A compound extracted from Ephedra equisetina, its preparation method and applications

Abstract

The present invention relates to a compound extracted from Ephedra equisetina Bunge, its preparation method and application, which can effectively solve the problem of drug use for allergic asthma. The technical solution is that the chemical structure of the compound extracted from Ephedra equisetina Bunge is shown as follows: The compound of the present invention can significantly inhibit the release of β-Hex in RBL-2H3 cells induced by C48 / 80, improve the degranulation phenomenon of RBL-2H3, and can be used as a drug for treating allergic asthma. Its drug composition can be a monomer or its derivative, which is an innovation in the compound extracted from Ephedra equisetina Bunge and its uses.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to a compound extracted from Ephedra equisetina, a preparation method thereof, and an application thereof. Background Art

[0002] Asthma is a chronic airway inflammatory disease involving multiple cells (eosinophils, mast cells, T lymphocytes, neutrophils, airway epithelial cells, etc.) and cellular components, mainly manifested as wheezing, chest tightness, coughing, and reversible airway obstruction. Asthma is one of the most common chronic diseases worldwide, with a high incidence and relatively difficult treatment, bringing a heavy economic burden to patients. Currently, studies have shown that the incidence and mortality of asthma patients are increasing year by year (Papi A, Brightling C, Pedersen S, et al. Asthma [J]. Lancet, 2018, 391(10122): 783). Mast cells are the core effector cells involved in asthma. During asthma, mast cell degranulation occurs, that is, the release of histamine, β-hexosaminidase, etc., which plays a key role in the pathogenesis of asthma (Martinsson J H, Enriquez E M. Mast cells and their progenitors in allergic asthma [J]. Front Immunol, 2019, 10: 821); and the rat basophilic leukemia cell line (RBL-2H3) can simulate mast cell activation and degranulation in asthma and can be used for screening the anti-asthmatic activity of compounds (Sun N, Zhou C, Zhou X, et al. Use of a rat basophil leukemia (RBL) cell-based immunological assay for allergen identification, clinical diagnosis of allergy, and identification of anti-allergy agents for use in immunotherapy [J]. J Immunotoxicol, 2015, 12(2): 199-205).

[0003] Ephedra equisetina has traditionally been used as medicine with its dried herbaceous stems. It was first recorded in "Shennong Ben Cao Jing" among the medical classics. It has the effects of inducing sweating to dispel cold, dispersing the lung qi to relieve asthma, and promoting diuresis to alleviate edema, and is used for symptoms such as wind-cold cold, chest distress and cough, and edema. Ephedra contains various types of compounds such as alkaloids, flavonoids, organic acids, tannins, volatile oils, and polysaccharides, among which alkaloids are the main components. Ephedra is an important component in various prescriptions for treating asthma-related diseases, such as Mahuang Shegan Decoction and Maxing Shigan Decoction (Zhang Shiyu, Huang Guirui, Hou Dan, et al. Network Meta-analysis of the Efficacy of 5 Commonly Used Classical Prescriptions in the Treatment of Acute Asthma Attacks in Children [J]. Chinese Journal of Experimental Traditional Medical Formulae, 2022, 28(02): 67-78).

[0004] Modern pharmacological activity studies have shown that the alkaloids in Ephedra play an anti-asthmatic role by promoting the release of neurotransmitters such as norepinephrine and epinephrine and blocking the release of allergic mediators (Miao S M, Zhang Q, Bi X B, et al. A review of the phytochemistry and pharmacological activities of Ephedra herb [J]. Chin J Nat Med, 2020, 18: 321-344), while the polysaccharides in Ephedra achieve the purpose of anti-asthma by shortening the cough time and prolonging the cough latency (Zhao Yunsheng, Mao Fuying, Yao Haihua, et al. Study on the sweating, antitussive and diuretic activities of Ephedra polysaccharide [J]. Traditional Medicine, 2014, 10(16): 11-13); in addition, tetramethylpyrazine and l-α-terpineol in Ephedra sinica also have certain anti-asthmatic activities (Miao S M, Zhang Q, Bi X B, et al. A review of the phytochemistry and pharmacological activities of Ephedra herb [J]. Chin J Nat Med, 2020, 18: 321-344). Ephedra plants and their monomeric compounds do have anti-asthmatic activities, but there are few studies on the anti-asthmatic activities of monomeric compounds of Ephedra equisetina. There have been no reports on the preparation methods and studies on anti-allergic asthma of the 3 novel compounds involved in this invention. Summary of the Invention

[0005] In view of the above situation, to solve the defects of the prior art, the purpose of this invention is to provide a compound extracted from Ephedra equisetina, its preparation method and application, which can effectively solve the problem of drug use for allergic asthma.

[0006] The technical solution solved by this invention is that the chemical structures of the compounds extracted from Ephedra equisetina are shown in Formulas 1-3:

[0007]

[0008] The preparation method of the compound of the present invention comprises the following steps:

[0009] 1) The stems of Ephedra equisetina Bunge. are refluxed and extracted 2 - 6 times with 5 - 15 times the amount of solvent, the extraction solutions are combined, filtered, and concentrated under reduced pressure to obtain a concentrated extract;

[0010] 2) The concentrate obtained in step 1) is suspended in water, and then successively extracted with petroleum ether, dichloromethane, ethyl acetate, and n - butanol to obtain a petroleum ether fraction, a dichloromethane fraction, an ethyl acetate fraction, an n - butanol fraction, and an aqueous fraction;

[0011] 3) The dichloromethane fraction in step 2) is subjected to silica gel column chromatography and eluted successively with petroleum ether:acetone in the ratios of 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, 1:1, 0:100, and 100% methanol to obtain 9 elution fractions Fr.1 - Fr.9;

[0012] 4) The concentrate of Fr.8 in step 3) is subjected to silica gel column chromatography and eluted successively with CH 2 Cl 2 :MeOH = 30:1, 20:1, 10:1 to obtain 6 elution fractions Fr.8 - 1 - Fr.8 - 6;

[0013] 5) The concentrate of Fr.8 - 2 in step 4) is subjected to Sephadex LH - 20 gel column chromatography and eluted with 100% methanol to obtain 6 elution fractions Fr.8 - 2 - 1 - Fr.8 - 2 - 6;

[0014] 6) The concentrate of Fr.8 - 2 - 2 in step 5) is subjected to silica gel column chromatography and eluted with CH 2 Cl 2 :MeOH = 200:1, 50:1, 20:1, 10:1, 5:1 to obtain 5 elution fractions Fr.8 - 2 - 2 - 1 - Fr.8 - 2 - 2 - 5;

[0015] 7) The Fr.8 - 2 - 2 - 4 obtained in step 6) is separated by C 18 ODS RP - HPLC to obtain the novel compound 1 (t R = 26.4 min);

[0016] 8) The concentrate of Fr.7 in step 3) is subjected to silica gel column chromatography and eluted with CH 2 Cl 2: Elution was carried out with MeOH = 50:1, 20:1, 10:1, 5:1, 3:1 to obtain 5 elution fractions Fr.7-1 to Fr.7-5;

[0017] 9) The concentrated solution of Fr.7-4 in step 8) was subjected to silica gel column chromatography and eluted with CH 2 Cl 2 : MeOH = 100:1, 50:1, 25:1, 10:1, 5:1 for gradient elution to obtain 10 elution fractions Fr.7-4-1 to Fr.7-4-10;

[0018] 10) The concentrated solution of Fr.7-4-7 in step 9) was subjected to silica gel column chromatography and eluted with CH 2 Cl 2 : MeOH = 30:1 for elution to obtain 5 elution fractions Fr.7-4-7-1 to Fr.7-4-7-5;

[0019] 11) Fr.7-4-7-2 obtained in step 10) was separated by C 18 ODS RP-HPLC to obtain 3 elution fractions Fr.7-4-7-2-1 to Fr.7-4-7-2-3;

[0020] 12) Fr.7-4-7-2-1 obtained in step 11) was separated by C 18 ODS RP-HPLC to obtain a novel compound 2 (t R = 16.0 min);

[0021] 13) The concentrated solution of Fr.7-3 in step 8) was subjected to silica gel column chromatography and eluted with CH 2 Cl 2 : MeOH = 50:1, 25:1 for gradient elution to obtain 2 elution fractions Fr.7-3-1 to Fr.7-3-2;

[0022] 14) The concentrated solution of Fr.7-3-2 in step 13) was subjected to Sephadex LH-20 gel column chromatography and eluted with 75% methanol to obtain 12 elution fractions Fr.7-3-2-1 to Fr.7-3-2-12;

[0023] 15) Fr.7-3-2-5 obtained in step 14) was separated by C 18 ODS RP-HPLC to obtain a novel compound 3 (t R = 27.6 min).

[0024] In the said step 7), C 18The conditions for ODS RP-HPLC preparation are 5 μm, 250×10 mm, 55% methanol, and 3 mL / min.

[0025] C in step 11) as described 18 The conditions for ODS RP-HPLC preparation are 5 μm, 250×10 mm, 65% methanol, and 3 mL / min.

[0026] C in step 12) as described 18 The conditions for ODS RP-HPLC preparation are 5 μm, 250×10 mm, 10% acetonitrile, and 3 mL / min.

[0027] C in step 15) as described 18 The conditions for ODS RP-HPLC preparation are 5 μm, 250×10 mm, 18% acetonitrile, and 3 mL / min.

[0028] Use of the compounds 1-3 of the present invention in the preparation of drugs for treating allergic asthma.

[0029] The compounds 1-3 of the present invention can significantly inhibit the release of β-Hex from RBL-2H3 cells induced by C48 / 80, improve the degranulation phenomenon of RBL-2H3, and can be used as drugs for treating allergic asthma. The drug composition can be monomers or their derivatives, which is an innovation in the compounds extracted from Ephedra equisetina and their uses. Description of the drawings

[0030] Figure 1 For compound 1 of the present invention 1 1H-NMR spectrum.

[0031] Figure 2 For compound 1 of the present invention 13 13C-NMR spectrum.

[0032] Figure 3 For the ECD spectrum of compound 1 of the present invention

[0033] Figure 4 For compound 2 of the present invention 1 1H-NMR spectrum.

[0034] Figure 5 For compound 2 of the present invention 13 13C-NMR spectrum.

[0035] Figure 6 For compound 3 of the present invention 1 1H-NMR spectrum.

[0036] Figure 7 For compound 3 of the present invention 1313C-NMR spectrum.

[0037] Figure 8 This is the ECD spectrum of Compound 3 of the present invention. Detailed implementation manners

[0038] The following further elaborates on the detailed implementation manners of the present invention in conjunction with the accompanying drawings and examples.

[0039] Example 1

[0040] When the present invention is specifically implemented, the preparation method of Compound 1 may include the following steps:

[0041] 1) Take 35 Kg of Ephedra equisetina stems, add 10 times the amount of water and reflux extract 3 times. Combine the extracts, filter, and concentrate under reduced pressure to obtain a concentrated extract;

[0042] 2) Suspend the concentrate obtained in step 1) in water, and then successively extract with petroleum ether, dichloromethane, ethyl acetate, and n-butanol to obtain a petroleum ether fraction, a dichloromethane fraction, an ethyl acetate fraction, an n-butanol fraction, and an aqueous fraction;

[0043] 3) Subject the dichloromethane fraction in step 2) to silica gel column chromatography and elute successively with petroleum ether:acetone = 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, 1:1, 0:100, and 100% methanol to obtain 9 elution fractions Fr.1 - Fr.9;

[0044] 4) Subject the concentrate of Fr.8 in step 3) to silica gel column chromatography and elute successively with CH 2 Cl 2 :MeOH = 30:1, 20:1, 10:1 to obtain 6 elution fractions Fr.8-1 - Fr.8-6;

[0045] 5) Subject the concentrate of Fr.8-2 in step 4) to Sephadex LH-20 gel column chromatography and elute with 100% methanol to obtain 6 elution fractions Fr.8-2-1 - Fr.8-2-6;

[0046] 6) Subject the concentrate of Fr.8-2-2 in step 5) to silica gel column chromatography and elute with CH 2 Cl 2 :MeOH = 200:1, 50:1, 20:1, 10:1, 5:1 to obtain 5 elution fractions Fr.8-2-2-1 - Fr.8-2-2-5;

[0047] 7) Separate Fr.8-2-2-4 obtained in step 6) by C 18 ODS RP-HPLC to obtain the novel Compound 1 (t R= 26.4 min).

[0048] Example 2

[0049] When the present invention is specifically implemented, the preparation method of Compound 2 may include the following steps:

[0050] 1) Take 20 Kg of Ephedra equisetina stems, add 5 times the amount of water and reflux extract 4 times. Combine the extracts, filter, and concentrate under reduced pressure to obtain a concentrated extract;

[0051] 2) Suspend the concentrate obtained in step 1) in water, and then extract successively with petroleum ether, dichloromethane, ethyl acetate, and n-butanol to obtain a petroleum ether fraction, a dichloromethane fraction, an ethyl acetate fraction, an n-butanol fraction, and a water fraction;

[0052] 3) Subject the dichloromethane fraction in step 2) to silica gel column chromatography, and elute successively with petroleum ether: acetone = 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, 1:1, 0:100, and 100% methanol to obtain 9 elution fractions Fr.1 - Fr.9;

[0053] 4) Subject the concentrate of Fr.7 in step 3) to silica gel column chromatography, and elute with CH 2 Cl 2 : MeOH = 50:1, 20:1, 10:1, 5:1, 3:1 to obtain 5 elution fractions Fr.7-1 - Fr.7-5;

[0054] 5) Subject the concentrate of Fr.7-4 in step 4) to silica gel column chromatography, and perform gradient elution with CH 2 Cl 2 : MeOH = 100:1, 50:1, 25:1, 10:1, 5:1 to obtain 10 elution fractions Fr.7-4-1 - Fr.7-4-10;

[0055] 6) Subject the concentrate of Fr.7-4-7 in step 5) to silica gel column chromatography, and elute with CH 2 Cl 2 : MeOH = 30:1 to obtain 5 elution fractions Fr.7-4-7-1 - Fr.7-4-7-5;

[0056] 7) Separate Fr.7-4-7-2 obtained in step 6) by C 18 ODS RP-HPLC to obtain 3 elution fractions Fr.7-4-7-2-1 - Fr.7-4-7-2-3;

[0057] 8) Subject Fr.7-4-7-2-1 obtained in step 7) to C 18Separation by ODS RP-HPLC gave the novel compound 2 (t R = 16.0 min).

[0058] Example 3

[0059] In the specific implementation of the present invention, the preparation method of compound 3 may include the following steps:

[0060] 1) Take 30 Kg of Ephedra equisetina stems, add 15 times the amount of water and reflux for extraction 6 times. Combine the extracts, filter, and concentrate under reduced pressure to obtain a concentrated extract;

[0061] 2) Suspend the concentrate obtained in step 1) in water, and then extract successively with petroleum ether, dichloromethane, ethyl acetate, and n-butanol to obtain a petroleum ether fraction, a dichloromethane fraction, an ethyl acetate fraction, an n-butanol fraction, and a water fraction;

[0062] 3) Subject the dichloromethane fraction in step 2) to silica gel column chromatography, and elute successively with petroleum ether: acetone = 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, 1:1, 0:100, and 100% methanol to obtain 9 elution fractions Fr.1 - Fr.9;

[0063] 4) Subject the concentrate of Fr.7 in step 3) to silica gel column chromatography, and elute with CH 2 Cl 2 : MeOH = 50:1, 20:1, 10:1, 5:1, 3:1 to obtain 5 elution fractions Fr.7-1 - Fr.7-5;

[0064] 5) Subject the concentrate of Fr.7-3 in step 4) to silica gel column chromatography, and perform gradient elution with CH 2 Cl 2 : MeOH = 50:1, 25:1 to obtain 2 elution fractions Fr.7-3-1 - Fr.7-3-2;

[0065] 6) Subject the concentrate of Fr.7-3-2 in step 5) to Sephadex LH-20 gel column chromatography, and elute with 75% methanol to obtain 12 elution fractions Fr.7-3-2-1 - Fr.7-3-2-12;

[0066] 7) Subject Fr.7-3-2-5 obtained in step 6) to C 18 ODS RP-HPLC separation to obtain the novel compound 3 (t R = 27.6 min).

[0067] The pharmacological activity results of these three compounds prepared from Ephedra equisetina Bge. showed that they could significantly inhibit the release of β-Hex in RBL-2H3 cells induced by C48 / 80, improve the degranulation phenomenon of RBL-2H3, and could be used as drugs for the treatment of allergic asthma. The relevant experimental data are as follows:

[0068] I. Structure identification of the compounds of the present invention:

[0069] For Compounds 1-3 1 1H-NMR and 13 13C-NMR data are shown in Table 1. The 1H-NMR spectrum, 1 13C-NMR spectrum, and ECD spectrum of the novel Compound 1 13 are shown in the appendix Figure 1-3 , and the 1H-NMR spectrum, 1 13C-NMR spectrum of the novel Compound 2 13 are shown in the appendix Figure 4-5 , and the 1H-NMR spectrum, 1 13C-NMR spectrum, and ECD spectrum of the novel Compound 3 13 are shown in the appendix Figure 6-8 .

[0070] Table 1. 1H-NMR(500MHz, CD 1 3OD) and 3 13C-NMR(125MHZ, CD3OD) data of Compounds 1-3 13

[0071]

[0072]

[0073] II. Anti-allergic asthma effects of Compounds 1-3 of the present invention

[0074] 1) Treatment of monomeric compounds

[0075] In the ultra-clean bench, the compounds were prepared into a stock solution with a concentration of 1 mM using PBS solution (DMSO < 0.01%), filtered and sterilized, and all the prepared drugs were stored in a 4°C refrigerator for later use.

[0076] 2) Preparation of C48 / 80

[0077] Take out C48 / 80 from the -20°C refrigerator and let it stand at room temperature for half an hour. Dissolve it with PBS in the ultra-clean workbench to prepare a stock solution with a concentration of 20 mg / mL, seal it with a label and store it in a 4°C refrigerator for later use.

[0078] 3) Preparation of 1% Trinton X-100

[0079] ​In a laminar flow hood, take 100 μL of Triton X-100, add it to 9.9 mL of tabletop solution and dissolve it to prepare 1% Trinton X-100. Prepare it immediately before use.

[0080] 4) Preparation of β-Hex substrate solution

[0081] Dissolve 4-nitrophenyl-N-acetyl-β-D-glucosamine in 1 M citrate buffer (pH = 4.5) to prepare a 1 mM substrate solution.

[0082] 5) Preparation of 1 M NaOH solution

[0083] Weigh 4 g of NaOH pellets and dissolve them in 100 mL of double-distilled water for adjusting the pH value of the solution.

[0084] 6) Na 2 CO 3 / NaHCO 3 Preparation of termination solution

[0085] Weigh 2.65 g of Na 2 CO 3 and 2.1 g of NaHCO 3 , dissolve them in 250 mL of double-distilled water, and adjust the solution to pH = 10.7 with 1 M NaOH.

[0086] 7) Cell culture

[0087] Take RBL-2H3 cells cryopreserved in liquid nitrogen and thaw them in a 37°C water bath until ice-water coexists. Centrifuge at 1000 rpm / min for 5 min, discard the supernatant, and transfer the cells into a culture dish containing DMEM medium with 10% FBS. Culture them in a 37°C constant temperature incubator with 5% CO 2 . When 80% of the cells cover the dish, passage them, and passage them once every 2 days. Then place the passaged cells in a 37°C, 5% CO 2 incubator and culture them until the logarithmic growth phase.

[0088] 8) Establishment of a C48 / 80-induced RBL-2H3 cell degranulation model

[0089] Take cells in the logarithmic growth phase and inoculate the cell suspension into a 96-well plate at a density of 2×10 4 / mL, 200 μL per well. Set 6 replicates for each group, and repeat the experiment at least 3 times each time; after culturing for 24 h, discard the medium, wash twice with PBS, and add a C48 / 80 solution with a final concentration of 20 μg / mL; add an equal amount of tabletop solution to the control group to establish a C48 / 80-induced RBL-2H3 cell degranulation model.

[0090] 9) Grouping and drug administration

[0091] Normal group (NC), model group (M, C48 / 80, 20 μg / mL), compound 1 (10 μM) + C48 / 80 (20 μg / mL) group, compound 2 (10 μM) + C48 / 80 (20 μg / mL) group, compound 3 (10 μM) + C48 / 80 (20 μg / mL) group, total enzyme group (1% Trinton X-100), and another 3 blank wells were set. After stimulation for 30 min, centrifugation was performed, and the supernatant was taken to detect the change levels of related indexes of RBL-2H3 cells.

[0092] 10) Determination of β-Hex release rate by substrate chromogenic method

[0093] Take 50 μL of cell supernatant, add 50 μL of 1 mM β-Hex substrate solution, incubate at 37 °C for 1 h, and add 150 μL of Na 2 CO 3 / NaHCO 3 Stop the reaction with the stop solution, detect its absorbance at 405 nm using an enzyme-linked immunosorbent assay (ELISA) reader, repeat 3 times, and calculate the β-Hex release rate. [β-Hex release rate = (OD value of the experimental group supernatant - OD value of the blank well supernatant) / (OD value of the total enzyme well supernatant - OD value of the blank well supernatant) × 100%]

[0094] 11) Experimental results

[0095] Compared with the C48 / 80 model group, the β-Hex release rate in the normal group was significantly decreased (P < 0.001); compared with the C48 / 80 model group, the novel compounds 1 - 3 at 10 μM could significantly decrease the β-Hex release rate of RBL-2H3 (P < 0.05 or P < 0.01). The experimental results are shown in Table 2.

[0096] Table 2. Effects of Ephedra equisetina monomer compounds on the β-Hex release rate of C48 / 80-induced RBL-2H3 cells

[0097]

[0098]

[0099] Note: Compared with group M, * P < 0.05, ** P < 0.01, ***P < 0.001

[0100] The present invention uses the stems of Ephedra equisetina Bunge of the genus Ephedra in the family Ephedraceae. Through steps such as solvent extraction, Toyopearl HW-40 column chromatography, silica gel column chromatography, and preparative high-performance liquid separation, three novel compounds 1-3 are obtained. The pharmacological activity results show that these three compounds can significantly reduce the β-Hex release rate of RBL-2H3 cells damaged by C48 / 80 induction, and can be used as drugs for treating anti-allergy. It is an innovation in novel compounds and their uses, and has good application and promotion value.

Claims

1. A compound extracted from Ephedra equisetina Bunge, characterized in that, the chemical structure of the compound is as follows:

2. The preparation method of the compound extracted from Ephedra equisetina Bunge according to claim 1, characterized in that, comprises the following steps: 1) Reflux extract the stems of Ephedra equisetina Bunge with 5 - 15 times the amount of solvent for 2 - 6 times, combine the extracts, filter, and concentrate under reduced pressure to obtain a concentrated extract; 2) Suspend the concentrate obtained in step 1) in water, and then successively extract with petroleum ether, dichloromethane, ethyl acetate, and n - butanol to obtain a petroleum ether fraction, a dichloromethane fraction, an ethyl acetate fraction, an n - butanol fraction, and a water fraction; 3) Subject the dichloromethane fraction in step 2) to silica gel column chromatography and elute successively with petroleum ether:acetone in the ratios of 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, 1:1, 0:100, and 100% methanol to obtain 9 elution fractions Fr.1 - Fr.9; 4) Concentrate the solution of Fr.7 in step 3) and subject it to silica gel column chromatography, eluting with CH 2 Cl 2 :MeOH = 50:1, 20:1, 10:1, 5:1, 3:1 to obtain 5 elution fractions Fr.7-1 to Fr.7-5; 5) Concentrate the solution of Fr.7-4 obtained in step 4) by silica gel column chromatography, and elute it with CH 2 Cl 2 : MeOH = 100:1, 50:1, 25:1, 10:1, 5:1 for gradient elution to obtain 10 elution fractions Fr.7-4-1 to Fr.7-4-10; 6) Concentrate the solution of Fr.7-4-7 obtained in step 5) by silica gel column chromatography, and elute with CH 2 Cl 2 :MeOH = 30:1 to obtain 5 elution fractions Fr.7-4-7-1 to Fr.7-4-7-5; 7) Use C to process Fr.7-4-7-2 obtained in step 6) 18 Perform ODS RP-HPLC separation on it to obtain three elution fractions, namely Fr.7-4-7-2-1 to Fr.7-4-7-2-3; 8) Separate the Fr.7-4-7-2-1 obtained in step 7) by C 18 ODS RP-HPLC to obtain the compound.

3. The preparation method of the compound extracted from Ephedra equisetina Bunge according to claim 2, characterized in that, C in step 7) described above 18 The conditions for ODS RP-HPLC preparation are 5 μm, 250×10 mm, 65% methanol, and 3 mL / min.

4. The preparation method of the compound extracted from Ephedra equisetina Bunge according to claim 2, characterized in that, C in step 8) described above 18 The conditions for ODS RP-HPLC preparation are 5 μm, 250×10 mm, 10% acetonitrile, and 3 mL / min.

5. Use of the compound extracted from Ephedra equisetina Bunge as claimed in claim 1 in the preparation of a medicament for treating allergic asthma.

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