disclosure of Invention

The invention aims to provide a new pharmaceutical application of protopanaxadiol A based on the current situation of the prior art, and particularly relates to an application of protopanaxadiol A (ProscillaridinA) (CAS466-06-8) in preparation of a medicament for treating liver cancer, in particular hepatocellular carcinoma (HCC).

The invention further aims to provide a medicament for treating liver cancer, in particular liver cell cancer, which takes the proclaim onion glycoside A as a prodrug.

The invention further aims to provide a pharmaceutical composition for treating liver cancer, in particular liver cell cancer, which comprises the procollagenin A or a medicament which takes the procollagenin A as a medicament precursor and a pharmaceutically acceptable carrier.

The structure of the alliin A (ProA) is shown as a formula 1, and the alliin A (ProA) is purchased from Sigma-Aldrich company with the product number R214310 and the specification of 1mg, is light yellow powder,

the molecular weight of the procollagen A is 530.7, the molecular formula is C30H42O8, and CAS 466-06-8.

The invention performs the experiment of the inhibition effect of the procollagen A on the proliferation of the hepatoma cell line cells, researches the toxic effect of the procollagen A on rat primary hepatocytes, performs the experiment of the procollagen A on promoting the apoptosis of the hepatoma cell and the experiment of the procollagen A on reducing the size of tumor of a tumor-bearing nude mouse, and shows that after the treatment of low-concentration medicament, the hepatoma cell obviously generates the growth inhibition effect, and can effectively promote the apoptosis of the hepatoma cell and influence the cell cycle process.

In the embodiment of the invention, the final concentration of the dissolved medicament is 10mM, and the dissolved medicament is stored at minus 80 ℃ for a long time and at minus 20 ℃ for a short time;

the medicine of the invention obviously inhibits cell growth after acting on a liver cancer cell line MHCC 97H;

the medicine of the invention obviously inhibits cell cycle and apoptosis after acting on a liver cancer cell line MHCC 97H;

the drug of the invention has no growth inhibition effect when acting on rat primary hepatocyte PRH.

The invention provides a new pharmaceutical application of procymidone A (Proscillaridin A) (CAS466-06-8), in particular to an application in preparing medicines for treating liver cancer, especially liver cell liver cancer (HCC). The procollagen A can be used as a prodrug to prepare a medicament for treating liver cancer, particularly hepatocellular carcinoma, and a pharmaceutical composition containing the procollagen A or the procollagen A as the prodrug and a pharmaceutically acceptable carrier for treating the hepatocellular carcinoma. The invention provides a new medicine and a method for treating liver cancer, in particular to hepatocellular carcinoma.

Compared with the existing medicament Sorafenib approved to be on the market for treating the advanced hepatocellular carcinoma, the medicament Sorafenib is easy to generate drug resistance, and the effective action concentration of the proto-echinacoside is obviously lower than that of Sorafenib.

Drawings

FIG. 1 is the molecular structural formula of raw onionsoside A.

FIG. 2 is the experimental results of the effect of different concentrations of proconiflorin A on the cell proliferation of liver cancer cell MHCC 97H;

FIG. 3 is the experimental results of the effect of different concentrations of euscaphioside A on the cell proliferation of hepatoma cell Huh 7;

FIG. 4 is the experimental results of the effect of the different concentrations of the raw onionside A on the cell proliferation of the liver cancer cell HepG 2; in FIGS. 2 to 4, the concentration (nM) of protopanaxaroside A is plotted on the abscissa, and the relative cell viability (relative cell viability) is plotted on the ordinate.

FIG. 5 shows the experimental results of the effect of different concentrations of raw onionside A on the proliferation of rat primary hepatocytes (PRH) cells; the concentration of protopanaxaroside A (. mu.M) is plotted on the abscissa, and the relative cell Viability (relative cell Viability) is plotted on the ordinate.

FIG. 6 and FIG. 7 are the flow analysis and the statistical analysis of different concentrations of proclaim onion glycoside A for promoting the apoptosis of MHCC97H of liver cancer cell;

FIG. 8 and FIG. 9 are the flow analysis and the statistical analysis experimental results of the pro-alliin A with different concentrations for promoting the apoptosis of the liver cancer cell Huh 7;

FIG. 10 and FIG. 11 are flow analysis and statistical analysis of the results of different concentrations of the protopanaxadiol A promoting apoptosis of liver cancer cell HepG 2;

in fig. 7, 9 and 11, the abscissa is the concentration of protopanaxacin a (nM), the ordinate is the apoptosis rate, p represents p <0.01, and p represents p < 0.001.

FIG. 12 is a graph showing the comparison of the volume of subcutaneous tumors in mice of the experimental group and the control group in example 4;

fig. 13 is a tumor redistribution graph of subcutaneous tumors of experimental and control mice in example 4, representing p < 0.001.

Detailed Description

In the process of drug screening, the invention discovers that the known chemical substance 'proallicin A' has obvious killing power on liver cancer cells, and can be applied to preparing drugs for treating liver cancer.

The alliin A is named Proscillaridin A by the name of Proscillaridin A and has the molecular formula of C30H42O8, and the specific molecular structure is shown in figure 1. The raw onionside A is odorless, white or nearly white crystal or powder in normal state, is soluble in dimethyl sulfoxide (DMSO), and is not easily soluble in water. Regarding the stability of the substance: in general, in bulk, the solution can be kept stable in light or at 60 deg.C, dissolved in DMSO at room temperature, and the concentration of the mother solution of dissolved procalingin A in DMSO can be adjusted to 10mM, stored at-80 deg.C for a long time, and stored at-20 deg.C for a short time.

As a member of cardiac glycoside drugs, the alliin A is also a Na⁺/K⁺ATPase inhibitors, structure, Na⁺/K⁺The ATP pump is a heterodimer consisting of an alpha subunit, which functions as a catalyst, and a glycosylated beta subunit. The alpha subunit contains ATP, Na +, K + and cardiac glycoside (e.g., digoxin, G-ouabain) binding sites.

Example 1: experiment of inhibition effect of proclaim onion glycoside A on cell proliferation of liver cancer cell line

Three different hepatoma cells were used in this experiment: MHCC97H, Huh7 and HepG2 cells are paved on a 96-well plate, a control group and six experimental groups according to the cell quantity of 5000 cells/well, each group is repeated for five times, and after 24 hours, a DMEM culture medium is mixed with a medicament to prepare mixed liquid of 10nM, 50nM, 100nM, 500nM, 1000nM and 5000 nM; adding equivalent DMSO into the blank group, respectively adding into each well, detecting the toxic effect of the drug on the cells by using a homonymous CCK-8 end-point method kit after 48 hours, and measuring the absorbance at 450nm by using an enzyme-labeling instrument after 30 minutes;

results as shown in FIGS. 2 to 4, FIG. 2 is a graph showing the experimental results of the effect of different concentrations of proclaitance A on the cell proliferation of MHCC97H of hepatocarcinoma cell; FIG. 3 is the experimental results of the effect of different concentrations of euscaphioside A on the cell proliferation of hepatoma cell Huh 7; fig. 4 is an experimental result of the influence of the procAliside A with different concentrations on the cell proliferation of the liver cancer cell HepG2, and the result shows that the ratio of the experimental group to the control group is smaller and smaller as the concentration of the drug is increased, shows that the number of living cells is sharply reduced as the concentration of the drug is increased, and can be calculated that the procAliside A has obvious toxic effects on various liver cancer cells.

Example 2: toxicity test of Proallicin A on rat Primary hepatocytes

Rat primary hepatocytes were isolated by hepatic venous perfusion of rats (detailed isolation method see doctor wizee thesis "study on molecular mechanism of hepatitis b virus infection model and its early invasion"), collagen-containing 96-well plates were plated and cultured using primary hepatocyte medium, and the remaining implementation methods were the same as the experimental methods described in example 1;

the results are shown in FIG. 5, and the number of viable cells in each experimental group did not decrease significantly compared to the control group at concentrations below 50. mu.M with the increase of the concentration of the protopanaxadiol A, which indicates that there is no cytotoxicity to rat primary hepatocytes under the concentration condition that the protopanaxadiol A has the effect of killing the hepatoma cells in example 1.

Example 3: experiment for promoting apoptosis of liver cancer cells by using procollagen A

And (3) apoptosis: MHCC97H, Huh7 and HepG2 cells were seeded in 12-well plates, and the corresponding concentration of procollagenin A was added, and the same amount of DMSO was added to the blank. Collecting cells after 48 hours, and detecting by using a homozygote apoptosis kit;

as shown in fig. 6 to 11, it is known that apoptotic cells are stained by the dyes anixv and PI, and located in the Q1+ Q2+ Q3 quadrants (fig. 6, 8 and 10), and the number of apoptotic cells increases with increasing drug concentration (fig. 6, 8 and 10), and data statistics (fig. 7, 9 and 11) show that the proapolinoside a can promote apoptosis.

Example 4: experiment for reducing tumor size of tumor-bearing nude mice by using proto-allicin A

MHCC97H cells were resuspended in PBS buffer and then 2X 10⁶The nude mice were inoculated subcutaneously at a concentration of 8 mice per group at a rate of 7mg/kg in the experimental group (sterile aqueous solution of procallin A) one week after cell inoculation, the nude mice were sacrificed 3 weeks after drug injection by injecting sterilized water every three days, and the volumes and tumor weights of the experimental group and the control group were measured;

as shown in fig. 12 and 13, the tumor volume and weight of the experimental group were reduced compared to the control group, demonstrating that the procalillarisin a has a tumor killing effect in mice.

The experimental result of the invention shows that after the treatment of the low-concentration medicament, the liver cancer cell obviously generates growth inhibition effect, and can effectively promote the apoptosis of the liver cancer cell and influence the cell cycle process; the invention provides application of the raw onionsoside A in liver cancer treatment, and further provides a new method and thought for research and development of new liver cancer drugs.