CN106176711B - Pharmaceutical comprising flavonoid compound composition and use thereof - Google Patents

Abstract

其中，R₁为羟基或

并且R₂为H或羟基。The present invention relates to the use of a flavonoid compound composition in the preparation of a drug for treating and/or preventing cancer, and also relates to a pharmaceutical composition for treating and/or preventing cancer. The flavonoid compound composition of the present invention can effectively improve the anti-cancer function, and each active ingredient has a good synergistic effect. The flavonoid compound composition comprises at least two flavonoid compounds represented by formula I as active ingredients:

Wherein, _R1 is hydroxyl or

And _R2 is H or hydroxyl.

Description

Medicine containing flavonoid composition and use thereof

technical field

The present invention belongs to the field of pharmacy, and in particular, relates to a medicament comprising a flavonoid compound composition, and the application of the flavonoid compound composition in preparing a medicament for treating and/or preventing cancer.

Background technique

In recent years, with the rapid economic growth, the deterioration of the living environment, and the changes in dietary structure and lifestyle, the incidence of cancer has exploded. Currently commonly used chemotherapy drugs are not only expensive, but also cause serious side effects in cancer patients, such as chemotherapy-induced anemia, decreased immune function, and decreased antioxidant function. Therefore, it is very important to develop a low-cost, less toxic and side effect drug for alleviating the suffering of cancer patients.

As a pure natural plant medicine, traditional Chinese medicine formulas usually have no toxic side effects and can be taken for a long time. Many studies have shown that many natural ingredients contained in botanical medicines have anticancer effects and can be used to prepare anticancer drugs, such as flavonoids and bioflavonoids. (See references "Flavonoids and cancer prevention: a review of theevidence. D.F. Romagnolo & O.I. Selmin, Journal of Nutrition in Gerontology and Geriatrics, 2013, 31:206-238" and "Anti-cancer potential of flavonoids: recenttrends and future perspectives" . P. Batra & A.K. Sharma, 3 Biotech, 2013, 3:439-459”). However, due to the complex composition of traditional Chinese medicine, usually composed of dozens or even hundreds of compounds, it is difficult to carry out pharmacological research and optimization on it. The quality control of Chinese medicinal materials is also particularly important. Factors such as origin, climate, and year can affect the quality of Chinese medicinal materials, thereby affecting the effectiveness and stability of the medicine. Therefore, it is of great significance for the development and promotion of traditional Chinese medicine to optimize traditional Chinese medicine and solve the problems of quality control.

The current research on the use of flavonoids in anticancer drugs has focused on the study of single flavonoids or flavonoid combination drugs in the treatment of cancer or cancer side effects, such as formononetin and vervasin on breast cancer when used alone It has certain inhibitory and therapeutic effects (see reference "Chen J, Lin C, Yong W, Ye Y, Huang Z. Calycosin and genistein induce apoptosis by inactivation of HOTAIR/p-Akt signaling pathway in human breastcancer MCF-7 cells. Cell Physiol Biochem. 2015;35(2):722-8";"J.Chen,J.Zeng,M.Xin,W.Huang,X.Chen.FormononetinInduces Cell Cycle Arrest of Human Breast Cancer Cells via IGF1/PI3K/AktPathways In Vitro and In Vivo. Horm Metab Res 2011, 43:681–686”;”Chen J, Zhao X, Ye Y, Wang Y, Tian J. Estrogen receptor beta-mediated proliferative inhibition and apoptosis in human breast cancer by calycosin and formononetin .CellPhysiol Biochem 2013;32(6):1790-7";"Chen J, Zhao X, Ye Y, Wang Y, Tian J.Estrogenreceptor beta-mediated proliferative inhibition and apoptosis in human breastcancer by calycosin and formononetin.Cell Physiol Biochem .2013;32(6):1790-7”). However, each component of traditional Chinese medicine has different targets for cancer treatment, and its efficacy is not significant when used alone. A more optimal method is to use multiple components as a combination drug, such as cyclophosphamide, salidroside The combination medicine of Versus isoflavone glucoside can effectively treat breast cancer under the combination of a specific ratio, (Xu Peng et al., Chinese Patent CN103933054(A), a pharmaceutical composition containing cyclophosphamide and its use in the treatment of breast cancer application), however, its therapeutic effects are mixed in different proportions of components. Since each drug acts on different targets, its synergistic effect can effectively improve the overall therapeutic effect. However, because the pharmacology of the components of traditional Chinese medicine is not completely clear, the optimization of the combination drug ratio requires a lot of experiments.

Astragalus has been used medicinally for more than 2,000 years and has the effect of tonifying qi and generating blood. Astragalus contains saponins, sucrose, polysaccharides, various amino acids, folic acid, selenium, zinc, copper and other trace elements, and has many biological and medical effects, such as liver protection, blood production, antioxidant, antihypertensive, and immune enhancement. Recent studies have shown that the flavonoids in astragalus can effectively stimulate the expression of erythropoietin in kidney cells, thereby increasing the level of red blood cells in the blood to improve and treat anemia. The main four flavonoid compounds that promote erythropoietin expression are formononetin, formononein, vervasin, and verbasin.

Formononetin is also known as 7-hydroxy-4'-methoxyphenyl isoflavone, formononetin; English name is Formononetin, 7-Hydroxy-3-(4-methoxyphenyl)chromone, 7-Hydroxy-4 '-methoxyisoflavone, or Dadein 4'-methyl ether; CAS number is 485-72-3; molecular formula is C ₁₆ H ₁₂ O ₄ ; molecular weight is 268.26; the chemical formula of formononetin is represented by the following formula III:

Formononetin is easily soluble in methanol, ethyl acetate, ether, dilute alkali solution, and insoluble in water. Formononetin can be extracted from plants or synthesized chemically. For example, synthetic methods can be found in reference: "J. Agric. FoodChem.. EN; 9.1994, 42, 1869-1871". At present, the known pharmacological effects of formononetin are as follows: (1) anticancer effect: can prevent and treat breast cancer, prostate cancer and colon cancer; (2) estrogen effect: can increase the weight of the uterus of animals such as mice, but estrogen The hormone effect is very weak, and the effect is not as good as the similar isoflavone genistein (genistein) and daidzein; (3) It has a hypolipidemic effect on hyperlipidemia in male albino rats caused by TritonWR-1339; (4) ) is clinically used as a diuretic.

Formononetin is also known as formononetin-7-glucoside; English name is Ononin, or Formononetin-7-O-beta-D-glucopyranoside; CAS number is 486-62-4; molecular formula is C ₂₂ H ₂₂ O ₉ ; molecular weight is 430.40; the chemical formula of formononein is represented by the following formula IV:

The English name of the isoflavone is Calycosin or 7,3'-dihydroxy-4'-methoxyisoflavone; the molecular formula is C ₁₆ H ₁₂ O ₅ ; the molecular weight is 284.26348; the chemical formula of the isoflavone is represented by the following formula II:

At present, the known pharmacological effects of mullein are as follows: (1) improve immune function; (2) enhance antioxidant, anti-radiation and anti-cancer effects; (3) protect cardiovascular and cerebrovascular, liver, kidney and lung effects; (4) protect Brain cells, improve memory; (5) antibacterial and antiviral effects; (6) lowering blood lipids, lowering blood sugar, reducing diabetes complications, etc.

Verascoside is also known as Verascoside glucoside, Verasco isoflavone-7-O-β-D-glucoside; English name is Calycosin-7-glucoside, Calycosin-7-O-β-D-glucoside, 3',7- Dihydroxy-4'-methoxyisoflavone-7-beta-D-glucopyranoside, Calycosin 7-O-beta-D-glucoside, Calycosin 7-beta-D-glucopyranoside, or Calycosin-7-O-beta-D-glucopyranoside; molecular weight is 446.40; the CAS number is 20633-67-4, and the chemical formula of verbascoside is represented by the following formula V:

Recent studies have shown that several flavonoid compounds isolated from Astragalus root are able to prevent and treat cancer when used alone (see Ref. "Formononetin induces cell cycle arrest of human breastcancer cells via IGF1/PI3K/Akt pathways in vitro and in vivo" .C.J.Zeng et al.,Hormone and Metabolic Research2011,43:681-686" and "Formononetin-inducedapoptosis of human prostate cancer cells through ERK1/2mitogen-activatedprotein kinase inactivation.Y.Ye et al.,Hormone and Metabolic Research,2012 , 44:263-267", and the patent document "The application of formononetin in the preparation of anti-breast cancer drugs", patent number CN103393638 A). However, the above studies all use a single flavonoid compound, and the current various flavonoid compounds have the defects of limited efficacy and large dosage when used alone.

SUMMARY OF THE INVENTION

In order to solve the above-mentioned problems in the prior art, the present invention provides a medicament comprising a flavonoid compound composition and an application of the flavonoid compound composition in preparing a medicament for treating and/or preventing cancer.

Specifically, the present invention provides:

(1) Use of a flavonoid composition in the preparation of a medicament for the treatment and/or prevention of cancer, wherein the flavonoid composition comprises at least two flavonoid compounds represented by formula I as active ingredients:

并且Wherein, R ₁ is hydroxyl or

and

R ₂ is H or hydroxy.

(2) The use according to (1), wherein, based on the total amount of the flavonoid compound composition, the total amount of the flavonoid compounds represented by formula I accounts for 1-100% by weight.

(3) The use according to (1), wherein the flavonoid composition includes formononetin, and further includes at least one selected from the group consisting of verbascoside, formononein, and verbascoside.

(4) The use according to (3), wherein the flavonoid compound composition comprises formononetin and vervasin.

(5) The application according to (4), wherein the molar ratio of the formononetin and the isoflavones is (0.5-100):(0.5-100).

(6) The use according to (3), wherein the flavonoid composition comprises formononetin and verbascoside.

(7) The application according to (6), wherein the molar ratio of formononetin and verbascoside is (0.5-100):(0.5-100).

(8) The use according to (3), wherein the flavonoid composition comprises formononetin, formononein and verbasin.

(9) The application according to (8), wherein the molar ratio of formononetin, formononetin and verbasco isoflavones is (0.5-100):(0.5-100):(0.5-100 ).

(10) The application according to (9), wherein the molar ratio of formononetin, formononetin and verbasco isoflavones is 2:25:1.

(11) The use according to (3), wherein the flavonoid composition comprises formononetin, formononein, and verbascoside.

(12) The application according to (11), wherein the molar ratio of formononetin, formononetin and verbascoside is (0.5-100):(0.5-100):(0.5- 100).

(13) The use according to (3), wherein the flavonoid composition comprises formononetin, verbascoside and verbascoside.

(14) The application according to (13), wherein the molar ratio of formononetin, verbascoside and verbascoside is (0.5-100):(0.5-100):(0.5-100) .

(15) The use according to (13), wherein the flavonoid composition comprises formononetin, formononein, verbasin, and verbasin.

(16) according to the application described in (15), wherein, the mol ratio of described formononetin, formononetin, verbasin and verbasin is (0.5-100): (0.5-100): (0.5-100): (0.5-100).

(17) The application according to (15), wherein the molar ratio of formononetin, formononein, verbasin and verbasin is 1:1:5:1.

(18) The use according to (1), wherein the drug further comprises other anticancer drugs compounded with the flavonoid composition, and the total molar amount of the flavonoid composition is the same as that of the flavonoid composition. The total molar ratio of other anticancer drugs is (1-400):(1-20).

(19) The use according to (1), wherein the drug also has the effect of improving immune function and/or antioxidant function.

(20) A pharmaceutical composition for treating and/or preventing cancer, comprising a flavonoid compound as an active ingredient, and a pharmaceutically acceptable excipient, wherein the flavonoid compound is at least two types of compounds represented by formula I Flavonoids:

并且Wherein, R ₁ is hydroxyl or

and

R ₂ is H or hydroxy.

(21) The pharmaceutical composition according to (20), wherein the pharmaceutical composition further comprises other anticancer drugs compounded with the flavonoid compound, and the total molar amount of the flavonoid compound is the same as that of the flavonoid compound. The total molar ratio of other anticancer drugs is (1-400):(1-20).

(22) The pharmaceutical composition according to (20), wherein, based on the total amount of the pharmaceutical composition, the total amount of the flavonoid compound accounts for 1-100% by weight.

(23) The pharmaceutical composition according to (20), wherein the dosage form of the pharmaceutical composition is an oral liquid or an injection.

Compared with the prior art, the present invention has the following advantages and positive effects:

The flavonoid composition of the present invention can effectively improve the anticancer function and inhibit the proliferation of cancer cells, so that it can be used for the prevention and treatment of cancer. Each active ingredient in the flavonoid compound composition of the present invention has good synergistic effect, and the dosage of each active ingredient is significantly reduced, the price is low, and the toxic and side effects are small.

In addition, the flavonoid composition of the present invention can also improve immune function and antioxidant function, and can alleviate and treat side effects such as decreased immune function and/or decreased antioxidant function caused by radiotherapy and/or chemotherapy.

Description of drawings

FIG. 1 shows the results of the inhibition rate of cancer cells by the composition of two flavonoid compounds in Example 1. FIG.

FIG. 2 shows the results of the inhibition rate of cancer cells by the composition of three flavonoid compounds 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.

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 composition of Preparation Example 7 in Example 2. FIG.

FIG. 6 shows the results of the inhibition rate of cancer cells by a single flavonoid drug in Comparative Example 1. FIG.

FIG. 7 shows the comparison of the best results of the single flavonoid compound and the flavonoid compound composition of the present invention on the inhibition rate of cancer cells.

FIG. 8 shows the results of detection of red blood cell count (RBC), white blood cell count (WBC), hematocrit (Ht), and hemoglobin (Hb) in Example 3. FIG.

FIG. 9 shows the results of detecting superoxide dismutase (SOD), total antioxidant capacity (T-AOC), and malondialdehyde (MDA) in Example 3. FIG.

FIG. 10 shows the results of detection of interleukin-2 (IL-2) in Example 3. FIG.

Detailed ways

The present invention will be further described below through the description of specific embodiments and with reference to the accompanying drawings, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements according to the basic idea of the present invention, but as long as they do not depart from the basic idea of the present invention The basic idea of the present invention is within the scope of the present invention.

As used herein, the term "treatment" refers to the process of blocking, relieving (or ameliorating) or eliminating a cause or lesion in order to restore or gain health or reduce suffering in a living human or animal body.

As used herein, the term "drug" includes human and veterinary drugs for human and animal applications. Furthermore, the term "drug" as used herein refers to any substance that provides prophylactic, therapeutic and/or beneficial effects. The term "drug" as used herein is not necessarily limited to substances requiring a marketing authorization.

As used herein, the term "purified form" refers to a pharmaceutically pure form. As used herein, the term "pharmaceutically pure" means substantially free of other compounds.

The present invention provides the use of a flavonoid compound composition in the preparation of a medicament for treating and/or preventing cancer.

The inventors of the present invention found through experiments that when two or more of verbascoside, formononetin, formononein, and verbascoside are combined, each component of the resulting composition exhibits With the unexpected synergistic effect, the obtained composition has a remarkable effect in improving the anticancer function, and can effectively prevent and treat cancer. On the basis of this discovery, the inventor further obtained the technical solution of the present invention.

Specifically, the present invention provides the use of a flavonoid composition in the preparation of a medicament for the treatment and/or prevention of cancer, wherein the flavonoid composition comprises at least two flavonoid compounds represented by formula I as Active ingredients:

并且Wherein, R ₁ is hydroxyl or

and

R ₂ is H or hydroxy.

Preferably, in preparing the flavonoid composition, the flavonoid is used in purified form.

Purified forms of the flavonoid compounds are commercially available (eg, from Sichuan Vikki Biotechnology Co., Ltd.), or can be prepared using purification methods known in the art.

Wherein, it should be noted that, in formula I, when R ₁ is hydroxyl and R ₂ is H, the flavonoid compound is formononetin.

In formula I, when R ₁ is a hydroxyl group and R ₂ is a hydroxyl group, the flavonoid compound is verbisoflavone.

R₂为H时，所述的类黄酮化合物为芒柄花苷。In formula I, when R ₁ is

When R ₂ is H, the flavonoid compound is formononein.

R₂为羟基时，所述的类黄酮化合物为毛蕊异黄酮苷。In formula I, when R ₁ is

When R ₂ is a hydroxyl group, the flavonoid compound is verbascoside.

Preferably, based on the total amount of the flavonoid compound composition, the total amount of the flavonoid compounds represented by formula I accounts for 1-100 wt %, more preferably 50-100 wt %, particularly preferably 90-100 wt % weight%. For example, based on the total amount of the flavonoid compound composition, the total amount of the flavonoid compound represented by Formula I may account for 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 %.

Preferably, the flavonoid composition of the present invention comprises formononetin, and further comprises at least one selected from the group consisting of verbascoside, formononein and verbascoside.

It is also preferred that the flavonoid composition of the present invention comprises formononetin and vervasin. Wherein, the molar ratio of Formononetin and Verbasidone is preferably (0.5-100):(0.5-100), more preferably (1-40):(0.5-100), particularly preferably (20):( 5-40).

It is also preferred that the flavonoid composition of the present invention comprises formononetin and verbascoside. Wherein, the molar ratio of formononetin and verbascoside is (0.5-100):(0.5-100), more preferably (1-40):(0.5-100), particularly preferably (20):( 0.5-5).

It is also preferred that the flavonoid composition of the present invention comprises formononetin, formononein and verbasin. Wherein, the molar ratio of Formononetin, Formononetin and Verbasidone is (0.5-100):(0.5-100):(0.5-100), more preferably (1-40):(0.5-100 ): (0.5-100), particularly preferably 2:25:1.

It is also preferred that the flavonoid composition of the present invention comprises formononetin, formononein and verbascoside. Wherein, the molar ratio of formononetin, formononetin and verbascoside is (0.5-100):(0.5-100):(0.5-100), more preferably (1-40):(0.5- 100):(0.5-100), particularly preferably 20:40:40.

It is also preferred that the flavonoid composition of the present invention comprises formononetin, verbascoside and verbascoside. Wherein, the molar ratio of formononetin, verbascoside and verbascoside is (0.5-100):(0.5-100):(0.5-100), more preferably (1-40):(0.5-100) : (0.5-100), particularly preferably 20:5:5.

It is also preferred that the flavonoid composition of the present invention comprises formononetin, formononein, verbasin and verbasin. Wherein, the molar ratio of formononetin, formononetin, verbascoside and verbascoside is (0.5-100):(0.5-100):(0.5-100):(0.5-100), particularly preferably (1-40):(0.5-100):(0.5-100):(0.5-100), most preferably 1:1:5:1.

In the medicament or pharmaceutical composition of the present invention, other anticancer medicaments or active ingredients compounded with the flavonoid compound may also be included. The ratio of the total molar amount of the flavonoid compound to the total molar amount of the other anticancer drugs or active ingredients is (1-400):(1-20), preferably 20:1. These other anticancer drugs or active ingredients are, for example, cisplatin, vitamin C, and the like. In the case of cisplatin, the molar ratio of cisplatin to the total amount of flavonoid compounds described in the present invention is, for example, (1-400):(1-20), preferably 20:1.

The flavonoid compound composition of the present invention can effectively improve the anti-cancer function, inhibit the proliferation of cancer cells, and can be used for the prevention and treatment of cancer. Therefore, the flavonoid composition of the present invention can be used to prepare a medicament for treating and/or preventing cancer.

In addition, the flavonoid composition of the present invention can also improve immune function and antioxidant function, and can alleviate and treat side effects such as decreased immune function and/or decreased antioxidant function caused by radiotherapy and/or chemotherapy. Therefore, preferably, the drug for treating and/or preventing cancer also has the effect of improving immune function and/or antioxidant function.

Preferably, the effective amount of the flavonoid composition of the present invention for treating and/or preventing cancer may be 0.05 μM-100 mM, more preferably 0.5 μM-1 mM, particularly preferably 20-60 μM. When the flavonoid composition of the present invention is used for therapeutic application, its therapeutically effective amount can be 0.05 μM-100 mM, more preferably 20-60 μM; when the flavonoid composition of the present invention is used for prophylactic application, its preventive An effective amount may be 0.05 μM-100 mM, more preferably 20-60 μM.

The present invention also provides a pharmaceutical composition for treating and/or preventing cancer, comprising the flavonoid compound of the present invention as an active ingredient, and a pharmaceutically acceptable excipient. Specifically, the flavonoid compound is at least two flavonoid compounds represented by formula I:

并且Wherein, R ₁ is hydroxyl or

and

R ₂ is H or hydroxy.

Preferably, based on the total weight of the pharmaceutical composition, the total amount of the flavonoid compounds is 1-100% by weight, more preferably 50-100% by weight. For example, based on the total amount of the pharmaceutical composition, the total amount of the flavonoid compound may account for 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% by weight.

Preferably, the pharmaceutical composition comprises formononetin, and further comprises at least one selected from the group consisting of verbascoside, formononein and verbascoside. The various combinations and corresponding ratios of flavonoids described above for the flavonoid composition also apply to the pharmaceutical composition.

Those skilled in the art can select the type and amount of pharmaceutically acceptable excipients according to needs (eg, route of administration and standard pharmaceutical practice) to prepare the desired drug. Pharmaceutical excipients known in the art can be used in the composition of the present invention, including but not limited to (for example) references: "Pharmaceuticals (Fourth Edition), edited by Bi Dianzhou, published by People's Medical Publishing House in 2001" Disclosed pharmaceutical excipients.

The pharmaceutically acceptable adjuvants include pharmaceutically acceptable carriers known in the art, such as polyethylene glycols, povidones, surfactants (such as poloxamer 188), organic acids (such as tartaric acid) , succinic acid, etc.), sugars (such as dextrose, galactose, sucrose, etc.) and alcohols (mannitol, sorbitol, xylitol, etc.), cellulose (such as ethyl cellulose, carboxymethyl ethyl cellulose, etc. ), polyacrylic resins, etc. The pharmaceutically acceptable adjuvants also include pharmaceutically acceptable diluents, examples of which include ethanol, glycerol, and water. The pharmaceutical compositions may also contain any suitable binders, lubricants, suspending agents, coatings, solubilizers, and the like.

The pharmaceutical compositions of the present invention may be administered orally or parenterally. Oral administration can be in the form of solid, liquid, etc., and non-oral administration can be in the form of injection.

The solid forms used for oral administration can include tablets, pills, capsules, powders, granules, etc., and those skilled in the art can select and use the following auxiliary materials to prepare as required: fillers (such as starch, dextrin, lactose, mannitol, etc.) , microcrystalline cellulose, etc.), absorbents (such as calcium sulfate, calcium hydrogen phosphate, etc.), wetting agents (such as water, ethanol, etc.), binders (such as hydroxymethyl cellulose, povidone, starch pulp, etc.), Disintegrants (such as croscarmellose sodium, crospovidone, etc.), lubricants (such as stearic acid, talc, polyethylene glycol, micropowder silica gel, etc.), flavoring agents (such as sweet Agents (such as sucrose, stevioside, aspartame, etc.), aromatics (such as spices and essences, etc.), colorants (such as beetroot, caramel, lemon yellow, etc.), etc.

The liquid form (oral liquid) used for oral administration includes solutions, emulsions, suspensions, syrups, etc., and those skilled in the art can select and use the following auxiliary materials to prepare as required: solvents (such as water, glycerol, dimethyl sulfoxide, etc.) (DMSO), ethanol, propylene glycol, polyethylene glycol, fatty oils, liquid paraffin, ethyl acetate, etc.), flavoring agents (such as sweeteners (such as sucrose, stevioside, aspartame, etc.), aromatics ( Such as spices and flavors), etc.), colorants (such as beetroot, caramel, tartrazine, etc.), preservatives (parabens, benzoic acid and sodium benzoate, sorbic acid, etc.), wetting agents (such as polysorbate) , polyoxyethylene fatty alcohol ethers, etc.), suspending agents (such as glycerol, gum trees, cellulose, diatomaceous earth, etc.), emulsifiers (such as surfactants, etc.), etc.

Preferably, the dosage form of the pharmaceutical composition of the present invention is an oral liquid.

The following examples are used to further explain or illustrate the content of the present invention, but these examples should not be construed as limiting the protection scope of the present invention.

In the following examples, the isoflavones, formononetin, formononetin, and isoflavone glycosides can be purchased from Sichuan Vikki Biotechnology Co., Ltd.

Fetal bovine serum (FBS) was purchased from life technologies.

Penicillin was purchased from Sigma Company.

Streptomycin was purchased from Sigma Company.

Cisplatin was purchased from Sigma Company.

MTT reagent was purchased from Sigma Company.

Test Example 1: Construction of an in vitro study model of cancer cell proliferation inhibition

In this experiment, 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 Chinese Academy of Medical Sciences Tumor Cell Bank) were used as cancer cell proliferation. Research models of inhibition. The above cells were seeded in a 96-well culture plate, the number of cells in each well was 4000, and 200 microliters of cell culture medium (DMEM medium containing 10% (v/v) fetal bovine serum (FBS) was added to each well. , 100U/ml penicillin and 100U/ml streptomycin), put into a 37°C constant temperature incubator, control the carbon dioxide concentration at 5% (v/v), and cultivate for 24 hours.

Preparation Example 1: Preparation of a flavonoid composition containing two active ingredients: formononein (F) + one of (formononein (O), verbascoside (C) and verbascoside (CG)) kind)

Formononetin, Formononetin, Verbasidone and Verbasidone glycosides were dissolved in DMSO, respectively, to prepare their respective DMSO stock solutions (3 concentrations for each stock solution: 50 μM, 500 μM, and 5 mM, respectively, for preparing different DMSO stock solutions. final concentration to improve experimental precision). Calculate and measure the required volume of DMSO stock solution according to the final concentration of different active ingredients, combine and dilute to 300 microliters with the cell culture medium described in Test Example 1. The final concentrations of each active ingredient were: formononetin 20 μM, formononetin, verbasin and verbasin 0.5, 1, 5, 10, 20, 40, 70, 100 μM, respectively.

Preparations 2-7 were carried out as in Preparation 1, but with the following final concentrations of active ingredients:

Preparation Example 2: Preparation of a flavonoid composition containing two active ingredients: F+ (one of O, C and CG)

Formononetin 40 μM, Formononetin, Verbasidone and Verbasidone glycosides were 0.5, 1, 5, 10, 20, 40, 70, 100 μM, respectively.

Preparation Example 3: Preparation of a flavonoid composition containing three active ingredients: F+ (two of O, C and CG)

Formononetin 20 μM, formononetin, verbascoside and verbascoside 0.5, 1, 5, 10, 20, 40, 70, 100 μM, respectively.

Preparation Example 4: Preparation of a flavonoid composition containing three active ingredients: F+ (two of O, C and CG)

Formononetin 40 μM, Formononetin, Verbasidone and Verbasidone glycosides were 0.5, 1, 5, 10, 20, 40, 70, 100 μM, respectively.

Preparation Example 5: Preparation of a flavonoid composition containing four active ingredients: F+O+C+CG

Formononetin 0.08 μM, Formononetin 0.08 μM, Verbasin 0.4 μM, and Verbasidin 0.08 μM. In addition, on the basis of this concentration, the flavonoid composition containing the four active ingredients in different concentrations was formulated, wherein the concentration of each active ingredient was 5 times, 10 times, 15 times, 25 times, 40 times, 60 times times, 80 times, 90 times, 100 times, 125 times, 300 times, 500 times, 800 times, and 1000 times the stated respective concentrations.

Preparation Example 6: Preparation of a flavonoid composition containing three active ingredients: F+O+C

Formononetin 0.016 μM, Formononetin 0.4 μM and Verbasin 0.08 μM. In addition, on the basis of this concentration, the flavonoid composition containing the three active ingredients in different concentrations was formulated, wherein the concentration of each active ingredient was 5 times, 10 times, 15 times, 25 times, 40 times, 60 times times, 80 times, 90 times, 100 times, 125 times, 300 times, 500 times, 800 times, and 1000 times the stated respective concentrations.

Preparation Example 7: Preparation of a flavonoid composition containing five active ingredients: F+O+C+CG+cisplatin

According to the ratio of formononetin:formononein:verbasin:verascoside 1:1:5:1, the total concentration of these four flavonoids is 12.5μM, 25μM, 50μM, 100μM, 200μM and 400μM, in the solutions of the above six concentrations, each solution was added with cisplatin 0, 1.25μM, 2.5μM, 5μM, 10μM and 20μM, a total of 36 kinds of concentration solutions were prepared.

Example 1: The effect of flavonoid composition in inhibiting the proliferation of cancer cells

The MCF-7 breast cancer cells and HeLa cervical cancer cells in Test Example 1 were cultured for 24 hours and then divided into a drug group and a control group. 200 microliters of the pharmaceutical compositions of Preparation Examples 2, 4, 5 and 6 were added to the MCF-7 cells of the drug group; 200 microliters of the pharmaceutical compositions of Preparation Examples 1, 3, 5 and 6 were respectively added to the HeLa cells of the drug group. liters; add medium + 10% v/v DMSO, 200 microliters to the control group. After culturing for 48 hours, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT reagent, final concentration 0.5 mg/mL) was added respectively. After an additional hour of incubation, the culture medium was removed and 100 microliters of DMSO was added. The OD value of each well at a wavelength of 570 nm was detected to determine the survival rate of cancer cells. Cellular responses were normalized by control OD values.

The viability of cancer cells treated with a composition containing two flavonoids is shown in Figure 1, where the concentrations of F were 20 μM (HeLa cells) and 40 μM (MCF-7 cells), and the concentrations of C and CG were 0.5, 1, respectively, respectively. , 5, 10, 20, 40, 70, 100 μM.

The survival rates of cancer cells treated with the composition containing the three flavonoids are shown in Figure 2, where the concentrations of F were 20 μM (HeLa cells) and 40 μM (MCF-7 cells), and the total concentrations of the other two flavonoids were each 0.5, 1, 5, 10, 20, 40, 70, 100 μM.

The survival rate of cancer cells treated with the flavonoid composition of Preparation Example 5 is shown in FIG. 3 .

The survival rate of cancer cells treated with the flavonoid composition of Preparation Example 6 is shown in FIG. 4 .

Example 2: The optimized flavonoid composition can enhance the anticancer effect of cisplatin

The multidrug-resistant A549-DDP lung cancer cells in Test Example 1 were cultured for 24 hours and then divided into a drug group and a control group. To the drug group, 200 microliters of the pharmaceutical composition of Preparation Example 7 was added; to the control group, 200 microliters of culture medium + cisplatin was added. After culturing for 48 hours, 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (3-(4,5-dimethyl-thiazol-2-yl )-2,5-diphenyltetrazolium bromide, namely MTT reagent, final concentration 0.5mg/mL). After an additional hour of incubation, the culture medium was removed and 100 microliters of DMSO was added. The OD value of each well at a wavelength of 570 nm was detected to determine the survival rate of cancer cells. Cellular responses were normalized by control OD values.

The results are shown in 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, thereby reducing the side effects caused by chemotherapy.

Comparative Example 1: Anticancer effect of a single flavonoid compound

The concentrations of the four single flavonoid compounds were respectively prepared at 0.05, 0.1, 0.15, 0.20, 0.30, 0.50, 1.0, 3, 10, 20, 40, 80 and 100 μM, and were added to the test according to the same steps as in Example 1. The OD value was detected after the cells of Example 1.

The survival rate of cancer cells treated with formononetin (F) is shown in FIG. 6 .

Figure 7 shows a comparison of the best results between a single flavonoid compound and the flavonoid compound combination drug of the present invention (the concentrations of various flavonoid compounds in Figure 7 are shown in Table 4 below). It can be seen that, for HeLa cervical cancer cells and MCF-7 breast cancer cells, better cancer cell inhibition rate can be obtained by using the combination medicine of the present invention. Thus, the present invention exhibits unexpected synergy.

The data of the above cell experiments are shown in Tables 1-4 below.

Table 1: Treatment of HeLa and MCF-7 cells with formononetin

Table 2: Combined effect of two flavonoids

Among them, formononetin is 20 μM (HeLa cells) or 40 μM (MCF-7 cells) per well, and the concentrations of other flavonoid compounds are shown in the second column from the left in the table.

As can be seen from Table 2, for the two cell lines, F+C and F+CG had the best effect.

Table 3: Combination effects of three flavonoid compounds

Among them, formononetin was 20 μM (HeLa cells) or 40 μM (MCF-7 cells) per well, and the concentration of each other flavonoid component was the same, as shown in the second column from the left in the table (for example, for HeLa cells, 20 μM F+70 μM C+70 μM CG)

As can be seen from Table 3, the composition comprising F and CG has the best effect.

Table 4: Comparison of Results for Different Flavonoid Compositions

Among them, the cell viability is the comparison value with the control well (no drug treatment, cell viability is 1)

From the above data, it can be seen that when the above flavonoids are used in combination, it is only necessary to use a lower concentration on the cell line than when a single flavonoid is used.

Test Example 2: Constructing an Animal Research Model Simulating the Side Effects of Chemotherapy

In this test example, male Sprague-Dawley rats (4 weeks, 180-220 g, Experimental Animal Center of China Pharmaceutical University, Nanjing) were used as animal research models. Rats were cultured in a constant temperature (25±2°C) environment with standard chow and water and provided with a 12/12 hour day-night cycle. Anemia, decreased immune function, oxidative stress, etc. were induced by injection of the antitumor drug cyclophosphamide (CYP) at 40 mg/kg/day for 3 days.

Example 3

Forty male Sprague-Dawley rats (4 weeks, 180-220 g, Experimental Animal Center of China Pharmaceutical University, Nanjing) were divided into 3 groups with 8 rats in each group. The normal group was provided with normal water and salt; the control group and the drug group were first treated with CYP according to the method described in the test example to simulate the side effects of chemotherapy, and then, the control group was provided with normal water and salt; the drug group was provided with mullein, thorn The mixture of formononetin, formononetin and vervasin (prepared according to the method of Preparation Example 1), its molar ratio is 20:4:4:4, and divided into low-dose group (FH) and high-dose group (FL) Two groups, the low doses were 1.4, 1.4, 7, 1.4 μmol/kg, and the high doses were 4, 4, 20, 4 μmol/kg, orally. The rats in the above three groups were fed for 14 days in the corresponding manner, and then the blood samples of the rats in each group were taken from the optic holes.

After the blood samples were obtained, an automatic biochemical analyzer (Biochemical Analysis Center, Zhongda Hospital, Nanjing) was used to detect the red blood cell count (RBC), white blood cell count (WBC), hematocrit (Ht), and hemoglobin (Hb) levels (see Fig. 8). The blood samples were centrifuged (3,000×g, 15 minutes, 4°C), and the upper serum was obtained for immunological detection and oxidative function tests, including the detection of superoxide dismutase (SOD), total antioxidant capacity (T-AOC) , malondialdehyde (MDA) and interleukin-2 (IL-2) levels (see Figures 9 and 10 for results). IL-2 is secreted by T lymphocytes and is an important parameter of immune function. It was detected by ELISA commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing) (the above detection methods can be found in the reference "Bernadette F.Rodak, George A.Fritsma, Kathryn Doig, Hematology: Clinical Principles and Applications, Elsevier Health Sciences, 2007").

The specific results are shown in Table 5.

Table 5. Parameters of hematopoietic function, immune function and antioxidant function of rat blood under the action of drugs

parameter normal 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

It can be seen from Table 5 that after the CYP effect of the rats in the control group, the number of red blood cells, the number of white blood cells, the hematocrit and the hemoglobin level in the blood were significantly reduced compared with the healthy rats in the normal group, showing a typical chemotherapy-induced effect. Symptoms of functional anemia. The parameters of the rats in the drug group are very close to those of the healthy rats in the normal group, indicating that the drug of the present invention can effectively improve the hematopoietic function and treat the side effects of anemia caused by chemotherapy. At the same time, the immune function parameter IL-2 of the rats in the control group was significantly lower than that in the normal group, showing symptoms of decreased immune function caused by chemotherapy. Compared with the control group, the IL-2 parameter of the rats in the drug group was significantly improved, even higher than that in the normal group, showing a great immune-enhancing effect, indicating that the drug of the present invention can treat the side effects of decreased immune function caused by chemotherapy. Similarly, in terms of antioxidant function, the drug group was able to effectively treat and improve chemotherapy-induced decreases in T-AOC and SOD parameters, as well as adverse side effects of increased MDA.

Comparative Example 2

In the aforementioned Example 3, the detection of the EPO group was carried out at the same time. After CYP treatment, the rats were injected with 75 IU/kg of EPO every day, and blood samples were taken 14 days later for the detection of the aforementioned parameters. The ordinary group, the control group, the drug group and the EPO group were compared, and the results are shown in Figures 8 to 10. As can be seen from FIG. 8 , the medicine of the present invention and EPO have similar effects in treating functional anemia caused by chemotherapy, and even in terms of parameters such as WBC, Hb, and Ht, the medicine of the present invention is better than EPO. Similarly, Figure 9 and Figure 10 show that the drug of the present invention is comparable to EPO in enhancing antioxidant function, and is better than EPO in enhancing immune function.

To sum up, from the results of the above examples and comparative examples, it can be seen that the flavonoid composition of the present invention has a very good synergistic effect on inhibiting the proliferation of cancer cells, and significantly reduces the dosage of each active component, At the same time, it can alleviate side effects such as decreased antioxidant function and decreased immune function caused by chemotherapy.

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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