Traditional Chinese medicine composition-derived extracellular vesicle particles as well as preparation method and application thereof

Abstract

The invention belongs to the field of traditional Chinese medicines, and relates to a traditional Chinese medicine composition for preventing or treating pulmonary fibrosis, a preparation method and application of the traditional Chinese medicine composition, and traditional Chinese medicine composition source extracellular vesicle particles. According to the idea of treatment based on syndrome differentiation in the traditional Chinese medicine, the effect of treating both symptoms and root causes is achieved, and the traditional Chinese medicine composition which is composed of rhodiola rosea, turtle shells, coix seeds, earthworms, poria cocos and pseudo-ginseng and is used for treating pulmonary fibrosis is obtained through reasonable compatibility. The invention also provides a traditional Chinese medicine composition-derived extracellular vesicle particle which can significantly improve the survival rate of patients with pulmonary fibrosis, reduce the lung index and significantly reduce the degree of pulmonary fibrosis. The traditional Chinese medicine composition and the traditional Chinese medicine composition-derived extracellular vesicle particles are exact in curative effect on pulmonary fibrosis, low in side effect and wide in medical application prospect.

Description

Technical Field

[0001] The invention belongs to the field of medicine and relates to use of a traditional Chinese medicine composition in preparing a medicine for preventing and treating pulmonary fibrosis, and extracellular vesicle particles derived from the traditional Chinese medicine composition. Background Art

[0002] Pulmonary fibrosis is a disease caused by various reasons, characterized by diffuse alveolitis and alveolar structural disorder, which eventually leads to pulmonary interstitial fibrosis. Its pathogenesis can be summarized into two stages: lung damage and pulmonary fibrosis. For a long time, pulmonary fibrosis, especially idiopathic pulmonary fibrosis, has been considered a progressive and basically irreversible pathological change.

[0003] Pulmonary fibrosis can be roughly divided into two categories: those with known causes (such as radiation, drugs, silicosis, etc.) and those with unknown causes (such as idiopathic pulmonary fibrosis). According to the classification of unknown causes, pulmonary fibrosis can be further divided into primary pulmonary fibrosis, secondary pulmonary fibrosis, idiopathic pulmonary fibrosis, pulmonary interstitial fibrosis and interstitial pneumonia.

[0004] Idiopathic pulmonary fibrosis is a chronic progressive fibrosing interstitial pneumonia of unknown cause. The cause of the disease in Western medicine is still unclear. It is mainly seen in middle-aged and elderly men and is limited to the lungs. Patients often have insidious onset. Clinically, they present with progressive dyspnea and deterioration of lung function. Histopathology and chest CT show characteristic common interstitial pneumonia. The disease is incurable. The goal of Western medicine treatment is to delay the progression of the disease, improve the quality of life, and prolong survival.

[0005] Idiopathic pulmonary fibrosis belongs to the category of "pulmonary atrophy and pulmonary paralysis" in traditional Chinese medicine. It is a disease with coughing and spitting turbid saliva as the main clinical manifestation. It is mostly caused by other lung diseases (such as long-term cough, long-term wheezing, etc.) that are not cured or are misdiagnosed, which consumes lung qi and burns lung fluid, resulting in lung deficiency, loss of fluid qi and lack of nourishment, leading to lung atrophy and disuse. It is a chronic wasting disease of the lung. The modern pathogenesis is mainly based on the theory of lung collaterals and collateral diseases, which can be specifically summarized as the theory of lung deficiency and collateral stasis, the theory of heat toxicity and collateral stagnation, and the theory of lung cold and collateral stagnation.

[0006] The accurate concept of pulmonary interstitial fibrosis is called interstitial lung disease, which is a group of diffuse lung diseases that mainly involve the pulmonary interstitium and alveolar cavity, leading to loss of alveolar and capillary function, and has a high morbidity and mortality rate. Currently, glucocorticoids combined with immunosuppressants are considered the main treatment for interstitial lung disease. However, this combination therapy increases the risk of Pneumocystis jiroveci pneumonia, which poses a huge challenge to clinicians.

[0007] Chinese patent medicine is made of Chinese medicinal materials and is also guided by the theory of traditional Chinese medicine. It is a dosage form that needs to be prepared according to the prescribed prescription and process. It has the advantages of being easy to take and carry. It is of great significance to study new drugs for the treatment of pulmonary fibrosis with good therapeutic effects and few toxic side effects. Summary of the invention

[0008] The invention discloses a Chinese medicine composition for preventing or treating pulmonary fibrosis, a preparation method and use thereof. The Chinese medicine composition of the invention has a significant therapeutic effect on treating pulmonary fibrosis, has readily available raw materials, is simple to prepare and has high safety, and overcomes the defects of existing drugs for treating pulmonary fibrosis, such as poor therapeutic effect and large toxic and side effects.

[0009] The present invention also provides a Chinese medicine composition-derived extracellular vesicle particle, a preparation method and use thereof, and the Chinese medicine composition-derived extracellular vesicle particle has a significant therapeutic effect on pulmonary fibrosis.

[0010] Specifically, the technical objectives of the present invention are achieved through the following technical solutions:

[0011] The first aspect of the present invention provides a traditional Chinese medicine composition for preventing or treating pulmonary fibrosis, wherein the traditional Chinese medicine composition is prepared from Rhodiola rosea, turtle shell, coix seed, earthworm, Poria cocos and Panax notoginseng.

[0012] Specifically, the Chinese medicine composition comprises the following raw materials in parts by weight:

[0013] 10-20 parts of Rhodiola rosea, 10-20 parts of turtle shell, 25-35 parts of coix seeds, 10-20 parts of earthworms, 10-20 parts of Poria cocos and 10-20 parts of Panax notoginseng.

[0014] In one embodiment, the Chinese medicine composition comprises the following raw materials by weight:

[0015] 15 parts of Rhodiola rosea, 15 parts of Carapax tricolor, 30 parts of Coix seeds, 15 parts of Pheretima, 15 parts of Poria cocos and 15 parts of Panax notoginseng.

[0016] The second aspect of the present invention provides a method for preparing the above-mentioned Chinese medicine composition for preventing or treating pulmonary fibrosis, comprising the following steps:

[0017] Weigh the raw materials, add purified water, soak, decoct twice, filter, and obtain a first decoction; add purified water to the residue, heat to boiling and keep it slightly boiling, filter, and obtain a second decoction; combine the first decoction and the second decoction to obtain the Chinese medicine composition of the present invention.

[0018] Furthermore, the preparation method of the Chinese medicine composition for preventing or treating pulmonary fibrosis of the present invention is as follows:

[0019] Weigh the above raw materials according to the ratio, add 5.0-7.5 times the weight of purified water, soak for 0.5-2h, heat to boiling and keep slightly boiling for 0.5-1.5h, filter to obtain a first decoction; add 5.0-7.5 times the weight of purified water to the residue, heat to boiling and keep slightly boiling for 2.5-3.5h, filter to obtain a second decoction; combine the first decoction and the second decoction to obtain the Chinese medicine composition of the present invention.

[0020] Furthermore, the preparation method of the Chinese medicine composition for preventing or treating pulmonary fibrosis of the present invention is as follows:

[0021] Weigh the above raw materials according to the ratio, add 6.0 times the weight of purified water, soak for 1.5 hours, heat to boiling and keep slightly boiling for 1 hour, filter to obtain a first decoction; add 6.5 times the weight of purified water to the residue, heat to boiling and keep slightly boiling for 3 hours, filter to obtain a second decoction; combine the first decoction and the second decoction to obtain the Chinese medicine composition of the present invention.

[0022] The third aspect of the present invention provides a pharmaceutical preparation of the above-mentioned traditional Chinese medicine composition for preventing or treating pulmonary fibrosis.

[0023] The Chinese medicine composition is added with conventional excipients and prepared according to conventional processes into clinically acceptable decoctions, pills, gels, tablets, mixtures, capsules, granules or powders, more preferably granules.

[0024] The conventional excipients are fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives and / or bases. The filler is selected from one or more of starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose or sucrose; the disintegrant is selected from one or more of starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose or cross-linked sodium carboxymethyl cellulose; the lubricant is selected from one or more of magnesium stearate, sodium lauryl sulfate, talc or silicon dioxide; the suspending agent is selected from one or more of polyvinyl pyrrolidone, microcrystalline cellulose, sucrose, agar or hydroxypropyl The invention relates to a novel novel food additive comprising: a first agent ...

[0025] The fourth aspect of the present invention provides use of the traditional Chinese medicine composition for preventing or treating pulmonary fibrosis in the preparation of a drug for preventing and treating pulmonary fibrosis.

[0026] Furthermore, the pulmonary fibrosis is idiopathic pulmonary fibrosis.

[0027] Furthermore, the pulmonary fibrosis is lung-spleen deficiency type pulmonary fibrosis.

[0028] The Chinese medicine composition of the present invention is based on the idea of ​​"differentiation and treatment" of traditional Chinese medicine, with rational use of medicines and proper compatibility, so as to achieve the effect of curing both the symptoms and the root cause.

[0029] Rhodiola rosea: sweet, bitter, neutral. Enters the lung and heart meridians. Astringent and hemostatic, dissipates blood stasis and relieves pain, relieves cough and asthma, replenishes qi and clears the lungs, suppresses the mind and nourishes the heart, and resists aging. Used for chest pain, heartache, stroke, hemiplegia, fatigue, asthma caused by qi deficiency, stomach cold, shortness of breath, fatigue, hot cough, hemoptysis, and trauma caused by qi deficiency and physical weakness.

[0030] Turtle shell: sweet, salty and cold in nature, it enters the liver and kidney meridians. It nourishes yin and suppresses yang, reduces fever and eliminates steam, softens and disperses nodules. Turtle shell is good at nourishing the yin of the liver and kidney, and is particularly suitable for yin deficiency internal heat, yin deficiency wind movement, yin deficiency and yang hyperactivity caused by liver and kidney yin deficiency.

[0031] Coix seeds: sweet, light, slightly cold. Enter the spleen, stomach, and lung meridians. It has the functions of promoting water, penetrating dampness, strengthening the spleen and stopping diarrhea, removing numbness, discharging pus, detoxifying and dispersing knots. It is mainly used to treat edema, beriberi, urinary incontinence, spleen deficiency diarrhea, dampness numbness and cramps, lung abscess, intestinal abscess; warts, cancer. It is often used clinically for spleen deficiency diarrhea, muscle soreness, joint pain, edema, beriberi, leucorrhea, lung abscess, appendicitis, etc.

[0032] Earthworm: salty in taste, cold in nature, enters the liver, spleen, and bladder meridians, clears away heat and stops convulsions; soothes the liver and extinguishes wind; unblocks meridians and activates collaterals; relieves asthma and promotes diuresis, and is used for fever and mania caused by febrile diseases; epilepsy and convulsions; liver yang headache; hemiplegia caused by stroke; rheumatic arthralgia; lung heat and cough; urinary obstruction, used for high fever and coma, epilepsy and convulsions, joint pain, limb numbness, hemiplegia, lung heat and cough, oliguria and edema, and hypertension.

[0033] Poria: light in taste, neutral in nature. Enters the heart, spleen, and kidney meridians. It promotes diuresis and eliminates dampness, strengthens the spleen and calms the mind. It is mainly used to treat edema, oliguria, phlegm and fluid, palpitations, spleen deficiency, poor appetite, loose stools, restlessness, palpitations, and insomnia.

[0034] Panax notoginseng: sweet, slightly bitter, warm in nature, enters the liver and stomach meridians, disperses blood stasis and stops bleeding, reduces swelling and relieves pain, used for hemoptysis, hematemesis, epistaxis, bloody stool, metrorrhagia, bleeding from trauma, stabbing pain in the chest and abdomen, and swelling and pain from falls.

[0035] The present invention fully considers the meridians and properties of each Chinese medicine, selects rhodiola rosea to replenish qi, selects coix seed and Poria cocos to strengthen the spleen and eliminate dampness, selects turtle shell to disperse knots, selects earthworm to resolve phlegm, and selects Panax notoginseng to remove blood stasis and dredge collaterals. The whole formula replenishes qi, replenishes spleen, resolves phlegm, activates blood circulation, removes blood stasis, and dredges collaterals. The present invention further selects the ratio between each Chinese medicine to obtain the Chinese medicine composition for treating pulmonary fibrosis with synergistic effect.

[0036] The fifth aspect of the present invention provides a Chinese medicine composition-derived extracellular vesicle particles, which are obtained by extraction and separation of a Chinese medicine composition, and the Chinese medicine composition consists of the following components in parts by weight: 10-20 parts of Rhodiola rosea, 10-20 parts of turtle shell, 25-35 parts of Coix seeds, 10-20 parts of earthworms, 10-20 parts of Poria cocos and 10-20 parts of Panax notoginseng.

[0037] Preferably, the Chinese medicine composition consists of the following components in parts by weight: 15 parts of Rhodiola rosea, 15 parts of turtle shell, 30 parts of coix seeds, 15 parts of earthworms, 15 parts of Poria cocos and 15 parts of Panax notoginseng.

[0038] The sixth aspect of the present invention provides a method for preparing the extracellular vesicle particles derived from the above-mentioned traditional Chinese medicine composition.

[0039] The method for preparing the extracellular vesicle particles derived from the traditional Chinese medicine composition of the present invention comprises the following steps:

[0040] Step S1: Preparation of decoction of Chinese medicine composition

[0041] Chop and mix Rhodiola rosea, turtle shell, coix seed, earthworm, Poria cocos and Panax notoginseng, extract with distilled water, and use a crude extraction system. The supernatants of the two extractions are homogenized to obtain the decoction extract of the Chinese medicine composition.

[0042] Step S2: Isolation of extracellular vesicle particles derived from traditional Chinese medicine composition

[0043] The traditional Chinese medicine composition-derived extracellular vesicles are separated from the traditional Chinese medicine composition decoction by differential centrifugation.

[0044] Furthermore, the method for preparing the extracellular vesicle particles derived from the traditional Chinese medicine composition of the present invention comprises the following steps:

[0045] Step S1: Preparation of decoction of Chinese medicine composition

[0046] Chop and mix Rhodiola rosea, turtle shell, coix seed, earthworm, Poria cocos and Panax notoginseng, extract with distilled water, and use a crude extraction system. The supernatants of the two extractions are homogenized to obtain the decoction extract of the Chinese medicine composition.

[0047] Step S2: Isolation of extracellular vesicle particles derived from traditional Chinese medicine composition

[0048] The Chinese medicine composition decoction extract collected in step S1 was centrifuged at 800×g for 10 min→2000×g for 20 min→5000×g for 35 min→10000×g for 60 min. The final supernatant was centrifuged at 150000×g for 1.5 h-2.5 h in a centrifuge using a sequential ultracentrifugation method, and the obtained particles were gently shaken in PBS and resuspended at 4°C for at least 1 h to obtain Chinese medicine composition-derived extracellular vesicle particles.

[0049] Furthermore, in step S1, the weight ratio of Rhodiola rosea, turtle shell, coix seed, earthworm, Poria cocos and Panax notoginseng is (10-20):(10-20):(25-35):(10-20):(10-20):(10-20).

[0050] Furthermore, in step S1, the crude extraction system is 95±5°C, the first extraction is 2-5h; and 95±5°C, the second extraction is 0.5-2h.

[0051] Furthermore, in step S1, the crude extraction system is 95° C. for 3 h for the first extraction and 95° C. for 1 h for the second extraction.

[0052] Furthermore, in step S2, centrifugation is performed at 800×g for 10 min.

[0053] Furthermore, in step S2, centrifugation is performed at 2000×g for 20 min.

[0054] Furthermore, in step S2, centrifugation is performed at 5000×g for 35 min.

[0055] Furthermore, in step S2, centrifugation is performed at 10,000×g for 60 min.

[0056] Furthermore, in step S2, centrifugation is performed at 150,000×g for 2 hours.

[0057] Furthermore, in step S2, the obtained particles are resuspended at 4° C. for 1-5 h.

[0058] The method for preparing the extracellular vesicle particles derived from the traditional Chinese medicine composition of the present invention specifically comprises the following steps:

[0059] S1: Preparation of decoction of Chinese medicine composition

[0060] Chop and mix Rhodiola rosea, turtle shell, coix seed, earthworm, Poria cocos and Panax notoginseng, extract with distilled water, use crude extraction system, 95±5℃, first extraction for 3h; 95±5℃, second extraction for 1h. The supernatant of the two extractions is homogenized to obtain the decoction extract of the Chinese medicine composition, which is stored in a sealed container.

[0061] S2: Isolation of extracellular vesicle particles derived from traditional Chinese medicine composition

[0062] The Chinese medicine composition decoction extract collected in step S1 was centrifuged at 800×g for 10 min→2000×g for 20 min→5000×g for 35 min→10000×g for 60 min to remove large particles and cell debris. Then, a sequential ultracentrifugation method was used, including centrifuging the final supernatant at 150,000×g for 2 h in a centrifuge, gently shaking the obtained particles in PBS, and resuspending them at 4°C for at least 1 h to obtain Chinese medicine composition-derived extracellular vesicle particles. The final sample of Chinese medicine composition-derived extracellular vesicles was stored at -80°C.

[0063] The seventh aspect of the present invention provides the use of the extracellular vesicle particles derived from the above-mentioned traditional Chinese medicine composition in the preparation of drugs for preventing and treating pulmonary fibrosis.

[0064] Preferably, the pulmonary fibrosis is primary pulmonary fibrosis, secondary pulmonary fibrosis, idiopathic pulmonary fibrosis, pulmonary interstitial fibrosis and interstitial pneumonia.

[0065] Furthermore, the pulmonary fibrosis is secondary pulmonary fibrosis, drug-induced pulmonary fibrosis or idiopathic pulmonary fibrosis.

[0066] Furthermore, the secondary pulmonary fibrosis is pulmonary fibrosis secondary to immune diseases such as rheumatoid arthritis, Sjögren's syndrome, etc.

[0067] Furthermore, the pulmonary interstitial fibrosis is virus-induced pulmonary interstitial fibrosis.

[0068] Furthermore, the virus is adenovirus, respiratory syncytial virus, influenza virus, parainfluenza virus and / or measles virus.

[0069] Furthermore, the virus is an adenovirus or an influenza virus.

[0070] An eighth aspect of the present invention provides a drug for preventing and treating pulmonary fibrosis, wherein the active ingredients of the drug include the extracellular vesicle particles derived from the above-mentioned traditional Chinese medicine composition.

[0071] The dosage form of the medicine for preventing and treating pulmonary fibrosis of the present invention is injection, oral preparation or external preparation.

[0072] Preferably, the drug for preventing and treating pulmonary fibrosis also includes pharmaceutically acceptable injection excipients, oral preparation excipients or external preparation excipients. The specific excipients and corresponding preparation methods can be conventionally selected according to the specific dosage form of the injection, oral preparation or external preparation, and do not adversely affect the pharmacological activity and physicochemical stability of the extracellular vesicle particles derived from the Chinese medicine composition of the present invention and meet the clinical medication requirements. The present invention does not limit this.

[0073] Preferably, the external preparation is a spray, an aerosol, a powder spray or an inhalant.

[0074] The efficacy example of the present invention uses intratracheal injection of bleomycin (BLM) to induce a mouse pulmonary fibrosis model to study the therapeutic effect of the Chinese medicine composition-derived extracellular vesicle particles on pulmonary fibrosis, especially pulmonary interstitial fibrosis. On the 30th day after modeling, the survival rate of mice in the Chinese medicine composition-derived extracellular vesicle group was 93.3%. The Chinese medicine composition-derived extracellular vesicles of the present invention can significantly improve the survival rate of mice with pulmonary fibrosis. The Chinese medicine composition-derived extracellular vesicles of the present invention reduce the lung index of mice with pulmonary fibrosis and significantly reduce the degree of pulmonary fibrosis.

[0075] The efficacy experiments show that the extracellular vesicle particles derived from the traditional Chinese medicine composition of the present invention have a significant therapeutic effect on pulmonary fibrosis, especially pulmonary interstitial fibrosis.

[0076] Compared with the prior art, the present invention has the following advantages:

[0077] (1) The Chinese medicine composition of the present invention improves the symptoms of pulmonary fibrosis, reduces the degree of pulmonary fibrosis, reduces the content of TNF-α and IL-8 in lung tissue, improves inflammatory changes in lung tissue, and plays a role in preventing and treating pulmonary fibrosis.

[0078] (2) The raw materials of the Chinese medicine composition of the present invention are easily available and the preparation is simple. The obtained Chinese medicine composition has a definite preventive and therapeutic effect on pulmonary fibrosis and is highly safe, and has great potential application value in the prevention and treatment of pulmonary fibrosis.

[0079] (3) The present invention uses a Chinese medicine composition as a raw material and adopts high speed and ultracentrifugation to separate the extracellular vesicle particles derived from the Chinese medicine composition. The extracellular vesicles derived from the Chinese medicine composition of the present invention can significantly improve the survival rate of mice with pulmonary fibrosis. The extracellular vesicles derived from the Chinese medicine composition of the present invention reduce the lung index of mice with pulmonary fibrosis and significantly reduce the degree of pulmonary fibrosis. The extracellular vesicles derived from the Chinese medicine composition of the present invention have the effect of preventing and / or treating pulmonary fibrosis diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 Comparison of pulmonary fibrosis scores in rats of each group

[0081] Compared with the blank control group, #### P<0.0001; compared with the model control group, * P<0.05, ** P<0.01, *** P < 0.001, **** P<0.0001; compared with Example 2 group, & P<0.05, &&P<0.01, &&& P < 0.001, &&&& P<0.0001.

[0082] Figure 2 Comparison of TNF-α content in lung tissue of rats in each group

[0083] Compared with the blank control group, #### P<0.0001; compared with the model control group, * P<0.05, ** P<0.01, *** P<0.001; compared with Example 2 group, & P<0.05, && P<0.01.

[0084] Figure 3 Comparison of IL-8 content in lung tissue of rats in each group

[0085] Compared with the blank control group, #### P<0.0001; compared with the model control group, * P<0.05, ** P<0.01, *** P < 0.001, **** P<0.0001; compared with Example 2 group, & P<0.05, && P<0.01, &&& P < 0.001, &&&& P<0.0001.

[0086] Figure 4 Effects of extracellular vesicles derived from traditional Chinese medicine combination on lung index in mice with pulmonary fibrosis

[0087] Compared with the blank control group, #### P<0.0001; compared with the model control group, * P<0.05, ** P<0.01, *** P < 0.001, **** P<0.0001; compared with the Example 5 group, & P<0.05, && P<0.01, &&& P<0.001.

[0088] Figure 5 Effects of extracellular vesicles derived from traditional Chinese medicine combination on the degree of pulmonary fibrosis in mice with pulmonary fibrosis

[0089] Compared with the blank control group, #### P<0.0001; compared with the model control group,* P<0.05, ** P<0.01, *** P < 0.001, **** P<0.0001; compared with the Example 5 group, & P<0.05, && P<0.01, &&& P<0.001. DETAILED DESCRIPTION

[0090] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0091] Example 1

[0092] A Chinese medicine composition for preventing or treating pulmonary fibrosis, comprising the following raw materials in parts by weight:

[0093] Rhodiola rosea 20g, turtle shell 10g, coix seed 35g, earthworm 10g, Poria cocos 10g, Panax notoginseng 20g.

[0094] The preparation method is as follows: weigh the above raw materials according to the ratio, add 6.0 times the weight of purified water, soak for 2.5 hours, heat to boiling and keep slightly boiling for 1.5 hours, filter to obtain a first decoction; add 6.5 times the weight of purified water to the residue, heat to boiling and keep slightly boiling for 2.5 hours, filter to obtain a second decoction; combine the first decoction and the second decoction, add pharmaceutically acceptable excipients through conventional procedures to prepare the Chinese medicine composition capsules of the present invention.

[0095] Example 2

[0096] A Chinese medicine composition for preventing or treating pulmonary fibrosis, comprising the following raw materials in parts by weight:

[0097] 15g of Rhodiola rosea, 15g of turtle shell, 30g of Coix seed, 15g of earthworm, 15g of Poria cocos, and 15g of Panax notoginseng.

[0098] The preparation method is as follows: weigh the above raw materials according to the ratio, add 6.0 times the weight of purified water, soak for 1.5 hours, heat to boiling and keep slightly boiling for 1 hour, filter to obtain a first decoction; add 6.5 times the weight of purified water to the residue, heat to boiling and keep slightly boiling for 3 hours, filter to obtain a second decoction; combine the first decoction and the second decoction, concentrate the filtrate to a relative density of 1.15, and then spray dry, the dried fine powder is passed through a 100-mesh sieve, granulated, and dried to obtain the granules of the Chinese medicine composition of the present invention.

[0099] Example 3

[0100] A Chinese medicine composition for preventing or treating pulmonary fibrosis, comprising the following raw materials in parts by weight:

[0101] 10g of Rhodiola rosea, 20g of turtle shell, 25g of coix seed, 20g of earthworm, 20g of Poria cocos, and 10g of Panax notoginseng.

[0102] The preparation method is as follows: weigh the above raw materials according to the ratio, add 5.0 times the weight of purified water, soak for 1.5 hours, heat to boiling and keep slightly boiling for 1.5 hours, filter to obtain a first decoction; add 7.5 times the weight of purified water to the residue, heat to boiling and keep slightly boiling for 2.5 hours, filter to obtain a second decoction; combine the first decoction and the second decoction, add pharmaceutically acceptable excipients through conventional procedures to prepare the Chinese medicine composition tablets of the present invention.

[0103] Example 4

[0104] A Chinese medicine composition for preventing or treating pulmonary fibrosis, comprising the following raw materials in parts by weight:

[0105] Rhodiola rosea 15g, turtle shell 20g, coix seed 32g, earthworm 20g, Poria cocos 10g, Panax notoginseng 15g.

[0106] The preparation method is as follows: weigh the above raw materials according to the ratio, add 7.5 times the weight of purified water, soak for 1.5 hours, heat to boiling and keep slightly boiling for 0.5 hours, filter to obtain a first decoction; add 5.0 times the weight of purified water to the residue, heat to boiling and keep slightly boiling for 2.5 hours, filter to obtain a second decoction; combine the first decoction and the second decoction, add pharmaceutically acceptable excipients through conventional procedures to prepare the Chinese medicine composition pills of the present invention.

[0107] Example 5

[0108] S1: Preparation of decoction of the Chinese medicine composition of the present invention

[0109] Chop and mix Rhodiola rosea, turtle shell, coix seed, earthworm, Poria cocos and Panax notoginseng, including 15g of Rhodiola rosea, 15g of turtle shell, 30g of coix seed, 15g of earthworm, 15g of Poria cocos and 15g of Panax notoginseng. Extract with distilled water, using a crude extraction system, 95℃, first extraction for 3h; 95℃, second extraction for 1h. The supernatants of the two extractions are homogenized to obtain the decoction extract of the Chinese medicine composition, which is stored in a sealed container.

[0110] S2: Isolation of extracellular vesicle particles derived from the Chinese medicine composition of the present invention

[0111] Extracellular vesicles were isolated from the decoction of the Chinese medicine composition by differential centrifugation. The extract of the decoction of the Chinese medicine composition collected in step S1 was centrifuged at 800×g for 10 min→2000×g for 20 min→5000×g for 35 min→10000×g for 60 min to remove large particles and cell debris. Then a sequential ultracentrifugation method was used, including centrifuging the final supernatant at 150000×g for 2 h in Himac ultracenfuge CP100NX, Ti45 rotor, and gently shaking the obtained particles in 600μL PBS and resuspending them at 4°C for 2 h to obtain Chinese medicine composition-derived extracellular vesicle particles. The final sample of Chinese medicine composition-derived extracellular vesicles was stored at -80°C.

[0112] Example 6

[0113] S1: Preparation of decoction of the Chinese medicine composition of the present invention

[0114] Chop and mix Rhodiola rosea, turtle shell, coix seed, earthworm, Poria cocos and Panax notoginseng, including 10g of Rhodiola rosea, 20g of turtle shell, 35g of coix seed, 10g of earthworm, 20g of Poria cocos and 10g of Panax notoginseng. Extract with distilled water, using a crude extraction system, 90℃, first extraction for 2h; 98℃, second extraction for 1.5h. The supernatant of the two extractions is homogenized to obtain the decoction extract of the Chinese medicine composition, which is stored in a sealed container.

[0115] S2: Isolation of extracellular vesicle particles derived from the Chinese medicine composition of the present invention

[0116] The decoction extract of the Chinese medicine composition collected in step S1 was centrifuged at 800×g for 15 min→2000×g for 15 min→5000×g for 35 min→10000×g for 65 min to remove large particles and cell debris. Then a sequential ultracentrifugation method was used, including centrifuging the final supernatant at 150000×g for 1.5 h in Himac ultracenfuge CP100NX, Ti45 rotor, gently shaking the obtained particles in 600 μL PBS, and resuspending them at 4°C for 1 h to obtain Chinese medicine composition-derived extracellular vesicle particles. The final sample of Chinese medicine composition-derived extracellular vesicles was stored at -80°C.

[0117] Example 7

[0118] S1: Preparation of decoction of the Chinese medicine composition of the present invention

[0119] Chop and mix Rhodiola rosea, turtle shell, coix seed, earthworm, Poria cocos and Panax notoginseng, including 20g of Rhodiola rosea, 10g of turtle shell, 25g of coix seed, 20g of earthworm, 10g of Poria cocos and 20g of Panax notoginseng. Extract with distilled water, using a crude extraction system, 98℃, 3.5h for the first extraction; 95℃, 1.5h for the second extraction. The supernatant of the two extractions is homogenized to obtain the decoction extract of the Chinese medicine composition, which is stored in a sealed container.

[0120] S2: Isolation of extracellular vesicle particles derived from the Chinese medicine composition of the present invention

[0121] The extract of the decoction of the Chinese medicine composition collected in step S1 was centrifuged at 800×g for 12 min→2000×g for 25 min→5000×g for 33 min→10000×g for 55 min to remove large particles and cell debris. Then, a sequential ultracentrifugation method was used, including centrifuging the final supernatant at 150000×g for 2 h in Himac ultracenfuge CP100NX, Ti45 rotor, and gently shaking the obtained particles in 600 μL PBS and resuspending them at 4°C for 3 h to obtain Chinese medicine composition-derived extracellular vesicle particles. The final sample of the Chinese medicine composition-derived extracellular vesicles was stored at -80°C.

[0122] Example 8

[0123] S1: Preparation of decoction of the Chinese medicine composition of the present invention

[0124] Chop and mix Rhodiola rosea, turtle shell, coix seed, earthworm, Poria cocos and Panax notoginseng, including 15g of Rhodiola rosea, 20g of turtle shell, 25g of coix seed, 20g of earthworm, 10g of Poria cocos and 10g of Panax notoginseng. Extract with distilled water, using a crude extraction system, 95℃, 3h for the first extraction; 98℃, 1.5h for the second extraction. The supernatant of the two extractions is homogenized to obtain the decoction extract of the Chinese medicine composition, which is stored in a sealed container.

[0125] S2: Isolation of extracellular vesicle particles derived from the Chinese medicine composition of the present invention

[0126] The extract of the decoction of the Chinese medicine composition collected in step S1 was centrifuged at 800×g for 10 min→2000×g for 20 min→5000×g for 35 min→10000×g for 65 min to remove large particles and cell debris. Then, a sequential ultracentrifugation method was used, including centrifuging the final supernatant at 150000×g for 2 h in Himac ultracenfuge CP100NX, Ti45 rotor, and gently shaking the obtained particles in 600 μL PBS, and resuspending them at 4°C for 1.5 h to obtain the Chinese medicine composition-derived extracellular vesicle particles. The final sample of the Chinese medicine composition-derived extracellular vesicles was stored at -80°C.

[0127] Comparative Example 1

[0128] A Chinese medicine composition for preventing or treating pulmonary fibrosis, comprising the following raw materials in parts by weight:

[0129] 15g of Rhodiola rosea, 15g of tortoise shell, 30g of Euryale ferox, 15g of earthworm, 15g of Poria cocos, and 15g of Panax notoginseng.

[0130] The preparation method is as follows: weigh the above raw materials according to the ratio, add 6.0 times the weight of purified water, soak for 1.5 hours, heat to boiling and keep slightly boiling for 1 hour, filter to obtain a first decoction; add 6.5 times the weight of purified water to the residue, heat to boiling and keep slightly boiling for 3 hours, filter to obtain a second decoction; combine the first decoction and the second decoction, concentrate the filtrate to a relative density of 1.15, and then spray dry, the dried fine powder is passed through a 100-mesh sieve, granulated, and dried to obtain the Chinese medicine composition granules.

[0131] Comparative Example 2

[0132] A Chinese medicine composition for preventing or treating pulmonary fibrosis, comprising the following raw materials in parts by weight:

[0133] Codonopsis pilosula 15g, turtle shell 15g, coix seed 30g, scorpion 15g, Poria 15g, Panax notoginseng 15g.

[0134] The preparation method is as follows: weigh the above raw materials according to the ratio, add 6.0 times the weight of purified water, soak for 1.5 hours, heat to boiling and keep slightly boiling for 1 hour, filter to obtain a first decoction; add 6.5 times the weight of purified water to the residue, heat to boiling and keep slightly boiling for 3 hours, filter to obtain a second decoction; combine the first decoction and the second decoction, concentrate the filtrate to a relative density of 1.15, and then spray dry, the dried fine powder is passed through a 100-mesh sieve, granulated, and dried to obtain the Chinese medicine composition granules.

[0135] Comparative Example 3

[0136] A Chinese medicine composition for preventing or treating pulmonary fibrosis, comprising the following raw materials in parts by weight:

[0137] 15g of Rhodiola rosea, 15g of turtle shell, 30g of coix seed, 15g of earthworm, 15g of Atractylodes macrocephala, and 15g of safflower.

[0138] The preparation method is as follows: weigh the above raw materials according to the ratio, add 6.0 times the weight of purified water, soak for 1.5 hours, heat to boiling and keep slightly boiling for 1 hour, filter to obtain a first decoction; add 6.5 times the weight of purified water to the residue, heat to boiling and keep slightly boiling for 3 hours, filter to obtain a second decoction; combine the first decoction and the second decoction, concentrate the filtrate to a relative density of 1.15, and then spray dry, the dried fine powder is passed through a 100-mesh sieve, granulated, and dried to obtain the Chinese medicine composition granules.

[0139] Comparative Example 4

[0140] A Chinese medicine composition for preventing or treating pulmonary fibrosis, comprising the following raw materials in parts by weight:

[0141] Rhodiola rosea 25g, turtle shell 25g, coix seed 20g, earthworm 25g, Poria cocos 5g, Panax notoginseng 5g.

[0142] The preparation method is as follows: weigh the above raw materials according to the ratio, add 6.0 times the weight of purified water, soak for 1.5 hours, heat to boiling and keep slightly boiling for 1 hour, filter to obtain a first decoction; add 6.5 times the weight of purified water to the residue, heat to boiling and keep slightly boiling for 3 hours, filter to obtain a second decoction; combine the first decoction and the second decoction, concentrate the filtrate to a relative density of 1.15, and then spray dry, the dried fine powder is passed through a 100-mesh sieve, granulated, and dried to obtain the Chinese medicine composition granules.

[0143] Comparative Example 5

[0144] S1: Preparation of decoction of the Chinese medicine composition of the present invention

[0145] Chop and mix Rhodiola rosea, turtle shell, coix seed, earthworm, Poria cocos and Panax notoginseng, including 25g of Rhodiola rosea, 10g of turtle shell, 15g of coix seed, 25g of earthworm, 25g of Poria cocos and 10g of Panax notoginseng. Extract with distilled water, using a crude extraction system, 95℃, first extraction for 3h; 95℃, second extraction for 1h. The supernatants of the two extractions are homogenized to obtain the decoction extract of the Chinese medicine composition, which is stored in a sealed container.

[0146] S2: Isolation of extracellular vesicle particles derived from the Chinese medicine composition of the present invention

[0147] The decoction extract of the traditional Chinese medicine composition collected in step S1 was centrifuged at 1000×g for 20 min→2000×g for 20 min→4000×g for 35 min→10000×g for 80 min to remove large particles and cell debris. Then, a sequential ultracentrifugation method was used, including centrifuging the final supernatant at 150000×g for 2 h in Himac ultracenfuge CP100NX, Ti45 rotor, and gently shaking the obtained particles in 600 μL PBS and resuspending them at 4°C for 2 h to obtain the extracellular vesicle particles of comparative example 5.

[0148] In vitro Example The inhibitory effect of the Chinese medicine composition of the present invention on the proliferation of human embryonic lung fibroblast cell line MRC-5

[0149] With reference to the CCK-8 method in Chinese patent CN111743903B, the proliferation inhibition effect of the Chinese medicine composition of the present invention on the human embryonic lung fibroblast cell line MRC-5 was determined.

[0150] For the powder of the Chinese medicine composition obtained in Example 2 and Comparative Examples 1-4, that is, the powder obtained by "combining the first decoction and the second decoction, concentrating the filtrate to a relative density of 1.15, and then spray drying, and passing the dried fine powder through a 100-mesh sieve" in the preparation method of Example 2 and Comparative Examples 1-4, the proliferation inhibitory effect of the Chinese medicine composition on the human embryonic lung fibroblast cell line MRC-5 was tested.

[0151] Human embryonic lung fibroblast cell line MRC-5 in logarithmic growth phase was obtained, digested and resuspended to make a cell culture medium with a density of 5×10 4 / mL of cell suspension was inoculated in a 96-well plate at an inoculum size of 200 μL per well. After adaptive culture for 24 h, the culture medium was replaced with a complete culture medium containing 5 μM of the Chinese medicine composition powder of Example 2, Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4, respectively. Three replicate wells were set for each concentration, and a culture solution without cells was set as a blank group. After continued culture in a constant temperature incubator at 37°C and 5% CO2 for 48 h, 10 μL of CCK-8 reagent was added to each well. After incubation for 4 h, the OD450 of each well was measured at a wavelength of 450 nm on an enzyme-linked immunosorbent assay device, and the average value of the three wells was taken to calculate the proliferation inhibition rate.

[0152] Table 1 The proliferation inhibition rate of the Chinese medicine composition of the present invention on the human embryonic lung fibroblast cell line MRC-5

[0153] Group Proliferation inhibition rate / % Example 2 Group 79.35±0.36 Comparative Example 1 42.74±0.33 Comparative Example 2 37.69±0.41 Comparative Example 3 46.11±0.34 Comparative Example 4 55.47±0.29

[0154] As can be seen from Table 1, the Chinese medicine composition of the present invention contains Rhodiola rosea, turtle shell, coix seed, earthworm, Poria cocos and Panax notoginseng in appropriate proportions, which play a synergistic role and have a significant inhibitory effect on human embryonic lung fibroblast cell line, indicating that the Chinese medicine composition of the present invention can be used to prevent and treat pulmonary fibrosis.

[0155] Efficacy Example 1 Study on the treatment of lung and spleen deficiency type pulmonary fibrosis by the Chinese medicine composition of the present invention

[0156] 1. Establishment of rat model of pulmonary fibrosis

[0157] 48 SPF SD rats, weighing 180-220g, were given free access to water and diet, and the room temperature was maintained at 25±1°C with a light-dark cycle of 12 hours. After one week of adaptive feeding, the rats were randomly divided into 8 groups according to body weight, with 6 rats in each group, namely, a blank control group, a model control group, Example 1 group, Example 2 group, Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group, and Comparative Example 4 group.

[0158] On the first day of the experiment, the rats in the model control group, embodiment group 1, embodiment group 2, comparative example group 1, comparative example group 2, comparative example group 3, and comparative example group 4 were anesthetized by intraperitoneal injection of 10% chloral hydrate (4mL / kg), and bleomycin liquid (5mg / kg) was injected into the trachea at one time to create a rat pulmonary fibrosis model. The model control group, embodiment group 1, embodiment group 2, comparative example group 1, comparative example group 2, comparative example group 3, and comparative example group 4 were irrigated with 2mL / rat of senna leaf water extract every day from the 2nd to the 16th day. During this period, the blank control group was fed normally, and the feeding time and feeding amount were parallel to the other 7 groups. On the 16th day of the experiment, the rat idiopathic pulmonary interstitial fibrosis lung and spleen deficiency type model was completed.

[0159] Starting from the 17th day of the experiment, the drug was administered once a day. The drug administration for each group was as follows:

[0160] The Example 1 group was intragastrically administered 0.01 mL / g (containing 10 g / kg of crude drug) of the Example 1 composition every day.

[0161] The Example 2 group was intragastrically administered with 0.01 mL / g (containing 10 g / kg of crude drug) of the Example 2 composition every day.

[0162] The comparative example 1 group was intragastrically administered with 0.01 mL / g (containing 10 g / kg of crude drug) of the comparative example 1 composition every day.

[0163] The comparative example 2 group was intragastrically administered with 0.01 mL / g (containing 10 g / kg of crude drug) of the comparative example 2 composition every day.

[0164] The comparative example 3 group was intragastrically administered with 0.01 mL / g (containing 10 g / kg of crude drug) of the comparative example 3 composition every day.

[0165] The comparative example 4 group was intragastrically administered with 0.01 mL / g (containing 10 g / kg of crude drug) of the comparative example 4 composition every day.

[0166] Rats in the blank control group and model control group were intragastrically administered with an equal volume of normal saline and maintained normally.

[0167] 2. Model observation and indicator detection

[0168] 2.1 Observe the general condition of rats after administration.

[0169] 2.2 On the 31st day of the experiment, 10% chloral hydrate was intraperitoneally injected into the 8 groups of rats, and the upper lobe of the right lung of the rats was removed, fixed with 10% formaldehyde, routinely dehydrated, paraffin-embedded, and sliced ​​to evaluate the degree of pulmonary fibrosis, and the grade data were converted into quantitative data. No alveolitis or pulmonary fibrosis (-, 1 point); mild alveolitis or pulmonary fibrosis (+, 2 points); moderate alveolitis or pulmonary fibrosis (++, 3 points); severe alveolitis or pulmonary fibrosis (+++, 4 points).

[0170] 2.3 Weigh 400-500 mg of the left lower lobe of the rat lung, prepare lung tissue homogenate in an ice bath, centrifuge at 4°C and 3000 r / min for 20 min, take the supernatant, and use ELISA to detect the content of TNF-α and IL-8 in the lung tissue homogenate.

[0171] 3. Statistical methods

[0172] SPSS 21.0 software was used for data processing, and the data were expressed as mean ± standard deviation. The data of each group were subjected to normal distribution test and variance homogeneity test, and the inter-group comparison was performed with one-way analysis of variance, with P < 0.05 as the difference being statistically significant.

[0173] 4. Experimental Results

[0174] 4.1 General Status

[0175] After modeling, the rats in the blank control group had normal physique, stretched body, quick reaction, agile movement, shiny fur, normal diet, weight gain, and even breathing; the rats in the model control group had long-term fatigue, listlessness, weight loss, slow movement, dull fur, reduced diet, loose stools, and rapid breathing.

[0176] After treatment, the rats in Example 1 and Example 2 groups showed corresponding improvement in symptoms compared with the model control group, with improved spirits, increased weight, more sensitive reactions, improved hair gloss, increased food intake, formed stools, and more even breathing.

[0177] The effects of Example 1 and Example 2 in improving the symptoms of rats were better than those of Comparative Example 1, Comparative Example 2, Comparative Example 3 and Comparative Example 4.

[0178] 4.2 Comparison of degree of pulmonary fibrosis

[0179] After modeling, the model control group had severe pulmonary fibrosis. After administration, compared with the model control group, the pulmonary fibrosis of Example 1 and Example 2 groups was significantly reduced. The effect of Example 2 on reducing the degree of pulmonary fibrosis was better than that of Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4. Figure 1 .

[0180] 4.3 Comparison of TNF-α and IL-8 contents

[0181] Compared with the blank control group, the content of TNF-α and IL-8 in the lung tissue homogenate of the model control group was significantly increased. After administration, compared with the model control group, the content of TNF-α and IL-8 in Example 1 and Example 2 was significantly reduced. The effect of Example 2 on reducing the content of TNF-α and IL-8 was better than that of Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4. Figure 2 , Figure 3 .

[0182] IL-8 is a pro-inflammatory cytokine, mainly secreted by alveolar macrophages, airway epithelial cells, neutrophils, lymphocytes, etc. It is a powerful neutrophil chemotactic factor. Through its chemotactic effect, it recruits inflammatory cells to migrate to the lung tissue for activation, releases some proteases to participate in the structural destruction and remodeling of the lung tissue, and plays a role in the occurrence of pulmonary fibrosis. It can not only engulf and kill invaders, but also induce and amplify inflammatory responses, leading to tissue destruction. Therefore, high levels of IL-8 play a role in initiating and amplifying inflammatory responses in the lungs, and through inflammatory responses, it can cause fibroblast proliferation and changes in lung structure.

[0183] TNF-α is a pro-inflammatory cytokine mainly produced by macrophages and monocytes, and is involved in normal inflammatory and immune responses. It can increase the adhesion factors synthesized by vascular endothelial cells, which is beneficial for the adhesion of inflammatory cells to the blood vessel wall, thereby recruiting neutrophils and lymphocytes to migrate and infiltrate from blood vessels to lung tissue, promoting chronic lung inflammation and even aggravating inflammatory reactions. Studies have shown that the level of TNF-α in lung tissue homogenates of bleomycin-induced pulmonary fibrosis is significantly increased.

[0184] The Chinese medicine composition of the present invention significantly reduces the content of TNF-α and IL-8 in lung tissue, indicating that the Chinese medicine composition of the present invention may weaken the chemotactic inflammatory effect of IL-8, improve the inflammatory changes in lung tissue and the degree of lung fibrosis, thereby playing a role in treating pulmonary fibrosis.

[0185] Efficacy Example 2 Study on the Effect of Extracellular Vesicles Derived from Traditional Chinese Medicine Composition on Pulmonary Fibrosis in Mice

[0186] 1. Animal Grouping and Dosing

[0187] Healthy ICR mice, SPF grade, male, 75, weighing (20±2) g, were randomly divided into 5 groups according to their weight, 15 in each group, namely normal control group, model control group, Example 5 group, Comparative Example 5 group, and Comparative Example 6 group. The mice were anesthetized by intraperitoneal injection of chloral hydrate 350 mg / kg, the normal control group was instilled with normal saline through the trachea, and the other 3 groups were instilled with bleomycin (5 mg / kg) through the trachea. Immediately after the instillation, the mice were rotated upright for 2 minutes to evenly distribute the drug solution in the lungs, and then returned to the cage for normal feeding.

[0188] Each group was given the following drugs:

[0189] Normal control group: gavage with an equal volume of normal saline;

[0190] Model control group: gavage with an equal volume of normal saline;

[0191] Group 5 of Example 5: intragastrically administered 300 mg / kg of the extracellular vesicles derived from the Chinese medicine composition of Example 5;

[0192] Comparative Example 5 group: intragastrically administered 300 mg / kg of the extracellular vesicle particles of Comparative Example 5;

[0193] Comparative Example 6 Group: Oral administration of 300 mg / kg of extracellular vesicles derived from Shenshi Sanjie recipe (the extracellular vesicles derived from Shenshi Sanjie recipe were prepared according to the method described in Example 1 of the patent with application number "2024103274204" and invention name "Application of extracellular vesicle particles derived from Shenshi Sanjie recipe in the preparation of drugs for preventing or treating pulmonary fibrosis").

[0194] The extracellular vesicles of Example 5, the extracellular vesicles of Comparative Example 5, and the extracellular vesicles of Comparative Example 6 were dissolved with normal saline and administered continuously from the second day after modeling, once a day. The normal control group and the model control group were given an equal volume of normal saline.

[0195] 2. Experimental methods and data processing

[0196] The weight of mice was measured daily, and the time of death of mice was recorded. On the 30th day after modeling, mice were anesthetized by intraperitoneal injection of 350 mg / kg chloral hydrate, and the mice were killed by bleeding from the abdominal aorta. The lungs of mice were taken and weighed. The lower lobe of the left lung of mice in each group was taken and fixed in 4% formaldehyde, and the remaining lung tissues were frozen and stored at -70°C.

[0197] 2.1 Statistics of mouse survival rate

[0198] The survival rate of mice on day 30 after model establishment was calculated.

[0199] 2.2 Calculation of mouse lung index

[0200] The lung index of mice in each group was calculated according to the following formula.

[0201]

[0202] 2.3 Calculation of the degree of pulmonary fibrosis in mice

[0203] After the lung tissue was fixed for 1 week, it was routinely dehydrated, embedded, and sliced. The degree of pulmonary fibrosis was evaluated according to the method of Szapiel et al. (Szapiel SV, Elson NA, Fulmer JD. Bleomycin-induced interstitial pulmonary disease in the nude, athymic mouse [J]. Am Rev Respir Dis, 1979, 120: 893), and the grade data were converted into quantitative data. No alveolitis or pulmonary fibrosis (-, 1 point); mild alveolitis or pulmonary fibrosis (+, 2 points); moderate alveolitis or pulmonary fibrosis (++, 3 points); severe alveolitis or pulmonary fibrosis (+++, 4 points).

[0204] 2.4 Data Statistics and Analysis

[0205] SPSS 21.0 software was used for data processing, and the data were expressed as mean ± standard deviation. The data of each group were subjected to normal distribution test and variance homogeneity test, and the inter-group comparison was performed with one-way analysis of variance, with P < 0.05 as the difference being statistically significant.

[0206] 3. Results and Discussion

[0207] 3.1 Effect of extracellular vesicles derived from the Chinese medicine composition of the present invention on the survival rate of mice with pulmonary fibrosis

[0208] Table 2 Effect of extracellular vesicles derived from the Chinese medicine composition of the present invention on the survival rate of mice with pulmonary fibrosis

[0209]

[0210]

[0211] The results in Table 2 show that on the 30th day after modeling, the survival rate of mice in the model control group was 53.3%; the survival rate of mice in the Example 5 group of the present invention was 93.3%, indicating that the extracellular vesicles derived from the Chinese medicine composition in Example 5 of the present invention can significantly improve the survival rate of mice with pulmonary fibrosis.

[0212] Compared with the comparative example 5 group, the effect of the present invention example 5 group on improving the survival rate of mice with pulmonary fibrosis is better. The effect of the present invention example 5 group on improving the survival rate of mice with pulmonary fibrosis is better than that of the comparative example 6 group.

[0213] 3.2 Effect of extracellular vesicles derived from the Chinese medicine composition of the present invention on the lung index of mice with pulmonary fibrosis

[0214] like Figure 4 As shown, Example 1 of the present invention reduced the lung index of mice with pulmonary fibrosis.

[0215] The effect of reducing the lung index of mice with pulmonary fibrosis in group 5 of the present invention is better than that in group 5 of the comparative example. The effect of reducing the lung index of mice with pulmonary fibrosis in group 5 of the present invention is better than that in group 6 of the comparative example.

[0216] 3.3 Effect of extracellular vesicles derived from the Chinese medicine composition of the present invention on lung tissue lesions in mice with pulmonary fibrosis

[0217] like Figure 5 As shown, on the 30th day after modeling, the degree of lung fibrosis in the model control group was very serious, and the extracellular vesicles in Example 5 of the present invention could significantly reduce the degree of lung fibrosis.

[0218] The 5th group of Example of the present invention significantly reduced the degree of pulmonary fibrosis, and the effect of the 5th group of Example of the present invention on reducing the degree of pulmonary fibrosis was better than that of the 5th group of Comparative Example. The 5th group of Example of the present invention on reducing the degree of pulmonary fibrosis was better than that of the 6th group of Comparative Example.

[0219] According to the method of the above-mentioned efficacy example, the inventors verified the effect of the extracellular vesicles derived from the Chinese medicine composition of Examples 6-8 on pulmonary fibrosis. The results showed that the extracellular vesicles derived from the Chinese medicine composition of Examples 6-8 significantly improved the survival rate of mice with pulmonary fibrosis, reduced the lung index, and reduced the degree of pulmonary fibrosis, and had a significant therapeutic effect on pulmonary fibrosis.

[0220] In summary, the extracellular vesicles derived from the traditional Chinese medicine composition of the present invention have the effect of preventing and / or treating pulmonary fibrosis.

[0221] The above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description, and the obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. A Chinese medicine composition for preventing or treating pulmonary fibrosis, characterized in that: The Chinese medicine composition is prepared from rhodiola rosea, turtle shell, coix seed, earthworm, poria and panax notoginseng; the Chinese medicine composition is prepared from the following raw materials in parts by weight: 10-20 parts of rhodiola rosea, 10-20 parts of turtle shell, 25-35 parts of coix seed, 10-20 parts of earthworm, 10-20 parts of poria and 10-20 parts of panax notoginseng.

2. The Chinese medicine composition according to claim 1, characterized in that: The Chinese medicine composition comprises the following raw materials in parts by weight: 15 parts of Rhodiola rosea, 15 parts of Carapax tricolor, 30 parts of Coix seeds, 15 parts of Pheretima, 15 parts of Poria cocos and 15 parts of Panax notoginseng.

3. The Chinese medicine composition according to any one of claims 1 or 2, characterized in that The preparation method of the Chinese medicine composition is as follows: Weigh the above raw materials according to the ratio, add 5.0-7.5 times the weight of purified water, soak for 0.5-2h, heat to boiling and keep slightly boiling for 0.5-1.5h, filter to obtain a first decoction; add 5.0-7.5 times the weight of purified water to the residue, heat to boiling and keep slightly boiling for 2.5-3.5h, filter to obtain a second decoction; combine the first decoction and the second decoction to obtain the Chinese medicine composition of the present invention.

4. The Chinese medicine composition according to any one of claims 1 or 2, characterized in that The dosage form of the Chinese medicine composition is decoction, pill, gel, tablet, mixture, capsule, granule or powder.

5. Use of the Chinese medicine composition according to any one of claims 1 or 2 in the preparation of a drug for preventing or treating pulmonary fibrosis.

6. A Chinese medicine composition-derived extracellular vesicle, characterized in that: The Chinese medicine composition-derived extracellular vesicle particles are obtained by extracting and separating the Chinese medicine composition according to claim 1, and the Chinese medicine composition consists of the following components in parts by weight: 10-20 parts of Rhodiola rosea, 10-20 parts of turtle shell, 25-35 parts of coix seeds, 10-20 parts of earthworms, 10-20 parts of Poria cocos and 10-20 parts of Panax notoginseng.

7. The Chinese medicine composition-derived extracellular vesicles according to claim 6, characterized in that: The traditional Chinese medicine composition consists of the following components in parts by weight: 15 parts of rhodiola rosea, 15 parts of turtle shell, 30 parts of coix seeds, 15 parts of earthworms, 15 parts of poria cocos and 15 parts of notoginseng.

8. The Chinese medicine composition-derived extracellular vesicles according to claim 6, characterized in that: The method for preparing the extracellular vesicle particles derived from the traditional Chinese medicine composition comprises the following steps: Step S1: Preparation of decoction of Chinese medicine composition Chop and mix Rhodiola rosea, turtle shell, coix seed, earthworm, Poria cocos and Panax notoginseng, extract with distilled water, use a crude extraction system, and homogenize the supernatants of the two extractions to obtain a decoction extract of the Chinese medicine composition; Step S2: Isolation of extracellular vesicle particles derived from traditional Chinese medicine composition The extract of the decoction of the traditional Chinese medicine composition collected in step S1 is centrifuged at 800×g for 5-15 min→2000×g for 15-25 min→5000×g for 25-35 min→10000×g for 50-70 min in sequence; the final supernatant is centrifuged at 150000×g in a centrifuge for 1.5h-2.5h using a sequential ultracentrifugation method, the obtained particles are gently shaken in PBS, and resuspended at 4°C for at least 1h to obtain the traditional Chinese medicine composition-derived extracellular vesicle particles.

9. The Chinese medicine composition-derived extracellular vesicles according to claim 6, characterized in that: In step S1, the weight ratio of Rhodiola rosea, turtle shell, coix seed, earthworm, Poria cocos and Panax notoginseng is (10-20):(10-20):(25-35):(10-20):(10-20):(10-20). In step S1, the crude extraction system is 95±5°C, the first extraction is 2-5h; 95±5°C, the second extraction is 0.5-2h; In the step S1, the crude extraction system is 95°C, the first extraction is 3h; 95°C, the second extraction is 1h; in the step S2, centrifugation is performed at 800×g for 10min; In step S2, centrifugation at 2000×g for 20 min; In step S2, centrifugation at 5000×g for 35 min; In the step S2, centrifugation at 10000×g for 60 min; In step S2, centrifuge at 150,000×g for 2 h; In step S2, the obtained particles are resuspended at 4° C. for 1-5 h.

10. Use of the extracellular vesicle particles derived from the traditional Chinese medicine composition according to claim 6 in the preparation of a drug for preventing or treating pulmonary fibrosis.

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