Traditional Chinese medicine composition for treating hypoxic pulmonary hypertension and preparation and application thereof

Abstract

The invention discloses a traditional Chinese medicine composition for treating hypoxic pulmonary hypertension and a preparation and application thereof, and belongs to the technical field of traditional Chinese medicine and network pharmacology. The traditional Chinese medicine composition comprises the following raw material medicines: choerospondias axillaris, myristica fragrans, sandalwood, salvia miltiorrhiza, ligusticum wallichii, angelica sinensis, codonopsis pilosula, cistanche deserticola, semen cuscutae, astragalus membranaceus, rhodiola rosea and herba epimedii. The traditional Chinese medicine composition disclosed by the invention is mainly used for regulating and controlling proliferation and migration processes of pulmonary artery smooth muscle cells and pulmonary artery endothelial cells and reaction to hypoxia and inflammation through multiple targets and multiple paths. Molecular docking results show that the core components of the traditional Chinese medicine composition and core intersection targets have good binding activity, molecular dynamics simulation verifies the molecular docking results, and the related action mechanism is clear. And meanwhile, through network pharmacology research, more choices and discussion bases are provided for clinical prescription application.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine and network pharmacology, and in particular to a traditional Chinese medicine composition for treating hypoxic pulmonary hypertension, and a preparation and application thereof. Background Art

[0002] Hypoxic pulmonary hypertension (HPH) is a type of pulmonary hypertension (PH) caused by chronic lung disease or diseases that cause hypoxemia. It belongs to type 3 PH. Due to the increase in pulmonary artery blood flow resistance caused by pulmonary vasoconstriction and abnormal proliferation of pulmonary artery smooth muscle and pulmonary artery endothelium under persistent hypoxic environment, persistent pulmonary hypertension can lead to increased right ventricular pressure, which can lead to right heart failure or even death in severe cases. Current estimates show that the prevalence of PH accounts for about 1% of the global population. Since PH has both cardiac and pulmonary causes, the prevalence is higher in individuals over 65 years old. Although modern medical treatments for HPH can improve patient symptoms, they cannot solve the underlying vascular lesions, cannot prevent or reverse the disease, and patients with pulmonary diseases have a worse prognosis and more adverse events. Therefore, it is very important to find a variety of safe and effective treatment methods. In recent years, more and more studies have confirmed the therapeutic effect of traditional Chinese medicine on HPH.

[0003] There is no record of the disease name "hypoxic pulmonary hypertension" in ancient Chinese medical books, but from the perspective of clinical symptoms and signs, as the disease progresses, HPH patients often show coughing and expectoration, wheezing, dyspnea, cyanosis of lips and nails, palpitations and edema, etc., which are similar to lung distension in Chinese medicine, so it is often classified as "lung distension", "wheezing syndrome", "cough" and "phlegm and fluid" in Chinese medicine. "Lingshu·Distension Theory" says "empty and full, wheezing and coughing"; Chao Yuanfang's "Zhubingyuanhoulun·Cough, Reverse and Shortness of Qi" says "lung deficiency is injured by slight cold, then cough, cough, then qi returns to the lungs, then lung distension, then qi reversal, and the lung is originally weak, qi is insufficient, and it is taken advantage of by evil, so it cannot be ventilated, so cough, reverse and shortness of breath." Zhu Danxi wrote in "Danxi Xinfa": "Lung distension and cough... This is phlegm with blood stasis, which hinders qi and causes illness." Ancient physicians believed that lung distension was a mixed symptom of deficiency and excess, with lung qi deficiency as the root cause and phlegm and blood stasis as the symptom. Modern scholars Li Qianqian et al. discussed HPH from the perspective of the lung's main regulation and believed that the course of HPH is the process of the lung's gradual failure to regulate qi, blood, and water. The onset of HPH is based on qi deficiency, and qi deficiency, blood stasis, and water retention are its important pathogenesis. In the early stage of HPH, qi deficiency is the main symptom, and the disease is located in the lung, with blood stasis and phlegm turbidity endogenous. The treatment focuses on replenishing qi and restoring the lung's regulating function on "qi". In the late stage, blood stasis and water retention are the main symptoms, from the lung to the heart, with water and fluid overflow, and qi deficiency is also present. The treatment focuses on promoting blood circulation and removing blood stasis, promoting diuresis and expelling fluid, and focusing on restoring the lung's regulating function on "blood and water" (see Li Qianqian et al. Exploring the TCM differentiation and treatment ideas of hypoxic pulmonary hypertension from the perspective of the lung's main regulation and regulation [J], Journal of Guangzhou University of Chinese Medicine. 2024, 41(07): 1905-1910). Liu Min et al. believe that the pathological process of HPH is qi deficiency, blood stasis, phlegm blocking local lung collaterals. Qi deficiency is the initiating factor, blood stasis and phlegm blocking are secondary pathological changes, and lung collaterals obstruction is the local pathological result. At the same time, qi deficiency runs through the entire course of the disease, and it evolves from lung qi deficiency to lung and kidney qi deficiency. Therefore, the basic treatment principles should be to tonify the lungs and kidneys, promote blood circulation and eliminate phlegm, and dredge the lung collaterals. Based on the opinions of ancient and modern doctors, it is generally believed that HPH is a disease of mixed deficiency and excess, with qi deficiency as the root cause and phlegm and blood stasis as the symptoms. The disease is mainly located in the lungs and may involve the heart, spleen and kidneys. The treatment should be to invigorate qi, activate blood circulation and dredge collaterals, warm yang, resolve phlegm and relieve asthma (see Liu Min et al. Clinical observation on the treatment of hypoxic pulmonary hypertension with Yifei Tongluo prescription [J]. Journal of Clinical Traditional Chinese Medicine, 2006, (03): 260-261; Liu Min et al. TCM understanding of hypoxic pulmonary vascular reconstruction [J]. Chinese Journal of Emergency in Traditional Chinese Medicine, 2006, (08): 869-870+938).

[0004] Although TCM has conducted some research on the pathology and treatment of HPH, the mechanism of action of the existing related Chinese patent medicines is unclear, and the effects of the medicines lack effective demonstration. There is no report on related Chinese medicine compositions that can meet the treatment needs of patients and have the effects of invigorating Qi, activating blood circulation, unblocking meridians, warming Yang, resolving phlegm and relieving asthma, and the mechanism of action is clear. Summary of the invention

[0005] Based on this, it is necessary to provide a Chinese medicine composition for treating hypoxic pulmonary hypertension, its preparation and application, in order to address the problems that the mechanism of action of the Chinese medicine formula currently used to treat hypoxic pulmonary hypertension is unclear and the effect of the medicine is lacking effective demonstration.

[0006] The invention provides a traditional Chinese medicine composition for treating hypoxic pulmonary hypertension. The traditional Chinese medicine composition comprises the following raw materials: jujube, nutmeg, santalin, salvia miltiorrhiza, chuanxiong, angelica, codonopsis, cistanche, dodder seed, astragalus, rhodiola rosea and epimedium.

[0007] Preferably, the traditional Chinese medicine composition comprises the following raw materials in parts by weight: 8-12 parts of Ziziphus jujuba, 4-12 parts of nutmeg, 4-8 parts of santalin, 8-12 parts of salvia miltiorrhiza, 8-12 parts of chuanxiong, 8-12 parts of angelica, 8-12 parts of codonopsis pilosula, 4-12 parts of cistanche, 4-12 parts of dodder seed, 4-18 parts of astragalus, 4-12 parts of rhodiola rosea, and 4-12 parts of epimedium.

[0008] The present invention also provides a traditional Chinese medicine preparation for treating hypoxic pulmonary hypertension, which is prepared from the traditional Chinese medicine composition for treating hypoxic pulmonary hypertension described in any of the aforementioned technical solutions.

[0009] Preferably, the Chinese medicine preparation is a decoction, and the decoction is prepared according to the following steps:

[0010] (1) Weigh the raw material according to weight, add 5-10 times the weight of water and soak for 30-120 minutes;

[0011] (2) heating and boiling the soaked API together with water, filtering, and collecting the filtrate;

[0012] (3) adding 5-10 times the weight of water to the residue, boiling, filtering, and collecting the filtrate;

[0013] (4) The filtrates obtained after the two decoctions are combined, heated and concentrated to obtain a decoction.

[0014] Preferably, the two decoctions are each 30-60 minutes in duration.

[0015] Preferably, the filtrates are combined and concentrated by heating to 10%-20% of the original volume.

[0016] In addition, the present invention also provides the use of the traditional Chinese medicine composition for treating hypoxic pulmonary hypertension described in any of the aforementioned technical solutions in the preparation of a drug for treating hypoxic pulmonary hypertension.

[0017] The Chinese medicine composition for treating hypoxic pulmonary hypertension of the present invention is explained as follows:

[0018] Hypoxic pulmonary hypertension is a mixed disease of deficiency and excess, with deficiency of qi as the root and phlegm and blood stasis as the symptom. The disease is mainly located in the lungs, which may involve the heart, spleen and kidneys. The treatment should be to invigorate qi, activate blood circulation and unblock collaterals, warm yang and resolve phlegm and relieve asthma. The heart governs blood vessels, while the lungs govern the regulation of qi, blood and water. The primordial qi in the lungs can help the heart circulate blood. The functions of the heart and lungs complement each other. If one organ is different, it will also affect the function of the other organ. Therefore, treatment cannot only treat a single organ. This prescription uses Astragalus, Codonopsis and Rhodiola to invigorate qi to replenish deficiency. Astragalus can also relieve stagnation and relieve numbness, and Rhodiola can also activate blood circulation and relieve asthma; Danshen and Angelica are used to activate blood circulation to resolve blood stasis, and Ziziphus jujuba and Chuanxiong are used to promote qi, activate blood circulation and resolve blood stasis. Ziziphus jujuba also has the effect of nourishing the heart; for those who treat phlegm and fluid, warming medicines should be used to harmonize them. Nutmeg and sandalwood are used to warm the middle and promote qi to remove phlegm; Cistanche, Cuscuta and Epimedium are used to warm the kidney yang to resolve phlegm. All the medicines are used together to treat both the symptoms and the root cause, and to coordinate the heart, lungs, kidneys and spleen, to achieve the effects of invigorating Qi, activating blood circulation, unblocking meridians, warming Yang, resolving phlegm and relieving asthma.

[0019] Specifically, each Chinese medicine raw material has the following pharmacological properties:

[0020] Choerospondias axillaris: It is the mature fruit of Choerospondias axillaris (Roxb.) Burtt et Hill, a plant of the Anacardiaceae family. It tastes sweet, sour, and flat. It has the effects of promoting qi and blood circulation, nourishing the heart and calming the mind.

[0021] Nutmeg: It is the dried seed kernel of the plant Myristica fragrans Houtt. of the Myristicaceae family. It is pungent and warm in taste. It enters the spleen, stomach, and large intestine meridians. It has the effects of warming the middle and promoting qi, astringing the intestines and stopping diarrhea.

[0022] Sandalwood: It is the dried heartwood of the trunk of Santalum album L., a plant of the Santalaceae family. It is pungent and warm in taste. It enters the spleen, stomach, heart, and lung meridians. It has the effects of promoting qi and warming the middle, stimulating appetite and relieving pain.

[0023] Danshen: It is the dried root and rhizome of Salvia miltiorrhiza Bge., a plant of the Labiatae family. It tastes bitter, slightly cold, and enters the heart and liver meridians. It has the effects of promoting blood circulation and removing blood stasis, regulating menstruation and relieving pain, clearing the heart and eliminating restlessness, cooling blood and eliminating carbuncle.

[0024] Chuanxiong: It is the rhizome of the plant Chuanxiong Hort. of the Umbelliferae family. It is pungent and warm in taste. It enters the liver, gallbladder, and pericardium meridians. It has the effects of promoting blood circulation and qi, dispelling wind and relieving pain.

[0025] Angelica sinensis: It is the dried root of Angelica sinensis (Oliv.) Diels, a plant of the Umbelliferae family. It is sweet and spicy, warm in nature, and enters the liver, heart, and spleen meridians. It has the effects of nourishing blood, activating blood circulation, regulating menstruation and relieving pain, and moistening the intestines and facilitating bowel movements.

[0026] Codonopsis: It is the dried root of Codonopsis pilosula (Franch.) Nannf., Codonopsis pilosula Nannf. var. modesta (Nannf.) LTShen or Codonopsis tangshen Oliv. of Campanulaceae. It is sweet, flat, and enters the spleen and lung meridians. It has the effects of tonifying the spleen and lungs, nourishing blood and promoting body fluid.

[0027] Cistanche deserticola: It is the dried fleshy stem with scaly leaves of Cistanche deserticola Y.c.Ma or Cistanche tubulosa (Schenk) wight of the Orobanchaceae family. It tastes sweet and salty, is warm, and enters the kidney and large intestine meridians. It has the effects of nourishing kidney yang, benefiting essence and blood, and moistening the intestines and relieving constipation.

[0028] Cuscuta: It is the dried mature seeds of Cuscuta australis R.Br or Cuscutachinensis Lam., a plant of the Convolvulaceae family. It is pungent, sweet, and neutral in taste. It enters the liver, kidney, and spleen meridians. It has the effects of nourishing the liver and kidney, consolidating semen and reducing urine, calming the fetus, improving eyesight, stopping diarrhea, and eliminating wind and removing spots when used externally.

[0029] Astragalus: It is the dried root of Astragalus membranaceus (Fisch.) Bge. var. Mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge. of the Leguminosae family. It is sweet and slightly warm. It enters the lung and spleen meridians. It has the effects of replenishing qi and raising yang, consolidating the exterior and stopping sweating, promoting diuresis and reducing swelling, promoting body fluid and nourishing blood, relieving stagnation and relieving numbness, expelling toxins and draining pus, and healing sores and promoting tissue regeneration.

[0030] Rhodiola rosea: It is the dried root and rhizome of Rhodiola crenulata (Hook.f.et Thoms.) H.Ohba, a plant of the Crassulaceae family. It tastes sweet and bitter, is neutral, and enters the lung and heart meridians. It has the effects of invigorating qi and promoting blood circulation, unblocking meridians and relieving asthma.

[0031] Epimedium: The dried leaves of Epimedium brevicornu Maxim., Epimedium sagittatum (Sieb. et Zucc.) Maxim., Epimedium pubescens Maxim. or Epimedium koreanum Nakai, all of which belong to the Berberidaceae family. It tastes spicy, sweet and warm, and enters the liver and kidney meridians. It has the effects of tonifying kidney yang, strengthening tendons and bones, and removing rheumatism.

[0032] The effective ingredients, targets, pathways, molecular docking and molecular simulations of the Chinese medicine composition for treating hypoxic pulmonary hypertension of the present invention are obtained through network pharmacology research as follows:

[0033] The active ingredients of the Chinese medicine composition of the present invention for treating HPH were preliminarily screened out by network pharmacology method as quercetin, kaempferol, luteolin, β-sitosterol, isorhamnetin, etc. Quercetin has antioxidant, anti-inflammatory and anti-cancer effects, can effectively inhibit coronary artery and pulmonary artery vasoconstriction and lower blood pressure, and inhibit PASMC (pulmonary artery smooth muscle) proliferation by regulating MMP-9, MMP-2, chemokine receptors CXCR4, integrinei, integrina5 and other proteins related to cell proliferation and migration and various functions and TrkA / AKT signal transduction pathway, and increase the ratio of Bax / Bcl-2 to promote its apoptosis. Kaempferol has anti-inflammatory, antioxidant, antibacterial and antiviral effects. It can improve pulmonary vascular remodeling in rats with chronic hypoxia-induced pulmonary hypertension by regulating the Akt-GSK3β-cyclin axis. It can reduce the phosphorylated protein levels of Akt and GSK3β, reduce the expression of pro-cell proliferation proteins and anti-apoptotic proteins, increase the expression of pro-apoptotic proteins, inhibit PASMC proliferation and promote cell apoptosis. Luteolin is a common flavonoid in medicinal plants, with strong anti-infection activity and effective anti-injury ability. AKT1 is one of the three closely related serine / threonine protein kinases, and is also a downstream signal of phosphatidylinositol 3-kinase (PI3K) to mediate the effects of various growth factors, such as epidermal growth factor (EGF). Smooth muscle mitogens stimulate G protein-coupled receptors and tyrosine kinases, thereby activating the downstream signal transduction pathway PI3K and inducing DNA synthesis in airway smooth muscle cells. Therefore, luteolin can inhibit the proliferation of pulmonary artery smooth muscle by inhibiting the phosphorylation level of AKT. Luteolin can also affect the activity of K+ channels in rat lung tissue and pulmonary artery by upregulating Kv1.5 and Ca in PASMCs. 2+ Activated K +Channels to dilate the pulmonary artery and reduce pulmonary hypertension. Secondly, luteolin can also downregulate CyclinD1, CDK4 and PCNA in the lung tissue of HPH rats, while upregulating the Bax / Bcl-2 / caspase-3 axis, reducing the proliferation of pulmonary artery smooth muscle and inducing apoptosis by regulating the cell cycle. β-Sitosterol is a natural steroid compound. β-Sitosterol is very similar to cholesterol in structure and has antibacterial, anti-inflammatory, anti-tumor, anti-hyperlipidemic, anti-atherosclerotic, serum cholesterol-lowering and platelet aggregation-inhibiting effects. In recent years, β-Sitosterol has shown a good inhibitory effect on liver cancer, gastric cancer, colorectal cancer, lung cancer and breast cancer. It has a good application prospect in the prevention and treatment of cancer, but research on HPH is still lacking. Pulmonary hypertension is also called a tumor of the cardiovascular system because its development mechanism is similar to that of tumors, both of which are caused by abnormal cell proliferation. β-Sitosterol exerts anti-tumor effects mainly by promoting cell cycle arrest, inducing cell apoptosis, inhibiting cancer cell migration and invasion, mediating immune response, regulating oxidative stress and inhibiting multidrug resistance of cancer cells. β-Sitosterol can inhibit the proliferation of cancer cells by regulating the expression levels of cycle-related proteins such as cyclin D1 and CDC25B, promoting cell cycle arrest and accelerating the accumulation of reactive oxygen species to damage DNA. Isorhamnetin is a natural flavonoid compound that exists in many natural plants. Oxidative stress is widely present in HPH. Many studies have found that flavonoid compounds have a good inhibitory effect on oxidative stress. The formation mechanism of pulmonary hypertension is related to cell damage caused by oxidative stress and pulmonary vascular remodeling caused by cell proliferation. Studies have shown that isorhamnetin can play an anti-oxidative stress role by increasing Nrf2 protein and inhibiting cell proliferation by inhibiting the pc-src / NOX1 signaling pathway, thereby playing a role in treating hypoxic pulmonary hypertension. Other studies have found that isorhamnetin can inhibit inflammation and reduce lung cell damage by inhibiting 1α-hemolysin, and can reduce oxidative stress in cardiomyocytes and inhibit TNF-α-induced HPASMC proliferation. These effects may be exerted through the BMP signaling pathway.

[0034] The molecular docking validation results also showed that quercetin, luteolin, kaempferol, β-sitosterol and isorhamnetin had strong binding activity with the main targets (AKT1, MAPK1, RELA, EGFR, TP53). Therefore, the Chinese medicine composition of the present invention may mainly regulate HPH through quercetin, luteolin, kaempferol, β-sitosterol and isorhamnetin.

[0035] The target of the Chinese medicine composition of the present invention for regulating HPH is mainly concentrated on AKT1. AKT1 is one of three closely related serine / threonine protein kinases, called AKT kinase, which can regulate many processes, including metabolism, proliferation, cell survival, growth and angiogenesis. In addition, AKT1 is also a signal downstream of phosphatidylinositol 3-kinase (PI3K) to mediate the effects of various growth factors, such as epidermal growth factor receptor EGFR. EGFR is also another main concentrated target of the Chinese medicine composition of the present invention for regulating HPH. The expression of TrkA gene is related to the proliferation, apoptosis, cell cycle and migration of hypoxic PASMCs. Hypoxia upregulates the expression activity of TrkA, and its downstream expression protein AKT also shows the same direction of change. In the main signal pathway PI3K-Akt signal pathway for regulating HPH by the Chinese medicine composition of the present invention, AKT1 is also one of the key links. Epidermal growth factor receptor (EGFR) is an important tyrosine kinase receptor that regulates cell growth and differentiation. Mutation or overexpression of EGFR will cause kinase activity disorder, thereby leading to cell proliferation. The signal pathway of resistance to EGFR tyrosine kinase inhibitors (EGFR tyrosine kinase inhibitor resistance) is also one of the main signal pathways for the Chinese medicine composition of the present invention to regulate HPH. In addition, the pathway with the highest degree value and the most gene enrichment in the network topology of the drug-target-pathway of the Chinese medicine composition of the present invention to regulate hypoxic pulmonary hypertension is the cancer signal pathway (Pathways incancer). Pulmonary hypertension is also called "cancer of the cardiovascular system" because of its mechanism of abnormal proliferation of pulmonary arterial smooth muscle and modern medical treatment methods can only improve symptoms but cannot prevent or reverse the disease. Cancer signal pathways play a key role in HPH, so it can be speculated that many active ingredients that play a role in cancer can also play a role in the treatment of HPH.

[0036] The molecular simulation results show that the RMSD of small molecules reaches stable fluctuations in the early stage of simulation, and fluctuates stably in the range of 0-1 angstroms (very small). The RMSF of most regional proteins is within 2 angstroms. The binding energy of AKT1-beta-sitosterol is -42.62±3.92kcal / mol, and the main contribution of binding is van der Waals energy, followed by electrostatic energy and non-polar solvation free energy. In summary, the complex binding movement process is stable, the protein is rigid after binding, and the binding affinity is strong, which is consistent with the molecular docking results.

[0037] Compared with the prior art, the present invention has the following advantages:

[0038] (1) The Chinese medicine composition of the present invention has low-cost raw materials, is simple to prepare, and is convenient to use. After many years of clinical application and pharmacological research, it has significant therapeutic effects in invigorating qi, promoting blood circulation, unblocking collaterals, warming yang, resolving phlegm, and relieving asthma.

[0039] (2) The main compounds of the Chinese medicine composition of the present invention for treating hypoxic pulmonary hypertension are quercetin, kaempferol, luteolin, β-sitosterol, isorhamnetin, etc. These compounds can participate in the regulation of cancer signaling pathways, PI3K-Akt, MAPK, epidermal growth factor receptor tyrosine kinase inhibitor resistance and other signaling pathways through targets such as AKT1, MAPK1, RELA, EGFR, TP53, etc., thereby exerting anti-inflammatory, antioxidant, cell proliferation and migration inhibition, and pulmonary hypertension reduction effects.

[0040] (3) The Chinese medicine composition of the present invention mainly regulates the proliferation and migration of pulmonary artery smooth muscle cells and pulmonary artery endothelial cells and the response to hypoxia and inflammation through multiple targets and multiple pathways. The results of molecular docking show that the core components of the Chinese medicine composition of the present invention have relatively good binding activity with the core intersection targets. The molecular dynamics simulation verifies the molecular docking results, and the relevant mechanism of action is clear. At the same time, through network pharmacology research, more choices and discussion bases are provided for clinical prescription and medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 A Venn diagram of potential drug targets and HPH targets for the Chinese medicine composition for treating hypoxic pulmonary hypertension according to an embodiment of the present invention;

[0042] Figure 2 This is a PPI interaction network diagram of potential therapeutic targets of the Chinese medicine composition according to the embodiment of the present invention;

[0043] Figure 3 This is a network diagram of drug-ingredient-HPH abnormal target points of the Chinese medicine composition according to an embodiment of the present invention;

[0044] Figure 4 GO-BP analysis diagram of target genes of the Chinese medicine composition according to the embodiment of the present invention;

[0045] Figure 5 GO-CC analysis diagram of the target gene of the Chinese medicine composition according to the embodiment of the present invention;

[0046] Figure 6 This is a GO-MF analysis diagram of the target gene of the Chinese medicine composition according to the embodiment of the present invention;

[0047] Figure 7 This is a KEGG pathway enrichment analysis diagram of the target gene of the Chinese medicine composition in the embodiment of the present invention;

[0048] Figure 8This is a network diagram of components of the Chinese medicine composition-HPH abnormality target-pathway in an embodiment of the present invention;

[0049] Fig. 9 This is a diagram showing the docking structure of the active ingredient of the Chinese medicine composition and the core target molecule in the embodiment of the present invention (wherein, Fig. 9 A is the molecular docking structure display diagram of AKT1-beta-sitosterol. Fig. 9 B is the diagram showing the MAPK1-luteolin molecular docking structure. Fig. 9 C is the diagram showing the docking structure of RELA-luteolin molecule. Fig. 9 D is the diagram showing the EGFR-quercetin molecular docking structure. Fig. 9 E is a diagram showing the docking structure of TP53-luteolin molecules);

[0050] Fig.10 This is an interaction diagram between the small molecule beta-sitosterol and AKT1 protein in an embodiment of the present invention;

[0051] Fig.11 RMSD diagram of the small molecule beta-sitosterol and AKT1 protein in the embodiment of the present invention;

[0052] Fig.12 This is the RMSF graph of the small molecule beta-sitosterol and AKT1 protein according to the examples of the present invention. DETAILED DESCRIPTION

[0053] A traditional Chinese medicine composition for treating hypoxic pulmonary hypertension, comprising the following raw materials: jujube, nutmeg, santalin, salvia miltiorrhiza, chuanxiong, angelica, codonopsis, cistanche, dodder seed, astragalus, rhodiola rosea, and epimedium.

[0054] In one of the preferred embodiments, the Chinese medicine composition comprises the following raw materials in parts by weight: 8-12 parts of Ziziphus jujuba, 4-12 parts of nutmeg, 4-8 parts of santalin, 8-12 parts of salvia miltiorrhiza, 8-12 parts of chuanxiong, 8-12 parts of angelica, 8-12 parts of codonopsis pilosula, 4-12 parts of cistanche, 4-12 parts of dodder seed, 4-18 parts of astragalus, 4-12 parts of rhodiola rosea, and 4-12 parts of epimedium.

[0055] A traditional Chinese medicine preparation for treating hypoxic pulmonary hypertension is prepared from the traditional Chinese medicine composition for treating hypoxic pulmonary hypertension described in any of the previous technical solutions.

[0056] In one preferred embodiment, the Chinese medicine preparation is a decoction, and the decoction is prepared according to the following steps:

[0057] (1) Weigh the raw material according to weight, add 5-10 times the weight of water and soak for 30-120 minutes;

[0058] (2) heating and boiling the soaked API together with water, filtering, and collecting the filtrate;

[0059] (3) adding 5-10 times the weight of water to the residue, boiling, filtering, and collecting the filtrate;

[0060] (4) The filtrates obtained after the two decoctions are combined, heated and concentrated to obtain a decoction.

[0061] In one preferred embodiment, the two decoctions are each 30-60 minutes in duration.

[0062] In one preferred embodiment, the filtrates are combined and then heated and concentrated to 10%-20% of the original volume.

[0063] The traditional Chinese medicine composition for treating hypoxic pulmonary hypertension can be used for preparing medicine for treating hypoxic pulmonary hypertension.

[0064] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0065] Example 1

[0066] Screening of effective ingredients of traditional Chinese medicine composition for treating hypoxic pulmonary hypertension

[0067] In the traditional Chinese medicine database TCMSP (http: / / lsp.Nwsuaf.edu.cn / tcmsp.php), the main compounds of nutmeg, sandalwood, salvia miltiorrhiza, chuanxiong, angelica, Codonopsis pilosula, Cistanche deserticola, Cuscuta australis, Astragalus membranaceus, and Epimedium were retrieved, and the compounds with OB≥30% and DL≥0.18 were retained; in addition, the drugs Rhodiola rosea and Ziziphus chinensis that could not be retrieved in the TCMSP database were supplemented by the ETCM database and literature, and the SMILES structures of the active compounds of Ziziphus chinensis and Rhodiola rosea were retrieved in the PubChem database, and ADME screening was performed through the Swiss website. The effective ingredients of nutmeg, sandalwood, salvia miltiorrhiza, chuanxiong, angelica, codonopsis, cistanche, cuscuta, astragalus and epimedium were preliminarily extracted through TCMSP, ETCM database and literature mining. According to drug ADME screening, 9 effective ingredients of nutmeg, 3 effective ingredients of sandalwood, 65 effective ingredients of salvia miltiorrhiza, 7 effective ingredients of chuanxiong, 2 effective ingredients of angelica, 21 effective ingredients of codonopsis, 5 effective ingredients of cistanche, 11 effective ingredients of cuscuta, 20 effective ingredients of astragalus, 23 effective ingredients of epimedium, 19 effective ingredients of jujube, and 23 effective ingredients of rhodiola rosea were extracted. After deduplication, a total of 166 effective ingredients were extracted, as shown in Table 1.

[0068] Table 1 Main active ingredients of Chinese medicine composition

[0069]

[0070]

[0071]

[0072]

[0073] Example 2

[0074] Screening of effective ingredient targets and disease targets of traditional Chinese medicine compositions for the treatment of hypoxic pulmonary hypertension

[0075] The protein targets of the active compounds of the traditional Chinese medicine composition except Rhodiola rosea and Ziziphus jujuba were obtained through the TCMSP database, the SMILES structures of the active compounds of Ziziphus jujuba and Rhodiola rosea were retrieved in the PubChem database, the corresponding targets were retrieved through the SwissTarget Prediction database, and the targets with "Probability greater than or equal to 0.1" were retained. Finally, 68 targets of nutmeg ingredients, 85 targets of santalin, 139 targets of salvia miltiorrhiza, 30 targets of ligusticum chuanxiong, 53 targets of angelica, 111 targets of codonopsis pilosula, 180 targets of cistanche deserticola, 222 targets of dodder seeds, 218 targets of astragalus, 227 targets of epimedium, 159 targets of jujube spinosa, and 247 targets of rhodiola rosea were obtained. After integration and deduplication, the protein targets of the compounds were converted into gene names using the Uiprot protein database, and a total of 426 targets were retained.

[0076] In the GeneCards database, the disease genes of HPH were searched with "hypoxic pulmonary hypertension" as the condition, and the species was selected as "Homo spaiens", and 5013 targets were obtained. Because there were too many disease targets, the quartile sites were selected, that is, the score value ≥ 13.1, and 1253 disease genes were screened out.

[0077] Example 3

[0078] Prediction of potential targets of traditional Chinese medicine combination for treating hypoxic pulmonary hypertension, construction of intersection gene PPI network and network diagram of traditional Chinese medicine combination drug-ingredient-HPH abnormal target

[0079] In order to identify the potential targets of TCM compositions for the treatment of HPH, 426 TCM composition drug targets and 1253 disease genes were introduced into Venny2.1 for Wayne analysis, and 169 potential therapeutic targets of TCM compositions for the treatment of HPH were screened out. The data were visualized. Figure 1 .

[0080] In order to clarify the interactions between potential targets of traditional Chinese medicine combination for HPH treatment, the 169 intersection genes obtained by Wayne analysis were imported into the STRING database, and the biological species was set as "Homosapiens" to obtain the PPI interaction network diagram of potential therapeutic targets. Figure 2 .

[0081] Based on the obtained effective ingredients, active ingredient genes, HPH targets and potential targets of TCM composition drugs for treating diseases, Network and Type tables were established and imported into CytoScape software. The network topology parameters between TCM composition drugs, TCM composition ingredients and intersection genes were analyzed using the built-in tools of CytoScape software. The core targets and the main active ingredients that exert the efficacy were determined according to the degree values, and a network diagram of TCM composition drugs-ingredients-HPH abnormal targets was constructed. Figure 3 .

[0082] Example 4

[0083] Function and pathway enrichment analysis of abnormal targets of traditional Chinese medicine composition for the treatment of hypoxic pulmonary hypertension-HPH

[0084] In order to clarify the role of a group of genes in different biological backgrounds and genes related to certain metabolic pathways in a gene set, the potential therapeutic targets of traditional Chinese medicine combination for HPH were imported into the DAVID database, and GO and KEGG enrichment analysis was performed on the intersection genes, where GO enrichment analysis included biological process BP, cell component CC and molecular function MF, and KEGG was pathway enrichment analysis. The results were visualized on the microbiological information platform.

[0085] 169 potential therapeutic targets were imported into the DAVID database for GO functional analysis, and 489 entries (BP384, CC36, MF69) were obtained with FDR < 0.05 as the screening condition. According to the FDR value, the top 20 BP, CC and MF data were imported into the microbial information platform and drawn into bubble charts for analysis. Figure 4-Figure 6 In terms of biological process BP, it involves negative regulation of apoptosis, positive regulation of gene expression, response to hypoxia, cell response to lipopolysaccharide, positive regulation of cell population proliferation, positive regulation of cell migration, positive regulation of smooth muscle cell proliferation, positive regulation of kinase activity, negative regulation of gene expression, inflammatory response, positive regulation of transcription by RNA polymerase II, positive regulation of protein phosphorylation, etc.; in terms of cellular component CC, it involves extracellular space, receptor complex, extracellular region, membrane raft, cell surface, plasma membrane, outer side of plasma membrane, etc.; in terms of molecular function MF, it involves enzyme binding, identical protein binding, transmembrane receptor protein tyrosine kinase activity, protein tyrosine kinase activity, protein binding, endopeptidase activity, etc.

[0086] KEGG pathway analysis was performed on 169 targets, and 162 pathways were obtained with FDR less than 0.05, involving the pathogenesis of cancer, AGE-RAGE signaling pathway in diabetic complications, blood lipids and atherosclerosis, fluid shear stress and atherosclerosis, PI3K-Akt signaling pathway, resistance to epidermal growth factor receptor tyrosine kinase inhibitors, MAPK signaling pathway, etc. The first 20 pathways were visualized and analyzed, see Figure 7 .

[0087] Example 5

[0088] Construction of a network diagram of components of a traditional Chinese medicine composition for the treatment of hypoxic pulmonary hypertension, abnormal targets of HPH, and pathways

[0089] According to the obtained active ingredients of the Chinese medicine composition, HPH abnormal targets and the top 20 pathways with the smallest FDR value, the Network and Type tables were established and imported into the Cytoscape software for visualization to obtain the Chinese medicine composition component-HPH abnormal target-pathway network diagram. The Cytoscape software was used to draw and analyze the relationship network diagram, and a total of 253 nodes (including 20 pathways, 64 active ingredients and 169 targets) and 1296 relationships were obtained, see Figure 8 .

[0090] The network topology parameters between the components of the Chinese medicine composition, HPH abnormal targets, and pathways were analyzed using the built-in tools of the CytoScape software. According to the dgree value, i.e., the node connectivity, the core components and core action targets were obtained. Cytoscape network analysis showed that the degree of connectivity of quercetin was 206, the betweenness centrality was 0.2615, and the closeness centrality was 0.4251. It was predicted that quercetin was the main component of the Chinese medicine composition of the present invention for the treatment of HPH, followed by luteolin (connectivity was 114, betweenness was 0.0304, and closeness was 0.3491), kaempferol, β-sitosterol, and isorhamnetin, as shown in Table 2.

[0091] Table 2 Main active ingredients of Chinese medicine compositions and characteristic parameters of network nodes

[0092]

[0093] Example 6

[0094] Component-target molecular docking of traditional Chinese medicine composition for the treatment of hypoxic pulmonary hypertension

[0095] After topological analysis of the network, the core compounds quercetin, luteolin, kaempferol, beta-sitosterol, and isorhamnetin were screened for molecular docking with the core intersection targets AKT1, MAPK1, RELA, EGFR, and TP53. The 3D structure mol2 format of the components in the TCMSP database was converted to pdbqt using Open Babel software. The 3D structure of the key targets in the PDB database was removed from water molecules and small molecule accessories using PyMoL software to obtain a pdbqt format file; then the key targets were hydrogenated using Autodock tools software; finally, the molecular docking was completed using Autodock vina 1.1.2, and the microscopic and macroscopic images of the docking mode were drawn using PyMol software and Adobe Illustrator software. The lower the binding energy of the molecular docking, the greater the binding potential of the two molecules. Most of the molecular docking results of the core compounds and core targets are less than -7.0 kcal / mol, and the remaining values ​​are also close to -7.0 kcal / mol, that is, most targets have good binding activity with the components and are tightly bound, as shown in Table 3.

[0096] Table 3 Molecular docking binding energy

[0097]

[0098] The five docking combinations of AKT1-beta-sitosterol (-11.9 kcal / mol), MAPK1-luteolin (-7.86 kcal / mol), RELA-luteolin (-8.696 kcal / mol), EGFR-quercetin (-9.913 kcal / mol), and TP53-luteolin (-8.39 kcal / mol) were selected to draw the docking model, corresponding to Fig. 9 In Fig. 9 A. Fig. 9 B. Fig. 9 C. Fig. 9 D and Fig. 9 E. The energy matching between ligand and receptor includes electrostatic interaction, hydrogen bonding, van der Waals interaction and hydrophobic interaction. The interaction between the two parties in the five docking groups is mainly through hydrogen bonding, and the length of hydrogen bonding is between 1 and Not equal. Fig. 9 A is the binding mode of AKT1-beta-sitosterol. Beta-sitosterol forms one hydrogen bond with SER-205 and ASN-204 respectively, and its length is Fig. 9 B is the binding mode of MAPK1-luteolin, with a total of 4 hydrogen bonds. Luteolin forms one hydrogen bond with GLN-22 and SER-202, respectively, with lengths of Forms 2 hydrogen bonds with HIS-299, both with length Fig. 9 C is the binding mode of RELA-luteolin, with a total of 5 hydrogen bonds. Luteolin forms one hydrogen bond with TYR-42, ALA-157, and LYS-158 respectively, with lengths of Forms two hydrogen bonds with ASP-41, with lengths of and Fig. 9 D is the binding mode of EGFR-quercetin, with a total of 7 hydrogen bonds. Quercetin forms one hydrogen bond with ILE-759 and LEU-788, with lengths of Form three hydrogen bonds with ASP-855, the lengths are The lengths of the two hydrogen bonds formed with THR-854 are Fig. 9 E is the binding mode of TP53-luteolin. There are 5 hydrogen bonds in total. Luteolin forms 1 hydrogen bond with MET-246, ASN-247 and GLY-244 respectively, with lengths of Forms two hydrogen bonds with SER-240, both with length

[0099] Fig.10 This is the interaction diagram of the small molecule beta-sitosterol and AKT1 protein. From the figure, we can see that beta-sitosterol forms a hydrogen bond with ASN-204 on the AKT1 protein, and the formation of hydrogen bonds makes the protein and the small molecule bind more tightly. It also forms a hydrophobic interaction with VAL-270, ASP-292, ILE-84, ASN-54, LEU-264, TYR-272, LEU-210, and TRP-80, providing a strong van der Waals force for the molecule.

[0100] Example 7

[0101] Molecular dynamics simulation of traditional Chinese medicine composition for treating hypoxic pulmonary hypertension

[0102] Based on the small molecule and protein complex obtained by docking, the structure with the best binding energy was selected as the initial structure, and all-atom molecular dynamics simulation was performed respectively. The simulation was performed using AMBER 22 software. Before the simulation, the charge of the small molecule was calculated by the antechamber module and the Hartree–Fock (HF) SCF / 6-31G* of the gaussian 09 software. Afterwards, the small molecule and protein were described by the GAFF2 small molecule force field and the ff14SB protein force field, respectively. Each system used the LEaP module to add hydrogen atoms to the system. Add a truncated octahedral TIP3P solvent box at a distance of 1.5 m and add Na + / Cl - Used to balance the system charge and finally output the topology and parameter files for simulation.

[0103] The molecular dynamics simulation was performed using AMBER 22 software. Before the simulation, the system was energy optimized, including 2500 steps of the steepest descent method and 2500 steps of the conjugate gradient method. After the system energy optimization was completed, the system was heated for 200ps at a fixed volume and constant heating rate to slowly increase the system temperature from 0K to 298.15K. While maintaining the system temperature at 298.15K, a 500ps NVT (isothermal isobaric) system simulation was performed to make the solvent molecules more evenly distributed in the solvent box. Finally, under the NPT (isothermal and isobaric) condition, the entire system was subjected to a 500ps equilibrium simulation. Finally, the two composite systems were subjected to 100ns NPT (isothermal and isobaric) system simulations under periodic boundary conditions. During the simulation, the non-bonded cutoff distance was set to The particle mesh Ewald (PME) method was used to calculate the long-range electrostatic interaction, the SHAKE method was used to limit the hydrogen atom bond length, and the Langevin algorithm was used for temperature control, where the collision frequency γ was set to 2ps. -1 The system pressure was 1 atm, the integration step was 2 fs, and the trajectory was saved every 10 ps for subsequent analysis.

[0104] The root mean square deviation of molecular dynamics simulation can reflect the movement process of the complex. The larger the RMSD and the more violent the fluctuation, the more violent the movement. On the contrary, the movement is stable. Fig.11 As shown in the figure, there is no large RMSD fluctuation during the protein simulation, which means that the protein structure has not collapsed, providing a basis for the binding of small molecules; the RMSD of small molecules reaches stable fluctuations in the early stage of the simulation, and fluctuates stably in the range of 0-1 angstroms (very small). This shows that the small molecule binds very closely to the protein and the binding stability of the small molecule is very good.

[0105] RMSF can reflect the flexibility of proteins during molecular dynamics simulation. Usually, after drugs bind to proteins, the flexibility of proteins decreases, thereby stabilizing the proteins and exerting the enzyme activity. Fig.12 As shown, except for the local region of the protein, the RMSF of the protein is within 2 angstroms, indicating that the main structure of the protein is very rigid, which may be achieved by combining with the small molecule beta-sitosterol.

[0106] Example 8

[0107] Calculation of binding free energy of a traditional Chinese medicine compound system for treating hypoxic pulmonary hypertension

[0108] The binding free energy between the protein and the ligand of all systems was calculated by the MM / GBSA method. In this application, the MD trajectory of 90-100ns was used for calculation, and the specific formula is as follows:

[0109] ΔG bind =ΔG complex –(ΔG receptor +ΔG ligand )

[0110] =ΔE internal +ΔE VDW +ΔE elec +ΔG GB +ΔG SA

[0111] In this formula, ΔE internal represents internal energy, ΔE VDW represents the van der Waals interaction and ΔE elec represents the electrostatic interaction, ΔG bind =ΔG complex –(ΔG receptor +ΔG ligand ) represents binding free energy, ΔG bind =ΔG complex –(ΔG receptor +ΔG ligand ) represents the free energy of the complex, ΔG receptor represents protein free energy, ΔG ligand represents the ligand free energy. The internal energy includes the bond energy (E bond ), angular energy (E angle ), and torsional energy (E torsion );ΔG GB and ΔG GA Collectively referred to as solvation free energy. GB is the polar solvation free energy, G SA is the nonpolar solvation free energy. GBIn this paper, the GB model developed by Nguyen et al. is used for calculation (igb = 2). Nonpolar solvation free energy (ΔG SA ) is calculated based on the product of surface tension (γ) and solvent accessible surface area (SA), ΔG SA =0.0072×ΔSASA. Entropy change is ignored in this application due to its high consumption of computing resources and low precision.

[0112] Based on the trajectory of molecular dynamics simulation, the MM-GBSA method was used to calculate the binding energy, which can more accurately reflect the binding effect of small molecules and target proteins. As shown in Table 4, the binding energy of AKT1-beta-sitosterol is -42.62±3.92kcal / mol. A negative value indicates that the two molecules have binding affinity with the target protein, and the lower the value, the stronger the binding. Obviously, the above calculations show that the binding affinity of AKT1-beta-sitosterol is very strong. Through residue decomposition, it can be seen that the main contribution to the binding of AKT1-beta-sitosterol is van der Waals energy, followed by electrostatic energy and non-polar solvation free energy.

[0113] Table 4 Binding free energy and energy components predicted by MM / GBSA

[0114]

[0115] Note:

[0116] ΔE vdw : Van der Waals

[0117] ΔE elec :Electrostatic Energy

[0118] ΔG GB :Polar Solvation Free Energy

[0119] ΔG SA : Nonpolar Solvation Free Energy

[0120] ΔG bind Binding free energy

[0121] Although the present invention has been described in detail in the embodiments through general explanations, specific implementation methods and experiments, modifications or improvements can still be made without departing from the core of the present invention, and all belong to the scope of protection required by the present invention.

Claims

1. A Chinese medicine composition for treating hypoxic pulmonary hypertension, characterized in that: The Chinese medicine composition comprises the following raw materials: jujube, nutmeg, sandalwood, salvia miltiorrhiza, chuanxiong, angelica, codonopsis, cistanche, dodder seed, astragalus, rhodiola rosea and epimedium.

2. The Chinese medicine composition for treating hypoxic pulmonary hypertension according to claim 1, characterized in that: The traditional Chinese medicine composition comprises the following raw materials in parts by weight: 8-12 parts of Ziziphus jujuba, 4-12 parts of nutmeg, 4-8 parts of santalin, 8-12 parts of salvia miltiorrhiza, 8-12 parts of chuanxiong, 8-12 parts of angelica, 8-12 parts of codonopsis pilosula, 4-12 parts of cistanche, 4-12 parts of dodder seeds, 4-18 parts of astragalus, 4-12 parts of rhodiola rosea, and 4-12 parts of epimedium.

3. A Chinese medicine preparation for treating hypoxic pulmonary hypertension, characterized in that: The Chinese medicine preparation is prepared from the Chinese medicine composition for treating hypoxic pulmonary hypertension according to any one of claims 1-2.

4. The Chinese medicine preparation for treating hypoxic pulmonary hypertension according to claim 3, characterized in that: The Chinese medicine preparation is a decoction, and the decoction is prepared according to the following steps: (1) Weigh the raw material according to weight, add 5-10 times the weight of water and soak for 30-120 minutes; (2) heating and boiling the soaked API together with water, filtering, and collecting the filtrate; (3) adding 5-10 times the weight of water to the residue, boiling, filtering, and collecting the filtrate; (4) The filtrates obtained after the two decoctions are combined, heated and concentrated to obtain a decoction.

5. The Chinese medicine preparation for treating hypoxic pulmonary hypertension according to claim 4, characterized in that: The decoction time for both times is 30-60 minutes.

6. The Chinese medicine preparation for treating hypoxic pulmonary hypertension according to claim 4, characterized in that: The filtrates were combined and concentrated by heating to 10%-20% of the original volume.

7. Use of the Chinese medicine composition for treating hypoxic pulmonary hypertension according to any one of claims 1 to 2 in the preparation of a medicament for treating hypoxic pulmonary hypertension.

Images