CN111467358A

CN111467358A - Pharmaceutical composition containing ginsenoside Rh3, PPD and Rh2

Info

Publication number: CN111467358A
Application number: CN202010243893.8A
Authority: CN
Inventors: 范代娣; 范翠英; 段志广; 马晓轩
Original assignee: Shaanxi Giant Biogene Technology Co ltd
Current assignee: Shaanxi Giant Biogene Technology Co ltd
Priority date: 2020-03-31
Filing date: 2020-03-31
Publication date: 2020-07-31
Anticipated expiration: 2040-03-31
Also published as: CN111467358B; AU2021248301A1; AU2021248301B2; WO2021197297A1

Abstract

The invention provides a pharmaceutical composition containing ginsenoside Rh3, PPD and Rh 2. The pharmaceutical composition is a rare ginsenoside pharmaceutical composition with an anti-tumor effect, and comprises effective treatment amounts of ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 as active ingredients, and a pharmaceutically acceptable carrier, wherein in the rare ginsenoside pharmaceutical composition, the weight ratio of the ginsenoside Rh3 to the ginsenoside PPD to the ginsenoside Rh2 is as follows: 1 (1-2) and (1-2). The experimental results show that: the pharmaceutical composition has significantly increased tumor cell inhibition rate compared with single ginsenoside group, and has Combination Index (CI) value of more than 0.8, which shows that the combined use of three ginsenosides has strong synergistic effect.

Description

Pharmaceutical composition containing ginsenoside Rh3, PPD and Rh2

Technical Field

The invention relates to the field of biological medicines, and in particular relates to a rare ginsenoside Rh3/PPD/Rh2 composition with anti-tumor activity.

Background

Tumors are one of the most serious malignant diseases in global fatality rate, and seriously jeopardize human life and health. The number of cancer diseases in China is more than 300 million per year, and the incidence of lung cancer, breast cancer and gastric cancer is increased year by year. Since 30 years, the mortality rate of breast cancer has increased by 96 percent, and the mortality rate of lung cancer has increased by 465 percent, so that the cancer prevention and treatment work in China is very slow and severe.

At present, the most common methods for treating cancer include surgical treatment, radiotherapy, chemotherapy, biological treatment and the like, which have obvious limitations and have certain side effects on the bodies of patients. The surgical treatment needs to consider various factors such as the physical bearing capacity of the patient, the resection effect and the like, and the postoperative recovery effect is not ideal. Radiotherapy utilizes the radiation effect of some rays to achieve the effect of killing tumor cells, but generally produces local myelosuppression, so that the occurrence of radioactive pericarditis is obviously increased, and the body of a patient who is irradiated by the rays is easy to cause irreversible damage. Chemotherapy is a relatively common cancer treatment means at present, and is an auxiliary treatment means for cancer patients by using some specific chemical drugs such as gefitinib, cyclophosphamide, methotrexate, cisplatin and the like. However, chemotherapy can only temporarily inhibit tumors, but cannot cure solid tumors at all, and has great side effects, so that the drugs often accumulate normal cells when acting on target cells, side effects such as low immune function, organ damage and the like are generated, and the most common adverse drug reactions include diarrhea, rash, vomiting, alopecia and the like. Therefore, there is a need for oral or injectable drugs with definite anti-tumor effects and reduced side effects.

The ginsenoside can be extracted from Panax ginseng C.A.Meyer, Panax notoginseng (Burk.) F.H.Chen, and Panax quinquefolium L.Chen of Araliaceae. Ginsenosides can be classified into panaxadiol saponins and panaxatriol saponins, wherein the panaxadiol saponins include Rb1, Rb2, Rb3, Rc, etc. The panaxadiol saponins can be hydrolyzed to obtain rare ginsenosides Rg3, Rk1, Rg5, Rh2, Rk2, Rh3, PPD and the like. Researches find that the ginsenoside Rg3, Rk1, Rg5 and the ginsenoside Rh2 have obvious treatment effects on promoting the apoptosis of tumor cells, inhibiting the proliferation of the tumor cells and inhibiting the invasion and metastasis. The ginsenosides Rh2, Rk2, Rh3 and PPD have less glycosyl groups and less molecular polarity than ginsenosides Rg3, Rk1 and Rg 5. The CN 106109483A patent discloses a diol/triol rare ginsenoside composition with antitumor activity, wherein, the ginsenoside Rk1, Rg5, Rk3 and Rh4 are combined together according to a specific proportion, and the antitumor effect is better than that of each ginsenoside independently. The CN 106109482 a patent discloses a diol group rare ginsenoside composition with antitumor activity, wherein the ginsenoside Rk1, Rg5 and Rg3 are combined together, and the antitumor effect is better than that of each ginsenoside individually. However, the antitumor effect of rare ginsenoside compositions with less glycosyl and less polarity has not been reported.

Disclosure of Invention

The invention aims to provide a rare ginsenoside composition which is based on rare ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2, has synergistic effect and has an anti-tumor effect. In addition, the invention also aims to provide the application of the rare ginsenoside composition in preparing antitumor drugs.

Through a large amount of experimental researches, the inventor verifies the in vitro anti-tumor effect of various rare ginsenosides by combining two kinds of saponins or three kinds of saponins, and specifically, detects the inhibition rate of tumor cells by adopting an MTT colorimetric method and calculates a combined index; detecting tumor cell apoptosis by adopting an AV/PI staining method; detecting the tumor cell cycle distribution by adopting a flow cytometry; western blotting is adopted to detect protein expression related to apoptosis of tumor cells. The experimental result shows that the inhibition rate of the rare ginsenoside Rh3/Rh2/PPD composition on tumor cells is obviously increased compared with that of a single ginsenoside group, the Combination Index (CI) values are all less than 0.7, and the combination of the three ginsenosides has stronger synergistic effect; the apoptosis rate of the rare ginsenoside Rh3/Rh2/PPD composition group is obviously higher than that of the single ginsenoside group; s-phase cells of the rare ginsenoside Rh3/Rh2/PPD composition group are obviously more than those of the single ginsenoside group; compared with the single ginsenoside group, the rare ginsenoside Rh3/Rh2/PPD composition group can obviously down-regulate the expression of Caspase-3, PARP and Bcl-2 proteins, thereby inducing cancer cell apoptosis. Therefore, the composition Rh3/PPD/Rh2 has better anti-tumor effect on gastric cancer, liver cancer and colon cancer than other compositions. Then, the inventor further carries out comparative study on the antitumor effect of various monomeric saponins in the Rh3/PPD/Rh2 composition under different proportions, and finally selects a composition with the best synergistic antitumor effect, thereby completing the invention.

Namely, the present invention comprises:

1. a rare ginsenoside pharmaceutical composition with anti-tumor effect (the rare ginsenoside pharmaceutical composition A of the invention) comprises ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 with effective treatment amount as active ingredients, and a pharmaceutically acceptable carrier,

wherein, in the rare ginsenoside pharmaceutical composition, the weight ratio of the ginsenoside Rh3 to the ginsenoside PPD to the ginsenoside Rh2 is as follows: 1 (1-2) and (1-2).

2. In the rare ginsenoside pharmaceutical composition, the weight ratio of the ginsenoside Rh3 to the ginsenoside PPD to the ginsenoside Rh2 is as follows:

1:(1～1.1):(1.9～2)。

3. the rare ginsenoside pharmaceutical composition is characterized in that the weight ratio of the ginsenoside Rh3 to the ginsenoside PPD to the ginsenoside Rh2 is 1:1: 2.

4. The rare ginsenoside pharmaceutical composition, wherein the rare ginsenoside pharmaceutical composition does not contain other ginsenosides.

5. The rare ginsenoside pharmaceutical composition comprises ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 as the only antitumor active ingredients.

6. Use of a composition comprising ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 in the preparation of an anti-tumor medicament, wherein,

the weight ratio of the ginsenoside Rh3 to the ginsenoside PPD to the ginsenoside Rh2 in the composition is as follows: 1 (1-2) and (1-2);

the antitumor drug contains ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 as active ingredients.

7. In the composition, the weight ratio of the ginsenoside Rh3, the ginsenoside PPD and the ginsenoside Rh2 is as follows: 1 (1-1.1) and (1.9-2).

8. In the composition, the weight ratio of the ginsenoside Rh3 to the ginsenoside PPD to the ginsenoside Rh2 in the composition is 1:1: 2.

9. The use of the foregoing, wherein the composition does not comprise other ginsenosides.

10. The use of the foregoing, wherein the composition comprises ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 as the sole anti-tumor active ingredients.

The inventor also finds that under a specific proportion (for example, the weight ratio of the ginsenoside Rh3 to the ginsenoside PPD to the ginsenoside Rh2 is 1 (1-2): (1-2), preferably 1 (1-1.1): (1.9-2), more preferably 1:1:2), the rare ginsenoside pharmaceutical composition A of the invention not only has the synergistic effect of the ginsenoside Rh3, the ginsenoside PPD and the ginsenoside Rh2, but also has the synergistic effect of any two of the three.

Therefore, the present invention also includes:

11. a rare ginsenoside pharmaceutical composition (the rare ginsenoside pharmaceutical composition B, C, D of the invention) with anti-tumor effect comprises ginsenoside Rh3 and ginsenoside PPD (or ginsenoside Rh3 and ginsenoside Rh2, or ginsenoside PPD and ginsenoside Rh2) as active ingredients in a therapeutically effective amount, and a pharmaceutically acceptable carrier,

in the rare ginsenoside pharmaceutical composition, the weight ratio of the ginsenoside Rh3 to the ginsenoside PPD is 1 (1-2) (or the weight ratio of the ginsenoside Rh3 to the ginsenoside Rh2 is 1 (1-2), or the weight ratio of the ginsenoside PPD to the ginsenoside Rh2 is (1-2): 1-2)).

12. The rare ginsenoside pharmaceutical composition of claim 11, wherein the weight ratio of ginsenoside Rh3 to ginsenoside PPD is 1 (1-1.1) (or the weight ratio of ginsenoside Rh3 to ginsenoside Rh2 is 1 (1.9-2), or the weight ratio of ginsenoside PPD to ginsenoside Rh2 is (1-1.1): 1.9-2)).

13. The rare ginsenoside pharmaceutical composition of claim 11 or 12, wherein the weight ratio of ginsenoside Rh3 to ginsenoside PPD is 1:1 (or the weight ratio of ginsenoside Rh3 to ginsenoside Rh2 is 1:2, or the weight ratio of ginsenoside PPD to ginsenoside Rh2 is 1:2 in the rare ginsenoside pharmaceutical composition.

14. The rare ginsenoside pharmaceutical composition of any one of claims 11-13, wherein no other ginsenosides are included in the rare ginsenoside pharmaceutical composition.

15. The rare ginsenoside pharmaceutical composition of any one of claims 11-14, wherein the rare ginsenoside pharmaceutical composition comprises ginsenoside Rh3 and ginsenoside PPD (or ginsenoside Rh3 and ginsenoside Rh2, or ginsenoside PPD and ginsenoside Rh2) as the only anti-tumor active ingredients.

16. Use of a composition comprising ginsenoside Rh3 and ginsenoside PPD (or ginsenoside Rh3 and ginsenoside Rh2, or ginsenoside PPD and ginsenoside Rh2) in the preparation of an antitumor drug, wherein,

in the composition, the weight ratio of the ginsenoside Rh3 to the ginsenoside PPD is 1 (1-2) (or the weight ratio of the ginsenoside Rh3 to the ginsenoside Rh2 is 1 (1-2), or the weight ratio of the ginsenoside PPD to the ginsenoside Rh2 is (1-2): 1-2));

the antitumor drug contains ginsenoside Rh3 and ginsenoside PPD (or ginsenoside Rh3 and ginsenoside Rh2, or ginsenoside PPD and ginsenoside Rh2) as active ingredients.

17. The use of item 16, wherein in the composition, the weight ratio of ginsenoside Rh3 to ginsenoside PPD is 1 (1-1.1) (or the weight ratio of ginsenoside Rh3 to ginsenoside Rh2 is 1 (1.9-2), or the weight ratio of ginsenoside PPD to ginsenoside Rh2 is (1-1.1): 1.9-2)).

18. The use of

item

16 or 17, wherein in the composition, the weight ratio of ginsenoside Rh3 to ginsenoside PPD is 1:1 (or the weight ratio of ginsenoside Rh3 to ginsenoside Rh2 is 1:2, or the weight ratio of ginsenoside PPD to ginsenoside Rh2 is 1: 2.

19. The use according to any one of claims 16 to 18, wherein no other ginsenosides are included in the composition.

20. The use of any one of items 16 to 19, wherein the composition comprises ginsenoside Rh3 and ginsenoside PPD (or ginsenoside Rh3 and ginsenoside Rh2, or ginsenoside PPD and ginsenoside Rh2) as the sole anti-tumor active ingredients.

In the present specification, the rare ginsenoside pharmaceutical composition a of the present invention and the rare ginsenoside pharmaceutical composition B, C, D of the present invention are collectively referred to as the rare ginsenoside pharmaceutical composition of the present invention in some cases.

The invention also relates to application of the rare ginsenoside pharmaceutical composition in preparing antitumor drugs. The tumor may be, for example, gastric cancer, liver cancer, pancreatic cancer.

The rare ginsenoside pharmaceutical composition of the present invention can be, for example, an oral preparation or an injection. The oral preparation can be, for example, hard capsules, soft capsules, sustained-release capsules, sugar-coated tablets, powders, granules, dripping pills, water-honeyed pills, syrup or oral liquid; the injection is in a solution type, a suspension type, an emulsion type or a freeze-dried powder type. The rare ginsenoside pharmaceutical composition can contain auxiliary materials or other pharmaceutically acceptable carriers. The auxiliary material can be one or more of sodium hyaluronate, sodium alginate, chitosan or collagen.

Drawings

Fig. 1 is a graph showing that ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2, and ginsenoside Rh3/PPD/Rh2 composition induce apoptosis of gastric cancer cells.

Fig. 2 is a graph showing the effect of ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2, and ginsenoside Rh3/PPD/Rh2 composition on gastric cancer cell cycle distribution.

FIG. 3 is a graph showing Western blotting detection of protein expression of ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2, and ginsenoside Rh3/PPD/Rh2 composition in gastric cancer cell apoptosis.

FIG. 4 is a graph showing the change in body weight and tumor volume of nude mice after 30 days of combined administration of ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2, and ginsenoside Rh3/PPD/Rh 2.

Figure 5 shows a graph of the effect on water intake, food intake and body weight of mice treated with the ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2, and ginsenoside Rh3/PPD/Rh2 compositions.

Figure 6 is a graph showing the effect on liver and kidney function of mice treated with the ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2, and ginsenoside Rh3/PPD/Rh2 compositions.

Detailed description of the invention

The present invention will be described in detail with reference to specific embodiments. In the absence of conflict, scientific terms used in this specification have the meaning commonly understood by one of ordinary skill in the art to which this specification pertains.

First, in one aspect, the present invention provides a rare ginsenoside pharmaceutical composition having an anti-tumor effect (the rare ginsenoside pharmaceutical composition of the present invention, hereinafter also referred to simply as the pharmaceutical composition or pharmaceutical composition of the present invention) comprising, as active ingredients, therapeutically effective amounts of ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2, and a pharmaceutically acceptable carrier,

In the present specification, ginsenoside Rh3 refers to a compound described in the following chemical formula 1.

[ chemical formula 1 ]

The ginsenoside Rh3 is a known compound, and can be prepared by a method known in the art, for example, ginsenoside Rh3 can be prepared by enzymatic hydrolysis of ginsenoside Rb 1.

In the present specification, ginsenoside PPD (protopanaxadiol) means a compound described in the following chemical formula 2.

[ chemical formula 2 ]

The above-mentioned ginsenoside PPD (protopanaxadiol) is a known compound and can be prepared by a method known in the art, for example, ginsenoside PPD (protopanaxadiol) can be prepared by enzymatically hydrolyzing ginsenoside Rb 1.

In the present specification, ginsenoside Rh2 refers to a compound described in the following chemical formula 3.

[ chemical formula 3 ]

The ginsenoside Rh2 is a known compound, and can be prepared by a method known in the art, for example, ginsenoside Rh2 can be prepared by enzymatic hydrolysis of ginsenoside Rb 1.

The inventors found that the combination of ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 in the ratio ranges defined in the present invention not only showed a significant synergistic effect in antitumor aspect (CI value less than 1, preferably CI value less than 0.7), but also showed low toxicity characteristics. The inventors have also found that in the above ratio range, ginsenoside Rh3 and ginsenoside PPD (or ginsenoside Rh3 and ginsenoside Rh2, or ginsenoside PPD and ginsenoside Rh2) are also synergistic and of low toxicity. Furthermore, the inventors found that there is no synergistic effect if the contents of ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 in the pharmaceutical composition are not within the ratio range defined in the present invention. In view of remarkable synergistic effect (CI value is less than 0.7), in the composition, the weight ratio of the ginsenoside Rh3 to the ginsenoside PPD to the ginsenoside Rh2 is preferably 1 (1-1.1) to 1.9-2, and more preferably 1:1: 2.

The pharmaceutical composition of the present invention may or may not contain other ginsenosides. In the case where another ginsenoside is contained in the pharmaceutical composition of the present invention, from the viewpoint of better exerting the synergistic effect, the content of the ginsenoside Rh3, the ginsenoside PPD, and the ginsenoside Rh2 (or the ginsenoside Rh3 and the ginsenoside PPD, or the ginsenoside Rh3 and the ginsenoside Rh2, or the ginsenoside PPD and the ginsenoside Rh2) 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 (i.e., the pharmaceutical composition contains only these three ginsenosides, or only two ginsenosides), based on 100 parts by weight of the total ginsenosides contained in the pharmaceutical composition. The purity of ginsenoside Rh3 used in the pharmaceutical composition can be more than 99%, the purity of ginsenoside PPD can be more than 95%, and the purity of ginsenoside Rh2 can be more than 98%.

The content of the total ginsenoside can be determined by vanillin method, and the content of ginsenoside Rh3, PPD and Rh2 can be determined by HP L C method.

The pharmaceutical composition of the present invention may or may not contain other antitumor active ingredients (i.e., ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 (or ginsenoside Rh3 and ginsenoside Rh2, or ginsenoside PPD and ginsenoside Rh2) as the only antitumor active ingredients).

On the other hand, the inventors found that the pharmaceutical composition of the present invention has a significant antitumor effect. Therefore, the invention also provides the application of the pharmaceutical composition in preparing antitumor drugs.

Pharmaceutically acceptable carriers, such as adjuvants, may be included in the pharmaceutical compositions of the invention. The excipients in the pharmaceutical composition of the present invention are not particularly limited, and for example, excipients generally used in pharmaceutical products or health products in the art may be used. Specifically, the auxiliary materials are starch, dextrin, lactose, mannitol, hydroxypropyl sodium methylcellulose, xanthan gum, aspartame and the like.

The dosage form of the pharmaceutical composition of the present invention 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.

When preparing a solid preparation for oral administration, an excipient and, if necessary, a binder, a disintegrant, a lubricant, a coloring agent, a corrigent, etc. are added to the main drug, and then the mixture is prepared into tablets, coated tablets, granules, fine granules, powders, capsules, etc. according to a conventional method.

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 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.

When preparing the injection, pH regulator, buffer, suspending agent, solubilizer, stabilizer, isotonic agent, preservative and the like can be added into the main drug according to the needs, and then the injection for intravenous injection, subcutaneous injection and intramuscular injection can be prepared according to the conventional method. In this case, a freeze-dried product may be prepared by a conventional method as needed.

Examples of the suspending aid include methyl cellulose, tween 80, hydroxyethyl cellulose, gum arabic, tragacanth powder, sodium carboxymethylcellulose, and polyoxyethylene sorbitol monolaurate.

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 of the present invention is administered to a subject, and tumors can be treated. The subject may be a mammal, for example, a human, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc., preferably a human.

The pharmaceutical composition of the present invention may be administered orally or non-orally. The amount to be administered 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 1 to 30000mg, preferably 10 to 3000mg, more preferably 100 to 2000mg, more preferably 1 to 1000mg, more preferably 10 to 500mg, more preferably 100 to 300mg (based on the total amount of ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 (or ginsenoside Rh3 and ginsenoside Rh2, or ginsenoside PPD and ginsenoside Rh 2)) per day for an adult (body weight 60Kg), and may be usually administered in 1 to 3 divided doses per day.

Examples

The following examples are given to facilitate understanding of the invention and are not intended to limit the scope of the claims of the invention in any way.

Example 1 inhibitory Effect of ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2, and ginsenoside Rh3/PPD/Rh2 composition, Rh3/PPD composition, PPD/Rh2 composition, Rh3/Rh2 composition on gastric cancer cells, liver cancer cells, pancreatic cancer cells

Preparing 150ug/m of Rh, PPD and Rh aqueous solution respectively, preparing 150ug/m of Rh, PPD and Rh aqueous solution respectively according to ginsenoside Rh: PPD: Rh weight ratio, and preparing 150ug/m of Rh, PPD and Rh aqueous solution respectively as composition 1, composition 2, composition 3, composition 4 and composition 5, preparing 150ug/m of Rh, PPD and Rh aqueous solution respectively according to ginsenoside Rh: PPD, PPD: Rh and Rh: Rh weight ratio, preparing 150ug/m of Rh and PPD, preparing 150ug/m of Rh aqueous solution respectively as PPD, Rh: Rh and Rh: Rh aqueous solution respectively as composition 6, composition 7 and composition 8, preparing 150 ug/Rh aqueous solution respectively as composition Rh: PPD, PPD: Rh and Rh: Rh, preparing 150 ug/Rh aqueous solution respectively as composition 150ug/m of Rh, preparing 150 ug/Rh aqueous solution respectively as composition 13, preparing 150ug/m of Rh and Rh, preparing 150 ug/Rh aqueous solution respectively as composition 1: PPD, preparing 1:1: 1:1, PPD, 1:1: 1: 1.5, Rh, 1:1: 1.

The method comprises inoculating human gastric cancer cells, liver cancer cells and pancreatic cancer cells into a sterile 96-well plate, adding 150ug/m L ginsenoside Rh3, 150ug/m L ginsenoside PPD, 150ug/m L ginsenoside Rh2 and 150ug/m L ginsenoside composition 1-14 (100 u 1640 culture solution per well for cell culture), setting three wells for each drug group concentration, adding equal volume of culture solution to a blank control group, placing in a 37 ℃ incubator for 48h, discarding supernatant, adding 100 μ L culture solution and 50 μ L MTT solution per well, shaking, placing in the incubator for 4h, sucking out the mixed solution 4h, adding 150 μ L purple crystal to dissolve, shaking for 10min, placing in an enzyme linked immunosorbent assay (ELISA) instrument, detecting absorbance value at 570 nm.

Inhibition was × 100% (blank OD — experimental OD) 100%/blank.

The interaction index CI ═ ABC/(a × B × C), T is the OD value of the cells of the experimental group, C is the OD value of the cells of the control group, ABC is the T/C value of the combination group, A, B, C is the T/C value of the group with ginsenoside alone (if two ginsenosides are combined, the formula becomes CI ═ AB/(a × B)). CI <1, indicates a synergistic effect in combination, and CI ≦ 0.7, the synergistic effect is very significant (see David h.kernel, Carol r.morgan, and Susanne u.hildebrand vivo interaction between cytokine and topotecan in ovo nccinoma systems [ J ]. Cancer Research,1988, li 48, and, synergy of daptomycin for inhibition of glioma and for combined studies of temozolomide [ university of temozolomide ], 2009, university of chinese medical science 2009, and university of synergy of tamycin

The inhibitory rates of the above ginsenosides Rh3, PPD, Rh2 and compositions 1 to 14 on various cancer cells are shown in Table 2. The combination index results for compositions 1-14 for various tumor cells are shown in Table 4.

TABLE 1 OD values of ginsenoside Rh3, PPD, Rh2 and compositions 1-14 on respective tumor cells

TABLE 2 inhibitory rates of ginsenoside Rh3, PPD, Rh2 and compositions 1-14 against various tumor cells

TABLE 3T/C values of compositions 1-14 against various tumor cells

TABLE 4 Combination Index (CI) results for compositions 1-14 on various tumor cells

From the results of example 1, it is clear that the inhibition rates of gastric cancer cells, gastric cancer cells and pancreatic cancer can be significantly improved by the

compositions

1, 2, 3, 6, 7 and 8 of the present invention compared with the Rh3, PPD and Rh2 monomers, and the combination index of all six compositions to gastric cancer cells, hepatic cancer cells and pancreatic cancer cells is less than 1. And by adjusting the weight ratio of Rh3, PPD and Rh2 monomers in the composition, better cancer cell inhibition effect can be obtained. The combination index of the composition 2 to gastric cancer, liver cancer and pancreatic cancer cells is less than 0.7, which shows that the combination of the three monomeric saponins in the weight ratio of 1:1:2 has extremely obvious inhibition synergistic effect on gastric cancer, liver cancer and pancreatic cancer cells. The combination indexes of the composition 6, the composition 7 and the composition 8 to the gastric cancer, liver cancer and pancreatic cancer cells are all less than 1, which shows that Rh3, Rh2 and Rh3 respectively have synergistic effect on the inhibition of the gastric cancer, liver cancer and pancreatic cancer cells. The combination indexes of the composition 4, the composition 5, the composition 9, the composition 10, the composition 11, the composition 12, the composition 13 and the composition 14 on gastric cancer, liver cancer and pancreatic cancer cells are all larger than 1, which indicates that the 8 compositions can not play a synergistic effect.

Example 2 effects of ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2, and ginsenoside Rh3/PPD/Rh2 on inducing apoptosis in gastric cancer cells in

compositions

2, 6, 7, and 8

Taking gastric cancer cells as an example, inoculating the cells into a sterile 6-well plate, culturing for 24 hours, adding ginsenoside Rh3 with the concentration of 150ug/m L, 150ug/m L ginsenoside PPD, 150ug/m L and ginsenoside Rh2 and 150ug/m L compositions, wherein the ginsenoside compositions are ginsenoside composition 2, composition 6, composition 7 and composition 8 in example 1, adding equivalent volume of 1640 culture solution into a control group, setting 5 repeated wells for each concentration, culturing for 24 hours, adding ice-cold PBS for washing for 2-3 times, adding 1 × Binding Buffer solution, blowing and beating uniformly, adding a proper amount of AV/PI mixed staining solution dropwise, incubating for 15 minutes in a dark place, and detecting apoptosis by using a flow cytometer.

Fig. 1 is a graph showing the results of experiments for detecting that ginsenoside Rh3 group, ginsenoside PPD group, ginsenoside Rh2 group, composition 2, composition 6, composition 7 and composition 8 induce gastric cancer cell apoptosis by using flow cytometry, and a histogram is obtained by forming four quadrants, wherein a region Q1 represents a cell with mechanical damage in the experimental operation process, a region Q2 represents a cell with late apoptosis, a region Q3 represents a cell with normal functional morphology, and a region Q4 represents a cell with early apoptosis, as shown in the graph, as the drug concentration of ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2 and ginsenoside Rh3/PPD/Rh2 group increases, the percentage of the cell with apoptosis represented by the region Q4 shows a gradually increasing trend, when the concentration of ginsenoside Rh3 is 150ug/m L, the early apoptosis rate of gastric cancer cell is 10.25% (the early apoptosis rate of the control group is 3.21%), the concentration of ginsenoside Rh is 150ug/m 3%, the synergistic effect of the composition on early apoptosis of gastric cancer cell is less than that when the synergistic effect of ginsenoside Rh 2/m on the composition is achieved by the control group, and the synergistic effect of ginsenoside Rh2/p 2 is < 3.21.26% (when the synergistic effect of the composition of ginsenoside Rh2 and the synergistic effect of the composition is achieved by the control group), when the control group is 150ug/m 3.21).

Meanwhile, the interaction index CI is ABC/A × B × C, T is the survival fraction of the experimental group after apoptosis, C is the survival fraction of the control group after apoptosis, ABC is the T/C value of the combination group, A, B, C is the T/C value of the ginsenoside single action group (if two ginsenosides are used together, the formula is changed into CI (AB/(A × B)), CI is less than 1, the combination has a synergistic effect, and if CI is less than or equal to 0.7, the synergistic effect is very obvious, the combination index CI value of the composition 2 is 0.62, and CI is less than 0.7, the synergistic effect is very obvious, and the synergistic effect is also realized on the gastric cancer apoptosis by the combination of two ginsenosides in the

compositions

6, 7 and 8.

TABLE 5 Combined Index (CI) results for gastric cancer apoptosis rate for ginsenoside Rh3, PPD, Rh2 and composition 2, composition 6, composition 7, composition 8

Example 3 Effect of ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2, and ginsenoside Rh3/PPD/Rh2 on gastric cancer cell cycle distribution of

compositions

2, 6, 7, and 8

For example, stomach cancer cells are treated with 1 × 10 cells in logarithmic growth phase⁵Inoculating each well in a 6-well plate, adding ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2, composition 2, composition 6, composition 7 and composition 8 with the final concentration of 150 mug/m L, adding RPMI-1640 culture solution with the same volume as that of a drug-adding group into a control group, setting 5 multiple wells for each concentration, continuously acting for 48 hours, digesting and centrifuging cells, removing supernatant, adding 4 ℃ precooled PBS (phosphate buffered saline) for washing for 2-3 times, adding 4 ℃ precooled 75% ethanol, fixing at 4 ℃ overnight, centrifuging for the next day, discarding supernatant, adding 4 ℃ precooled PBS for washing for 2-3 times, dropwise adding an appropriate amount of PI solution, incubating at room temperature in dark for 30min, and detecting and analyzing cell cycle distribution by using a flow cytometer.

FIG. 2 Effect of ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2, and ginsenoside Rh3/PPD/Rh2 composition 2, composition 6, composition 7, composition 8 on gastric cancer cell cycle distribution. Compared with the control group, the composition 2, the ginsenoside Rh3, the ginsenoside PPD and the ginsenoside Rh2 group have the advantages that the proportion of cells in the G1 stage is obviously reduced, the proportion of cells in the S stage is obviously increased, and the proportion of cells in the G2 stage is not obviously changed. Ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2 and composition 2, composition 6, composition 7, and composition 8 increased the S-phase cell ratio to 33.9%, 36.49%, 33.49%, 46.49%, 45.02%, 44.80%, and 42.85%, respectively. The results show that the gastric cancer cells acted by the composition 2, the composition 6, the composition 7, the composition 8, the ginsenoside Rh3, the ginsenoside PPD and the ginsenoside Rh2 are blocked in the S phase.

TABLE 6 Effect of ginsenoside Rh3, PPD, Rh2 and composition 2, composition 6, composition 7, composition 8 on gastric carcinoma cells in S phase

Example 4 Western blotting detection of protein expression of gastric cancer cell apoptosis-related protein by ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2 and ginsenoside Rh3/PPD/Rh2 composition 2

Taking gastric cancer cells as an example, 150 mu g/m L of ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2 and the composition are respectively acted on the cells for 48 hours, the cells are collected and added with lysate to extract total protein, a BCA protein analysis detection kit is used for detecting the protein concentration, the cells are separated by 10 percent SDS-PAGE and transferred onto a PVDF membrane, after 2 percent skim milk powder is closed for 2 hours in a room temperature shaking table, GAPDH, Caspase-3, PARP and Bcl-2 primary antibody (1:1000) are respectively added, the cells are incubated overnight at 4 ℃, TBST is washed for 3 times in the next day, a secondary antibody rabbit anti-IgG (1:10000) is added for incubation for 2 hours at room temperature, the membrane is washed for 3 times, then the prepared EC L luminescence solution is added to soak the PVDF membrane, a chemiluminescence gel imager is used for scanning, and the result is used for carrying out optical density analysis on protein bands by using Image J software.

FIG. 3 is a graph showing the results of Western blotting detection of protein expression experiments of ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2 and ginsenoside Rh3/PPD/Rh2 composition 2 related to gastric cancer cell apoptosis. Compared with the ginsenoside Rh3, the ginsenoside PPD and the ginsenoside Rh2, the expression levels of proteins related to apoptosis, such as Caspase-3, Bcl-2 and PARP, are obviously reduced in the composition 2, so that the composition 2, the ginsenoside Rh3, the ginsenoside PPD and the ginsenoside Rh2 can induce the apoptosis of gastric cancer cells by regulating and controlling the down regulation of the proteins.

Example 5 inhibitory Effect of ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2, and ginsenoside composition 2, composition 6, composition 7, and composition 8 on gastric cancer tumor

Referring to example 6 of Chinese patent CN 109045052A, with slight modification, human gastric cancer cells were xenogeneically inoculated into female BA L B/c nude mice, each nude mouse was injected with 3-4 × 10⁶And (4) cells. After 20 days of inoculation, the nude mice were observed to have tumor mass growing under the skin of the left axilla, only in the size of rice grains. After the nude mice are continuously raised for 4 days, the tumor size exceeds 100mm³In the meantime, nude mice were randomly divided into a blank control group, a ginsenoside Rh3 treatment group, a ginsenoside PPD treatment group, a ginsenoside Rh2 treatment group and a ginsenoside Rh3/PPD/Rh2 composition treatment group, which were ginsenoside composition 2, composition 6, composition 7 and composition 8 in example 1. The blank group is injected with physiological saline solution in the abdominal cavity according to the body weight, the ginsenoside Rh3 treatment group, the ginsenoside PPD treatment group, the ginsenoside Rh2 treatment group and the ginsenoside Rh3/PPD/Rh2 composition group are injected with physiological saline solution containing Rh3, PPD and Rh2 with the concentration of 30mg/kg/d in the abdominal cavity according to the body weight once a day for 30 days continuously.

The results of this example are shown in FIG. 4. Compared with the control group, the weight of the mice of the ginsenoside Rh3, PPD, Rh2 and the composition treatment group is in a slow increasing trend. The xenograft tumor volumes of the ginsenoside Rh3, PPD, Rh2 treated group and the ginsenoside composition group were lower than the control group, and the xenograft tumor volumes of the ginsenoside composition 2, composition 6, composition 7, and composition 8 were lower than the ginsenoside Rh3, PPD, Rh2 treated group alone.

Meanwhile, calculating an interaction index CI/(A × B × C), wherein T is the tumor inhibition rate of an experimental group, C is the tumor inhibition rate of a control group, ABC is the T/C value of a combination group, A, B, C is the T/C value of three ginsenosides when the three ginsenosides act independently (if two ginsenosides are used together, the formula is changed into CI/(A × B)), and CI is less than 1, so that the composition has a synergistic effect, and when CI is less than or equal to 0.7, the synergistic effect is very obvious.

TABLE 7 Combined Index (CI) results for tumor suppression rates for gastric cancer for ginsenoside Rh3, PPD, Rh2 and composition 2, composition 6, composition 7, and composition 8 (taking tumor volume data on day 35 as an example)

Example 6 evaluation of in vivo safety of ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2, and ginsenoside Rh3/PPD/Rh2 composition

The male healthy ICR mice are raised in a room temperature environment, are fed freely, have relative humidity of 50-60% and are 12h day/12 h night. Feeding for 1 week, and after acclimatization, fasting for 12 h. After 12h, the mice were randomized into 4 groups: (1) a control group; (2)30mg/kg ginsenoside Rh3 group; (3)30mg/kg ginsenoside PPD group; (4)30mg/kg ginsenoside Rh 2; (5) 30mg/kg ginsenoside Rh3/PPD/Rh 2. The above 5 groups were administered by intragastric administration. After 6h of administration, fasting was removed and the mice were fed normally for 15 days. Mice were observed for drinking, ingestion, weight and changes in liver and kidney function.

The changes in body weight, food intake and food intake of the mice within 15 days after the administration are shown in fig. 5. As can be seen from the figure, the weight, food intake and water intake of mice in the ginsenoside Rh3 group, the ginsenoside PPD group, the ginsenoside Rh2 group and the ginsenoside Rh3/PPD/Rh2 group were not significantly different from those of the control group. As shown in fig. 6, after 15 days of administration of the mice with the ginsenoside Rh3, the liver and kidney weights, and the contents of glutamic-pyruvic transaminase and creatinine in the serum of the mice in the ginsenoside Rh3 treatment group, the ginsenoside PPD group, the ginsenoside Rh2 group and the ginsenoside Rh3/PPD/Rh2 group were not significantly different from those in the blank group. This indicates that the ginsenoside Rh3/PPD/Rh2 composition is safe for in vivo treatment of mice.

It is to be noted that any feature or combination of features described as a constituent of one embodiment in the present specification can be applied to other embodiments as well, without significantly departing from the spirit of the present invention; further, the technical features described as the constituent elements of the different technical aspects may be combined in any manner to constitute the other technical aspects, without significantly departing from the gist of the present invention. The present invention also includes technical means obtained by combining the above cases, and these technical means are described in the present specification.

While the present invention has been described with respect to the specific embodiments and examples, it will be understood by those skilled in the art that these are not intended to limit the scope of the present invention, which should be determined from the claims.

Industrial applicability

The invention provides a rare ginsenoside pharmaceutical composition containing rare ginsenoside Rh3, PPD and Rh2, which has synergistic effect and anti-tumor effect.

Claims

1. A rare ginsenoside pharmaceutical composition with anti-tumor effect comprises ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 as active ingredients in therapeutically effective amount, and pharmaceutically acceptable carrier,

2. A rare ginsenoside pharmaceutical composition of claim 1, wherein the weight ratio of ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 in the rare ginsenoside pharmaceutical composition is: 1 (1-1.1) and (1.9-2).

3. A rare ginsenoside pharmaceutical composition of claim 1, wherein the weight ratio of ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 in the rare ginsenoside pharmaceutical composition is 1:1: 2.

4. A rare ginsenoside pharmaceutical composition of claim 1, wherein no other ginsenosides are included in the rare ginsenoside pharmaceutical composition.

5. A rare ginsenoside pharmaceutical composition of claim 1, wherein the rare ginsenoside pharmaceutical composition comprises ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 as the only anti-tumor active ingredients.

7. The use according to claim 6, wherein the weight ratio of ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 in the composition is: 1 (1-1.1) and (1.9-2).

8. The use according to claim 6, wherein in the composition, the weight ratio of ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 in the composition is 1:1: 2.

9. The use according to claim 6, wherein the composition does not comprise other ginsenosides.

10. The use according to claim 6, wherein the composition comprises ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 as the only anti-tumor active ingredients.