CN114081879B - New medical application of safflower Xiaoyao preparation monomer active ingredient - Google Patents
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Abstract
The invention relates to the technical field of medicines, provides a new medical application of a safflower Xiaoyao preparation monomer active ingredient, and particularly relates to an application of formononetin in preparing a medicine for treating hyperplasia of mammary glands. Furthermore, the invention also provides a pharmaceutical composition for treating hyperplasia of mammary glands, which consists of the safflower Xiaoyao preparation monomer active ingredients and pharmaceutically acceptable auxiliary materials. A mouse model experiment shows that after formononetin is administrated, hyperplasia of mammary glands of a rat is improved, and the volume of lobule of the mammary gland, the number of acinus and secretion of a lumen are obviously reduced; cell experiments show that formononetin can obviously inhibit the T level increase in the thecal cells caused by dexamethasone, which means that the formononetin has an inhibition effect on the testosterone production of the thecal cells and relieves the hyperplasia of mammary glands generated by the stimulation of estrogen by inhibiting the production of androgen. Therefore, the invention provides a new basis for the safflower Xiaoyao preparation monomer active ingredient to treat hyperplasia of mammary glands.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to application of a safflower Xiaoyao preparation monomer active ingredient in preparation of a medicine for treating hyperplasia of mammary glands and a pharmaceutical composition containing the safflower Xiaoyao preparation monomer active ingredient.
Background
Mastoproliferation (MGH), also known as mammary dysplasia, is a common mammary disease with lumps in the breast, breast pain, and nipple discharge as the main symptoms, and is a degenerative and progressive lesion of the structure of the duct and lobules of the breast. Hyperplasia of mammary glands is not only in the category of tumors, but also in the scope of inflammation, but rather in the structural changes of the lobules and ducts of the mammary glands. According to the pathology, the traditional Chinese medicine can be divided into simple mammary epithelial hyperplasia, cystic hyperplasia of breast and mastopathy. The main clinical features of hyperplasia of mammary glands are breast lumps and periodic swelling pain of the breast. The mastosis accounts for more than 70 percent of all mastosis, is the most common mastosis of women of childbearing age, accounts for about 50 percent of the mastosis of women of childbearing age, has a trend of increasing prevalence year by year in recent years, and greatly influences the normal life of patients. The disease has the risk of developing into breast cancer to a certain extent, seriously threatens the health of women, and has important practical significance in researching the current situation of the drug therapy of the disease and effectively preventing and treating the disease.
In order to solve the technical problems, the prior art carries out a lot of exploration, for example, the chinese patent application with the patent number CN110507708A, which was earlier applied by the applicant, discloses a safflower Xiaoyao preparation for treating hyperplasia of mammary glands and a preparation method and application thereof, wherein the safflower Xiaoyao preparation comprises the following raw materials in parts by weight: 18-67 parts of angelica, 100-150 parts of white peony root, 30-55 parts of bighead atractylodes rhizome, 105-135 parts of poria cocos, 110-160 parts of safflower, 20-35 parts of spina gleditsiae, 80-140 parts of bupleurum chinense, 45-85 parts of mint and 20-75 parts of liquorice; the safflower Xiaoyao preparation prepared by the specific preparation method can well play the effective role of the medicine, and has good regulation effect on female endocrine.
Chinese patent application with the patent number of CN101954021A discloses a quality detection method of a traditional Chinese medicine composition for treating hyperplasia of mammary glands, the traditional Chinese medicine composition is prepared from hawthorn, fried malt, caulis spatholobi and ricepaperplant pith, the method comprises two parts of identification and content measurement, wherein the identification comprises identification by adopting a thin-layer chromatography and respectively using a formononetin reference substance, a hawthorn reference medicinal material and a citric acid reference substance; the content measurement is determined by epicatechin content, and is not less than 0.70mg/g based on epicatechin.
Formononetin (FMN, 7-hydroxy-4' -methoxy isoflavone), also known as Formononetin, exist widely in leguminous plants such as radix astragali, licorice, radix Puerariae, red triaxial grass, etc., it is a kind of phytoestrogen substance separated and purified from the leguminous plant clover, belong to isoflavonoid, it is a kind of micro-component in safflower ease preparation too, it is apt to dissolve in methanol, ethyl acetate, ether, dilute alkali solution, difficult to dissolve in water. Modern pharmacological research shows that formononetin has the effects of resisting tumor, reducing blood fat, resisting arrhythmia, resisting bacteria, relieving spasm, resisting lipid peroxidation, resisting estrogen and the like, can improve atherosclerotic lesions, also has the effects of scavenging oxygen free radicals, inhibiting proliferation of vascular smooth muscle cells and the like, is widely applied to clinic and has obvious curative effect (modern research progress of Wangjian, sunyu, chenyu, qiansheng and formononetin, university of Shanxi traditional Chinese medicine and pharmacology, 2017-05-026). Studies have shown that formononetin has a cardiotonic effect on isolated toad heart (Erlenmeyer's wort, hanzhengkang, the influence of formononetin on blood pressure and electrocardio of rats, proceedings of Nanjing university of agriculture 1990, P126-127).
In the current literature reports, the relation research between formononetin and hyperplasia diseases of mammary glands is not found.
Disclosure of Invention
The invention is based on the research, and aims to provide a novel medical application of a safflower Xiaoyao preparation monomer active ingredient and a pharmaceutical composition containing the safflower Xiaoyao preparation monomer active ingredient.
The invention provides the application of the safflower Xiaoyao preparation monomer active ingredient in preparing the medicine for treating hyperplasia of mammary glands, wherein the safflower Xiaoyao preparation monomer active ingredient is formononetin and derivatives thereof; the medicine for treating hyperplasia of mammary glands is a medicine for improving the level of sex hormone disorder.
Preferably, the agent for improving the level of sex hormone disorder is an agent for reducing the level of estrogen in the body by inhibiting androgen synthesis, specifically, by inhibiting testosterone production.
The formononetin derivative in the invention refers to formononetin compounds capable of exerting pharmacological action, such as formononetin amino ether derivatives and the like.
In order to verify the treatment effect of formononetin on hyperplasia of mammary glands, the invention verifies through two aspects of in vivo experiments of mice and in vitro experiments of cytology.
For the in vivo experiments in mice, 24 female rats were randomly selected and equally divided into 3 groups: control group (sham), model group (Mod), formononetin group (FMN). After the molding is finished, the FMN group is perfused with the FMN solution and continuously administrated for 3 weeks; perfusing a blank solvent by a Sham group and a Mod group; for cytological in vitro experiments, thecal cells (thecal cells) are extracted and purified from ovaries of female rats aged 3-4 weeks for culture, LH, DEX, HHXYT, FMN, PA are added into different groups of wells at different concentrations, and the T or E2 content in the cell culture medium is detected.
Experiments show that after FMN administration, hyperplasia of mammary glands of rats is improved, and the volume of lobule of mammary glands, the number of acinus and luminal secretion are obviously reduced; cell experiments show that FMN (3.2, 6.3 and 12.5 mu M) can obviously inhibit DEX (10 mu M) caused T level increase in the thecal cells, which means that the T level increase has an inhibitory effect on the production of testosterone by the thecal cells, and the generation of hyperplasia of mammary glands stimulated by estrogen is relieved by inhibiting the production of androgen.
The medicine for treating hyperplasia of mammary glands provided by the invention takes formononetin as the only active ingredient or is a medicine composition containing the formononetin.
The pharmaceutical composition can be prepared into any dosage form with pharmaceutically common auxiliary materials, for example, the pharmaceutical composition can be tablets, dispersible tablets, buccal tablets, orally disintegrating tablets, sustained release tablets, capsules, soft capsules, dripping pills, microcapsule microsphere preparations, suppositories, ointments, sprays, targeted preparations, granules, injections, powder injections or aerosols.
The second aspect of the invention provides a pharmaceutical composition for treating hyperplasia of mammary glands, which consists of formononetin serving as an active ingredient of a safflower Xiaoyao preparation monomer and pharmaceutically acceptable auxiliary materials.
The safflower Xiaoyao preparation monomer active component is used for treating hyperplasia of mammary glands, and the administration mode is not limited to oral administration, injection and the like.
Action and effects of the invention
The mouse model experiment shows that after FMN administration, hyperplasia of mammary glands of rats is improved, and the volume of lobule of mammary gland, the number of acinus and the secretion of lumen are obviously reduced; cell experiments show that FMN can obviously inhibit T level increase in thecal cells caused by dexamethasone, which means that the FMN has an inhibitory effect on testosterone production of the thecal cells and can relieve hyperplasia of mammary glands produced by estrogen stimulation by inhibiting androgen production. Therefore, the invention provides a new basis for the safflower Xiaoyao preparation monomer active ingredient to treat the hyperplasia of mammary glands and also provides a new clinical medicine for treating the hyperplasia of mammary glands.
Drawings
Figure 1 is the improved result of formononetin on mammary gland hyperplasia: a is Sham group, B is model group, C is FMN group mammary gland section HE staining (100 x: A-1, B-1, C-1 x: A-2, B-2, C-2; d is the result of 2 nd pair of teat diameters.
FIG. 2 is the effect of formononetin on testosterone synthesis in the cal cells: (A) the proliferative effects of LH and FSH on the cal cells. (B) the effects of LH on T and E2 production by the cal cells. Effects of DEX (C), HHXYT, FMN and PA (D) on T production by the focal cells compared to control group (n =3, # # p <0.01; p <0.05, # p <0.01 compared to DEX group). (E) The pathways for T and E2 synthesis by theca and granulosa cells.
FIG. 3 is a graph showing the effect of HHXYT (A), FMN (B), and PA (C) on the survival rate of the cal cells.
Detailed Description
The present invention will be described in detail below with reference to examples and the accompanying drawings. The following examples should not be construed as limiting the scope of the invention.
Example 1: formononetin for improving mammary lobular volume, acinus number and luminal secretion of rat
1. Materials and methods
1. Chemicals and reagents
TABLE 1 summary of materials and reagents used in the invention
2. Animal experiments and sample preparation
All sexually mature and non-pregnant female Sprague-Dawley rats were from the center of the animal in Zhejiang province. Raising in stable environment of free drinking water and feed for 1 week at room temperature of 25 + -2 deg.C and relative humidity of 55 + -5% for 12 hr each in light and dark.
24 female rats were randomly divided into 3 groups: control group (sham), model group (Mod), administration group (FMN). Method for establishing hyperplasia of mammary glands (M) by combining estradiol benzoate and progesterone for intramuscular injectionGH) model [1] . The rats except Sham were injected intramuscularly with estradiol benzoate (0.5 mg/kg/d) for 25 days, followed by progesterone (5 mg/kg/d) for 5 days, while Sham was injected with saline injection (0.5 mg/kg/d).
After the molding is finished, the administration group is used for intragastric administration of formononetin suspended in 0.5% ethyl acetate water solution, the dosage is 4.5g/kg/d, and the continuous administration is carried out for 3 weeks. Sham and Mod groups gavage a 0.5% ethyl acetate aqueous blank.
3 weeks after administration, rats were anesthetized by intraperitoneal injection of 10% chloral hydrate, and the 2 nd pair of teat diameters were measured. Collecting blood sample from abdominal aorta, centrifuging at 3500rpm and 4 deg.C for 15min; serum was separated from the upper layer and stored at-80 ℃. And collecting the 2 nd mammary gland on the left side of the breast, fixing the tissue fixing solution for 24h, then carrying out paraffin section HE staining, and carrying out optical microscopy.
3. Results of the experiment
The results are shown in fig. 1, where histological changes of the breast tissue are visualized using HE staining method. The Sham results showed no abnormal hyperplasia of mammary tissue, a small number of acini, almost no dilatation, and no secretion of mammary ducts and lumens (fig. 1, a-1 and a-2). The Mod group mammary tissue is obviously hyperplastic, and the volume of mammary lobules is increased; the number of acini is increased and they are fused close to each other; the acini and lumen dilate and the secretion is excessive (FIGS. 1, B-1 and B-2). After the administration of the administration group (FMN), the hyperplasia of mammary glands of rats is obviously improved, and the volume of lobule of mammary glands, the number of acinus and luminal secretion are obviously reduced (figure 1, C-1 and C-2).
The increase in diameter of the right papilla of rats 2 caused by mammary gland hyperplasia was significantly inhibited after FMN treatment compared to the Mod group (FIG. 1D, p-were-woven 0.01).
Example 2: formononetin inhibits production of testosterone by thecal cells
1. Experimental methods
Oocyst cells (thecal cells) were extracted and purified from ovaries of 3 to 4 weeks old female rats [2] . Will be 4X 10 4 The individual cells were added to 48-well plates/well in McCoy5A medium containing 10% FBS and 1% penicillin-streptomycin. 37 ℃ and 5% of CO 2 After 36h of incubation, different groups of wells were addedCulturing at the concentration of LH, DEX, HHXYT, FMN, and PA for 24h [3] . Detection of T or E in cell culture medium by ELISA kit 2 And (4) content.
2. Results of the experiment
Among follicles, thecal cells (thecal cells) and granulosa cells (granulosa cells) are predominant. As shown in FIG. 2E, the thecal cells possess LH receptors and synthesize T, while granulosa cells possess FSH receptors and produce E 2 . Under LH stimulation, the thecal cells promote cell proliferation and testosterone production [2] . When LH was added to the purified cells, the cell proliferation rate and the level of T increased significantly. However, after addition of FSH, cell number and E 2 The levels did not change significantly (fig. 2A and B). The results show that the purified cells were predominantly the cal cells, with no interference from granulosa cells.
It was investigated whether the thecal cells inhibited T production after addition of HHXYT, FMN, PA. DEX promotes T synthesis by the focal cells and is used as a model set [3] (FIG. 2C). FIG. 2D shows that HHXYT (50 ng/mL) and FMN (3.2, 6.3 and 12.5. Mu.M) significantly inhibited DEX (10. Mu.M) induced increases in T levels in the thecal cells, which means that they had an inhibitory effect on the production of testosterone by the thecal cells.
The cytotoxicity test results of different concentrations of HHXYT, FMN, PA are shown in FIG. 3: when the concentration of the safflower Xiaoyao tablet (HHXYT) is lower than 50ng/mL, the cell survival rate is higher than 90 percent; the cell survival rate of the Formononetin (FMN) is still higher than 90% when the concentration of the formononetin is 1.56 mu M; the cell viability was also higher than 90% at a Protocatechuic Acid (PA) concentration of 6.25. Mu.M. The safety of Formononetin (FMN) and Protocatechuic Acid (PA) is higher than that of safflower Xiaoyao tablet (HHXYT).
Results and conclusions
MGH is the most common breast disease, with breast cancer [4] Are closely related. Mammary gland is the target organ of sex hormone, and hypothalamus-pituitary-gonad axis and mammary tissue hyperplasia and menstrual cycle change [5] Are highly correlated. Sex hormone disorders are the main etiological and important features of MGH, especially estrogen and progestin disorders: the estrogen can accelerate hyperplasia of mammary glands, and the progestational hormone can promote growth and maturation of acinar cells of mammary glands and resist estrogen [6] The influence on hyperplasia of mammary glands. E 2 Excessive levels or insufficient levels of P lead to prolonged stimulation of the mammary epithelium by estrogens without progesterone protection, ultimately leading to mammary tissue [7] A large amount of hyperproliferation.
The sex hormone synthesis pathway is shown in figure 2E: synthesizing androgen and estrogen by using cholesterol as raw material, wherein the androgen is synthesized estrogen [8] A precursor of (2). Cholesterol is converted to pregnenolone by CYP11A1 in the thecal cells, and pregnenolone is converted to androgens under the catalysis of HSD3B2 and CYP17A1, particularly CYP17 A1. Androgen production in granulosa cells by CYP19A1 is then converted to estrogen [9,10] 。
It is therefore postulated that HHXYT and FMN may reduce estrogen levels in vivo by inhibiting androgen synthesis, thereby protecting the breast from estrogen-stimulated hyperproliferation. Then, we extracted cells from ovaries, and observed HHXYT and monomer FMN vs T and E using dexamethasone as model group 2 Whether or not there is an inhibitory effect on the synthesis of (A). The results show that HHXYT and FMN significantly inhibit T production, E 2 Not detected due to too low a content.
In conclusion, HHXYT has obvious therapeutic effect on MGH and can remarkably improve hormone disorder caused by MGH. HHXYT may act through inhibition of androgen production and reduction of estrogen stimulation leading to mammary gland hyperplasia.
Reference to the literature
1.Wang L,Zhao D,Di L,Cheng D,Zhou X,Yang X,Liu Y:The anti-hyperplasia of mammary gland effect of Thladiantha dubia root ethanol extract in rats reduced by estrogen and progestogen.Journal of ethnopharmacology 2011,134(1):136-140.
2.Li SK,Hearn MT:Isolation of thecal cells:an assessment of purity and steroidogenic potential.Journal of biochemical and biophysical methods 2000,45(2):169-181.
3.Ong M,Cheng J,Jin X,Lao W,Johnson M,Tan Y,Qu X:Paeoniflorin extract reverses dexamethasone-induced testosterone over-secretion through downregulation of cytochrome P450 17A1 expression in primary murine theca cells.Journal of ethnopharmacology 2019,229:97-103.
4.Basree MM,Shinde N,Koivisto C,Cuitino M,Kladney R,Zhang J,Stephens J,Palettas M,Zhang A,Kim HK et al:Abrupt involution induces inflammation,estrogenic signaling,and hyperplasia linking lack of breastfeeding with increased risk of breast cancer.Breast cancer research:BCR 2019,21(1):80.
5.Li X,Xin P,Wang C,Wang Z,Wang Q,Kuang H:Mechanisms of Traditional Chinese Medicine in the Treatment of Mammary Gland Hyperplasia.The American journal of Chinese medicine 2017,45(3):443-458.
6.Chang XJ,Zhou J,Zhang S,Chen J,Chen CM,Wang ZZ,Xiao W:[Effects of guizhi fuling capsule on sex hormone levels and breast issue morphology of mammary gland hyperplasia model rats].Zhongguo Zhong yao za zhi＝Zhongguo zhongyao zazhi＝China journal of Chinese materia medica 2014,39(21):4139-4142.
7.Liu J,Zhang JF,Gong GH,Zhang B,Wei CX:Therapeutic Effect of Mongolian Medicine RuXian-I on Hyperplasia of Mammary Gland Induced by Estrogen/Progesterone through CRYAB-Promoted Apoptosis.Evidence-based complementary and alternative medicine:eCAM 2020,2020:5707106.
8.Williams K,McKinnell C,Saunders PT,Walker M,Fisher JS,Turner KJ,Atanassova N,Sharpe M:Neonatal exposure to potent and environmental oestrogens and abnormalities of the male reproductive system in the rat:evidence for importance of the androgen-oestrogen balance and assessment of the relevance to man.Human reproduction update 2001,7(3):236-247.
9.Burris-Hiday SD,Scott EE:Steroidogenic cytochrome P450 17A1 structure and function.Molecular and cellular endocrinology 2021,528:111261.
10.Imamichi Y,Sekiguchi T,Kitano T,Kajitani T,Okada R,Inaoka Y,Miyamoto K,Uwada J,Takahashi S,Nemoto T et al:Diethylstilbestrol administration inhibits theca cell androgen and granulosa cell estrogen production in immature rat ovary.Sci Rep 2017,7(1):8374.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (5)
1. The application of the safflower Xiaoyao preparation monomer active ingredient in the preparation of the medicine for treating the hyperplasia of mammary glands is disclosed, wherein the medicine for treating the hyperplasia of mammary glands is a medicine composition taking formononetin as the only active ingredient.
2. The use of the safflower Xiaoyao preparation monomer active ingredient of claim 1 in the preparation of a medicament for treating hyperplasia of mammary glands is characterized in that:
wherein, the medicine for treating hyperplasia of mammary glands is a medicine for improving the level of sex hormone disorder.
3. The application of the safflower Xiaoyao preparation monomer active ingredient in the preparation of the medicine for treating hyperplasia of mammary glands according to claim 2 is characterized in that:
wherein the drug for improving the level of the sex hormone disorder is a drug for reducing the level of estrogen in vivo by inhibiting androgen synthesis.
4. The use of the safflower Xiaoyao preparation monomer active ingredient of claim 2 in the preparation of a medicament for treating hyperplasia of mammary glands is characterized in that:
wherein the drug for ameliorating the sex hormone disorder is a drug for inhibiting testosterone production.
5. The use of the safflower Xiaoyao preparation monomer active ingredient of claim 1 in the preparation of a medicament for treating hyperplasia of mammary glands is characterized in that:
the medicine for treating hyperplasia of mammary glands is tablet, capsule, pill, microcapsule microsphere preparation, suppository, ointment, spray, targeting preparation, granule, injection, powder injection or aerosol.
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