WO2021197372A1 - Anti-tumor composition containing rare ginsenosides rk2, ck, and ppt - Google Patents

Anti-tumor composition containing rare ginsenosides rk2, ck, and ppt Download PDF

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WO2021197372A1
WO2021197372A1 PCT/CN2021/084352 CN2021084352W WO2021197372A1 WO 2021197372 A1 WO2021197372 A1 WO 2021197372A1 CN 2021084352 W CN2021084352 W CN 2021084352W WO 2021197372 A1 WO2021197372 A1 WO 2021197372A1
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ginsenoside
composition
ppt
rare
tumor
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PCT/CN2021/084352
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French (fr)
Chinese (zh)
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范代娣
董杨芳
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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Publication of WO2021197372A1 publication Critical patent/WO2021197372A1/en

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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
    • 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 Rk2/CK/PPT composition with anti-tumor activity and synergy.
  • cancer As one of the most serious and malignant diseases in the world, cancer has seriously threatened human health and quality of life. Its morbidity and mortality are second only to cardiovascular and cerebrovascular diseases, and it is one of the most important causes of human death. one.
  • the incidence of cancer across the country is grim, and the incidence and mortality of different cancers such as lung cancer, breast cancer, and colon cancer continue to increase.
  • the more popular treatment methods for cancer include chemotherapy, radiation therapy and surgical treatment. Although these traditional treatment methods can alleviate the condition, they have serious side effects and cause great physical and mental pain to patients. Therefore, natural medicines Its anti-tumor properties have gained more and more attention.
  • Natural medicine is an important resource with great potential, which can be used to treat tumors and other diseases.
  • Ginseng, Panax notoginseng, American ginseng and other plants of the genus Ginseng, as a kind of precious Chinese medicinal materials, have been used to treat various diseases, such as cancer.
  • people have discovered more than 60 kinds of ginsenosides, most of which belong to prototype ginsenosides, including R1, Rb1, Rb2, Rb2, Rd, Re, Rf, Rg1, etc. They are easily soluble in water and are used by plants of the genus Ginseng.
  • the leading components of various pharmacological effects have pharmacological effects such as calming nerves, anti-oxidation, and regulating immune function, but they have not shown good anti-tumor effects.
  • the rare ginsenoside monomer For the rare ginsenoside monomer, its content in Panax genus plants is very small, and it must be processed before it can exist and is not easy to obtain, mainly including Rg3, Rh2, Rk1, Rg5, Rk3, Rh4, CK, Rk2, Rh3, PPD, PPT, etc., have poor water solubility, and only show good solubility in organic solvents such as ethanol and ethyl acetate.
  • the above-mentioned rare ginsenoside monomers usually exhibit different anti-tumor effects under the influence of sugars in the structure, and the law of their anti-tumor activity is: aglycon>monoglycoside>diglycoside>triglycoside>tetraglycoside.
  • rare ginsenoside monomers exhibit obvious anti-tumor effects, which can block tumor cell cycle distribution, induce tumor cell apoptosis, and inhibit tumor angiogenesis.
  • rare ginseng Saponin monomers such as the dozens of rare ginsenoside monomers mentioned above, have not shown particularly significant anti-tumor effects.
  • the purpose of the present invention is to provide a rare ginsenoside composition based on rare ginsenoside Rk2, ginsenoside CK and ginsenoside PPT, 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 present invention is based on more than a dozen rare ginsenoside monomers, such as Rg3, Rh2, Rk1, Rg5, Rk3, Rh4, CK, Rk2, Rh3, PPD, PPT, etc., which are combined in two or three or three or three combinations.
  • the MTT method is used to detect its inhibitory effect on tumor cells and the joint index is calculated;
  • the flow cytometry is used to detect its effect on the distribution of tumor cell cycle;
  • the AV/PI kit is used to detect its apoptosis effect on tumor cells.
  • Western blotting was used to detect its effect on the expression of tumor cell apoptosis-related proteins.
  • the results of flow cytometry showed that the rare ginsenoside Rk2/CK/PPT composition blocked significantly more tumor cells in the G1 phase than the single-use group, showing a strong synergistic effect.
  • the AV/PI kit test results show that the rare ginsenoside Rk2/CK/PPT composition induces tumor cell apoptosis, and its early apoptosis rate is significantly better than that of the single-use group.
  • Western blot detection results show that the ginsenoside Rk2/CK/PPT composition promotes the activation of Caspase-3 and PARP proteins, down-regulates the expression of Bcl-2 protein, and finally induces tumor cell apoptosis.
  • the present invention includes:
  • a rare ginsenoside pharmaceutical composition with anti-tumor effect (the rare ginsenoside pharmaceutical composition 1 of the present invention), which contains as active ingredients a therapeutically effective amount of ginsenoside Rk2, ginsenoside CK and ginsenoside PPT, And a pharmaceutically acceptable carrier.
  • the weight ratio of the ginsenoside Rk2, ginsenoside CK and ginsenoside PPT is 1:(1-2):(1-2).
  • the rare ginsenoside pharmaceutical composition according to item 1 wherein, in the rare ginsenoside pharmaceutical composition, the weight ratio of the ginsenoside Rk2, ginsenoside CK and ginsenoside PPT is: 1:(1 ⁇ 1.1 ): (1 ⁇ 1.1).
  • the rare ginsenoside pharmaceutical composition according to item 1 or 2 wherein, in the rare ginsenoside pharmaceutical composition, the weight ratio of the ginsenoside Rk2, ginsenoside CK and ginsenoside PPT is 1:1:1 .
  • the rare ginsenoside pharmaceutical composition according to any one of items 1 to 4, wherein the rare ginsenoside pharmaceutical composition comprises ginsenoside Rk2, ginsenoside CK and ginsenoside PPT as the sole anti-tumor active ingredients.
  • composition comprising ginsenoside Rk2, ginsenoside CK and ginsenoside PPT in the preparation of anti-tumor drugs, wherein:
  • the weight ratio of ginsenoside Rk2, ginsenoside CK and ginsenoside PPT in the composition is: 1:(1-2):(1-2);
  • the anti-tumor drug contains ginsenoside Rk2, ginsenoside CK and ginsenoside PPT as active ingredients.
  • composition comprises ginsenoside Rk2, ginsenoside CK and ginsenoside PPT as the sole anti-tumor active ingredients.
  • the inventors also found that under a specific ratio (for example, the weight ratio of ginsenoside Rk2, ginsenoside CK and ginsenoside PPT is 1:(1 ⁇ 2):(1 ⁇ 2), preferably 1:(1 ⁇ 1.1): (1 to 1.1), more preferably 1:1:1), in the rare ginsenoside pharmaceutical composition 1 of the present invention, not only ginsenoside Rk2, ginsenoside CK, and ginsenoside PPT are synergistic, and the above three Any two of these are also synergistic.
  • a specific ratio for example, the weight ratio of ginsenoside Rk2, ginsenoside CK and ginsenoside PPT is 1:(1 ⁇ 2):(1 ⁇ 2), preferably 1:(1 ⁇ 1.1): (1 to 1.1), more preferably 1:1:1
  • the present invention also includes:
  • a rare ginsenoside pharmaceutical composition with anti-tumor effect (the rare ginsenoside pharmaceutical composition 2-4 of the present invention), which contains as active ingredients a therapeutically effective amount of ginsenoside Rk2 and ginsenoside CK (or ginsenoside CK) Saponin Rk2 and ginsenoside PPT, or ginsenoside CK and ginsenoside PPT), and a pharmaceutically acceptable carrier,
  • the weight ratio of ginsenoside Rk2 and ginsenoside CK is 1:(1-2) (or the weight ratio of ginsenoside Rk2 and ginsenoside PPT is 1:(1-2). ), or the weight ratio of ginsenoside CK to ginsenoside PPT 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 Rk2 and ginsenoside CK (or ginsenoside Rk2 and ginsenoside PPT, or ginsenoside Rk2 and ginsenoside CK). Saponin CK and ginsenoside PPT) are the only anti-tumor active ingredients.
  • composition comprising ginsenoside Rk2 and ginsenoside CK (or ginsenoside Rk2 and ginsenoside PPT, or ginsenoside CK and ginsenoside PPT) in the preparation of anti-tumor drugs, wherein,
  • the weight ratio of ginsenoside Rk2 and ginsenoside CK is 1:(1-2) (or the weight ratio of ginsenoside Rk2 and ginsenoside PPT is 1:(1-2), or ginsenoside
  • the weight ratio of CK to ginsenoside PPT is (1 ⁇ 2):(1 ⁇ 2));
  • the anti-tumor drug contains ginsenoside Rk2 and ginsenoside CK (or ginsenoside Rk2 and ginsenoside PPT, or ginsenoside CK and ginsenoside PPT) as active ingredients.
  • composition comprises ginsenoside Rk2 and ginsenoside CK (or ginsenoside Rk2 and ginsenoside PPT, or ginsenoside CK and ginsenoside PPT) as The only anti-tumor active ingredient.
  • the rare ginsenoside pharmaceutical composition 1 of the present invention and the rare ginsenoside pharmaceutical composition 2-4 of the present invention are collectively referred to as the rare ginsenoside pharmaceutical composition of the present invention.
  • the rare ginsenoside pharmaceutical composition of the present invention is administered to the subcutaneous tumor-bearing mice, and its effect on the tumor volume and body weight of the tumor-bearing mice is detected and observed.
  • the results show that the single-use group can inhibit the increase in tumor volume.
  • the rare ginsenoside pharmaceutical composition of the present invention exhibits a significant synergistic effect in inhibiting tumor growth; there is no significant difference in the body weight of nude mice in all groups, showing low Toxic characteristics. Therefore, the present invention also relates to the use of the rare ginsenoside pharmaceutical composition of the present 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 the effects of rare ginsenoside Rk2/CK/PPT composition 1, composition 6, composition 9 and composition 12 on the cycle distribution of colon cancer cells
  • Figure 2 is a graph showing the apoptosis-inducing effect of rare ginsenoside Rk2/CK/PPT composition 1, composition 6, composition 9 and composition 12 on colon cancer cells
  • Figure 3 is a graph showing the effect of rare ginsenoside Rk2/CK/PPT composition 1 on the expression of Bcl-2 family proteins, Caspase-3 and PARP proteins in colon cancer cells.
  • Figure 4 is a graph showing the inhibitory effects of rare ginsenoside Rk2/CK/PPT composition 1, composition 6, composition 9 and composition 12 on colon cancer xenograft tumor models.
  • Figure 5 is a graph showing the effect of rare ginsenoside Rk2/CK/PPT composition 1 on the water intake, food intake and body weight changes of healthy mice.
  • Fig. 6 is a graph showing the effect of rare ginsenoside Rk2/CK/PPT composition 1 on liver function and kidney function of healthy mice.
  • 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 Rk2, ginsenoside CK and ginsenoside PPT (or ginsenoside Rk2 and ginsenoside CK, or ginsenoside Rk2 and ginsenoside PPT, or ginsenoside CK and ginsenoside PPT), And a pharmaceutically acceptable carrier,
  • the weight ratio of ginsenoside Rk2 and ginsenoside CK is 1:(1-2) (or the weight ratio of ginsenoside Rk2 and ginsenoside PPT is 1:(1-2). ), or the weight ratio of ginsenoside CK to ginsenoside PPT is (1-2):(1-2)).
  • ginsenoside PPT protopanaxatriol
  • PPT protopanaxatriol
  • PPT protopanaxatriol
  • PPT can be prepared by enzymatically hydrolyzing ginsenoside Re.
  • ginsenoside C-K refers to the compound described in the following chemical formula 2.
  • ginsenoside C-K is a known compound and can be prepared by methods known in the art.
  • ginsenoside C-K can be prepared by hydrolysis of ginsenoside Rb1 catalyzed by pectinase.
  • ginsenoside Rk2 refers to the compound described in the following chemical formula 3.
  • ginsenoside Rk2 is a known compound, which can be prepared by methods known in the art, for example, ginsenoside Rk2 can be prepared by catalyzing ginsenoside Rb1 at high temperature in an aqueous sodium hydroxide solution.
  • the inventor also found that if the content of ginsenoside Rk2, ginsenoside CK and ginsenoside PPT in the pharmaceutical composition is not within the ratio range defined by the present invention, it does not have a synergistic effect. Considering the significant synergistic effect (CI value is less than 0.7), in the composition, the weight ratio of ginsenoside Rk2, ginsenoside CK and ginsenoside PPT is preferably 1:(1 ⁇ 1.1):( 1 to 1.1), more preferably 1:1:1.
  • the pharmaceutical composition of the present invention may or may not contain other ginsenosides.
  • the ginsenoside Rk2, ginsenoside CK and ginsenoside PPT (or ginsenoside Rk2 and ginsenoside
  • the content of CK, or ginsenoside Rk2 and ginsenoside PPT, or ginsenoside CK and ginsenoside PPT) 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 The above two ginsenosides), based on 100 parts by weight of the total ginsen
  • the content of the total ginsenosides can be determined by the vanillin method, and the content of the ginsenosides Rk2, CK and PPT 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 Rk2, ginsenoside CK and ginsenoside PPT (or ginsenoside Rk2 and ginsenoside CK, or Ginsenoside Rk2 and ginsenoside PPT, or ginsenoside CK and ginsenoside PPT) as the only anti-tumor active ingredients).
  • anti-tumor active ingredients ie, ginsenoside Rk2, ginsenoside CK and ginsenoside PPT (or ginsenoside Rk2 and ginsenoside CK, or Ginsenoside Rk2 and ginsenoside PPT, or ginsenoside CK and ginsenoside PPT
  • 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)
  • the total amount of saponin Rk2 and ginsenoside PPT, or ginsenoside CK and ginsenoside PPT) is usually divided into 1 to 3 times a day.
  • Example 1 Inhibitory effect of rare ginsenoside Rk2/CK/PPT composition on the proliferation of three different tumor cells
  • composition 1, composition 2, composition 3, composition 4, and composition 5 weigh appropriate amounts of ginsenoside Rk2, ginsenoside CK, and ginsenoside PPT to prepare an aqueous solution with a final concentration of 100 ⁇ g/mL, which is called Rk2, CK, and PPT in turn.
  • the aqueous solution containing ginsenoside Rk2, ginsenoside CK, and ginsenoside PPT with a final concentration of 100 ⁇ g/mL is called composition 1, composition 2, composition 3, composition 4, and composition 5 in sequence.
  • Ginsenoside Rk2 and ginsenoside CK were mixed in a weight ratio of 1:1, 1:0.5, and 1:3 to prepare an aqueous solution containing ginsenoside Rk2 and ginsenoside CK at a final concentration of 100 ⁇ g/mL, and called the composition 6.
  • Composition 7 and Composition 8 Mix ginsenoside Rk2 and ginsenoside PPT in a weight ratio of 1:1, 1:0.5, and 1:3 to prepare an aqueous solution containing ginsenoside Rk2 and ginsenoside PPT with a final concentration of 100 ⁇ g/mL, which is called the composition 9.
  • composition 12 Mix ginsenoside CK and ginsenoside PPT in a weight ratio of 1:1, 1:0.5, and 1:3 to prepare an aqueous solution containing ginsenoside CK and ginsenoside PPT with a final concentration of 100 ⁇ g/mL, which is called a composition 12.
  • Composition 13 and Composition 14
  • Inhibition rate (1-administration group average OD value/control group average OD value) ⁇ 100%
  • composition two-drug combination index CI (refer to David H. Kern, Carol R. Morgan, and Susanne U. Hildebrand-Zanki. In vitro and in vivo interaction between cisplatin and topotecan in ovarian cancer systems[J]. Cancer Research, 1988,48. and Li Xingqi. The inhibitory effect of lidamycin on glioma and the synergistic effect of combined temozolomide[D]. Peking Union Medical College, 2009.):
  • T is the OD value of the experimental group
  • C is the OD value of the control group
  • AB is the T/C value of the saponin combination group
  • a and B are the T/C values of the saponin single-use group.
  • composition three-drug combination index CI Composition three-drug combination index CI:
  • T is the OD value of the experimental group
  • C is the OD value of the control group
  • ABC is the T/C value of the saponin combination group
  • A, B, and C are the T/C values of the saponin single-use group.
  • ginsenoside Rk2, ginsenoside CK and ginsenoside PPT all show certain inhibitory effects on the three types of tumor cells.
  • the combination of ginsenoside Rk2, ginsenoside CK and ginsenoside PPT can effectively enhance their Compared with the control, the inhibition rate of 3 kinds of tumor cells showed statistical significance (P ⁇ 0.05).
  • the anti-tumor effect is more obvious, and the combination index is less than 1.
  • composition 4 and composition 5 did not show a synergistic effect (CI>1).
  • composition 1 showed a more significant inhibitory effect than other compositions. The above results indicate that the composition of the present invention exerts a synergistic effect.
  • the value in the table is the inhibition rate, in%
  • the value in the table is the inhibition rate, in%
  • Example 2 The effect of rare ginsenoside Rk2/CK/PPT composition 1, composition 6, composition 9 and composition 12 on the cycle distribution of colon cancer cells
  • ginsenoside Rk2 ginsenoside CK
  • ginsenoside PPT composition 1, composition 6, composition 9 and composition at a concentration of 100 ⁇ g/mL 12.
  • an equal volume of RPMI-1640 culture medium was added, and 5 replicate wells were set for each concentration.
  • add ice-cold PBS to wash 2-3 times add 75% ice ethanol, adjust the cells to a suitable density and place them in a refrigerator at 4°C overnight.
  • add ice-cold PBS to wash 2-3 times add an appropriate amount of PI solution, incubate for 30 minutes in the dark, and use flow cytometry to detect the cell cycle distribution.
  • Figure 1 is a graph showing the experimental results of using flow cytometry to detect the effects of rare ginsenoside composition 1, composition 6, composition 9 and composition 12 on the cycle distribution of colon cancer cells.
  • Colon cancer cells were treated with ginsenoside Rk2, ginsenoside CK, ginsenoside PPT, composition 1, composition 6, composition 9, and composition 12 respectively, and compared with the control, the proportion of cells in the G1 phase increased significantly.
  • the proportion of cells in S phase decreased, and the proportion of cells in G2 phase did not change significantly.
  • Ginsenoside Rk2, ginsenoside CK, ginsenoside PPT, composition 1, composition 6, composition 9, composition 12 increase the proportion of colon cancer cells in G1 stage to 49.97%, 48.09%, 49.39%, 68.23%, respectively. 55.98%, 55.68%, 56.10%.
  • composition 1 composition 6, composition 9, and composition 12 on blocking the cycle distribution of colon cancer cells
  • Example 3 The effect of rare ginsenoside Rk2/CK/PPT composition 1, composition 6, composition 9 and composition 12 on colon cancer cells inducing apoptosis
  • T is the cell survival score of the experimental group
  • C is the cell survival score of the control group
  • AB is the T/C value of the saponin combination group
  • a and B are the survival score T/C values of the saponin alone group.
  • composition three-drug combination index CI Composition three-drug combination index CI:
  • T is the cell survival score of the experimental group
  • C is the cell survival score of the control group
  • ABC is the T/C value of the saponin combination group
  • A, B, and C are the survival score T/C values of the saponin alone group.
  • Figure 2 is a graph showing the experimental results of using flow cytometry to detect the rare ginsenoside composition 1, composition 6, composition 9 and composition 12 inducing apoptosis of colon cancer cells, obtaining a cell histogram composed of four quadrants, area Q1 It indicates the cells that have been mechanically damaged during the experimental operation.
  • the area Q2 indicates the cells that have undergone late apoptosis
  • the area Q3 indicates the cells that have normal functional morphology
  • the area Q4 indicates the cells that have undergone early apoptosis.
  • Ginsenoside Rk2, ginsenoside CK and ginsenoside PPT can induce certain apoptosis of colon cancer cells.
  • the rare ginsenoside composition 1, composition 6, composition 9 and composition 12 of the present invention promote the apoptosis of colon cancer cells.
  • the percentage of dead cells increased significantly.
  • the early apoptosis rates of ginsenoside Rk2, ginsenoside CK, and ginsenoside PPT on colon cancer cells were 11.32%, 10.01%, and 12.73%, respectively; composition 1, composition 6, composition 9 and composition 12 of the present invention
  • the early apoptosis rates of colon cancer cells were 40.38%, 24.34%, 27.67%, and 25.89%.
  • Example 4 The effect of composition 1 on the expression of Bcl-2 family proteins, Caspase-3 and PARP proteins in colon cancer cells
  • ginsenoside Rk2 ginsenoside Rk2
  • ginsenoside CK ginsenoside PPT
  • composition 1 a concentration of 100 ⁇ g/mL.
  • trypsin to digest the cells
  • RIPA lysate After 30 minutes, centrifuge to obtain the target sample.
  • gel preparation, SDS-PAGE electrophoresis, membrane transfer, blocking and incubation, and DAB color development the apoptosis-related protein expression map is obtained.
  • Figure 3 shows the results of detecting the expression of related proteins induced by ginsenoside Rk2, ginsenoside CK, ginsenoside PPT and composition 1 to induce apoptosis of colon cancer cells by Western blotting.
  • the expression of Caspase-3, PARP, and Bcl-2 proteins all decreased; after treatment with composition 1, the expression of the above three proteins changed more. obvious. It can be seen from this that Composition 1 finally induces cell apoptosis through Caspase-3 activation and Bcl-2 protein down-regulation.
  • Example 5 Inhibition of rare ginsenoside Rk2/CK/PPT composition 1, composition 6, composition 9 and composition 12 on colon cancer xenograft tumor model
  • the male BALB/c nude mice were reared at room temperature, and after they had adapted to the environment, they were inoculated with colon cancer cells, and each nude mouse was injected with 3-4 ⁇ 10 6 cells. Seven days after the inoculation, the left axillary of the nude mouse was observed under the skin, and it was found that nodules the size of rice grains were formed at the inoculation site of the nude mouse. When the tumor size exceeds 100mm 3 , the nude mice with large differences in tumor size and weight will be eliminated, and the remaining tumor-bearing nude mice will be randomly divided into groups.
  • T is the tumor weight of the experimental group
  • C is the tumor weight of the control group
  • AB is the T/C value of the two saponin combination group
  • a and B are the survival score T/C values of the saponin alone group.
  • T is the tumor weight of the experimental group
  • C is the tumor weight of the control group
  • ABC is the T/C value of the saponin combination group
  • A, B, and C are the T/C values of the saponin single-use group.
  • the ginsenoside Rk2 group, ginsenoside CK group, and ginsenoside PPT group had 27.15% tumor inhibition rates on colon cancer nude mice after 30 days of administration. , 33.18% and 34.05%, indicating that the ginsenoside Rk2 group, ginsenoside CK group and ginsenoside PPT group have a certain inhibitory effect on colon cancer transplanted tumors in nude mice.
  • the composition 1, composition 6, composition 9 and composition 12 groups can significantly reduce the tumor weight of tumor-bearing mice, and the corresponding tumor inhibition rates are 79.74%, 64.65%, 65.51%, and 67.24%, respectively.
  • the rare ginsenoside Rk2/CK/PPT composition can significantly hinder the growth of colon cancer transplanted tumors in nude mice, and its inhibition rate is significantly higher than that of the ginsenoside Rk2 group, ginsenoside CK group and ginsenoside PPT group.
  • Nude mice exhibit low toxicity and side effects, and the rare ginsenoside Rk2/CK/PPT composition provided by the present invention exhibits a synergistic effect.
  • Table 8 The combined index (CI index) of composition 1, composition 6, composition 9, composition 12 on tumor re-growth inhibition of colon cancer tumor-bearing mice
  • 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) blank group; (2) 40mg/kg ginsenoside Rk2 group; (3) 40mg/kg ginsenoside CK group; (4) 40mg/kg ginsenoside PPT group ; (5) 1 group of Rk2/CK/PPT composition. 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 14 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 ginsenoside Rk2, ginsenoside CK and ginsenoside PPT, which is synergistic and has anti-tumor effects.

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Abstract

An anti-tumor composition containing the rare ginsenosides Rk2, CK, and PPT, taking a therapeutically effective dose of ginsenoside Rk2, ginsenoside CK, and ginsenoside PPT as active ingredients, and further containing a pharmaceutically acceptable carrier. In the rare ginsenoside pharmaceutical composition, the mass ratio between the ginsenoside Rk2, the ginsenoside CK, and the ginsenoside PPT is: 1:(1-2):(1-2). Research has shown that when the mass ratio between the ginsenoside Rk2, the ginsenoside CK, and the ginsenoside PPT is a particular ratio, in the present anti-tumor composition, not only does a synergistic effect occur between the ginsenoside Rk2, the ginsenoside CK, and the ginsenoside PPT, but a synergistic effect also occurs between any two of the three.

Description

包含稀有人参皂苷Rk2、CK和PPT的抗肿瘤组合物Anti-tumor composition containing rare ginsenoside Rk2, CK and PPT 技术领域Technical field
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、协同增效的稀有人参皂苷Rk2/CK/PPT组合物。The invention relates to the field of biomedicine, in particular to a rare ginsenoside Rk2/CK/PPT composition with anti-tumor activity and synergy.
背景技术Background technique
癌症作为世界上最严重、恶性程度最强的疾病之一，已经严重威胁到人类的身体健康和生活质量，其发病率和死亡率仅次于心脑血管疾病，是人类死亡的最主要因素之一。全国癌症发病形势严峻，且肺癌、乳腺癌、结肠癌等不同癌症的发病率与死亡率持续保持上升趋势，每年新发癌症病例约400万，因癌症死亡约250万。目前，对于癌症比较流行的治疗手段有化学治疗、辐射治疗及手术治疗等，这些传统治疗方法虽能缓解病情，但却具有严重的副作用，给病人带来极大的身心痛苦，因此，天然药物的抗肿瘤特性取得了越来越多的关注。As one of the most serious and malignant diseases in the world, cancer has seriously threatened human health and quality of life. Its morbidity and mortality are second only to cardiovascular and cerebrovascular diseases, and it is one of the most important causes of human death. one. The incidence of cancer across the country is grim, and the incidence and mortality of different cancers such as lung cancer, breast cancer, and colon cancer continue to increase. There are about 4 million new cancer cases each year, and about 2.5 million cancer deaths. At present, the more popular treatment methods for cancer include chemotherapy, radiation therapy and surgical treatment. Although these traditional treatment methods can alleviate the condition, they have serious side effects and cause great physical and mental pain to patients. Therefore, natural medicines Its anti-tumor properties have gained more and more attention.
天然药物是一种很有潜力的重要资源，可以用其治疗肿瘤等疾病。人参、三七、西洋参等人参属植物，作为一类珍贵的中药材，已经被用于治疗各种疾病，如癌症。目前，人们从中发现的人参皂苷达60多种，绝大多数属于原型人参皂苷，包括R1、Rb1、Rb2、Rb2、Rd、Re、Rf、Rg1等，其易溶于水，是人参属植物发挥各种药理学作用的主导成分，具有安神、抗氧化、调节免疫功能等药理作用，但是并没有表现出较好的抗肿瘤效果。Natural medicine is an important resource with great potential, which can be used to treat tumors and other diseases. Ginseng, Panax notoginseng, American ginseng and other plants of the genus Ginseng, as a kind of precious Chinese medicinal materials, have been used to treat various diseases, such as cancer. At present, people have discovered more than 60 kinds of ginsenosides, most of which belong to prototype ginsenosides, including R1, Rb1, Rb2, Rb2, Rd, Re, Rf, Rg1, etc. They are easily soluble in water and are used by plants of the genus Ginseng. The leading components of various pharmacological effects have pharmacological effects such as calming nerves, anti-oxidation, and regulating immune function, but they have not shown good anti-tumor effects.
对于稀有人参皂苷单体，其在人参属植物中含量微乎其微，必须将其经过加工处理后才可能存在且不易获得，主要包括Rg3、Rh2、Rk1、Rg5、Rk3、Rh4、CK、Rk2、Rh3、PPD、PPT等，水溶性差，只有在乙醇、乙酸乙酯等有机溶剂中才表现出较好的溶解性。上述稀有人参皂苷单体通常受结构中的糖影响表现出不同的抗肿瘤作用，其抗肿瘤活性强弱规律是：苷元＞单糖苷＞二糖苷＞三糖苷＞四糖苷。目前，大量文献表明稀有人参皂苷单体表现出明显的抗肿瘤作用，其能够阻断肿瘤细胞周期分布、诱导肿瘤细胞凋亡、抑制肿瘤血管的生成等，但是现有的单一的一种稀有人参皂苷单体，例如上述十几种稀有人参皂苷单体并没有表现出特别显著的抗肿瘤效果。For the rare ginsenoside monomer, its content in Panax genus plants is very small, and it must be processed before it can exist and is not easy to obtain, mainly including Rg3, Rh2, Rk1, Rg5, Rk3, Rh4, CK, Rk2, Rh3, PPD, PPT, etc., have poor water solubility, and only show good solubility in organic solvents such as ethanol and ethyl acetate. The above-mentioned rare ginsenoside monomers usually exhibit different anti-tumor effects under the influence of sugars in the structure, and the law of their anti-tumor activity is: aglycon>monoglycoside>diglycoside>triglycoside>tetraglycoside. At present, a large number of documents show that rare ginsenoside monomers exhibit obvious anti-tumor effects, which can block tumor cell cycle distribution, induce tumor cell apoptosis, and inhibit tumor angiogenesis. However, there is a single rare ginseng Saponin monomers, such as the dozens of rare ginsenoside monomers mentioned above, have not shown particularly significant anti-tumor effects.
发明内容Summary of the invention
本发明的目的在于，提供一种以稀有人参皂苷Rk2、人参皂苷CK和人参皂苷PPT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。The purpose of the present invention is to provide a rare ginsenoside composition based on rare ginsenoside Rk2, ginsenoside CK and ginsenoside PPT, which is synergistic and has anti-tumor effects. In addition, 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.
本发明是在十几种稀有人参皂苷单体中，如Rg3、Rh2、Rk1、Rg5、Rk3、Rh4、CK、Rk2、Rh3、PPD、PPT等，将其进行两两组合或者三三组合后作用于肿瘤细胞并进行相关检测。其中，采用MTT法检测其对肿瘤细胞的抑制作用并计算联合指数；采用流式细胞术检测其对肿瘤细胞周期分布的影响；采用AV/PI试剂盒检测其对肿瘤细胞的凋亡作用并得出其凋亡率；采用蛋白印迹法检测其对肿瘤细胞凋亡相关蛋白表达的影响。流式细胞术检测结果显示稀有人参皂苷Rk2/CK/PPT组合物阻断在G1期的肿瘤细胞明显多于单用组，表现出较强的协同效应。AV/PI试剂盒检测结果显示稀有人参皂苷Rk2/CK/PPT组合物诱导肿瘤细胞凋亡，其早期凋亡率明显优于单用组。蛋白印迹法检测结果显示人参皂苷Rk2/CK/PPT组合物促使Caspase-3和PARP蛋白活化，下调Bcl-2蛋白表达，最终诱导肿瘤细胞凋亡。大量实验结果表明，将人参皂苷Rk2、人参皂苷CK和人参皂苷PPT按特定比例进行组合，不仅具有协同增效的抗肿瘤作用，而且表现出低毒性特点，从而完成了本发明。The present invention is based on more than a dozen rare ginsenoside monomers, such as Rg3, Rh2, Rk1, Rg5, Rk3, Rh4, CK, Rk2, Rh3, PPD, PPT, etc., which are combined in two or three or three or three combinations. On tumor cells and perform related tests. Among them, the MTT method is used to detect its inhibitory effect on tumor cells and the joint index is calculated; the flow cytometry is used to detect its effect on the distribution of tumor cell cycle; the AV/PI kit is used to detect its apoptosis effect on tumor cells. To determine its apoptosis rate; Western blotting was used to detect its effect on the expression of tumor cell apoptosis-related proteins. The results of flow cytometry showed that the rare ginsenoside Rk2/CK/PPT composition blocked significantly more tumor cells in the G1 phase than the single-use group, showing a strong synergistic effect. The AV/PI kit test results show that the rare ginsenoside Rk2/CK/PPT composition induces tumor cell apoptosis, and its early apoptosis rate is significantly better than that of the single-use group. Western blot detection results show that the ginsenoside Rk2/CK/PPT composition promotes the activation of Caspase-3 and PARP proteins, down-regulates the expression of Bcl-2 protein, and finally induces tumor cell apoptosis. A large number of experimental results show that the combination of ginsenoside Rk2, ginsenoside CK and ginsenoside PPT in a specific ratio not only has a synergistic anti-tumor effect, but also exhibits low toxicity characteristics, thus completing the present invention.
即，本发明包括：That is, the present invention includes:
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1)，其包含作为活性成分的治疗有效量的人参皂苷Rk2、人参皂苷CK和人参皂苷PPT，以及药学可接受的载体。1. A rare ginsenoside pharmaceutical composition with anti-tumor effect (the rare ginsenoside pharmaceutical composition 1 of the present invention), which contains as active ingredients a therapeutically effective amount of ginsenoside Rk2, ginsenoside CK and ginsenoside PPT, And a pharmaceutically acceptable carrier.
其中，该稀有人参皂苷药物组合物中，所述人参皂苷Rk2、人参皂苷CK和人参皂苷PPT的重量比为：1:(1～2):(1～2)。Wherein, in the rare ginsenoside pharmaceutical composition, the weight ratio of the ginsenoside Rk2, ginsenoside CK and ginsenoside PPT is 1:(1-2):(1-2).
2.根据项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Rk2、人参皂苷CK和人参皂苷PPT的重量比为：1:(1～1.1):(1～1.1)。2. The rare ginsenoside pharmaceutical composition according to item 1, wherein, in the rare ginsenoside pharmaceutical composition, the weight ratio of the ginsenoside Rk2, ginsenoside CK and ginsenoside PPT is: 1:(1～1.1 ): (1～1.1).
3.根据项1或2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Rk2、人参皂苷CK和人参皂苷PPT的重量比为1:1:1。3. The rare ginsenoside pharmaceutical composition according to item 1 or 2, wherein, in the rare ginsenoside pharmaceutical composition, the weight ratio of the ginsenoside Rk2, ginsenoside CK and ginsenoside PPT is 1:1:1 .
4.根据项1～3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。4. The rare ginsenoside pharmaceutical composition according to any one of items 1 to 3, wherein the rare ginsenoside pharmaceutical composition does not contain other ginsenosides.
5.根据项1～4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Rk2、人参皂苷CK和人参皂苷PPT作为唯一抗肿瘤活性成分。5. The rare ginsenoside pharmaceutical composition according to any one of items 1 to 4, wherein the rare ginsenoside pharmaceutical composition comprises ginsenoside Rk2, ginsenoside CK and ginsenoside PPT as the sole anti-tumor active ingredients.
6.包含人参皂苷Rk2、人参皂苷CK和人参皂苷PPT的组合物在制备抗肿瘤药物中的用途，其中，6. Use of a composition comprising ginsenoside Rk2, ginsenoside CK and ginsenoside PPT in the preparation of anti-tumor drugs, wherein:
所述组合物中人参皂苷Rk2、人参皂苷CK和人参皂苷PPT的重量比为：1:(1～2):(1～2)；The weight ratio of ginsenoside Rk2, ginsenoside CK and ginsenoside PPT in the composition is: 1:(1-2):(1-2);
所述抗肿瘤药物包含人参皂苷Rk2、人参皂苷CK和人参皂苷PPT作为活性成分。The anti-tumor drug contains ginsenoside Rk2, ginsenoside CK and ginsenoside PPT as active ingredients.
7.根据项6所述的用途，其中，所述组合物中，所述人参皂苷Rk2、人参皂苷CK和人参皂苷PPT的重量比为：1:(1～1.1):(1～1.1)。7. The use according to item 6, wherein, in the composition, the weight ratio of the ginsenoside Rk2, ginsenoside CK and ginsenoside PPT is 1: (1 to 1.1): (1 to 1.1).
8.根据项6或7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Rk2、人参皂苷CK和人参皂苷PPT的重量比为1:1:1。8. The use according to item 6 or 7, wherein, in the composition, the weight ratio of the ginsenoside Rk2, ginsenoside CK and ginsenoside PPT in the composition is 1:1:1.
9.根据项6～8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。9. The use according to any one of items 6 to 8, wherein the composition does not contain other ginsenosides.
10.根据项6～9中任一项所述的用途，其中，所述组合物包含人参皂苷Rk2、人参皂苷CK和人参皂苷PPT作为唯一抗肿瘤活性成分。10. The use according to any one of items 6 to 9, wherein the composition comprises ginsenoside Rk2, ginsenoside CK and ginsenoside PPT as the sole anti-tumor active ingredients.
发明人还发现，在特定的比例下(例如人参皂苷Rk2、人参皂苷CK和人参皂苷PPT的重量比为1:(1～2):(1～2)、优选1:(1～1.1):(1～1.1)、更优选1:1:1)，本发明的稀有人参皂苷药物组合物1中，不但人参皂苷Rk2、人参皂苷CK和人参皂苷PPT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。The inventors also found that under a specific ratio (for example, the weight ratio of ginsenoside Rk2, ginsenoside CK and ginsenoside PPT is 1:(1～2):(1～2), preferably 1:(1～1.1): (1 to 1.1), more preferably 1:1:1), in the rare ginsenoside pharmaceutical composition 1 of the present invention, not only ginsenoside Rk2, ginsenoside CK, and ginsenoside PPT are synergistic, and the above three Any two of these are also synergistic.
因此，本发明还包括：Therefore, the present invention also includes:
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2-4)，其包含作为活性成分的治疗有效量的人参皂苷Rk2和人参皂苷CK(或者人参皂苷Rk2和人参皂苷PPT，或者人参皂苷CK和人参皂苷PPT)，以及药学可接受的载体，11. A rare ginsenoside pharmaceutical composition with anti-tumor effect (the rare ginsenoside pharmaceutical composition 2-4 of the present invention), which contains as active ingredients a therapeutically effective amount of ginsenoside Rk2 and ginsenoside CK (or ginsenoside CK) Saponin Rk2 and ginsenoside PPT, or ginsenoside CK and ginsenoside PPT), and a pharmaceutically acceptable carrier,
其中，该稀有人参皂苷药物组合物中，所述人参皂苷Rk2和人参皂苷CK的重量比为1:(1～2)(或者人参皂苷Rk2和人参皂苷PPT的重量比为1:(1～2)，或者人参皂苷CK和人参皂苷PPT的重量比为(1～2):(1～2))。Wherein, in the rare ginsenoside pharmaceutical composition, the weight ratio of ginsenoside Rk2 and ginsenoside CK is 1:(1-2) (or the weight ratio of ginsenoside Rk2 and ginsenoside PPT is 1:(1-2). ), or the weight ratio of ginsenoside CK to ginsenoside PPT is (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Rk2和人参皂苷CK的重量比为1:(1～1.1)(或者人参皂苷Rk2和人参皂苷PPT的重量比为1:(1～1.1)，或者人参皂苷CK和人参皂苷PPT的重量比为(1～1.1):(1～1.1))。12. The rare ginsenoside pharmaceutical composition according to item 11, wherein, in the rare ginsenoside pharmaceutical composition, the weight ratio of the ginsenoside Rk2 and ginsenoside CK is 1:(1～1.1) (or ginsenoside The weight ratio of Rk2 and ginsenoside PPT is 1: (1 to 1.1), or the weight ratio of ginsenoside CK to ginsenoside PPT is (1 to 1.1): (1 to 1.1)).
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Rk2和人参皂苷CK的重量比为1:1(或者人参皂苷Rk2和人参皂苷PPT的重量比为1:1，或者人参皂苷CK和人参皂苷PPT的重量比为1:1)。13. The rare ginsenoside pharmaceutical composition according to item 11 or 12, wherein, in the rare ginsenoside pharmaceutical composition, the weight ratio of ginsenoside Rk2 and ginsenoside CK is 1:1 (or ginsenoside Rk2 and The weight ratio of ginsenoside PPT is 1:1, or the weight ratio of ginsenoside CK and ginsenoside PPT is 1:1).
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。14. 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.
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Rk2和人参皂苷CK(或者人参皂苷Rk2和人参皂苷PPT，或者人参皂苷CK和人参皂苷PPT)作为唯一抗肿瘤活性成分。15. The rare ginsenoside pharmaceutical composition according to any one of items 11 to 14, wherein the rare ginsenoside pharmaceutical composition comprises ginsenoside Rk2 and ginsenoside CK (or ginsenoside Rk2 and ginsenoside PPT, or ginsenoside Rk2 and ginsenoside CK). Saponin CK and ginsenoside PPT) are the only anti-tumor active ingredients.
16.包含人参皂苷Rk2和人参皂苷CK(或者人参皂苷Rk2和人参皂苷PPT，或者人参皂苷CK和人参皂苷PPT)的组合物在制备抗肿瘤药物中的用途，其中，16. Use of a composition comprising ginsenoside Rk2 and ginsenoside CK (or ginsenoside Rk2 and ginsenoside PPT, or ginsenoside CK and ginsenoside PPT) in the preparation of anti-tumor drugs, wherein,
所述组合物中，所述人参皂苷Rk2和人参皂苷CK的重量比为1:(1～2)(或者人参皂苷Rk2和人参皂苷PPT的重量比为1:(1～2)，或者人参皂苷CK和人参皂苷PPT的重量比为(1～2):(1～2))；In the composition, the weight ratio of ginsenoside Rk2 and ginsenoside CK is 1:(1-2) (or the weight ratio of ginsenoside Rk2 and ginsenoside PPT is 1:(1-2), or ginsenoside The weight ratio of CK to ginsenoside PPT is (1～2):(1～2));
所述抗肿瘤药物包含人参皂苷Rk2和人参皂苷CK(或者人参皂苷Rk2和人参皂苷PPT，或者人参皂苷CK和人参皂苷PPT)作为活性成分。The anti-tumor drug contains ginsenoside Rk2 and ginsenoside CK (or ginsenoside Rk2 and ginsenoside PPT, or ginsenoside CK and ginsenoside PPT) as active ingredients.
17.根据项16所述的用途，其中，所述组合物中，所述人参皂苷Rk2和人参皂苷CK的重量比为1:(1～1.1)(或者人参皂苷Rk2和人参皂苷PPT的重量比为1:(1～1.1)，或者人参皂苷CK和人参皂苷PPT的重量比为(1～1.1):(1～1.1))。17. The use according to item 16, wherein, in the composition, the weight ratio of ginsenoside Rk2 and ginsenoside CK is 1:(1～1.1) (or the weight ratio of ginsenoside Rk2 and ginsenoside PPT) It is 1:(1～1.1), or the weight ratio of ginsenoside CK and ginsenoside PPT is (1～1.1):(1～1.1)).
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Rk2和人参皂苷CK(或者人参皂苷Rk2和人参皂苷PPT，或者人参皂苷CK和人参皂苷PPT)的重量比1:1。18. The use according to item 16 or 17, wherein, in the composition, the weight of the ginsenoside Rk2 and ginsenoside CK (or ginsenoside Rk2 and ginsenoside PPT, or ginsenoside CK and ginsenoside PPT) Ratio 1:1.
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。19. The use according to any one of items 16 to 18, wherein the composition does not contain other ginsenosides.
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Rk2和人参皂苷CK(或者人参皂苷Rk2和人参皂苷PPT，或者人参皂苷CK和人参皂苷PPT)作为唯一抗肿瘤活性成分。20. The use according to any one of items 16 to 19, wherein the composition comprises ginsenoside Rk2 and ginsenoside CK (or ginsenoside Rk2 and ginsenoside PPT, or ginsenoside CK and ginsenoside PPT) as The only anti-tumor active ingredient.
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1和本发明的稀有人参皂苷药物组合物2-4统称为本发明的稀有人参皂苷药物组合物。In this specification, in some cases, the rare ginsenoside pharmaceutical composition 1 of the present invention and the rare ginsenoside pharmaceutical composition 2-4 of the present invention are collectively referred to as the rare ginsenoside pharmaceutical composition of the present invention.
将本发明的稀有人参皂苷药物组合物给药于皮下荷瘤小鼠，检测并观察其对荷瘤小鼠肿瘤体积和体重的影响。结果显示，单用组能抑制其肿瘤体积的增加，本发明的稀有人参皂苷药物组合物在抑制肿瘤生长方面表现出明显的协同效应；所有组的裸鼠体重之间没有明显差异，表现出低毒性特点。因此，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。The rare ginsenoside pharmaceutical composition of the present invention is administered to the subcutaneous tumor-bearing mice, and its effect on the tumor volume and body weight of the tumor-bearing mice is detected and observed. The results show that the single-use group can inhibit the increase in tumor volume. The rare ginsenoside pharmaceutical composition of the present invention exhibits a significant synergistic effect in inhibiting tumor growth; there is no significant difference in the body weight of nude mice in all groups, showing low Toxic characteristics. Therefore, the present invention also relates to the use of the rare ginsenoside pharmaceutical composition of the present invention in the preparation of anti-tumor drugs. The tumor may be, for example, stomach cancer, liver cancer, pancreatic cancer.
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。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.
附图说明Description of the drawings
图1为显示稀有人参皂苷Rk2/CK/PPT组合物1、组合物6、组合物9及组合物12对结肠癌细胞周期分布的影响的图Figure 1 is a graph showing the effects of rare ginsenoside Rk2/CK/PPT composition 1, composition 6, composition 9 and composition 12 on the cycle distribution of colon cancer cells
图2为显示稀有人参皂苷Rk2/CK/PPT组合物1、组合物6、组合物9及组合物12对结肠癌细胞诱导凋亡作用的图Figure 2 is a graph showing the apoptosis-inducing effect of rare ginsenoside Rk2/CK/PPT composition 1, composition 6, composition 9 and composition 12 on colon cancer cells
图3为显示稀有人参皂苷Rk2/CK/PPT组合物1对结肠癌细胞Bcl-2家族蛋白、Caspase-3及PARP蛋白表达的影响的图。Figure 3 is a graph showing the effect of rare ginsenoside Rk2/CK/PPT composition 1 on the expression of Bcl-2 family proteins, Caspase-3 and PARP proteins in colon cancer cells.
图4为显示稀有人参皂苷Rk2/CK/PPT组合物1、组合物6、组合物9及组合物12对结肠癌移植瘤模型的抑制作用的图。Figure 4 is a graph showing the inhibitory effects of rare ginsenoside Rk2/CK/PPT composition 1, composition 6, composition 9 and composition 12 on colon cancer xenograft tumor models.
图5为显示稀有人参皂苷Rk2/CK/PPT组合物1对健康小鼠饮水量、摄食量和体重变化的影响的图。Figure 5 is a graph showing the effect of rare ginsenoside Rk2/CK/PPT composition 1 on the water intake, food intake and body weight changes of healthy mice.
图6为显示稀有人参皂苷Rk2/CK/PPT组合物1对健康小鼠肝功能和肾功能的影响的图。Fig. 6 is a graph showing the effect of rare ginsenoside Rk2/CK/PPT composition 1 on liver function and kidney function of healthy mice.
在上述图1-6中，如无特殊说明，未标注表示无显著差异，*表示有显著差异，P<0.05；**表示有显著差异，P<0.01；***表示有显著差异，P<0.001。In the above Figure 1-6, if there is no special description, unmarked indicates no significant difference, * indicates a significant difference, P<0.05; ** indicates a significant difference, P<0.01; *** indicates a significant difference, P <0.001.
发明的具体实施方式Specific embodiments of the invention
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。The present invention will be described in detail below in conjunction with specific embodiments. In the absence of conflicts, the scientific terms in this specification have the meanings commonly understood by those skilled in the art, and in case of conflict, the definitions in this specification shall prevail.
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Rk2、人参皂苷CK和人参皂苷PPT(或者人参皂苷Rk2和人参皂苷CK，或者人参皂苷Rk2和人参皂苷PPT，或者人参皂苷CK和人参皂苷PPT)，以及药学可接受的载体，First, in one aspect, 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 Rk2, ginsenoside CK and ginsenoside PPT (or ginsenoside Rk2 and ginsenoside CK, or ginsenoside Rk2 and ginsenoside PPT, or ginsenoside CK and ginsenoside PPT), And a pharmaceutically acceptable carrier,
其中，该稀有人参皂苷药物组合物中，所述人参皂苷Rk2和人参皂苷CK的重量比为1:(1～2)(或者人参皂苷Rk2和人参皂苷PPT的重量比为1:(1～2)，或者人参皂苷CK和人参皂苷PPT的重量比为(1～2):(1～2))。Wherein, in the rare ginsenoside pharmaceutical composition, the weight ratio of ginsenoside Rk2 and ginsenoside CK is 1:(1-2) (or the weight ratio of ginsenoside Rk2 and ginsenoside PPT is 1:(1-2). ), or the weight ratio of ginsenoside CK to ginsenoside PPT is (1-2):(1-2)).
在本说明书中，人参皂苷PPT(原人参三醇)是指下述化学式1所述的化合物。In this specification, ginsenoside PPT (protopanaxatriol) refers to the compound described in the following chemical formula 1.
【化学式1】【Chemical formula 1】
Figure PCTCN2021084352-appb-000001
Figure PCTCN2021084352-appb-000001
上述原人参三醇(PPT)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Re来制备原人参三醇(PPT)。The above-mentioned protopanaxatriol (PPT) is a known compound and can be prepared by methods known in the art, for example, protopanaxatriol (PPT) can be prepared by enzymatically hydrolyzing ginsenoside Re.
在本说明书中，人参皂苷C-K是指下述化学式2所述的化合物。In this specification, ginsenoside C-K refers to the compound described in the following chemical formula 2.
【化学式2】【Chemical formula 2】
Figure PCTCN2021084352-appb-000002
Figure PCTCN2021084352-appb-000002
上述人参皂苷C-K是已知化合物，可以采用本技术领域已知的方法来制备，例如可以通过果胶酶催化人参皂苷Rb1水解来制备人参皂苷C-K。The above-mentioned ginsenoside C-K is a known compound and can be prepared by methods known in the art. For example, ginsenoside C-K can be prepared by hydrolysis of ginsenoside Rb1 catalyzed by pectinase.
在本说明书中，人参皂苷Rk2是指下述化学式3所述的化合物。In this specification, ginsenoside Rk2 refers to the compound described in the following chemical formula 3.
【化学式3】【Chemical formula 3】
Figure PCTCN2021084352-appb-000003
Figure PCTCN2021084352-appb-000003
上述人参皂苷Rk2是已知化合物，可以采用本技术领域已知的方法来制备，例如可以通过氢氧化钠水溶液高温催化人参皂苷Rb1来制备人参皂苷Rk2。The above-mentioned ginsenoside Rk2 is a known compound, which can be prepared by methods known in the art, for example, ginsenoside Rk2 can be prepared by catalyzing ginsenoside Rb1 at high temperature in an aqueous sodium hydroxide solution.
发明人发现，将人参皂苷Rk2、人参皂苷CK和人参皂苷PPT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Rk2和人参皂苷CK、或者人参皂苷Rk2和人参皂苷PPT、或者人参皂苷CK和人参皂苷PPT也是协同增效和低毒性的。而且，发明人还发现，如果所述药物组合物中人参皂苷Rk2、人参皂苷CK和人参皂苷PPT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Rk2、人参皂苷CK和人参皂苷PPT的重量比为优选为1:(1～1.1):(1～1.1)，更优选为1:1:1。The inventor found that combining ginsenoside Rk2, ginsenoside CK, and ginsenoside PPT in the ratio range defined by the present invention not only showed significant synergy in anti-tumor effects (CI value less than 1, preferably CI value less than 0.7) , And also show low toxicity characteristics. The inventor also found that within the above ratio range, ginsenoside Rk2 and ginsenoside CK, or ginsenoside Rk2 and ginsenoside PPT, or ginsenoside CK and ginsenoside PPT are also synergistic and low-toxic. Moreover, the inventor also found that if the content of ginsenoside Rk2, ginsenoside CK and ginsenoside PPT in the pharmaceutical composition is not within the ratio range defined by the present invention, it does not have a synergistic effect. Considering the significant synergistic effect (CI value is less than 0.7), in the composition, the weight ratio of ginsenoside Rk2, ginsenoside CK and ginsenoside PPT is preferably 1:(1~1.1):( 1 to 1.1), more preferably 1:1:1.
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Rk2、人参皂苷CK和人参皂苷PPT(或者人参皂苷Rk2和人参皂苷CK，或者人参皂苷Rk2和人参皂苷PPT，或者人参皂苷CK和人参皂苷PPT)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者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Rk2的纯度可以大于98％，人参皂苷CK的纯度可以大于98％，人参皂苷PPT的纯度可以大于95％。The pharmaceutical composition of the present invention may or may not contain other ginsenosides. In the case where the pharmaceutical composition of the present invention contains other ginsenosides, from the perspective of better synergistic effects, the ginsenoside Rk2, ginsenoside CK and ginsenoside PPT (or ginsenoside Rk2 and ginsenoside The content of CK, or ginsenoside Rk2 and ginsenoside PPT, or ginsenoside CK and ginsenoside PPT) 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 The above two ginsenosides), based on 100 parts by weight of the total ginsenosides contained in the pharmaceutical composition. The purity of ginsenoside Rk2 used in the pharmaceutical composition of the present invention can be greater than 98%, the purity of ginsenoside CK can be greater than 98%, and the purity of ginsenoside PPT can be greater than 95%.
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Rk2、CK和PPT的含量均可以采用HPLC法测定。The content of the total ginsenosides can be determined by the vanillin method, and the content of the ginsenosides Rk2, CK and PPT can all be determined by the HPLC method.
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Rk2、人参皂苷CK和人参皂苷PPT(或者人参皂苷Rk2和人参皂苷CK，或者人参皂苷Rk2和人参皂苷PPT，或者人参皂苷CK和人参皂苷PPT)作为唯一抗肿瘤活性成分)。The pharmaceutical composition of the present invention may contain other anti-tumor active ingredients, or may not contain anti-tumor active ingredients (ie, ginsenoside Rk2, ginsenoside CK and ginsenoside PPT (or ginsenoside Rk2 and ginsenoside CK, or Ginsenoside Rk2 and ginsenoside PPT, or ginsenoside CK and ginsenoside PPT) as the only anti-tumor active ingredients).
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。On the other hand, the inventor found that the pharmaceutical composition of the present invention has a significant anti-tumor effect. Therefore, the present invention also provides the use of the pharmaceutical composition of the present invention in the preparation of anti-tumor drugs.
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。The pharmaceutical composition of the present invention may contain pharmaceutically acceptable carriers, such as excipients. There are no special restrictions on the excipients in the pharmaceutical composition of the present invention. For example, the excipients commonly used in medicines or health care products in this technical field can be used. Specifically, the auxiliary materials are starch, dextrin, lactose, mannitol, sodium hypromellose, xanthan gum, protein sugar and the like.
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。There is no particular limitation on the dosage form of the pharmaceutical composition of the present invention, for example, it 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.
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。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.
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。As excipients, for example, lactose, corn starch, white sugar, glucose, sorbitol, crystalline cellulose, silicon dioxide, etc. can be used; as binders, for example, polyvinyl alcohol, ethyl cellulose, and methyl cellulose can be used. As 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. Of course, the above-mentioned tablets and granules can also be coated with sugar coating, gelatin coating, and other necessary outer coatings.
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。When preparing injections, 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 . At this time, if necessary, a freeze-dried product can also be prepared by a conventional method.
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。Examples of the suspending aid include methyl cellulose, Tween 80, hydroxyethyl cellulose, gum arabic, tragacanth powder, sodium carboxymethyl cellulose, polyoxyethylene sorbitol monolaurate, and the like.
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。Examples of the solubilizer include polyoxyethylene hydrogenated castor oil, Tween 80, nicotinamide, polyoxyethylene sorbitol monolaurate, polyethylene glycol, and castor oil fatty acid ethyl ester.
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。In addition, examples of stabilizers include sodium sulfite and sodium metasulfite; examples of preservatives include methyl paraben, ethyl paraben, sorbic acid, phenol, cresol, and chlorocresol.
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。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.
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Rk2、人参皂苷CK和人参皂苷PPT(或者人参皂苷Rk2和人参皂苷PPT，或者人参皂苷CK和人参皂苷PPT)总量计)，上述施用量通常可每日分1～3次施用。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) Daily 1μg～30000mg, preferably 10μg～3000mg, more preferably 100μg～2000mg, more preferably 1mg～1000mg, more preferably 10mg～500mg, more preferably 100mg～300mg (with ginsenoside Rk2, ginsenoside CK and ginsenoside PPT (or ginsenoside PPT) The total amount of saponin Rk2 and ginsenoside PPT, or ginsenoside CK and ginsenoside PPT), is usually divided into 1 to 3 times a day.
实施例Example
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。The examples given below are for facilitating the understanding of the present invention, and do not limit the scope of the claims of the present invention in any way.
实施例1稀有人参皂苷Rk2/CK/PPT组合物对3种不同肿瘤细胞增殖的抑制作用Example 1 Inhibitory effect of rare ginsenoside Rk2/CK/PPT composition on the proliferation of three different tumor cells
分别称取适量的人参皂苷Rk2、人参皂苷CK和人参皂苷PPT，配制终浓度为100μg/mL的水溶液，并依次称作Rk2，CK，PPT。将人参皂苷Rk2、人参皂苷CK和人参皂苷PPT分别按照重量比1:1:1、1:1.5:1.5、1:2:2、1:0.5:0.5、1:3:3的比例混合，配制终浓度为100μg/mL的包含人参皂苷Rk2、人参皂苷CK和人参皂苷PPT的水溶液，依次称作组合物1、组合物2、组合物3、组合物4及组合物5。将人参皂苷Rk2和人参皂苷CK按照重量比1:1、1:0.5、1:3的比例混合，配制终浓度为100μg/mL的包含人参皂苷Rk2和人参皂苷CK的水溶液，并称作组合物6、组合物7及组合物8。将人参皂苷Rk2和人参皂苷PPT按照重量比1:1、1:0.5、1:3的比例混合，配制终浓度为100μg/mL的包含人参皂苷Rk2和人参皂苷PPT的水溶液，并称作组合物9、组合物 10及组合物11。将人参皂苷CK和人参皂苷PPT按照重量比1:1、1:0.5、1:3的比例混合，配制终浓度为100μg/mL的包含人参皂苷CK和人参皂苷PPT的水溶液，并称作组合物12、组合物13及组合物14。Weigh appropriate amounts of ginsenoside Rk2, ginsenoside CK, and ginsenoside PPT to prepare an aqueous solution with a final concentration of 100 μg/mL, which is called Rk2, CK, and PPT in turn. Mix ginsenoside Rk2, ginsenoside CK, and ginsenoside PPT at the weight ratios of 1:1:1, 1:1.5:1.5, 1:2:2, 1:0.5:0.5, and 1:3:3 to prepare The aqueous solution containing ginsenoside Rk2, ginsenoside CK, and ginsenoside PPT with a final concentration of 100 μg/mL is called composition 1, composition 2, composition 3, composition 4, and composition 5 in sequence. Ginsenoside Rk2 and ginsenoside CK were mixed in a weight ratio of 1:1, 1:0.5, and 1:3 to prepare an aqueous solution containing ginsenoside Rk2 and ginsenoside CK at a final concentration of 100 μg/mL, and called the composition 6. Composition 7 and Composition 8. Mix ginsenoside Rk2 and ginsenoside PPT in a weight ratio of 1:1, 1:0.5, and 1:3 to prepare an aqueous solution containing ginsenoside Rk2 and ginsenoside PPT with a final concentration of 100 μg/mL, which is called the composition 9. Composition 10 and composition 11. Mix ginsenoside CK and ginsenoside PPT in a weight ratio of 1:1, 1:0.5, and 1:3 to prepare an aqueous solution containing ginsenoside CK and ginsenoside PPT with a final concentration of 100 μg/mL, which is called a composition 12. Composition 13 and Composition 14.
以肺癌细胞、乳腺癌细胞、结肠癌细胞为实验细胞系，将3种癌细胞依次接种于无菌96孔板中，用RPMI-1640培养液培养48h后，分别加入上述17种皂苷水溶液，每孔体积100μL。对照组加入等体积的RPMI-1640培养液，每个浓度设置5个复孔。培养48h后，每孔加入5μg/mL MTT，继续培养4h。加入150μL DMSO，缓慢振荡10min，使其溶解生成的蓝紫色结晶Formazan。在570nm处检测每个孔对应的吸光度A值，并利用以下公式来计算细胞抑制率：Using lung cancer cells, breast cancer cells, and colon cancer cells as the experimental cell lines, three types of cancer cells were sequentially inoculated into a sterile 96-well plate. The pore volume is 100 μL. In the control group, an equal volume of RPMI-1640 culture medium was added, and 5 replicate wells were set for each concentration. After culturing for 48 hours, add 5μg/mL MTT to each well and continue culturing for 4 hours. Add 150μL of DMSO and shake slowly for 10min to dissolve the formed blue-purple crystal Formazan. Detect the absorbance A value corresponding to each well at 570nm, and use the following formula to calculate the cell inhibition rate:
抑制率＝(1-给药组平均OD值/对照组平均OD值)×100％Inhibition rate=(1-administration group average OD value/control group average OD value)×100%
同时计算组合物两药联合指数CI(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.)：At the same time calculate the composition two-drug combination index CI (refer to David H. Kern, Carol R. Morgan, and Susanne U. Hildebrand-Zanki. In vitro and in vivo interaction between cisplatin and topotecan in ovarian cancer systems[J]. Cancer Research, 1988,48. and Li Xingqi. The inhibitory effect of lidamycin on glioma and the synergistic effect of combined temozolomide[D]. Peking Union Medical College, 2009.):
CI＝AB/(A×B)CI=AB/(A×B)
其中T为实验组的OD值，C为对照组的OD值，AB为皂苷联合组的T/C值，A、B为皂苷单用组的T/C值。CI＜1时，两者有协同作用，CI≤0.7时，协同作用非常显著。Among them, T is the OD value of the experimental group, C is the OD value of the control group, AB is the T/C value of the saponin combination group, and A and B are the T/C values of the saponin single-use group. When CI<1, the two have a synergistic effect, and when CI≤0.7, the synergy is very significant.
组合物三药联合指数CI：Composition three-drug combination index CI:
CI＝ABC/(A×B×C)CI=ABC/(A×B×C)
其中T为实验组的OD值，C为对照组的OD值，ABC为皂苷联合组的T/C值，A、B、C为皂苷单用组的T/C值。CI＜1时，三者有协同作用，CI≤0.7时，协同作用非常显著。上述Rk2，CK，PPT和组合物1-14对3种肿瘤细胞的抑制作用及联合指数如表1-4所示。Where T is the OD value of the experimental group, C is the OD value of the control group, ABC is the T/C value of the saponin combination group, and A, B, and C are the T/C values of the saponin single-use group. When CI<1, the three have a synergistic effect, and when CI≤0.7, the synergistic effect is very significant. The inhibitory effects of the above-mentioned Rk2, CK, PPT and composition 1-14 on the three types of tumor cells and the combination index are shown in Table 1-4.
由表1-4可知，人参皂苷Rk2、人参皂苷CK和人参皂苷PPT均对3种肿瘤细胞表现出一定的抑制作用，将人参皂苷Rk2，人参皂苷CK和人参皂苷PPT组合后，可以有效提升其对3种肿瘤细胞的抑制率，且和对照相比，表现出统计学意义(P<0.05)。通过改变组合物中人参皂苷Rk2、人参皂苷CK和人参皂苷PPT的特定重量比例(组合物1-3)，其抗肿瘤作用更加明显，且联合指数均小于1。而组合物4和组合物5并没有表现出协同效应(CI＞1)。It can be seen from Table 1-4 that ginsenoside Rk2, ginsenoside CK and ginsenoside PPT all show certain inhibitory effects on the three types of tumor cells. The combination of ginsenoside Rk2, ginsenoside CK and ginsenoside PPT can effectively enhance their Compared with the control, the inhibition rate of 3 kinds of tumor cells showed statistical significance (P<0.05). By changing the specific weight ratio of ginsenoside Rk2, ginsenoside CK and ginsenoside PPT in the composition (compositions 1-3), the anti-tumor effect is more obvious, and the combination index is less than 1. However, composition 4 and composition 5 did not show a synergistic effect (CI>1).
将人参皂苷Rk2，人参皂苷CK和人参皂苷PPT任意二者以特定比例组合后，其抑制率明显增加，且联合指数均小于1(结果见表3-4)。另外，组合物1表现出比其他组合物更显著的抑制作用。以上结果说明本发明的组合物发挥了协同增效的作用。After combining any two of ginsenoside Rk2, ginsenoside CK and ginsenoside PPT in a specific ratio, the inhibition rate was significantly increased, and the combination index was all less than 1 (see Table 3-4 for the results). In addition, Composition 1 showed a more significant inhibitory effect than other compositions. The above results indicate that the composition of the present invention exerts a synergistic effect.
表1 Rk2，CK，PPT和组合物1-5对3种肿瘤细胞的抑制作用Table 1 The inhibitory effects of Rk2, CK, PPT and composition 1-5 on 3 kinds of tumor cells
Figure PCTCN2021084352-appb-000004
Figure PCTCN2021084352-appb-000004
表中的数值为抑制率，单位％The value in the table is the inhibition rate, in%
与空白对照组比较： *P<0.05， **P<0.01； Compared with the blank control group: * P<0.05, ** P<0.01;
表2 组合物1-5对3种肿瘤细胞的联合指数(CI指数)Table 2 Combination index (CI index) of composition 1-5 to 3 kinds of tumor cells
Figure PCTCN2021084352-appb-000005
Figure PCTCN2021084352-appb-000005
表3 Rk2，CK，PPT和组合物6-14对3种肿瘤细胞的抑制作用Table 3 The inhibitory effect of Rk2, CK, PPT and composition 6-14 on 3 kinds of tumor cells
Figure PCTCN2021084352-appb-000006
Figure PCTCN2021084352-appb-000006
表中的数值为抑制率，单位％The value in the table is the inhibition rate, in%
与空白对照组比较： *P<0.05， **P<0.01； Compared with the blank control group: * P<0.05, ** P<0.01;
表4 组合物6-14对3种肿瘤细胞的联合指数(CI指数)Table 4 Combination Index (CI Index) of Compositions 6-14 to 3 Kinds of Tumor Cells
Figure PCTCN2021084352-appb-000007
Figure PCTCN2021084352-appb-000007
Figure PCTCN2021084352-appb-000008
Figure PCTCN2021084352-appb-000008
实施例2稀有人参皂苷Rk2/CK/PPT组合物1、组合物6、组合物9及组合物12对结肠癌细胞周期分布的影响Example 2 The effect of rare ginsenoside Rk2/CK/PPT composition 1, composition 6, composition 9 and composition 12 on the cycle distribution of colon cancer cells
将结肠癌细胞接种于无菌6孔板中，培养24h后，加入浓度为100μg/mL的人参皂苷Rk2、人参皂苷CK、人参皂苷PPT、组合物1、组合物6、组合物9及组合物12。对照组加入等体积的RPMI-1640培养液，每个浓度设置5个复孔。继续培养48h后，加入冰冷的PBS洗涤2-3次，加入75％冰乙醇，调整细胞至合适密度放置4℃冰箱过夜。再加入冰冷的PBS洗涤2-3次，滴加适量的PI溶液，避光孵育30min，使用流式细胞术检测细胞周期分布情况。Inoculate colon cancer cells in a sterile 6-well plate, culture for 24 hours, add ginsenoside Rk2, ginsenoside CK, ginsenoside PPT, composition 1, composition 6, composition 9 and composition at a concentration of 100 μg/mL 12. In the control group, an equal volume of RPMI-1640 culture medium was added, and 5 replicate wells were set for each concentration. After culturing for 48 hours, add ice-cold PBS to wash 2-3 times, add 75% ice ethanol, adjust the cells to a suitable density and place them in a refrigerator at 4°C overnight. Then add ice-cold PBS to wash 2-3 times, add an appropriate amount of PI solution, incubate for 30 minutes in the dark, and use flow cytometry to detect the cell cycle distribution.
图1为采用流式细胞术检测稀有人参皂苷组合物1、组合物6、组合物9及组合物12对结肠癌细胞周期分布影响的实验结果图。结肠癌细胞分别经人参皂苷Rk2、人参皂苷CK、人参皂苷PPT、组合物1、组合物6、组合物9及组合物12作用后，和对照相比，处于G1期的细胞比例均明显增加，S期的细胞比例均减少，G2期细胞比例均没有明显变化。人参皂苷Rk2、人参皂苷CK、人参皂苷PPT、组合物1、组合物6、组合物9、组合物12将处于G1期结肠癌细胞比例分别提高到49.97％、48.09％、49.39％、68.23％、55.98％、55.68％、56.10％。和对照组相比，均表现出统计学差异(P<0.01)；以上数据显示，经人参皂苷Rk2、人参皂苷CK和人参皂苷PPT处理后，结肠癌细胞被阻断在G1期，通过其特定比例组合的组合物表现出明显的阻断效果。Figure 1 is a graph showing the experimental results of using flow cytometry to detect the effects of rare ginsenoside composition 1, composition 6, composition 9 and composition 12 on the cycle distribution of colon cancer cells. Colon cancer cells were treated with ginsenoside Rk2, ginsenoside CK, ginsenoside PPT, composition 1, composition 6, composition 9, and composition 12 respectively, and compared with the control, the proportion of cells in the G1 phase increased significantly. The proportion of cells in S phase decreased, and the proportion of cells in G2 phase did not change significantly. Ginsenoside Rk2, ginsenoside CK, ginsenoside PPT, composition 1, composition 6, composition 9, composition 12 increase the proportion of colon cancer cells in G1 stage to 49.97%, 48.09%, 49.39%, 68.23%, respectively. 55.98%, 55.68%, 56.10%. Compared with the control group, both showed statistical differences (P<0.01); the above data showed that after treatment with ginsenoside Rk2, ginsenoside CK, and ginsenoside PPT, colon cancer cells were blocked in the G1 phase, through their specific The composition of the ratio combination shows obvious blocking effect.
表5组合物1、组合物6、组合物9、组合物12对结肠癌细胞周期分布阻断的影响Table 5 The influence of composition 1, composition 6, composition 9, and composition 12 on blocking the cycle distribution of colon cancer cells
Figure PCTCN2021084352-appb-000009
Figure PCTCN2021084352-appb-000009
与空白对照组比较： *P<0.05， **P<0.01。 Compared with the blank control group: * P<0.05, ** P<0.01.
实施例3稀有人参皂苷Rk2/CK/PPT组合物1、组合物6、组合物9及组合物12对结肠癌细胞诱导凋亡作用Example 3 The effect of rare ginsenoside Rk2/CK/PPT composition 1, composition 6, composition 9 and composition 12 on colon cancer cells inducing apoptosis
将结肠癌细胞接种于无菌6孔板中，培养24h后，加入浓度为100μg/mL的人参皂苷Rk2、人参皂苷CK、人参皂苷PPT、组合物1、组合物6、组合物9及组合物12。对照组加入等体积的RPMI-1640培养液，每个浓度设置5个复孔。继续培养48h后，加入冰冷的PBS洗涤2-3次，加入1×Binding Buffer缓冲液，吹打均匀，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并计算组合物两药联合指数CI：Inoculate colon cancer cells in a sterile 6-well plate, culture for 24 hours, add ginsenoside Rk2, ginsenoside CK, ginsenoside PPT, composition 1, composition 6, composition 9 and composition at a concentration of 100 μg/mL 12. In the control group, an equal volume of RPMI-1640 culture medium was added, and 5 replicate wells were set for each concentration. After continuing to incubate for 48 hours, add ice-cold PBS to wash 2-3 times, add 1×Binding Buffer, pipette evenly, add an appropriate amount of AV/PI mixed dye solution, incubate in the dark for 15 minutes, and use flow cytometry to detect cell apoptosis. Die and calculate the combination index CI of the two drugs of the composition:
CI＝(1-A×B)/(1-AB)CI=(1-A×B)/(1-AB)
其中T为实验组的细胞存活分数，C为对照组的细胞存活分数，AB为皂苷联合组的T/C值，A、B为皂苷单独作用组的存活分数T/C值。CI＜1时，两者有协同作用，CI≤0.7时，协同作用非常显著。Where T is the cell survival score of the experimental group, C is the cell survival score of the control group, AB is the T/C value of the saponin combination group, and A and B are the survival score T/C values of the saponin alone group. When CI<1, the two have a synergistic effect, and when CI≤0.7, the synergy is very significant.
组合物三药联合指数CI：Composition three-drug combination index CI:
CI＝(1-A×B×C)/(1-ABC)CI=(1-A×B×C)/(1-ABC)
其中T为实验组的细胞存活分数，C为对照组的细胞存活分数，ABC为皂苷联合组的T/C值，A、B、C为皂苷单独作用组的存活分数T/C值。CI＜1时，三者有协同作用，CI≤0.7时，协同作用非常显著。Where T is the cell survival score of the experimental group, C is the cell survival score of the control group, ABC is the T/C value of the saponin combination group, and A, B, and C are the survival score T/C values of the saponin alone group. When CI<1, the three have a synergistic effect, and when CI≤0.7, the synergistic effect is very significant.
图2为采用流式细胞术检测稀有人参皂苷组合物1、组合物6、组合物9及组合物12诱导结肠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。人参皂苷Rk2、人参皂苷CK和人参皂苷PPT能够诱导结肠癌细胞发生一定的凋亡作用，本发明的稀有人参皂苷组合物1、组合物6、组合物9及组合物12促使结肠癌细胞的凋亡细胞百分率明显提升。人参皂苷Rk2、人参皂苷CK、人参皂苷PPT作用于结肠癌细胞的早期凋亡率分别为11.32％、10.01％、12.73％；本发明的组合物1、组合物6、组合物9及组合物12作用于结肠癌细胞的早期凋亡率分别为40.38％、24.34％、27.67％、25.89％。和对照组相比，表现出明显的统计学差异(P<0.01)，且组合物1、组合物6、组合物9及组合物12诱导结肠癌细胞凋亡的联合指数分别为0.59、0.67、0.68、0.68(结果见表6)，表现出协同效应。上述结果显示人参皂苷Rk2/CK/PPT组合物促使结肠癌细胞加速凋亡，从而起到治疗结肠癌的作用。Figure 2 is a graph showing the experimental results of using flow cytometry to detect the rare ginsenoside composition 1, composition 6, composition 9 and composition 12 inducing apoptosis of colon cancer cells, obtaining a cell histogram composed of four quadrants, area Q1 It indicates the cells that have been mechanically damaged during the experimental operation. The area Q2 indicates the cells that have undergone late apoptosis, the area Q3 indicates the cells that have normal functional morphology, and the area Q4 indicates the cells that have undergone early apoptosis. Ginsenoside Rk2, ginsenoside CK and ginsenoside PPT can induce certain apoptosis of colon cancer cells. The rare ginsenoside composition 1, composition 6, composition 9 and composition 12 of the present invention promote the apoptosis of colon cancer cells. The percentage of dead cells increased significantly. The early apoptosis rates of ginsenoside Rk2, ginsenoside CK, and ginsenoside PPT on colon cancer cells were 11.32%, 10.01%, and 12.73%, respectively; composition 1, composition 6, composition 9 and composition 12 of the present invention The early apoptosis rates of colon cancer cells were 40.38%, 24.34%, 27.67%, and 25.89%. Compared with the control group, there was a significant statistical difference (P<0.01), and the combined index of composition 1, composition 6, composition 9 and composition 12 in inducing colon cancer cell apoptosis was 0.59, 0.67, 0.68, 0.68 (see Table 6 for the results), showing a synergistic effect. The above results show that the ginsenoside Rk2/CK/PPT composition promotes the accelerated apoptosis of colon cancer cells, thereby playing a role in the treatment of colon cancer.
表6组合物1、组合物6、组合物9、组合物12诱导结肠癌细胞凋亡的联合指数(CI指数)Table 6 Combination index (CI index) of composition 1, composition 6, composition 9, composition 12 inducing apoptosis of colon cancer cells
Figure PCTCN2021084352-appb-000010
Figure PCTCN2021084352-appb-000010
与空白对照组比较： *P<0.05， **P<0.01。 Compared with the blank control group: * P<0.05, ** P<0.01.
实施例4组合物1对结肠癌细胞Bcl-2家族蛋白、Caspase-3及PARP蛋白表达的影响Example 4 The effect of composition 1 on the expression of Bcl-2 family proteins, Caspase-3 and PARP proteins in colon cancer cells
将结肠癌细胞接种于6孔板中，加入浓度为100μg/mL的人参皂苷Rk2、人参皂苷CK、人参皂苷PPT和组合物1，培养48h后，加入胰酶将细胞消化，加入适量RIPA裂解液，30min后，离心得到目标样品。接下来经过制胶、SDS-PAGE电泳、转膜、封闭和孵育及DAB显色等步骤后得到凋亡相关蛋白表达图。Inoculate colon cancer cells in a 6-well plate, add ginsenoside Rk2, ginsenoside CK, ginsenoside PPT, and composition 1 at a concentration of 100 μg/mL. After culturing for 48 hours, add trypsin to digest the cells, and add appropriate amount of RIPA lysate After 30 minutes, centrifuge to obtain the target sample. Next, after gel preparation, SDS-PAGE electrophoresis, membrane transfer, blocking and incubation, and DAB color development, the apoptosis-related protein expression map is obtained.
图3为采用蛋白印迹法检测经人参皂苷Rk2、人参皂苷CK、人参皂苷PPT和组合物1诱导结肠癌细胞凋亡的相关蛋白表达结果。与对照相比，经人参皂苷Rk2、人参皂苷CK和人参皂苷PPT处理后，Caspase-3、PARP、Bcl-2蛋白表达量均减少；经组合物1处理后，以上3种蛋白表达量变化更明显。由此看出，组合物1通过Caspase-3活化作用和Bcl-2蛋白下调最终诱导细胞凋亡。Figure 3 shows the results of detecting the expression of related proteins induced by ginsenoside Rk2, ginsenoside CK, ginsenoside PPT and composition 1 to induce apoptosis of colon cancer cells by Western blotting. Compared with the control, after treatment with ginsenoside Rk2, ginsenoside CK, and ginsenoside PPT, the expression of Caspase-3, PARP, and Bcl-2 proteins all decreased; after treatment with composition 1, the expression of the above three proteins changed more. obvious. It can be seen from this that Composition 1 finally induces cell apoptosis through Caspase-3 activation and Bcl-2 protein down-regulation.
实施例5稀有人参皂苷Rk2/CK/PPT组合物1、组合物6、组合物9及组合物12对结肠癌移植瘤模型的抑制作用Example 5 Inhibition of rare ginsenoside Rk2/CK/PPT composition 1, composition 6, composition 9 and composition 12 on colon cancer xenograft tumor model
将雄性BALB/c裸鼠于室温下饲养，待其适应环境后，向其接种结肠癌细胞，每只裸鼠注射3-4×10 6个细胞。接种7天后，观察裸鼠左腋窝皮下，发现裸鼠接种部位有米粒大小的结节生成。继续饲养，肿瘤尺寸超过100mm 3时，剔除肿瘤尺寸和体重差异较大的裸鼠，并将剩余的荷瘤裸鼠进行随机分组。本实验将所有荷瘤裸鼠分为以下4组：(1)模型组；(2)10mg/kg Rk2组；(3)10mg/kg CK组；(4)10mg/kg PPT组；(4)组合物1组(10mg/kg)；(5)组合物6组(10mg/kg)；(6)组合物9组(10mg/kg)；(7)组合物12组(10mg/kg)。模型组给予聚乙二醇和水的混合液(体积比为1:1)。其余组均按照荷瘤裸鼠的体重灌胃给药，每天给药一次。每隔5天测量荷瘤裸鼠的体重、肿瘤体积及饮食变化，并及时观察记录。等到30天以后，停止 给药，处死所有小鼠，剥离肿瘤块，逐一称重、记录并收集。同时计算第30天时两药或三药联合指数CI： The male BALB/c nude mice were reared at room temperature, and after they had adapted to the environment, they were inoculated with colon cancer cells, and each nude mouse was injected with 3-4×10 6 cells. Seven days after the inoculation, the left axillary of the nude mouse was observed under the skin, and it was found that nodules the size of rice grains were formed at the inoculation site of the nude mouse. When the tumor size exceeds 100mm 3 , the nude mice with large differences in tumor size and weight will be eliminated, and the remaining tumor-bearing nude mice will be randomly divided into groups. In this experiment, all tumor-bearing nude mice were divided into the following 4 groups: (1) model group; (2) 10mg/kg Rk2 group; (3) 10mg/kg CK group; (4) 10mg/kg PPT group; (4) Composition 1 group (10 mg/kg); (5) Composition 6 group (10 mg/kg); (6) Composition 9 group (10 mg/kg); (7) Composition 12 group (10 mg/kg). The model group was given a mixture of polyethylene glycol and water (volume ratio 1:1). The other groups were administered intragastrically according to the body weight of the tumor-bearing nude mice, once a day. The body weight, tumor volume and diet changes of tumor-bearing nude mice were measured every 5 days, and timely observation and records were made. After 30 days, the administration was stopped, all the mice were sacrificed, the tumor masses were stripped, weighed, recorded and collected one by one. At the same time calculate the two-drug or three-drug combination index CI on the 30th day:
两药联合Two-drug combination
CI＝AB/(A×B)CI=AB/(A×B)
其中T为实验组的肿瘤瘤重，C为对照组的肿瘤瘤重，AB为两种皂苷联合组的T/C值，A、B为皂苷单用组的存活分数T/C值。CI＜1时，两者有协同作用，CI≤0.7时，协同作用非常显著。Among them, T is the tumor weight of the experimental group, C is the tumor weight of the control group, AB is the T/C value of the two saponin combination group, and A and B are the survival score T/C values of the saponin alone group. When CI<1, the two have a synergistic effect, and when CI≤0.7, the synergy is very significant.
三药联合Three-drug combination
CI＝ABC/(A×B×C)CI=ABC/(A×B×C)
其中T为实验组的肿瘤瘤重，C为对照组的肿瘤瘤重，ABC为皂苷联合组的T/C值，A、B、C为皂苷单用组的T/C值。CI＜1时，三者有协同作用，CI≤0.7时，协同作用非常显著。Among them, T is the tumor weight of the experimental group, C is the tumor weight of the control group, ABC is the T/C value of the saponin combination group, and A, B, and C are the T/C values of the saponin single-use group. When CI<1, the three have a synergistic effect, and when CI≤0.7, the synergistic effect is very significant.
由图4A和表7-8可知，和模型组相比，给药30天时，人参皂苷Rk2组、人参皂苷CK组和人参皂苷PPT组对结肠癌裸鼠移植瘤的抑瘤率分别为27.15％、33.18％和34.05％，表明人参皂苷Rk2组、人参皂苷CK组和人参皂苷PPT组对结肠癌裸鼠移植瘤具有一定的抑制作用。组合物1组、组合物6组、组合物9组及组合物12组均可明显减少荷瘤小鼠的瘤重，相应的抑瘤率分别为79.74％、64.65％、65.51％、67.24％，具有显著效果(P<0.01)，且其联合指数分别为0.63、0.73、0.72、0.74(结果见表7-8)。和模型组相比，人参皂苷Rk2组、人参皂苷CK组、人参皂苷PPT组、组合物1组、组合物6组、组合物9组及组合物12组的裸鼠体重呈现缓慢增加的趋势，表现出低毒性特点，安全性高(图4B)。综上所述，稀有人参皂苷Rk2/CK/PPT组合物能够明显阻碍结肠癌裸鼠移植瘤的生长，其抑制率明显高于人参皂苷Rk2组、人参皂苷CK组和人参皂苷PPT组，并对裸鼠表现出低毒副作用，本发明提供的稀有人参皂苷Rk2/CK/PPT组合物表现出协同效应。It can be seen from Figure 4A and Table 7-8 that, compared with the model group, the ginsenoside Rk2 group, ginsenoside CK group, and ginsenoside PPT group had 27.15% tumor inhibition rates on colon cancer nude mice after 30 days of administration. , 33.18% and 34.05%, indicating that the ginsenoside Rk2 group, ginsenoside CK group and ginsenoside PPT group have a certain inhibitory effect on colon cancer transplanted tumors in nude mice. The composition 1, composition 6, composition 9 and composition 12 groups can significantly reduce the tumor weight of tumor-bearing mice, and the corresponding tumor inhibition rates are 79.74%, 64.65%, 65.51%, and 67.24%, respectively. It has a significant effect (P<0.01), and its combined index is 0.63, 0.73, 0.72, 0.74, respectively (see Table 7-8 for the results). Compared with the model group, the weight of nude mice in the ginsenoside Rk2 group, ginsenoside CK group, ginsenoside PPT group, composition 1 group, composition 6 group, composition 9 group and composition 12 group showed a slow increase in body weight. It exhibits low toxicity and high safety (Figure 4B). In summary, the rare ginsenoside Rk2/CK/PPT composition can significantly hinder the growth of colon cancer transplanted tumors in nude mice, and its inhibition rate is significantly higher than that of the ginsenoside Rk2 group, ginsenoside CK group and ginsenoside PPT group. Nude mice exhibit low toxicity and side effects, and the rare ginsenoside Rk2/CK/PPT composition provided by the present invention exhibits a synergistic effect.
表7组合物1、组合物6、组合物9、组合物12对结肠癌荷瘤小鼠生长的抑制作用Table 7 Inhibitory effects of composition 1, composition 6, composition 9, and composition 12 on the growth of colon cancer tumor-bearing mice
Figure PCTCN2021084352-appb-000011
Figure PCTCN2021084352-appb-000011
与模型组比较： *P<0.05， **P<0.01。 Compared with the model group: * P<0.05, ** P<0.01.
表8组合物1、组合物6、组合物9、组合物12对结肠癌荷瘤小鼠瘤重生长抑制的联合指数(CI指数)Table 8 The combined index (CI index) of composition 1, composition 6, composition 9, composition 12 on tumor re-growth inhibition of colon cancer tumor-bearing mice
Figure PCTCN2021084352-appb-000012
Figure PCTCN2021084352-appb-000012
实施例6人参皂苷Rk2/CK/PPT组合物1的体内安全性评价Example 6 In vivo safety evaluation of ginsenoside Rk2/CK/PPT composition 1
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)空白组；(2)40mg/kg人参皂苷Rk2组；(3)40mg/kg人参皂苷CK组；(4)40mg/kg人参皂苷PPT组；(5)Rk2/CK/PPT组合物1组。以上5组均以灌胃方式给药。给药6h后，取消禁食，正常喂养小鼠14天。观察小鼠饮水、摄食、体重及肝肾功能变化。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) blank group; (2) 40mg/kg ginsenoside Rk2 group; (3) 40mg/kg ginsenoside CK group; (4) 40mg/kg ginsenoside PPT group ; (5) 1 group of Rk2/CK/PPT composition. 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 14 days. Observe the changes of mice drinking water, food intake, body weight and liver and kidney function.
由图5可知，和空白组相比，经染毒后，人参皂苷Rk2组、人参皂苷CK组和人参皂苷PPT组小鼠的饮水量、摄食量和体重未发生明显改变，Rk2/CK/PPT组合物1组小鼠的饮水量、摄食量和体重未发生明显改变。It can be seen from Figure 5 that compared with the blank group, the water intake, food intake and body weight of mice in the ginsenoside Rk2 group, ginsenoside CK group, and ginsenoside PPT group did not change significantly after exposure, and Rk2/CK/PPT The water intake, food intake and body weight of mice in the composition 1 group did not change significantly.
由图6可知，和对照组相比，人参皂苷Rk2组、人参皂苷CK组和人参皂苷PPT组小鼠的肝脏重量、肾脏重量、血清中谷丙转氨酶含量及血清中肌氨酸酐含量均未发生明显改变，Rk2/CK/PPT组合物1组小鼠的肝脏重量、肾脏重量、血清中谷丙转氨酶含量及血清中肌氨酸酐含量均未发生明显改变。由此可见，Rk2/CK/PPT组合物1对健康小鼠表现出一定的安全性。It can be seen from Figure 6 that compared with the control group, the liver weight, kidney weight, serum alanine aminotransferase content and serum creatinine content of mice in the ginsenoside Rk2 group, ginsenoside CK group, and ginsenoside PPT group were not significant. Changed, the liver weight, kidney weight, serum alanine aminotransferase content and serum creatinine content of mice in the Rk2/CK/PPT composition group 1 did not change significantly. It can be seen that the Rk2/CK/PPT composition 1 shows a certain degree of safety in healthy mice.
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。It should be noted that 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.
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。The present invention has been described above through specific embodiments and examples, but those skilled in the art should understand that these are not intended to limit the scope of the present invention, and the scope of the present invention should be determined by the claims.
工业实用性Industrial applicability
本发明提供了一种包含稀有人参皂苷Rk2、人参皂苷CK和人参皂苷PPT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。The present invention provides a rare ginsenoside pharmaceutical composition containing rare ginsenoside Rk2, ginsenoside CK and ginsenoside PPT, which is synergistic and has anti-tumor effects.

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Rk2、人参皂苷CK和人参皂苷PPT，以及药学可接受的载体，A rare ginsenoside pharmaceutical composition with anti-tumor effect, which comprises a therapeutically effective amount of ginsenoside Rk2, ginsenoside CK and ginsenoside PPT as active ingredients, and a pharmaceutically acceptable carrier,
    其中，该稀有人参皂苷药物组合物中，所述人参皂苷Rk2、人参皂苷CK和人参皂苷PPT的重量比为：1:(1～2):(1～2)。Wherein, in the rare ginsenoside pharmaceutical composition, the weight ratio of the ginsenoside Rk2, ginsenoside CK and ginsenoside PPT is 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Rk2、人参皂苷CK和人参皂苷PPT的重量比为：1:(1～1.1):(1～1.1)。The rare ginsenoside pharmaceutical composition according to claim 1, wherein, in the rare ginsenoside pharmaceutical composition, the weight ratio of the ginsenoside Rk2, ginsenoside CK and ginsenoside PPT is 1:(1~1.1) :(1～1.1).
  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Rk2、人参皂苷CK和人参皂苷PPT的重量比为1:1:1。The rare ginsenoside pharmaceutical composition according to claim 1, wherein, in the rare ginsenoside pharmaceutical composition, the weight ratio of the ginsenoside Rk2, ginsenoside CK and ginsenoside PPT is 1:1:1.
  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。The rare ginsenoside pharmaceutical composition of claim 1, wherein the rare ginsenoside pharmaceutical composition does not contain other ginsenosides.
  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Rk2、人参皂苷CK和人参皂苷PPT作为唯一抗肿瘤活性成分。The rare ginsenoside pharmaceutical composition according to claim 1, wherein the rare ginsenoside pharmaceutical composition comprises ginsenoside Rk2, ginsenoside CK and ginsenoside PPT as the only anti-tumor active ingredients.
  6. 包含人参皂苷Rk2、人参皂苷CK和人参皂苷PPT的组合物在制备抗肿瘤药物中的用途，其中，Use of a composition comprising ginsenoside Rk2, ginsenoside CK and ginsenoside PPT in the preparation of an anti-tumor drug, wherein:
    所述组合物中人参皂苷Rk2、人参皂苷CK和人参皂苷PPT的重量比为：1:(1～2):(1～2)；The weight ratio of ginsenoside Rk2, ginsenoside CK and ginsenoside PPT in the composition is: 1:(1-2):(1-2);
    所述抗肿瘤药物包含人参皂苷Rk2、人参皂苷CK和人参皂苷PPT作为活性成分。The anti-tumor drug contains ginsenoside Rk2, ginsenoside CK and ginsenoside PPT as active ingredients.
  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Rk2、人参皂苷CK和人参皂苷PPT的重量比为：1:(1～1.1):(1～1.1)。The use according to claim 6, wherein the weight ratio of the ginsenoside Rk2, ginsenoside CK and ginsenoside PPT in the composition is 1:(1~1.1):(1~1.1).
  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Rk2、人参皂苷CK和人参皂苷PPT的重量比为1:1:1。The use according to claim 6, wherein, in the composition, the weight ratio of the ginsenoside Rk2, ginsenoside CK and ginsenoside PPT in the composition is 1:1:1.
  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。The use according to claim 6, wherein the composition does not contain other ginsenosides.
  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Rk2、人参皂苷CK和人参皂苷PPT作为唯一抗肿瘤活性成分。The use according to claim 6, wherein the composition comprises ginsenoside Rk2, ginsenoside CK and ginsenoside PPT as the sole anti-tumor active ingredients.
PCT/CN2021/084352 2020-03-31 2021-03-31 Anti-tumor composition containing rare ginsenosides rk2, ck, and ppt WO2021197372A1 (en)

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