HK1195725B - A composition for promoting erythropoiesis and uses thereof - Google Patents

Description

Composition for promoting erythropoiesis and use thereof

Technical Field

The present invention belongs to the field of pharmaceuticals, and in particular relates to a pharmaceutical composition for promoting erythropoiesis and preventing and treating anemia and/or hypoxia, and uses thereof.

Background Art

Red blood cells are primarily produced in the human bone marrow (specifically red bone marrow). Stem cells in the bone marrow first differentiate into erythroid progenitor cells, which then mature into mature red blood cells under the influence of erythropoietin (also known as erythropoietin). Erythropoietin is a hormone-like substance secreted by the kidneys and liver.

Anemia is a common clinical symptom characterized by a decrease in the volume of red blood cells (RBCs) in the human peripheral blood, below the lower limit of the normal range. Anemia includes chemotherapy-induced anemia (CIA) and renal anemia. In CIA, chemotherapy drugs, while killing a large number of cancer cells, also damage normal tissue cells, causing varying degrees of bone marrow suppression. Thus, chemotherapy drugs cause anemia by inhibiting bone marrow hematopoietic cells. Over 60% of cancer patients develop anemia after chemotherapy, severely impairing their function and quality of life. Most chemotherapy drugs can cause stomach discomfort, leading to vomiting and nausea, severely affecting appetite and causing weight loss. One cause of this is CIA. Renal anemia, caused by various factors that result in insufficient renal erythropoietin production or by toxins in uremic plasma that interfere with RBC production and metabolism, is a common complication of end-stage chronic renal failure. Reduced RBC production, shortened RBC lifespan, and increased RBC loss impair blood metabolism. The usual treatments for chemotherapy-induced anemia and renal anemia are blood transfusion and dialysis, but blood transfusion may cause serious clinical or subclinical side effects, and patients may suffer great pain during the treatment.

Hypoxia (also known as hypoxia) refers to a pathological process in which tissue metabolism, function, and morphological structure undergo abnormal changes due to insufficient oxygen supply or impaired oxygen utilization. Symptoms of hypoxia include dizziness, headache, tinnitus, blurred vision, and weakness in the limbs; or nausea, vomiting, palpitations, shortness of breath, rapid breathing, and a rapid and weak heartbeat. Red blood cells are the primary means of transporting oxygen through the blood in vertebrates, so increasing the number of red blood cells in the body can effectively improve hypoxia (see references: "Analysis of the Efficacy of Erythropoietin in the Treatment of Neonatal Hypoxic-Ischemic Encephalopathy, Guo Zhimei et al., Journal of Clinical Military Medicine, 2008, 5" and "Effect of EPO on NSE Expression in Neonatal Rats with Hypoxic-Ischemic Brain Damage, Liu Qing, Zeng Zhi et al., Journal of Jinan University: Natural Science and Medicine, 2011, 6").

Currently, the most effective clinical treatments for chemotherapy-induced anemia, renal anemia, and hypoxia are erythropoiesis-stimulating agents (ESAs), primarily recombinant erythropoietin (rHuEPO). Erythropoietin (EPO) is the primary regulatory factor for erythropoiesis in mammals, primarily produced by the kidneys and liver. Failure to increase EPO levels in the blood under hypoxic conditions can lead to anemia. EPO synthesis and expression are primarily regulated by hypoxia-inducible factor (HIF), which regulates multiple processes, including erythropoiesis, angiogenesis, and cellular metabolism. Under hypoxic conditions, HIF selectively binds to the hypoxia response element (HRE) of the EPO gene, thereby initiating transcriptional expression of the EPO gene. For related information, please refer to the reference: "Flavonoids from radix astragali induce the expression of erythropoietin in cultured cells: a signaling mediated via the accumulation of hypoxia-inducible factor-1α, Ken Y.Z.Zheng et al, Journal of Agricultural and Food Chemistry, 2011, 59, 1697-1704".

ESAs treat chemotherapy-induced anemia and renal anemia primarily through subcutaneous administration of EPO, which increases the patient's red blood cell count and effectively reduces the need for blood transfusions. Numerous clinical trials have demonstrated that subcutaneous administration of 5000 IU/day of recombinant erythropoietin (EPO) effectively increases red blood cell counts and reduces the need for blood transfusions. Currently, several EPO-based drugs are commercially available. Amgen (Thousand Oaks, California) holds multiple EPO patents, and one of its products, epoetin alfa, is the most widely used EPO drug. However, the use of EPO is plagued by high costs and serious side effects, such as heart disease, blood clot formation, hypertension, and allergic reactions. The U.S. Food and Drug Administration (FDA) has issued numerous reports on ESA dosage control to improve the safety of ESA use. Therefore, developing a low-cost drug with minimal toxicity and side effects is crucial for alleviating the suffering of cancer and kidney disease patients.

Traditional Chinese medicine (TCM) has a long history of use in the treatment of anemia. As pure natural botanicals, TCM blood-tonifying formulas are generally non-toxic and have no side effects, allowing for long-term use. Astragalus membranaceus is often used as the main ingredient in TCM blood-tonifying formulas for anemia treatment. Astragalus membranaceus has been used medicinally for over 2,000 years and has the benefits of replenishing qi and promoting blood production. Containing saponins, sucrose, polysaccharides, various amino acids, folic acid, and trace elements such as selenium, zinc, and copper, Astragalus membranaceus has numerous biological and medical benefits, such as protecting the liver, promoting blood production, providing antioxidants, combating hypertension, and enhancing immunity. However, due to the complex composition of TCM, which often consists of dozens or even hundreds of compounds, pharmacological research and optimization are difficult. Quality control of TCM ingredients is also particularly important. Factors such as origin, climate, and year can affect the quality of TCM ingredients, thereby affecting the effectiveness and stability of the drug. Although there are many traditional Chinese medicines for treating anemia on the domestic market, their therapeutic effects vary greatly. Therefore, optimizing traditional Chinese medicine and solving problems such as quality control are of great significance to the development and promotion of traditional Chinese medicine.

Recent studies have shown that the flavonoids in Astragalus membranaceus can effectively stimulate the expression of erythropoietin in kidney cells, thereby increasing red blood cell levels in the blood and improving and treating anemia. The four main flavonoids that promote erythropoietin expression are formononetin, formononetin, calycosin, and calycosin. Recent studies have shown that several flavonoids isolated from Astragalus membranaceus, when used alone, can enhance the transcriptional activity of the cellular hypoxia response element (HRE), thereby promoting the expression of erythropoietin and increasing red blood cell levels in the blood (see reference: "Flavonoids from radix astragali induce the expression of erythropoietinin in cultured cells: a signaling mediated via the accumulation of hypoxia-inducible factor-1α, Ken Y.Z.Zheng et al, Journal of Agricultural and Food Chemistry, 2011, 59, 1697-1704"). However, current flavonoids have the disadvantages of limited efficacy and high dosage when used alone.

Summary of the Invention

In order to solve the above problems in the prior art, the present invention provides a composition for promoting erythropoiesis and its use.

Specifically, the present invention provides:

(1) A composition for promoting erythropoiesis, comprising two or more flavonoid compounds as active ingredients; wherein the flavonoid compounds are represented by the following formula I:

Wherein, _R1 is hydroxyl or

_R2 is H or hydroxyl;

Furthermore, when preparing the composition, the flavonoid compound is used in a purified form.

(2) The composition according to (1), wherein the composition comprises calycosin and at least one selected from formononetin, formononetin and calycosin glycosides.

(3) The composition according to (2), wherein the composition comprises calycosin and calycosin glycosides.

(4) The composition according to (3), wherein the molar ratio of calycosin to calycosin glycoside is (0.016-2):(0.016-2).

(5) The composition according to (4), wherein the molar ratio of calycosin to calycosin glycoside is 1:1.

(6) The composition according to (2), wherein the composition comprises calycosin and formononetin.

(7) The composition according to (6), wherein the molar ratio of calycosin to formononetin is (0.016-2):(0.016-2).

(8) The composition according to (7), wherein the molar ratio of calycosin to formononetin is 1:1.

(9) The composition according to (2), wherein the composition comprises calycosin, formononetin and formononetin.

(10) The composition according to (9), wherein the molar ratio of the isoflavones, formononetin and formononetin is (4-6):(0.5-1.5):(20-30).

(11) The composition according to (10), wherein the molar ratio of the isoflavones, formononetin and formononetin is 5:1:25.

(12) The composition according to (2), wherein the composition comprises calycosin, formononetin, formononetin and calycosin glycosides.

(13) The composition according to (12), wherein the molar ratio of the calycosin, formononetin, formononetin and calycosin is (4-6):(0.5-1.5):(0.5-1.5):(0.5-1.5).

(14) The composition according to (13), wherein the molar ratio of calycosin, formononetin, formononetin and calycosin is 5:1:1:1.

(15) The composition according to any one of (1) to (14), wherein the composition is a pharmaceutical composition and contains pharmaceutical excipients.

(16) The composition according to (15), wherein the composition is in the form of an oral solution.

(17) Use of the composition according to any one of (1) to (16) in the preparation of a drug for promoting erythropoiesis.

(18) The use according to (17), wherein the drug is used to prevent and/or treat hypoxia and/or anemia.

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

The composition of the present invention has a good synergistic effect in enhancing the transcriptional activity of HRE, thereby promoting erythropoiesis, and preventing and treating anemia and/or hypoxia. It also significantly reduces the dosage of each active ingredient, is inexpensive, and has minimal toxic and side effects. For example, in a preferred composition of the present invention, HRE transcriptional activity can even be increased by approximately 3 times. The enhancement of HRE transcriptional activity can directly stimulate the expression of EPO, thereby achieving the purpose of promoting erythropoiesis and preventing and treating anemia and hypoxia. Compared to the effects of each active ingredient acting alone and the effects of an aqueous extract of Astragalus membranaceus, the composition of the present invention can enhance their efficacy by 2 times, and the required dosage can be as low as approximately 0.6 μM, less than 1/10 of the previous dosage.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram showing the effect of the Astragalus membranaceus aqueous extract components in Comparative Example 1 on HRE transcriptional activity when applied to pHRE-Luc transfected HEK 293T kidney cells;

FIG2 is a schematic diagram showing the effects of the four compounds in Comparative Example 2 on HRE transcriptional activity when acting alone on pHRE-Luc transfected HEK 293T kidney cells;

FIG3 is a schematic diagram of the pHRE-Luc vector in Experimental Example 1.

DETAILED DESCRIPTION

The present invention will be further illustrated below through the description of specific implementation methods 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 based on the basic idea of the present invention, but as long as they do not deviate from the basic idea of the present invention, they are all within the scope of the present invention.

The treatment mentioned herein refers to the process of blocking, alleviating (or improving) or eliminating the cause or focus of disease in order to restore or gain health or reduce suffering in a living human or animal body.

The English name of the calycosin described herein 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 calycosin is shown in the following Chemical Formula II:

The currently known pharmacological effects of calycosin are as follows: (1) improving immune function; (2) enhancing antioxidant, anti-radiation and anti-cancer effects; (3) protecting cardiovascular, liver, kidney and lung functions; (4) protecting brain cells and improving memory; (5) antibacterial and antiviral effects; (6) lowering blood lipids, lowering blood sugar, and reducing diabetic complications.

The formononetin described herein is also known as 7-hydroxy-4'-methoxyisoflavone, formononetin, and its English names include Formononetin, 7-Hydroxy-3-(4-methoxyphenyl)chromone, 7-Hydroxy-4'-methoxyisoflavone, or Dadein 4'-methyl ether. Its CAS number is 485-72-3, its molecular formula is C ₁₆ H ₁₂ O ₄ , and its molecular weight is 268.26. The chemical formula of formononetin is shown in Chemical Formula III below:

Formononetin is easily soluble in methanol, ethyl acetate, ether, and dilute alkaline solutions, but poorly soluble in water. It can be extracted from plants or synthesized chemically. For example, the synthesis method can be found in the reference "J. Agric. Food Chem. EN; 9. 1994, 42, 1869-1871." Currently, the known pharmacological effects of formononetin are as follows: (1) Anticancer effect: It can prevent and treat breast cancer, prostate cancer, and colon cancer; (2) Estrogenic effect: It can increase the weight of the uterus in mice and other animals, but the estrogenic effect is very weak, not as strong as that of similar isoflavones genistein (genistein) and daidzein; (3) It has a lipid-lowering effect on hyperlipidemia in male albino rats induced by Triton WR-1339; (4) It is used clinically as a diuretic.

The formononetin described herein is also known as formononetin-7-glucoside; its English name is Ononin or Formononetin-7-O-beta-D-glucopyranoside; its CAS number is 486-62-4; its molecular formula is C ₂₂ H ₂₂ O ₉ ; and its molecular weight is 430.40. The chemical formula of formononetin is shown in the following Chemical Formula IV:

The calycosin isoflavone glycoside described herein is also called calycosin isoflavone glucoside, calycosin isoflavone-7-O-β-D-glucoside; its 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; its molecular weight is 446.40; its CAS number is 20633-67-4. The chemical formula of calycosin isoflavone glycoside is shown in the following chemical formula V:

The object of the present invention is to provide a composition containing flavonoid compounds and use thereof.

The inventors have discovered through experiments that when two or more of calycosin, formononetin, formononetin, and calycosin glycosides are combined, the resulting composition exhibits unexpected synergistic effects, resulting in a composition that is more effective than any of the components alone in increasing red blood cell levels. Based on this discovery, the inventors further developed the technical solution of the present invention.

Specifically, the present invention provides a composition for promoting erythropoiesis, which contains two or more flavonoid compounds as active ingredients; wherein the flavonoid compounds are represented by the following formula I:

Wherein, _R1 is hydroxyl or

_R2 is H or hydroxyl;

Furthermore, when preparing the composition, the flavonoid compound is used in a purified form.

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

It is particularly noted that, in Formula I, when R ₁ is a hydroxyl group 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 calycosin;

In formula I, when R ₁ is R ₂ is H, the flavonoid compound is formononetin;

In formula I, when R ₁ is R ₂ is hydroxy, the flavonoid compound is calycosin.

In the composition of the present invention, the concentrations of calycosin, formononetin, formononetin and calycosin glycosides can be 0.016-10 μM, respectively.

Preferably, the composition of the present invention comprises calycosin and at least one selected from the group consisting of formononetin, formononetin and calycosin glycosides.

Preferably, the composition of the present invention comprises calycosin and calycosin glycosides. More preferably, the molar ratio of calycosin to calycosin glycosides is (0.016-2):(0.016-2), and more preferably 1:1.

Preferably, the composition of the present invention comprises calycosin and formononetin. More preferably, the molar ratio of calycosin to formononetin is (0.016-2):(0.016-2), more preferably 1:1.

Preferably, the composition of the present invention comprises isoflavones, formononetin and formononetin. More preferably, the molar ratio of isoflavones, formononetin and formononetin is (4-6):(0.5-1.5):(20-30), more preferably 5:1:25.

Preferably, the composition of the present invention comprises calycosin, formononetin, formononetin and calycosin. More preferably, the molar ratio of calycosin, formononetin, formononetin and calycosin is (4-6):(0.5-1.5):(0.5-1.5):(0.5-1.5), more preferably 5:1:1:1.

Preferably, the composition of the present invention may further comprise other active ingredients, such as vitamin C.

Preferably, the composition of the present invention is a pharmaceutical composition and contains pharmaceutical excipients.

Those skilled in the art can select the type and amount of pharmaceutical excipients as needed to prepare the desired medicine. The composition of the present invention can adopt pharmaceutical excipients well known in the art, including but not limited to (for example) the pharmaceutical excipients disclosed in the reference: "Pharmaceutics (4th edition), edited by Bi Dianzhou, published by People's Medical Publishing House in 2001".

The pharmaceutical excipients 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 composition of the present invention can be administered orally or parenterally. Those skilled in the art will appreciate that the dosage varies depending on age, body weight, symptoms, therapeutic effect, administration method, treatment time, and the like. Oral administration can be in the form of a solid or liquid, while parenteral administration can be in the form of an injection.

Solid forms for oral administration may include tablets, pills, capsules, powders, granules, etc., and those skilled in the art may select the following excipients for preparation as needed: fillers (such as starch, dextrin, lactose, mannitol, 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 slurry, etc.), disintegrants (such as cross-linked sodium carboxymethyl cellulose, cross-linked povidone, etc.), lubricants (such as stearic acid, talc, polyethylene glycol, micropowdered silica gel, etc.), flavoring agents (such as sweeteners (such as sucrose, stevioside, aspartame, etc.), aromatics (such as spices and flavors, etc.), colorants (such as beet red, caramel, tartrazine, etc.), etc.

Liquid forms for oral administration (oral liquids) include solutions, emulsions, suspensions, syrups, and the like. Those skilled in the art can select the following excipients for preparation as needed: solvents (such as water, glycerol, dimethyl sulfoxide (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 beet red, caramel, tartrazine, etc.), preservatives (parabens, benzoic acid and sodium benzoate, sorbic acid, etc.), wetting agents (such as polysorbates, polyoxyethylene fatty alcohol ethers, etc.), suspending agents (such as glycerol, gums, celluloses, diatomaceous earth, etc.), emulsifiers (such as surfactants, etc.), etc.

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

The present invention also provides the use of any of the above-mentioned compositions in the preparation of a medicament for promoting erythropoiesis. Preferably, the medicament is used to improve and/or treat hypoxia and/or anemia (more preferably chemotherapy-induced anemia and renal anemia).

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

In the following examples, calycosin, formononetin, formononetin, and calycosin glycosides can all be purchased from Sichuan Weikeqi Biotechnology Co., Ltd.

Experimental Example 1: Construction of an in vitro research model for EPO expression regulation

This experiment used HEK293T cells transfected with the pHRE-Luc vector as an in vitro model for studying EPO expression regulation. The construction process of HEK293T cells transfected with the pHRE-Luc vector can be found in the reference "Flavonoids from radix astragali induce the expression of erythropoietin in cultured cells: a signaling mediated via the accumulation of hypoxia-inducible factor-1α, Ken Y.Z.Zheng et al, Journal of Agricultural and Food Chemistry, 2011, 59, 1697-1704." The details are as follows:

Human embryonic kidney (HEK) 293T cell line was obtained from the American Type Culture Collection (ATCC) in Manassas, Virginia, USA. The cells were added to a cell culture medium (DMEM medium containing 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin, and 100 U/ml streptomycin) and cultured in a 37°C incubator with a carbon dioxide concentration of 5% (v/v) for 24 hours.

The hypoxia response element (HRE) (5'-TCG AGG CCCTAC GTG CTG TCT CAC ACA GCC TGT CTG ACG-3') derived from the human erythropoietin (EPO) gene contains a highly conserved hypoxia-inducible factor-1 (HIF-1) binding target site (5'-TAC GTG-3'). Six HRE sequences were synthesized and tandemly cloned into the pBI-GL vector (available from BD Biosciences Clontech, San Jose, USA) (for details, see the following reference: "Generation of bidirectional hypoxia/HIF-responsive expression vectors to target gene expression to hypoxic cells. Post, D.E., and Van-Meir, E.G., 2001. Gene Therapy 8, 1801-1807") to generate the pHRE-Luc vector (see Figure 3). The pBI-GL vector itself contains a reporter gene: the firefly luciferase gene. pHRE-Luc was transfected into cultured HEK293T cells using calcium phosphate cell transfection technology (for specific steps, please refer to the following document: "Regulation of a transcript encoding the proline-rich membrane anchor of globular muscle acetylcholinesterase: the suppressive roles of myogenesis and innervating nerves. Xie, H.Q., et al., 2007. Journal of Biological Chemistry, 282, 11765-11775."), with a transfection efficiency of over 80%.

Example 1

The calycosin, cyperus sclerotin, cyperus sclerotin and cyperus sclerotin glycosides were dissolved in DMSO and prepared into their own DMSO mother solutions (each DMSO mother solution can be 3 concentrations: 50μM, 500μM, 5mM, used to prepare different final concentrations to improve the accuracy of the experiment). For each composition of the present invention shown in Table 1, according to its components and final concentration, the required volume of DMSO mother solution was calculated and measured, combined, and diluted to 300 microliters with the cell culture medium described in Test Example 1 for the following 3 parallel experiments. After the resulting composition solution was applied to the transfected HEK293T cells constructed in Test Example 1, the resulting changes in luciferase activity were measured. The specific steps are as follows:

Transfected HEK293T cells were seeded in a 24-well culture plate, with 30,000 cells per well and a cell culture medium volume of 500 μl. One day later, the original cell culture medium was aspirated and 400 μl of fresh cell culture medium was added. Three hours later, 100 μl of the cell culture medium containing the composition of the present invention was added, bringing the total volume per well to 500 μl. The concentrations of the components of each composition in the 500 μl cell culture medium were the final concentrations shown in Table 1. Each composition was repeated three times. Two days after application of the composition of the present invention, cells were lysed and 300 μl of a lysis reagent (a buffer containing 0.2% Triton X-100, 1 mM dithiothreitol, and 100 mM potassium phosphate (pH 7.8)) was added to each well. After shaking for ten minutes, the cell lysate was collected and centrifuged at 13,500 rpm for 10 minutes. The supernatant was collected and its fluorescence intensity was measured. Luciferase activity in the cell lysate was measured using a FLUOstar OPTIMA (available from BMG Labtech, Ortenberg, Germany). Fluorescence intensity was internally standardized using protein content, which was determined by the Bradford method. The kit used was available from Bio-Rad Laboratories, Hercules, California, USA. This measured value reflects the effect of the drug on HRE transcriptional activity. The control group was a cell response without any drug. The drug effect was evaluated using ((fluorescence intensity of the experimental group - fluorescence intensity of the control group) / fluorescence intensity of the control group) × 100%.

See Table 1 for specific results.

Table 1 Effects of the composition of the present invention on the transcriptional activity of HRE

In the above table, a concentration of 0 means that the corresponding compound is not contained in the composition.

As can be seen from Table 1, the composition of the present invention can effectively increase HRE transcriptional activity. Furthermore, the composition comprising calycosin and at least one of formononetin, formononetin and calycosin can effectively increase HRE transcriptional activity.

Among them, the composition consisting of calycosin and formononetin (molar ratio 1:1), and the composition consisting of calycosin and calycosin glycoside (molar ratio 1:1) can both increase the HRE transcription activity by about one-fold at most.

Furthermore, combinations of calycosin and two of formononetin, formononetin, and calycosin glycosides each increased HRE transcriptional activity by approximately twofold. In particular, a combination of 0.08 μM calycosin, 0.016 μM formononetin, and 0.4 μM formononetin (at a molar ratio of 5:1:25) increased HRE transcriptional activity by 215%, with a combined molar concentration of only approximately 0.6 μM.

Furthermore, a combination of calycosin, formononetin, formononetin, and calycosin glycosides can increase HRE transcriptional activity by approximately 1-3 fold. In particular, a combination of 0.4 μM calycosin, 0.08 μM formononetin, 0.08 μM formononetin, and 0.08 μM calycosin glycosides (at a molar ratio of 5:1:1:1) can increase HRE transcriptional activity by 333%, with a total molar concentration of only approximately 0.6 μM.

Comparative Example 1

Preparation of the mother liquor of the Astragalus membranaceus aqueous extract: Mix Astragalus membranaceus with water in a weight ratio of 1:8, decoct twice, each time for 2 hours, combine the two decoctions, and dry the resulting aqueous extract to obtain a powdered solid. Upon use, the powder was dissolved in the cell culture medium described in Experimental Example 1 at the corresponding concentration. The resulting change in luciferase activity was measured using the same method as in Example 1. The final concentrations were: 0.01, 0.1, 0.3, 0.5, 1, 1.5, and 10 mg/ml, respectively. See Figure 1 for specific results. As can be seen from Figure 1, in order to achieve a better effect of enhancing HRE transcriptional activity, the effective concentration of the Astragalus water extract components required is relatively high, basically 1-10 mg/mL (wherein, when the final concentration of the Astragalus water extract components is 10 mg/mL, it can be measured that it contains about 6.9 μM of calycosin, about 5.6 μM of formononetin, about 1.6 μM of formononetin, and about 5.13 μM of calycosin, totaling about 19 μM). Moreover, at this high concentration, the enhancement of HRE transcriptional activity is also limited, and the maximum HRE transcriptional activity can be enhanced by about 80%.

Comparative Example 2

DMSO stock solutions of calycosin, formononetin, formononetin, and calycosin were prepared. Upon use, the resulting stock solutions were dissolved in the cell culture medium described in Experimental Example 1 at the corresponding concentrations. The resulting changes in luciferase activity were measured using the same method as in Example 1. The final concentrations were 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, and 30 μM, respectively. See Figure 2 for specific results. As can be seen from Figure 2, when each component is used alone, the effective final concentration required to achieve a significant effect in enhancing HRE transcriptional activity is relatively high, typically between 1 and 30 μM. Furthermore, even at these high concentrations, the enhanced HRE transcriptional activity is limited, with a maximum increase of approximately 80%. At concentrations below 1 μM, the enhanced HRE transcriptional activity is generally low.

In summary, it can be seen from the results of the above examples and comparative examples that the composition of the present invention has a good synergistic effect on enhancing the transcriptional activity of HRE, thereby promoting erythropoiesis, and preventing and treating anemia and/or hypoxia, and significantly reduces the dosage of each active component.

Claims (6)

1. A composition for promoting erythropoiesis, comprising versicolor, styracin, styracin glycoside and versicolor glycoside, wherein the versicolor, styracin, styracin glycoside and versicolor glycoside are used in purified form, and the molar ratio of the versicolor, styracin, styracin glycoside and versicolor glycoside is 5:1:1:1. 2. A composition for promoting erythropoiesis, comprising vernix caseosa, argentin and argentin, wherein the vernix caseosa, argentin and argentin are used in purified form, and the molar ratio of the vernix caseosa, argentin and argentin is 5:1:25. 3. The composition according to claim 1 or 2, wherein the composition is a pharmaceutical composition and comprises pharmaceutical excipients. 4. The composition according to claim 3, wherein the dosage form of the composition is an oral liquid. 5. Use of the composition according to any one of claims 1-4 in the preparation of a medicament for promoting erythropoiesis. 6. The use according to claim 5, wherein the drug is used for the prevention and/or treatment of hypoxia and/or anemia.