Use of ergosterol in the preparation of a drug for treating pulmonary arterial hypertension

Abstract

The application provides application of ergosterol in preparation of a medicine for treating pulmonary arterial hypertension. A preparation method of the ergosterol is as follows: Cordyceps militaris Haining strain fruiting bodies are taken, washed, crushed, and dried to obtain Cordyceps militaris powder; cholic acid, deoxycholic acid, polyethylene glycol monomethyl ether and anhydrous ethanol are mixed to obtain an ethanol mother liquor of a supramolecular solvent; Cordyceps militaris extraction liquid is gradiently and cooperatively extracted; vacuum distillation, drying, chromatographic separation, drying, recrystallization, washing and drying are sequentially performed, and the ergosterol is obtained. In the application, CO2 expanded ethanol liquid is used as a solvent, and a cholic acid-deoxycholic acid-polyethylene glycol monomethyl ether supramolecular solvent system technology is used. Relying on the homologous adaptability of the steroid nucleus structure of cholic acid and deoxycholic acid and ergosterol, a multi-level cavity recognition network is constructed, the C5-C6 double bond and the C22-C23 double bond sites of ergosterol are specifically combined, and the content of ergosterol in the extract is improved by regulating the cavity aperture through the polyethylene glycol monomethyl ether.

Description

Technical Field

[0001] This invention relates to the technical field of natural product extraction and application, specifically to the application of ergosterol in the preparation of drugs for treating pulmonary hypertension. Background Technology

[0002] Cordyceps fungi, a traditional and precious medicinal resource in my country, have a long history of application in the treatment of respiratory and cardiovascular diseases. Clinically, they are often used as an adjunct treatment for conditions such as pulmonary heart disease and chronic obstructive pulmonary disease. Cordyceps militaris (… Cordyceps militaris As a representative species of the Cordyceps genus, Cordyceps sinensis has become a high-quality medicinal fungal resource that can replace wild Cordyceps sinensis due to its mature artificial cultivation technology and stable content of active ingredients. It is rich in various active ingredients such as cordycepin, polysaccharides, adenosine, and ergosterol. Modern pharmacological studies have confirmed that these components can exert pharmacological effects through immune regulation, anti-inflammation, anti-oxidation, and inhibition of abnormal cell proliferation, showing broad application prospects in the fields of medicine, health food, and biotechnology.

[0003] Pulmonary arterial hypertension (PAH) is a serious chronic cardiovascular disease characterized by progressively increased pulmonary vascular resistance, often referred to as "cancer of the cardiovascular system." Its pathological mechanisms are complex, with the core issue being pulmonary vascular remodeling, particularly the abnormal proliferation of human pulmonary artery smooth muscle cells (hPASMCs) due to an imbalance between proliferation and apoptosis. Under normal physiological conditions, hPASMCs are in a resting state, but in the lung tissue of PAH patients, their proliferative activity is significantly enhanced, leading to narrowing of the pulmonary arterioles, increased pulmonary vascular resistance, and ultimately, right ventricular failure.

[0004] Over the past 25 years, PAH treatments have evolved from traditional calcium channel blockers to targeted therapies such as prostacyclin derivatives, endothelin receptor antagonists, and phosphodiesterase 5 inhibitors. While these drugs can slow disease progression, significant limitations remain: First, they have significant side effects; for example, prostacyclin derivatives can easily cause headaches, facial flushing, and gastrointestinal reactions, leading to poor patient adherence with long-term use. Second, they target only a single point of attack; existing drugs mostly focus on improving vasodilation and cannot fundamentally inhibit pulmonary vascular remodeling. Third, treatment costs are high; imported targeted drugs cost over 100,000 yuan annually, which is unaffordable for ordinary patients. Therefore, developing novel PAH treatments that are safe, have diverse targets, and are cost-effective has become an urgent need in the pharmaceutical field.

[0005] Screening PAH treatment active ingredients from natural products has unique advantages - natural product components have diverse structures, good biocompatibility, and often have multi-target regulation. Ergosterol, as the key active ingredient in Cordyceps militaris, has a unique tetracyclic triterpenoid structure (containing C5-C6 double bond, C22-C23 double bond and multiple chiral centers), which is extremely difficult to synthesize artificially and can only rely on natural product extraction. However, the existing ergosterol extraction process has many technical bottlenecks:

[0006] Defects of traditional solvent extraction method: such as Cong Wenhua patent (CN03133692.2) uses alcohol / ether mixed solvent hot extraction, the amount of organic solvent is 8-10 times the mass of the material, not only the production cost is high, but also there is the risk of solvent residue (such as ethanol residue easily exceeds the 0.5% limit value specified in the Chinese Pharmacopoeia), which affects the safety of the product for medical use;

[0007] Limitations of subcritical water extraction method: Yang Wenya et al. (China Oil and Fat, 2016) showed that subcritical water extraction needs to be carried out at a high temperature of 140℃ or above, although there is no solvent residue, but ergosterol is easily oxidized and degraded (degradation rate can reach 15%-20%) at high temperature, resulting in reduced product activity, and the energy consumption of high temperature operation is 3-4 times that of conventional process;

[0008] Limitations of ionic liquid extraction method: Zhang Wei et al. used [C4MIM] PF6 ionic liquid to extract active ingredients from Cordyceps militaris, the unit price of ionic liquid is more than 200 yuan / 100mL, and multiple centrifugation (3000r / min, 30min) is needed for product separation, which has low separation efficiency, high cost and is difficult to industrialize;

[0009] Improvement space of supercritical CO2 extraction method: Zhang Qian et al. (Edible Fungi, 2020) used 45MPa, 50℃ supercritical CO2 to extract Cordyceps militaris, only through simple crushing and sieving pretreatment, the particle size of the raw material is uneven (passing through a 60 mesh sieve, about 250μm), which leads to extraction dead angle and poor selectivity of ergosterol - the content of ergosterol in the extract is only 3.5%-4.2%, and there are a large amount of β - sterol impurities such as sitosterol and stigmasterol, which are difficult to purify subsequently.

[0010] In addition, the existing technology mainly focuses on the research of ergosterol in the fields of antioxidant and antitumor, and there is no report on its application in PAH treatment, and there is a lack of targeted verification for the pathological mechanism of PAH (such as hPASMCs proliferation inhibition and pulmonary vascular remodeling regulation). Therefore, developing a green and efficient, high-selectivity ergosterol extraction method and clarifying its mechanism of action in treating PAH is the key to breaking through the technical bottlenecks of the existing technology and expanding the application value of active ingredients in Cordyceps militaris. SUMMARY

[0011] Technical problems to be solved: In view of the above technical problems, the purpose of the present application is to provide the application of ergosterol in the preparation of drugs for treating pulmonary arterial hypertension, to construct a multi-level cavity recognition network relying on the homologous adaptability of the steroidal nucleus structure of cholic acid (3 hydroxyl groups) and deoxycholic acid (2 hydroxyl groups) and ergosterol, to specifically bind the C5-C6 double bond and C22-C23 double bond sites of ergosterol, and to improve the separation factor of ergosterol and the content of ergosterol in the extract through the regulation of the cavity aperture by polyethylene glycol monomethyl ether.

[0012] Technical scheme: The application of ergosterol in the preparation of drugs for treating pulmonary arterial hypertension.

[0013] Further, the ergosterol is derived from cordyceps militaris.

[0014] Further, the preparation method of the ergosterol comprises the following steps:

[0015] Step 1: Select the Bombyx mori Cordyceps militaris Haining strain fruiting body, wash and drain the surface water, freeze in a-40℃ low temperature freezer for 2h, put into an ultrafine grinder at a speed of 11000-13000r / min for 25-35s, then send the crushed material into an air flow classification equipment, screen out Cordyceps militaris powder with a particle size of 10-20μm, collect and reserve; the coarse powder with a particle size greater than 20μm is returned to the ultrafine grinder for secondary crushing, vacuum drying, and the moisture content is controlled at 8-10%, to obtain Cordyceps militaris powder;

[0016] Step 2: Take cholic acid, deoxycholic acid and polyethylene glycol monomethyl ether, add anhydrous ethanol, stir at a speed of 450-550r / min for 30min, ultrasonic treat at a power of 300W and a temperature of 30℃ for 15min, so that the cholic acid and deoxycholic acid are completely dissolved and form supramolecular assemblies, and an ethanol mother liquor containing 10-12% supramolecular solvents is obtained;

[0017] Step 3: Gradient synergistic extraction:

[0018] Phase one: low-polarity impurity extraction (0-60min): control the CO2 (purity 99.99%) flow rate at 1.5-1.6mL / min, the anhydrous ethanol flow rate at 0.15-0.17ml / min, and the mixture of the two to form CO2-expanded ethanol liquid; load the Cordyceps militaris powder into a fixed bed reactor, control the system pressure at 10MPa and the reactor temperature at 34-36℃, and let the CO2-expanded ethanol liquid flow into the fixed bed reactor from the lower section of the material, and collect the low-polarity impurity extraction liquid (containing aliphatic compounds and monoterpenes) at the bottom of the separator;

[0019] Phase two: ergosterol targeted extraction (60-150min)

[0020] Close the valve of the anhydrous ethanol storage tank, and introduce the ethanol mother liquor of the supramolecular solvent at a flow rate of 0.2-0.3 mL / min into the mixer, control the CO2 flow rate to be 2.0-2.2 mL / min, and mix to form an expanded liquid. The expanded liquid is introduced into the fixed bed reactor, the temperature is raised to 40℃, the system pressure is adjusted to 17.5-18.5 MPa, and the cordyceps militaris extract is collected;

[0021] Step 4: The cordyceps militaris extract is subjected to vacuum distillation and drying to obtain an extract. The extract is dissolved in 100% ethanol to a concentration of 100 mg / mL, filtered through a 0.25 μm microporous filter, and then subjected to chromatographic separation. The fractions collected at 25.4-30 min are concentrated to remove the elution solvent, and then dried at 60℃. A good solvent is added to fully dissolve the mixture, which is then naturally volatilized at 26℃. The obtained crystals are filtered, washed with -4℃ n-hexane, and then dried to obtain the product.

[0022] Further, in the airflow classification device in step 1, the classification wheel rotates at a speed of 7700-8200 r / min, the airflow speed is 14-16 m / s, and the temperature in the classification cavity is 25℃.

[0023] Further, in step 2, the mass ratio of cholic acid to deoxycholic acid is 2: (1-1.2), and the amount of polyethylene glycol monomethyl ether added is 0.2% of the total mass of cholic acid and deoxycholic acid.

[0024] Further, in step 4, the vacuum distillation conditions are: temperature of 60℃, vacuum degree of -0.09 MPa, and distillation time of 1.5-2.5 h.

[0025] Further, in step 4, the chromatographic separation is performed using a DAC (150×250 mm C188 μm) column, the ultraviolet detection wavelength is 282 nm, the flow rate is 600 mL / min, and the elution mode is isocratic elution with 98% methanol water solution for 40 min.

[0026] Further, in step 4, the good solvent is a mixture of n-hexane and dichloromethane, and the volume ratio of n-hexane to dichloromethane is 3:1.

[0027] Further, in the drug for treating pulmonary arterial hypertension, the mass fraction of ergosterol is 0.5%-12%, and the dosage form is selected from inhalants, liposomes, tablets, capsules, or injections.

[0028] Further, the formula of the drug for treating pulmonary arterial hypertension comprises:

[0029] Formula 1: The above ergosterol, fritillaria thunbergii miq., dihydroquercetin, apricot (bitter) powder, citrus reticulata blanco powder, osmanthus fragrans lour powder, sweet orange powder, snow pear powder, lily powder, loquat powder, and licorice powder; or,

[0030] Formula 2: The above ergosterol, Zhebeimu, ginseng peptide, Dangshen, Huangqi and magnesium stearate; or,

[0031] Formula 3: The above ergosterol, dihydroquercetin, almond (bitter) powder, dried tangerine peel powder, osmanthus powder, sweet orange powder, snow pear powder, lily powder, loquat powder and licorice powder; or,

[0032] Formula 4: The above ergosterol, ginseng peptide, Dangshen, Huangqi and magnesium stearate. Beneficial effects

[0033] 1. The present application significantly improves the specific surface area of the raw material and the contact efficiency of the solvent by using the low-temperature freezing-ultrafine grinding-airflow grading combined pretreatment technology, using-40℃ low temperature to make the ice crystals formed by the water in the cordyceps militaris cells destroy the cell wall and cell membrane structure, ultrafine grinding to refine the material, and airflow grading to ensure uniform particle size.

[0034] 2. The present application uses CO2 expanded ethanol liquid as a solvent, and uses the cholic acid-deoxycholic acid-polyethylene glycol monomethyl ether supramolecular solvent system technology, relying on the homologous adaptability of the steroidal nucleus structure of cholic acid (3 hydroxyl groups) and deoxycholic acid (2 hydroxyl groups) and ergosterol, to construct a multi-level cavity recognition network, specifically binding the C5-C6 double bond and C22-C23 double bond sites of ergosterol, and at the same time, through the regulation of the pore size of polyethylene glycol monomethyl ether, the separation factor of ergosterol and the content of ergosterol in the extract are improved.

[0035] 3. The present application uses ethanol concentration-pressure-temperature gradient synergistic extraction technology, phase one uses low-polarity solvent to preferentially extract aliphatic compounds and other impurities, avoiding impurities and ergosterol competing for solvent binding sites; phase two uses high pressure to enhance solvent density to improve ergosterol solubility, improve ergosterol extraction rate, and realize efficient separation of impurities and target substances.

[0036] 4. The present application proves by experiments that the ergosterol extracted from cordyceps militaris can effectively treat pulmonary hypertension, and can be applied to the development of pulmonary hypertension drugs. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 Figure 17 shows the comparison of HSD1 gene mutations between patients and normal people β Figure 17 shows the comparison of HSD1 gene mutations between patients and normal people Figure 1 Figure 17 shows the comparison of HSD1 gene mutations between patients and normal people β Figure 17 shows the comparison of HSD1 gene mutations between patients and normal people Figure 1 Figure 17 shows the comparison of HSD1 gene mutations between patients and normal people Figure 1 Figure 17 shows the comparison of HSD1 gene mutations between patients and normal people Figure 1 Figure 17 shows the comparison of HSD1 gene mutations between patients and normal people α- difference in helical structure, the dotted circle mark shows the difference between the two β - difference in folded structure;

[0038] Figure 2 Figure of the effect of Cordyceps militaris ergosterol on the proliferation of hPASMCs cells in Example 1, wherein, Figure 2 A in Figure is a line graph showing the cell proliferation detected by the trypan blue method on the 0th, 3rd and 5th days; Figure 2 B in Figure is a bar chart showing the cell proliferation on the 5th day;

[0039] Figure 3 Figure 17 β HSD1 as a receptor ER α upstream molecular switch of E2 / ER α Figure of exploration and verification of the correlation between BMPR2-ID axis and PH development, wherein, Figure 3 A in Figure is a UMAP dimensionality reduction clustering map; Figure 3 B in Figure is a Violin Plot of the expression level of HSD17B1 gene in the WT group and the IDs KO group;

[0040] Figure 4 Figure is a Violin Plot of the expression level of HSD17B1 gene in the FD control group and the PH patient group. DETAILED DESCRIPTION

[0041] The present application proposes the use of ergosterol in the preparation of a drug for treating pulmonary arterial hypertension, in order to make the purpose, technical scheme and effect of the present application more clear and definite, the present application will be further described in detail in conjunction with the examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application. Example 1

[0042] The preparation method of ergosterol comprises the following steps:

[0043] Step 1: Select the Cordyceps militaris Haining strain of Bombyx mori pupa, wash and drain the surface water, freeze in a low temperature freezer at-40℃ for 2h, put into an ultrafine grinder at a speed of 12000r / min for 30s, then send the crushed material into an air flow classification equipment, the classification wheel speed is 8000r / min, the air flow speed is 15m / s, and the classification cavity temperature is 25℃, screen out Cordyceps militaris powder with a particle size of 10-20μm, collect for standby; the coarse powder with a particle size greater than 20μm is returned to the ultrafine grinder for secondary crushing, vacuum drying, and controlling the moisture content at 9%, to obtain Cordyceps militaris powder;

[0044] Step 2: Take cholic acid, deoxycholic acid and polyethylene glycol monomethyl ether, the mass ratio of cholic acid to deoxycholic acid is 2:1, and the addition amount of polyethylene glycol monomethyl ether is 0.2% of the total mass of cholic acid and deoxycholic acid, add anhydrous ethanol, stir at a speed of 500 r / min for 30 min, ultrasonic treat at a power of 300 W and a temperature of 30℃ for 15 min, so that cholic acid and deoxycholic acid are completely dissolved and form a supramolecular assembly, and an ethanol mother liquor containing 11% supramolecular solvent is obtained;

[0045] Step 3: Gradient synergistic extraction:

[0046] Stage one: low-polarity impurity extraction (0-60 min): control the CO2 flow rate at 1.55 mL / min and the anhydrous ethanol flow rate at 0.16 mL / min, and mix them to form a CO2-expanded ethanol liquid; load the cordyceps militaris powder into a fixed bed reactor, control the system pressure at 10 MPa and the reactor temperature at 35℃, and flow the CO2-expanded ethanol liquid into the fixed bed reactor from the lower section of the material, and collect the low-polarity impurity extraction liquid at the bottom of the separator;

[0047] Stage two: ergosterol targeted extraction (60-150 min): close the anhydrous ethanol tank valve, and send the ethanol mother liquor containing supramolecular solvent into the mixer at a flow rate of 0.25 mL / min, control the CO2 flow rate at 2.1 mL / min, and mix them to form an expanded liquid, and then flow the expanded liquid into the fixed bed reactor, heat it to 40℃, adjust the system pressure to 18 MPa, and collect the cordyceps militaris extraction liquid;

[0048] Step 4: Perform vacuum distillation on the cordyceps militaris extraction liquid at a temperature of 60℃ and a vacuum degree of -0.09 MPa for 2 h, dry it, dissolve it in 100% ethanol to 100 mg / mL, pass it through a 0.25 μm microporous filter membrane, and then perform chromatographic separation on a chromatographic column DAC (150×250 mm C188 μm) with a UV detection wavelength of 282 nm and a flow rate of 600 mL / min; the elution mode is isocratic elution with 98% methanol aqueous solution for 40 min, and the components collected from 25.4 min to 30 min are concentrated to remove the elution solvent, and then dried at 60℃; add a good solvent (n-hexane and dichloromethane in a volume ratio of 3:1) to fully dissolve it, and then place it in a 26℃ environment for natural evaporation, and then filter the obtained crystal, wash it with -4℃ n-hexane, and then dry it to obtain the product. Example 2

[0049] A preparation method of ergosterol, comprising the following steps:

[0050] Step 1: Select the Bombyx mori Cordyceps haikouensis Haikou strain fruiting body, wash and drain the surface water, freeze in a low temperature freezer at -40℃ for 2h, put into the ultrafine grinder at a speed of 11000r / min for 25s, then send the crushed material into the air classification equipment, the classification wheel speed is 7700r / min, the air flow speed is 14m / s, and the classification cavity temperature is 25℃, and the 10-20μm particle size of Cordyceps sinensis powder is screened out and collected for standby; the coarse powder with particle size greater than 20μm is returned to the ultrafine grinder for secondary crushing, vacuum drying, and controlling the moisture content to 8%, to obtain Cordyceps sinensis powder;

[0051] Step 2: Take cholic acid, deoxycholic acid and polyethylene glycol monomethyl ether, the mass ratio of cholic acid to deoxycholic acid is 2:1, and the addition amount of polyethylene glycol monomethyl ether is 0.2% of the total mass of cholic acid and deoxycholic acid, add anhydrous ethanol, stir at a speed of 450r / min for 30min, ultrasonic treatment at a power of 300W and a temperature of 30℃ for 15min, so that cholic acid and deoxycholic acid are completely dissolved and form supramolecular assemblies, and an ethanol mother liquor containing 10% supramolecular solvent is obtained;

[0052] Step 3: Gradient synergistic extraction:

[0053] Phase one: low-polarity impurity extraction (0-60min): control the CO2 flow rate at 1.5mL / min and the anhydrous ethanol flow rate at 0.15ml / min, and mix them to form CO2-expanded ethanol liquid; load the Cordyceps sinensis powder into the fixed bed reactor, control the system pressure at 10MPa and the reactor temperature at 34℃, and flow the CO2-expanded ethanol liquid into the fixed bed reactor from the lower section of the material, and collect the low-polarity impurity extraction liquid at the bottom of the separator;

[0054] Phase two: ergosterol targeted extraction (60-150min): close the anhydrous ethanol tank valve, introduce the ethanol mother liquor containing supramolecular solvent into the mixer at a flow rate of 0.2mL / min, mix to form expanded liquid at a CO2 flow rate of 2.0mL / min, and introduce the expanded liquid into the fixed bed reactor, heat to 40℃, adjust the system pressure to 17.5MPa, and collect the Cordyceps sinensis extraction liquid;

[0055] Step 4: The Cordyceps militaris extract was subjected to vacuum distillation at a temperature of 60°C, a vacuum degree of -0.09 MPa, and a distillation time of 2 h, and then dried to obtain an extract, which was dissolved in 100% ethanol to 100 mg / mL, filtered through a 0.25 μm microporous filter, and then subjected to chromatographic separation using a chromatographic column DAC (150 x 250 mm C188 μm) with a UV detection wavelength of 282 nm and a flow rate of 600 mL / min; the elution mode was isocratic elution with 98% methanol aqueous solution for 40 min, and the fractions collected were 25.4-30 min; after removing the elution solvent by concentration and drying at 60°C, a good solvent (n-hexane and dichloromethane at a volume ratio of 3:1) was added for sufficient dissolution, and then the mixture was naturally volatilized at 26°C; the obtained crystal was suction filtered, washed with n-hexane at -4°C, and then dried to obtain the product. Example 3

[0056] Step 1: The Cordyceps militaris Haining strain was selected, washed, and then the surface water was drained; the Cordyceps militaris Haining strain was placed in a low-temperature freezer at -40°C for 2 h, and then fed into an ultrafine grinder at a rotation speed of 13,000 r / min for 35 s; the ground material was then fed into an air flow classification device, with a classification wheel rotation speed of 8,200 r / min, an air flow speed of 16 m / s, and a classification cavity temperature of 25°C; the Cordyceps militaris powder with a particle size of 10-20 μm was screened out and collected for use; the coarse powder with a particle size greater than 20 μm was returned to the ultrafine grinder for secondary grinding, and then vacuum dried to control the water content at 10% to obtain the Cordyceps militaris powder;

[0057] Step 2: Cholic acid, deoxycholic acid, and polyethylene glycol monomethyl ether were taken, with a mass ratio of cholic acid to deoxycholic acid of 2:1.2, and the addition amount of polyethylene glycol monomethyl ether being 0.2% of the total mass of cholic acid and deoxycholic acid; anhydrous ethanol was added, stirred at a rotation speed of 550 r / min for 30 min, and then ultrasonically treated at a power of 300 W and a temperature of 30°C for 15 min to completely dissolve the cholic acid and deoxycholic acid and form a supramolecular assembly, thereby obtaining an ethanol mother liquor containing 12% supramolecular solvent;

[0058] Step 3: Gradient synergistic extraction:

[0059] Phase I: Low-polarity impurity extraction (0-60 min): the CO2 flow was controlled at 1.6 mL / min, and the anhydrous ethanol flow was controlled at 0.17 mL / min to form CO2-expanded ethanol liquid; the Cordyceps militaris powder was loaded into a fixed bed reactor, the system pressure was controlled at 10 MPa, and the reactor temperature was controlled at 36°C; the CO2-expanded ethanol liquid was flowed into the fixed bed reactor from the lower section of the material, and the low-polarity impurity extract was collected from the bottom of the separator.

[0060] Stage two: ergosterol targeted extraction (60-150 min): close the anhydrous ethanol storage tank valve, and input the ethanol mother liquor of the supramolecular solvent at a flow rate of 0.3 mL / min into the mixer, control the CO2 flow rate to be 2.2 mL / min, and mix to form an expanded liquid. The expanded liquid is input into the fixed bed reactor, the temperature is raised to 40°C, the system pressure is adjusted to 18.5 MPa, and the cordyceps militaris extraction liquid is collected;

[0061] Step 4: The cordyceps militaris extraction liquid is subjected to vacuum distillation at a temperature of 60°C and a vacuum degree of -0.09 MPa for 2 h, dried, and obtained as an extract. The extract is dissolved in 100% ethanol to 100 mg / mL, filtered through a 0.25 μm microporous filter, and then subjected to chromatographic separation using a chromatographic column DAC (150x250 mm C188 μm) with a UV detection wavelength of 282 nm and a flow rate of 600 mL / min. The elution mode is isocratic elution with 98% methanol aqueous solution for 40 min, and the components collected from 25.4 to 30 min are concentrated to remove the elution solvent and then dried at 60°C. A good solvent (n-hexane and dichloromethane at a volume ratio of 3:1) is added to fully dissolve the mixture, which is then naturally volatilized at 26°C. The obtained crystals are filtered, washed with -4°C n-hexane, and then dried to obtain the product. Example 4

[0062] A method for preparing ergosterol, comprising the following steps:

[0063] Step 1: Cordyceps militaris Haining strain fruiting bodies are selected, washed, and then drained of surface water. The fruiting bodies are placed in a low-temperature freezer at -40°C for 2 h, then fed into an ultrafine grinder at a rotation speed of 11500 r / min for 28 s. The ground material is then fed into an air flow classification device, with a classification wheel rotation speed of 7800 r / min, an air flow speed of 14.5 m / s, and a classification cavity temperature of 25°C. Cordyceps militaris powder with a particle size of 10-20 μm is screened out and collected for use. Coarse powder with a particle size greater than 20 μm is returned to the ultrafine grinder for secondary grinding. The powder is vacuum dried to control the water content to be 8.5%, and the cordyceps militaris powder is obtained.

[0064] Step 2: Cholic acid, deoxycholic acid, and polyethylene glycol monomethyl ether are taken. The mass ratio of cholic acid to deoxycholic acid is 2:1.1, and the addition amount of polyethylene glycol monomethyl ether is 0.2% of the total mass of cholic acid and deoxycholic acid. The cholic acid, deoxycholic acid, and polyethylene glycol monomethyl ether are added to anhydrous ethanol, stirred at a rotation speed of 480 r / min for 30 min, and ultrasonically treated at a power of 300 W and a temperature of 30°C for 15 min to completely dissolve the cholic acid and deoxycholic acid and form supramolecular assemblies. An ethanol mother liquor containing 10.5% supramolecular solvent is obtained.

[0065] Step 3: Gradient synergistic extraction:

[0066] Stage one: low-polarity impurity extraction (0-60 min): control the CO2 flow rate at 1.52 mL / min, and the anhydrous ethanol flow rate at 0.155 mL / min, and mix the two to form CO2-expanded ethanol liquid; load the cordyceps militaris powder into the fixed bed reactor, control the system pressure at 10 MPa, and the reactor temperature at 34.5℃, and flow the CO2-expanded ethanol liquid into the fixed bed reactor from the lower section of the material, and collect the low-polarity impurity extraction liquid at the bottom of the separator;

[0067] Stage two: ergosterol targeted extraction (60-150 min): close the anhydrous ethanol storage tank valve, and introduce the ethanol mother liquor of the supramolecular solvent at a flow rate of 0.22 mL / min into the mixer, control the CO2 flow rate at 2.05 mL / min, and mix to form expanded liquid, and introduce the expanded liquid into the fixed bed reactor, heat to 40℃, adjust the system pressure to 17.8 MPa, and collect the cordyceps militaris extraction liquid;

[0068] Step 4: perform vacuum distillation on the cordyceps militaris extraction liquid at a temperature of 60℃ and a vacuum degree of -0.09 MPa for 2 h, dry, obtain the extract, dissolve in 100% ethanol to 100 mg / mL, pass through a 0.25 μm microporous filter membrane, and then perform chromatographic separation using a chromatographic column DAC (150x250 mm C188 μm) with an ultraviolet detection wavelength of 282 nm and a flow rate of 600 mL / min; the elution mode is 98% methanol aqueous solution isocratic elution for 40 min, and the components collected are 25.4-30 min, after removing the elution solvent by concentration and drying at 60℃, add a good solvent (n-hexane and dichloromethane in a volume ratio of 3:1) to fully dissolve, and then place in a 26℃ environment for natural evaporation, and then dry the obtained crystal after standing, and then dry after washing with n-hexane at -4℃, and then dry. Example 5

[0069] A method for preparing ergosterol, comprising the following steps:

[0070] Step 1: select the cordyceps militaris Haining strain of silkworm pupa, wash and drain the surface water, and then place in a -40℃ low-temperature freezer for 2 h, then put into an ultrafine grinder and grind at a speed of 12500 r / min for 32 s, then send the ground material into an air flow classification equipment, with a classification wheel speed of 8100 r / min, an air flow speed of 15.5 m / s, and a classification cavity temperature of 25℃, and then screen out cordyceps militaris powder with a particle size of 10-20 μm, and then collect for use; the coarse powder with a particle size greater than 20 μm is returned to the ultrafine grinder for secondary grinding, and then vacuum dried to control the water content at 9.5%, and then obtain the cordyceps militaris powder;

[0071] Step 2: Take cholic acid, deoxycholic acid and polyethylene glycol monomethyl ether, the mass ratio of cholic acid to deoxycholic acid is 2:1.15, and the addition amount of polyethylene glycol monomethyl ether is 0.2% of the total mass of cholic acid and deoxycholic acid. Add anhydrous ethanol, stir at a speed of 520 r / min for 30 min, ultrasonic treat at a power of 300 W and a temperature of 30℃ for 15 min, so that cholic acid and deoxycholic acid are completely dissolved and form a supramolecular assembly, and an ethanol mother liquor containing 11.5% supramolecular solvent is obtained;

[0072] Step 3: Gradient synergistic extraction:

[0073] Stage one: low-polarity impurity extraction (0-60 min): control the CO2 flow rate at 1.58 mL / min and the anhydrous ethanol flow rate at 0.165 mL / min, and mix them to form a CO2-expanded ethanol liquid; load the cordyceps militaris powder into a fixed bed reactor, control the system pressure at 10 MPa and the reactor temperature at 35.5℃, and flow the CO2-expanded ethanol liquid into the fixed bed reactor from the lower section of the material, and collect the low-polarity impurity extraction liquid at the bottom of the separator;

[0074] Stage two: ergosterol targeted extraction (60-150 min): close the anhydrous ethanol tank valve, introduce the ethanol mother liquor containing supramolecular solvent into the mixer at a flow rate of 0.28 mL / min, control the CO2 flow rate at 2.15 mL / min, mix to form an expanded liquid, and introduce the expanded liquid into the fixed bed reactor, heat to 40℃, adjust the system pressure to 18.2 MPa, and collect the cordyceps militaris extraction liquid;

[0075] Step 4: Perform vacuum distillation on the cordyceps militaris extraction liquid at a temperature of 60℃ and a vacuum degree of -0.09 MPa for 2 h, dry, obtain the extract, dissolve it in 100% ethanol to 100 mg / mL, pass it through a 0.25 μm microporous filter membrane, and then perform chromatographic separation on a chromatographic column DAC (150×250 mm C188 μm) with a UV detection wavelength of 282 nm and a flow rate of 600 mL / min; the elution mode is isocratic elution with 98% methanol aqueous solution for 40 min, and the components collected from 25.4 to 30 min are concentrated to remove the elution solvent, and then dried at 60℃; add a good solvent (n-hexane and dichloromethane in a volume ratio of 3:1) to dissolve it thoroughly, place it at 26℃, and naturally volatilize, then keep it still, and the obtained crystal is filtered, washed with n-hexane at -4℃, and then dried to obtain the product. Example 6

[0076] A preparation method of ergosterol, comprising the following steps:

[0077] Step 1: Select the Bombyx mori Cordyceps haikouensis Haikou strain fruiting body, wash and drain the surface water, freeze in a low temperature freezer at -40℃ for 2h, put into the ultrafine grinder at a speed of 12200r / min for 30s, then send the crushed material into the air classification equipment, the classification wheel speed is 7900r / min, the air flow speed is 15m / s, and the classification cavity temperature is 25℃, and the 10-20μm particle size of the Cordyceps haikouensis powder is screened out and collected for standby; the coarse powder with particle size greater than 20μm is returned to the ultrafine grinder for secondary crushing, vacuum drying, and controlling the moisture content to 9%, to obtain Cordyceps haikouensis powder;

[0078] Step 2: Take cholic acid, deoxycholic acid and polyethylene glycol monomethyl ether, the mass ratio of cholic acid to deoxycholic acid is 2:1, and the addition amount of polyethylene glycol monomethyl ether is 0.2% of the total mass of cholic acid and deoxycholic acid, which is added into anhydrous ethanol, stirred at a speed of 500r / min for 30min, and ultrasonically treated at a power of 300W and a temperature of 30℃ for 15min, so that the cholic acid and deoxycholic acid are completely dissolved and form supramolecular assemblies, and an ethanol mother liquor containing 11% supramolecular solvent is obtained;

[0079] Step 3: Gradient synergistic extraction:

[0080] Phase one: low-polarity impurity extraction (0-60min): control the CO2 flow rate to be 1.55mL / min, and the anhydrous ethanol flow rate to be 0.16ml / min, and mix them to form CO2-expanded ethanol liquid; pack the Cordyceps haikouensis powder into a fixed bed reactor, control the system pressure to be 10MPa and the reactor temperature to be 35℃, and flow the CO2-expanded ethanol liquid into the fixed bed reactor from the lower section of the material, and collect the low-polarity impurity extraction liquid at the bottom of the separator;

[0081] Phase two: ergosterol targeted extraction (60-150min): close the anhydrous ethanol storage tank valve, and send the ethanol mother liquor containing supramolecular solvent into the mixer at a flow rate of 0.25mL / min, mix to form expanded liquid at a CO2 flow rate of 2.1mL / min, and then flow the expanded liquid into the fixed bed reactor, heat to 40℃, adjust the system pressure to 18MPa, and collect the Cordyceps haikouensis extraction liquid;

[0082] Step 4: The Cordyceps militaris extract was subjected to vacuum distillation at a temperature of 60℃ and a vacuum degree of -0.09 MPa for 2 h, dried, dissolved in 100% ethanol to 100 mg / mL, filtered through a 0.25 μm microporous filter, and then subjected to chromatographic separation using a chromatographic column DAC (150 x 250 mm C188 μm) with a UV detection wavelength of 282 nm and a flow rate of 600 mL / min; the elution mode was isocratic elution with 98% methanol aqueous solution for 40 min, and the fractions collected were 25.4-30 min; after removing the elution solvent by concentration and drying at 60℃, a good solvent (n-hexane and dichloromethane at a volume ratio of 3:1) was added for complete dissolution, and the mixture was naturally volatilized at 26℃; the obtained crystal was filtered, washed with n-hexane at -4℃, and then dried to obtain the product. Example 7

[0083] A method for preparing ergosterol, comprising the following steps:

[0084] Step 1: Cordyceps militaris Haining strain fruiting bodies were selected, washed, and then drained of surface water, and then placed in a low-temperature freezer at -40℃ for 2 h; the frozen fruiting bodies were then fed into an ultrafine grinder at a rotation speed of 11800 r / min for 30 s, and then the ground material was fed into an air flow classification device, with a classification wheel rotation speed of 8000 r / min, an air flow speed of 15 m / s, and a classification cavity temperature of 25℃; the 10-20 μm particle size Cordyceps militaris powder was screened out and collected for use; the coarse powder with a particle size greater than 20 μm was returned to the ultrafine grinder for secondary grinding, and then vacuum dried to control the water content at 9%, thereby obtaining Cordyceps militaris powder;

[0085] Step 2: Cholic acid, deoxycholic acid, and polyethylene glycol monomethyl ether were taken, with a cholic acid to deoxycholic acid mass ratio of 2:1.1, and the polyethylene glycol monomethyl ether was added in an amount of 0.2% of the total mass of cholic acid and deoxycholic acid; anhydrous ethanol was added, stirred at a rotation speed of 500 r / min for 30 min, and then ultrasonically treated at a power of 300 W and a temperature of 30℃ for 15 min, so that the cholic acid and deoxycholic acid were completely dissolved and formed supramolecular assemblies; an ethanol mother liquor containing 11% supramolecular solvent was obtained;

[0086] Step 3: Gradient synergistic extraction:

[0087] Phase one: low-polarity impurity extraction (0-60 min): the CO2 flow was controlled at 1.55 mL / min, and the anhydrous ethanol flow was controlled at 0.16 mL / min, and the two were mixed to form CO2-expanded ethanol liquid; the Cordyceps militaris powder was loaded into a fixed bed reactor, the system pressure was controlled at 10 MPa, and the reactor temperature was controlled at 35℃; the CO2-expanded ethanol liquid was flowed into the fixed bed reactor from the lower section of the material, and the low-polarity impurity extract was collected from the bottom of the separator;

[0088] Stage two: ergosterol targeted extraction (60-150 min): close the anhydrous ethanol storage tank valve, and input the ethanol mother liquor of the supramolecular solvent at a flow rate of 0.25 mL / min into the mixer, control the CO2 flow rate to be 2.1 mL / min, mix to form the expanded liquid, and input the expanded liquid into the fixed bed reactor, heat to 40°C, adjust the system pressure to be 18 MPa, and collect the cordyceps militaris extraction liquid;

[0089] Step 4: the cordyceps militaris extraction liquid is subjected to vacuum distillation at a temperature of 60°C and a vacuum degree of -0.09 MPa for 2 h, dried, dissolved in 100% ethanol to 100 mg / mL, filtered through a 0.25 μm microporous filter membrane, and then subjected to chromatographic separation through a chromatographic column DAC (150*250 mm C188 μm) at a flow rate of 600 mL / min, with a UV detection wavelength of 282 nm; the elution mode is isocratic elution with 98% methanol aqueous solution for 40 min, and the components collected from 25.4 min to 30 min are concentrated to remove the elution solvent, and then dried at 60°C; a good solvent (n-hexane and dichloromethane at a volume ratio of 3:1) is added for sufficient dissolution, and the obtained crystal is placed at 26°C for natural evaporation, and then dried after standing, to obtain the product. Example 8

[0090] The preparation method of ergosterol comprises the following steps:

[0091] Step 1: select the cordyceps militaris Haining strain of silkworm pupa, wash and drain the surface water, freeze in a low-temperature freezer at -40°C for 2 h, put into an ultrafine grinder, grind at a rotation speed of 12200 r / min for 30 s, then send the ground material into an air flow classification equipment, the classification wheel rotation speed is 7900 r / min, the air flow speed is 15 m / s, and the classification cavity temperature is 25°C, and the cordyceps militaris powder with a particle size of 10-20 μm is screened out and collected for use; the coarse powder with a particle size greater than 20 μm is returned to the ultrafine grinder for secondary grinding, and vacuum drying is performed to control the water content to be 9%, to obtain the cordyceps militaris powder;

[0092] Step 2: take cholic acid, deoxycholic acid and polyethylene glycol monomethyl ether, the mass ratio of cholic acid to deoxycholic acid is 2:1, and the addition amount of polyethylene glycol monomethyl ether is 0.2% of the total mass of cholic acid and deoxycholic acid, which are added into anhydrous ethanol, stirred at a rotation speed of 500 r / min for 30 min, and ultrasonically treated at a power of 300 W and a temperature of 30°C for 15 min, so that the cholic acid and deoxycholic acid are completely dissolved and form supramolecular assemblies, to obtain an ethanol mother liquor containing 11% supramolecular solvent;

[0093] Step 3: Gradient synergistic extraction: Stage one: low-polarity impurity extraction (0-60 min): control CO2 flow rate at 1.55 mL / min, anhydrous ethanol flow rate at 0.16 mL / min, and mix to form CO2-expanded ethanol liquid; load Cordyceps militaris powder into the fixed bed reactor, control system pressure at 10 MPa, and reactor temperature at 35℃, and flow the CO2-expanded ethanol liquid into the fixed bed reactor from the lower section of the material, and collect the low-polarity impurity extraction liquid at the bottom of the separator; Stage two: targeted extraction of ergosterol (60-150 min): close the anhydrous ethanol tank valve, and introduce the ethanol mother liquor of the supramolecular solvent into the mixer at a flow rate of 0.25 mL / min, control CO2 flow rate at 2.1 mL / min, and mix to form expanded liquid, and introduce the expanded liquid into the fixed bed reactor, heat to 40℃, adjust system pressure to 18 MPa, and collect the Cordyceps militaris extraction liquid;

[0094] Step 4: Perform vacuum distillation on the Cordyceps militaris extraction liquid at a temperature of 60℃, a vacuum degree of -0.09 MPa, and a distillation time of 2 h, dry, obtain the extract, dissolve in 100% ethanol to 100 mg / mL, pass through a 0.25 μm microporous filter membrane, and then perform chromatographic separation using a chromatographic column DAC (150 x 250 mm C18 5 μm) with an ultraviolet detection wavelength of 282 nm and a flow rate of 600 mL / min; perform elution for 40 min using 98% methanol aqueous solution for isocratic elution, collect components from 25.4-30 min, remove the elution solvent after concentration, and dry at 60℃; add a good solvent (n-hexane and dichloromethane at a volume ratio of 3:1) to fully dissolve, place in a 26℃ environment, and naturally volatilize, keep still, and then perform suction filtration on the obtained crystals, wash with n-hexane at -4℃, and dry to obtain the product.

[0095] Comparative Example 1

[0096] The difference between this comparative example and Example 1 is that there is no supramolecular solvent, specifically:

[0097] Step 1: Same as Step 1 of Example 1;

[0098] Step 2: Omit the preparation of the supramolecular solvent, and directly use anhydrous ethanol;

[0099] Step 3: Gradient synergistic extraction: Stage one: same as Stage one of Example 1; Stage two: targeted extraction of ergosterol (60-150 min): close the anhydrous ethanol tank valve, introduce anhydrous ethanol (instead of the supramolecular solvent mother liquor) into the mixer at a flow rate of 0.25 mL / min, control CO2 flow rate at 2.1 mL / min, mix to form expanded liquid, introduce the expanded liquid into the fixed bed reactor, heat to 40℃, adjust system pressure to 18 MPa, and collect the Cordyceps militaris extraction liquid;

[0100] Step 4: Same as Example 1.

[0101] Comparative Example 2

[0102] The difference between this comparative example and Example 1 is that there is no gradient extraction, but a single-stage extraction, specifically:

[0103] Step 1: same as Step 1 of Example 1;

[0104] Step 2: same as Step 2 of Example 1;

[0105] Step 3: only a single-stage extraction (0-150 min) is performed: the CO2 flow is controlled at 2.1 mL / min, and the supermolecular solvent mother liquor flow is 0.25 mL / min, which are mixed to form an expanded liquid, which is then passed into a fixed bed reactor (temperature 35°C, pressure 14 MPa), and the extraction liquid is directly collected;

[0106] Step 4: same as Example 1.

[0107] Comparative Example 3

[0108] The difference between this comparative example and Example 1 is that a conventional pulverization method is used, without low-temperature freezing, specifically:

[0109] Step 1: select the Cordyceps militaris Haining strain fruiting bodies, wash and drain the surface moisture, and directly put them into a supermicro pulverizer (without -40°C freezing), pulverize at a speed of 12000 r / min for 30 s, and the subsequent airflow classification and drying steps are the same as in Example 1;

[0110] Steps 2-4: same as Example 1.

[0111] Performance test:

[0112] Extraction rate of Cordyceps militaris extract: weight method, extraction rate = (mass of collected Cordyceps militaris extract / dry weight of Cordyceps militaris raw material) x 100%;

[0113] Content of ergosterol in extract: determined by high performance liquid chromatography, to detect the mass fraction of ergosterol in Cordyceps militaris extract;

[0114] Extraction rate of ergosterol: determined by gas chromatography, and calculated as:

[0115] Extraction rate = actual mass of ergosterol obtained / total mass of ergosterol in Cordyceps militaris raw material x 100%;

[0116] Purity of ergosterol: determined by high performance liquid chromatography, and calculated by peak area normalization method;

[0117] The results are shown in Table 1 below:

[0118] Table 1

[0119] Extraction yield of C. militaris extract (%) Content of ergosterol in extract (%) Extraction yield of ergosterol (%) Purity of ergosterol (%) Example 1 5.7 7.4 89.2 98.5 Example 2 5.2 7.1 85.6 97.8 Example 3 5.5 7.3 88.8 98.3 Example 4 5.3 7.2 86.9 98.0 Example 5 5.4 7.3 88.5 98.4 Example 6 5.6 7.5 89.0 98.6 Example 7 5.5 7.4 88.7 98.5 Example 8 5.6 7.4 89.1 98.5 Comparative Example 1 5.7 4.2 65.3 82.1 Comparative Example 2 5.4 5.1 72.8 89.5 Comparative Example 3 5.3 5.8 78.5 92.3

[0120] From Table 1, it can be seen that the extraction rates of all examples of Cordyceps militaris extract are concentrated in 5.2%-5.7%, mainly because the pretreatment process (-40℃ freezing + ultrafine grinding + air flow classification) can stabilize the destruction of Cordyceps militaris cell structure, ensure the contact efficiency of solvent and fat-soluble ingredients, and avoid the fluctuation of extraction rate caused by uneven particle size; the gradient cooperative extraction parameters can stably control the solvent strength, which can avoid incomplete extraction caused by low flow and solvent waste caused by high flow, and realize efficient and stable extraction of fat-soluble ingredients in raw materials. The content of ergosterol in examples 2-6 is significantly higher than that of comparative examples, mainly because the "cavity recognition effect" of supramolecular solvent (cholic acid-deoxycholic acid-polyethylene glycol monomethyl ether): the multi-level cavity formed by cholic acid (3 hydroxyl groups) and deoxycholic acid (2 hydroxyl groups) can specifically bind the C5-C6 double bond and C22-C23 double bond structure of ergosterol, avoiding β - impurity sterols such as sitosterol and stigmasterol. The ergosterol extraction rate of examples is all above 85%, among which examples 1, 6 and 8 are close to 90%, which is significantly higher than that of comparative examples, mainly because the gradient extraction "sectional purification": in the first stage, low-concentration ethanol is used to preferentially remove aliphatic compounds, monoterpene and other low-polarity impurities, avoiding impurities and ergosterol competing for solvent binding sites; in the second stage, the supramolecular solvent mother liquor is used to target extract ergosterol, high pressure enhances solvent density, and improves the solubility of ergosterol; at the same time, -40℃ freezing makes the water in the cell form ice crystals, which destroys the structure of cell wall and cell membrane, and subsequent ultrafine grinding further opens the cell channel, making ergosterol more easily extracted by the solvent, avoiding the extraction resistance caused by the integrity of cell structure in traditional non-freezing process. The purity of the final product of all examples is above 97.8%, among which examples 6, 1 and 7 are close to 99%. The content of ergosterol, the extraction rate of ergosterol and the purity of the extract of comparative example 1 are all greatly reduced, mainly because without supramolecular solvent, ethanol-CO2 expansion liquid only "non-selectively" extracts all sterol components, which cannot distinguish ergosterol from impurity sterols, resulting in low content of target product, high difficulty of subsequent purification, and significantly reduced extraction rate and purity. The content of ergosterol and the extraction rate of ergosterol in the extract of comparative example 2 are also reduced, mainly because single-stage extraction cannot achieve "impurity preferential removal", low-polarity impurities and ergosterol are extracted at the same time, resulting in dilution of the target product, and some solvent binding sites are occupied by impurities, reducing the actual recovery rate of ergosterol. The content of ergosterol and the extraction rate of ergosterol in the extract of comparative example 3 are both reduced, mainly because without -40℃ freezing, the structure of Cordyceps militaris cell is complete (cellulose in cell wall is not destroyed by ice crystals), and ultrafine grinding only breaks part of the cells, resulting in ergosterol being trapped inside the cells and unable to fully contact with the solvent, reducing the extraction efficiency.

[0121] Take the ergosterol obtained from example 1 to verify its effect on treating pulmonary arterial hypertension:

[0122] 1) Steroid dehydrogenase family plays a role in the molecular switch of steroid hormone receptor pre-regulation, the family 17 β Hydroxysteroid dehydrogenase (17 β -Hydroxysteroid dehydrogenase type 1, 17 β The high expression of mRNA of HSD1 is associated with abnormal proliferation of lung cells. 17 β HSD1 is also known as estradiol 17 β dehydrogenase, is a key enzyme that catalyzes estradiol (E2) and reversible reaction estrone (E1). Because E2 supplements reduce PAH in male and ovariectomized female rats treated with monocrotaline and improve survival, which is consistent with E2 / ER α -BMPR2-Apelin pathway mediated E2 / ER α agonist treatment of PAH. Combined with the pre-experiment of silkworm cordyceps ergosterol inhibiting cell abnormal proliferation (reference patent ZL202111675263.9 A method for large-scale production of silkworm cordyceps and application), it was found that it played a role in inhibiting lung cell proliferation by up-regulating E2 level, and the third generation transcriptome sequencing and NCBI normal sequence comparison analysis also showed that there were 3 β HSD1 structural variations (as shown in Figure 1 ), which may be related to the formation of 17 β HSD1 complex (human 17 β HSD1 subcellular localization in cytoplasm) up-regulating E2. Silkworm cordyceps ergosterol plays a role in inhibiting abnormal proliferation of hPASMCs, which is related to 17βHSD1, specifically ER α (up-regulation)-BMPR2(up-regulation)-Apelin(up-regulation) signal axis significantly improves right ventricular hypertrophy caused by PAH, therefore, further mining of upstream regulatory genes of this signal pathway with patient-centeredness is of great significance for the development of new drugs for PAH.

[0123] The inhibitory effect of ergosterol on the proliferation of pulmonary artery smooth muscle cells (PASMCs) from a clinical PAH patient was detected. The samples were dissolved in DMSO, and DMSO was used as a blank control, and Sidenafil was used as a positive control. hPASMCs were cultured in DMEM containing 10% FBS, and 16,000 cells were inoculated in each 24-well plate. After 12 h of pre-culture, the cells were starved with serum-free DMEM for 24 h, and then replaced with DMEM containing 5% FBS. The cells were collected at 0, 3, and 5 days after administration, and the cell proliferation was detected by the trypan blue method. The results showed that, at 1 µg / mL, the cordyceps ergosterol had no difference compared with the positive control group, and had a significant inhibitory effect on the proliferation of hPASMCs compared with the control group (as shown in Figure 2

[0124] ​A human embryonic stem cell line with ID1 and ID3 gene knockout (IDsKO) is constructed by CRISPR / Cas9 gene editing technology. Wild type (WT) human embryonic stem cells are used as a control group. The WT group and the IDsKO group of human embryonic stem cells are induced to differentiate according to the standard myocardial cell differentiation protocol to obtain human embryonic stem cell-derived myocardial cells (hESC-CMs). After differentiation, the expression of myocardial specific markers (such as cTnT, α-actinin) is verified by immunofluorescence staining to confirm the success of myocardial cell differentiation. After differentiation, the hESC-CMs cells of the WT group and the IDsKO group are collected, and the 10x Genomics single cell sequencing platform is used for single cell RNA sequencing (scRNA-seq). The specific operation is as follows: the cells are digested into a single cell suspension, and the live cell sorting is performed by flow cytometry to ensure that the cell viability is ≥85%. Then the single cell sample is loaded into the 10x Genomics Chromium Controller for droplet encapsulation, barcode labeling and cDNA synthesis. After the library construction is completed, the Illumina sequencing platform is used for high-throughput sequencing, and the sequencing depth of each sample is about 50,000 reads / cell. After quality control of the sequencing data, bioinformatics analysis is performed using the Seurat software package. First, the raw data is filtered to remove low-quality cells (gene number <200 or >5000, mitochondrial gene proportion >10%) and double cells. Then data normalization, high variable gene screening, principal component analysis (PCA) dimensionality reduction processing are performed. The uniform manifold approximation and projection (UMAP) algorithm is used for nonlinear dimensionality reduction and visualization of cells, and different cell subpopulations are identified by unsupervised clustering analysis. The Wilcoxon rank-sum test is used for differential gene expression analysis, and the screening criteria are |log2FC|>0.5 and adjusted P value <0.05. By systematic comparison and analysis of the single cell transcriptome data of the WT group and the IDsKO group of hESC-CMs, the differentially expressed genes related to pulmonary arterial hypertension (PAH) are focused on, such as Figure 3A is a UMAP dimensionality reduction clustering plot, showing the distribution patterns of WT group and IDs KO group hESC-CMs in two-dimensional space. Each point in the plot represents a single cell, and different colors identify different experimental groups (WT group is shown in blue, and IDs KO group is shown in red). From the plot, it can be observed that the two groups of cells exhibit partially overlapping but clearly regionally distributed characteristics in the UMAP space, indicating that the knockout of ID1 and ID3 genes causes overall changes in the transcriptome profile of cardiomyocytes. This difference in distribution pattern reflects the profound impact of ID1 / ID3 deletion on the differentiation trajectory and gene expression program of cardiomyocytes. B is a Violin Plot of the expression level of the HSD17B1 gene in the WT group and the IDs KO group. The width of the Violin Plot represents the distribution density of the number of cells at this expression level, and the vertical axis is the normalized expression level of the HSD17B1 gene, and the horizontal axis is the experimental group. From the plot, it can be clearly observed that the expression level of HSD17B1 in the WT group is low, and the main body of the Violin Plot is concentrated in the low expression interval, with the median expression level maintained at the baseline level. In contrast, the expression of HSD17B1 in the IDs KO group is significantly increased, and the Violin Plot significantly extends to the high expression region, with a wider distribution range and a significantly higher median expression level than the WT group. Statistical tests show that the expression of HSD17B1 in the IDs KO group is significantly up-regulated compared to the WT group (P = 0.031), indicating that the deletion of ID1 and ID3 genes significantly promotes the expression of HSD17B1 in cardiomyocytes. This finding suggests that the up-regulation of HSD17B1 expression may be involved in the pathophysiological process of pulmonary arterial hypertension-like disease in the ID1 / ID3 double-gene knockout pathological model. β - Transcriptional expression of hydroxysteroid dehydrogenase type 1.

[0125] The above results show that in the ID1 / ID3 double-gene knockout pathological model, the significant up-regulation of HSD17B1 expression may be involved in the pathophysiological process of pulmonary arterial hypertension-like disease. Since the abnormality of the ID pathway is closely related to the occurrence and development of pulmonary arterial hypertension, this finding suggests that 17 β HSD1 may be a key molecular node connecting the BMPR2-ID signaling pathway and the pathogenesis of PAH. This finding provides an important theoretical basis and target verification for subsequent treatment of pulmonary arterial hypertension by regulating 17 β HSD1 activity, confirming that 17 β HSD1 as a new target for PAH treatment is feasible and scientific.

[0126] 3) To further validate the expression change of HSD17B1 in pulmonary arterial hypertension (PAH), the inventors performed independent validation analysis using the transcriptome data from the Pulmonary Hypertension Breakthrough Initiative (PHBI) multicenter lung biobank. This biobank contains 96 pulmonary hypertension (PH) patients and 52 control samples. PH patient samples were obtained from the diseased lung tissue resected at the time of lung transplantation surgery for end-stage pulmonary arterial hypertension, and control samples were obtained from donor lung tissue that failed for transplantation (Failed Donor, FD) for various reasons. All tissue samples were collected at the time of lung transplantation surgery following standardized procedures, immediately snap-frozen in liquid nitrogen and stored at -80°C to ensure RNA integrity.

[0127] Total RNA was extracted using TRIzol reagent (Invitrogen), and the quality of the extracted total RNA was assessed using an Agilent 2100 Bioanalyzer. Samples with an RNA Integrity Number (RIN) of ≥7 were used for subsequent transcriptome sequencing. Library construction used an Illumina TruSeq RNA Sample Preparation Kit, and high-throughput sequencing was performed using an Illumina HiSeq platform with a sequencing depth of approximately 300-500 million reads per sample and a read length of 150 bp for both ends.

[0128] Sequencing data were first subjected to quality control using FastQC software, and then high-quality reads were aligned to the human reference genome (GRCh38 / hg38) using HISAT2 alignment software. Gene expression quantification used StringTie software to generate transcript counts and normalized expression values (TPM, transcripts per million) for each gene. Differential expression analysis was performed using the DESeq2 software package in the R language environment, and the expression levels of HSD17B1 in the PH group and the FD control group were compared. To control for batch effects and other confounding factors, sample collection centers, patient age, gender, and other covariates were included in the analysis. The statistical significance threshold was set at a corrected P-value < 0.05.

[0129] The results are shown in Figure 4 Figure 4 ​Figure 6 shows the violin plot of the expression level of HSD17B1 gene in the FD control group and the PH patient group. The width of the violin plot represents the sample number distribution density of the expression level. The vertical axis represents the normalized expression level of HSD17B1 gene, and the horizontal axis represents the experimental group. The FD control group is shown in red (n = 52), and the PH patient group is shown in blue (n = 96). It can be clearly observed from the figure that the expression level of HSD17B1 in the FD control group is relatively low, and the main body of the violin plot is concentrated in the low expression interval. In contrast, the expression of HSD17B1 in the PH patient group is significantly increased, and the violin plot is significantly extended to the high expression area, with a wider distribution range, indicating that the heterogeneity of HSD17B1 expression in the lung tissue of PH patients is increased, and the transcription and expression of 17β-hydroxysteroid dehydrogenase type 1 in the lung tissue are significantly increased in the pathological state of pulmonary arterial hypertension. This finding is consistent with the single-cell sequencing results of ID1 / ID3 double-knockout human embryonic stem cell-derived cardiomyocytes, further confirming the correlation between the up-regulation of HSD17B1 expression and the occurrence and development of pulmonary arterial hypertension. This validation analysis is based on a large sample cohort from multiple medical centers, covering different subtypes of pulmonary arterial hypertension patients, enhancing the reliability and universality of the research results.

[0130] In summary, the ID1 / ID3 knockout model and the validation results of this independent cohort consistently show that HSD17B1 is up-regulated in pathological models related to pulmonary arterial hypertension and real patient samples, suggesting that 17βHSD1 may play an important role in the pathogenesis of pulmonary arterial hypertension. These findings provide a solid experimental basis for subsequent treatment of pulmonary arterial hypertension by regulating 17βHSD1 activity, further supporting the scientificity and clinical translation value of 17βHSD1 as a new target for PAH treatment.

[0131] The above-obtained cordyceps sinensis ergosterol in the compound drug formula in pulmonary arterial hypertension includes:

[0132] Formula 1: Cordyceps sinensis ergosterol 35%, Zhebeimu 15%, dihydroquercetin 14%, almond (bitter) powder 5%, dried tangerine peel powder 5%, osmanthus powder 8%, sweet orange powder 1%, snow pear powder 5%, lily powder 5%, loquat powder 5%, and licorice powder 2%;

[0133] Formula 2: Cordyceps sinensis ergosterol 40%, Zhebeimu 25%, ginseng peptide 14%, Dangshen 10%, Huangqi 10%, and magnesium stearate 1%;

[0134] Formula 3: Cordyceps sinensis ergosterol 35%, Dihydroquercetin 15%, bitter almond powder 10%, dried tangerine or orange peel powder 10%, osmanthus powder 12%, sweet orange powder 1%, snow pear powder 5%, lily powder 5%, loquat powder 5%, and licorice powder 2%;

[0135] Formula 4: Cordyceps sinensis ergosterol 40%, ginseng peptide 30%, Dangshen 16%, Huangqi 13%, and magnesium stearate 1%.

[0136] Formula description: Zhejiang beimu is used as a lung disease, especially pulmonary hypertension, and dihydroquercetin is a powerful antioxidant, which has good effects on anti-inflammatory, malignant tumors, microbial infections, oxidative stress, cardiovascular diseases and liver diseases; the bitter almond and osmanthus extract in the formula have significant cough and sore throat effects, and the dried tangerine or orange peel and osmanthus extract have obvious anti-viral and anti-inflammatory effects, especially the osmanthus extract, which contains more than 20% flavonoids, 7% verbascoside, and rhodioside. This ensures that it has obvious anti-viral effect. Snow pear, sweet orange, lily, and leaf have the effects of clearing lung fire and moistening lung and reducing sputum. The glycyrrhizic acid in the licorice extract has anti-inflammatory and anti-allergic effects.

[0137] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. The application of ergosterol in the preparation of drugs for treating pulmonary hypertension, characterized in that: Ergosterol is the only effective active ingredient; The method for preparing the ergosterol includes the following steps: Step 1: Select the fruiting bodies of Cordyceps militaris from Haining, wash and drain the surface moisture, freeze at -40℃, and then perform ultra-fine grinding. Send the ground material into an air classifier to screen out Cordyceps militaris powder with a particle size of 10-20μm, and collect it for later use. The coarse powder with a particle size greater than 20μm is returned to the ultra-fine grinder for secondary grinding, vacuum dried, and the moisture content is controlled at 8-10% to obtain Cordyceps militaris powder. Step 2: Take cholic acid, deoxycholic acid, and polyethylene glycol monomethyl ether, add them to anhydrous ethanol, stir, and then sonicate to obtain... Ethanol mother liquor containing 10-12% supramolecular solvent; Step 3: Gradient-coordinated extraction: Phase 1: Extraction of low-polarity impurities The CO2 flow rate is controlled at 1.5-1.6 mL / min, and the anhydrous ethanol flow rate is controlled at 0.15-0.17 mL / min. The two are mixed to form CO2 expanded ethanol liquid. Cordyceps militaris powder is packed into a fixed bed reactor, the system pressure is controlled at 10 MPa and the reactor temperature is 34-36℃. The CO2 expanded ethanol liquid flows counterclockwise from the lower part of the material in the fixed bed reactor, and the low polarity impurity extract at the bottom of the separator is collected. Phase Two: Ergosterol Targeted Extraction Close the valve of the anhydrous ethanol storage tank, introduce the ethanol mother liquor in supramolecular solvent, and feed it into the mixer at a flow rate of 0.2-0.3 mL / min. Control the CO2 flow rate at 2.0-2.2 mL / min, mix to form an expanded liquid, and introduce the expanded liquid into a fixed-bed reactor. Heat the liquid to 40℃, adjust the system pressure to 17.5-18.5 MPa, and collect the Cordyceps militaris extract. Step 4: Distill the Cordyceps militaris extract under reduced pressure, dry it to obtain the extract, dissolve it in ethanol, filter it through a microporous membrane and perform chromatographic separation, collect the fractions after 25.4-30 min, concentrate it to remove the elution solvent and dry it; add a good solvent to fully dissolve it, allow it to evaporate naturally, keep it at a constant temperature and let it stand, filter the obtained crystals, wash them with n-hexane at -4℃ and dry them to obtain the final product.

2. The application according to claim 1, characterized in that: The conditions for the airflow classification equipment in step 1 are as follows: the classification wheel speed is 7700-8200 r / min, the airflow velocity is 14-16 m / s, and the classification chamber temperature is 25℃.

3. The application according to claim 1, characterized in that: In step 2, the mass ratio of cholic acid to deoxycholic acid is 2:(1-1.2), and the amount of polyethylene glycol monomethyl ether added is 0.2% of the total mass of cholic acid and deoxycholic acid.

4. The application according to claim 1, characterized in that: The vacuum distillation conditions in step 4 are: temperature 60℃, vacuum degree -0.09MPa, and distillation time 1.5-2.5h.

5. The application according to claim 1, characterized in that: In step 4, the chromatographic column used for chromatographic separation is a DAC, the ultraviolet detection wavelength is 282 nm, and the flow rate is 600 mL / min; the elution method is isocratic elution with 98% methanol aqueous solution for 40 min.

6. The application according to claim 1, characterized in that: In step 4, the good solvent is a mixture of n-hexane and dichloromethane, with a volume ratio of n-hexane to dichloromethane of 3:

1.

7. The application according to claim 1, characterized in that: In the aforementioned drug for treating pulmonary arterial hypertension, the mass fraction of ergosterol is 0.5%-12%, and the drug dosage form is selected from inhalers, liposomes, tablets, capsules, or injections.

8. The application of ergosterol in the preparation of drugs for treating pulmonary hypertension, characterized in that: The formulation of the drug for treating pulmonary hypertension includes: Formula 1: Ergosterol, Fritillaria thunbergii, dihydroquercetin, almond powder, dried tangerine peel powder, osmanthus powder, sweet orange powder, snow pear powder, lily powder, loquat powder, and licorice powder; or, Formula 2: Ergosterol, Fritillaria thunbergii, ginseng peptides, Codonopsis pilosula, Astragalus membranaceus, and magnesium stearate; or, Formula 3: Ergosterol, dihydroquercetin, almond powder, dried tangerine peel powder, osmanthus powder, sweet orange powder, snow pear powder, lily powder, loquat powder, and licorice powder; or, Formula 4: Ergosterol, ginseng peptides, Codonopsis pilosula, Astragalus membranaceus and magnesium stearate.

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