Method for extracting triterpenoid A2 from oriental oriental waterplantain rhizome and application of triterpenoid A2

Abstract

A preparation method of the triterpenoid A2 comprises the following steps: drying and crushing the oriental oriental waterplantain rhizome, dipping the oriental oriental waterplantain rhizome in acetone at room temperature, crushing and extracting, filtering, merging filtrate, and concentrating under reduced pressure to obtain an extract; sequentially extracting the extract with petroleum ether, dichloromethane, ethyl acetate and n-butyl alcohol with the same amount, concentrating and drying to obtain a petroleum ether part, a dichloromethane part, an ethyl acetate part, an n-butyl alcohol part and a water part; dissolving the dichloromethane part with methanol, stirring silica gel, packing and eluting for four times to obtain a target fraction; and loading the target fractions on a chromatographic column, collecting fractions within the retention time, concentrating and drying to obtain the target fractions. The raw materials are rich, a unique preparation method is adopted, and the compound A2 can improve airway inflammation and airway hyperresponsiveness of OVA-induced allergic asthma, can be used for preparing drugs for treating allergic asthma, develops the medicinal value and economic value of oriental rhizoma alismatis, and has huge economic and social benefits.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a method for extracting triterpene compound A2 from oriental alisma orientalis and application thereof. Background Art

[0002] The Chinese medicine Alismatis rhizoma is the dried tuber of the Alisma planta go-aquatica or Alisma orientate, which was first recorded in Shennong's Herbal Classic. It has the effects of promoting diuresis and dispelling dampness, relieving heat, clearing turbidity and reducing lipids. It has been widely used in clinical compound medicines and various Chinese patent medicines for more than 3,000 years. It is a medicinal material for both medicinal and edible use recognized by the State Food and Drug Administration. The 2020 edition of the Chinese Pharmacopoeia lists oriental Alisma as the source plant of the Chinese medicine Alisma.

[0003] Asthma is a chronic inflammatory disease of the airways involving multiple cells and cell components, with recurrent wheezing, shortness of breath, chest tightness or cough as the main clinical symptoms. According to the Global Initiative for Asthma (GINA), asthma affects more than 300 million people worldwide and is growing rapidly at a rate of 20%-25% every decade, making it one of the most common diseases worldwide.

[0004] Traditional Chinese medicine, with its advantage of overall regulation, has a definite and stable effect in preventing and treating asthma recurrence and aggravation, and has relatively few adverse reactions, and has become a hot topic of research for domestic and foreign scientists. Traditional Chinese medicine has complex ingredients and multi-directional targets. So, can a new active ingredient be extracted from Oriental Alisma for use in the treatment of allergic asthma and its application in the preparation of drugs for the treatment of allergic asthma? Therefore, this application adopts a unique preparation method to extract the triterpene compound A2 (oriterpenoid A2) from Oriental Alisma, and evaluate the efficacy of oriterpenoid A2 through the OVA-induced allergic asthma mouse model, and further explore its mechanism with the help of multifactorial joint analysis, flow cytometry and other biochemical methods. The results show that oriterpenoid A2 can significantly prolong the airway hyperresponsiveness and serum IgE, HIS, MTC-β, and LTC4 levels of asthmatic mice; significantly reduce lung damage and inflammatory infiltration in asthmatic mice; significantly reverse the levels of cytokines IgE, PGD2, IL-4, IL-5, IL-10, IL-13, IL-17A, TNF-α, GM-CSF, CXCL1, CCL11, and mMCP-1 in serum and bronchoalveolar lavage fluid; and maintain the balance of Th17 and Treg in asthmatic mice. Oriterpenoid A2 can improve airway inflammation and airway hyperresponsiveness in OVA-induced allergic asthmatic mice. Its mechanism of action may be related to the regulation of Th17 / Treg immune imbalance by oriterpenoid A2. Its structure and mechanism of action have not been publicly reported so far. Summary of the invention

[0005] In view of the above situation, in order to overcome the shortcomings of the prior art, the purpose of the present invention is to provide a method for extracting triterpenoid A2 from Oriental Alisma and its application, which can effectively solve the problem of extracting triterpenoid A2 (oriterpenoid A2) from Oriental Alisma and realize its application in the preparation of drugs for the treatment of allergic asthma.

[0006] The technical solution provided by the present invention is: a method for extracting a triterpene compound A2 from oriental oriental rhizome, wherein the molecular structure of the compound is:

[0007]

[0008] The preparation method thereof is:

[0009] (1) Dry and crush oriental rhizome, soak with acetone at room temperature, crush and extract, filter, combine the filtrate, and concentrate under reduced pressure to obtain an extract;

[0010] (2) dissolving the extract in water, and extracting with equal amounts of petroleum ether, dichloromethane, ethyl acetate, and n-butanol in sequence, concentrating and drying to obtain a petroleum ether fraction, a dichloromethane fraction, an ethyl acetate fraction, an n-butanol fraction, and a water fraction;

[0011] (3) Dissolve the dichloromethane fraction with methanol, mix the sample with silica gel and load it onto the column, and perform gradient elution with dichloromethane:methanol to obtain the first fraction C-6;

[0012] (4) Dissolve the first fraction C-6 in methanol, load onto a column and elute to obtain the second fraction C-6-2;

[0013] (5) Dissolve the second fraction C-6-2 in methanol, load onto a column and elute to obtain the third fraction C-6-2-1;

[0014] (6) Dissolve the third fraction C-6-2-1 in methanol, load onto a column and elute to obtain the fourth fraction C-6-2-1-1;

[0015] (7) The fourth fraction C-6-2-1-1 was separated by semi-preparative HPLC and loaded onto a chromatographic column. The retention time t R = The fraction from 29.0 to 30.5 min was concentrated and dried to obtain triterpenoid A2 (sample number: DFZX-145).

[0016] The triterpene compound A2 prepared by the method of the present invention can significantly improve airway inflammation and airway hyperresponsiveness of OVA-induced allergic asthma, can be used to prepare anti-allergic asthma drugs, and can be used in the preparation of drugs for treating allergic asthma.

[0017] The invention has abundant raw materials and adopts a unique preparation method. The oriterpenoid A2 is extracted by crushing tissue with 50% aqueous acetone, and a new triterpenoid compound is separated and identified from the dichloromethane part of oriental ... BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The molecular structure diagram of the compound of the present invention.

[0019] Figure 2 Compound oriterpenoid A2 1H-NMR spectrum (500 MHz, CD3OD).

[0020] Figure 3 Compound oriterpenoid A2 13 C-NMR spectrum (125 MHz, CD3OD).

[0021] Figure 4 DEPT 135 spectrum of compound oriterpenoid A2.

[0022] Figure 5 Compound oriterpenoid A2 1 H- 1 H COSY spectrum.

[0023] Figure 6 HSQC spectrum of compound oriterpenoid A2.

[0024] Figure 7 HMBC spectrum of compound oriterpenoid A2.

[0025] Figure 8 NOESY spectrum of compound oriterpenoid A2.

[0026] Fig. 9 HR-ESI-MS spectrum of compound oriterpenoid A2.

[0027] Fig.10 UV spectrum of compound oriterpenoid A2.

[0028] Fig.11 IR spectrum of compound oriterpenoid A2.

[0029] Fig.12 Figure 3 Effects of oriterpenoid A2 on asthma phenotype and lung damage in asthmatic mice.

[0030] Fig.13 Effects of the compound oriterpenoid A2 on cytokine levels in asthmatic mice.

[0031] Fig.14 Effect of oriterpenoid A2 on Th17 / Treg balance in asthmatic mice. DETAILED DESCRIPTION

[0032] The specific implementation modes of the present invention are described in detail below in combination with embodiments and specific situations.

[0033] The present invention provides a method for extracting triterpene compound A2 from oriental oriental rhizome, comprising the following steps:

[0034] (1) Take 45 kg of Oriental oriental rhizome, dry and crush it, soak it in 50% acetone at room temperature for 24 hours, crush and extract it three times, filter it, combine the filtrate, and concentrate it under reduced pressure to obtain 9.8 kg of extract;

[0035] (2) Dissolve 9.8 kg of the extract in 16 L of water, and extract with equal amounts of petroleum ether, dichloromethane, ethyl acetate, and n-butanol for 6 times, respectively, and concentrate and dry to obtain a petroleum ether fraction, a dichloromethane fraction, an ethyl acetate fraction, an n-butanol fraction, and a water fraction;

[0036] (3) Dissolve the dichloromethane fraction (280.6 g) in methanol, mix with 200-300 mesh silica gel and load onto the column. Perform gradient elution with a volume ratio of dichloromethane:methanol = 100:0 (8 L), 80:1 (10 L), 30:1 (9 L), 15:1 (11 L), 8:1 (15 L), 5:1 (13 L), 3:1 (12 L), 2:1 (9 L) and 1:1 (10 L). The flow rate is 10 ml / min. The sample is tested every 200 ml. The amount of each gradient mobile phase is determined by thin layer testing with anisaldehyde-concentrated sulfuric acid. The elution is completed in 8 days. Combine the fractions with dichloromethane:methanol = 5:1 and mark them as the first fraction C-6.

[0037] (4) Dissolve the first fraction C-6 in methanol, pass it through a Sephadex LH-20 column, and elute it with 50% methanol by volume at a flow rate of 0.6 ml / min. The amount of mobile phase is 1800 ml. Use anisaldehyde-concentrated sulfuric acid thin layer chromatography to detect. Combine 41-68 ml of the fraction and label it as the second fraction C-6-2.

[0038] (5) The second fraction C-6-2 was dissolved in methanol and passed through a Toyopearl HW-40C column. It was eluted with 30% methanol by volume at a flow rate of 0.6 ml / min. The amount of mobile phase was 450 ml. It was detected by thin layer chromatography of anisaldehyde-concentrated sulfuric acid. The fractions of 20-42 ml were combined and labeled as the third fraction C-6-2-1.

[0039] (6) The third fraction C-6-2-1 was dissolved in methanol, mixed with 200-300 mesh silica gel and loaded into the column, eluted with a volume ratio of dichloromethane:methanol = 25:1, the flow rate was 4.5 ml / min, and detected with anisaldehyde-concentrated sulfuric acid thin layer chromatography, and 35-95 ml of the fractions were combined and marked as the fourth fraction C-6-2-1-1;

[0040] (7) The fourth fraction C-6-2-1-1 was separated by semi-preparative HPLC on a YMC-Pack ODS-AA column with a specification of 250×10 mm, a particle size of 5 μm, and a pore size of 12 nm. The mobile phase was acetonitrile and a trifluoroacetic acid aqueous solution with a volume ratio of 45:55. The flow rate was 3 ml / min. The retention time t was collected. R = The fraction from 29.0 to 30.5 min was concentrated to dryness to obtain triterpenoid A2.

[0041] The triterpene compound A2 prepared by the method of the present invention can significantly improve airway inflammation and airway hyperresponsiveness of OVA-induced allergic asthma, can be used to prepare anti-allergic asthma drugs, and can be used in the preparation of drugs for treating allergic asthma.

[0042] According to the composition given in the above embodiment, any amount of triterpene compound A2 can be prepared as needed. The given embodiment is only used to illustrate the specific implementation mode of the present invention, rather than to limit the protection scope of the present invention. The technical core protected by the present invention is triterpene compound A2 and its preparation method and application.

[0043] The triterpenoid compound prepared by the unique preparation method of the present invention, i.e., oriterpenoid A2, can significantly prolong the airway hyperresponsiveness and serum IgE, HIS, MTC-β, and LTC4 levels of asthmatic mice; significantly reduce the lung damage and inflammatory infiltration of asthmatic mice; significantly reverse the levels of cytokines IgE, PGD2, IL-4, IL-5, IL-10, IL-13, IL-17A, TNF-α, GM-CSF, CXCL1, CCL11, and mMCP-1 in serum and bronchoalveolar lavage fluid, and maintain the balance of Th17 and Treg in asthmatic mice, and can improve the airway inflammation and airway hyperresponsiveness of OVA-induced allergic asthmatic mice. The mechanism of action may be related to the regulation of oriterpenoid A2 on Th17 / Treg immune imbalance. The relevant experimental data are as follows:

[0044] 1. Instruments and test drugs

[0045] 1.1 Main experimental instruments

[0046] Table 1 List of main instruments

[0047]

[0048]

[0049] 1.2 Experimental Reagents

[0050] Ovalbumin II (62-88%, A5253) and ovalbumin V (≥98%) were purchased from Sigma, USA; Inject TMAlum adjuvant (77161) was purchased from Thermo Fisher Scientific, USA; histamine phosphate (H8530) and acetylcholine chloride (C12410057) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; interleukin-4 (Interleukins-4), interleukin-5 (Interleukins-5), interleukin-10 (Interleukins-10), interleukin-13 (Interleukins-13), interleukin-17A (Interleukins-17A), tumor necrosis factor-ɑ (Tumor necrosis factor-ɑTNF-α), granulocyte-macrophage colony-stimulating factor (granulocyte-macrophage colony-stimulating factor, GM-CSF), chemokine CXC ligand 1 (CXCL1), chemokine CCL11 (Chemokine (C-Cmotif) ligand 11, CCL11), mutant monocyte chemotactic protein-1 (mMCP-1) multifactor combined analysis kit (ABplexMouse 10-Plex Custom Panel, Abplex-100) were purchased from Wuhan ABclonal Company; Prostaglandin D2 (PGD2) was purchased from Meimian Company, China (MM-43727M1); Immunoglobulin E (Immunoglobulin E, IgE) was purchased from Chondrex Company, USA (3004); Anti-Mouse CD4 FITC (11-0041-82), Anti-Mouse CD25 PE (12-0251-83), Anti-Mouse Foxp3APC (17-5773-82) and Anti-MouseIL-17AAPC (17-7177-81) flow cytometry antibodies were purchased from eBioscience, USA. Chromatographic acetonitrile, petroleum ether, ethyl acetate, dichloromethane, n-butanol, and analytical methanol were purchased from Tianjin Fuyu Fine Chemical Co., Ltd. Column chromatography fillers Toyopearl HW-40C (TOSOH, Japan), Sephadex LH-20 (Sigma-Aldrich, Germany), thin layer chromatography silica gel (particle range 10-40 μm) (Qingdao Ocean Chemical Plant), column chromatography silica gel H (100-200 mesh, 200-300 mesh, Qingdao Ocean Chemical).

[0051] 1.3 Experimental Materials

[0052] Oriental Alisma was collected from Jiyang Town, Fujian Province in June 2022 and was identified as the dried tuber of Oriental Alisma (Alismatis rhizoma) of the Alismataceae family. The specimen (20220618B) was stored in the Engineering and Technology Research Center for Traditional Chinese Medicine Development, Henan University of Traditional Chinese Medicine, Henan Province.

[0053] The triterpene compound A2 (oriterpenoid A2) prepared by the method of the present invention.

[0054] 2. Structural Identification

[0055] Physical analysis, nuclear magnetic resonance and spectrometer showed that the product of the present invention was a light yellow solid (CH3OH). HR-ESI-MS gave a quasi-molecular ion peak of m / z 503.3730 [M+Na] + ,(calcd.For C 31 H 51 O5Na503.3731), and its molecular formula is determined to be C 31 H 51 O5; UV(MeOH)λ max :198(0.37), 244(0.79); IR(KBr)ν max cm -1 :3433, 2935, 1685, 1631, 1460, 1379, 1243, 1153, 1020cm -1 ; Its chemical molecular structure is:

[0056]

[0057] Table 2 NMR data of compound oriterpenoid A2 (in CD3OD)

[0058]

[0059]

[0060] 3. Activity Test

[0061] 3.1 Establishment of OVA-induced asthma mouse model and intervention plan

[0062] After 7 days of adaptive feeding, 6-7 week-old BALB / c male mice were randomly divided into 5 groups, namely normal control group (NC), model group (OVA), positive drug montelukast sodium group (Mon, 2.6 mg kg -1 ), oriterpenoid A2 low-dose group (OrA-L, 5 mg kg -1), oriterpenoid A2 high-dose group (OrA-H, 10 mg kg -1 ), 10 mice in each group. As shown in Table 3, asthma mouse models were established by intraperitoneal injection of OVA and aluminum hydroxide gel and OVA aerosol stimulation. Except for the normal control group, the rest of the experimental mice were given intraperitoneal injection of 0.2 mL of chicken ovalbumin (OVA) and aluminum hydroxide (Al2(OH)3) suspension for sensitization (OVA 50 μg, Al2(OH)3 2 mg dissolved in 0.2 mL sterile saline) on days 0, 7, and 14, and 1% OVA dissolved in saline was given for aerosol stimulation (100 mg OVA dissolved in 10 mL saline) from day 21 to day 27, 30 min / time / d. The model group, the positive drug montelukast sodium group, and the low and high dose groups of OrA were gavaged 30 min before stimulation. The normal control group was sensitized by intraperitoneal injection of 0.2 mL aluminum hydroxide (Al2(OH)3) suspension (2 mg Al2(OH)3 dissolved in 0.2 mL sterile saline) on days 0, 7, and 14, and was challenged with saline nebulization for 30 minutes from day 21 to 27, and an equal volume of saline was given 30 minutes before the challenge. 24 hours after the last nebulization challenge, the lung function of mice in each group was tested and samples were collected.

[0063] Table 3 Establishment of asthma mouse model

[0064]

[0065] 3.2 Airway responsiveness testing

[0066] 24 hours after the last challenge, the mice were placed in the WBP plethysmography chamber in turn, the baseline value was adjusted with PBS, and the basal value was recorded for about 2 minutes. Subsequently, each mouse was given a nebulized inhalation of methacholine (Mech) at increasing concentrations (0, 3.125 mg / mL, 6.25 mg / mL, 12.5 mg / mL, 25 mg / mL, 50 mg / mL), each dose was nebulized for 30 seconds, and the recording was continuous for 5 minutes. Airway responsiveness (Penh) was measured at different concentrations.

[0067] 3.3 Collection of serum and bronchoalveolar lavage fluid samples

[0068] After the airway responsiveness test was completed, the mice were anesthetized with isoflurane, and blood was collected by eye removal. The blood was centrifuged at 4000 rpm for 10 min to obtain serum samples, which were stored at -80°C for later use. After blood collection, tracheal intubation was performed to obtain bronchoalveolar lavage fluid (BALF), and the whole lung was lavaged with sterile saline for 3 times, 0.5 mL / time. The supernatant was collected by centrifugation at 4000 rpm for 10 min and stored at -80°C for later use.

[0069] 3.4 Lung histopathological evaluation

[0070] Three mice were randomly selected from each group, and the left lungs without lavage were taken and fixed in 4% paraformaldehyde for 24 hours, and H&E, PAS and Masson staining were performed according to the operating standards.

[0071] 3.5 Serum IgE and airway PGD2 level detection

[0072] The levels of IL-4, IL-5, IL-10, IL-13, IL-17A, TNF-α, GM-CSF, CXCL1, CCL11, and mMCP-1 in mouse serum and BALF samples were carefully operated according to the multifactorial combined analysis procedures; the levels of IgE and PGD2 in serum and BALF samples were strictly carried out according to the ELISA instructions.

[0073] 3.6 Detection of Th17 and Treg cell levels in lung tissue and peripheral blood

[0074] Single cell suspensions of peripheral blood and lung tissue were collected and placed in flow cytometry tubes. Th17 cells were labeled with CD4 and IL-17A antibodies, and Treg cells were labeled with CD4, CD25, and Foxp3 antibodies. The Aria III flow cytometer was carefully loaded according to the staining steps and operating instructions.

[0075] 3.7 Statistical analysis

[0076] The experimental data are SPSS 20.0 was used for one-way ANOVA statistical analysis. All statistical analysis results were presented using GraphPad Prism 8.0 (GraphPad Software Inc., USA). P < 0.05 was considered to be significantly different, and P < 0.01 was considered to be extremely significantly different.

[0077] IV. Experimental Results

[0078] In this study, an OVA-induced asthma mouse model was established to evaluate the efficacy of oriterpenoid A2 (OrA) in the treatment of asthma. WBP analysis showed that airway hyperresponsiveness (Penh) was significantly reduced in the OrA-treated group (10 or 20 mg / kg) compared with the model group (M). The levels of mast cell degranulation markers, including β-MCT, LTC4, and HIS in lung tissue, and the asthma marker IgE in serum were quantified by enzyme-linked immunosorbent assay. The results showed that the levels of β-MCT, LTC4, HIS, and IgE were significantly decreased after treatment with OrA (10 or 20 mg / kg). Histological evaluation by H&E and Masson staining showed that the alveolar structure of the control mice was preserved, the bronchial wall was thinned, and there were no obvious pathological changes. In contrast, the alveolar structure of the M group mice was destroyed, the airway smooth muscle was significantly hyperplastic and hypertrophic, the bronchial wall was thickened, the bronchial and blood vessels were infiltrated with inflammatory cells, and collagen was widely deposited. Administration of OrA significantly alleviated these pathological changes. In addition, montelukast sodium, which was used as a positive control, also had a certain therapeutic effect. These data collectively indicate that OrA can effectively improve the asthma phenotype and lung damage in the asthma mouse model. Fig.12 shown.

[0079] Asthma is characterized by the involvement of multiple hallmark inflammatory factors, mainly interleukins (such as IL-2, IL-4, IL-6, IL-13, IL-17), tumor necrosis factor-α (TNF-α), chemokines (such as CXCL1, CCL11), and other inflammatory mediators such as GM-CSF. These factors interact through various mechanisms to contribute to the pathogenesis of asthma, causing airway inflammation and related clinical symptoms. To elucidate the effect of OrA on inflammation in asthmatic mice, we measured several cytokines in serum and bronchoalveolar lavage fluid (BALF). It was shown that treatment with OrA and montelukast sodium significantly downregulated CCL4, IL-13, TNF-α, CCL2, IL-6, CXCL1, CCL11, GM-CSF, and IL-17A, while increasing the levels of IL-2 and IL-10 in BALF. Similarly, serum analysis showed that after treatment with OrA and montelukast sodium, interleukins (IL-4, IL-6, IL-13, IL-17A), chemokines (CXCL1, CCL2, CCL4, CCL11), TNF-α, and GM-CSF were significantly downregulated, and IL-2 and IL-10 were upregulated. In addition, compared with the model group, OrA significantly reduced the level of Th17 cells in peripheral blood and lung tissue, while increasing the level of Treg cells, as shown in Figure 2. Figure 13-14 These findings suggest that OrA can effectively alleviate inflammation and regulate the balance of Th17 / Treg immune cells in asthmatic mice.

[0080] In summary, this application is the first to isolate a new triterpenoid compound oriterpenoid A2 from Alisma orientalis, which can significantly improve airway inflammation and airway hyperresponsiveness in OVA-induced allergic asthma mice. Its mechanism of action may be related to the regulation of oriterpenoid A2 on Th17 / Treg immune imbalance.

[0081] The invention has abundant raw materials and adopts a unique preparation method. The oriterpenoid A2 is separated and identified from the dichloromethane part of the oriental orientalis by crushing the tissue with 50% aqueous acetone. The oriterpenoid A2 can improve the airway inflammation and airway hyperresponsiveness of allergic asthma induced by OVA, can be used to prepare drugs for treating allergic asthma, and has expanded the medicinal value and economic value of the oriental orientalis, with huge economic and social benefits.

Claims

1. A triterpene compound A2 extracted from Oriental oriental rhizome, the molecular structure formula is: 。 2. A method for extracting triterpenoid compound A2 from Orientalis orientalis, characterized in that: Here are the steps: (1) Take 45 kg of Oriental oriental rhizome, dry and crush it, soak it in 50% acetone at room temperature for 24 h, crush and extract it 3 times, filter it, combine the filtrate, and concentrate it under reduced pressure to obtain 9.8 kg of extract; (2) Dissolve 9.8 kg of the extract in 16 L of water, and extract with equal amounts of petroleum ether, dichloromethane, ethyl acetate, and n-butanol for 6 times respectively, concentrate and dry to obtain a petroleum ether fraction, a dichloromethane fraction, an ethyl acetate fraction, a n-butanol fraction, and a water fraction; (3) Dissolve the dichloromethane fraction (280.6 g) in methanol, mix with 200-300 mesh silica gel and load onto the column. Perform gradient elution with a volume ratio of dichloromethane:methanol = 100:0 (8 L), 80:1 (10 L), 30:1 (9 L), 15:1 (11 L), 8:1 (15 L), 5:1 (13 L), 3:1 (12 L), 2:1 (9 L) and 1:1 (10 L). The flow rate was 10 ml / min and the sample was tested every 200 ml. The amount of each gradient mobile phase was determined by thin layer testing with anisaldehyde-concentrated sulfuric acid. The elution was completed in 8 days. The fractions with dichloromethane:methanol = 5:1 were combined and marked as the first fraction C-6. (4) Dissolve the first fraction C-6 in methanol and pass it through a Sephadex LH-20 column. Elute with 50% methanol by volume at a flow rate of 0.6 ml / min. The amount of mobile phase is 1800 ml. Detect with anisaldehyde-concentrated sulfuric acid thin layer chromatography. Combine the fractions of 41-68 ml and label them as the second fraction C-6-2. (5) The second fraction C-6-2 was dissolved in methanol and passed through a Toyopearl HW-40C column. It was eluted with 30% methanol by volume at a flow rate of 0.6 ml / min. The amount of mobile phase was 450 ml. It was detected by thin layer chromatography with anisaldehyde-concentrated sulfuric acid. The fractions of 20-42 ml were combined and labeled as the third fraction C-6-2-1. (6) The third fraction C-6-2-1 was dissolved in methanol, mixed with 200-300 mesh silica gel and loaded into the column. The column was eluted with a volume ratio of dichloromethane:methanol = 25:1 at a flow rate of 4.5 ml / min. The column was identified with anisaldehyde-concentrated sulfuric acid thin layer chromatography. The fractions of 35-95 ml were combined and labeled as the fourth fraction C-6-2-1-1. (7) The fourth fraction C-6-2-1-1 was separated by semi-preparative HPLC on a YMC-Pack ODS-AA column with a specification of 250×10 mm, a particle size of 5 μm, and a pore size of 12 nm. The mobile phase was acetonitrile and a trifluoroacetic acid aqueous solution with a volume ratio of 45:

55. The flow rate was 3 ml / min and the retention time was collected. t R = 29.0 ~ 30.5min fraction was concentrated and dried to obtain triterpene compound A2.

3. Use of the triterpene compound A2 according to claim 1 in the preparation of a drug for treating allergic asthma.

4. Use of the triterpene compound A2 according to claim 1 in the preparation of a drug for improving airway inflammation and airway hyperresponsiveness in OVA-induced allergic asthma.

Images