A method for extracting triterpenoid compound A2 from Alisma plantago-aquatica and its application

Abstract

The application relates to a triterpenoid compound A2 extracted from oriental alisma, and a preparation method thereof. The preparation method is as follows: oriental alisma is dried and crushed, soaked with acetone at room temperature, broken and extracted, filtered, and the filter liquor is combined and concentrated under reduced pressure to obtain an extract; the extract is extracted with equal amounts of petroleum ether, dichloromethane, ethyl acetate and n-butanol in sequence, concentrated and dried to obtain a petroleum ether part, a dichloromethane part, an ethyl acetate part, an n-butanol part and a water part; the dichloromethane part is dissolved in methanol, mixed with silica gel, loaded on a column and eluted four times to obtain a target flow fraction; the target flow fraction is loaded on a chromatographic column, the flow fraction in the retention time is collected, concentrated and dried, and the triterpenoid compound A2 is obtained. The raw material is rich, the unique preparation method is adopted, the compound A2 can improve airway inflammation and airway hyperresponsiveness of OVA-induced allergic asthma, can be used for preparing a medicine for treating allergic asthma, expands the medicinal value and economic value of the oriental alisma, and has great economic and social benefits.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to a method for extracting triterpenoid compound A2 from Alisma plantago-aquatica and its application. Background Technology

[0002] Alismatis rhizoma, a traditional Chinese medicine, is the dried tuber of Alismatis planta go-aquatica or Alismatis orientate. First recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), it possesses diuretic, heat-clearing, turbidity-resolving, and lipid-lowering effects. For over 3000 years, it has been widely used in traditional Chinese medicine compound prescriptions and various prepared medicines. It is recognized by the State Food and Drug Administration as a medicinal and edible herb. The 2020 edition of the *Chinese Pharmacopoeia* lists Alismatis orientate as the source plant of the traditional Chinese medicine Alismatis.

[0003] Asthma is a chronic inflammatory disease of the airways involving multiple cells and cellular components. Clinically, it is characterized by recurrent episodes of wheezing, shortness of breath, chest tightness, or cough. Data from the Global Initiative for Asthma (GINA) shows that asthma affects over 300 million people worldwide and is growing rapidly at a rate of 20%-25% per decade, making it one of the most common diseases globally.

[0004] Traditional Chinese medicine (TCM), with its holistic regulatory advantages, has demonstrated definite and stable effects in preventing and treating asthma recurrence and exacerbation, with relatively few adverse reactions, making it a hot research topic for scientists both domestically and internationally. Given the complex composition and multi-target nature of TCM, the question arises: can a novel active ingredient be extracted from *Alisma plantago-aquatica* (Oriental Alisma) for use in treating allergic asthma and its application in the preparation of drugs for treating allergic asthma? Therefore, this application employs a unique preparation method to extract triterpenoid compound A2 (oriterpenoid A2) from *Alisma plantago-aquatica*. The efficacy of orientpenoid A2 was evaluated using an OVA-induced allergic asthma mouse model, and its mechanism was further explored using multifactorial combined analysis, flow cytometry, and other biochemical methods. The results show that orientpenoid... A2 significantly prolonged airway hyperresponsiveness and serum IgE, HIS, MTC-β, and LTC4 levels in asthmatic mice; significantly reduced lung damage and inflammatory infiltration in asthmatic mice; significantly reversed serum and bronchoalveolar lavage fluid levels of cytokines IgE, PGD2, IL-4, IL-5, IL-10, IL-13, IL-17A, TNF-α, GM-CSF, CXCL1, CCL11, and mMCP-1; and maintained the Th17 / Treg balance in asthmatic mice. Oriterpenoid A2 improved 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, but its structure and mechanism of action have not been publicly reported to date. Summary of the Invention

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

[0006] The technical solution provided by this invention is: a triterpenoid compound A2 extracted from Alisma plantago-aquatica, the molecular structural formula of which is:

[0007]

[0008] Its preparation method is as follows:

[0009] (1) Dry and pulverize the Oriental Ze Xie, soak it in acetone at room temperature, crush and extract it, filter it, combine the filtrates, and concentrate it under reduced pressure to obtain the extract.

[0010] (2) Dissolve the extract in water and extract it in sequence with equal amounts of petroleum ether, dichloromethane, ethyl acetate and n-butanol. Concentrate and dry to obtain petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction, n-butanol fraction and water fraction.

[0011] (3) Dissolve the dichloromethane fraction in methanol, pack the sample with silica gel, and elute with a gradient of dichloromethane:methanol to obtain the first fraction C-6.

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

[0013] (5) Dissolve the second fraction C-6-2 in methanol, pack it into 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, pack it into a column and elute to obtain the fourth fraction C-6-2-1-1;

[0015] (7) Separate the fourth fraction C-6-2-1-1 using semi-preparative HPLC, load it onto a chromatographic column, and collect the fraction with retention time t. R The fraction with a flow rate of 29.0–30.5 min was concentrated and dried to obtain triterpenoid compound A2 (sample number: DFZX-145).

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

[0017] This invention utilizes abundant raw materials and a unique preparation method. The tissue is broken down with 50% hydrated acetone for extraction, and a new triterpenoid compound, orientalis A2, is isolated and identified from the dichloromethane fraction. The efficacy of orientalis A2 is evaluated using an OVA-induced allergic asthma mouse model, and its mechanism is further explored using multifactorial analysis, flow cytometry, and other biochemical techniques. This new triterpenoid compound, A2, can improve airway inflammation and airway hyperresponsiveness in OVA-induced allergic asthma and can be used to prepare drugs for treating allergic asthma. This expands the medicinal and economic value of orientalis, yielding significant economic and social benefits. Attached Figure Description

[0018] Figure 1 This is a molecular structural diagram of the compound of the present invention.

[0019] Figure 2 The compound orterpenoid A2 1H-NMR spectrum (500MHz, CD3OD).

[0020] Figure 3 The compound orterpenoid A2 13 C-NMR spectrum (125MHz, CD3OD).

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

[0022] Figure 5 The compound orterpenoid 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] Figure 9 HR-ESI-MS spectrum of compound oriterpenoid A2.

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

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

[0029] Figure 12 The effect of compound oriterpenoid A2 on asthma phenotype and lung injury in asthmatic mice.

[0030] Figure 13 The effect of compound oriterpenoid A2 on cytokine levels in asthmatic mice.

[0031] Figure 14 The effect of compound oriterpenoid A2 on the Th17 / Treg balance in asthmatic mice. Detailed Implementation

[0032] The specific implementation of the present invention will be described in detail below with reference to examples and specific circumstances.

[0033] This invention discloses a method for extracting triterpenoid compound A2 from Alisma plantago-aquatica, comprising the following steps:

[0034] (1) Take 45kg of dried and pulverized Alisma plantago-aquatica, soak it in 50% acetone at room temperature for 24h, extract it 3 times, filter it, combine the filtrates, and concentrate it under reduced pressure to obtain 9.8kg of extract.

[0035] (2) Dissolve 9.8 kg of extract in 16 L of water, and extract it 6 times each with equal amounts of petroleum ether, dichloromethane, ethyl acetate and n-butanol, concentrate and dry to obtain petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction, n-butanol fraction and water fraction.

[0036] (3) Dissolve the dichloromethane fraction (280.6g) in methanol, mix with 200-300 mesh silica gel and pack into a column. Perform gradient elution with volume ratios of dichloromethane:methanol = 100:0 (8L), 80:1 (10L), 30:1 (9L), 15:1 (11L), 8:1 (15L), 5:1 (13L), 3:1 (12L), 2:1 (9L) and 1:1 (10L) at a flow rate of 10ml / min. Check every 200ml. The amount of each gradient mobile phase is determined by anisaldehyde-concentrated sulfuric acid thin-layer chromatography. After elution for 8 days, combine the dichloromethane:methanol = 5:1 fraction and label it as the first fraction C-6.

[0037] (4) Dissolve the first fraction C-6 in methanol, pass it through a Sephadex LH-20 column, elute with 50% methanol at a flow rate of 0.6 ml / min, use 1800 ml of mobile phase, identify with anisaldehyde-concentrated sulfuric acid thin layer chromatography, combine 41-68 ml fractions and label them as the second fraction C-6-2.

[0038] (5) The second fraction C-6-2 was dissolved in methanol, passed through a Toyopearl HW-40C column, eluted with 30% methanol at a flow rate of 0.6 ml / min, and the mobile phase volume was 450 ml. It was identified by anisaldehyde-concentrated sulfuric acid thin-layer chromatography. 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 packed into a column. It was eluted with a volume ratio of dichloromethane:methanol = 25:1 at a flow rate of 4.5 ml / min. It was identified by thin-layer chromatography with anisaldehyde-concentrated sulfuric acid. The fractions of 35-95 ml were combined and labeled 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 using a YMC-Pack ODS-AA column with a specification of 250×10mm, particle size of 5μm, and pore size of 12nm. The mobile phase was an aqueous solution of acetonitrile and trifluoroacetic acid with a volume ratio of 0.03% = 45:55, the flow rate was 3ml / min, and the retention time was t. R The fraction with a flow rate of 29.0–30.5 min was concentrated and dried to obtain triterpenoid compound A2 (oriterpenoid A2).

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

[0042] The compositions given in the above embodiments can be used to prepare any amount of triterpenoid compound A2 as needed. The given embodiments are only for illustrating specific implementation methods of the present invention and are not intended to limit the scope of protection of the present invention. The core technology protected by the present invention is triterpenoid compound A2 and its preparation method and application.

[0043] This invention utilizes a unique preparation method to produce a triterpenoid compound, oriterpenoid A2, which significantly prolongs airway hyperresponsiveness and serum IgE, HIS, MTC-β, and LTC4 levels in asthmatic mice; significantly reduces lung damage and inflammatory infiltration in asthmatic mice; significantly reverses 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 maintains the Th17 / Treg balance in asthmatic mice. It can also 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. Relevant experimental data are as follows:

[0044] I. Instruments and Reagents

[0045] 1.1 Main Experimental Instruments

[0046] Table 1 List of Main Instruments

[0047]

[0048]

[0049] 1.2 Experimental Reagents

[0050] Oval albumin grade II (62-88%, A5253) and oval albumin grade V (≥98%) were purchased from Sigma-Aldrich, USA. 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, interleukin-5, interleukin-10, interleukin-13, interleukin-17A, tumor necrosis factor-α (TNF-α), granulocyte-macrophage colony-stimulating factor (GM-CSF), chemokine CXCL1 (CXC ligand 1), and chemokine CCL11 (Chemokine (C-Cmotif) ligand 1) were purchased from Shanghai Maclean Biochemical Technology Co., Ltd. 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 (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. Acetonitrile, petroleum ether, ethyl acetate, dichloromethane, n-butanol, and analytical methanol for chromatography were all purchased from Tianjin Fuyu Fine Chemical Co., Ltd. Column chromatography packing materials included Toyopearl HW-40C (TOSOH, Japan), Sephadex LH-20 (Sigma-Aldrich, Germany), thin-layer chromatography silica gel (particle size range 10–40 μm) (Qingdao Haiyang Chemical Plant), and column chromatography silica gel H (100–200 mesh, 200–300 mesh, Qingdao Haiyang Chemical).

[0051] 1.3 Experimental Materials

[0052] Oriental Ze Xie was harvested in June 2022 from Jiyang Town, Fujian Province. It was identified as the dried tuber of Oriental Ze Xie (Alismatis rhizoma), a plant of the Alismataceae family. The specimen (20220618B) is stored at the Engineering Technology Research Center for the Development of Traditional Chinese Medicine, Henan University of Traditional Chinese Medicine, Henan Province.

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

[0054] II. Structural Identification

[0055] Physical analysis, including nuclear magnetic resonance (NMR) and spectroscopy, revealed that the product of this invention is a pale yellow solid (CH3OH). HR-ESI-MS analysis showed a quasi-molecular ion peak at m / z 503.3730 [M+Na]. + ,(calcd.For C 31 H 51 O5Na5O3.3731), its molecular formula was 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] III. Activity Test

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

[0062] After 7 days of acclimatization feeding, 6-7 week old male BALB / c rats were randomly divided into 5 groups: normal control group (NC), model group (OVA), positive control group (Mon, 2.6 mg / kg), and positive control group (Mon, 2.6 mg / kg). -1 low-dose orterpenoid A2 group (OrA-L, 5 mg·kg) -1high-dose orterpenoid A2 group (OrA-H, 10 mg·kg) -1 Ten mice were included in each group. As shown in Table 3, an asthma mouse model was established using intraperitoneal injection of OVA and aluminum hydroxide gel, and OVA nebulization challenge. Except for the normal control group, the other experimental mice were sensitized by intraperitoneal injection of 0.2 mL of chicken ovalbumin (OVA) and aluminum hydroxide (Al2(OH)3) suspension (OVA 50 μg, Al2(OH)3 2 mg dissolved in 0.2 mL sterile physiological saline) on days 0, 7, and 14. From day 21 to day 27, they were challenged by nebulization with 1% OVA dissolved in physiological saline (100 mg OVA dissolved in 10 mL physiological saline), 30 min / time / day. The model group, the positive control group, and the low- and high-dose OrA groups were administered the drugs by gavage 30 min before challenge. The normal control group was sensitized by intraperitoneal injection of 0.2 mL of aluminum hydroxide (Al2(OH)3) suspension (2 mg Al2(OH)3 dissolved in 0.2 mL sterile saline) on days 0, 7, and 14. From days 21 to 27, mice were challenged by saline nebulization for 30 min, with an equal volume of saline administered 30 min before the challenge. Lung function was assessed and samples were collected 24 h after the last nebulization challenge.

[0063] Table 3 Establishment of the asthma mouse model

[0064]

[0065] 3.2 Airway responsiveness testing

[0066] Twenty-four hours after the last challenge, mice were sequentially placed into the WBP plethysmography chamber, and baseline values ​​were adjusted with PBS. Baseline values ​​were recorded for approximately 2 minutes. Subsequently, each mouse was administered methacholine (Mech) via nebulization at fold-increase concentrations (0, 3.125 mg / mL, 6.25 mg / mL, 12.5 mg / mL, 25 mg / mL, and 50 mg / mL), with each dose nebulized for 30 seconds, and continuous recording for 5 minutes. Airway responsiveness (Penh) at different concentrations was measured.

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

[0068] After the airway responsiveness test was completed, mice were anesthetized with isoflurane, and blood was collected by enucleation. The blood samples were centrifuged at 4000 rpm for 10 min to obtain serum samples, which were then stored at -80℃ for later use. Following blood collection, bronchoalveolar lavage fluid (BALF) was obtained through endotracheal intubation. The entire lungs were lavaged three times with sterile saline, 0.5 mL per lavage. The BALF was centrifuged at 4000 rpm for 10 min, and the supernatant was collected and stored at -80℃ for later use.

[0069] 3.4 Pathological assessment of lung tissue

[0070] Three mice were randomly selected from each group. The left lung of the mice was taken without irrigation and fixed in 4% paraformaldehyde for 24 hours. H&E, PAS and Masson staining were performed according to the standard procedure.

[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 analyzed according to the multifactorial combined analysis procedure; the levels of IgE and PGD2 in serum and BALF samples were strictly analyzed 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 from peripheral blood and lung tissue were placed in flow cytometry tubes. Th17 cells were labeled with CD4 and IL-17A antibodies, while Treg cells were labeled with CD4, CD25, and Foxp3 antibodies. The cells were then carefully run on an Aria III flow cytometer following the staining procedures and operating instructions.

[0075] 3.7 Statistical Analysis

[0076] Experimental data The results were analyzed using SPSS 20.0 using one-way ANOVA. All statistical analysis results were presented using GraphPad Prism 8.0 (GraphPad Software Inc., USA). A p-value < 0.05 was considered statistically significant, and a p-value < 0.01 was considered highly significant.

[0077] IV. Experimental Results

[0078] This study established an OVA-induced asthma mouse model to evaluate the efficacy of oriterpenoid A2 (OrA) in treating asthma. WBP analysis showed that airway hyperresponsiveness (Penh) was significantly reduced in the OrA treatment groups (10 or 20 mg / kg) compared to the model group (M). Enzyme-linked immunosorbent assay (ELISA) was used to quantify mast cell degranulation markers (including β-MCT, LTC4, and HIS in lung tissue) and serum asthma marker IgE levels. The results indicated that treatment with OrA (10 or 20 mg / kg) significantly reduced the levels of β-MCT, LTC4, HIS, and IgE. Histological evaluation by H&E and Masson staining showed that the alveolar structure of the control group mice was preserved, the bronchial walls were thinned, and there were no obvious pathological changes. In contrast, the alveolar structure of the M group mice was disrupted, with significant airway smooth muscle hyperplasia and hypertrophy, thickened bronchial walls, inflammatory cell infiltration in the bronchi and blood vessels, and extensive collagen deposition. OrA administration significantly alleviated these pathological changes. In addition, montelukast sodium, used as a positive control, also showed some therapeutic effect. These data collectively indicate that OrA can effectively improve the asthma phenotype and lung injury in an asthmatic mouse model, see [link to relevant data]. Figure 12 As shown.

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

[0080] In summary, this application is the first to isolate a novel triterpenoid compound, oriterpenoid A2, from Alisma plantago-aquatica. It can significantly 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.

[0081] This invention utilizes abundant raw materials and employs a unique preparation method. The tissue is broken down and extracted using 50% hydrated acetone. A novel triterpenoid compound, oriterpenoid A2, was isolated and identified from the dichloromethane fraction of *Alisma plantago-aquatica*. This new triterpenoid compound, oriterpenoid A2, can improve airway inflammation and airway hyperresponsiveness in OVA-induced allergic asthma and can be used to prepare drugs for treating allergic asthma. This invention expands the medicinal and economic value of *Alisma plantago-aquatica*, resulting in significant economic and social benefits.

Claims

1. The application of a triterpenoid compound A2 extracted from Alisma plantago-aquatica in the preparation of a drug for treating allergic asthma. The molecular structural formula of triterpenoid compound A2 is as follows: 。 2. The use of the triterpenoid compound A2 according to claim 1 in the preparation of a drug for improving airway inflammation and airway hyperresponsiveness in OVA-induced allergic asthma.

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