CN106176711B - Pharmaceutical comprising flavonoid compound composition and use thereof - Google Patents
Pharmaceutical comprising flavonoid compound composition and use thereof Download PDFInfo
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Abstract
The invention relates to application of a flavonoid compound composition in preparing a medicament for treating and/or preventing cancer, and also relates to a pharmaceutical composition for treating and/or preventing cancer. The flavonoid compound composition can effectively improve the anticancer function, and each active component has good synergistic effect. The flavonoid compound composition comprises at least two flavonoid compounds represented by formula I as active ingredients:
wherein R is1Is hydroxy or
And R is2Is H or hydroxyl.
Description
Technical Field
The invention belongs to the field of pharmacy, and particularly relates to a medicament containing a flavonoid compound composition and application of the flavonoid compound composition in preparing a medicament for treating and/or preventing cancer.
Background
In recent years, the incidence of cancer has increased explosively with rapid economic growth, deterioration of living environment, and changes in dietary structure and lifestyle. The current chemotherapy drugs are not only expensive, but also cause serious side effects of cancer patients, such as chemotherapy-induced anemia, immune function, and antioxidant function reduction. Therefore, the development of a medicament with low price and small toxic and side effects has very important significance for relieving the pain of cancer patients.
As a pure natural plant medicine, the traditional Chinese medicine formula generally has no toxic or side effect and can be taken for a long time. Many studies have shown that many natural ingredients contained in plant drugs have anticancer effects, and can be used for preparing anticancer drugs such as flavonoids and bioflavonoids. (see references "Flavonoids and cancer preservation: a review of the evaluation. D.F.Romagnolo & O.I.Selmin, Journal of Nutrition in Geronology and Geriatics, 2013,31: 206-. However, since the ingredients of the traditional Chinese medicine are complex and are usually composed of dozens or even hundreds of compounds, the pharmacological research and optimization of the traditional Chinese medicine are difficult. The quality control of the traditional Chinese medicine is also particularly important, and factors such as producing areas, climate, years and the like can influence the quality of the traditional Chinese medicine, thereby influencing the effectiveness and stability of the medicine. Therefore, the optimization of the traditional Chinese medicine and the solving of the quality control and other problems have important significance for the development and popularization of the traditional Chinese medicine.
The research on flavonoid compounds used as anticancer drugs focuses on the research on the single flavonoid compound or flavonoid compound combination drug In the treatment of Cancer or side effects of Cancer, such as the inhibition and treatment of Breast Cancer when formononetin and calycosin are used alone (see references "Chen J, L In C, Yong W, Ye Y, Huang Z. Calycosin and genistein introduction by inhibition of TAIR/p-Akt signalizing milk In Human branched Biochem. 2015; 35(2): 722-8"; J. 201n, J. Zeng, M. Xin, W. Chen et al; the research, X. formanec, J. Zeng Cycle, J. Zephyne, J. Xin et al, the synergistic effect of various drugs In combination drugs such as vitamin A, vitamin J, vitamin E, vitamin D, vitamin E, vitamin D. A, vitamin E. D. C. D. C. D. C. D. C.
The astragalus root has been used for more than 2000 years and has the efficacy of tonifying qi and generating blood. Radix astragali contains saponin, sucrose, polysaccharide, amino acids, folic acid, and trace elements such as selenium, zinc, and copper, and has many biological and medical effects, such as protecting liver, promoting hematopoiesis, resisting oxidation, resisting hypertension, and enhancing immunity. Recent studies have shown that the flavonoid component of astragalus effectively stimulates the expression of erythropoietin in kidney cells, thereby increasing the level of red blood cells in the blood to ameliorate and treat anemia. The four main flavonoid compounds with erythropoietin expression promoting effect are formononetin, calycosin and calycosin glycoside.
Formononetin is also called 7-hydroxy-4' -methoxy isoflavone and formononetin; the English name is Formononetin, 7-Hydroxy-3- (4-methoxyphenyl) chromone, 7-Hydroxy-4 '-methoxyisolavone, or Dadein 4' -methyl ether; CAS number is 485-72-3; molecular formula C16H12O4(ii) a The molecular weight is 268.26; the chemical formula of formononetin is represented by the following formula III:
formononetin is easily dissolved in methanol, ethyl acetate, ether and dilute alkali solution, and is insoluble in water. The formononetin can be extracted from plants or synthesized by a chemical method. For example, synthetic methods can be found in references: "j.agric.foodchem.en; 9.1994,42,1869-1871". The pharmacological actions known for formononetin at present are as follows: (1) the anticancer effect is as follows: can be used for preventing and treating breast cancer, prostatic cancer and colon cancer; (2) estrogenic effects: can increase the weight of uterus of animals such as mice, but the estrogen action is very weak and is not as good as that of isoflavone genistein (genistein) and daidzein of the same kind; (3) the blood fat reducing agent has the function of reducing the blood fat of rat with male albinism caused by TritonWR-1339; (4) it is used clinically as diuretic.
Formononetin is also known as formononetin-7-glucoside; the English name is Onone, or Formononetin-7-O-beta-D-glucopyranoside; CAS number 486-62-4; molecular formula C22H22O9(ii) a The molecular weight is 430.40; the chemical formula of formononetin is represented by the following formula IV:
calycosin or 7,3'-dihydroxy-4' -methoxyisolavone, the british name of Calycosin; molecular formula C16H12O5(ii) a The molecular weight is 284.26348; the chemical formula of calycosin is represented by the following formula II:
the known pharmacological actions of calycosin are as follows: (1) improving the immune function; (2) enhancing antioxidant, antiradiation and anticancer effects; (3) protecting cardiovascular and cerebrovascular, liver, kidney and lung; (4) protecting brain cells and improving memory; (5) antibacterial and virus inhibiting effects; (6) reducing blood lipid, reducing blood glucose, and reducing diabetic complications.
Calycosin glycosides are also known as Calycosin glucoside, Calycosin-7-O- β -D glucoside, known in English as Calycosin-7-glucoside, Calycosin-7-O- β -D-glucoside, 3',7-Dihydroxy-4' -methoxy isoflavanone-7-beta-D-glucopyranoside, Calycosin 7-O-beta-D-glucoside, Calycosin 7-beta-D-glucopyranoside, or Calycosin-7-O-beta-D-glucopyranoside, having a molecular weight of 446.40, CAS number 20633-67-4, and the chemical formula of Calycosin glycoside is represented by formula V below:
recent studies have shown that several flavonoid compounds isolated from astragalus membranaceus can prevent and treat cancer when used alone (see references "on Hormone receptors cell cycle extract of human Research cells IGF1/PI3K/Akt pathway in vitro and in vivo. c. j. zeng et al, Hormone and Metabolic Research2011,43:681 686" and "on Hormone-induced apoptosis of human Hormone cancer cells tissue error ERK1/2 mitogen-actevidencin kinase activation. y. ye et al, Hormone and Metabolic Research 2012,44: 263", and patent publication "application of stem extract in preparation of anti-flavin drug", n. 3638 CN). However, the above studies all use one flavonoid compound alone, and the existing flavonoid compounds have the disadvantages of limited efficacy and large dosage when used alone.
Disclosure of Invention
In order to solve the problems in the prior art, the present invention provides a medicament comprising the flavonoid compound composition and the use of the flavonoid compound composition in the preparation of a medicament for the treatment and/or prevention of cancer.
Specifically, the present invention provides:
(1) use of a flavonoid compound composition for the manufacture of a medicament for the treatment and/or prevention of cancer, wherein said flavonoid compound composition comprises as active ingredients at least two flavonoid compounds represented by formula I:
wherein R is1Is hydroxy or
And is
R2Is H or hydroxyl.
(2) The use according to (1), wherein the total amount of the flavonoid compound represented by formula I is 1 to 100% by weight based on the total amount of the flavonoid compound composition.
(3) The use according to (1), wherein the flavonoid compound composition comprises formononetin and further comprises at least one selected from the group consisting of calycosin, formononetin and calycosin glycoside.
(4) The use of (3), wherein the flavonoid composition comprises formononetin and calycosin.
(5) The use according to (4), wherein the molar ratio of formononetin to calycosin is (0.5-100): 0.5-100).
(6) The use of (3), wherein the flavonoid composition comprises formononetin and calycosin glycoside.
(7) The use of (6), wherein the molar ratio of formononetin to calycosin glycoside is (0.5-100): (0.5-100).
(8) The use of (3), wherein the flavonoid composition comprises formononetin, formononetin and calycosin.
(9) The use according to (8), wherein the molar ratio of formononetin, formononetin and calycosin is (0.5-100): 0.5-100.
(10) The use of (9), wherein the molar ratio of formononetin, formononetin and calycosin is 2:25: 1.
(11) The use of (3), wherein the flavonoid composition comprises formononetin, formononetin and calycosin glycoside.
(12) The application of the compound preparation in the step (11), wherein the molar ratio of formononetin to verbascoflavone glycoside is (0.5-100) to (0.5-100): (0.5-100).
(13) The use of (3), wherein the flavonoid composition comprises formononetin, calycosin and calycosin glycoside.
(14) The use of (13), wherein the molar ratio of formononetin, calycosin and calycosin glycoside is (0.5-100): (0.5-100).
(15) The use of (13), wherein the flavonoid composition comprises formononetin, calycosin and calycosin glycoside.
(16) The use of (15), wherein the molar ratio of formononetin, calycosin and calycosin glycoside is (0.5-100): (0.5-100).
(17) The use of (15), wherein the molar ratio of formononetin, calycosin and calycosin glycoside is 1:1:5: 1.
(18) The use of (1), wherein the medicament further comprises an additional anticancer drug compounded with the flavonoid compound composition, and the ratio of the total molar amount of the flavonoid compound composition to the total molar amount of the additional anticancer drug is (1-400): 1-20.
(19) The use according to (1), wherein the medicament further has an effect of improving an immune function and/or an antioxidant function.
(20) A pharmaceutical composition for treating and/or preventing cancer, comprising a flavonoid compound as an active ingredient, and pharmaceutically acceptable excipients, wherein the flavonoid compound is at least two flavonoid compounds represented by formula I:
wherein R is1Is hydroxy or
And is
R2Is H or hydroxyl.
(21) The pharmaceutical composition of (20), wherein the pharmaceutical composition further comprises an additional anticancer drug compounded with the flavonoid compound, and the ratio of the total molar amount of the flavonoid compound to the total molar amount of the additional anticancer drug is (1-400): 1-20.
(22) The pharmaceutical composition of (20), wherein the total amount of flavonoid compounds is 1-100 wt% based on the total amount of the pharmaceutical composition.
(23) The pharmaceutical composition according to (20), wherein the pharmaceutical composition is in the form of oral liquid or injection.
Compared with the prior art, the invention has the following advantages and positive effects:
the flavonoid compound composition of the present invention can effectively improve anticancer functions and inhibit cancer cell proliferation, and thus can be used for preventing and treating cancer. The flavonoid compound composition has good synergistic effect of the active ingredients, obviously reduces the dosage of the active ingredients, and has low price and small toxic and side effect.
In addition, the flavonoid compound composition can improve the immune function and the antioxidant function, and can relieve and treat side effects such as the reduction of the immune function and/or the reduction of the antioxidant function caused by radiotherapy and/or chemotherapy.
Drawings
FIG. 1 shows the results of the inhibition rate of cancer cells by the composition of two flavonoids in example 1.
FIG. 2 shows the results of the inhibition rate of cancer cells by the composition of three flavonoids in example 1.
FIG. 3 shows the results of the inhibition rate of cancer cells by the flavonoid compound composition of preparation example 5 in example 1.
FIG. 4 shows the results of the inhibition rate of cancer cells by the flavonoid compound composition of preparation example 6 in example 1.
FIG. 5 shows the results of the inhibition rate of cancer cells by the flavonoid compound composition of preparation example 7 in example 2.
Fig. 6 shows the results of the inhibition rate of the single flavonoid drug on cancer cells in comparative example 1.
Figure 7 shows the optimal results of single flavonoid and flavonoid compositions of the present invention versus cancer cell inhibition rate.
Fig. 8 shows the results of detecting the red blood cell count (RBC), white blood cell count (WBC), hematocrit (Ht), and hemoglobin (Hb) in example 3.
FIG. 9 shows the results of detecting superoxide dismutase (SOD), total antioxidant capacity (T-AOC), Malondialdehyde (MDA) in example 3.
FIG. 10 shows the results of detecting interleukin-2 (I L-2) in example 3.
Detailed Description
The present invention is further described in the following description of the embodiments with reference to the drawings, which are not intended to limit the invention, and those skilled in the art may make various modifications or improvements based on the basic idea of the invention, but within the scope of the invention, unless departing from the basic idea of the invention.
As used herein, the term "treatment" refers to the process of blocking, alleviating (or ameliorating) or eliminating a cause or lesion in order to restore or gain health or reduce pain in a living human or animal body.
As used herein, the term "medicament" includes human and veterinary medicaments for human and animal applications. Further, the term "drug" as used herein refers to any substance that provides a prophylactic, therapeutic, and/or beneficial effect. The term "drug" as used herein is not necessarily limited to substances that require marketing approval.
In this context, the term "purified form" refers to a pharmaceutically pure form. As used herein, the term "pharmaceutically pure" means substantially free of other compounds.
The invention provides the use of a flavonoid composition in the manufacture of a medicament for the treatment and/or prevention of cancer.
The inventor of the invention finds out through experiments that: when two or more of calycosin, formononetin and calycosin glycoside are combined, the components of the obtained composition show unexpected synergistic effect, and the obtained composition has remarkable effect on improving anticancer function and can effectively prevent and treat cancer. Based on the findings, the inventors further obtained the technical solution of the present invention.
Specifically, the present invention provides use of a flavonoid compound composition for the preparation of a medicament for the treatment and/or prevention of cancer, wherein the flavonoid compound composition comprises at least two flavonoid compounds represented by formula I:
wherein R is1Is hydroxy or
And is
R2Is H or hydroxyl.
Preferably, in preparing the flavonoid composition, the flavonoid is used in purified form.
Purified forms of the flavonoid compounds are commercially available (e.g., available from Sichuan Veckqi Biotechnology, Inc.) and can also be prepared using purification methods known in the art.
Wherein, in formula I, when R is1Is hydroxy, R2And when the number is H, the flavonoid compound is formononetin.
In the formula I, when R1Is hydroxy, R2In the case of hydroxyl, the flavonoid is calycosin.
In the formula I, when R1Is composed of
R2And when the number of the flavonoid compounds is H, the flavonoid compound is formononetin.
In the formula I, when R1Is composed of
R2In the case of hydroxyl, the flavonoid is calycosin glycoside.
Preferably, the total amount of the flavonoid compound represented by formula I is 1 to 100% by weight, more preferably 50 to 100% by weight, particularly preferably 90 to 100% by weight, based on the total amount of the flavonoid compound composition. For example, the total amount of the flavonoid compound represented by formula I may comprise 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 wt% based on the total amount of the flavonoid compound composition.
Preferably, the flavonoid compound composition of the present invention comprises formononetin and further comprises at least one selected from the group consisting of calycosin, formononetin and calycosin glycoside.
It is also preferred that the flavonoid composition of the present invention comprises formononetin and calycosin. Wherein, the mol ratio of formononetin to calycosin is preferably (0.5-100): 0.5-100), more preferably (1-40): 0.5-100, and particularly preferably (20): 5-40.
It is also preferred that the flavonoid composition of the present invention comprises formononetin and calycosin glycoside. Wherein the molar ratio of formononetin to calycosin glycoside is (0.5-100): (0.5-100), more preferably (1-40): (0.5-100), and particularly preferably (20): (0.5-5).
It is also preferred that the flavonoid composition of the present invention comprises formononetin, formononetin and calycosin. Wherein the molar ratio of formononetin, formononetin and calycosin is (0.5-100): 0.5-100), more preferably (1-40): 0.5-100), and particularly preferably 2:25: 1.
It is also preferred that the flavonoid compositions of the present invention comprise formononetin, formononetin and calycosin glycosides. Wherein the molar ratio of formononetin, formononetin and calycosin is (0.5-100): (0.5-100): 0.5-100), more preferably (1-40): 0.5-100, and particularly preferably 20:40: 40.
It is also preferred that the flavonoid compositions of the present invention comprise formononetin, calycosin, and calycosin glycosides. Wherein the molar ratio of formononetin, calycosin and calycosin glycoside is (0.5-100): 0.5-100), more preferably (1-40): 0.5-100), and particularly preferably 20:5: 5.
It is also preferred that the flavonoid compositions of the present invention comprise formononetin, calycosin, and calycosin glycosides. Wherein the molar ratio of formononetin, calycosin and calycosin is (0.5-100): 0.5-100), especially preferred is (1-40): 0.5-100, most preferred is 1:1:5: 1.
The drug or the pharmaceutical composition of the invention can also comprise other anticancer drugs or active ingredients compounded with the flavonoid compound. The ratio of the total molar amount of the flavonoid compound to the total molar amount of the other anticancer drug or active ingredient is (1-400): 1-20, preferably 20: 1. Such other anticancer drugs or active ingredients are, for example, cisplatin, vitamin C, etc. In the case of cisplatin, the molar ratio of cisplatin to the total amount of flavonoids described in the present invention is, for example, (1-400) to (1-20), preferably 20: 1.
The flavonoid compound composition can effectively improve the anticancer function and inhibit the proliferation of cancer cells, and can be used for preventing and treating cancers. Therefore, the flavonoid compound composition provided by the invention can be used for preparing a medicament for treating and/or preventing cancers.
In addition, the flavonoid compound composition can improve the immune function and the antioxidant function, and can relieve and treat side effects such as the reduction of the immune function and/or the reduction of the antioxidant function caused by radiotherapy and/or chemotherapy. Therefore, it is preferable that the agent for treating and/or preventing cancer further has an effect of improving immune function and/or antioxidant function.
Preferably, the flavonoid compound composition of the present invention may be used in an amount effective for the treatment and/or prevention of cancer of 0.05. mu.M-100 mM, more preferably 0.5. mu.M-1 mM, particularly preferably 20-60. mu.M. When the flavonoid composition of the present invention is used in therapeutic applications, its therapeutically effective amount may be in the range of 0.05 μ M to 100mM, more preferably 20 to 60 μ M; when the flavonoid compound composition of the present invention is used for prophylactic applications, the prophylactically effective amount thereof may be 0.05. mu.M-100 mM, more preferably 20-60. mu.M.
The invention also provides a pharmaceutical composition for treating and/or preventing cancer, which comprises the flavonoid compound provided by the invention as an active ingredient and pharmaceutically acceptable auxiliary materials. Specifically, the flavonoid compound is at least two flavonoid compounds represented by formula I:
wherein R is1Is hydroxy or
And is
R2Is H or hydroxyl.
Preferably, the total amount of flavonoid compound is 1 to 100 wt. -%, more preferably 50 to 100 wt. -%, based on the total weight of the pharmaceutical composition. For example, the total amount of flavonoid compound may comprise 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 wt% based on the total amount of the pharmaceutical composition.
Preferably, the pharmaceutical composition comprises formononetin and further comprises at least one selected from the group consisting of calycosin, formononetin and calycosin glycoside. Various combinations of flavonoids and the corresponding ratios of flavonoids described above for the flavonoid composition are also applicable to the pharmaceutical composition.
The type and amount of pharmaceutically acceptable excipients can be selected by those skilled in the art as desired (e.g., route of administration and standard pharmaceutical practice) to produce the desired drug. Pharmaceutical excipients well known in the art may be employed in the compositions of the present invention, including but not limited to, for example, the references: pharmaceutic adjuvants as disclosed in "pharmacy (fourth edition), edited by the university of Pitaya, published by the people's health Press in 2001".
The pharmaceutically acceptable excipients include pharmaceutically acceptable carriers known in the art, such as polyethylene glycols, povidone, surfactants (e.g., poloxamer 188), organic acids (e.g., tartaric acid, succinic acid, etc.), saccharides (e.g., dextrose, galactose, sucrose, etc.), alcohols (mannitol, sorbitol, xylitol, etc.), celluloses (e.g., ethyl cellulose, carboxyethyl cellulose, etc.), polyacrylics, etc. The pharmaceutically acceptable auxiliary materials also comprise pharmaceutically acceptable diluents, and examples of the pharmaceutically acceptable auxiliary materials include ethanol, glycerol, water and the like. The pharmaceutical composition may also comprise any suitable binders, lubricants, suspending agents, coating agents, solubilizing agents, and the like.
The pharmaceutical compositions of the present invention may be administered orally or non-orally. The oral administration can be solid or liquid, and the non-oral administration can be injection.
Solid forms for oral administration may include tablets, pills, capsules, powders, granules and the like, and those skilled in the art may optionally employ the following excipients: fillers (e.g., starch, dextrin, lactose, mannitol, microcrystalline cellulose, etc.), absorbents (e.g., calcium sulfate, calcium hydrogen phosphate, etc.), humectants (e.g., water, ethanol, etc.), binders (e.g., hydroxymethyl cellulose, povidone, starch slurry, etc.), disintegrants (e.g., croscarmellose sodium, crospovidone, etc.), lubricants (e.g., stearic acid, talc, polyethylene glycol, colloidal silica, etc.), flavorants (e.g., sucrose, stevioside, aspartame, etc.), fragrances (e.g., flavors and essences, etc.), colorants (e.g., beet red, caramel, lemon yellow, etc.), and the like.
The liquid forms (oral liquid) for oral administration include solutions, emulsions, suspensions, syrups and the like, and the skilled person can select the following auxiliary materials to prepare the oral liquid according to needs: solvents (such as water, glycerin, dimethyl sulfoxide (DMSO), ethanol, propylene glycol, polyethylene glycol, fatty oil, liquid paraffin, ethyl acetate, etc.), flavoring agents (such as sweetening agents (such as sucrose, stevioside, aspartame, etc.), flavoring agents (such as flavors and essences), coloring agents (such as beet red, caramel, lemon yellow, etc.), preservatives (such as parabens, benzoic acid and sodium benzoate, sorbic acid, etc.), wetting agents (such as polysorbates, polyoxyethylene fatty alcohol ethers, etc.), suspending agents (such as glycerin, gums, celluloses, diatomaceous earth, etc.), emulsifying agents (such as surfactants, etc.), etc.
Preferably, the dosage form of the pharmaceutical composition of the present invention is oral liquid.
The present invention will be further explained or illustrated by way of examples, which should not be construed as limiting the scope of the invention.
In the following examples, calycosin, formononetin and calycosin glycoside are available from VICKEQI biotechnology, Sichuan.
Fetal Bovine Serum (FBS) was purchased from life technologies.
Penicillin was purchased from Sigma.
Streptomycin was purchased from Sigma.
Cisplatin was purchased from Sigma.
MTT reagent was purchased from Sigma.
Test example 1: construction of in vitro model for cancer cell proliferation inhibition
In this test example, MCF-7 breast cancer cell line, Hela cervical cancer cell line (purchased from Sigma) and multidrug resistant A549-DDP lung cancer cell line (purchased from tumor cell bank of Chinese academy of medicine science) were used as the study models for the inhibition of cancer cell proliferation. The cells were seeded in a 96-well culture plate, the number of cells in each well was 4000, 200. mu.l of a cell culture medium (DMEM medium containing 10% (v/v) Fetal Bovine Serum (FBS), 100U/ml penicillin and 100U/ml streptomycin) was added to each well, and the mixture was placed in a 37 ℃ incubator and cultured for 24 hours while controlling the carbon dioxide concentration at 5% (v/v).
Preparation example 1: preparation of a flavonoid composition containing two active ingredients: formononetin (F) + (one of formononetin (O), calycosin (C) and Calycosin (CG))
Dissolving formononetin, calycosin and calycosin glycoside in DMSO respectively to prepare respective DMSO mother liquor (each mother liquor has 3 concentrations: 50 μ M, 500 μ M and 5mM respectively, and is used for preparing different final concentrations to improve experimental precision). The required volume of DMSO stock was calculated and measured according to the final concentration of the different active ingredients, combined and diluted to 300. mu.l with the cell culture solution described in test example 1. The final concentration of each active ingredient is as follows: formononetin 20 μ M, formononetin, calycosin and calycosin glycoside 0.5, 1, 5, 10, 20, 40, 70 and 100 μ M respectively.
Preparation examples 2 to 7 were carried out according to the method of preparation example 1, but such that the final concentrations of the active ingredients were as follows:
preparation example 2: preparation of a flavonoid composition containing two active ingredients: f + (O, C or CG)
Preparation example 3: preparation of a flavonoid composition containing three active ingredients: f + (O, C and CG, two kinds)
Preparation example 4: preparation of a flavonoid composition containing three active ingredients: f + (O, C and CG, two kinds)
Preparation example 5: a flavonoid composition was prepared containing four active ingredients: f + O + C + CG
Formononetin 0.08 μ M, calycosin 0.4 μ M, and calycosin 0.08 μ M. In addition, on the basis of this concentration, flavonoid compound compositions containing these four active ingredients at different concentrations, each at 5-fold, 10-fold, 15-fold, 25-fold, 40-fold, 60-fold, 80-fold, 90-fold, 100-fold, 125-fold, 300-fold, 500-fold, 800-fold, and 1000-fold of the respective concentration, were reformulated.
Preparation example 6: preparation of a flavonoid composition containing three active ingredients: f + O + C
Formononetin 0.016. mu.M, formononetin 0.4. mu.M and calycosin 0.08. mu.M. In addition, on the basis of this concentration, flavonoid compound compositions containing these three active ingredients at different concentrations, each at 5-fold, 10-fold, 15-fold, 25-fold, 40-fold, 60-fold, 80-fold, 90-fold, 100-fold, 125-fold, 300-fold, 500-fold, 800-fold, and 1000-fold of the respective concentration, were reformulated.
Preparation example 7: preparation of a flavonoid composition containing five active ingredients: f + O + C + CG + cisplatin
The preparation method comprises the steps of preparing formononetin, calycosin and calycosin according to the ratio of 1:1:5:1, so that the total concentration of the four flavonoid compounds is 12.5 mu M, 25 mu M, 50 mu M, 100 mu M, 200 mu M and 400 mu M, and respectively adding cisplatin 0, 1.25 mu M, 2.5 mu M, 5 mu M, 10 mu M and 20 mu M into each solution of the six concentrations to prepare 36 concentrations of solutions.
Example 1: flavonoid compound composition having effect of inhibiting proliferation of cancer cells
MCF-7 breast cancer cells and He L a cervical cancer cells in test example 1 were divided into a drug group and a control group after 24 hours of culture, 200. mu.L of the pharmaceutical compositions of preparation examples 2,4, 5 and 6 were added to drug group MCF-7cells, respectively, 200. mu.L of the pharmaceutical compositions of preparation examples 1, 3, 5 and 6 were added to drug group He L a cells, respectively, 200. mu.L of the pharmaceutical compositions of preparation examples 1, 3, 5 and 6 were added to control group, medium + 10% v/vDMSO was added to control group, 200. mu.L of culture was continued for 48 hours, 3- (4, 5-dimethyltetrazolium-2-yl) -2,5-diphenyltetrazolium bromide (MTT reagent, final concentration 0.5mg/m L) was added to control group, culture was continued for one hour, the medium was removed and 100. mu.L of DMSO was added to measure the OD value at a wavelength of 570nm per well, thereby determining the survival rate of cancer cells, and the response of the cells was normalized by the OD value of the control group.
The survival of cancer cells treated with a composition containing two flavonoids is shown in FIG. 1, where F is at a concentration of 20 μ M (He L a cells) and 40 μ M (MCF-7 cells) and C and CG are at a concentration of 0.5, 1, 5, 10, 20, 40, 70, 100 μ M, respectively.
The survival of cancer cells treated with a composition containing three flavonoids is shown in FIG. 2, wherein the concentration of F is 20. mu.M (He L a cells) and 40. mu.M (MCF-7 cells), and the total concentration of the other two flavonoids is 0.5, 1, 5, 10, 20, 40, 70, 100. mu.M, respectively.
The survival rate of cancer cells treated with the flavonoid compound composition of preparation example 5 is shown in FIG. 3.
The survival rate of cancer cells treated with the flavonoid compound composition of preparation example 6 is shown in FIG. 4.
Example 2: the optimized flavonoid compound composition can enhance anticancer effect of cisplatin
After culturing the multidrug-resistant A549-DDP lung cancer cells in Experimental example 1 for 24 hours, the cells were divided into a drug group and a control group, 200. mu.l of the pharmaceutical composition of preparation example 7 was added to each drug group, 200. mu.l of the culture medium + cisplatin drug was added to the control group, and after further culturing for 48 hours, 3- (4, 5-dimethylthiazole-2) -2,5-diphenyltetrazolium bromide (3- (4, 5-dimethyl-thiazole-2-yl) -2, 5-phenyltetrazolium bromide, MTT reagent, final concentration 0.5mg/m L) was added, and after further culturing for one hour, the culture medium was removed and 100. mu.l of DMSO was added, and the OD value of each well at 570nm was measured, thereby determining the survival rate of cancer cells, and the cell response was normalized by the OD value of the control group.
Referring to fig. 5, it can be seen that the pharmaceutical composition of the present invention can greatly improve the cancer treatment effect of cisplatin, and effectively reduce the required dose thereof, thereby reducing the side effects caused by chemotherapy.
Comparative example 1: anticancer effects of single flavonoids
The concentrations of the four single flavonoids were each adjusted to 0.05, 0.1, 0.15, 0.20, 0.30, 0.50, 1.0, 3, 10, 20, 40, 80 and 100. mu.M, and OD values were measured after adding the cells of test example 1, respectively, in the same procedure as in example 1.
The survival of cancer cells treated with formononetin (F) is shown in FIG. 6.
Figure 7 shows the best results comparison of single flavonoids with the flavonoid combination of the present invention (the concentrations of the individual flavonoids in figure 7 are shown in table 4 below). it can be seen that for He L a cervical cancer cells and MCF-7 breast cancer cells, better cancer cell inhibition is achieved with the combination of the present invention.
The data of the above cell experiments are shown in tables 1-4 below.
TABLE 1 treatment of He L a and MCF-7cells with formononetin
Table 2: combined Effect of two flavonoid Compounds
Wherein formononetin is 20 μ M (He L a cells) or 40 μ M (MCF-7 cells) per well, and the concentrations of other flavonoid components are shown in the second column from left
As can be seen from Table 2, F + C and F + CG showed the best results for both cell lines.
Table 3: combined Effect of three flavonoid Compounds
Wherein formononetin is 20 μ M (He L a cells) or 40 μ M (MCF-7 cells) per well, and the concentration of each other flavonoid component is the same as shown in the second column from the left in the table (e.g., 20 μ M F +70 μ M C +70 μ MCG for He L a cells)
As can be seen from table 3, the composition comprising F and CG had the best effect.
Table 4: comparison of results for different flavonoid compositions
Wherein the cell activity is compared with that of a control well (no drug treatment, cell activity of 1)
From the above data, it can be seen that when the above flavonoids are used in combination, only lower concentrations need to be used on the cell line than when only one flavonoid is used.
Test example 2: animal research model for simulating side effect of chemotherapy
In this test example, male Sprague-Dawley rats (4 weeks, 180-. Rats were cultured in a thermostated (25 + -2 deg.C) environment, fed standard food and water, and provided a day and night cycle of 12/12 hours. Anemia, immune function decline, oxidative stress, etc. are induced by the injection of 3 days of 40 mg/kg/day antitumor drug Cyclophosphamide (CYP).
Example 3
40 male Sprague-Dawley rats (4 weeks, 180-.
After the blood sample was obtained, the red blood cell count (RBC), white blood cell count (WBC), hematocrit (Ht), and level of hemoglobin (Hb) were measured using an automated biochemical analyzer (miyao biochemical analysis center, Nanjing) (see results in fig. 8). blood samples were centrifuged (3,000 × g,15 minutes, 4 ℃) to obtain supernatant serum for immunological tests and oxidative function tests, including measuring the levels of superoxide dismutase (SOD), total antioxidant capacity (T-AOC), Malondialdehyde (MDA), and interleukin-2 (I L-2) (see results in fig. 9 and 10). I L-2 is secreted from T lymphocytes and is an important immune function parameter, and was measured by the E L ISA commercial suite (Nanjing Biochemical engineering institute, Nanjing) using the reference "Berneaddete F. Rodak, George A. friendly, Kathr, Hematology Doig, clinical research: Applications, and printers, Prime, Inc. 2007").
The specific results are shown in Table 5.
TABLE 5 parameters of hematopoietic function, immune function and antioxidant function of rat blood under drug action
| Parameter(s) | General group | Control group | Drug group |
| RBC(1012cells/L) | 7.23±0.50 | 4.26±0.92 | 6.37±0.53 |
| WBC(109cells/L) | 11.81±1.57 | 3.09±0.82 | 11.85±4.94 |
| Hb(g/L) | 135.52±9.71 | 69.77±19.64 | 120.14±15.61 |
| Ht(％) | 43.41±2.96 | 30.91±2.77 | 38.47±3.69 |
| T-AOC(U/mol) | 6.14±0.715 | 3.69±0.54 | 5.39±0.56 |
| MDA(nmol/ml) | 1.24±0.161 | 3.41±0.27 | 1.82±0.10 |
| SOD(U/ml) | 168.57±2.44 | 155.04±10.37 | 163.45±4.72 |
| IL-2(pg/ml) | 71.82±4.65 | 50.32±4.04 | 82.82±4.58 |
As can be seen from Table 5, after the rats in the control group are subjected to CYP action, the number of red blood cells, the number of white blood cells, the hematocrit and the hemoglobin level in blood are remarkably reduced compared with the ordinary healthy rats in the group, and the functional anemia symptoms caused by typical chemotherapy are displayed.
Comparative example 2
In the aforementioned example 3, the EPO group was tested simultaneously, and after CYP treatment, the rats were injected with 75IU/kg of EPO daily, and 14 days later blood samples were taken for testing of the aforementioned parameters. The results of comparison of the normal group, the control group, the drug group and the EPO group are shown in FIGS. 8 to 10. As can be seen from FIG. 8, the drug of the present invention has similar effect to EPO in treating chemotherapy-induced functional anemia, and even in WBC, Hb, Ht and other parameters, the drug of the present invention is better than EPO. Also, fig. 9 and 10 show that the drug of the present invention has the effect of enhancing antioxidant function comparable to that of EPO and is superior to EPO in enhancing immune function.
In conclusion, it can be seen from the results of the above examples and comparative examples that the flavonoid compound composition of the present invention has a very good synergistic effect on inhibiting cancer cell proliferation, and significantly reduces the amount of each active ingredient, and simultaneously can alleviate the side effects of chemotherapy, such as reduction in antioxidant function and reduction in immune function.
Claims (7)
1. Use of a flavonoid compound composition in the preparation of a medicament for the treatment and/or prevention of cervical cancer, wherein the active ingredients of said flavonoid compound composition are formononetin and calycosin, and the molar ratio of formononetin to calycosin is 20 (5-100).
2. Use according to claim 1, wherein the total amount of active ingredients is 1-100 wt% based on the total amount of the flavonoid composition.
3. The use of claim 1, wherein the medicament further comprises an additional anticancer drug compounded with the flavonoid composition, and the ratio of the total molar amount of the flavonoid composition to the total molar amount of the additional anticancer drug is (1-400): 1-20.
4. The use according to claim 1, wherein the medicament further has an effect of improving immune function and/or antioxidant function.
5. The use according to claim 1, wherein the medicament for the treatment and/or prevention of cervical cancer further comprises a pharmaceutically acceptable excipient.
6. The use according to claim 1, wherein the total amount of the flavonoid composition is 1-100% by weight, based on the total amount of the medicament.
7. The use of claim 1, wherein the medicament is in the form of oral liquid or injection.
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| CN1985876A (en) * | 2006-12-30 | 2007-06-27 | 中国人民解放军总医院 | Application of astragalus flavonid in preparing antitumor medicine |
| CN101775418A (en) * | 2010-01-28 | 2010-07-14 | 东北林业大学 | Method for extracting, separating and purifying formononetin and calycosin from Astragalus mongholicus waste residue |
| CN103690523A (en) * | 2012-09-27 | 2014-04-02 | 香港科技大学 | Composition used for promoting erythropoiesis, and its use |
| CN104523733A (en) * | 2014-12-16 | 2015-04-22 | 同济大学 | Pharmaceutical composition with anti-aging effect |
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| CN1985876A (en) * | 2006-12-30 | 2007-06-27 | 中国人民解放军总医院 | Application of astragalus flavonid in preparing antitumor medicine |
| CN101775418A (en) * | 2010-01-28 | 2010-07-14 | 东北林业大学 | Method for extracting, separating and purifying formononetin and calycosin from Astragalus mongholicus waste residue |
| CN103690523A (en) * | 2012-09-27 | 2014-04-02 | 香港科技大学 | Composition used for promoting erythropoiesis, and its use |
| CN104523733A (en) * | 2014-12-16 | 2015-04-22 | 同济大学 | Pharmaceutical composition with anti-aging effect |
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