Andrographolide compounds and their application and pharmaceutical composition

Abstract

The present invention discloses an andrographolide compound as an estrogen receptor agonist, including the above-mentioned andrographolide compound and the application of andrographolide compound, wherein the andrographolide compound is deoxyandrographolide or 14-deoxy-11,12-didehydroandrographolide. In combination with the contents of the embodiment part of the specification, this andrographolide compound has a pro-proliferation effect on breast cancer cells MCF-7 (estrogen receptor positive cells), and in addition, in combination with Figures 1 and 2, it is verified by a molecular docking calculation method that andrographolide compounds have a strong affinity with estrogen receptors, thereby indicating that andrographolide compounds can be used as estrogen receptor agonists.

Description

Technical Field

[0001] The present invention relates to the medical field, and in particular to an andrographolide compound as an estrogen receptor agonist and its application and pharmaceutical composition. Background Art

[0002] Estrogen is the primary female hormone responsible for the development and regulation of the female reproductive system and secondary female sexual characteristics. Estrogen also plays a role in protein synthesis, blood coagulation, lipid balance, fluid balance, melanin, gastrointestinal function, lung function, cognition, immune response and heart disease, among others.

[0003] Estrogen receptors are ligand-activated transcriptional regulatory proteins that mediate the induction of a variety of biological effects through interaction with endogenous estrogens.

[0004] Estrogen can bind to estrogen receptors in the body and play an important physiological role by activating estrogen receptors. It plays an important role in maintaining the functions of multiple systems such as the reproductive system, endocrine system, skeletal system, nervous system and cardiovascular system. When the body's estrogen level decreases and the estrogen receptor cannot be activated normally, it is easy to cause osteoporosis, irregular menstruation, infertility, menopausal syndrome, Alzheimer's disease and other diseases. Estrogen receptor is an important member of the nuclear receptor superfamily. It can bind to estrogen response elements to regulate the DNA transcription process. It plays an important regulatory role in the growth, development and metabolism of the body. It is also an important drug target for the treatment of osteoporosis, gynecological and obstetric diseases, neurodegenerative diseases, cardiovascular and cerebrovascular diseases, autoimmune diseases, skin diseases, hair loss, cancer and other diseases, as well as anti-aging.

[0005] Andrographolide compounds have good anti-inflammatory and bactericidal, heat-clearing and detoxifying effects, and are not only widely used in clinical practice, but are also often used as ingredients in Chinese patent medicine preparations. There is no relevant report in the prior art on whether andrographolide compounds can be used as estrogen receptor agonists. Summary of the invention

[0006] Based on this, it is necessary to provide an andrographolide compound that can serve as an estrogen receptor agonist.

[0007] In addition, it is also necessary to provide a pharmaceutical composition comprising the andrographolide compounds and the application of the andrographolide compounds.

[0008] An andrographolide compound as an estrogen receptor agonist, wherein the andrographolide compound is deoxyandrographolide or 14-deoxy-11,12-didehydroandrographolide, and its chemical formula is as follows:

[0009]

[0010] In one embodiment, the effective concentration of the andrographolide compound is 0.01 μM to 1 μM.

[0011] A pharmaceutical composition comprises the andrographolide compound, its stereoisomer, its pharmaceutically acceptable salt, its solvate or its prodrug.

[0012] In one embodiment, the pharmaceutical composition is used to prevent and treat osteoporosis, gynecological diseases, neurodegenerative diseases, cardiovascular and cerebrovascular diseases, autoimmune diseases, skin diseases, hair loss and cancer.

[0013] Application of the andrographolide compound, its stereoisomer, its pharmaceutically acceptable salt, its solvate or its prodrug in the fields of medicine, health food and cosmetics.

[0014] Combined with the contents of the embodiment of the specification, this andrographolide compound has a proliferative effect on breast cancer cells MCF-7 (estrogen receptor positive cells). Figure 1 and Figure 2 , the molecular docking calculation method verified that andrographolide compounds have a strong affinity for estrogen receptors, indicating that andrographolide compounds can be used as estrogen receptor agonists. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] in:

[0017] Figure 1 Diagram of the bonding pattern between deoxyandrographolide and estrogen receptor.

[0018] Figure 2 This is a diagram of the bonding pattern between 14-deoxy-11,12-didehydroandrographolide and the estrogen receptor.

[0019] Figure 3 This is a graph showing the effect of deoxyandrographolide on MCF-7 cell proliferation in Example 1.

[0020] Figure 4 This is a graph showing the effect of 14-deoxy-11,12-didehydroandrographolide on the proliferation of MCF-7 cells in Example 2. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] The present invention discloses an andrographolide compound as an estrogen receptor agonist. The andrographolide compound is deoxyandrographolide or 14-deoxy-11,12-didehydroandrographolide, and its chemical formula is as follows:

[0023]

[0024] Combined with the contents of the examples in the specification, this andrographolide compound has a proliferation-promoting effect on breast cancer cells MCF-7 (estrogen receptor positive cells).

[0025] In order to study the estrogenic activity of andrographolide compounds, the present invention analyzes the binding conformation between estrogen receptor and andrographolide compounds by molecular docking software, and further analyzes the conformation of the interaction between small molecules and estrogen receptor.

[0026] Combination Figure 1 and Figure 2 The oxygen atoms of deoxyandrographolide and 14-deoxy-11,12-didehydroandrographolide form hydrogen bonds with the hydrogen atoms of the key amino acid residue Arg394 of the estrogen receptor. The oxygen atom of 14-deoxy-11,12-didehydroandrographolide can also form hydrogen bonds with the hydrogen atoms of the key amino acid residue His524 of the estrogen receptor, indicating that the two can bind to the estrogen receptor through hydrogen bond interactions.

[0027] It should be noted that hydrogen bonding refers to a chemical bond of the form XH formed by a hydrogen atom and an atom X with high electronegativity and small radius combined by a special intermolecular force.

[0028] Therefore, combined with the data in the examples and the molecular docking calculation method, it is verified that andrographolide compounds have a strong affinity for estrogen receptors, thereby indicating that andrographolide compounds can be used as estrogen receptor agonists.

[0029] Preferably, the effective concentration of andrographolide compounds is 0.01 μM to 1 μM. The effective concentration of andrographolide compounds is moderate.

[0030] The present invention also discloses a pharmaceutical composition, comprising the andrographolide compound, its stereoisomer, its pharmaceutically acceptable salt, its solvate or its prodrug.

[0031] When the body's estrogen level drops and the estrogen receptors cannot be activated normally, it can easily lead to osteoporosis, irregular menstruation, infertility, menopausal syndrome, Alzheimer's disease and other diseases.

[0032] Based on this, andrographolide compounds that can act as estrogen receptor agonists can theoretically be used to prevent and treat the above-mentioned related diseases.

[0033] Specifically, the pharmaceutical composition is used to prevent and treat osteoporosis, gynecological diseases, neurodegenerative diseases, cardiovascular and cerebrovascular diseases, autoimmune diseases, skin diseases, hair loss and cancer.

[0034] The present invention also discloses the use of the andrographolide compound, its stereoisomer, its pharmaceutically acceptable salt, its solvate or its prodrug in the fields of medicine, health food and cosmetics.

[0035] The following are specific examples. In the specific examples, breast cancer cells MCF-7 were purchased from the cell bank of the Chinese Academy of Sciences, fetal bovine serum was a product of Hangzhou Sijiqing Biotechnology Co., Ltd., China, and bispecific antibodies were a product of Gibco, USA.

[0036] Example 1

[0037] Effect of deoxyandrographolide on the growth of breast cancer cell line MCF-7

[0038] (1) Activated carbon treatment of four seasons green fetal bovine serum:

[0039] 1) Add 1 g of activated carbon to a beaker containing 100 mL of fetal bovine serum, place it in a 56°C water bath and stir it continuously for 2 hours, and then centrifuge it at 17,000 rpm for 20 minutes in an ultracentrifuge; 2) Collect the supernatant in a new beaker, add 1 g of activated carbon, place it in a 37°C water bath and stir it continuously for 2 hours, and then centrifuge it at 17,000 rpm for 20 minutes in an ultracentrifuge; 3) Collect the supernatant in a new beaker, add 1 g of activated carbon, place it in a 4°C ice bath and stir it continuously for 3 hours, and then centrifuge it at 17,000 rpm for 30 minutes in an ultracentrifuge; 4) Finally, filter the collected supernatant with a 0.22 μm sterile filter membrane, and store it in a -80°C refrigerator for later use.

[0040] (2) Determination of the proliferation of breast cancer cells MCF-7 by deoxyandrographolide (crystal violet staining method):

[0041] 1) MCF-7 cells were cultured in RPMI1640 medium without phenol red (containing 10% fetal bovine serum treated with activated carbon and 1% double antibody) for 3 days; 2) The cells were digested and 5×10 4 100 μL of MCF-7 cell suspension with a concentration of cells / mL was added to a 96-well plate and cultured for 24 hours. The cell culture medium in the culture plate was aspirated, and different concentrations of deoxyandrographolide diluted with the cell culture medium were added, each dilution concentration contained 6 replicate wells, and the plate was placed in a 37°C carbon dioxide incubator for culture for 72 hours. The cell culture medium was discarded, and newly prepared cell culture medium with different concentrations of deoxyandrographolide was added, and the plate was placed in a 37°C carbon dioxide incubator for another 72 hours; 3) The cell culture medium was discarded, 100 μL of 1% glutaraldehyde was added for fixation for 30 minutes, the cells were washed 3 times with PBS, and after being completely dried at room temperature, 50 μL of 0.5% crystal violet solution was used for staining for 15 minutes, the staining solution was discarded, and excess crystal violet was washed with clean water, and the plate was placed at room temperature for complete drying. After 100 μL of 0.5% 100X-Triton solution was dissolved overnight, the OD values ​​at 405 nm and 560 nm were detected by an enzyme marker.

[0042] Calculate the proliferation effect of deoxyandrographolide on breast cancer cell MCF-7: cell density = OD value (650) - OD value (405), the result Figure 3 shown.

[0043] Depend on Figure 3 It can be seen that deoxyandrographolide has a significant proliferation-promoting effect on breast cancer cells MCF-7.

[0044] Specifically, the effective concentration of deoxyandrographolide is 0.01 μM to 1 μM, which is a mild concentration.

[0045] Example 2

[0046] Effect of 14-deoxy-11,12-didehydroandrographolide on the growth of breast cancer cell line MCF-7

[0047] (1) Activated carbon treatment of four-season fetal bovine serum:

[0048] 1) Add 1 g of activated carbon to a beaker containing 100 mL of fetal bovine serum, place it in a 56°C water bath and stir it continuously for 2 hours, and then centrifuge it at 17,000 rpm for 20 minutes in an ultracentrifuge; 2) Collect the supernatant in a new beaker, add 1 g of activated carbon, place it in a 37°C water bath and stir it continuously for 2 hours, and then centrifuge it at 17,000 rpm for 20 minutes in an ultracentrifuge; 3) Collect the supernatant in a new beaker, add 1 g of activated carbon, place it in a 4°C ice bath and stir it continuously for 3 hours, and then centrifuge it at 17,000 rpm for 30 minutes in an ultracentrifuge; 4) Finally, filter the collected supernatant with a 0.22 μm sterile filter membrane, and store it in a -80°C refrigerator for later use.

[0049] (2) Determination of the effect of 14-deoxy-11,12-didehydroandrographolide on the proliferation of breast cancer cells MCF-7 (crystal violet staining method):

[0050] 1) MCF-7 cells were cultured in RPMI1640 medium without phenol red (containing 10% fetal bovine serum treated with activated carbon and 1% double antibody) for 3 days; 2) The cells were digested and 5×10 4 100 μL of MCF-7 cell suspension with a concentration of 100 μL / mL was added to a 96-well plate and cultured for 24 h. The cell culture medium in the culture plate was aspirated and different concentrations of 14-deoxy-11,12-didehydroandrographolide diluted with the cell culture medium were added. Each dilution concentration contained 6 replicate wells and the plate was placed in a 37°C carbon dioxide incubator for 72 h. The cell culture medium was discarded and new cell culture medium with different concentrations of 14-deoxy-11,12-didehydroandrographolide was added and placed in a 37°C carbon dioxide incubator for 30 h. The cells were cultured in a carbon dioxide incubator for another 72 h; 3) the cell culture medium was discarded, 100 μL of 1% glutaraldehyde was added for fixation for 30 min, the cells were washed 3 times with PBS, and after being completely dried at room temperature, 50 μL of 0.5% crystal violet solution was used for staining for 15 min, the staining solution was discarded, and excess crystal violet was washed with clean water, and after being completely dried at room temperature, 100 μL of 0.5% 100X-Triton solution was used for dissolution overnight, and the OD values ​​at 405 nm and 560 nm were detected by an enzyme marker.

[0051] Calculate the proliferation effect of 14-deoxy-11,12-didehydroandrographolide on breast cancer cell MCF-7: cell density = OD value (650) - OD value (405). Figure 4 shown.

[0052] Depend on Figure 4 It can be seen that 14-deoxy-11,12-didehydroandrographolide has a significant pro-proliferation effect on breast cancer cells MCF-7.

[0053] Specifically, the effective concentration of 14-deoxy-11,12-didehydroandrographolide is 0.01 μM to 1 μM, which is a mild concentration.

[0054] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An application of an andrographolide compound in the preparation of an estrogen receptor agonist, wherein the andrographolide compound is deoxyandrographolide or 14-deoxy-11,12-didehydroandrographolide, and its chemical formula is as follows: The effective concentration of the andrographolide compound is 0.01 μM to 1 μM.

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