A Chinese medicine composition for enhancing macrophage phagocytosis and preparation method thereof

Abstract

The invention discloses a Chinese medicine composition for enhancing the phagocytic ability of macrophages and a preparation method thereof, and belongs to the field of medical technology. The Chinese medicine composition of the invention comprises quince glycoside, aurantin glucoside, strychnosin, arctiin, tectoriusin and tectoriusin. The Chinese medicine composition prepared by the invention has excellent activity of enhancing the phagocytic function of macrophages.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to a traditional Chinese medicine composition for enhancing the phagocytic ability of macrophages and a preparation method thereof. Background Art

[0002] The first aspect of phagocytes is non-specific phagocytosis. Phagocytes can engulf pathogenic microorganisms that enter the human body. Including bacteria, viruses, parasites, protozoa, etc. At the same time, phagocytes can also engulf aging cells in the human body, as well as fragments of various cells. The second aspect of the role of phagocytes is specific immune action. After phagocytes engulf pathogenic microorganisms, they can transmit the information of pathogenic microorganisms to the human body's B lymphocytes; stimulate B lymphocyte proliferation and transformation into plasma cells. At the same time, plasma cells will produce special antibodies against pathogenic microorganisms and play a humoral immune role. Macrophages belong to phagocytes and play an important role in the body's immune function. Existing studies have shown that macrophages in biological organisms can change their phenotype and immune function with changes in the microenvironment, and constantly change their physiological state and immune activity according to the stimulation they receive, so as to better ensure the health of the body and tissues of the organism. Different stimuli, including the body's own protein molecules, small molecules, fungal and bacterial extracts, can stimulate and activate macrophages, thereby enhancing the immune response and phagocytic ability of macrophages. In most vertebrate cells, phagocytosis is a measure to protect the body in a healthy state rather than a means of ingestion. The strength of phagocytosis is an important indicator of the strength and activity of macrophage immune function. Through the phagocytosis of immune cells, we can directly study the related roles of macrophages in immunology and deeply reveal the dynamic process of cell phagocytosis.

[0003] Chinese patent application CN201510148058.5 discloses the use of dodecyl decenoic acid BDSF in improving the phagocytic ability of RAW264.7 macrophages. When the concentration of BDSF is 3-300 μmol / L, the ability of RAW264.7 macrophages to phagocytose C.albicans can be improved. The action time of BDSF in improving the phagocytosis of C.albicans by RAW264.7 macrophages is 2h, 3h, 4h, 5h, and 6h. BDSF has low biological toxicity and has a significant effect on improving the phagocytic ability of immune cells RAW264.7 macrophages.

[0004] Chinese patent application CN202011077884.2 discloses a soluble powder of Chinese medicine for improving immunity and anti-virus and its preparation process, wherein the soluble powder comprises the following raw materials in parts by weight: 5-8 parts of malt, 25-35 parts of astragalus, 2-6 parts of turmeric, 6-10 parts of kudzu root, 5-10 parts of scutellaria, 10-15 parts of trichosanthes, 1-5 parts of houttuynia, 30-40 parts of sodium bicarbonate, and 4-8 parts of dextrin. The present invention discloses a soluble powder of Chinese medicine for improving immunity and anti-virus and its preparation process, which does not contain antibiotics and hormones, can enhance the phagocytic ability of monocytes and macrophages, clear away heat and detoxify, enhance the body's immunity, and improve the anti-virus ability.

[0005] At present, there are few studies on traditional Chinese medicine for improving the phagocytic ability of macrophages. More relevant drugs need to be developed in a targeted manner to meet the needs of clearing heat and detoxifying and enhancing immunity. Summary of the invention

[0006] The purpose of the present invention is to provide a Chinese medicine composition for enhancing the phagocytic ability of macrophages and a preparation method thereof. The preparation method is simple and easy and can significantly improve the phagocytic ability of macrophages.

[0007] To achieve the above-mentioned purpose of the invention, the technical solution of the present invention is as follows:

[0008] In one aspect, the present invention provides a traditional Chinese medicine composition for enhancing the phagocytic ability of macrophages, comprising quince glycoside, aurantin glucoside, strychnosin, arctiin, tectoriusin and tectoriusin.

[0009] Preferably, the Chinese medicine composition comprises, by weight percentage, 0-20% of acanthoside, 0-20% of aurantifolia glucoside, 0-20% of strychnoside, 0-30% of arctiin, 0-20% of tectoriusin and 0-20% of tectoriusin.

[0010] Further preferably, the Chinese medicine composition comprises, by weight percentage, 3-12.26% of quinceanine, 4.62-10.37% of aurantifoliin glucoside, 7.24-16.08% of strychnosin, 5.84-34.01% of arctiin, 7.85-18.63% of tectoriusin and 5.05-13.21% of tectoriusin.

[0011] Most preferably, the Chinese medicine composition comprises, by weight percentage, 11.97% of acanthoside, 9.05% of aurantifolia glucoside, 15.37% of strychnoside, 6.01% of arctiin, 18.63% of tectoriusin and 10.46% of tectoriusin.

[0012] In another aspect, the present invention provides a method for preparing the above-mentioned Chinese medicine composition, comprising the following steps:

[0013] The burdock fruit, belamcanda chinensis, platycodon grandiflorum, white peony root, perilla leaf, honeysuckle, charred hawthorn, astragalus, knotweed, liquorice, chrysanthemum and black tea are prepared into a mixture.

[0014] Preferably, the ingredients by weight are 5-15 parts of burdock seeds, 5-15 parts of belamcanda, 5-15 parts of platycodon, 5-15 parts of white peony root, 5-20 parts of perilla leaves, 5-15 parts of honeysuckle, 5-10 parts of charred hawthorn, 5-15 parts of astragalus, 5-10 parts of knotweed, 5-15 parts of licorice, 3-12 parts of chrysanthemum, and 40-80 parts of black tea.

[0015] Further preferably, the ingredients in parts by weight are 10 parts of burdock seeds, 10 parts of belamcanda vine, 10 parts of platycodon grandiflorum, 10 parts of white peony root, 10-15 parts of perilla leaves, 10 parts of honeysuckle, 8 parts of charred hawthorn, 10 parts of astragalus, 10 parts of knotweed, 8-10 parts of licorice, 5-10 parts of chrysanthemum, and 60 parts of black tea.

[0016] Preferably, the mixture also includes Fritillaria thunbergii.

[0017] Further preferably, the ingredients by weight are 5-15 parts of burdock seeds, 5-15 parts of belamcanda, 5-15 parts of platycodon, 5-15 parts of white peony root, 5-20 parts of perilla leaves, 5-15 parts of honeysuckle, 5-10 parts of charred hawthorn, 5-15 parts of astragalus, 5-10 parts of knotweed, 5-15 parts of licorice, 10-20 parts of thunbergii, 3-12 parts of chrysanthemum, and 40-80 parts of black tea.

[0018] More preferably, the ingredients in parts by weight are 10 parts of burdock seeds, 10 parts of belamcanda vine, 10 parts of platycodon grandiflorum, 10 parts of white peony root, 10-15 parts of perilla leaves, 10 parts of honeysuckle, 8 parts of charred hawthorn, 10 parts of astragalus, 10 parts of knotweed, 8-10 parts of licorice, 15 parts of thunbergii, 5-10 parts of chrysanthemum, and 60 parts of black tea.

[0019] Preferably, the mixture is prepared by:

[0020] Water was added to the formula raw materials for reflux extraction, and after cooling, the weight loss was supplemented, centrifuged, and the supernatant was collected.

[0021] Further preferably, the amount of water added is 8-20 times, more preferably 10 times, of the formula raw materials.

[0022] Further preferably, the reflux extraction time is 30-180 min, further preferably 50-70 min, most preferably 60 min.

[0023] Further preferably, the centrifugation condition is: 2000-6000 rpm for 5-20 minutes. More preferably, the centrifugation condition is: 4000 rpm for 10 minutes.

[0024] As a specific embodiment of the present invention, the preparation method of the mixture is: accurately weigh 1 part of each single herbal powder into a round-bottom flask, add 10 times the amount of ultrapure water (weigh and record); reflux extraction for 1 hour, weigh and record after cooling to make up for the weight loss. Centrifuge at 4000rpm for 10 minutes, take the supernatant for use, and prepare a 100mg / ml mother solution.

[0025] In another aspect, the present invention provides another method for preparing the above-mentioned Chinese medicine composition, comprising the following steps:

[0026] (1) preparing a mixture of burdock fruit, belamcanda chinensis, platycodon grandiflorum, white peony root, perilla leaf, honeysuckle, charred hawthorn, astragalus, knotweed, liquorice, chrysanthemum, and black tea;

[0027] (2) applying the mixture obtained in step (1) to a column, using water and acetonitrile as the mobile phase, and gradient elution, wherein the elution conditions are: 0 min: 4-6% acetonitrile, 15 min: 13-17% acetonitrile, 30 min: 18-22% acetonitrile, 45 min: 28-40% acetonitrile, 55 min: 90-100% acetonitrile, 70 min: 95-100% acetonitrile;

[0028] (3) taking the 30-45min and 45-60min elution fractions, concentrating them under reduced pressure, and removing acetonitrile to obtain the traditional Chinese medicine composition.

[0029] Preferably, in step (1), the ingredients are 5-15 parts by weight of burdock seeds, 5-15 parts by beam dryness, 5-15 parts by platycodon, 5-15 parts by white peony root, 5-20 parts by perilla leaf, 5-15 parts by honeysuckle, 5-10 parts by charred hawthorn, 5-15 parts by astragalus, 5-10 parts by knotweed, 5-15 parts by liquorice, 3-12 parts by chrysanthemum, and 40-80 parts by weight of black tea.

[0030] Further preferably, in step (1), the ingredients in parts by weight are 10 parts of burdock seeds, 10 parts of belamcanda vine, 10 parts of platycodon grandiflorum, 10 parts of white peony root, 10-15 parts of perilla leaves, 10 parts of honeysuckle, 8 parts of charred hawthorn, 10 parts of astragalus, 10 parts of knotweed, 8-10 parts of licorice, 5-10 parts of chrysanthemum, and 60 parts of black tea.

[0031] Preferably, in step (1), the mixture also includes Fritillaria thunbergii.

[0032] Further preferably, in step (1), the weight portions include 5-15 parts of burdock seeds, 5-15 parts of belamcanda, 5-15 parts of platycodon, 5-15 parts of white peony root, 5-20 parts of perilla leaves, 5-15 parts of honeysuckle, 5-10 parts of charred hawthorn, 5-15 parts of astragalus, 5-10 parts of knotweed, 5-15 parts of liquorice, 10-20 parts of thunbergii, 3-12 parts of chrysanthemum, and 40-80 parts of black tea.

[0033] More preferably, in step (1), the ingredients in parts by weight are 10 parts of burdock seeds, 10 parts of belamcanda vine, 10 parts of platycodon grandiflorum, 10 parts of white peony root, 10-15 parts of perilla leaves, 10 parts of honeysuckle, 8 parts of charred hawthorn, 10 parts of astragalus, 10 parts of knotweed, 8-10 parts of licorice, 15 parts of thunbergii, 5-10 parts of chrysanthemum, and 60 parts of black tea.

[0034] Preferably, in step (1), the mixture is prepared by:

[0035] Water was added to the formula raw materials for reflux extraction, and after cooling, the weight loss was supplemented, centrifuged, and the supernatant was collected.

[0036] Further preferably, the amount of water added is 8-20 times, more preferably 10 times, of the formula raw materials.

[0037] Further preferably, the reflux extraction time is 30-180 min, further preferably 50-70 min, most preferably 60 min.

[0038] Further preferably, the centrifugation condition is: 2000-6000 rpm for 5-20 minutes. More preferably, the centrifugation condition is: 4000 rpm for 10 minutes.

[0039] As a specific embodiment of the present invention, the preparation method of the mixture is: accurately weigh 1 part of each single herbal powder into a round-bottom flask, add 10 times the amount of ultrapure water (weigh and record); reflux extraction for 1 hour, weigh and record after cooling to make up for the weight loss. Centrifuge at 4000rpm for 10 minutes, take the supernatant for use, and prepare a 100mg / ml mother solution.

[0040] Preferably, before sampling and analysis, an appropriate amount of the mother solution is diluted to 25 mg / ml, centrifuged at 14,000 rpm in a high-speed centrifuge for 10 min, and the supernatant is taken for testing.

[0041] Preferably, in step (2), the column used is selected from Agilent InfinityLab Poroshell 120HPH-C18 (21.2×150 mm, 5 μm), Agilent ZORBAX SB-C18 (21×250 mm, 7 μm), Polaris Any of the C18-A (50.0 x 250 mm, 10 μm) columns, more preferably an Agilent ZORBAX SB-C18 (21 x 250 mm, 7 μm) column.

[0042] Preferably, in step (2), the elution conditions are: 0 min-5% acetonitrile, 15 min-15% acetonitrile, 30 min-20% acetonitrile, 45 min-30% acetonitrile, 55 min-100% acetonitrile, 70 min-100% acetonitrile.

[0043] Preferably, in step (3), the 30-45 min fraction is collected.

[0044] On the other hand, the present invention provides a traditional Chinese medicine composition for enhancing the phagocytic ability of macrophages, comprising the following ingredients: burdock fruit, belamcanda chinensis, platycodon grandiflorum, white peony root, perilla leaf, honeysuckle, charred hawthorn, astragalus, knotweed, licorice, thunbergia thunbergii, chrysanthemum, and black tea.

[0045] Preferably, the composition comprises, by weight, 5-15 parts of burdock seeds, 5-15 parts of belamcanda vine, 5-15 parts of platycodon grandiflorum, 5-15 parts of white peony root, 5-20 parts of perilla leaves, 5-15 parts of honeysuckle, 5-10 parts of charred hawthorn, 5-15 parts of astragalus, 5-10 parts of knotweed, 5-15 parts of liquorice, 10-20 parts of thunbergii fritillary, 3-12 parts of chrysanthemum, and 40-80 parts of black tea.

[0046] Further preferably, the composition comprises, by weight, 10 parts of burdock seeds, 10 parts of belamcanda vine, 10 parts of platycodon grandiflorum, 10 parts of white peony root, 10-15 parts of perilla leaves, 10 parts of honeysuckle, 8 parts of charred hawthorn, 10 parts of astragalus, 10 parts of knotweed, 8-10 parts of licorice, 15 parts of thunbergii, 5-10 parts of chrysanthemum, and 60 parts of black tea.

[0047] Finally, the present invention provides a medicine, the effective ingredient of which is any one of the above-mentioned Chinese medicine compositions.

[0048] The drug can be prepared into dosage forms such as pills, capsules, granules, oral liquids, powders, tablets, lozenges, and lozenges, and suitable drug carriers in the art can be selected for different dosage forms.

[0049] The pharmaceutical carrier used can be solid, liquid or gas. Examples of solid carriers include lactose, kaolin, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate and stearic acid. Examples of liquid carriers include syrup, peanut oil, olive oil and water. Examples of gaseous carriers include carbon dioxide and nitrogen.

[0050] When preparing the composition for oral dosage form, any convenient pharmaceutical medium can be used. For example, water, ethanol, oil, alcohol, flavoring agent, preservative, coloring agent, etc. can be used to form oral liquid preparations, such as suspensions, elixirs and solutions; while carriers, such as starch, sugars, microcrystalline cellulose, diluents, granulating agents, emulsifiers, lubricants, binders, disintegrants can be used to form oral solid preparations, such as powders, capsules and tablets. Tablets and capsules are preferred oral dosage units using solid pharmaceutical carriers due to their ease of administration. Tablets can be coated using standard aqueous or non-aqueous techniques.

[0051] Tablets containing the Chinese medicine composition of the present invention can be prepared by tableting or molding, and one or more auxiliary ingredients or adjuvants can be used. The active ingredient can be tableted in a free-flowing form (such as powder or granules) in a suitable machine, and can be mixed with an adhesive, a lubricant, an inert diluent, a surfactant or a dispersant to prepare a tablet. Molded tablets can be molded in a suitable machine, i.e., a powdered compound mixture moistened with an inert liquid diluent. Each tablet preferably contains about 0.05 mg to about 5 g of active ingredient, and each sachet or capsule preferably contains about 0.05 mg to about 5 g of active ingredient. For example, a preparation intended for oral administration to humans may contain about 0.5 mg to about 5 g of active drug, mixed with an appropriate and convenient carrier material, which may account for about 5% to 95% of the total composition. The unit dosage form usually contains about 1 mg to about 2 g of active ingredient, usually 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg or 1000 mg.

[0052] The pharmaceutical composition suitable for parenteral administration of the present invention can be prepared as an aqueous solution or suspension of the active compound. Suitable surfactants such as hydroxypropylcellulose can be included. Dispersions can also be prepared in glycerol, liquid polyethylene glycol and oil mixtures thereof. In addition, preservatives can be added to prevent the harmful growth of microorganisms.

[0053] The drugs suitable for injection use in the present invention include sterile aqueous solutions or dispersions. In addition, the drug can be in the form of sterile powders for the extemporaneous preparation of such sterile injections or dispersions. In all cases, the final injection form must be sterile and must be an effective liquid so that the injectable drug component must remain stable under production and storage conditions; therefore, it is best to preserve it to prevent the contamination of microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium, for example, containing water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils and suitable mixtures thereof.

[0054] Medicine of the present invention can be in the form suitable for topical use, for example aerosol, cream, ointment, lotion, powder or the like. In addition, composition can be in the form suitable for transdermal administration device. Can use Chinese medicine composition of the present invention, prepare these prescriptions by conventional processing method. For example, by mixing hydrophilic material and water, and about 5wt% to about 10wt% of compound, prepare cream or ointment with required consistency.

[0055] The medicine of the present invention can be in a form suitable for rectal administration, wherein the carrier is a solid. Preferably, the mixture is made into a unit dose suppository. Suitable carriers include cocoa butter and other materials commonly used in the art. Suppositories can be prepared by first forming a composition containing a softened or melted carrier, followed by cooling and shaping in a mold.

[0056] In addition to the above-mentioned carrier components, the above-mentioned pharmaceutical preparations may include (if applicable) one or more additional carrier components, such as diluents, buffers, flavoring agents, adhesives, surfactants, thickeners, lubricants, preservatives (including antioxidants), etc. In addition, other excipients, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc., colorants and flavoring agents, etc., may be added. The preparation is made isotonic with the blood of the intended recipient. The components containing the Chinese medicine composition of the present invention can also be prepared in the form of powder or concentrate.

[0057] In the present invention, the structural formula of Ammijin is as follows:

[0058]

[0059] The structural formula of Aurantio-obtusin beta-D-glucoside is as follows:

[0060]

[0061] The structural formula of Secologanin is as follows:

[0062]

[0063] The structural formula of Arctiin is as follows:

[0064]

[0065] The structural formula of Irisflorentin is as follows:

[0066]

[0067] The structural formula of Iridin is as follows:

[0068]

[0069] The beneficial effects of the present invention are:

[0070] (1) The Chinese medicine composition prepared by the present invention has excellent activity in enhancing the phagocytic function of macrophages. Mouse experiments show that the Chinese medicine composition prepared by the present invention can improve the initial immune function of immunodeficient mice to a certain extent, specifically manifested in the significant enhancement of the phagocytic function of peritoneal macrophages. At the same time, co-culturing the Chinese medicine composition with the macrophage RAW264.7 cell line significantly enhanced the phagocytic function of RAW cells.

[0071] (2) The Chinese medicine composition of the present invention can be prepared by a simple extraction method and by selecting a specific elution segment, and the raw materials are widely available. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 The results of the neutral red phagocytosis assay in mouse peritoneal macrophages;

[0073] Figure 2 The results of fluorescent microsphere phagocytosis assay of mouse peritoneal macrophages;

[0074] Figure 3 The results of fluorescent microsphere phagocytosis assay of RAW cells. DETAILED DESCRIPTION

[0075] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is merely an exemplary description of the scope of protection claimed in this application, and those skilled in the art can make various changes and modifications to the invention of this application based on the disclosed content, and they should also fall within the scope of protection claimed in this application.

[0076] The present invention is further described below by way of specific examples. The various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels unless otherwise specified. The contents described below are all by mass.

[0077] In the following embodiments, burdock seeds are from Taihe, Anhui (190915), belamcanda chinensis is from Anguo, Hebei (2005011), platycodon grandiflorum is from Haozhou, Anhui (190601), white peony root is from Chifeng, Inner Mongolia (2004082), perilla leaf is from Taihe, Anhui (2001032), honeysuckle is from Anguo, Hebei (20200701), charred hawthorn is from Linyi, Shandong (200301), astragalus is from Anguo, Hebei (2004065), knotweed is from Taihe, Anhui (2003149), liquorice is from Dingxi, Gansu (200601), fritillaria thunbergii is from Jinhua, Zhejiang (20200415), chrysanthemum is from Haozhou, Anhui (191101), and black tea is from Haozhou, Anhui.

[0078] Example 1 Preparation of Chinese medicine composition

[0079] (1) Prepare a mixture of 10 parts of burdock seeds, 10 parts of belamcanda, 10 parts of platycodon, 10 parts of white peony root, 15 parts of perilla leaf, 10 parts of honeysuckle, 8 parts of charred hawthorn, 10 parts of astragalus, 10 parts of knotweed, 8 parts of liquorice, 15 parts of thunbergii, 5 parts of chrysanthemum, and 60 parts of black tea according to the following method: weigh and mix the medicinal materials, crush at 4800r / min for 60 minutes, sieve into the coarsest powder, but mix with no more than 40% of the coarse powder that can pass through the No. 4 sieve. After sieving, mix in a trough mixer for 30 minutes for total mixing, and sterilize with 60Co irradiation. Weigh the medicinal powder and add ten times the amount of ultrapure water (weigh and record); reflux extraction for 1 hour, and weigh to make up for the weight loss after cooling. Centrifuge at 4000rpm for 10 minutes, and take the supernatant to obtain the mother solution for testing;

[0080] (2) The mixture obtained in step (1) was loaded onto an Agilent ZORBAX SB-C18 (21×250 mm, 7 μm) column with a mobile phase of water and acetonitrile and a gradient elution. The elution conditions were: 0 min-5% acetonitrile, 15 min-15% acetonitrile, 30 min-20% acetonitrile, 45 min-30% acetonitrile, 55 min-100% acetonitrile, 70 min-100% acetonitrile;

[0081] (3) taking the 30-45 min elution fraction, concentrating it under reduced pressure to near dryness, and evaporating the acetonitrile to obtain the traditional Chinese medicine composition.

[0082] Example 2 Preparation of Chinese medicine composition

[0083] The difference from Example 1 is that in step (1), the mixture prescription is: 10 parts of burdock seeds, 10 parts of belamcanda, 10 parts of platycodon, 10 parts of white peony root, 10 parts of perilla leaf, 10 parts of honeysuckle, 8 parts of charred hawthorn, 10 parts of astragalus, 10 parts of knotweed, 10 parts of liquorice, 15 parts of thunbergii, 10 parts of chrysanthemum, and 60 parts of black tea. The rest are the same.

[0084] Example 3 Preparation of Chinese medicine composition

[0085] The difference from Example 1 is that in step (3), the 45-60 min elution fraction is taken, concentrated under reduced pressure to near dryness, and the acetonitrile is evaporated to obtain the traditional Chinese medicine composition.

[0086] Example 4 Preparation of Chinese medicine composition

[0087] The mixture was prepared according to step (1) of Example 1.

[0088] Example 5 Preparation of Chinese medicine composition

[0089] The mixture was prepared according to step (1) of Example 2.

[0090] Comparative Example 1 Preparation of Chinese medicine composition

[0091] The difference from Example 1 is that in step (3), the 0-15 min elution fraction is taken, concentrated to near dryness under reduced pressure, and the acetonitrile is evaporated to obtain the Chinese medicine composition. The rest is the same.

[0092] Comparative Example 2 Preparation of Chinese medicine composition

[0093] The difference from Example 1 is that in step (3), the elution fraction of 15-30 min is taken, concentrated to near dryness under reduced pressure, and the acetonitrile is evaporated to obtain the Chinese medicine composition. The rest is the same.

[0094] Comparative Example 3 Preparation of Chinese medicine composition

[0095] The difference from Example 1 is that in step (1), Fritillaria thunbergii is not added to the mixture. The rest are the same.

[0096] Test Example 1: Component Determination

[0097] The chemical composition of the Chinese medicine composition prepared in Example 1 was analyzed by mass spectrometry:

[0098] The results are as follows:

[0099]

[0100] Test Example 2: Drug Efficacy Determination

[0101] 1. After 54 male Balb / c mice were adaptively fed, 100mg / kg cyclophosphamide (CTX) solution was intraperitoneally injected, once a day, for 3 consecutive days, with a dosage of 10mL / kg, causing systemic immunodeficiency in mice, and then supplemented with cyclophosphamide once every 3 days. After modeling, all mice were randomly divided into 2 groups, 6 in each group, including a control group, an example 1 administration group, an example 2 administration group, an example 3 administration group, an example 4 administration group, an example 5 administration group, a comparative example 1 administration group, a comparative example 2 administration group, and a comparative example 3 administration group. The mice in each administration group were gavaged with 600mg / kg for pre-administration for one week, and the administration was given once a day at a dosage of 10mL / kg. At the same time, the mice in the control group were gavaged with normal saline at a dosage of 10mL / kg. After the last administration, the primary peritoneal macrophages of the experimental mice were separated 6 hours later, collected and cultured, and starved until all the cells were attached to the wall. A portion of the cells were lysed after two hours of culture with a neutral red solution, and the phagocytic ability of macrophages to neutral red dye was observed by detecting the absorbance. Another part of the starved macrophages were added with L3030 fluorescent microspheres, and the phagocytic effect of macrophages on the fluorescent microspheres was observed by high-content imaging.

[0102] Results: Figure 1 As shown, compared with the control group, the OD value of the cells in the drug administration groups of Examples 1-5 and Comparative Example 3 increased significantly after phagocytizing the neutral red dye. Figure 2 As shown, in the fluorescent microsphere phagocytosis experiment, the average fluorescence intensity of peritoneal macrophages in mice administered with Examples 1-5 and Comparative Example 3 was significantly enhanced, indicating that the number of phagocytosed fluorescent microspheres increased significantly, and Example 1 had the strongest activity, indicating that the embodiments of the present invention can effectively enhance the phagocytic ability of macrophages in immunodeficient mice.

[0103] 2. Digest and collect RAW264.7 cells in the logarithmic growth phase, and adjust the cell number to 1×10 after cell counting. 5 / mL, seeded into a black 96-well plate at 100μL / well, set up a control group in the drug group, and set up 3 replicates in each group. The cells in the control group were not treated with any drugs, and the drug group was added with the drug to be screened and incubated for 48h. The old culture medium in the plate was discarded and washed with PBS three times. Serum-free DMEM high-glucose medium was added to starve the cells for 12h, the culture medium was removed and 100μL of fluorescent microsphere working solution was added, and the cells were cultured for 2h at 37℃, 5% CO2 and away from light. The remaining fluorescent microsphere working solution was removed and washed with PBS, and then 4% paraformaldehyde solution was added to fix the cells for 40min. The cells were washed with PBS three times, and 100μL of hoechst dye solution (1:1000) was added to each well. After incubation at room temperature for 15min, PBS was washed three times, and then photographed and recorded under a fluorescence microscope. Three fields of view were observed on each slide, and the number of macrophages and the number of fluorescent microspheres in each field of view were counted. The average number of phagocytic cells in each field of view was used to represent the phagocytic ability. The samples were stored in a 4℃ environment away from light.

[0104] Results: Figure 3 As shown, compared with the control group, the number of fluorescent microspheres phagocytosed by the RAW cells treated in Examples 1-5 and Comparative Example 3 increased significantly, proving that the RAW cells induced by the embodiments of the present invention have stronger phagocytic ability.

[0105] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A Chinese medicine composition for enhancing the phagocytic ability of macrophages, characterized in that: The Chinese medicine composition comprises, by weight percentage, 3-12.26% of amaranthin, 4.62-10.37% of aurantium glucoside, 7.24-16.08% of strychnine, 5.84-34.01% of arctiin, 7.85-18.63% of tectoriusin and 5.05-13.21% of tectoriusin; The preparation method of the Chinese medicine composition comprises: Prepare a mixture from 5-15 parts of burdock seeds, 5-15 parts of belamcanda, 5-15 parts of platycodon, 5-15 parts of white peony root, 5-20 parts of perilla leaf, 5-15 parts of honeysuckle, 5-10 parts of charred hawthorn, 5-15 parts of astragalus, 5-10 parts of knotweed, 5-15 parts of liquorice, 10-20 parts of thunbergii, 3-12 parts of chrysanthemum, and 40-80 parts of black tea; The preparation method of the mixture is as follows: adding water to the formula raw materials for reflux extraction, cooling to make up for the weight loss, centrifuging, and taking the supernatant; The added weight of the water is 10 times the weight of the formula raw materials.

2. The Chinese medicine composition according to claim 1, characterized in that: The Chinese medicine composition comprises, by weight percentage, 11.97% of adiantum glycoside, 9.05% of aurantium glucoside, 15.37% of strychnosin, 6.01% of arctiin, 18.63% of tectoriusin and 10.46% of tectoriusin.

3. The method for preparing the Chinese medicine composition according to any one of claims 1 to 2, characterized in that: The following steps are involved: According to weight parts, prepare a mixture with 5-15 parts of burdock seeds, 5-15 parts of belamcanda, 5-15 parts of platycodon, 5-15 parts of white peony root, 5-20 parts of perilla leaves, 5-15 parts of honeysuckle, 5-10 parts of charred hawthorn, 5-15 parts of astragalus, 5-10 parts of knotweed, 5-15 parts of liquorice, 10-20 parts of thunbergii, 3-12 parts of chrysanthemum, and 40-80 parts of black tea; The preparation method of the mixture is as follows: adding water to the formula raw materials for reflux extraction, cooling to make up for the weight loss, centrifuging, and taking the supernatant; The added weight of the water is 10 times the weight of the formula raw materials.

4. The preparation method according to claim 3, characterized in that: The preparation method further comprises: applying the obtained mixture to a column, using water and acetonitrile as the mobile phase, and gradient eluting, wherein the elution conditions are: 0 min-5% acetonitrile, 15 min-15% acetonitrile, 30 min-20% acetonitrile, 45 min-30% acetonitrile, 55 min-100% acetonitrile, and 70 min-100% acetonitrile; Taking the 30-45min elution fraction, concentrating under reduced pressure, and removing acetonitrile to obtain the traditional Chinese medicine composition; The upper column is an Agilent ZORBAX SB-C18 with a diameter of 21×250 mm and a diameter of 7 μm.

5. The preparation method according to claim 3, characterized in that: In parts by weight, the ingredients include 10 parts of burdock seeds, 10 parts of belamcanda, 10 parts of platycodon, 10 parts of white peony root, 10-15 parts of perilla leaves, 10 parts of honeysuckle, 8 parts of charred hawthorn, 10 parts of astragalus, 10 parts of knotweed, 8-10 parts of licorice, 15 parts of thunbergii, 5-10 parts of chrysanthemum, and 60 parts of black tea.

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