Traditional Chinese medicine composition as well as preparation method and application thereof

Abstract

The invention relates to the field of traditional Chinese medicine, and particularly discloses a traditional Chinese medicine composition and a preparation method and application thereof.The traditional Chinese medicine composition is prepared from, by weight, 15-30 parts of herba ardisiae japonicae, 2-3 parts of hypericum perforatum, 3-10 parts of cortex magnoliae officinalis, 3-10 parts of platycodon grandiflorum, 3-10 parts of exocarpium citri rubrum, 9-15 parts of longan aril, 2-6 parts of kadsura longepedunculata, 1-3 parts of indigo naturalis, 6-12 parts of cortex mori radicis, 3-6 parts of fructus amomi, 9-30 parts of radix pseudostellariae, 9-15 parts of peach twig, 3-10 parts of herba elsholtziae and 6-12 parts of rhizoma anemarrhenae. The prepared traditional Chinese medicine composition can regulate and control related signal channels of the chronic obstructive pulmonary disease, inhibit expression of inflammation genes related to the chronic obstructive pulmonary disease, down-regulate cell aging channels, reduce expression of genes related to aging marker genes and improve cell viability of a bronchitis cell model.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine, and more specifically, to a traditional Chinese medicine composition and a preparation method and application thereof. Background Art

[0002] Chronic obstructive pulmonary disease (COPD), abbreviated as COPD, is a lung disease characterized by airflow limitation. The main causes include smoking, occupational dust exposure, indoor and outdoor air pollution, etc. These factors lead to airway inflammation, alveolar destruction, and small airway fibrosis, which ultimately lead to airflow limitation. Common symptoms are cough, sputum, and difficulty breathing.

[0003] Currently, drugs for the treatment of COPD include bronchodilators, glucocorticoids and antibiotics. However, long-term use of bronchodilators and glucocorticoids leads to increased drug tolerance and side effects. For example, glucocorticoids may cause increased blood sugar and osteoporosis. During the use of antibiotics, drug-resistant bacteria may appear, and choosing appropriate antibiotics becomes a challenge. Summary of the invention

[0004] In view of this, the present invention provides a Chinese medicine composition and a preparation method and application thereof. The Chinese medicine composition provided by the present invention is prepared into an extract according to a ratio, which can downregulate the expression of inflammatory genes related to lung aging and lung aging diseases such as chronic obstructive pulmonary disease. The present invention also provides a Chinese medicine formula that can reverse the disease signal pathway of COPD patients in lung fibroblasts and macrophages, and can regulate the abnormal signal pathway of IL-1β in human bronchial epithelial cells.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a traditional Chinese medicine composition, comprising the following components in proportion by weight: 15-30 parts of dwarf camellia, 2-3 parts of Hypericum perforatum, 3-10 parts of Magnolia officinalis, 3-10 parts of Platycodon grandiflorum, 3-10 parts of Citrus aurantium, 9-15 parts of Longan pulp, 2-6 parts of Schisandra chinensis, 1-3 parts of Indigo naturalis, 6-12 parts of Morus alba bark, 3-6 parts of Amomum villosum, 9-30 parts of Pseudostellaria pseudoginseng, 9-15 parts of Peach twig, 3-10 parts of Elsholtzia ciliata and 6-12 parts of Rhizoma Anemarrhenae.

[0007] Specifically, in some embodiments of the present invention, the above-mentioned Chinese medicine composition comprises the following components in proportion by weight: 15 parts of dwarf camellia, 3 parts of Hypericum perforatum, 9 parts of Magnolia officinalis, 9 parts of Platycodon grandiflorum, 10 parts of Citrus aurantium, 10 parts of Longan pulp, 6 parts of Schisandra chinensis, 3 parts of Indigo naturalis, 10 parts of Morus alba bark, 5 parts of Amomum villosum, 15 parts of Pseudostellaria pseudoginseng, 10 parts of Peach twig, 9 parts of Elsholtzia ciliata, and 9 parts of Anemarrhena asphodeloides; or

[0008] 30 parts of Camellia davidiana, 2 parts of Hypericum perforatum, 10 parts of Magnolia officinalis, 10 parts of Platycodon grandiflorum, 3 parts of Citrus aurantium, 9 parts of Longan pulp, 2 parts of Schisandra chinensis, 2 parts of Indigo naturalis, 6 parts of Morus alba bark, 3 parts of Amomum villosum, 9 parts of Pseudostellaria pseudoginseng, 15 parts of Peach twig, 10 parts of Elsholtzia ciliata, 6 parts of Anemarrhena asphodeloides; or

[0009] 20 parts of Camellia davidiana, 2.5 parts of Hypericum perforatum, 3 parts of Magnolia officinalis, 3 parts of Platycodon grandiflorum, 6 parts of Dangpi, 15 parts of Longan pulp, 4 parts of Schisandra chinensis, 1 part of Indigo naturalis, 12 parts of Morus alba bark, 6 parts of Amomum villosum, 30 parts of Pseudostellaria pseudoginseng, 9 parts of Peach twig, 3 parts of Elsholtzia ciliata, and 12 parts of Rhizoma Anemarrhenae.

[0010] The present invention also provides a method for preparing the above-mentioned Chinese medicine composition, comprising the following steps:

[0011] S1: Weigh the powder components in the formula, add purified water to the powder components for soaking, and use ultrasonic extraction or soaking extraction to obtain an extract;

[0012] S2: After the extract is concentrated under reduced pressure, purified water is added, and freeze-dried to prepare freeze-dried powder, and the freeze-dried powder is dissolved in distilled water to obtain an extract mother solution.

[0013] Specifically, in some embodiments of the present invention, in S1, the volume ratio of the powder component to purified water is 1:15-20, the purified water soaking time is 30 minutes, the ultrasonic extraction time is 30-60 minutes, and the soaking extraction time is 48 hours.

[0014] Specifically, in some embodiments of the present invention, in S2, the freeze-drying time is 24 to 48 hours, and the storage temperature of the extract mother solution is -15 to 20°C.

[0015] The present invention also provides the use of the above-mentioned Chinese medicine composition and / or the Chinese medicine composition obtained by the preparation method in the preparation of drugs and / or preparations for preventing and / or treating COPD.

[0016] The present invention also provides the use of the above-mentioned Chinese medicine composition and / or the Chinese medicine composition obtained by the preparation method in the preparation of drugs and / or preparations for regulating and / or inhibiting the expression of cytokines and / or genes related to the treatment of chronic obstructive pulmonary disease.

[0017] Specifically, in some embodiments of the present invention, the cytokines and / or genes include: one or more of IL-6, IL-8, IL17, NF-kB, TNF, and TGF-β.

[0018] The present invention also provides the use of the above-mentioned Chinese medicine composition and / or the Chinese medicine composition obtained by the preparation method in improving the vitality of senescent cells.

[0019] The present invention also provides the use of the above-mentioned Chinese medicine composition and / or the Chinese medicine composition obtained by the preparation method in reducing the expression of aging marker genes.

[0020] In summary, this application has the following beneficial effects:

[0021] 1. The present invention creatively combines 14 kinds of traditional Chinese medicines in a reasonable way, so that the components can be synergistically regulated. In bronchial epithelial cells, lung fibroblasts, and macrophage cell models, the COPD-related signaling pathways can be regulated, and the expression of COPD-related inflammatory genes can be inhibited;

[0022] 2. The Chinese medicine composition of the present invention focuses on overall conditioning and treatment based on syndrome differentiation. It can relieve clinical symptoms, improve lung function and immune function, and improve the quality of life through expectorant, bronchial dilation and immune regulation. It can improve respiratory symptoms, inhibit inflammation, and improve lung function or lung fibrosis. It can be used for the related treatment or auxiliary treatment of COPD. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The present invention shows the regulation of each single drug in the Chinese medicine composition on the abnormal signal pathway of COPD;

[0024] Figure 2 The regulation of abnormal signal pathways of COPD by Examples 1-3 and Comparative Examples 1-3 is shown;

[0025] Figure 3 It is shown that the Chinese medicine composition of the present application inhibits the key signaling pathway of COPD;

[0026] Figure 4 The present invention shows the inhibition of the cell senescence pathway (HSA04218: cellular senescence) after each single drug in the Chinese medicine composition of the present application is treated with THP1, A549, BEAS2B and MRC5 cell lines;

[0027] Figure 5 The inhibition of each single drug in the Chinese medicine composition of the present application on COPD and aging-related genes is shown, wherein the specific value is the logarithm of the gene change value (log2FoldChange), and a negative value indicates that the expression of the related gene is inhibited;

[0028] Figure 6 The results of CCK8 test on the cytotoxicity of the Chinese medicine composition of the present application to A549 are shown;

[0029] Figure 7 The present invention shows the results of cell activity detection of the Chinese medicine composition in the bronchial epithelial cell inflammation model. DETAILED DESCRIPTION

[0030] The invention discloses a traditional Chinese medicine composition and a preparation method and application thereof.

[0031] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0032] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0033] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0034] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0035] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0036] The raw materials and reagents used in the present invention can be purchased from the market.

[0037] The invention provides a traditional Chinese medicine composition, comprising the following components in proportion by weight: 15-30 parts of dwarf camellia, 2-3 parts of Hypericum perforatum, 3-10 parts of Magnolia officinalis, 3-10 parts of Platycodon grandiflorum, 3-10 parts of Citrus aurantium, 9-15 parts of Longan pulp, 2-6 parts of Schisandra chinensis, 1-3 parts of Indigo naturalis, 6-12 parts of Morus alba bark, 3-6 parts of Amomum villosum, 9-30 parts of Pseudostellaria pseudoginseng, 9-15 parts of Peach twig, 3-10 parts of Elsholtzia ciliata and 6-12 parts of Rhizoma Anemarrhenae.

[0038] The traditional Chinese medicine composition provided by the present invention comprises 15 to 30 parts by weight of Camellia oleifera, preferably 16 to 29 parts by weight, and more preferably 17 to 28 parts by weight.

[0039] The dwarf tea belongs to the whole plant of the plant Myrsinaceae, Ardisia japonica. It is flat in nature, spicy and slightly bitter in taste, and belongs to the lung and liver meridians. In traditional Chinese medicine, dwarf tea has the effects of resolving phlegm and relieving cough, promoting dampness, and promoting blood circulation. It is used to treat symptoms such as cough, blood in sputum, chronic bronchitis, damp-heat jaundice, and injuries caused by falls. Modern pharmacological studies have shown that dwarf tea has antitussive, expectorant and asthmatic, antibacterial and antiviral effects.

[0040] The traditional Chinese medicine composition provided by the invention comprises 2-3 parts by weight of Hypericum perforatum.

[0041] Hypericum perforatum belongs to the perennial herbaceous plant of the genus Hypericum in the family Garcinia. In traditional Chinese medicine, the dried aerial parts of Hypericum perforatum are used as medicine, which has the effects of soothing the liver and relieving depression, clearing away heat and dampness, and relieving swelling and pain. It is mainly used to treat symptoms such as qi stagnation, joint swelling and pain, and difficulty urinating.

[0042] The traditional Chinese medicine composition provided by the present invention comprises 3 to 10 parts by weight of magnolia bark, preferably 4 to 9 parts by weight, and more preferably 5 to 8 parts by weight.

[0043] Magnolia officinalis is the dried bark, root bark and branch bark of Magnolia officinalis, a plant of the Magnoliaceae family. In traditional Chinese medicine, Magnolia officinalis has the effects of drying dampness and eliminating phlegm, and removing fullness from the lower qi. It is mainly used to treat symptoms such as dampness stagnation, abdominal distension, vomiting and diarrhea, food accumulation and qi stagnation, abdominal distension and constipation, phlegm and cough. Modern pharmacological studies have shown that Magnolia officinalis contains a variety of chemical components, including phenolic compounds, alkaloids and volatile oils, among which magnolol and honokiol are the main active substances. These components have obvious pharmacological activities on the digestive system, nervous system, cardiovascular system and respiratory system. Magnolia officinalis also has anti-inflammatory, analgesic, anti-pathogenic microorganism, antibacterial, anti-tumor and antioxidant effects.

[0044] The traditional Chinese medicine composition provided by the present invention comprises 3 to 10 parts by weight of Platycodon grandiflorum, preferably 4 to 9 parts by weight, and more preferably 5 to 8 parts by weight.

[0045] Platycodon grandiflorum belongs to the genus Platycodon grandiflorum of the family Campanulaceae. Its dried roots are used in traditional Chinese medicine to clear the lungs, remove phlegm, relieve sore throat and discharge pus. It is mainly used to treat cough with phlegm, chest tightness, sore throat, aphonia and pulmonary abscess with pus. Modern pharmacological studies have shown that Platycodon grandiflorum contains saponin compounds, flavonoid compounds, phenolic compounds, sterol compounds and polysaccharide compounds, which have pharmacological effects such as expectorant, antitussive and anti-inflammatory.

[0046] The traditional Chinese medicine composition provided by the present invention comprises 3 to 10 parts by weight of tangerine peel, preferably 4 to 9 parts by weight, and more preferably 5 to 8 parts by weight.

[0047] Tangerine peel belongs to the Rutaceae family and is the outer red part of the peel of many citrus fruits such as the Fuju or Zhuju in the genus Citrus. In traditional Chinese medicine, tangerine peel has the effects of eliminating phlegm, promoting qi, relaxing the stomach, and dispersing nodules. It is mainly used to treat symptoms such as cold and phlegm cough, nausea, vomiting, chest pain and stuffiness. Modern pharmacological studies have shown that tangerine peel contains ingredients such as naringin, which has multiple pharmacological effects such as anti-inflammatory, anti-tumor, anti-asthma, and neuroprotection. In terms of application, in addition to being used as a traditional Chinese medicine, its ingredients are also used in the fields of food nutrition and drug research.

[0048] The traditional Chinese medicine composition provided by the present invention comprises 9 to 15 parts by weight of longan pulp, preferably 10 to 14 parts by weight, and more preferably 11 to 13 parts by weight.

[0049] Longan pulp belongs to the genus Longan of the Sapindaceae family. In traditional Chinese medicine, longan pulp is used as medicine with its aril, which is warm in nature and sweet in taste. It enters the heart and spleen meridians and has the effects of nourishing the heart and spleen, nourishing blood and calming the mind. It is mainly used to treat symptoms such as insufficient qi and blood, palpitations, forgetfulness and insomnia, and blood deficiency and chlorosis.

[0050] The traditional Chinese medicine composition provided by the present invention comprises 2 to 6 parts by weight of Schisandra chinensis, preferably 3 to 5 parts by weight, and more preferably 4 parts by weight.

[0051] Schisandra chinensis is the dried mature fruit of Schisandra sphenanthera, a plant of the Magnoliaceae family. In traditional Chinese medicine, Schisandra chinensis has the effects of astringency, invigorating qi and promoting body fluid, and tonifying the kidney and calming the mind. It is mainly used to treat chronic cough, asthma, nocturnal emission, frequent urination, chronic diarrhea, spontaneous sweating, night sweats, thirst due to loss of body fluid, internal heat and thirst, palpitations and insomnia.

[0052] The traditional Chinese medicine composition provided by the present invention comprises 1 to 3 parts by weight of Indigo Naturalis, preferably 2 parts by weight.

[0053] Indigo is derived from the leaves or stems of the Acanthaceae plant Indigofera tinctoria, the Polygonaceae plant Indigofera tinctoria, the Leguminosae plant Indigofera tinctoria, and the Cruciferae plant Isatis indigotica. In traditional Chinese medicine, Indigo has the effects of clearing away heat and detoxifying, cooling blood and stopping bleeding, clearing the liver and purging fire, and calming the nerves.

[0054] The traditional Chinese medicine composition provided by the present invention comprises 6 to 12 parts by weight of Morus alba bark, preferably 7 to 11 parts by weight, and more preferably 8 to 10 parts by weight.

[0055] Morus alba bark is the dried root bark of the mulberry plant of the Moraceae family, and its scientific name is Morus alba L. In traditional Chinese medicine, Morus alba bark has the effects of purging the lungs and relieving asthma, promoting diuresis and reducing swelling. It is mainly used to treat symptoms such as lung heat, asthma, cough, edema, oliguria, and puffy face and skin. Modern pharmacological studies have shown that Morus alba bark contains a variety of chemical components such as flavonoids, morifurans, and umbelliferones, which have diuretic, blood pressure lowering, heart contraction force and frequency inhibition, smooth muscle excitation, and certain antibacterial effects.

[0056] The traditional Chinese medicine composition provided by the present invention comprises 3 to 6 parts by weight of Amomum villosum, preferably 4 to 5 parts by weight.

[0057] Amomum villosum is a plant of the ginger family. Its TCM effects include removing dampness and promoting qi, warming the middle and stopping diarrhea, and calming the fetus. It is often used to treat symptoms such as dampness blocking the middle jiao, spleen and stomach qi stagnation, and spleen and stomach deficiency and cold. Modern pharmacological studies have shown that Amomum villosum contains volatile oils and other ingredients, which have multiple pharmacological effects such as anti-inflammatory, antioxidant, and anti-tumor. In terms of application, in addition to being used as a traditional Chinese medicine, Amomum villosum's ingredients are also used in food seasoning and health products.

[0058] The traditional Chinese medicine composition provided by the present invention comprises 9 to 30 parts by weight of Pseudostellariae Radix, preferably 10 to 29 parts by weight, and more preferably 11 to 28 parts by weight.

[0059] Pseudostellariae belongs to the genus Pseudostellariae of the Caryophyllaceae family. In traditional Chinese medicine, Pseudostellariae has the effects of invigorating qi and strengthening the spleen, promoting fluid production and moistening the lungs. It is mainly used to treat symptoms such as spleen deficiency, fatigue, loss of appetite, weakness after illness, insufficient qi and yin, spontaneous sweating and thirst, dry cough due to lung dryness, etc. Modern pharmacological studies have shown that Pseudostellariae contains volatile oils, polysaccharides, cyclic peptides, alkaloids, saponins, phenols and other ingredients, and has pharmacological effects such as lowering blood sugar, anti-inflammatory, anti-cancer, protecting cells, inhibiting tyrosinase, and immunomodulating.

[0060] The traditional Chinese medicine composition provided by the present invention comprises 9 to 15 parts by weight of peach branches, preferably 10 to 14 parts by weight, and more preferably 11 to 13 parts by weight.

[0061] Peach branches are the dried branches of the peach plant Prunus persica (L.) Batsch of the Rosaceae family. In traditional Chinese medicine, peach branches have the effects of promoting blood circulation, unblocking meridians, detoxifying and killing insects. They are mainly used to treat symptoms such as heart and abdomen stabbing pain, rheumatic pain, traumatic injuries, and sores and ringworms. Modern pharmacological studies have shown that peach branches contain flavonoids, such as quercetin and its glucoside, kaempferol and its glucoside, etc., which have multiple pharmacological effects such as anti-inflammatory, cough and expectorant, antioxidant, lipid-lowering, free radical elimination and anti-tumor.

[0062] The traditional Chinese medicine composition provided by the present invention comprises 3 to 10 parts by weight of Elsholtzia ciliata, preferably 4 to 9 parts by weight, and more preferably 5 to 8 parts by weight.

[0063] Elsholtzia belongs to the genus Elsholtzia of the family Lamiaceae. In traditional Chinese medicine, the above-ground part of Elsholtzia is used as medicine. It is slightly warm in nature, pungent in taste, and enters the lung and stomach meridians. Its main functions are to induce sweating and relieve exterior symptoms, and to harmonize the middle and eliminate dampness. It is used to treat symptoms such as summer-heat cold, aversion to cold and fever, headache without sweating, abdominal pain, vomiting and diarrhea, and urinary difficulties. Modern pharmacological studies have shown that Elsholtzia has antipyretic and analgesic effects, inhibits intestinal function, enhances immunity, has a broad-spectrum antibacterial and certain antiviral effects, and may also have the effects of lowering blood pressure and cholesterol.

[0064] The traditional Chinese medicine composition provided by the present invention comprises 6 to 12 parts by weight of Anemarrhena asphodeloides, preferably 7 to 11 parts by weight, and more preferably 8 to 10 parts by weight.

[0065] Anemarrhena belongs to the Liliaceae family. It is bitter and cold in nature and enters the lung, stomach and kidney meridians. It has the TCM effects of clearing away heat and purging fire, promoting the production of body fluids and moistening dryness. Modern pharmacological studies have shown that Anemarrhena contains a variety of chemical components, including Anemarrhena polysaccharides, Anemarrhena saponins, flavonoids, etc., which have the effects of lowering blood sugar and blood lipids, improving Alzheimer's disease, relieving fever, anti-inflammatory, preventing and treating osteoporosis. In terms of application, Anemarrhena is widely used to treat symptoms such as exogenous febrile diseases, high fever and thirst, lung heat and dry cough, bone steaming and hot flashes, internal heat and thirst, and dry intestines and constipation.

[0066] In one preferred embodiment of the present invention, the Chinese medicine composition includes the following raw materials in parts by weight: 16-29 parts of dwarf camellia, 2 parts of Hypericum perforatum, 2-9 parts of Magnolia officinalis, 2-9 parts of Platycodon grandiflorum, 2-9 parts of Citrus aurantium, 10-11 parts of longan pulp, 3-5 parts of Schisandra chinensis, 2-3 parts of Indigo naturalis, 7-11 parts of Morus alba bark, 4-5 parts of Amomum villosum, 10-29 parts of Pseudostellaria pseudoginseng, 10-14 parts of peach twigs, 4-9 parts of Elsholtzia ciliata, and 7-11 parts of Anemarrhena asphodeloides.

[0067] In one preferred embodiment of the present invention, the Chinese medicine composition includes the following raw materials in parts by weight: 17-28 parts of dwarf camellia, 3 parts of Hypericum perforatum, 3-8 parts of Magnolia officinalis, 3-8 parts of Platycodon grandiflorum, 3-8 parts of Citrus aurantium, 10 parts of longan pulp, 4 parts of Schisandra chinensis, 3 parts of Indigo naturalis, 8-10 parts of Morus alba bark, 4 parts of Amomum villosum, 11-28 parts of Pseudostellaria pseudoginseng, 11-13 parts of peach twigs, 5-8 parts of Elsholtzia ciliata, and 8-10 parts of Anemarrhena asphodeloides.

[0068] In one preferred embodiment of the present invention, the Chinese medicine composition comprises the following raw materials in parts by weight: 15 parts of dwarf camellia, 3 parts of Hypericum perforatum, 9 parts of Magnolia officinalis, 9 parts of Platycodon grandiflorum, 10 parts of Citrus aurantium, 10 parts of Longan pulp, 6 parts of Schisandra chinensis, 3 parts of Indigo naturalis, 10 parts of Morus alba bark, 5 parts of Amomum villosum, 15 parts of Pseudostellaria pseudoginseng, 10 parts of Peach twig, 9 parts of Elsholtzia ciliata, and 9 parts of Anemarrhena asphodeloides.

[0069] In one preferred embodiment of the present invention, the Chinese medicine composition comprises the following raw materials in parts by weight: 30 parts of dwarf camellia, 2 parts of Hypericum perforatum, 10 parts of Magnolia officinalis, 10 parts of Platycodon grandiflorum, 3 parts of Citrus aurantium, 9 parts of Longan pulp, 2 parts of Schisandra chinensis, 2 parts of Indigo naturalis, 6 parts of Morus alba bark, 3 parts of Amomum villosum, 9 parts of Pseudostellaria pseudoginseng, 15 parts of peach twigs, 10 parts of Elsholtzia ciliata, and 6 parts of Anemarrhena asphodeloides.

[0070] In one preferred embodiment of the present invention, the Chinese medicine composition comprises the following raw materials in parts by weight: 20 parts of dwarf camellia, 2.5 parts of Hypericum perforatum, 3 parts of Magnolia officinalis, 3 parts of Platycodon grandiflorum, 6 parts of Citrus aurantium, 15 parts of longan pulp, 4 parts of Schisandra chinensis, 1 part of Indigo naturalis, 12 parts of Morus alba bark, 6 parts of Amomum villosum, 30 parts of Pseudostellaria pseudoginseng, 9 parts of peach twigs, 3 parts of Elsholtzia ciliata, and 12 parts of Anemarrhena asphodeloides.

[0071] The present invention creatively combines the above 14 kinds of Chinese medicines in a reasonable way, so that the components can be synergistically regulated, and the COPD-related signal pathways can be regulated in bronchial epithelial cells, lung fibroblasts, and macrophage models. The Chinese medicine composition of the present invention focuses on overall conditioning and syndrome differentiation and treatment. It can relieve clinical symptoms, improve lung function and immune function, and improve the quality of life through expectoration, bronchial dilation and immune regulation. It can improve respiratory symptoms, inhibit inflammation, and improve lung function or lung fibrosis, and can be used for the related treatment or adjuvant treatment of COPD.

[0072] The present invention provides a method for preparing a traditional Chinese medicine composition as described in any one of the above technical schemes, comprising: mixing and soaking powders of dwarf camellia, Hypericum perforatum, Magnolia officinalis, Platycodon grandiflorum, Citrus aurantium, longan pulp, Schisandra chinensis, Indigo naturalis, Morus alba bark, Amomum villosum, Pseudostellaria sibiricum, Prunus persica, Elsholtzia ciliata, and Anemarrhena asphodeloides with purified water, extracting by ultrasonic extraction or soaking extraction to obtain an extract; concentrating the extract under reduced pressure, adding purified water, freeze-drying to obtain freeze-dried powder, and dissolving the freeze-dried powder in distilled water to obtain an extract mother liquor.

[0073] The above components and proportions provided by the present invention have been clearly described and will not be repeated here.

[0074] In some preferred embodiments of the present invention, the volume ratio of the powder component to purified water is 1:15-20, the purified water soaking time is 30 minutes, the ultrasonic extraction time is 30-60 minutes, and the soaking extraction time is 48 hours.

[0075] The freeze-drying time is 24 to 48 hours, and the storage temperature of the extract mother solution is -15 to 20°C.

[0076] The present invention provides use of any one of the above-mentioned Chinese medicine compositions and / or Chinese medicine compositions obtained by the preparation methods in the preparation of medicines and / or preparations for preventing and / or treating chronic obstructive pulmonary disease.

[0077] The present invention provides the use of any one of the above-mentioned Chinese medicine compositions and / or Chinese medicine compositions obtained by the preparation methods in the preparation of drugs and / or preparations for regulating and / or inhibiting the expression of cytokines and / or genes related to the treatment of COPD.

[0078] The cytokines and / or genes include: one or more of IL-6, IL-8, IL17, NF-kB, TNF, and TGF-β.

[0079] The present invention also provides the use of any one of the above-mentioned Chinese medicine compositions and / or the Chinese medicine composition obtained by the preparation method in improving the vitality of senescent cells.

[0080] The present invention also provides the use of any one of the above-mentioned Chinese medicine compositions and / or the Chinese medicine compositions obtained by the preparation methods in reducing the expression of aging marker genes.

[0081] The dosage form of the present invention includes, but is not limited to, one or more of granules, decoctions, powders, oral liquids, tablets, pills, pastes or capsules.

[0082] In order to further illustrate the present invention, a Chinese medicine composition provided by the present invention and its preparation method and application are described in detail below in conjunction with embodiments.

[0083] Example

[0084] Table 1 Chinese medicine composition examples and comparative examples by weight

[0085]

[0086] Weigh the powder formula according to the dosage, add purified water to the formula powder, the volume ratio of powder formula to purified water is 1:15-20, soak for 30 minutes, ultrasonically extract for 30-60 minutes or continue to soak for 48 hours, collect the supernatant, add appropriate amount of purified water after decompression concentration, use a low-temperature vacuum dryer to freeze-dry for 24-28 hours to make the extract freeze-dried powder, weigh a certain mass of freeze-dried powder, dissolve it in distilled water to make the extract mother solution, and store it at -15-20℃.

[0087] Verification Example 1

[0088] The Chinese medicine composition regulates and improves the disease signal pathway of chronic obstructive pulmonary disease. The experiment includes the following steps:

[0089] Step 1: Obtain COPD signaling pathways

[0090] Publicly published data were retrieved from the GEO database on the official website of NCBI. Specifically, the transcriptome sequencing data of COPD tissue (GSE27597 / GSE57148) were retrieved from the NCBI (https: / / www.ncbi.nlm.nih.gov / ) database, and the sequencing data were subjected to KEGG pathway gene set enrichment analysis (GSEA), and abnormal signaling pathways related to COPD were selected as the signaling pathway changes in lung tissue-related cells (lung epithelial cells, bronchial epithelial cells, and lung fibroblasts) in COPD patients; the macrophage transcriptome data of bronchoalveolar lavage fluid of COPD patients (GSE183973) were selected, and the KEGG pathway gene set enrichment analysis was performed, and the related abnormal signaling pathways were selected as the pathway changes in airway macrophages in COPD patients.

[0091] Step 2: Transcriptome sequencing of A549, MRC5, BEAS2B, and THP1 cells treated with Chinese medicine composition

[0092] 1) Extraction of Chinese herbal medicine composition

[0093] Fourteen single drugs, the Chinese medicine compositions of Examples 1-3 and Comparative Examples 1-3 were extracted with water by ultrasonic extraction, and freeze-dried powder was prepared using a freeze dryer. An appropriate amount of freeze-dried powder was weighed and dissolved in phosphate buffer saline (PBS) to prepare an extract mother solution with a concentration of 50 mg / mL.

[0094] 2) Cell plating and drug addition

[0095] For adherent cells such as A549, MRC5, and BEAS2B, when they are in the logarithmic growth phase, take them out from the incubator, discard the culture medium, and place the cell culture flask with adherent cells. Add 1 mL of trypsin to the culture flask, gently shake the culture flask so that the entire cell surface is covered with trypsin, and put it into the incubator for digestion until the cells partially float. Add 2 mL of complete culture medium to terminate the digestion, use a Pasteur pipette to blow the culture medium to make the cells fall off, centrifuge, discard the supernatant, add 2 mL of culture medium, gently blow the cells to mix, and count them using a cell counting plate.

[0096] A549 was seeded in a 384-well plate at a seeding density of 3000 / well. After overnight attachment, 200ug / mL of drug was added for treatment for 24h.

[0097] MRC5 was seeded in a 384-well plate at a seeding density of 3000 / well. After overnight attachment, the cells were starved for 24 hours and treated with 10 ng / mL TGF-β1 and 200 ug / mL drug for 48 hours.

[0098] BEAS2B was seeded in a 384-well plate at a seeding density of 2500 / well. After overnight attachment, the inflammatory model was constructed by stimulating with 5 ng / mL of IL-1β for 24 h, and then treated with 200 μg / mL of the drug extract for 24 h.

[0099] For THP1 cells, when the cells are in the logarithmic growth phase, collect the cells by centrifugation, add 2 mL of culture medium, gently pipette and mix the cells, and count them using a cell counting plate.

[0100] THP1 was inoculated in a 384-well plate at a seeding density of 8000 in RPMI1640 complete medium containing 100 ng / mL PMA. After directional induction for 24 to 48 hours, the cells were treated with 200 μg / mL of the drug extract for 24 hours.

[0101] 3) Transcriptome sequencing and analysis

[0102] The lysis, reverse transcription and amplification of drug screening cells were carried out according to the instructions of the relevant kits. The samples were sequenced on the machine, and 2M reads were measured for each sample. The data off the machine were analyzed. Gene set enrichment analysis (GSEA) was used to perform enrichment analysis of signal pathways (FDR<0.25, the pathway was considered to be significantly regulated).

[0103] Depend on Figure 1 The results show that 14 single drugs regulate abnormal signaling pathways of COPD in A549, MRC5, BEAS2B and THP1, with blue representing negative regulation and red representing positive regulation. It can be seen that Camellia ovata, Hypericum perforatum, Magnolia officinalis, Platycodon grandiflorum, Citrus aurantium, Longan pulp, Schisandra chinensis, Indigo naturalis, Morus alba, Amomum villosum, Pseudostellaria baicalensis, Peach twig, Elsholtzia ciliata, and Anemarrhena asphodeloides regulate at least one signaling pathway in four cell lines.

[0104] Depend on Figure 2As can be seen from the results, it shows the heat map of the abnormal signaling pathways of COPD regulated by Examples 1-3 and Comparative Examples 1-3. The results show that Example 1-3 completely regulates 30 COPD-related pathways in A549, MRC5, BEAS2B and THP1 cells, and can downregulate the NF-kB signaling pathway, IL17 signaling pathway, and TNF signaling pathway that are closely related to COPD. Example 1-3 inhibits the TNF-α inflammatory mediator that affects the occurrence and development of airway inflammation by regulating the TNF signaling pathway, thereby inhibiting the inflammatory response and alleviating the symptoms of COPD. Example 1-3 can inhibit the NF-kB signaling pathway, and inhibit its activation by intervening at different sites of the NF-kB signaling pathway, and is used to treat COPD. Example 1-3 can inhibit the IL17 signaling pathway, reduce IL-17 expression, inhibit the secretion of proinflammatory cytokines, and has therapeutic effects in reducing neutrophil aggregation and reducing inflammation, and thus can treat COPD. Comparative Example 1 had no significant effect on the TNF signaling pathway and the C-type lectin receptor signaling pathway in A549; and had no significant effect on the PPAR signaling pathway in THP1 cells. Comparative Example 2 had no significant effect on ECM receptor interaction in A549; had no significant effect on oxidative phosphorylation in BEAS2B; and had no significant effect on the NF-kB signaling pathway in MRC5. Comparative Example 3 had no significant effect on the NOD-like receptor signaling pathway and the Wnt signaling pathway in A549; had no significant effect on the C-type lectin receptor signaling pathway, the IL-17 signaling pathway, and the NOD-like receptor signaling pathway in BEAS2B; and had no significant effect on the MAPK signaling pathway in THP1.

[0105] Depend on Figure 3 The results show that the Chinese medicine composition of the present invention can down-regulate the NF-kB, TNF, IL-17 and TGF-β signaling pathways in patients with COPD. Among them, the TGF-β signaling pathway is closely related to the progression of pulmonary fibrosis. Down-regulating the TGF-β signaling pathway can reduce the expansion of the alveolar cavity and delay the progression of emphysema and COPD.

[0106] In summary, the composition composed of 14 medicinal materials in Examples 1-3 can act on 30 abnormal signal pathways related to COPD, and the components cooperate with each other to produce a synergistic effect, thereby playing a role in treating COPD.

[0107] Verification Example 2

[0108] The Chinese medicine composition regulates the cell aging signaling pathway, and the experiment includes the following steps:

[0109] Step 1: Obtaining aging-related pathway signals

[0110] The GEO database on the NCBI official website was used to search for publicly published data.

[0111] The transcriptome sequencing data of the elderly population (GSE193141) were retrieved from the (https: / / www.ncbi.nlm.nih.gov / ) database, and the sequencing data were subjected to KEGG pathway gene set enrichment analysis (GSEA). The abnormal signaling pathways related to aging were selected, and the cellular senescence pathway (HSA04218: cellular senescence) was used as an example for the experiment.

[0112] Step 2: Transcriptome sequencing of A549, MRC5, BEAS2B, and THP1 cells treated with Chinese medicine composition

[0113] The specific sequencing method is the same as that of Verification Example 1.

[0114] Depend on Figure 4 As a result, it is shown that the 14 single drugs of the Chinese medicine composition of the present application treat A549, MRC5, BEAS2B and THP1 cells, and the effects on the pathways related to the cellular senescence pathway (HSA04218: cellular senescence) are shown. The results show that dwarf tea, Hypericum perforatum, longan meat and peach branch can inhibit the senescence pathway of BEAS2B cells, Magnolia officinalis can inhibit the senescence pathway of A549 cells, Tangerine peel can inhibit the senescence pathway of THP1 cells and A549 cells, Schisandra chinensis and Anemarrhena asphodeloides can inhibit the senescence pathway of THP1 cells, Pseudostellaria chinensis can inhibit the senescence pathway of MRC5 cells, and Elsholtzia ciliata can inhibit the senescence pathway of THP1 cells and MRC5 cells. It can be seen that dwarf tea, Hypericum perforatum, Magnolia officinalis, Tangerine peel, longan meat, Schisandra chinensis, Pseudostellaria chinensis, Peach branch, Elsholtzia ciliata and Anemarrhena asphodeloides can inhibit the senescence pathway of THP1 cells to varying degrees, showing good anti-aging potential.

[0115] By screening and analyzing key genes and inflammatory cytokines related to COPD and aging, it was found that each single drug in the Chinese medicine composition of the present application can reduce the expression of related genes, such as Figure 5 As shown, the specific value is the logarithm of the gene change value (log2FoldChange). If it is a negative value, it means that inflammatory secretion is inhibited. The results show that the Chinese medicine composition of the present invention can reduce the expression of proinflammatory cytokines IL6, IL8, and TGF-β, and can also reduce the expression of aging marker genes CDKN1A, CDKN2B, PLAU, RB1, SERPINE1, SERPINB2, ABL1, BCL2L1, EFNB1, NOTCH3, CTCF, and TNFRSF10D, showing anti-aging effects.

[0116] It should be noted that there is a complex interaction between lung aging, lung aging diseases such as COPD, inflammatory genes, and aging. With age, lung aging, decreased immunity and lung function, and increased susceptibility to infection will interact with each other and promote lung aging and disease.

[0117] Certain inflammatory cytokines such as IL6, IL8, and TGF-β play an important role in the development of COPD. Among them, IL-6 is a proinflammatory cytokine that can reflect airflow obstruction and its severity. The serum IL-6 level of COPD patients is higher than that of healthy people. The IL-6 level is also related to the susceptibility of patients to lung infection and is an important factor in causing chronic respiratory inflammation. In addition, IL-6 is involved in the regulation of airway remodeling and the formation of pulmonary fibrosis, and is negatively correlated with the patient's lung function. Inhibiting IL6 expression can improve the airflow limitation of COPD caused by excessive airway mucus secretion induced by IL-6; IL-8 (interleukin 8) is one of the main inflammatory mediators, and multiple factors can lead to its significant increase in COPD patients. The Chinese medicine composition of the present invention can inhibit IL8 expression and improve the inflammatory condition of COPD; TGF-β can induce inflammatory cells to infiltrate the lungs, blood vessels and respiratory tract, promote fibroblast chemotaxis, produce collagen and fibronectin, inhibit collagen degradation, and promote fibrosis. The Chinese medicine composition of the present invention can inhibit TGF-β expression and improve and relieve the severity of COPD.

[0118] Senescent cells often show significant changes in their secretome, a phenomenon known as the senescence-associated secretory phenotype (SASP). SASP upregulates and releases many pro-inflammatory cytokines, such as IL-6 / IL-1β. The Chinese medicine composition of the present invention improves the cell viability of senescent cells by inhibiting the expression of IL-6 and IL-1β.

[0119] Verification Example 3

[0120] The cytotoxicity test of the Chinese medicine composition on lung epithelial cells A549 comprises the following steps:

[0121] Step 1: Cell plating and drug treatment

[0122] When A549 cells were in the logarithmic growth phase, A549 cells were seeded in a 384-well plate at a seeding density of 3000 / well. After overnight attachment, the extracts of the prescriptions described in Example 1, Example 2, and Example 3 were added for treatment for 24 hours.

[0123] Step 2: Take out the drug-treated A549 cells from the incubator, slowly discard the culture medium, add 110 μL of culture medium containing CCK-8 reagent, continue to culture for 3 hours, measure the absorbance of each well with a microplate reader (wavelength 450 nm), and calculate the cell survival rate according to the reagent instructions.

[0124] Depend on Figure 6 The results showed that under the treatment of less than 1600 μg / mL of the extract, the Chinese medicine compositions of Example 1, Example 2, and Example 3 had no obvious cytotoxicity to the lung epithelial cells A549.

[0125] Verification Example 4

[0126] The cell activity detection of the bronchial epithelial cell inflammation model by the Chinese medicine composition comprises the following steps:

[0127] Step 1: Cell plating and drug treatment

[0128] BEAS2B were seeded in 384-well plates at a seeding density of 2500 / well. After overnight attachment, the inflammatory model was constructed by stimulating with 5 ng / mL IL-1β for 24 h, and then treated with 800 μg / mL of the drug extract for 24 h;

[0129] Step 2: Take the drug-treated BEAS2B cells out of the incubator, slowly discard the culture medium, add 110 μL of culture medium containing CCK-8 reagent, continue to culture for 3 hours, measure the absorbance of each well with a microplate reader (wavelength 450 nm), and calculate the cell activity according to the reagent instructions.

[0130] Depend on Figure 7 The results showed that under the treatment of 800 μg / mL of the extract, the Chinese medicine compositions of Example 1, Example 2, and Example 3 were able to increase the cell activity of the bronchial epithelial cell inflammation model.

[0131] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A Chinese medicine composition, characterized in that: The invention comprises the following components in proportion by weight: 15-30 parts of dwarf camellia, 2-3 parts of Hypericum perforatum, 3-10 parts of magnolia bark, 3-10 parts of platycodon, 3-10 parts of tangerine peel, 9-15 parts of longan pulp, 2-6 parts of schisandra chinensis, 1-3 parts of indigo, 6-12 parts of mulberry bark, 3-6 parts of amomum villosum, 9-30 parts of pseudoginseng, 9-15 parts of peach branch, 3-10 parts of elsholtzia and 6-12 parts of anemarrhena.

2. The Chinese medicine composition according to claim 1, characterized in that: The invention comprises the following components in proportion by weight: 15 parts of dwarf tea, 3 parts of Hypericum perforatum, 9 parts of Magnolia officinalis, 9 parts of Platycodon grandiflorum, 10 parts of orange peel, 10 parts of longan pulp, 6 parts of Schisandra chinensis, 3 parts of Indigo naturalis, 10 parts of Morus alba bark, 5 parts of Amomum villosum, 15 parts of Pseudostellaria heterophylla, 10 parts of Peach branch, 9 parts of Elsholtzia ciliata, and 9 parts of Rhizoma Anemarrhenae; or 30 parts of Camellia davidiana, 2 parts of Hypericum perforatum, 10 parts of Magnolia officinalis, 10 parts of Platycodon grandiflorum, 3 parts of Citrus aurantium, 9 parts of Longan pulp, 2 parts of Schisandra chinensis, 2 parts of Indigo naturalis, 6 parts of Morus alba bark, 3 parts of Amomum villosum, 9 parts of Pseudostellaria pseudoginseng, 15 parts of Peach twig, 10 parts of Elsholtzia ciliata, 6 parts of Anemarrhena asphodeloides; or 20 parts of Camellia davidiana, 2.5 parts of Hypericum perforatum, 3 parts of Magnolia officinalis, 3 parts of Platycodon grandiflorum, 6 parts of Dangpi, 15 parts of Longan pulp, 4 parts of Schisandra chinensis, 1 part of Indigo naturalis, 12 parts of Morus alba bark, 6 parts of Amomum villosum, 30 parts of Pseudostellaria pseudoginseng, 9 parts of Peach twig, 3 parts of Elsholtzia ciliata, and 12 parts of Rhizoma Anemarrhenae.

3. A method for preparing the Chinese medicine composition according to claim 1 or 2, characterized in that: The method comprises the following steps: S1: Weigh the powder components in the formula, add purified water to the powder components for soaking, and use ultrasonic extraction or soaking extraction to obtain an extract; S2: After the extract is concentrated under reduced pressure, purified water is added, and freeze-dried to prepare freeze-dried powder, and the freeze-dried powder is dissolved in distilled water to obtain an extract mother solution.

4. The preparation method according to claim 3, characterized in that: In S1, the volume ratio of the powder component to purified water is 1:15-20, the purified water soaking time is 30 minutes, the ultrasonic extraction time is 30-60 minutes, and the soaking extraction time is 48 hours.

5. The preparation method according to claim 3, characterized in that: In S2, the freeze-drying time is 24 to 48 hours, and the storage temperature of the extract mother solution is -15 to 20°C.

6. Use of the Chinese medicine composition according to claim 1 or 2 and / or the Chinese medicine composition obtained according to the preparation method according to any one of claims 3 to 5 in the preparation of drugs and / or preparations for preventing and / or treating chronic obstructive pulmonary disease.

7. Use of the Chinese medicine composition according to claim 1 or 2 and / or the Chinese medicine composition obtained according to the preparation method according to any one of claims 3 to 5 in the preparation of drugs and / or preparations for regulating and / or inhibiting the expression of cytokines and / or genes related to the treatment of chronic obstructive pulmonary disease.

8. The use according to claim 7, characterized in that: The cytokines and / or genes include: one or more of IL-6, IL-8, IL17, NF-kB, TNF, and TGF-β.

9. Use of the traditional Chinese medicine composition according to claim 1 or 2 and / or the traditional Chinese medicine composition obtained according to the preparation method according to any one of claims 3 to 5 in improving the vitality of senescent cells.

10. Use of the traditional Chinese medicine composition according to claim 1 or 2 and / or the traditional Chinese medicine composition obtained according to the preparation method according to any one of claims 3 to 5 in reducing the expression of aging marker genes.

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