WO2021197297A1 - 包含人参皂苷Rh3、PPD和Rh2的药物组合物 - Google Patents

包含人参皂苷Rh3、PPD和Rh2的药物组合物 Download PDF

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WO2021197297A1
WO2021197297A1 PCT/CN2021/083840 CN2021083840W WO2021197297A1 WO 2021197297 A1 WO2021197297 A1 WO 2021197297A1 CN 2021083840 W CN2021083840 W CN 2021083840W WO 2021197297 A1 WO2021197297 A1 WO 2021197297A1
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ginsenoside
ppd
composition
pharmaceutical composition
rare
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PCT/CN2021/083840
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English (en)
French (fr)
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范代娣
范翠英
段志广
严建亚
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陕西巨子生物技术有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to the field of biomedicine, in particular to a rare ginsenoside Rh3/PPD/Rh2 composition with anti-tumor activity.
  • Tumor is one of the malignant diseases with the highest fatality rate in the world, which seriously endangers human life and health.
  • the incidence of cancer in my country exceeds 3 million every year, and the incidence of lung cancer, breast cancer, and gastric cancer is increasing year by year.
  • the mortality rate of breast cancer has risen by 96%, and the mortality rate of lung cancer has risen by 465%. Therefore, my country's cancer prevention and control work is urgent and the situation is serious.
  • Radiotherapy uses the radiation effect of some rays to achieve the effect of killing tumor cells, but generally it will produce local bone marrow suppression, leading to a significant increase in the incidence of radiation pericarditis. Patients who have experienced radiation exposure will easily cause irreversible damage to their bodies. Chemotherapy is currently the most commonly used cancer treatment method.
  • Ginsenosides can be obtained by extracting ginseng, Panax notoginseng, American ginseng, etc. from the Araliaceae family. Ginsenosides can be divided into glycol group ginsenosides and triol group ginsenosides, among which glycol group ginsenosides include Rb1, Rb2, Rb3, Rc, etc. The diol group ginsenosides can be hydrolyzed to obtain rare ginsenosides Rg3, Rk1, Rg5, Rh2, Rk2, Rh3, PPD, etc.
  • ginsenosides Rg3, Rk1, Rg5 and ginsenoside Rh2 have significant therapeutic effects in promoting tumor cell apoptosis, inhibiting tumor cell proliferation, invasion and metastasis.
  • ginsenosides Rg3, Rk1 and Rg5 Compared with ginsenosides Rg3, Rk1 and Rg5, ginsenosides Rh2, Rk2, Rh3, and PPD have fewer sugar groups and lower molecular polarity.
  • CN 106109483 A patent discloses a glycol group/triol group rare ginsenoside composition with anti-tumor activity, which combines ginsenosides Rk1, Rg5, Rk3, and Rh4 in a specific ratio, and the anti-tumor effect is better than The anti-tumor effect of each ginsenoside alone.
  • CN 106109482 A patent discloses a glycol group rare ginsenoside composition with anti-tumor activity. Combining ginsenosides Rk1, Rg5, and Rg3 together, the anti-tumor effect is also better than the anti-tumor effect of each ginsenoside alone. Tumor effect. However, the anti-tumor effect of rare ginsenoside compositions with fewer sugar groups and less polarity has not been reported yet.
  • the purpose of the present invention is to provide a rare ginsenoside composition based on rare ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2, which is synergistic and has anti-tumor effects.
  • another object of the present invention is to provide the use of the above-mentioned rare ginsenoside composition in the preparation of anti-tumor drugs.
  • the inventors verified the anti-tumor effect of a variety of rare ginsenosides through two saponins or three saponins combinations in vitro. Specifically, the MTT colorimetric method was used to detect the inhibition rate of tumor cells and calculate the combination index ; Use AV/PI staining method to detect tumor cell apoptosis; use flow cytometry to detect tumor cell cycle distribution; use Western blotting to detect tumor cell apoptosis-related protein expression.
  • the experimental results show that the inhibitory rate of rare ginsenoside Rh3/Rh2/PPD composition on tumor cells is significantly higher than that of the ginsenoside alone group, and the combination index (CI) value is less than 0.7, indicating that the combination of three ginsenosides has a strong synergy Effect; the cell apoptosis rate of the rare ginsenoside Rh3/Rh2/PPD composition group was significantly higher than that of the ginsenoside alone group; the S phase cells of the rare ginsenoside Rh3/Rh2/PPD composition group were significantly more than that of the ginsenoside alone group; Compared with the ginsenoside alone group, the saponin Rh3/Rh2/PPD composition group can significantly down-regulate the expression of Caspase-3, PARP, and Bcl-2 proteins, thereby inducing cancer cell apoptosis.
  • the composition Rh3/PPD/Rh2 has superior anti-tumor effects to gastric cancer, liver cancer, and colon cancer than other compositions. Then, the inventors further conducted a comparative study on the anti-tumor effects of various monomer saponins in the Rh3/PPD/Rh2 composition under different ratios, and finally selected the synergistic and best anti-tumor effect. , And completed the present invention.
  • the present invention includes:
  • a rare ginsenoside pharmaceutical composition with anti-tumor effect (the rare ginsenoside pharmaceutical composition A of the present invention), which contains as active ingredients a therapeutically effective amount of ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2, And a pharmaceutically acceptable carrier,
  • the weight ratio of the ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 is 1:(1-2):(1-2).
  • the aforementioned rare ginsenoside pharmaceutical composition wherein, in the rare ginsenoside pharmaceutical composition, the weight ratio of the ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 is 1:(1 ⁇ 1.1):(1.9) ⁇ 2).
  • the aforementioned rare ginsenoside pharmaceutical composition wherein, in the rare ginsenoside pharmaceutical composition, the weight ratio of the ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 is 1:1:2.
  • the aforementioned rare ginsenoside pharmaceutical composition wherein the rare ginsenoside pharmaceutical composition contains ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 as the only anti-tumor active ingredients.
  • composition comprising ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 in the preparation of an anti-tumor drug, wherein:
  • the weight ratio of ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 in the composition is: 1:(1-2):(1-2);
  • the anti-tumor drug contains ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 as active ingredients.
  • composition does not contain other ginsenosides.
  • composition contains ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 as the only anti-tumor active ingredients.
  • the inventors also found that under a specific ratio (for example, the weight ratio of ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 is 1:(1 ⁇ 2):(1 ⁇ 2), preferably 1:(1 ⁇ 1.1): (1.9-2), more preferably 1:1:2), in the rare ginsenoside pharmaceutical composition A of the present invention, not only ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 are synergistically effective, and the above three Any two of these are also synergistic.
  • a specific ratio for example, the weight ratio of ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 is 1:(1 ⁇ 2):(1 ⁇ 2), preferably 1:(1 ⁇ 1.1): (1.9-2), more preferably 1:1:2)
  • the present invention also includes:
  • a rare ginsenoside pharmaceutical composition with anti-tumor effect (the rare ginsenoside pharmaceutical composition B, C, D of the present invention), which contains as active ingredients a therapeutically effective amount of ginsenoside Rh3 and ginsenoside PPD ( Or ginsenoside Rh3 and ginsenoside Rh2, or ginsenoside PPD and ginsenoside Rh2), and a pharmaceutically acceptable carrier,
  • the weight ratio of ginsenoside Rh3 and ginsenoside PPD is 1:(1-2) (or the weight ratio of ginsenoside Rh3 and ginsenoside Rh2 is 1:(1-2) ), or the weight ratio of ginsenoside PPD and ginsenoside Rh2 is (1-2):(1-2)).
  • the rare ginsenoside pharmaceutical composition according to any one of items 11 to 13, wherein the rare ginsenoside pharmaceutical composition does not contain other ginsenosides.
  • the rare ginsenoside pharmaceutical composition according to any one of items 11 to 14, wherein the rare ginsenoside pharmaceutical composition comprises ginsenoside Rh3 and ginsenoside PPD (or ginsenoside Rh3 and ginsenoside Rh2, or ginsenoside Rh3). Saponin PPD and ginsenoside Rh2) are the only anti-tumor active ingredients.
  • 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,
  • the weight ratio of ginsenoside Rh3 and ginsenoside PPD is 1:(1-2) (or the weight ratio of ginsenoside Rh3 and ginsenoside Rh2 is 1:(1-2), or ginsenoside
  • the weight ratio of PPD and ginsenoside Rh2 is (1 ⁇ 2):(1 ⁇ 2));
  • the anti-tumor drug contains ginsenoside Rh3 and ginsenoside PPD (or ginsenoside Rh3 and ginsenoside Rh2, or ginsenoside PPD and ginsenoside Rh2) as active ingredients.
  • the weight ratio of ginsenoside Rh3 and ginsenoside PPD is 1:(1 to 1.1) (or the weight ratio of ginsenoside Rh3 and ginsenoside Rh2) It is 1:(1.9 ⁇ 2), or the weight ratio of ginsenoside PPD and ginsenoside Rh2 is (1 ⁇ 1.1):(1.9 ⁇ 2)).
  • 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 ingredient.
  • the rare ginsenoside pharmaceutical composition A of the present invention and the rare ginsenoside pharmaceutical composition B, C, and D of the present invention are collectively referred to as the rare ginsenoside pharmaceutical composition of the present invention.
  • the invention also relates to the use of the rare ginsenoside pharmaceutical composition of the invention in the preparation of anti-tumor drugs.
  • the tumor may be, for example, stomach cancer, liver cancer, pancreatic cancer.
  • the rare ginsenoside pharmaceutical composition of the present invention may be, for example, an oral agent or an injection.
  • the oral preparation can be, for example, hard capsules, soft capsules, sustained-release capsules, sugar-coated tablets, powders, granules, dripping pills, honey pills, syrups or oral liquids; the injections are in the form of solutions, suspensions, and emulsions. Turbid liquid type or freeze-dried powder.
  • the rare ginsenoside pharmaceutical composition of the present invention may contain excipients or other pharmaceutically acceptable carriers.
  • the adjuvant may be, for example, one or more of sodium hyaluronate, sodium alginate, chitosan or collagen.
  • Figure 1 is a graph showing that the composition of ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2, and ginsenoside Rh3/PPD/Rh2 induces apoptosis of gastric cancer cells.
  • Figure 2 is a graph showing the effects of ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2, and ginsenoside Rh3/PPD/Rh2 composition on the cycle distribution of gastric cancer cells.
  • Figure 3 is a diagram showing Western blotting to detect the expression of ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2, and ginsenoside Rh3/PPD/Rh2 composition on the expression of proteins related to apoptosis in gastric cancer cells.
  • Figure 4 is a graph showing the changes in body weight and tumor volume of nude mice 30 days after administration of the ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2, and ginsenoside Rh3/PPD/Rh2 composition group.
  • Figure 5 shows a graph showing the effects of ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2, and ginsenoside Rh3/PPD/Rh2 composition on the water intake, food intake, and body weight of mice after treatment.
  • Figure 6 is a graph showing the effect of mice on liver and kidney functions after treatment with ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2, and ginsenoside Rh3/PPD/Rh2 composition.
  • the present invention provides a rare ginsenoside pharmaceutical composition with anti-tumor effects (the rare ginsenoside pharmaceutical composition of the present invention, hereinafter also referred to as the pharmaceutical composition or the pharmaceutical composition of the present invention), It contains as active ingredients a therapeutically effective amount of ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2, and a pharmaceutically acceptable carrier,
  • the weight ratio of the ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 is 1:(1-2):(1-2).
  • ginsenoside Rh3 refers to the compound described in the following chemical formula 1.
  • ginsenoside Rh3 is a known compound and can be prepared by methods known in the art, for example, ginsenoside Rh3 can be prepared by enzymatically hydrolyzing ginsenoside Rb1.
  • ginsenoside PPD protopanaxadiol
  • ginsenoside PPD protopanaxadiol
  • ginsenoside PPD protopanaxadiol
  • ginsenoside PPD can be prepared by enzymatically hydrolyzing ginsenoside Rb1.
  • ginsenoside Rh2 refers to the compound described in the following chemical formula 3.
  • ginsenoside Rh2 is a known compound and can be prepared by methods known in the art, for example, ginsenoside Rh2 can be prepared by enzymatically hydrolyzing ginsenoside Rb1.
  • the weight ratio of the ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 is preferably 1:(1 ⁇ 1.1):( 1.9-2), more preferably 1:1:2.
  • the pharmaceutical composition of the present invention may or may not contain other ginsenosides.
  • the ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 (or ginsenoside Rh3 and ginsenoside Rh2)
  • the content of PPD, or ginsenoside Rh3 and ginsenoside Rh2, or ginsenoside PPD and 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 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, more preferably 100 parts by weight (that is, the pharmaceutical composition contains only these three ginsenosides, or only Containing the above two ginsenosides), based on 100 parts by weight of the total
  • the content of the total ginsenosides can be determined by the vanillin method, and the content of the ginsenosides Rh3, PPD and Rh2 can all be determined by the HPLC method.
  • the pharmaceutical composition of the present invention may contain other anti-tumor active ingredients, or may not contain anti-tumor active ingredients (ie, ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 (or ginsenoside Rh3 and ginsenoside Rh2, or Ginsenoside PPD and ginsenoside Rh2) as the only anti-tumor active ingredients).
  • anti-tumor active ingredients ie, ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 (or ginsenoside Rh3 and ginsenoside Rh2, or Ginsenoside PPD and ginsenoside Rh2
  • the present invention also provides the use of the pharmaceutical composition of the present invention in the preparation of anti-tumor drugs.
  • the pharmaceutical composition of the present invention may contain pharmaceutically acceptable carriers, such as excipients.
  • pharmaceutically acceptable carriers such as excipients.
  • excipients there are no special restrictions on the excipients in the pharmaceutical composition of the present invention.
  • the excipients commonly used in medicines or health care products in this technical field can be used.
  • the auxiliary materials are starch, dextrin, lactose, mannitol, sodium hypromellose, xanthan gum, protein sugar and the like.
  • the dosage form of the pharmaceutical composition of the present invention may be an oral dosage form or an injection dosage form.
  • the oral dosage form may be a liquid dosage form or a solid dosage form.
  • the oral dosage form may be, for example, a hard capsule, a soft capsule, a sustained-release capsule, a compressed tablet, a sugar-coated tablet, a powder, a granule, a dripping pill, a honey pill, a syrup, or an oral liquid;
  • the injection dosage form may be, for example, a solution type, Suspension type, emulsion type or lyophilized powder.
  • the administration mode of the pharmaceutical composition for improving sleep can be, for example, oral, drip or injection.
  • tablets When preparing solid preparations for oral administration, after adding excipients and optional binders, disintegrating agents, lubricants, coloring agents, flavoring agents, etc. to the main drug, tablets can be prepared according to conventional methods. , Coated tablets, granules, fine granules, powders, capsules, etc.
  • lactose corn starch, white sugar, glucose, sorbitol, crystalline cellulose, silicon dioxide, etc.
  • binders for example, polyvinyl alcohol, ethyl cellulose, and methyl cellulose can be used.
  • a lubricant for example, magnesium stearate, talc, silicon dioxide, etc. can be used; as a coloring agent, it can be used and allowed to be added to medicines.
  • the coloring agent as the flavoring agent, cocoa powder, menthol, aromatic acid, peppermint oil, borneol, cinnamon powder can be used.
  • the above-mentioned tablets and granules can also be coated with sugar coating, gelatin coating, and other necessary outer coatings.
  • pH regulators, buffers, suspending aids, solubilizers, stabilizers, isotonic agents, preservatives, etc. can be added to the main drug as needed, and then made into intravenous, subcutaneous, and intramuscular injections according to conventional methods .
  • a freeze-dried product can also be prepared by a conventional method.
  • suspending aid examples include methyl cellulose, Tween 80, hydroxyethyl cellulose, gum arabic, tragacanth powder, sodium carboxymethyl cellulose, polyoxyethylene sorbitol monolaurate, and the like.
  • solubilizer examples include polyoxyethylene hydrogenated castor oil, Tween 80, nicotinamide, polyoxyethylene sorbitol monolaurate, polyethylene glycol, and castor oil fatty acid ethyl ester.
  • examples of stabilizers include sodium sulfite and sodium metasulfite; examples of preservatives include methyl paraben, ethyl paraben, sorbic acid, phenol, cresol, and chlorocresol.
  • the pharmaceutical composition of the present invention is administered to a subject to treat tumors.
  • the subject may be a mammal, for example, a human, a rat, a rabbit, a sheep, a pig, a cow, a cat, a dog, a monkey, etc., preferably a human.
  • the pharmaceutical composition of the present invention can be administered orally or parenterally.
  • the dosage varies depending on the degree of symptoms, patient age, sex, weight, differences in sensitivity, application method, application period, application interval, nature of the pharmaceutical preparation, types of active ingredients, etc., and there is no particular limitation, but usually adults (weight 60Kg)
  • Example 1 Ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2, and ginsenoside Rh3/PPD/Rh2 composition, Rh3/PPD composition, PPD/Rh2 composition, Rh3/Rh2 composition is effective against gastric cancer cells, liver cancer cells, Inhibition of pancreatic cancer cells
  • Rh3, PPD, and Rh2 aqueous solutions with a concentration of 150ug/mL respectively.
  • the weight ratio of ginsenoside Rh3:PPD:Rh2 as 1:1:1, 1:1:2, 1:2:1, 1:0.5:0.5, 1:3:3, the total concentration of 150ug/mL containing Rh3 is prepared.
  • the aqueous solution is called composition 9, composition 10, composition 11; the total concentration is prepared according to the weight ratio of ginsenoside Rh3:PPD, PPD:Rh2, Rh3:Rh2, respectively, 1:3, 3:3, and 1:3 A 150ug/mL aqueous solution
  • ginsenosides Rh3, PPD, Rh2 and compositions 1-14 were used to act on gastric cancer, liver cancer, and pancreatic cancer cells to perform cancer cell suppression experiments.
  • the specific steps are as follows: inoculate human gastric cancer cells, gastric cancer, liver cancer, and pancreatic cancer cells in a sterile 96-well plate, and add 150ug/mL ginsenoside Rh3, 150ug/mL ginsenoside PPD, 150ug/mL ginsenoside Rh2, and 150ug/mL, respectively Ginsenoside composition 1-14 (100uL per well, cell culture using RPMI 1640 medium).
  • Inhibition rate (blank group OD-experimental group OD) ⁇ 100%/blank group.
  • Table 2 shows the inhibition rates of the above-mentioned ginsenosides Rh3, PPD, Rh2 and compositions 1-14 on various cancer cells.
  • Table 4 shows the combination index results of compositions 1-14 on various tumor cells.
  • the composition 1, composition 2, composition 3, composition 6, composition 7, and composition 8 of the present invention can significantly improve gastric cancer. Inhibition rates of cellular gastric cancer, liver cancer, and pancreatic cancer, and the combined index of the six compositions for gastric cancer, liver cancer, and pancreatic cancer are all less than 1. And by adjusting the weight ratio of Rh3, PPD, and Rh2 monomers in the composition, a better cancer cell inhibitory effect can be achieved.
  • the combination index of composition 2 on gastric cancer, liver cancer, and pancreatic cancer cells is less than 0.7, indicating that the combined use of the three monomer saponins at a weight ratio of 1:1:2 has an extremely synergistic inhibitory effect on gastric cancer, liver cancer, and pancreatic cancer cells.
  • the combination index of composition 4, composition 5, composition 9, composition 10, composition 11, composition 12, composition 13, and composition 14 for gastric cancer, liver cancer, and pancreatic cancer cells are all greater than 1, indicating that 8 This composition does not play a synergistic effect.
  • Example 2 The effect of ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2, and ginsenoside Rh3/PPD/Rh2 composition 2, composition 6, composition 7, and composition 8 on inducing apoptosis of gastric cancer cells
  • ginsenoside composition is ginsenoside composition 2, composition 6, composition 7, and composition 8 in Example 1.
  • An equal volume of 1640 culture medium was added to the control group, with 5 replicate wells for each concentration.
  • Figure 1 is a graph showing the experimental results of using flow cytometry to detect ginsenoside Rh3 group, ginsenoside PPD group, ginsenoside Rh2 group, and composition 2, composition 6, composition 7, and composition 8 inducing apoptosis of gastric cancer cells, obtained
  • the cell histogram is composed of four quadrants.
  • Area Q1 indicates cells that have undergone mechanical damage during the experimental operation
  • area Q2 indicates cells that have undergone late apoptosis
  • area Q3 indicates cells that have normal functional morphology
  • area Q4 indicates cells that have undergone early apoptosis.
  • the percentage of apoptotic cells represented by area Q4 shows a gradual increase the trend of.
  • the concentration of ginsenoside Rh3 is 150ug/mL
  • the early apoptosis rate of gastric cancer cells is 10.25% (the early apoptosis rate of the control group is 3.21%)
  • the early apoptosis rate of gastric cancer cells when the concentration of ginsenoside PPD is 150ug/mL It is 12.76% (the early apoptotic rate of the control group is 3.21%).
  • the concentration of ginsenoside Rh2 is 150ug/mL
  • the early apoptotic rate of gastric cancer cells is 13.30% (the early apoptotic rate of the control group is 3.21%).
  • the early apoptosis rate of gastric cancer cells corresponding to the composition 2 group was 55.28%.
  • composition group 2 has a synergistic effect on the apoptosis of gastric cancer, CI ⁇ 0.7, the synergistic effect is significant; composition 6, composition 7, composition 8
  • the apoptotic synergistic index CI for gastric cancer is ⁇ 1; indicating that the pairwise use of the three saponins also has a synergistic effect on the apoptosis of gastric cancer cells.
  • T is the survival score after apoptosis in the experimental group
  • C is the survival score after apoptosis in the control group
  • ABC is the T/C value of the combination group
  • CI ⁇ 1 it indicates that the combination has a synergistic effect, and when CI ⁇ 0.7, the synergy is very significant.
  • the calculation shows that the CI value of the combination index of composition 2 is 0.62, CI ⁇ 0.7, indicating that the synergistic effect is very significant; the combination of saponin in composition 6, composition 7, and composition 8 also has a synergistic effect on the apoptosis of gastric cancer. Synergistic effect.
  • Example 3 Effects of ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2, and ginsenoside Rh3/PPD/Rh2 composition 2, composition 6, composition 7, and composition 8 on the cycle distribution of gastric cancer cells
  • composition 6 cells in the logarithmic growth phase were seeded in a 6-well plate at 1 ⁇ 10 5 cells/well, and the final concentration of 150 ⁇ g/mL ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2 and composition 2 were added.
  • composition 7 composition 8
  • the control group was added with the same volume of RPMI-1640 culture medium as the drug-adding group, and 5 replicate holes were set for each concentration.
  • the cells were digested and centrifuged, the supernatant was removed, washed 2-3 times with 4°C pre-cooled PBS, and 4°C pre-cooled 75% ethanol was added, and the cells were fixed overnight at 4°C.
  • Figure 2 The effect of ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2, and ginsenoside Rh3/PPD/Rh2 composition 2, composition 6, composition 7, and composition 8 on the cycle distribution of gastric cancer cells.
  • the composition 2 and ginsenoside Rh3, ginsenoside PPD, and ginsenoside Rh2 groups had a significant decrease in the proportion of cells in the G1 phase, a significant increase in the proportion of cells in the S phase, and no significant changes in the proportion of cells in the G2 phase.
  • Ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2 and composition 2, composition 6, composition 7, and composition 8 increase the proportion of S-phase cells to 33.9%, 36.49%, 33.49%, 46.49%, 45.02%, respectively. 44.80%, 42.85%.
  • the results showed that the gastric cancer cells treated with composition 2, composition 6, composition 7, composition 8, ginsenoside Rh3, ginsenoside PPD and ginsenoside Rh2 were all blocked in S phase.
  • Example 4 Western blotting detection of ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2, and ginsenoside Rh3/PPD/Rh2 composition 2 on the expression of apoptosis-related proteins in gastric cancer cells
  • ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2 and the composition were respectively acted on the cells for 48 hours.
  • the cells were collected and added to the lysate to extract the total protein, and the protein concentration was detected with the BCA protein analysis and detection kit . It was separated by 10% SDS-PAGE and transferred to PVDF membrane. After 2% skimmed milk powder was sealed in a room temperature shaker for 2 hours, GAPDH, Caspase-3, PARP, and Bcl-2 primary antibodies (1:1000) were added respectively, and incubated overnight at 4°C.
  • the membrane was washed 3 times with TBST, and the secondary antibody rabbit anti-IgG (1:10000) was added to incubate at room temperature for 2 hours, and then the membrane was washed 3 times. Then the PVDF membrane was infiltrated with the freshly prepared ECL luminescent solution, and then scanned with a chemiluminescence gel imager. As a result, the protein bands were analyzed by the optical density using Image J software.
  • Fig. 3 is a diagram showing the experimental results of Western blotting detecting ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2, and ginsenoside Rh3/PPD/Rh2 composition 2 on the expression of proteins related to apoptosis in gastric cancer cells.
  • composition 2 Compared with the ginsenoside Rh3, ginsenoside PPD, and ginsenoside Rh2 groups, the expression levels of apoptosis-related proteins such as Caspase-3, Bcl-2, and PARP were significantly reduced in composition 2, indicating that composition 2 and ginsenoside Rh3 , Ginsenoside PPD, Ginsenoside Rh2 may induce the apoptosis of gastric cancer cells by regulating the down-regulation of these proteins.
  • apoptosis-related proteins such as Caspase-3, Bcl-2, and PARP were significantly reduced in composition 2, indicating that composition 2 and ginsenoside Rh3 , Ginsenoside PPD, Ginsenoside Rh2 may induce the apoptosis of gastric cancer cells by regulating the down-regulation of these proteins.
  • Example 5 Inhibitory effects of ginsenoside Rh3, ginsenoside PPD, ginsenoside Rh2, and ginsenoside composition 2, composition 6, composition 7, and composition 8 on gastric cancer tumors
  • Example 6 of Chinese Patent CN 109045052 A with a slight modification.
  • Human gastric cancer cells were xeno-inoculated into female BALB/c nude mice, and each nude mouse was injected with 3-4 ⁇ 10 6 cells. Twenty days after the inoculation, tumor masses were observed under the skin of the left axillary of nude mice, only the size of rice grains.
  • the nude mice After continuing to raise the nude mice for 4 days, when the tumor size exceeds 100mm 3 , the nude mice are 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 combination
  • the treatment group of this composition is ginsenoside composition 2, composition 6, composition 7, and composition 8 in Example 1.
  • the blank group was intraperitoneally injected with physiological saline solution according to body weight, ginsenoside Rh3 treatment group, ginsenoside PPD treatment group, ginsenoside Rh2 treatment group, and ginsenoside Rh3/PPD/Rh2 composition group were injected intraperitoneally with Rh3, PPD, Rh2 composition according to body weight A physiological saline solution with a concentration of 30 mg/kg/d once a day for 30 consecutive days.
  • the interaction index CI ABC/(A ⁇ B ⁇ C)
  • T is the tumor inhibition rate of the experimental group
  • C is the tumor inhibition rate of the control group
  • ABC is the T/C value of the combination group
  • CI is less than 1, it indicates that the composition has a synergistic effect, and when CI is less than or equal to 0.7, the synergistic effect is very significant.
  • composition index CI of composition group 2 composition 6, composition group 7, and composition 8 are 0.72, 0.75, 0.78, and 0.74, respectively, indicating that the combination of three saponins in the composition and two of the three saponins Both combinations have synergistic effects.
  • mice Male healthy ICR mice were raised in a room temperature environment, eating freely, with a relative humidity of 50-60%, 12h day/12h night. They were raised for 1 week, and after they had adapted to the environment, they were fasted for 12 hours. After 12h, the mice were randomly divided into 4 groups: (1) control group; (2) 30mg/kg ginsenoside Rh3 group; (3) 30mg/kg ginsenoside PPD group; (4) 30mg/kg ginsenoside Rh2; (5) 30mg/kg ginsenoside Rh3/PPD/Rh2 group. The above 5 groups were all administered by gavage. After 6 hours of administration, the fasting was cancelled and the mice were fed normally for 15 days. Observe the changes of mice drinking water, food intake, body weight and liver and kidney function.
  • any technical feature or combination of technical features described in this specification as a constituent part of a technical solution can also be applied to other technical features, provided that it can be implemented and does not obviously violate the gist of the present invention.
  • Technical solutions; and, on the premise that it can be implemented and does not obviously violate the gist of the present invention the technical features described as constituent parts of different technical solutions can also be combined in any manner to form other technical solutions.
  • the present invention also includes technical solutions obtained by combining in the above cases, and these technical solutions are equivalent to being described in this specification.
  • the present invention provides a rare ginsenoside pharmaceutical composition containing rare ginsenosides Rh3, PPD and Rh2, which is synergistic and has anti-tumor effects.

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Abstract

一种具有抗肿瘤作用的稀有人参皂苷药物组合物，包含作为活性成分的治疗有效量的人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2以及药学可接受的载体，人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2的重量比为1：（1-2）：（1-2）。实验结果显示：该药物组合物对肿瘤细胞的抑制率较单独人参皂苷组明显增加，三种人参皂苷联合使用具有较强的协同效应。

Description

包含人参皂苷Rh3、PPD和Rh2的药物组合物 技术领域
本发明涉及生物医药领域，具体涉及一种具有抗肿瘤活性的稀有人参皂苷Rh3/PPD/Rh2组合物。
背景技术
肿瘤是全球致死率最高的恶性疾病之一，严重危害人类的生命与健康。我国癌症的发病人数每年超过300万人，且肺癌、乳腺癌、胃癌的发病率逐年增长。30年以来，乳腺癌的死亡率上升了96％，肺癌的死亡率猛增465％，因此我国的癌症防治工作刻不容缓且形势严峻。
目前，治疗癌症的最常见的方法有手术治疗、放疗、化疗和生物治疗等，这些手段都有明显的局限性，并且对病人的身体均有一定的副作用。手术疗法要考虑到患者的身体承受能力、切除效果等多方面因素，且术后恢复效果也不尽理想。放射疗法是利用一些射线的辐射效应来达到杀伤肿瘤细胞的效果，但是一般会产生局部的骨髓抑制，导致放射性心包炎的发生明显增加，经历过放射线照射的患者，身体都会容易造成不可逆的损伤。化疗是目前比较常用的癌症治疗手段，一般是利用一些特定的化学药物如吉非替尼、环磷酰胺、甲氨蝶呤、顺铂等对癌症患者的一种辅助治疗手段。但是化学疗法只能暂时抑制肿瘤，根本无法治愈实体性肿瘤，同时副作用作用很大，药物在作用于靶细胞时往往累计正常细胞，产生免疫功能低下、脏器受损等副作用，最常见的药物不良反应有腹泻、皮疹、呕吐、脱发等。因此，临床上上急需抗肿瘤效果确切、副作用小的口服或者注射类药物。
人参皂苷可通过五加科植物人参、三七、西洋参等提取获得。人参皂苷可以分为二醇组人参皂苷和三醇组人参皂苷，其中二醇组人参皂苷包括Rb1、Rb2、Rb3、Rc等。二醇组人参皂苷经过水解可以获得稀有人参皂苷Rg3、Rk1、 Rg5、Rh2、Rk2、Rh3、PPD等。研究发现，人参皂苷Rg3、Rk1、Rg5以及人参皂苷Rh2在促肿瘤细胞凋亡、抑制肿瘤细胞增殖和侵袭转移方面有明显的治疗效果。人参皂苷Rh2、Rk2、Rh3、PPD与人参皂苷Rg3、Rk1、Rg5相比，糖基更少，分子极性更小。CN 106109483 A专利公开了一种具有抗肿瘤活性的二醇组/三醇组稀有人参皂苷组合物，将人参皂苷Rk1、Rg5、Rk3、Rh4按照特定的比例组合在一起，抗肿瘤效果要优于单独的每一种人参皂苷的抗肿瘤效果。CN 106109482 A专利公开了一种具有抗肿瘤活性的二醇组稀有人参皂苷组合物，将人参皂苷Rk1、Rg5、Rg3组合在一起，抗肿瘤效果也要优于单独的每一种人参皂苷的抗肿瘤效果。但是对于糖基更少、极性更小的稀有人参皂苷组合物的抗肿瘤效果还未有人报道。
发明内容
本发明的目的在于，提供一种以稀有人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2为基础的、协同增效的、具有抗肿瘤作用的稀有人参皂苷组合物。此外，本发明的另一目的在于提供上述稀有人参皂苷组合物在制备抗肿瘤药物中的用途。
本发明人经过大量的实验研究，将多种稀有人参皂苷通过两种皂苷组合或者三种皂苷组合进行体外抗肿瘤效果验证，具体地，采用MTT比色法检测肿瘤细胞的抑制率并计算联合指数；采用AV/PI染色法检测肿瘤细胞凋亡；采用流氏细胞术检测肿瘤细胞周期分布；采用Western blotting检测肿瘤细胞与凋亡相关的蛋白表达。实验结果显示，稀有人参皂苷Rh3/Rh2/PPD组合物对肿瘤细胞的抑制率较单独人参皂苷组明显增加，联合指数(CI)值均小于0.7，显示三种人参皂苷联合使用具有较强的协同效应；稀有人参皂苷Rh3/Rh2/PPD组合物组的细胞凋亡率明显高于单独人参皂苷组；稀有人参皂苷Rh3/Rh2/PPD组合物组S期细胞明显多于单独人参皂苷组；稀有人参皂苷Rh3/Rh2/PPD组合物组与单独人参皂苷组相比可以明显下调Caspase-3、 PARP、Bcl-2蛋白的表达，从而诱导癌细胞凋亡。因此，组合物Rh3/PPD/Rh2对胃癌、肝癌、结肠癌具有比其他组合物优效的抗肿瘤效果。然后，发明人又进一步对Rh3/PPD/Rh2组合物中各种单体皂苷在不同配比下的抗肿瘤效果进行了对比研究，最终优选出协同增效的、抗肿瘤效果最好的组合物，进而完成了本发明。
即，本发明包括：
1.一种具有抗肿瘤作用的稀有人参皂苷药物组合物(本发明的稀有人参皂苷药物组合物A)，其包含作为活性成分的治疗有效量的人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2，以及药学可接受的载体，
其中，该稀有人参皂苷药物组合物中，所述人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2的重量比为：1:(1～2):(1～2)。
2.前述的稀有人参皂苷药物组合物，其中，该稀有人参皂苷药物组合物中，所述人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2的重量比为：1:(1～1.1):(1.9～2)。
3.前述的稀有人参皂苷药物组合物，其中，该稀有人参皂苷药物组合物中，所述人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2的重量比为1:1:2。
4.前述的稀有人参皂苷药物组合物，其中，该稀有人参皂苷药物组合物中不包含其它人参皂苷。
5.前述的稀有人参皂苷药物组合物，其中，该稀有人参皂苷药物组合物包含人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2作为唯一抗肿瘤活性成分。
6.包含人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2的组合物在制备抗肿瘤药物中的用途，其中，
所述组合物中人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2的重量比为：1:(1～2):(1～2)；
所述抗肿瘤药物包含人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2作为活性成分。
7.前述的用途，其中，所述组合物中，所述人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2的重量比为：1:(1～1.1):(1.9～2)。
8.前述的用途，其中，所述组合物中，所述组合物中，所述人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2的重量比为1:1:2。
9.前述的用途，其中，所述组合物中不包含其它人参皂苷。
10.前述的用途，其中，所述组合物包含人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2作为唯一抗肿瘤活性成分。
发明人还发现，在特定的比例下(例如人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2的重量比为1:(1～2):(1～2)、优选1:(1～1.1):(1.9～2)、更优选1:1:2)，本发明的稀有人参皂苷药物组合物A中，不但人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2三者是协同增效的，而且上述三者中的任意两者也是协同增效的。
因此，本发明还包括：
11.一种具有抗肿瘤作用的稀有人参皂苷药物组合物(本发明的稀有人参皂苷药物组合物B、C、D)，其包含作为活性成分的治疗有效量的人参皂苷Rh3和人参皂苷PPD(或者人参皂苷Rh3和人参皂苷Rh2，或者人参皂苷PPD和人参皂苷Rh2)，以及药学可接受的载体，
其中，该稀有人参皂苷药物组合物中，所述人参皂苷Rh3和人参皂苷PPD的重量比为1:(1～2)(或者人参皂苷Rh3和人参皂苷Rh2的重量比为1:(1～2)，或者人参皂苷PPD和人参皂苷Rh2的重量比为(1～2):(1～2))。
12.根据项11所述的稀有人参皂苷药物组合物，其中，该稀有人参皂苷药物组合物中，所述人参皂苷Rh3和人参皂苷PPD的重量比为1:(1～1.1)(或者人参皂苷Rh3和人参皂苷Rh2的重量比为1:(1.9～2)，或者人参皂苷PPD和人参皂苷Rh2的重量比为(1～1.1):(1.9～2))。
13.根据项11或12所述的稀有人参皂苷药物组合物，其中，该稀有人参皂苷药物组合物中，所述人参皂苷Rh3和人参皂苷PPD的重量比为1:1(或者人 参皂苷Rh3和人参皂苷Rh2的重量比为1:2，或者人参皂苷PPD和人参皂苷Rh2的重量比为1:2。
14.根据项11～13中任一项所述的稀有人参皂苷药物组合物，其中，该稀有人参皂苷药物组合物中不包含其它人参皂苷。
15.根据项11～14中任一项所述的稀有人参皂苷药物组合物，其中，该稀有人参皂苷药物组合物包含人参皂苷Rh3和人参皂苷PPD(或者人参皂苷Rh3和人参皂苷Rh2，或者人参皂苷PPD和人参皂苷Rh2)作为唯一抗肿瘤活性成分。
16.包含人参皂苷Rh3和人参皂苷PPD(或者人参皂苷Rh3和人参皂苷Rh2，或者人参皂苷PPD和人参皂苷Rh2)的组合物在制备抗肿瘤药物中的用途，其中，
所述组合物中，所述人参皂苷Rh3和人参皂苷PPD的重量比为1:(1～2)(或者人参皂苷Rh3和人参皂苷Rh2的重量比为1:(1～2)，或者人参皂苷PPD和人参皂苷Rh2的重量比为(1～2):(1～2))；
所述抗肿瘤药物包含人参皂苷Rh3和人参皂苷PPD(或者人参皂苷Rh3和人参皂苷Rh2，或者人参皂苷PPD和人参皂苷Rh2)作为活性成分。
17.根据项16所述的用途，其中，所述组合物中，所述人参皂苷Rh3和人参皂苷PPD的重量比为1:(1～1.1)(或者人参皂苷Rh3和人参皂苷Rh2的重量比为1:(1.9～2)，或者人参皂苷PPD和人参皂苷Rh2的重量比为(1～1.1):(1.9～2))。
18.根据项16或17所述的用途，其中，所述组合物中，所述人参皂苷Rh3和人参皂苷PPD的重量比为1:1(或者人参皂苷Rh3和人参皂苷Rh2的重量比为1:2，或者人参皂苷PPD和人参皂苷Rh2的重量比为1:2。
19.根据项16～18中任一项所述的用途，其中，所述组合物中不包含其它人参皂苷。
20.根据项16～19中任一项所述的用途，其中，所述组合物包含人参皂苷Rh3和人参皂苷PPD(或者人参皂苷Rh3和人参皂苷Rh2，或者人参皂苷PPD和人参皂苷Rh2)作为唯一抗肿瘤活性成分。
本说明书中，在有些情况下将本发明的稀有人参皂苷药物组合物A和本发明的稀有人参皂苷药物组合物B、C、D统称为本发明的稀有人参皂苷药物组合物。
本发明还涉及本发明的稀有人参皂苷药物组合物在制备抗肿瘤药物中的用途。所述肿瘤可以为例如胃癌、肝癌、胰腺癌。
本发明的稀有人参皂苷药物组合物可以是例如口服剂或者注射剂。所述口服剂可以是例如硬胶囊、软胶囊、缓控释放胶囊、糖衣片、粉剂、颗粒剂、滴丸、水蜜丸、糖浆或口服液；所述注射剂为溶液型、混悬液型、乳浊液型或冻干粉。本发明的稀有人参皂苷药物组合物中可包含辅料或其它药学可接受的载体。所述辅料可以为例如透明质酸钠、海藻酸钠、壳聚糖或者胶原蛋白中的一种或几种。
附图说明
图1为显示人参皂苷Rh3、人参皂苷PPD、人参皂苷Rh2、以及人参皂苷Rh3/PPD/Rh2组合物诱导胃癌细胞凋亡的图。
图2为显示人参皂苷Rh3、人参皂苷PPD、人参皂苷Rh2、以及人参皂苷Rh3/PPD/Rh2组合物对胃癌胃癌细胞周期分布的影响的图。
图3为显示Western blotting检测人参皂苷Rh3、人参皂苷PPD、人参皂苷Rh2、以及人参皂苷Rh3/PPD/Rh2组合物对胃癌细胞与凋亡相关的蛋白表达的图。
图4为显示人参皂苷Rh3、人参皂苷PPD、人参皂苷Rh2、以及人参皂苷Rh3/PPD/Rh2组合物组用药30天后裸鼠体重和肿瘤体积的变化的图。
图5显示小鼠经人参皂苷Rh3、人参皂苷PPD、人参皂苷Rh2、以及人参皂苷Rh3/PPD/Rh2组合物处理后对其饮水量、摄食量和体重的影响的图。
图6为显示小鼠经人参皂苷Rh3、人参皂苷PPD、人参皂苷Rh2、以及人参皂苷Rh3/PPD/Rh2组合物处理后对其肝功能和肾功能的影响的图。
具体实施方式
下面结合具体实施方式对本发明进行详细的说明。在无冲突的情况下，本说明书中的科学术语具有本领域技术人员通常理解的含义，如有冲突应以本说明书中的定义为准。
首先，在一个方面中，本发明提供一种具有抗肿瘤作用的稀有人参皂苷药物组合物(本发明的稀有人参皂苷药物组合物，以下也简称为本发明的药物组合物或药物组合物)，其包含作为活性成分的治疗有效量的人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2，以及药学可接受的载体，
其中，该稀有人参皂苷药物组合物中，所述人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2的重量比为：1:(1～2):(1～2)。
在本说明书中，人参皂苷Rh3是指下述化学式1所述的化合物。
【化学式1】
Figure PCTCN2021083840-appb-000001
上述人参皂苷Rh3是已知化合物，可以采用本技术领域已知的方法来制备，例如可以通过酶解人参皂苷Rb1来制备人参皂苷Rh3。
在本说明书中，人参皂苷PPD(原人参二醇)是指下述化学式2所述的化合物。
【化学式2】
Figure PCTCN2021083840-appb-000002
上述人参皂苷PPD(原人参二醇)是已知化合物，可以采用本技术领域已知的方法来制备，例如可以通过酶解人参皂苷Rb1来制备人参皂苷PPD(原人参二醇)。
在本说明书中，人参皂苷Rh2是指下述化学式3所述的化合物。
【化学式3】
Figure PCTCN2021083840-appb-000003
上述人参皂苷Rh2是已知化合物，可以采用本技术领域已知的方法来制备，例如可以通过酶解人参皂苷Rb1来制备人参皂苷Rh2。
发明人发现，将人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2按本发明限定的比例范围进行组合，不仅在抗肿瘤方面表现出显著的协同增效(CI值小于1、优选CI值小于0.7)，而且还表现出低毒性特点。发明人还发现，在上述比例范围内，人参皂苷Rh3和人参皂苷PPD(或者人参皂苷Rh3和人参皂苷Rh2，或者人参皂苷PPD和人参皂苷Rh2)也是协同增效和低毒性的。而且，发明人发现，如果所述药物组合物中人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2的含量不在本发明限定的比例范围内，则不具有协同增效作用。从协同增效作用显著的角度考虑(CI值小于0.7)，所述组合物中，所述人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2的重量比为优选为1:(1～1.1):(1.9～2)，更优选为1:1:2。
本发明的药物组合物中，可以包含其它人参皂苷，也可以不包含其它人参皂苷。在本发明的药物组合物中包含其它人参皂苷的情况下，从更好地发挥协同增效作用的角度考虑，所述人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2(或者人参皂苷Rh3和人参皂苷PPD、或者人参皂苷Rh3和人参皂苷Rh2，或者人参皂苷PPD和人参皂苷Rh2)的含量优选为50重量份以上、更优选为60重量份以上、更优选为70重量份以上、更优选为80重量份以上、更优选为90重量份以上、更优选为95重量份以上、更优选为99重量份以上、更优选100重量份(即所述药物组合物中仅包含这三种人参皂苷、或仅包含上述两种人参皂苷)，以该药物组合物中包含的总人参皂苷为100重量份计。本发明的药物组合物使用的人参皂苷Rh3纯度可以为99％以上，人参皂苷PPD纯度可以为95％以上，人参皂苷Rh2纯度可以为98％以上。
所述总人参皂苷的含量可以采用香草醛法测定，所述人参皂苷Rh3、PPD和Rh2的含量均可以采用HPLC法测定。
本发明的药物组合物中，可以包含其它抗肿瘤活性成分，也可以不包含抗肿瘤活性成分(即，以人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2(或者人 参皂苷Rh3和人参皂苷Rh2，或者人参皂苷PPD和人参皂苷Rh2)作为唯一抗肿瘤活性成分)。
另一方面，发明人发现本发明的药物组合物具有显著的抗肿瘤效果。因此，本发明也提供本发明的药物组合物在制备抗肿瘤药物中的用途。
本发明的药物组合物中可以包含药物可接受的载体，例如辅料。对于本发明的药物组合物中的辅料，没有特殊限制，例如可以使用本技术领域通常用于药品或保健品的辅料。具体地，所述辅料为淀粉、糊精、乳糖、甘露醇、羟丙甲纤维素钠、黄原胶、蛋白糖等。
对本发明的药物组合物的剂型没有特殊限制，例如可以是口服剂型或注射剂型。所述口服剂型可以是液体剂型，也可以是固体剂型。所述口服剂型可以为例如硬胶囊、软胶囊、缓控释放胶囊、压片、糖衣片、粉剂、颗粒剂、滴丸、水蜜丸、糖浆或口服液；所述注射剂型可以为例如溶液型、混悬液型、乳浊液型或冻干粉。所述用于改善睡眠的药物组合物的给药方式可以为例如口服、滴注或注射。
制备口服用固体制剂时，可以在向主药中加入赋形剂以及视需要而定的粘合剂、崩解剂、滑润剂、着色剂、矫味剂等后，按照常规方法制成片剂、包衣片剂、颗粒剂、细粒剂、散剂、胶囊剂等。
作为赋形剂，可使用例如乳糖、玉米淀粉、白糖、葡萄糖、山梨糖醇、结晶纤维素、二氧化硅等；作为粘合剂，可使用例如聚乙烯醇、乙基纤维素、甲基纤维素、阿拉伯胶、羟基丙基纤维素、羟基丙基甲基纤维素等；作为滑润剂，可使用例如硬脂酸镁、滑石、二氧化硅等；作为着色剂，可使用允许在药品中添加的着色剂；作为矫味剂，可使用可可粉、薄荷脑、芳香酸、薄荷油、龙脑、桂皮粉。当然，也可以在上述片剂、颗粒剂上包覆糖衣、明胶衣、以及其它的必要外衣。
制备注射剂时，可以根据需要向主药中添加pH调节剂、缓冲剂、悬助剂、增溶剂、稳定剂、等渗剂、防腐剂等，再按照常规方法制成静脉、皮下、肌肉内注射剂。此时，也可以根据需要，利用常规方法制成冷冻干燥物。
作为悬助剂，可列举例如甲基纤维素、吐温80、羟基乙基纤维素、阿拉伯胶、黄蓍树胶粉、羧甲基纤维素钠、聚氧乙烯山梨糖醇单月桂酸盐等。
作为增溶剂，可列举例如聚氧化乙烯氢化蓖麻油、吐温80、烟酰胺、聚氧乙烯山梨糖醇单月桂酸盐、聚乙二醇、蓖麻油脂肪酸乙酯等。
另外，作为稳定剂，可列举例如亚硫酸钠、偏亚硫酸钠等；作为防腐剂，可列举例如对羟基苯甲酸甲酯、对羟基苯甲酸乙酯、山梨酸、苯酚、甲酚、氯甲酚等。
将本发明的药物组合物施用于对象，可以治疗的肿瘤。所述对象可以是哺乳动物，例如可以是人、大鼠、兔、羊、猪、牛、猫、狗、猴等，优选为人。
本发明的药物组合物，可以口服或非口服施用。施用量因症状程度、患者年龄、性别、体重、敏感性差异、施用方法、施用时期、施用间隔、药物制剂的性质、有效成分的种类等而异，无特殊限制，但通常成人(体重60Kg)每日1μg～30000mg、优选10μg～3000mg、更优选100μg～2000mg、更优选1mg～1000mg、更优选10mg～500mg、更优选100mg～300mg(以人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2(或者人参皂苷Rh3和人参皂苷Rh2，或者人参皂苷PPD和人参皂苷Rh2)总量计)，上述施用量通常可每日分1～3次施用。
实施例
下面给出的实施例，是为了便于理解本发明，并不以任何方式限定本发明的权利要求的范围。
实施例1 人参皂苷Rh3、人参皂苷PPD、人参皂苷Rh2、以及人参皂苷Rh3/PPD/Rh2组合物、Rh3/PPD组合物、PPD/Rh2组合物、Rh3/Rh2组合物对胃癌细胞、肝癌细胞、胰腺癌细胞的抑制作用
分别配制浓度150ug/mL的Rh3、PPD、Rh2的水溶液。按人参皂苷Rh3：PPD：Rh2重量比分别为1:1:1、1:1:2、1:2:1、1:0.5:0.5、1:3:3配制总浓度150ug/mL的包含Rh3、PPD、Rh2的水溶液，将其称作组合物1、组合物2、组合物3、组合物4、组合物5；按人参皂苷Rh3：PPD、PPD：Rh2、Rh3：Rh2重量比分别为1:1:、1:2、1:2配制总浓度150ug/mL的包含Rh3、PPD；PPD：Rh2、Rh3：Rh2的水溶液，将其称作组合物6、组合物7、组合物8；按人参皂苷Rh3：PPD、PPD：Rh2、Rh3：Rh2重量比分别为1:0.5、0.5:0.5、1:0.5配制总浓度150ug/mL的包含Rh3、PPD；PPD：Rh2、Rh3：Rh2的水溶液的水溶液，将其称作组合物9、组合物10、组合物11；按人参皂苷Rh3：PPD、PPD：Rh2、Rh3：Rh2重量比分别为1:3、3:3、1:3配制总浓度150ug/mL的包含Rh3、PPD；PPD：Rh2、Rh3：Rh2的水溶液，将其称作组合物12、组合物13、组合物14。
使用上述人参皂苷Rh3、PPD、Rh2和组合物1-14，分别作用于胃癌、肝癌、胰腺癌细胞进行癌细胞抑制实验。具体步骤如下：将人胃癌细胞胃癌、肝癌、胰腺癌细胞接种于无菌96孔板中，分别加入150ug/mL人参皂苷Rh3、150ug/mL人参皂苷PPD、150ug/mL人参皂苷Rh2以及150ug/mL人参皂苷组合物1-14(每孔100uL，细胞培养使用RPMI 1640培养液)。每个药物组浓度设置三个复孔，空白对照组加入等体积的培养液，置于37℃培养箱培48h。弃上清，每孔加入100μL培养液和50μLMTT溶液，摇匀，放进培养箱中孵育4h，4h后吸出混合液，加入150μL DMSO来溶解紫色晶体甲瓒，震荡10min，放进酶标仪中，检测波长570nm，测定每孔的吸光度值。
抑制率＝(空白组OD-实验组OD)×100％/空白组。
同时计算相互作用指数CI＝ABC/(A×B×C)，T为实验组细胞的OD值，C为对照组细胞的OD值，ABC为联用组的T/C值，A、B、C为人参皂苷单独作用组的T/C值(若两种人参皂苷联用时，则公式变成CI＝AB/(A×B))。CI＜1时，表明联用有协同作用，CI≤0.7时，协同作用非常显著。(参照David H.Kern,Carol R.Morgan,and Susanne U.Hildebrand-Zanki.In vitro and in vivo interaction between cisplatin and topotecan in ovarian carcinoma systems[J].Cancer Research,1988,48.，以及，李兴起.力达霉素对神经胶质瘤抑制作用及联合替莫唑胺的协同作用研究[D].中国协和医科大学,2009.)
上述人参皂苷Rh3、PPD、Rh2和组合物1-14对各种癌细胞的抑制率如表2所示。组合物1-14对各种肿瘤细胞的联合用药指数结果如表4所示。
表1 人参皂苷Rh3、PPD、Rh2和组合物1-14作用于各肿瘤细胞的OD值
Figure PCTCN2021083840-appb-000004
表2 人参皂苷Rh3、PPD、Rh2和组合物1-14对各种肿瘤细胞的抑制率
Figure PCTCN2021083840-appb-000005
表3 组合物1-14对各种肿瘤细胞的T/C值
Figure PCTCN2021083840-appb-000006
Figure PCTCN2021083840-appb-000007
表4 组合物1-14对各种肿瘤细胞的联合指数(CI)结果
Figure PCTCN2021083840-appb-000008
由上述实施例1的结果可知，与Rh3、PPD、Rh2单体相比，本发明的组合物1、组合物2、组合物3、组合物6、组合物7、组合物8能够显著提高胃癌细胞胃癌、肝癌、胰腺癌抑制率，六种组合物对胃癌、肝癌、胰腺癌联合指数均小于1。且通过调整组合物中Rh3、PPD、Rh2单体的重量比，能够取得更佳的癌细胞抑制效果。其中组合物2对胃癌、肝癌、胰腺癌细胞 的联用指数均小于0.7，表明三种单体皂苷在1:1:2这个重量比联用对胃癌、肝癌、胰腺癌细胞的抑制协同作用极显著。组合物6、组合物7、组合物8对胃癌、肝癌、胰腺癌细胞的联用指数均小于1，表明Rh3:PPD＝1:1、PPD:Rh2＝1:2、Rh3:PPD＝1:2对胃癌、肝癌、胰腺癌细胞的抑制具有协同作用。组合物4、组合物5、组合物9、组合物10、组合物11、组合物12、组合物13、组合物14对胃癌、肝癌、胰腺癌细胞的联用指数均大于1，表明此8种组合物并不能起到协同增效的作用。
实施例2 人参皂苷Rh3、人参皂苷PPD、人参皂苷Rh2、以及人参皂苷Rh3/PPD/Rh2组合物2、组合物6、组合物7、组合物8诱导胃癌细胞凋亡的作用
以胃癌细胞为例，将细胞接种于无菌6孔板中，培养24h后，加入浓度为150ug/mL的人参皂苷Rh3；150ug/mL人参皂苷PPD；150ug/mL的人参皂苷Rh2和150ug/mL人参皂苷组合物，人参皂苷组合物为实施例1中的人参皂苷组合物2、组合物6、组合物7、组合物8。对照组加入等体积的1640培养液，每个浓度设置5个复孔。培养24h后，加入冰冷的PBS洗涤2-3次，加入1×Binding Buffer缓冲液，吹打均匀，再滴加适量的AV/PI混合染液，避光孵育15min，使用流式细胞仪检测细胞凋亡。
图1为采用流式细胞术检测人参皂苷Rh3组、人参皂苷PPD组、人参皂苷Rh2组以及组合物2、组合物6、组合物7、组合物8诱导胃癌细胞凋亡的实验结果图，得到由四个象限组成细胞直方图。区域Q1表示实验操作过程中出现机械损伤的细胞，区域Q2表示发生晚期凋亡的细胞，区域Q3表示功能形态正常的细胞，区域Q4表示发生早期凋亡的细胞。从图中可以看出，随着人参皂苷Rh3、人参皂苷PPD、人参皂苷Rh2以及人参皂苷Rh3/PPD/Rh2组合物组药物浓度的增加，区域Q4所代表的发生凋亡的细胞百分率呈现逐渐增加的趋势。当人参皂苷Rh3浓度为150ug/mL时，胃癌细 胞的早期凋亡率为10.25％(对照组的早期凋亡率为3.21％)，人参皂苷PPD浓度为150ug/mL时胃癌细胞的早期凋亡率为12.76％(对照组的早期凋亡率为3.21％),人参皂苷Rh2浓度为150ug/mL时，胃癌细胞的早期凋亡率为13.30％(对照组的早期凋亡率为3.21％)，此时组合物2组对应的胃癌细胞早期凋亡率为55.28％。由表8数据可知，人参皂苷Rh3/PPD/Rh2组合物2组对胃癌的凋亡具有协同增效的作用，CI<0.7，协同作用及其显著；组合物6、组合物7、组合物8对胃癌的凋亡协同指数CI<1；表明三种皂苷中两两联用对胃癌细胞的凋亡也具有协同增效的作用。
同时计算相互作用指数CI＝ABC/A×B×C，T为实验组细胞凋亡后存活分数，C为对照组细胞凋亡后存活分数；ABC为联用组的T/C值，A、B、C为人参皂苷单独作用组的T/C值(若两种人参皂苷联用时，则公式变成CI＝AB/(A×B))。CI＜1时，表明联用有协同作用，CI≤0.7时，协同作用非常显著。经计算可知，组合物2联用指数CI值为0.62，CI＜0.7，表明协同作用非常显著；组合物6、组合物7、组合物8中皂苷两两联用对胃癌的凋亡也具有协同增效作用。
表5 人参皂苷Rh3、PPD、Rh2以及组合物2、组合物6、组合物7、组合物8对胃癌细胞凋亡率的联合指数(CI)结果
Figure PCTCN2021083840-appb-000009
实施例3 人参皂苷Rh3、人参皂苷PPD、人参皂苷Rh2、以及人参皂苷Rh3/PPD/Rh2组合物2、组合物6、组合物7、组合物8对胃癌细胞周期分布的影响
以胃癌细胞为例，将对数生长期的细胞以1×10 5个/孔接种于6孔板，加入终浓度为150μg/mL人参皂苷Rh3、人参皂苷PPD、人参皂苷Rh2和组合物2、组合物6、组合物7、组合物8，对照组加入与加药组等体积的RPMI-1640培养液，每个浓度设置5个复孔。继续作用48h后，细胞消化离心，去掉上清液，加入4℃预冷的PBS洗涤2-3次，再加入4℃预冷的75％乙醇，4℃固定过夜。次日，离心弃上清，再加入4℃预冷的PBS洗涤2-3次，滴加适量的PI溶液，室温避光孵育30min，使用流式细胞仪检测分析细胞周期分布。
图2参皂苷Rh3、人参皂苷PPD、人参皂苷Rh2、以及人参皂苷Rh3/PPD/Rh2组合物2、组合物6、组合物7、组合物8对胃癌细胞周期分布的影响。组合物2和人参皂苷Rh3、人参皂苷PPD、人参皂苷Rh2组与对照组相比，处于G1期的细胞比例均明显减少，S期的细胞比例均明显增加，G2期细胞比例均没有明显变化。人参皂苷Rh3、人参皂苷PPD、人参皂苷Rh2和组合物2、组合物6、组合物7、组合物8将S期细胞比例分别提高到33.9％、36.49％、33.49％、46.49％、45.02％、44.80％、42.85％。结果表明，组合物2、组合物6、组合物7、组合物8、人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2作用过的胃癌细胞均被阻断在S期。
表6 人参皂苷Rh3、PPD、Rh2以及组合物2、组合物6、组合物7、组合物8对胃癌细胞S期细胞影响
Figure PCTCN2021083840-appb-000010
Figure PCTCN2021083840-appb-000011
实施例4 Western blotting检测人参皂苷Rh3、人参皂苷PPD、人参皂苷Rh2、以及人参皂苷Rh3/PPD/Rh2组合物2对胃癌细胞与凋亡相关的蛋白表达
以胃癌细胞为例，将150μg/mL人参皂苷Rh3、人参皂苷PPD、人参皂苷Rh2以及组合物分别作用于该细胞48h，收集细胞加入裂解液提取总蛋白，用BCA蛋白分析检测试剂盒检测蛋白浓度。经10％SDS-PAGE分离，转移至PVDF膜上。2％脱脂奶粉室温摇床封闭2h后，分别加入GAPDH、Caspase-3、PARP、Bcl-2一抗(1:1000)，在4℃环境下孵育过夜。次日，TBST洗膜3次，加入二抗兔抗IgG(1:10000)室温孵育2h，再洗膜3次。随后加入现配的ECL发光液浸润PVDF膜后，用化学发光凝胶成像仪扫描，结果用Image J软件对蛋白条带进行光密度分析。
图3为Western blotting检测人参皂苷Rh3、人参皂苷PPD、人参皂苷Rh2、以及人参皂苷Rh3/PPD/Rh2组合物2对胃癌细胞与凋亡相关的蛋白表达实验结果图。组合物2与人参皂苷Rh3、人参皂苷PPD、人参皂苷Rh2组相比，与凋亡相关的蛋白如Caspase-3、Bcl-2、PARP的表达量均明显减少，表明组合物2与人参皂苷Rh3、人参皂苷PPD、人参皂苷Rh2可能通过调控这些蛋白的下调，来诱导胃癌细胞的凋亡。
实施例5 人参皂苷Rh3、人参皂苷PPD、人参皂苷Rh2、以及人参皂苷组合物2、组合物6、组合物7、组合物8对胃癌肿瘤的抑制作用
参考中国专利CN 109045052 A实施例6，稍作修改。将人胃癌细胞异种接种于雌性BALB/c裸鼠，每只裸鼠注射3-4×10 6个细胞。接种20天后，观察到裸鼠左腋窝皮下有肿瘤块长出，仅米粒大小。继续饲养裸鼠4天后， 肿瘤尺寸超过100mm 3时，将裸鼠随机分为空白对照组、人参皂苷Rh3治疗组、人参皂苷PPD治疗组、人参皂苷Rh2治疗组以及人参皂苷Rh3/PPD/Rh2组合物治疗组，此组合物治疗组为实施例1中的人参皂苷组合物2、组合物6、组合物7、组合物8。空白组按体重腹腔注射生理盐水溶液，人参皂苷Rh3治疗组、人参皂苷PPD治疗组、人参皂苷Rh2治疗组以及人参皂苷Rh3/PPD/Rh2组合物组按体重腹腔注射含Rh3、PPD、Rh2组合物浓度为30mg/kg/d的生理盐水溶液一天一次，连续30天。
本实施例结果如图4所示。与对照组相比，人参皂苷Rh3、PPD、Rh2以及组合物处理组小鼠体重呈缓慢增长趋势。人参皂苷Rh3、PPD、Rh2处理组以及人参皂苷组合物组的异种移植肿瘤体积低于对照组，且人参皂苷组合物2、组合物6、组合物7、组合物8的异种移植肿瘤体积要低于单独的人参皂苷Rh3、PPD、Rh2处理组。
同时计算相互作用指数CI＝ABC/(A×B×C)，T为实验组肿瘤抑制率，C为对照组肿瘤抑制率，ABC为联用组的T/C值，A、B、C为三种人参皂苷单独作用时的T/C值(若两种人参皂苷联用时，则公式变成CI＝AB/(A×B))。CI＜1时，表明组合物有协同作用，CI≤0.7时，协同作用非常显著。经计算可知，组合物组2、组合物6、组合物组7、组合物8联用指数CI分别为0.72、0.75、0.78、0.74，表明组合物中三种皂苷联用以及三种皂苷中两两联用都具有协同增效作用。
表7 人参皂苷Rh3、PPD、Rh2以及组合物2、组合物6、组合物7、组合物8对胃癌肿瘤抑制率的联合指数(CI)结果(以第35天肿瘤体积数据为例)
Figure PCTCN2021083840-appb-000012
Figure PCTCN2021083840-appb-000013
实施例6 人参皂苷Rh3、人参皂苷PPD、人参皂苷Rh2、以及人参皂苷Rh3/PPD/Rh2组合物体内安全性评价
将雄性健康ICR小鼠饲养于室温环境中，自由饮食，相对湿度为50～60％，12h白天/12h黑夜。饲养1周，待其适应环境后，禁食12h。12h后，将小鼠随机分为4组：(1)对照组；(2)30mg/kg人参皂苷Rh3组；(3)30mg/kg人参皂苷PPD组；(4)30mg/kg人参皂苷Rh2；(5))30mg/kg人参皂苷Rh3/PPD/Rh2组。以上5组均灌胃方式给药。给药6h后，取消禁食，正常喂养小鼠15天。观察小鼠饮水、摄食、体重及肝肾功能变化。
小鼠在用药后15天内体重、摄食量和饮食量的变化见图5。由图可知，人参皂苷Rh3组、人参皂苷PPD组、人参皂苷Rh2组以及人参皂苷Rh3/PPD/Rh2组小鼠体重、摄食量和饮水量都，与对照都没有明显差异。如图6所示，小鼠经人参皂苷Rh3给药15天后，人参皂苷Rh3治疗组、人参皂苷PPD组、人参皂苷Rh2组以及人参皂苷Rh3/PPD/Rh2组小鼠的肝肾重量、血清中谷丙转氨酶和肌酸酐的含量相对于空白组来说也并没有明显的差异。这说明，人参皂苷Rh3/PPD/Rh2组合物对小鼠的体内治疗是安全的。
需要说明的是，在可实施且不明显违背本发明的主旨的前提下，在本说明书中作为某一技术方案的构成部分所描述的任一技术特征或技术特征的组合同样也可以适用于其它技术方案；并且，在可实施且不明显违背本发明的主旨的前提下，作为不同技术方案的构成部分所描述的技术特征之间也可以以任意方式进行组合，来构成其它技术方案。本发明也包含在上述情况下通过组合而得到的技术方案，并且这些技术方案相当于记载在本说明书中。
以上通过具体实施方式和实施例对本发明进行了说明，但本领域技术人员应该理解的是，这些并非意图对本发明的范围进行限定，本发明的范围应由权利要求书确定。
工业实用性
本发明提供了一种包含稀有人参皂苷Rh3、PPD和Rh2的、协同增效的、具有抗肿瘤作用的稀有人参皂苷药物组合物。

Claims (10)

  1. 一种具有抗肿瘤作用的稀有人参皂苷药物组合物，其包含作为活性成分的治疗有效量的人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2，以及药学可接受的载体，
    其中，该稀有人参皂苷药物组合物中，所述人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2的重量比为：1:(1～2):(1～2)。
  2. 根据权利要求1所述的稀有人参皂苷药物组合物，其中，该稀有人参皂苷药物组合物中，所述人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2的重量比为：1:(1～1.1):(1.9～2)。
  3. 根据权利要求1所述的稀有人参皂苷药物组合物，其中，该稀有人参皂苷药物组合物中，所述人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2的重量比为1:1:2。
  4. 根据权利要求1所述的稀有人参皂苷药物组合物，其中，该稀有人参皂苷药物组合物中不包含其它人参皂苷。
  5. 根据权利要求1所述的稀有人参皂苷药物组合物，其中，该稀有人参皂苷药物组合物包含人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2作为唯一抗肿瘤活性成分。
  6. 包含人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2的组合物在制备抗肿瘤药物中的用途，其中，
    所述组合物中人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2的重量比为：1:(1～2):(1～2)；
    所述抗肿瘤药物包含人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2作为活性成分。
  7. 根据权利要求6所述的用途，其中，所述组合物中，所述人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2的重量比为：1:(1～1.1):(1.9～2)。
  8. 根据权利要求6所述的用途，其中，所述组合物中，所述组合物中，所述人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2的重量比为1:1:2。
  9. 根据权利要求6所述的用途，其中，所述组合物中不包含其它人参皂苷。
  10. 根据权利要求6所述的用途，其中，所述组合物包含人参皂苷Rh3、人参皂苷PPD和人参皂苷Rh2作为唯一抗肿瘤活性成分。
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