Application of composition in preparing iron supplement preparation

Abstract

The present invention discloses the use of a composition for preparing an iron-supplementing preparation. The composition comprises cornus officinalis and / or a cornus officinalis extract, and mangosteen and / or a mangosteen extract. By combining the active ingredients, the present invention provides a composition that can promote the body's absorption of iron by promoting intestinal iron absorption and / or inhibiting hepcidin, thereby providing an iron-supplementing effect. The composition can further be used to treat functional iron deficiency and other indications, providing a more effective preparation for improving iron deficiency. Furthermore, the preparation prepared using the composition also has the effects of regulating physical constitution, replenishing deficiency and invigorating qi, and regulating the body's health.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine preparations, and in particular to application of a composition in preparing an iron supplement preparation. Background Art

[0002] From single-celled bacteria to multicellular organisms like humans, iron is one of the most essential metals for sustaining life. Iron plays a crucial role in several cellular processes, including DNA synthesis, nucleic acid repair, mitochondrial cell respiration, cell growth, and cell death. It also contributes to host defense and cell signaling. Most of the iron in the human body is associated with red blood cell hemoglobin. Iron deficiency can lead to anemia, which in turn can cause a range of health problems, including insufficient Qi and blood, and symptoms such as cold hands and feet.

[0003] Iron absorbed by enterocytes can be directly used in the cell's internal metabolic machinery, stored in ferritin, or released through the basal outer membrane for systemic iron delivery. Most of the iron absorbed in the body is obtained from food. 3+ Must first be reduced to Fe 2+ Iron absorbed by the intestine is then transported to the liver for storage. Hepcidin, released by the liver, is a key regulator of iron homeostasis. It binds to FPN, the only known cellular iron exporter, mediating its ubiquitination, internalization, and degradation. It also directly blocks this channel, thereby preventing the export of iron from cells to the plasma. When serum iron levels rise, hepcidin expression is upregulated, resulting in the obstruction of iron transport to the plasma via FPN, thus providing a negative feedback reaction to prevent potential toxic iron accumulation in the body. Decreased plasma iron levels lead to decreased transferrin saturation, resulting in reduced hepcidin synthesis. As a result, hepcidin expression is enhanced, downregulating the expression of ferroportin and reducing the body's absorption of iron. When the body is iron deficient, the above processes undergo the opposite changes, thereby maintaining iron homeostasis.

[0004] Therefore, in order to improve the series of adverse effects caused by iron deficiency in the body, it becomes more important to explore safe and effective iron-supplementing bioactive substances. Summary of the Invention

[0005] Based on this, it is necessary to provide a composition with excellent iron supplementation effect for use in the preparation of iron supplementation preparations.

[0006] In a first aspect, the present invention provides an application of a composition in the preparation of an iron supplement preparation, wherein the composition comprises Cornus officinalis and Mangosteen, wherein the Cornus officinalis comprises Cornus officinalis medicinal materials and / or Cornus officinalis extracts, and wherein the Mangosteen comprises Mangosteen medicinal materials and / or Mangosteen extracts.

[0007] In some embodiments, the composition satisfies at least one of the following (1) to (3):

[0008] (1) The mass ratio of Cornus officinalis to Mangosteen in the composition is (10-20): (1-5);

[0009] (2) The Cornus officinalis extract is selected from a water extract and / or an alcohol extract;

[0010] (3) The mangosteen extract is selected from a water extract and / or an alcohol extract.

[0011] In some embodiments, the composition further comprises 2'-methoxymatrine and / or pyralidinol A.

[0012] In some embodiments, the composition satisfies the following (1) and / or (2):

[0013] (1) The amount of 2'-methoxymatrine added to the composition is 0.1-1 μM;

[0014] (2) The amount of Panlongol A added to the composition is 0.05~0.5 μM.

[0015] In some embodiments, the composition comprises 10-20 μg / mL Cornus officinalis extract, 1-5 μg / mL Mangosteen extract, 0.1-1 μM 2'-methoxymatrine and 0.05-0.5 μM Penlongol A.

[0016] In some embodiments, the iron supplement preparation achieves iron supplementation by promoting intestinal iron absorption and / or inhibiting hepcidin.

[0017] In a second aspect, the present invention also provides a complex, the components of which include Cornus officinalis and Mangosteen, the Cornus officinalis includes Cornus officinalis medicinal materials and / or Cornus officinalis extracts, the mangosteen includes Mangosteen medicinal materials and / or Mangosteen extracts, and also contains 2'-methoxymatrine and / or panlongol A.

[0018] In some embodiments, the compound satisfies at least one of the following (1) to (3):

[0019] (1) The content ratio of Cornus officinalis, Mangosteen and 2'-methoxymatrine in the composite is (10-20) μg / mL: (1-5) μg / mL: (0.1-1) μM;

[0020] (2) The mass ratio of Cornus officinalis, Mangosteen and Panlongol A in the composite is (10-20) μg / mL: (1-5) μg / mL: (0.05-0.5) μM;

[0021] (3) The mass ratios of Cornus officinalis, Mangosteen, 2'-methoxymatrine and Panlongol A in the complex are (10-20) μg / mL: (1-5) μg / mL: (0.1-1) μM: (0.05-0.5) μM.

[0022] In some embodiments, the compound satisfies at least one of the following (1) to (5):

[0023] (1) The mass ratio of Cornus officinalis, Mangosteen and 2'-methoxymatrine in the composite is (15-18) μg / mL: (3-4) μg / mL: (0.1-0.5) μM;

[0024] (2) The mass ratio of Cornus officinalis, Mangosteen and Panlongol A in the composite is (15-18) μg / mL: (3-4) μg / mL: (0.05-0.2) μM;

[0025] (3) The mass ratio of Cornus officinalis, Mangosteen, 2'-methoxymatrine and Panlongol A in the composite is (15-18) μg / mL: (3-4) μg / mL: (0.1-0.5) μM: (0.05-0.2) μM;

[0026] (4) The Cornus officinalis extract is selected from a water extract and / or an alcohol extract;

[0027] (5) The mangosteen extract is selected from a water extract and / or an alcohol extract.

[0028] In some embodiments, the composition of the complex includes 10-20 μg / mL Cornus officinalis extract, 1-5 μg / mL Mangosteen extract, 0.1-1 μM 2'-methoxymatrine and 0.05-0.5 μM Penlongol A.

[0029] In a third aspect, the present invention further provides a drug for treating anemia, wherein the drug comprises the complex and pharmaceutically acceptable excipients.

[0030] In some embodiments, the drug satisfies at least one of the following (1) to (4):

[0031] (1) The drug is in the form of tablets, granules, capsules, solutions, powders, suspensions, emulsions, pastes or aerosols;

[0032] (2) The dosage form of the drug includes oral dosage, injection, patch, liniment, spray, inhalant, drops, lotion or suppository;

[0033] (3) The anemia is iron deficiency anemia, autoimmune hemolytic anemia or nausea anemia;

[0034] (4) The drug is used to treat anemia by promoting intestinal iron absorption and / or inhibiting hepcidin.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention makes full use of the characteristics of oral medicinal and edible substances, and based on the advantage of direct oral administration, combines the traditional Chinese medicine raw materials of Cornus officinalis and Mangosteen and / or the active ingredients extracted therefrom. The obtained composition has a better ability to promote iron absorption, significantly improves the body's absorption efficiency of iron elements, and plays an iron-replenishing role. The composition can promote intestinal iron absorption and / or inhibit hepcidin, and then the composition is used to prepare a drug for treating anemia indications, which can be used to treat anemia indications such as iron deficiency anemia, autoimmune hemolytic anemia or nausea anemia. At the same time, the drug prepared by using the composition also has significant effects of regulating physical constitution, replenishing deficiency and invigorating qi, and regulating the health status of the body, and has broader application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The effects of each single substance and combination formulas 1 to 6 in Experimental Example 1 on the survival rate of Caco-2 cells are shown.

[0038] Figure 2 The effects of each single substance and combination formulas 1 to 6 in Experimental Example 1 on the survival rate of HepG2 cells.

[0039] Figure 3 The data of each single substance and combination formulas 1 to 6 in Experimental Example 2 on the absorption of iron ions by Caco-2 cells are statistically shown.

[0040] Figure 4 The data of each single substance and combination formulas 1 to 6 in Experimental Example 3 on the mRNA expression of Hepcidin gene in HepG2 cells are statistically shown.

[0041] Figure 5 The data of each single substance and combination formulas 1 to 6 in Experimental Example 4 on the expression level of Hepcidin protein in HepG2 cells are statistically shown. DETAILED DESCRIPTION

[0042] To facilitate understanding of the present invention, the present invention will be described more fully below. Preferred embodiments of the present invention are provided, but the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, the purpose of providing these embodiments is to provide a more thorough and comprehensive understanding of the disclosure of the present invention.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention pertains. The terms used in this specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0044] It should be noted that the experimental methods in the following examples of the present invention, where specific conditions are not specified, are generally carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0045] Cornus officinalis (scientific name: Cornus officinalis Sieb. et Zucc.), also known as cornus meat, yam meat, and cornus meat, is the dried mature flesh of the cornus officinalis Sieb. et Zucc. plant, a member of the Cornaceae family. It has a sour and astringent flavor and a slightly warm nature, and enters the liver and kidney meridians. It has the effects of nourishing the liver and kidneys, as well as astringing and strengthening the body.

[0046] Mangosteen (Garcinia mangostana L.), also known as mangosteen, mangosteen, or mangosteen, has a neutral nature, with a sweet and slightly sour taste. Mangosteen peel has a cooling nature, a bitter and astringent taste, and is known to invigorate the spleen and promote fluid production, while also stopping diarrhea. Mangosteen peel is also known for its anti-inflammatory and analgesic properties, and is primarily used to treat spleen deficiency diarrhea, dry mouth, burns, scalds, eczema, and stomatitis.

[0047] In a first aspect, the present invention provides an application of a composition in the preparation of an iron supplement preparation, wherein the composition comprises Cornus officinalis and Mangosteen, wherein the Cornus officinalis comprises Cornus officinalis medicinal materials and / or Cornus officinalis extracts, and wherein the Mangosteen comprises Mangosteen medicinal materials and / or Mangosteen extracts.

[0048] The present invention combines the above-mentioned ingredients and finds that the multiple ingredients in each formula can synergistically promote iron absorption, thereby playing the role of iron supplementation for the body.

[0049] In some embodiments, the content ratio of Cornus officinalis to Mangosteen in the composition is (10-20): (1-5), including but not limited to 10:1, 10:2, 10:3, 10:4, 10:5, 12:1, 14:1, 16:1, 18:1, 18.2:1, 20:1, 12:2, 14:2, 16:2, 18:2, 20:2, 12:3, 14:3, 15:3, 16:3.2, 18:3, 20:3, 12:4, 14:4, 15:4, 16:4, 18:4, 20:4, 12:5, 14:5, 14.2:5, 16:5, 18:5, 20:5 or any range formed by any two of the foregoing and any ratio within the range.

[0050] In some embodiments, the Cornus officinalis medicinal material includes the pulp, peel, flower, bark and other parts of Cornus officinalis.

[0051] In some embodiments, the cornus officinalis extract includes substances extracted from the pulp, peel, flower, bark, etc. of cornus officinalis;

[0052] Optionally, the cornus officinalis extract includes substances such as alkaloids, polysaccharides, saponins, flavonoids and polyphenols.

[0053] In some embodiments, the Cornus officinalis extract is selected from a water extract and / or an alcohol extract.

[0054] In some embodiments, the mangosteen medicinal material includes mangosteen pulp, mangosteen peel, mangosteen flower, mangosteen bark root, stem and other parts.

[0055] In some embodiments, the mangosteen extract includes substances extracted from mangosteen pulp, mangosteen peel, mangosteen flower, mangosteen bark, root, stem and other parts;

[0056] Optionally, the mangosteen extract includes substances such as alkaloids, polysaccharides, saponins, flavonoids and polyphenols.

[0057] In some embodiments, the mangosteen extract is selected from a water extract and / or an alcohol extract.

[0058] In some embodiments, the composition further comprises 2'-methoxymatrine and / or pyralidinol A.

[0059] In some embodiments, the added amount of 2'-methoxymatrine in the composition is 0.1~1 μM, including but not limited to 0.1 μM, 0.15 μM, 0.2 μM, 0.25 μM, 0.3 μM, 0.35 μM, 0.4 μM, 0.45 μM, 0.5 μM, 0.55 μM, 0.6 μM, 0.65 μM, 0.7 μM, 0.75 μM, 0.8 μM, 0.85 μM, 0.9 μM, 0.95 μM, 1 μM or any range formed by any two of the foregoing and any value within the range.

[0060] In some embodiments, the added amount of pyrocyanine ginseng A in the composition is 0.05~0.5μM, including but not limited to 0.05μM, 0.08μM, 0.1μM, 0.12μM, 0.15μM, 0.18μM, 0.2μM, 0.22μM, 0.25μM, 0.28μM, 0.3μM, 0.32μM, 0.35μM, 0.38μM, 0.4μM, 0.42μM, 0.45μM, 0.48μM, 0.5μM or any range formed by any two of the foregoing and any value within the range.

[0061] In some embodiments, the amount of mangosteen added to the composition is 1-5 μg / mL, including but not limited to 1 μg / mL, 1.2 μg / mL, 1.5 μg / mL, 1.8 μg / mL, 2 μg / mL, 2.2 μg / mL, 2.5 μg / mL, 2.8 μg / mL, 3 μg / mL, 3.2 μg / mL, 3.5 μg / mL, 3.8 μg / mL, 4 μg / mL, 4.2 μg / mL, 4.5 μg / mL, 4.8 μg / mL, 5 μg / mL or any range formed by any two of the foregoing and any value within the range.

[0062] In some embodiments, the amount of Cornus officinalis added to the composition is 10-20 μg / mL, including but not limited to 10 μg / mL, 11 μg / mL, 12 μg / mL, 13 μg / mL, 14 μg / mL, 14.2 μg / mL, 15 μg / mL, 16 μg / mL, 17 μg / mL, 18 μg / mL, 18.2 μg / mL, 19 μg / mL, 20 μg / mL or a range formed by any two of the foregoing and any value within the range.

[0063] In some embodiments, the composition comprises 10-20 μg / mL Cornus officinalis extract, 1-5 μg / mL Mangosteen extract, and 0.1-1 μM 2'-methoxymatrine.

[0064] In some embodiments, the composition comprises 10-20 μg / mL Cornus officinalis extract, 1-5 μg / mL Mangosteen extract, and 0.05-0.5 μM Penicillin A.

[0065] In some embodiments, the composition comprises 10-20 μg / mL Cornus officinalis extract, 1-5 μg / mL Mangosteen extract, 0.1-1 μM 2'-methoxymatrine and 0.05-0.5 μM Penlongol A.

[0066] Within the composition and proportion range of the above-mentioned compositions, the positive effect of treating anemia can be achieved. Furthermore, the composition comprising 16 μg / mL Cornus officinalis extract, 3.2 μg / mL mangosteen extract, 0.3 μM 2'-methoxymatrine and 0.1 μM pyralidinol A can better achieve the therapeutic effect.

[0067] In some embodiments, the iron supplement preparation achieves iron supplementation by promoting intestinal iron absorption and / or inhibiting hepcidin.

[0068] In a second aspect, the present invention also provides a complex, the components of which include Cornus officinalis and Mangosteen, the Cornus officinalis includes Cornus officinalis medicinal materials and / or Cornus officinalis extracts, the mangosteen includes Mangosteen medicinal materials and / or Mangosteen extracts, and also contains 2'-methoxymatrine and / or panlongol A.

[0069] In some embodiments, the cornus officinalis can be the flower of cornus officinalis, the bark, root, stem and other parts of cornus officinalis and their extracts, and the cornus officinalis extract includes alkaloids, polysaccharides, saponins, flavonoids and polyphenols;

[0070] In some embodiments, the Cornus officinalis extract is selected from a water extract and / or an alcohol extract.

[0071] In some embodiments, the mangosteen and / or mangosteen extract can be mangosteen pulp, mangosteen peel, mangosteen flower, mangosteen bark, root, stem and other parts and extracts thereof, and the mangosteen extract includes alkaloids, polysaccharides, saponins, flavonoids and polyphenols;

[0072] In some embodiments, the mangosteen extract is selected from a water extract and / or an alcohol extract.

[0073] In some embodiments, the content ratio of Cornus officinalis, Mangosteen and 2'-methoxymatrine in the composite is (10-20) μg / mL: (1-5) μg / mL: (0.1-1) μM, including but not limited to 10:1:0.1, 10:2:0.3, 10:3:0.4, 10:4:0.5, 10:5:1, 12:1:0.1, 14:1:0.3, 16:1:0.4, 18:1:0.5, 18.2:1:0.1, 18.2:1:0.3, 20:1:0.1, 12:2:0.1, 14:2:0.3, and 16:2:0. 4, 18:2:0.5, 20:2:0.1, 12:3:0.1, 14:3:0.2, 15:3:0.3, 16:3.2:0.3, 18:3:0.4, 20:3:1, 12:4:0.1, 14:4:0.2, 15:4:0.3, 16:4:0.3, 18:4:0.5, 20:4:1, 12:5:0.1, 14:5:0.2, 14.2:5:0.1, 14.2:5:0.3, 16:5:0.3, 18:5:0.5, 20:5:1, or any range formed by any two of the foregoing, and any ratio within the range.

[0074] In some embodiments, the content ratio of Cornus officinalis, Mangosteen and Panlongol A in the composite is (10-20) μg / mL: (1-5) μg / mL: (0.05-0.5) μM, including but not limited to 10:1:0.05, 10:2:0.1, 10:3:0.2, 10:4:0.3, 10:5:0.5, 12:1:0.05, 14:1:0.1, 16:1:0.2, 18:1:0.3, 18.2:1:0.1, 18.2:1:0.3, 20:1:0.5, 12:2:0.05, 14:2:0.1, 16:2:0.2, : 18:2:0.3, 20:2:0.5, 12:3:0.05, 14:3:0.1, 15:3:0.2, 16:3.2:0.3, 18:3:0.3, 20:3:0.5, 12:4:0.1, 14:4:0.2, 15:4:0.3, 16:4:0.3, 18:4:0.3, 20:4:0.5, 12:5:0.05, 14:5:0.1, 14.2:5:0.1, 14.2:5:0.3, 16:5:0.2, 18:5:0.3, 20:5:0.5, or a range formed by any two of the foregoing, and any ratio within the range.

[0075] In some embodiments, the mass ratio of Cornus officinalis, Mangosteen, 2'-methoxymatrine and Panlongol A in the complex is (10-20) μg / mL: (1-5) μg / mL: (0.1-1) μM: (0.05-0.5) μM, including but not limited to 10:1:0.1:0.05, 10:2:0.3:0.1, 10:3:0.3:0.2, 10:4:0.5:0.3, 10:5:1:0.5, 12:1 :0.1:0.05, 14:1:0.3:0.1, 16:1:0.3:0.3, 18:1:0.5:0.3, 18.2:1:0.3:0.1, 18.2:1:0.1:0.3, 20:1:1:0.5, 12:2:0.1:0.05, 14:2:0.3:0.1, 16:2:0.3:0.3, 18:2:0.5:0.3 、20:2:1:0.5、12:3:0.1:0.05、14:3:0.3:0.1、15:3:0.3:0.3、16:3.2:0.5:0.3、18:3:1:0.5、20:3:0.1:0.5、12:4:0.2:0.05、14:4:0.3:0.1、15:4:0.3:0.2、16:4:0.3:0 .3, 18:4:0.5:0.4, 20:4:1:0.5, 12:5:0.1:0.05, 14:5:0.3:0.1, 14.2:5:0.3:0.1, 14.2:5:0.1:0.3, 16:5:0.3:0.3, 18:5:0.5:0.3, 20:5:1:0.5 or the range formed by any two of the foregoing and any ratio within the range.

[0076] In some embodiments, the mass ratio of Cornus officinalis, Mangosteen and 2'-methoxymatrine in the composite is (15-18) μg / mL: (3-4) μg / mL: (0.1-0.5) μM.

[0077] In some embodiments, the mass ratio of Cornus officinalis, Mangosteen and Panlongol A in the complex is (15-18) μg / mL: (3-4) μg / mL: (0.05-0.2) μM.

[0078] In some embodiments, the mass ratio of Cornus officinalis, Mangosteen, 2'-methoxymatrine and Panlongol A in the complex is (15-18) μg / mL: (3-4) μg / mL: (0.1-0.5) μM: (0.05-0.2) μM.

[0079] In some embodiments, the composition of the complex includes 10-20 μg / mL Cornus officinalis extract, 1-5 μg / mL mangosteen extract, 0.1-1 μM 2'-methoxymatrine, and 0.05-0.5 μM pyrocalyx quinoline A. The complex has good compatibility and is effective in treating anemia. Furthermore, the composition of the complex including 16 μg / mL Cornus officinalis extract, 3.2 μg / mL mangosteen extract, 0.3 μM 2'-methoxymatrine, and 0.1 μM pyrocalyx quinoline A can better achieve the above effects.

[0080] In a third aspect, the present invention further provides a drug for treating anemia, wherein the drug comprises the complex and pharmaceutically acceptable excipients.

[0081] In some embodiments, the pharmaceutically acceptable excipients include natural excipients, semi-synthetic and / or synthetic excipients, such as one or more of solvents, propellants, solubilizers, suspending agents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, glidants, compression aids, flavoring agents, preservatives, suspending agents, coating agents and fragrances.

[0082] In some embodiments, the drug is in the form of tablets, granules, capsules, solutions, powders, suspensions, emulsions, pastes or aerosols.

[0083] In some embodiments, the dosage form of the drug includes oral dosage, injection, patch, liniment, spray, inhalant, drops, lotion or suppository.

[0084] In some embodiments, the anemia is iron deficiency anemia, autoimmune hemolytic anemia or nausea anemia. Furthermore, the complex has the activity of reversing functional iron deficiency, thereby improving the body's iron deficiency abnormalities. Therefore, the complex has a better therapeutic effect on iron deficiency anemia.

[0085] In some embodiments, the drug is for treating anemia by promoting intestinal iron absorption and / or inhibiting hepcidin.

[0086] For experimental parameters not specified in the following specific examples, reference is made to the guidance given in this application document, and reference may also be made to experimental manuals in the art or other experimental methods known in the art, or to the experimental conditions recommended by the manufacturer.

[0087] The raw materials and reagents involved in the following specific examples can be obtained commercially, or can be prepared by those skilled in the art according to known methods.

[0088] Human colon adenocarcinoma Caco-2 cells were purchased from Xiamen Yimo Biotechnology Co., Ltd., China; human hepatoma HepG2 cells were purchased from Saiye Biotechnology Co., Ltd., China; DMEM medium and FBS fetal bovine serum were purchased from Gibco, USA; Cell Counting Kit (CCK-8) cell proliferation-toxicity detection kit was purchased from Tongren Chemical Research Institute; Taq Pro Universal SYBR qPCR Master Mix was purchased from Nanjing Novozymes Biotechnology Co., Ltd., China; RNA-easy Isolation Reagent was purchased from Nanjing Novozymes Biotechnology Co., Ltd., China; FastKing one-step genomic cDNA first-strand synthesis premix was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., China; the divalent iron ion detection probe - FerroOrange kit was purchased from Dojindo Chemical Research Institute, Japan; 2'-methoxymatrine (CAS: 270249-38-2) and synthetic pyroquinone A (CAS: 126192-35-6) were purchased from Tianjin WuXi AppTec; Cornus officinalis extract (water-soluble extract of Cornus officinalis fruit pulp, 10:1) and mangosteen extract (mangosteen peel extract 10:1) were purchased from Fufeng Snot Biotechnology Co., Ltd.

[0089] The composition formula provided in this embodiment is as follows:

[0090] Formula 1: 18.2μg / mL Cornus officinalis extract and 1μg / mL Mangosteen extract.

[0091] Formula 2: 16μg / mL Cornus officinalis extract and 3.2μg / mL Mangosteen extract.

[0092] Formula 3: 16 μg / mL Cornus officinalis extract, 3.2 μg / mL Mangosteen extract and 0.3 μM 2'-methoxymatrine.

[0093] Formula 4: 16μg / mL Cornus officinalis extract, 3.2μg / mL Mangosteen extract and 0.1μM Panax notoginseng A.

[0094] Formula 5: 16 μg / mL Cornus officinalis extract, 3.2 μg / mL Mangosteen extract, 0.3 μM 2'-methoxymatrine and 0.1 μM Panax notoginseng A.

[0095] Formula 6: 14.2 μg / mL Cornus officinalis extract, 5 μg / mL Mangosteen extract, 0.1 μM 2'-methoxymatrine and 0.3 μM Panax notoginseng A.

[0096] The compositions of the above six groups of formulas all have the effect of replenishing iron.

[0097] In this test example, the CCK-8 method was used to screen the concentration of the composition that had no significant inhibitory effect on the viability of Caco-2 cells and HepG2 cells. The specific method was as follows:

[0098] Caco-2 cells and HepG2 cells in the logarithmic growth phase were obtained, digested, and prepared into cell suspensions with complete culture medium. The cells were inoculated into 96-well plates, with 100 μL of cell suspension in each well and a cell number of 1.2 × 10 4 / well, cultured overnight in a constant temperature incubator at 37°C and 5% CO2. When the cell confluence was about 80%, the following test substances were added: the six groups of formula compositions provided in Example 1, 16 μg / mL Cornus officinalis extract, 3.2 μg / mL Mangosteen extract, 0.3 μM 2'-methoxymatrine, and 0.1 μM Panlongol A to treat the two types of cells.

[0099] A blank group containing culture medium and cells without the test substance was designated as the control group, and a control group containing culture medium and cells with the test substance was designated as the experimental group. Each group was then incubated in a 37°C, 5% CO2 incubator for 24 hours. The original culture medium was replaced with culture medium containing 10% CCK-8. The 96-well plate was then placed in the incubator for 4 hours. The OD value of each well was measured at a wavelength of 450 nm, and the cell proliferation and survival rate of the different test substances was calculated according to formula (I). Three replicates were used for each concentration.

[0100] (I)

[0101] The statistical results of the effects of each group on the survival rates of Caco-2 cells and HepG2 cells obtained by the above test method are shown in Table 1. Figure 1 and Figure 2 As shown:

[0102] Table 1: Effects of the test substances on the viability of Caco-2 cells and HepG2 cells ( ±s, n=3)

[0103]

[0104] From Table 1 and Figure 1 and Figure 2 The results showed that Cornus officinalis extract, mangosteen extract, 2'-methoxymatrine, panlongol A and the six combination formulas had no significant inhibitory effect on the survival rate of Caco-2 cells and HepG2 cells.

[0105] Experimental Example 2: Fe uptake by Caco-2 cells of the composition 2+ Impact

[0106] This test method uses FerroOrange fluorescent probe to detect the effect of the composition on promoting the absorption of iron ions Fe by Caco-2 cells. 2+ The specific test methods are as follows:

[0107] Caco-2 cells in the logarithmic growth phase were obtained, digested, and prepared into a cell suspension with complete medium. The cell suspension was inoculated into a 96-well plate, with 100 μL of cell suspension in each well and a cell number of 1.2 × 10 4 / well, cultured in a constant temperature incubator at 37°C and 5% CO2. When the cell confluence was about 80%, 16μg / mL Cornus officinalis extract, 3.2μg / mL Mangosteen extract, 0.3μM 2'-methoxy-matrine, 0.1μM Panlong phenol A and the six groups of composition formulas provided in Example 1 were added to treat Caco-2 cells respectively. After 12 hours of treatment, the culture medium was discarded and 100μL of complete culture medium containing 4μg / mL ferric citrate and 10μM ferrous sulfate was added to each well. After overnight culture, the culture medium was discarded, washed 3 times with Hank's salt solution, and 1μM FerroOrange working solution was added and placed in a constant temperature incubator at 7°C and 5% CO2 for incubation for 30 minutes. After the incubation, Hoechst 33342 dye was added and incubated for 5 minutes. The fluorescence value was detected on the machine, and the results are shown in Table 2 and Figure 3 shown.

[0108] Table 2: Effects of the test substances on Fe uptake by Caco-2 cells 2+ The impact of

[0109]

[0110] Note: Compared with a single substance, * represents p < 0.05, ** represents p < 0.01;

[0111] From Table 2 and Figure 3 The results showed that compared with a single substance, the six groups of compositions enhanced the ability of Caco-2 cells to take up iron ions, which was statistically significant compared with a single substance.

[0112] Experimental Example 3: Effect of the Composition on Hepcidin mRNA Expression in HepG2 Cells

[0113] In this experiment, HepG2 cells were treated with 16 μg / mL Cornus officinalis extract, 3.2 μg / mL Mangosteen extract, 0.3 μM 2'-methoxymatrine, 0.1 μM Panlongphenol A, and the six composition formulas provided in Example 1. RNA was extracted and reverse transcribed, and qPCR experiments were performed to evaluate the mRNA expression level of the Hepcidin gene in HepG2 cells. The specific method is as follows:

[0114] HepG2 cells in the logarithmic growth phase were digested and prepared into a cell suspension using DMEM complete medium containing 15% fetal bovine serum and 1% double antibody. 1×10 6 Cultured in 6cm cell culture dishes, 5mL per well, in a constant temperature incubator at 37°C and 5% CO2. After 72h of adaptive culture, the control group was replaced with fresh complete culture medium, and the intervention group was replaced with complete culture medium containing the corresponding monomers and compositions. After 12h of culture in a constant temperature incubator at 37°C and 5% CO2, the RNA of each group of cells was extracted according to the instructions of RNA-easy Isolation Reagent, and the RNA concentration was detected. According to the instructions of FastKing one-step genomic cDNA first-chain synthesis premix reagent, the extracted RNA of each group was reverse transcribed into cDNA at a concentration of 100ng / μL. Finally, the experimental system was prepared according to the instructions of TaqPro Universal SYBR qPCR Master Mix to detect the mRNA expression level of the Hepcidin gene. The results are shown in Table 3 and Figure 4 shown.

[0115] Table 3: Effects of the test substances on the mRNA expression of the Hepcidin gene

[0116]

[0117] Note: * indicates p < 0.05 compared with a single substance;

[0118] From Table 3 and Figure 4 The results showed that compared with a single substance, the six groups of compositions could significantly reduce the mRNA expression of Hepcidin, which was statistically significant compared with a single substance.

[0119] Experimental Example 4: Effect of the Composition on Hepcidin Protein Expression in HepG2 Cells

[0120] In this experiment, 16 μg / mL Cornus officinalis extract, 3.2 μg / mL Mangosteen extract, 0.3 μM 2'-methoxymatrine, 0.1 μM Panlongol A and the six compositions provided in Example 1 were used to treat HepG2 cells respectively. After protein extraction, Western blot experiments were performed to evaluate the expression level of Hepcidin protein in HepG2 cells. The specific method is as follows:

[0121] HepG2 cells in the logarithmic growth phase were obtained and digested, and then cell suspension was prepared using DMEM complete medium containing 10% fetal bovine serum and 1% double antibody. 1×10 6The cells were cultured in 6 cm cell culture dishes, 5 mL per well, in a constant temperature incubator at 37°C and 5% CO2. After 72 h of adaptive culture, the control group was replaced with fresh complete culture medium, and the intervention group was replaced with complete culture medium containing the above monomers and mixtures. After 12 h of culture in a constant temperature incubator at 37°C and 5% CO2, the cells were scraped off with a cell spatula, centrifuged at 1000 rpm for 5 min, lysed on ice to extract total cell protein, and the protein concentration was determined by BCA kit; 30 μg of protein was taken from each group for SDS-PAGE electrophoresis, transferred to PVDF membrane, blocked with 5% BSA, added with Hepcidin and GAPDH primary antibodies, and incubated overnight at 4°C in a shaker. The membrane was washed with TBS-T washing solution, and HRP-labeled secondary antibody was added and incubated at room temperature for 1 h. The membrane was washed with TBS-T washing solution and photographed with a chemiluminescence imaging system. The data results are shown in Table 4 and Figure 5 shown.

[0122] Table 4: Effects of the test substances on the protein expression of Hepcidin

[0123]

[0124] Note: * indicates p < 0.05 compared with a single substance;

[0125] From Table 4 and Figure 5 The results showed that compared with a single substance, the six groups of compositions could significantly reduce the protein expression of Hepcidin, which was statistically significant compared with a single substance.

[0126] GraphPad Prism 9.3.0 statistical software was used for data processing, and some experimental results were expressed as Mean ± SD.

[0127] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0128] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. The use of the composition in preparing an iron supplement preparation, characterized in that: The composition comprises 16 μg / mL Cornus officinalis extract, 3.2 μg / mL Mangosteen extract, 0.3 μM 2'-methoxymatrine and 0.1 μM Penlongol A.

2. The use according to claim 1, characterized in that The composition satisfies the following (1) (2) At least one of the following: (1) The Cornus officinalis extract is selected from a water extract and / or an alcohol extract; (2) The mangosteen extract is selected from a water extract and / or an alcohol extract.

3. The use according to claim 1 or 2, characterized in that The iron supplement preparation achieves the iron supplement effect by promoting intestinal iron absorption and / or inhibiting hepcidin.

4. A composite, characterized in that The components of the complex are cornus officinalis extract, mangosteen extract, 2'-methoxymatrine and panlongol A; The mass ratio of the cornus officinalis extract, the mangosteen extract, 2'-methoxymatrine and pyralidinol A in the complex is 16 μg / mL: 3.2 μg / mL: 0.3 μM: 0.1 μM.

5. The composite according to claim 4, characterized in that The compound satisfies at least one of the following (1) to (2): (1) The Cornus officinalis extract is selected from a water extract and / or an alcohol extract; (2) The mangosteen extract is selected from a water extract and / or an alcohol extract.

6. A drug for treating anemia, characterized in that: The composition of the drug comprises the complex according to any one of claims 4 to 5, and pharmaceutically acceptable excipients.

7. The drug for treating anemia according to claim 6, characterized in that The drug satisfies at least one of the following (1) to (4): (1) The drug is in the form of tablets, granules, capsules, solutions, powders, suspensions, emulsions, pastes or aerosols; (2) The dosage form of the drug includes oral dosage, injection, spray, inhalation, drops or suppository; (3) The anemia is iron deficiency anemia or autoimmune hemolytic anemia; (4) The drug is used to treat anemia by promoting intestinal iron absorption and / or inhibiting hepcidin.

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