CN111759853A

CN111759853A - Pharmaceutical composition and application thereof

Info

Publication number: CN111759853A
Application number: CN202010693107.4A
Authority: CN
Inventors: 范代娣; 殷诗玉; 段志广; 马晓轩
Original assignee: Shaanxi Giant Biogene Technology Co ltd
Current assignee: Shaanxi Giant Biogene Technology Co ltd
Priority date: 2020-07-17
Filing date: 2020-07-17
Publication date: 2020-10-13
Anticipated expiration: 2040-07-17
Also published as: WO2022012635A9; AU2021310228B2; AU2021310228A1; WO2022012635A1; CN111759853B

Abstract

The present application discloses a pharmaceutical composition, comprising: ginsenoside monomer and bicyclol. The pharmaceutical composition has remarkable curative effects on chemoprevention and treatment of liver cancer, and has remarkable synergistic and synergistic effects compared with the single use of ginsenoside monomer or single use of bicyclol. The application also discloses application of the ginsenoside monomer and the bicyclol in preparing a pharmaceutical composition for preventing and/or treating tumors, and application of the ginsenoside monomer in promoting the effect of the pharmaceutical composition for preventing and/or treating tumors.

Description

Pharmaceutical composition and application thereof

Technical Field

The application belongs to the technical field of medicines, and particularly relates to a pharmaceutical composition and application thereof.

Background

Primary liver cancer is one of the most common malignant tumors worldwide. The national cancer center predicts 46.6 million liver cancer morbidity and 42.2 million liver cancer mortality in 2015 years, and the liver cancer is the fourth most common malignant tumor and the third most lethal cause of the tumor in China, thereby seriously threatening the life and health of people in China.

Early stage liver cancer patients generally have no obvious symptoms, most of the patients have mid-stage and late-stage liver cancer when being diagnosed, and the average survival time of untreated primary liver cancer after being diagnosed is less than 4 months. Currently, the best treatment for liver cancer is surgical resection and liver transplantation. However, in the case of liver transplantation, it is difficult to find a donor; in the case of surgical resection, patients who can undergo surgery account for only 20% of all patients, and thus are not suitable as a treatment for all patients with liver cancer, and the recurrence rate of advanced liver cancer after surgical resection or in situ liver transplantation is high.

Other treatment methods include systemic anticancer therapy, hepatic artery chemo-injection therapy, hepatic artery chemoembolization, radiotherapy and the like, but the treatment methods can kill liver cancer cells and normal cells at the same time, and have fatal side effects. In addition, sorafenib, a representative liver cancer-targeting drug, is reported to have side effects such as pancreas shrinkage and drug resistance problems when taken for a long period of time. Multidrug resistance (MDR) is a major obstacle in tumor therapy and its mechanism is complex. Researches show that tumor cells almost have different degrees of drug resistance to all single drugs, but the combined administration of a plurality of drugs can simultaneously act on a plurality of target sites or signal pathways of cancer cells, can effectively overcome the defect of single drug resistance, provides a new method and hope for preventing and/or treating tumors, and also becomes a future trend of tumor prevention and/or treatment.

Ginsenoside monomers are main active ingredients of traditional Chinese medicinal materials such as ginseng, American ginseng, pseudo-ginseng and the like, and more than 30 ginsenoside monomers are separated from ginseng, and the effects of resisting tumor activity, enhancing immunity activity, reducing blood sugar, improving memory and the like are hot spots of development. Ginsenoside CK is a metabolite of natural diol type ginsenoside monomer in human intestinal tract, and is an entity of ginseng exerting pharmacological activity in vivo. Ginsenoside CK is used for treating rheumatoid arthritis and is currently in clinical trial. The anti-tumor related research shows that the ginsenoside CK can obviously inhibit the in vitro proliferation of colon cancer HCT-116 cells, SW-480 cells and HT-29 cells and the proliferation and invasion of liver cancer MHCC97-H cells, and can inhibit the growth and liver metastasis of tumors such as colon cancer, liver cancer and the like in mice; the CK of the ginsenoside is related to a PI3K/Akt signal channel, and the apoptosis of the MCF-7 of the breast cancer cell is promoted; ginsenoside CK up-regulates the expression of p53/p21, FoxO3a-p27/p15 and Smad3 in HCT-116 cells, down-regulates the expression of cdc25A, CDK4/6 and cyclinD1/3, induces the apoptosis and G1 phase block of HCT-116 cells; the ginsenoside CK influences the interaction and nuclear co-localization of Annexin A2 protein and NF-kappa B p50 subunit in liver cancer HepG2 cells, inhibits the transcriptional activation of NF-kappa B and the expression of downstream anti-apoptosis genes X-IAP, c-IAP1, c-IAP2 and Survivin, promotes the activation of Caspase 9 and the apoptosis of HepG2 cells, and inhibits the expression level of downstream MMP-2/9, thereby inhibiting the metastasis of tumor cells.

Bicyclol (trade name: Poisono) is the first new anti-hepatitis medicine with proprietary intellectual property rights in China, and clinical test results show that the serum neutralization level of chronic hepatitis B and chronic hepatitis C patients can be remarkably reduced, and hepatitis B virus indexes of partial patients can be turned negative. Pharmacological research results show that the bicyclol has the effects of resisting liver injury caused by chemical toxicants, resisting hepatic fibrosis, inhibiting hepatocyte apoptosis caused by immune injury and the like. According to the reports of the literature, bicyclol can obviously inhibit the damage of a chemical mutagen to a cell genome, recover the inhibition of the function of the Gap Junction (GJIC) of a liver epithelial cell caused by a carcinogen, and pay more and more attention to the relation between the function of the Gap Junction (GJIC) and the tumorigenesis. Bicyclol can inhibit 3MC/TPA combined induced in vitro malignant transformation of rat liver epithelial WB-F344 cells, and prevent liver cancer. The bicyclol has obvious inhibition effect on adhesion of high metastasis human liver cancer cell strains MHCC97-H, LN and FN, reduces expression of VEGF mRNA of MHCC97-H cells, and inhibits angiogenesis, thereby inhibiting invasion and metastasis of tumors, and suggesting that the bicyclol has certain potential value in the aspects of preventing and inhibiting occurrence and development of liver cancer.

Patent document 1 discloses a liver cancer targeted ginsenoside CK-loaded chitosan polymer micelle drug delivery system, which is characterized by comprising: targeting headgroups, basal carriers, and anti-cancer drugs; wherein the targeting head group is A54 polypeptide, the basic carrier is amphiphilic polymer, and the anticancer drug is ginsenoside CK; the targeting head group is connected with the hydrophobic end of the basic carrier through a connecting group to form a drug delivery carrier; the drug delivery carrier is used for encapsulating ginsenoside CK to form a liver cancer targeted ginsenoside CK-loaded chitosan polymer micelle drug delivery system.

Patent document 2 discloses a composition comprising the following components: ginsenoside Rk1, ginsenoside Rg5, ginsenoside CK, and total ginsenoside extract, wherein the composition is Interleukin (IL) -6 regulator.

Patent document 3 discloses the use of bicyclol and pharmaceutically acceptable derivatives thereof for the preparation of a medicament for the prevention or treatment of pharmacological liver injury.

In the prior art, ginsenoside monomers or bicyclic alcohol is used, however, the effect of preventing and treating tumors is limited, and the prior art does not disclose that the ginsenoside monomers can promote the effect of a pharmaceutical composition for preventing and/or treating tumors, and does not disclose that the ginsenoside monomers and the bicyclic alcohol are used together to achieve synergistic effect and improve the effect of preventing and/or treating tumors.

Documents of the prior art

Patent document 1CN109793711A publication

Patent document 2CN110090236A publication

Patent document 3CN109106711A publication

Disclosure of Invention

In order to solve the problem of limited tumor prevention and treatment effects caused by the single use of a ginsenoside monomer or the single use of bicyclol in the prior art, the application provides a synergistic antitumor drug composition, which can prevent and treat tumors, particularly liver cancer, more safely and effectively.

The specific technical scheme of the application is as follows:

1. a pharmaceutical composition, comprising: ginsenoside monomer and bicyclol.

2. The pharmaceutical composition according to item 1, wherein the weight ratio of the ginsenoside monomer to the bicyclol is 1: 0.3-3.

3. The pharmaceutical composition according to item 2, wherein the weight ratio of the ginsenoside monomer to the bicyclol is 1:1.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the ginsenoside monomer is one or more selected from the group consisting of: ginsenoside CK, ginsenoside 20- (S, R) -Rg3, ginsenoside 20- (S, R) -Rh2, ginsenoside 20- (S, R) -PPD, ginsenoside Rk1, ginsenoside Rg5, ginsenoside Rk3 or ginsenoside Rh 4;

preferably, the ginsenoside monomer comprises ginsenoside CK.

5. The application of ginsenoside monomer and bicyclol in preparing a pharmaceutical composition for preventing and/or treating tumors is provided.

6. The use according to item 5, wherein the weight ratio of the ginsenoside monomer to the bicyclol is 1: 0.3-3.

7. The use according to item 6, wherein the weight ratio of the ginsenoside monomer to the bicyclol is 1:1.

8. The use according to any one of claims 5 to 7, wherein the ginsenoside monomer is selected from one or more of the following: ginsenoside CK, ginsenoside 20- (S, R) -Rg3, ginsenoside 20- (S, R) -Rh2, ginsenoside 20- (S, R) -PPD, ginsenoside Rk1, ginsenoside Rg5, ginsenoside Rk3 and ginsenoside Rh 4;

preferably, the ginsenoside monomer comprises ginsenoside CK.

9. The use according to any one of claims 5 to 8, wherein the tumor is liver cancer.

10. The application of ginsenoside monomer in promoting the action of pharmaceutical composition for preventing and/or treating tumor is provided.

11. The use according to item 10, wherein the ginsenoside monomer is one or more selected from the group consisting of: ginsenoside CK, ginsenoside 20- (S, R) -Rg3, ginsenoside 20- (S, R) -Rh2, ginsenoside 20- (S, R) -PPD, ginsenoside Rk1, ginsenoside Rg5, ginsenoside Rk3 or ginsenoside Rh 4;

preferably, the ginsenoside monomer comprises ginsenoside CK.

12. The use according to item 10 or 11, wherein the pharmaceutical composition for the prevention and/or treatment of tumors comprises bicyclol.

13. Use according to any one of claims 10 to 12, characterized in that the effect is a synergistic effect.

Effect of application

The pharmaceutical composition containing ginsenoside monomer and bicyclol, especially the pharmaceutical composition containing ginsenoside CK and bicyclol, can inhibit the proliferation of tumor cells, especially liver cancer cells; inhibiting the malignant transformation of rat liver epithelioid stem cells induced by chemical mutagen to liver cancer cells; has obvious inhibition effect on the growth of liver cancer in a nude mouse; can be used for preventing hepatocarcinoma induced by carcinogen. Compared with single medication, the pharmaceutical composition containing the ginsenoside CK and the bicyclol has obvious synergistic and synergistic effects, and is an efficient and low-toxicity antitumor pharmaceutical composition. The molecular biological research related to the application also suggests that the drug combination has a remarkable synergistic molecular mechanism and theoretical basis. Bicyclol can restore the inhibition of the function of the gap communication link (GJIC) of the liver epithelial cells caused by chemical carcinogens, protect the genomic DNA of the cells, and inhibit the malignant transformation of the liver epithelial cells and the occurrence and the development of liver tumors induced by chemical carcinogens. Ginsenoside CK is related to multiple signal pathways such as PI3K/Akt signal pathway, NF-kB signal pathway, cell cycle regulation signal pathway and the like, and can inhibit tumor cell growth and metastasis. The ginsenoside CK and the bicyclol respectively act on different targets and signal paths in different stages of liver cancer occurrence, so that a stronger curative effect of preventing and/or treating liver cancer is achieved.

Drawings

FIGS. 1(a) - (b) are the cell morphology of WB-F344 after 3MC/TPA combined induction in the blank control and model groups, respectively.

Fig. 2(a) - (E) are respectively H & E staining pathological observation graphs of SD rat liver cancer prevention experiment livers of a blank control group, a model group, a ginsenoside CK group, a bicyclol group and a ginsenoside CK/bicyclol combined group.

Detailed Description

The present application will be described in detail below.

It is to be understood that throughout the specification and claims, the terms "including" and "comprising" are used in an open-ended fashion, and thus should be interpreted to mean "including, but not limited to. The description which follows is a preferred embodiment of the present application, but is made for the purpose of illustrating the general principles of the application and not for the purpose of limiting the scope of the application. The protection scope of the present application shall be subject to the definitions of the appended claims.

The present application provides a pharmaceutical composition, wherein the pharmaceutical composition comprises: ginsenoside monomer and bicyclol.

The ginsenoside monomer of the present application may be a commercially available ginsenoside monomer, or a ginsenoside monomer separated from ginseng cultivated or collected in nature, or another ginsenoside monomer converted from one isolated ginsenoside monomer, or a ginsenoside monomer synthesized by a synthesis method, however, any ginsenoside monomer showing the liver cancer preventing or treating effect of the present application may be used without limitation. The ginsenoside monomer can be one or more of monomers selected from the group consisting of ginsenoside CK, ginsenoside 20- (S, R) -Rg3, ginsenoside 20- (S, R) -Rh2, ginsenoside 20- (S, R) -PPD, ginsenoside Rk1, ginsenoside Rg5, ginsenoside Rk3 and ginsenoside Rh4, and preferably comprises ginsenoside CK.

Ginsenoside CK has a chemical name of 20-0- β -D-glucopyranosyl-20-protopanaxadiol (ginsenoside CK) and a molecular formula of C₃₆H₆₂O₈Molecular weight: 622.87, noneSmelly, white powder. Soluble in methanol, ethanol, slightly soluble with ethyl acetate, poorly water soluble, insoluble in chloroform, diethyl ether, CAS No.: 39262-14-1, which is one of saponins as a main active ingredient of ginseng such as ginseng or red ginseng, refers to a ginsenoside monomer belonging to protopanaxadiol (PPT) group. Ginsenoside CK for experiments in the application is high-activity rare ginsenoside CK with purity of more than 98% obtained by extracting ginseng from a common ginsenoside monomer through a directional biotransformation technology and then separating and purifying. The structural formula of the ginsenoside CK is as follows:

"ginsenoside 20- (S, R) -Rg 3", molecular formula: c₄₂H₇₀O₁₂Molecular weight 767.0, white-like powder, dissolved in methanol, CAS No.: 14197-60-5, the structural formula is:

"ginsenoside 20- (S, R) -Rh 2", molecular formula: c₃₆H₆₂O₈Molecular weight 622.87, colorless needle crystals, readily soluble in methanol, CAS No.: 78214-33-2, the structural formula is:

"ginsenoside 20- (S, R) -PPD", molecular formula: c₃₀H₅₂O₃Molecular weight 460.74, white crystal, soluble in solvents such as acetone and DMSO. CAS number: 30636-90-9, the structural formula is:

"ginsenoside Rk 1", molecular formula: c₄₂H₇₀O₁₂Molecular weight 767.0, odorless, white powder. Can be used forSoluble in methanol and ethanol, slightly soluble in ethyl acetate, poorly water soluble, insoluble in chloroform and diethyl ether, CAS No.: 494753-69-4, the structural formula is:

the formula of the ginsenoside Rg5 is as follows: c₄₂H₇₀O₁₂Molecular weight: 767.0, odorless, white powder. Soluble in methanol, ethanol, slightly soluble in ethyl acetate, poorly water soluble, insoluble in chloroform, diethyl ether, CAS No.: 74964-14-0, the structural formula is:

"ginsenoside Rk 3", molecular formula: c₃₆H₆₀O₈Molecular weight: 620.9, tasteless, white powder. Readily soluble in water, methanol, ethanol, CAS No.: 364779-15-7, the structural formula is:

"ginsenoside Rh 4", molecular formula: c₃₆H₆₀O₈620.4 molecular weight, 6-O- β -D-glucopyranosyl-20(-HO) -trans-protopanaxatriol, odorless white powder, CAS No. 174721-08-5, wherein the CAS No. is:

the chemical name of the bicyclol is 4, 4 '-dimethoxy-5, 6, 5', 6 '-bis (methylenedioxy) -2-hydroxymethyl-2' -methoxycarbonyl biphenyl, and the molecular formula is C₁₉H₁₈O₉It can be used for treating aminotransferase increase caused by chronic hepatitis. The bicyclol is a biphenyldicarboxylate analogueHas effects in resisting hepatitis virus and liver cell injury.

The pharmaceutical composition comprises effective dose of ginsenoside monomer and dicyclic alcohol as active ingredients. The effective dose refers to the dose of the pharmaceutical composition used when the pharmaceutical composition performs a pharmaceutical function.

In one embodiment, the combination of ginsenoside monomer and bicyclol in the ratio ranges defined herein shows synergistic effects in inhibiting proliferation of liver cancer cells in vitro, inhibiting malignant transformation of liver cancer precursor cells in vitro, treating liver cancer in vivo, and preventing liver cancer in rats. The inventors have also found that the ginsenoside monomer and bicyclic alcohol are also of low toxicity in the above ratio range. In view of significant synergistic effect, in the pharmaceutical composition, the weight ratio of the ginsenoside monomer to the bicyclol is preferably 1:0.3-3, specifically 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, and preferably 1:1.

In one embodiment, the concentration of the ginsenoside monomer in the pharmaceutical composition can be 3-30 μ M, specifically 3 μ M, 4 μ M, 5 μ M, 6 μ M, 7 μ M, 8 μ M, 9 μ M, 10 μ M, 11 μ M, 12 μ M, 13 μ M, 14 μ M, 15 μ M, 16 μ M, 17 μ M, 18 μ M, 19 μ M, 20 μ M, 21 μ M, 22 μ M, 23 μ M, 24 μ M, 25 μ M, 26 μ M, 27 μ M, 28 μ M, 29 μ M, 30 μ M, preferably 5 μ M-20 μ M during the process of inhibiting the proliferation of cancer cells in vitro or inhibiting the malignant transformation of normal cells into cancer cells in vitro; the concentration of bicyclol may be 1-20 μ M, specifically 1 μ M, 2 μ M, 3 μ M, 4 μ M, 5 μ M, 6 μ M, 7 μ M, 8 μ M, 9 μ M, 10 μ M, 11 μ M, 12 μ M, 13 μ M, 14 μ M, 15 μ M, 16 μ M, 17 μ M, 18 μ M, 19 μ M, 20 μ M, preferably 3 μ M-15 μ M.

In one embodiment, the pharmaceutical compositions of the present application may be administered orally or non-orally. The administration amount varies depending on the degree of symptoms, age, sex, body weight, sensitivity of patients, administration method, administration period, administration interval, properties of pharmaceutical preparations, kinds of active ingredients, and the like, and is not particularly limited, but is usually 10mg to 1.5g, preferably 50mg to 900mg, more preferably 100mg to 600mg, and still more preferably 300mg to 500mg (based on the total weight of ginsenoside CK) per day for an adult (body weight 60kg), and the administration amount may be usually administered in 1 to 3 times per day.

In one embodiment, in the pharmaceutical composition of the present application, the ginsenoside monomer is selected from one or two or three or four or five or more of the following: ginsenoside CK, ginsenoside 20- (S, R) -Rg3, ginsenoside 20- (S, R) -Rh2, ginsenoside 20- (S, R) -PPD, ginsenoside Rk1, ginsenoside Rg5, ginsenoside Rk3 and ginsenoside Rh 4.

In a specific embodiment, in the pharmaceutical composition of the present application, the ginsenoside monomer comprises ginsenoside CK. Besides the ginsenoside CK, the ginsenoside compound also can comprise one or more than two other ginsenoside monomers selected from the following group: ginsenoside 20- (S, R) -Rg3, ginsenoside 20- (S, R) -Rh2, ginsenoside 20- (S, R) -PPD, ginsenoside Rk1, ginsenoside Rg5, ginsenoside Rk3 and ginsenoside Rh4, or other ginsenoside monomers may not be included. In the case where another ginsenoside monomer is included in the pharmaceutical composition of the present application, the content of the ginsenoside CK is preferably 50 parts by weight or more, more preferably 60 parts by weight or more, more preferably 70 parts by weight or more, more preferably 80 parts by weight or more, more preferably 90 parts by weight or more, more preferably 95 parts by weight or more, more preferably 99 parts by weight or more, and more preferably 100 parts by weight, based on 100 parts by weight of the total ginsenoside monomer included in the pharmaceutical composition, from the viewpoint of better exerting the synergistic effect (i.e., the ginsenoside monomer included in the pharmaceutical composition is only ginsenoside CK). The ginsenoside monomer used in the pharmaceutical composition of the present application may have a purity of 98% or more.

In a specific embodiment, a pharmaceutically acceptable carrier, such as an adjuvant, may be included in the pharmaceutical composition of the present application. The pharmaceutical composition of the present application is not particularly limited, and for example, those commonly used in the art for pharmaceutical products or health products can be used. Specifically, the auxiliary materials are starch, dextrin, lactose, mannitol, hydroxypropyl sodium methylcellulose, xanthan gum, aspartame and the like.

In a specific embodiment, the dosage form of the pharmaceutical composition of the present application is not particularly limited, and may be, for example, an oral dosage form or an injectable dosage form. The oral dosage form may be a liquid dosage form or a solid dosage form. The oral dosage form can be, for example, hard capsules, soft capsules, sustained-release capsules, tablets, sugar-coated tablets, powders, granules, pills, water-honeyed pills, syrups or oral liquids; the injection formulation may be, for example, a solution type, a suspension type, an emulsion type or a lyophilized powder. The administration mode of the pharmaceutical composition for improving sleep may be, for example, oral, instillation or injection.

In one embodiment, when a solid preparation for oral administration is prepared, an excipient and, if necessary, a binder, a disintegrant, a lubricant, a colorant, a flavoring agent, etc. may be added to the base drug, and then the mixture may be prepared into tablets, coated tablets, granules, fine granules, powders, capsules, etc. according to a conventional method.

In the above embodiments, as the excipient, for example, lactose, corn starch, white sugar, glucose, sorbitol, crystalline cellulose, silicon dioxide, or the like; as the binder, for example, polyvinyl alcohol, ethyl cellulose, methyl cellulose, gum arabic, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, and the like; as the disintegrant, for example, dry starch, sodium carboxymethyl starch, polyvinylpyrrolidone, crosslinked polymer of sodium carboxymethyl cellulose, or the like; as the lubricant, for example, magnesium stearate, talc, silica, etc.; as the colorant, a colorant that allows addition in a pharmaceutical product; as correctant, cocoa powder, Mentholum, aromatic acid, oleum Menthae Dementholatum, Borneolum Syntheticum, and cortex Cinnamomi Japonici powder can be used. Of course, sugar coating, gelatin coating, and other necessary coatings may be applied to the tablets and granules.

In one embodiment, when preparing an injection, a pH adjuster, a buffer, a suspending aid, a solubilizer, a stabilizer, an isotonic agent, a preservative, and the like may be added to the main drug as needed, and then the injection may be prepared into intravenous, subcutaneous, and intramuscular injections according to a conventional method. In this case, a freeze-dried product may be prepared by a conventional method as needed.

In the above embodiment, examples of the suspension aid include methyl cellulose, tween 80, hydroxyethyl cellulose, gum arabic, tragacanth powder, sodium carboxymethylcellulose, polyoxyethylene sorbitol monolaurate, and the like. Examples of the solubilizer include polyoxyethylene hydrogenated castor oil, tween 80, niacinamide, polyoxyethylene sorbitol monolaurate, polyethylene glycol, and castor oil fatty acid ethyl ester. Examples of the stabilizer include sodium sulfite, sodium metabisulfite, and the like; examples of the preservative include methyl paraben, ethyl paraben, sorbic acid, phenol, cresol, chlorocresol, and the like.

The pharmaceutical composition can be used for preventing and/or treating liver cancer by being applied to a subject. The subject may be a mammal, for example, a human, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc., preferably a human.

On the other hand, the inventors found that the pharmaceutical composition of the present application has a significant effect of preventing and/or treating tumors, in particular preventing and/or treating liver cancer. Therefore, the application also provides the application of the pharmaceutical composition in the preparation of the pharmaceutical composition for preventing and/or treating tumors. Further, the tumor is preferably liver cancer.

The application also provides application of the ginsenoside monomer in promoting the effect of the pharmaceutical composition for preventing and/or treating tumors.

The pharmaceutical composition shows synergistic and even obvious synergistic effects in the aspects of inhibiting in-vitro proliferation of liver cancer cells, inhibiting in-vitro malignant transformation of liver cancer precursor cells, treating liver cancer in vivo and preventing liver cancer. Specifically, the CI value of the interaction index of the ginsenoside monomer and the bicyclol is less than 1, and preferably less than or equal to 0.7. Furthermore, the ginsenoside monomer and the bicyclol are combined according to the proportion limited in the application, so that the pharmaceutical composition with more excellent effect of preventing and/or treating liver cancer can be obtained.

Examples

The content of the present application is further illustrated by the following examples. The technical means used in the examples are conventional means well known to those skilled in the art and commercially available common instruments, unless otherwise specified.

Example 1 combination of ginsenoside CK and Bicyclol inhibits in vitro proliferation of HepG2 cells from liver cancer

In the embodiment, the MTT method is adopted to detect the inhibition effect of ginsenoside CK on the proliferation of liver cancer HepG2 cells. The chemical name of MTT is 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazole bromide, which is a yellow dye. The "MTT method", also known as "MTT colorimetric method", is a method for detecting cell survival and growth. The detection principle is that succinate dehydrogenase in mitochondria of living cells can enable exogenous MTT to be reduced into water-insoluble blue-violet crystal Formazan (Formazan) and deposited in the cells, and dead cells do not have the function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, an enzyme-labeling instrument is used for measuring the absorbance value of the formazan at 490nm wavelength, and the MTT crystal formation amount is in direct proportion to the number of cells within a certain cell number range. The number of living cells is judged according to the measured absorbance value (OD value), and the larger the OD value is, the stronger the cell activity is (if the drug toxicity is measured, the lower the drug toxicity is). The method is widely used for activity detection of some bioactive factors, large-scale screening of anti-tumor drugs, cytotoxicity test, tumor radiosensitivity determination and the like. It features high sensitivity and economy.

Firstly, HepG2 cells with good growth state and cell density of more than 80% are taken, trypsinized and adjusted to 1 × 10 cell density⁵Per mL, 100. mu.L per well volume, was added to a 96-well plate and incubated at 37 ℃ in a 5% incubator. After 24h, the supernatant was discarded, and a set of ginsenoside CK, bicyclol and a combined set of ginsenoside CK/bicyclol was set, and 200 μ L of ginsenoside CK, bicyclol and ginsenoside CK/bicyclol at different concentrations was added to each drug-added set as shown in table 1. Accordingly, the blank control group was added with 200. mu.L of 1640 culture solution, and 6 duplicate wells were set for each concentration. After the time of 48 hours, the reaction kettle is cooled,adding a mixture of 150 mu LMTT and cell culture solution into each well at a volume ratio of 1:2, continuing the culture for 4h, carefully removing the culture medium, adding 150 mu L DMSO, slowly shaking for 10min to dissolve the formed purple blue crystal formazan, finally detecting the absorbance OD value corresponding to each well at 490nm, and calculating the cell inhibition rate by using the following formula (1-average OD value of dosing group/average OD value of control group) × 100%.

The combination effect is judged by two monomer interaction indexes CI, wherein CI is AB/(A multiplied by B), T is the OD value of the cells of the drug-adding group, C is the OD value of the cells of the control group, AB is the T/C value of the combination group of the two monomers, and A, B is the T/C value of the single drug action group. When CI is less than 1, the synergistic effect of the two monomers is shown, and when CI is less than or equal to 0.7, the synergistic effect is very obvious.

Experimental data are expressed in x ± s and analyzed using SPSS17.0 statistical software. The LSD method was used for pairwise comparisons between groups. P <0.05 indicates significant difference, and P <0.01 indicates significant difference.

The results of the inhibitory effect of ginsenoside CK and bicyclol on the in vitro proliferation of hepatoma HepG2 cells measured by the MTT method are shown in Table 1, and the inhibitory effect of ginsenoside CK single drug and bicyclol single drug with different concentrations on the growth of tumor cells to a certain extent are shown in the results of concentration dependent effect. Compared with single-use medicaments, the combined use of the ginsenoside CK and the bicyclol has stronger inhibition effect on HepG2, compared with single-use medicaments, the combined use of two monomers has significant difference (P is less than 0.05), and the CI index also shows that the combined use of the two monomers has obvious synergistic effect. And when the total concentration of the CK and the bicyclol is not changed, the CI index is low when the ratio of the CK to the bicyclol is in the range of 1:0.3-3, and the CI index is minimum when the ratio is 1:1, so that the synergistic effect is most obvious.

TABLE 1 inhibitory Effect of ginsenoside CK and Bicyclol on in vitro proliferation of hepatoma HepG2 cells (x + -s)

Example 2 combination of ginsenoside CK and Bicyclol inhibits 3 MC/TPA-induced in vitro malignant transformation of WB-F344 cells into cancer cells

The models for studying chemopreventive drugs for tumors are mainly in vitro systems and in vivo systems. The in vitro system directly or indirectly induces normal cells to generate malignant transformation by using various carcinogens, has an effective index of inhibiting the malignant transformation of the cells, and has the advantages of being capable of well simulating a multi-stage process of in vivo tumorigenesis, easily controlling experimental conditions and the like. The rat liver epithelioid stem cell (WB-F344 cell) is from a normal adult isogenic male rat, is a liver immature epithelial cell, has the property of a liver stem cell, has a certain relation with the occurrence of liver cancer, and is a liver cancer precursor cell.

Model group: the WB-F344 cells were seeded in 6-well plates at 1500 cells/well, 2mL of culture per well. 24h after cell inoculation, adding a carcinogen 3-methylcholanthrene (3MC) into a culture solution to enable the final concentration to be 2 mug/mL, removing the 3MC after 96h of culture to replace a fresh culture medium, continuously culturing for 3 days, adding phorbol myristoleate (TPA) for 8-12 days, enabling the TPA concentration to be 50ng/mL for induction, then enabling the TPA concentration to be 80ng/mL for induction, removing the TPA on 25 days, enabling the 30 th day, finishing the experiment, washing the cells with PBS (phosphate buffered saline) solution, carrying out Wright-Giemsa staining, and observing and counting the number of transformative foci under a mirror.

Blank control group: on a model group basis, 3MC and TPA were replaced with 0.2% DMSO.

Adding a medicine group: on the basis of the model group, a drug treatment was performed 24 hours after cell inoculation, and a ginsenoside CK single administration group (concentration of 5 μ M, 7.8 μ M, 2.5 μ M, 2 μ M), a bicyclol single administration group (concentration of 5 μ M, 2.2 μ M, 7.5 μ M, 8 μ M), a ginsenoside CK/bicyclol combination group 1 (concentration of 5 μ M/5 μ M), a ginsenoside CK/bicyclol combination group 2 (concentration of 7.8 μ M/2.2 μ M), a ginsenoside CK/bicyclol combination group 3 (concentration of 2.5 μ M/7.5 μ M), a ginsenoside CK/bicyclol combination group 4 (concentration of 2 μ M/8 μ M) were set, and the other treatments were performed in the same model group until the end of the experiment. After TPA is added to the cells, the cells are changed into liquid every 1-3 days according to the cell state.

Calculating the liver cancer cell inhibition rate of the drug adding group by using the following formula: inhibition (%) × (1-number of foci in drug-added group/number of foci in blank control group) × 100%. The combined effect is judged by two monomer interaction indexes CI, wherein CI is AB/(A multiplied by B), T is the number of the transformation focus of the drug-added group cells, C is the number of the transformation focus of the blank control group cells, AB is the T/C value of the two monomer combined group, and A, B is the T/C value of the two monomer single action group. When CI is less than 1, the synergistic effect of the two monomers is shown, and when CI is less than or equal to 0.7, the synergistic effect is very obvious.

After the experiment, the observation results of the cell morphology of the blank control group and the model group are shown in fig. 1, and fig. 1(a) is the cell morphology of the blank control group, and it can be seen that the WB-F344 cells growing normally are regular in morphology, polygonal in shape, well spread on the substrate, abundant in cytoplasm and contact inhibitory. FIG. 1(b) is a graph showing the cell morphology of the model group, and it can be seen that transformed WB-F344 cells have irregular morphology, are fusiform or polygonal, have different cell sizes, have no growth polarity, lose contact inhibition, often have overlapping growth, and are scattered in the foci. The results of the counts of the foci are shown in table 2 below, at the end of the experiment, the model group formed significant foci, the number of the foci was 58.6 ± 7.2, and the blank control group had cells cultured for 30 days, also had less formation of the foci, and had a very significant difference (P <0.05) compared with the model group. The ginsenoside CK group, the bicyclol group, the ginsenoside CK/bicyclol combination group 1, the ginsenoside CK/bicyclol combination group 2, the ginsenoside CK/bicyclol combination group 3 and the ginsenoside CK/bicyclol combination group 4 have inhibition effects on the formation of a transformation focus (specific results are shown in table 2), the CI values of two monomer interaction indexes are less than 1, and the synergistic effect on the inhibition of the in-vitro malignant transformation of WB-F344 cells is achieved.

TABLE 2 Effect of ginsenoside CK and Bicyclol on 3MC/TPA combination induced in vitro malignant transformation of WB-F344 cells (x. + -. s)

In comparison to the blank set, the data is,^#P<0.05，^##P<0.01; compared with model group<0.05，**P<0.01，***P<0.01。

Example 3 evaluation of in vivo efficacy of ginsenoside CK and Bicyclol in combination for treatment of liver cancer

Establishing a human liver cancer nude mouse xenograft tumor model, namely establishing a 4-5-week-old female BALB/c nude mouse, inoculating Hep-3B cells subcutaneously in the left armpit, and injecting 4 × 10 into each nude mouse⁶And (4) cells. The tumor volume reaches 100-300 mm³At this time, nude mice were randomly divided into 16 groups of 10 animals each.

16 groups are respectively 1 model group and 15 medicine adding groups, and the 15 medicine adding groups are respectively: (1)5 ginsenoside CK groups are administrated by intragastric administration, and the administration dose is respectively 60mg/kg, 30mg/kg, 90mg/kg, 100mg/kg and 24 mg/kg; (2)5 bicyclol groups are administrated by gastric lavage, and the administration dose is respectively 60mg/kg, 90mg/kg, 30mg/kg, 20mg/kg and 96 mg/kg; (3)5 ginsenoside CK/bicyclol combination groups are administered by intragastric administration, and the administration doses of the ginsenoside CK/bicyclol are respectively 60mg/kg/60mg/kg, 30mg/kg/90mg/kg, 90mg/kg/30mg/kg, 100mg/kg/20mg/kg and 24mg/kg/96 mg/kg. The dosing volume of each dosing group was 0.2mL, once daily for 21 consecutive days. The animals are observed 1 time a day, the clinical state of the animals is recorded, and the observation contents comprise death, dying, appearance, mental condition, activity condition and the like. Tumor length and tumor width were measured twice weekly, and tumor volume and tumor inhibition (%) were calculated as follows. The tumor volume V is 0.5 × a × b × b, where a is the length of the tumor and b is the width of the tumor. The tumor inhibition rate (%) is (model group V-addition group V)/model group V × 100%.

The combined effect is judged by two monomer interaction indexes CI, wherein CI is AB/(A multiplied by B), T is the tumor volume of a dosing group, C is the tumor volume of a model group, AB is the T/C value of a two-monomer combined group, and A, B is the T/C value of a two-monomer single-acting group. When CI is less than 1, the synergistic effect of the two monomers is shown, and when CI is less than or equal to 0.7, the synergistic effect is very obvious.

The animal has no abnormal clinical symptoms after being continuously administrated for 21 days. The results of the tumor volume test are shown in Table 3, the CI index of the combination of the two monomers is 0.68(CI is less than 0.7), and the combination has obvious synergistic effect. And when the total concentration of the ginsenoside CK and the bicyclol is not changed, when the ratio of the CK to the bicyclol is in the range of 1:0.3-3, the CI index is lower than that when the ratio of the CK to the bicyclol is out of the range, and when the ratio is 1:1, the CI index is minimum, and the synergistic effect is most obvious.

TABLE 3 Effect of ginsenoside CK and Bicyclol on tumor volume in nude mice model of human hepatoma xenograft tumor

P <0.05, P <0.01 compared to model group.

Example 4 prevention of DEN-induced liver cancer in rats by combination of ginsenoside CK and Bicyclol

Model establishment and administration: 100 SD rats were purchased, and after one week acclimation in the animal house, the animals were randomly divided into 5 groups, namely a blank control group, a model group, a ginsenoside CK group, a bicyclol group, and a ginsenoside CK/bicyclol combination group. The drug administration dose of the ginsenoside CK group and the bicyclol group is 60mg/kg, the drug administration dose of the ginsenoside CK/bicyclol combined group is 60mg/kg/60mg/kg, the drug administration dose of the blank control group and the model group is equal to that of the blank control group and the model group, and the drug administration is continuously performed for 20 weeks by intragastric administration. One week after gavage, animals in each group were injected intraperitoneally with the chemical mutagen Diethylnitrosamine (DEN) for 2 consecutive weeks, 40mg/kg for 2 consecutive weeks, 60mg/kg for 2 consecutive weeks, and 80mg/kg for 2 consecutive weeks, except for the blank control group.

Detection of indexes: after 20 weeks of gavage, the animals were sacrificed. Rats were weighed before sacrifice. The rats were anesthetized by intraperitoneal injection of a pentobarbital sodium solution, the abdominal wall was dissected open, the liver was exposed, and the size, shape, color, presence or absence of nodules and the like of the liver were observed. The abdominal aorta is sampled for detection of alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST). The liver was completely removed, liver weight was measured, and liver coefficient was calculated as liver weight/body weight 100%. A part of liver sample is taken and fixed by 4% formaldehyde, and H & E staining pathological section is prepared. H & E staining is a common staining method for pathological sections, and is Hematoxylin-Eosin (Hematoxylin-Eosin) staining. This method can be used for any tissue fixed by a fixing solution and for sections to which various embedding methods are applied. Hematoxylin is a basic dye, and can stain basophilic substances in tissues into blue, such as chromatin in cell nuclei and the like; eosin is an acid dye that stains eosinophilic material in tissues red, e.g., the cytoplasm, nucleoli, etc., of most cells appear red in H & E stained sections.

The experimental results are as follows:

(1) serum ALT, AST and ALP levels in rats as models for preventing liver cancer

The results are shown in Table 4, compared with the blank control group, the serum ALT, AST and ALP levels of the model group rats are remarkably increased (P <0.05), which indicates that the chemical mutagen DEN is accompanied with the change of the serum liver function indexes ALT, AST and ALP levels in the process of inducing the liver cancer of the SD rats. Compared with the model group, the serum ALT, AST and ALP levels of the ginsenoside CK group and the bicyclol group are both obviously reduced (P is less than 0.05), while the effect of the ginsenoside CK/bicyclol combination group is obviously better than that of the single medicine group, and the serum ALT, AST and ALP levels are close to that of the blank control group.

TABLE 4 Effect of ginsenoside CK and Bicyclol on serum ALT, AST and ALP levels (x + -s) in rats with liver cancer prevention model

# P <0.05 compared to blank; p <0.05 compared to model group.

(2) Body weight, liver weight and liver index of liver cancer prevention model rat

After 20 weeks of gavage, the body weight of the model rats was significantly lower than that of the blank control group, and both the liver weight and the liver coefficient were significantly higher than that of the blank control group (P <0.05), as shown in Table 5. The organ coefficient change generally reflects the swelling, congestion, hyperplasia and hypertrophy of organs and the like, and is an index of liver injury, and the liver coefficient of a rat can be increased by injecting DEN into the abdominal cavity, so that the rat liver is presumed to be possibly injured. The weight of rats in the combined group of the ginsenoside CK group, the bicyclol group and the ginsenoside CK/bicyclol is obviously higher than that in the model group (P is less than 0.05), the liver coefficient is obviously lower than that in the model group (P is less than 0.05), and the effect of the combined group of the ginsenoside CK/bicyclol is obviously better than that of the single administration group.

TABLE 5 Effect of ginsenoside CK and Bicyclol on rat body weight, liver weight and liver index (x + -s)

Compared with the blank control group, the composition of the composition,^#P<0.05; comparison with model group<0.05。

(3) H & E staining pathological section of liver

All rats were sacrificed 20 weeks after gavage, as shown in fig. 2(a) - (E), the liver tissues of the rats in the blank control group were deep red, normal in size, soft and fragile, smooth and non-abnormal in liver surface, and regular liver lobules and liver cell chordae structure were visible on H & E stained sections; the liver of the animal in the model group has serious pathological changes, the texture of the liver becomes hard, each leaf is scattered on cancer nodules with different sizes and is in a huge block shape, and H & E staining proves that the liver cancer is hepatocellular carcinoma or moderate and severe cirrhosis; the CK group of ginsenoside and bicyclo-ethanol group also have a part of animal livers with a plurality of grey white nodules, and the number of liver cancer occurrence cases and the degree of liver pathological changes are lighter than those of the model group; and the combined group of the ginsenoside CK and the bicyclol only has a small amount of grey-white nodules in the liver of individual animals, and the incidence of liver cancer and the pathological change degree of the liver are obviously lower than those of the single administration group.

The embodiment shows that the combination of the ginsenoside CK and the bicyclol disclosed by the application shows extremely strong drug effect when being applied to a liver cancer model induced by a carcinogen DEN, is obviously superior to single drug application, does not show obvious adverse reaction, and has good safety.

The foregoing is merely a preferred embodiment of the present application and is not intended to limit the present application in any way, as any person skilled in the art can, using the teachings of the present disclosure, modify or adapt for various equivalent embodiments. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present application still belong to the protection scope of the technical solution of the present application.

Claims

1. A pharmaceutical composition, comprising: ginsenoside monomer and bicyclol.

2. The pharmaceutical composition according to claim 1, wherein the weight ratio of the ginsenoside monomer to the bicyclol is 1: 0.3-3.

3. The pharmaceutical composition of claim 2, wherein the weight ratio of ginsenoside monomer to bicyclol is 1:1.

4. The pharmaceutical composition of any one of claims 1-3, wherein the ginsenoside monomer is selected from one or more of the following: ginsenoside CK, ginsenoside 20- (S, R) -Rg3, ginsenoside 20- (S, R) -Rh2, ginsenoside 20- (S, R) -PPD, ginsenoside Rk1, ginsenoside Rg5, ginsenoside Rk3 and ginsenoside Rh 4;

preferably, the ginsenoside monomer comprises ginsenoside CK.

6. The use as claimed in claim 5, wherein the weight ratio of the ginsenoside monomer to the bicyclol is 1: 0.3-3.

7. The use according to claim 6, wherein the weight ratio of ginsenoside monomer to bicyclol is 1:1.

8. The use according to any one of claims 5 to 7, wherein the ginsenoside monomer is selected from one or more of the following: ginsenoside CK, ginsenoside 20- (S, R) -Rg3, ginsenoside 20- (S, R) -Rh2, ginsenoside 20- (S, R) -PPD, ginsenoside Rk1, ginsenoside Rg5, and ginsenoside Rh 4;

preferably, the ginsenoside monomer comprises ginsenoside CK.

9. Use according to any one of claims 5 to 8, wherein the tumour is liver cancer.