A method for extracting a compound alisma organic alkali A from alisma orientalis and application thereof

Abstract

This invention relates to a method for extracting compound Alisma alkaloid A from Alisma plantago-aquatica and its application, effectively solving the problem of extracting Alisma alkaloids from Alisma plantago-aquatica and enabling its application in the preparation of drugs for treating lung cancer. This compound, Alisma alkaloid A, significantly reduces cell viability and Bcl2 expression, induces apoptosis and mitochondrial membrane depolarization in A549 lung cancer cells, and significantly increases the expression of Bax, Caspase3, and Caspase9, without affecting normal human lung epithelial cells BEAS-2B. It has the effect of inducing apoptosis in A549 cells through the mitochondrial apoptosis pathway, thus realizing the application of Alisma alkaloid A in the preparation of drugs for treating lung cancer. The method of this invention is scientifically sound, novel, unique, and easy to operate, effectively extracting compound Alisma alkaloid A from Alisma plantago-aquatica. This compound has the effect of inducing apoptosis in A549 cells through the mitochondrial apoptosis pathway, realizing the application of Alisma alkaloid A in the preparation of drugs for treating lung cancer, and expanding the medicinal and commercial value of Alisma plantago-aquatica.

Description

Technical Field

[0001] This invention relates to medicine, and in particular to a method for extracting the compound alisma alkaloid A 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. It is widely used in traditional Chinese medicine compound prescriptions and various prepared Chinese medicines, and is recognized as a health food ingredient by the State Food and Drug Administration. The 2020 edition of the *Chinese Pharmacopoeia* lists Alismatis orientate as the source plant for Alismatis rhizoma, but how to extract the modern pharmacological activity and active ingredients from Alismatis rhizoma remains a technical issue requiring further research. Traditional Chinese medicine has unique advantages in treating various diseases, especially intractable diseases such as various cancers, particularly lung cancer. According to WHO statistics, lung cancer is one of the diseases with the highest incidence and mortality rates in the world. Lung cancer can be divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC is the most common, accounting for more than 85% of all lung cancers, seriously threatening human health. Currently, the treatment of lung cancer mostly involves surgery and chemotherapy. To reduce the adverse reactions of chemotherapy drugs, the search for anti-cancer active ingredients with low toxicity and high efficacy from natural plants has been a key focus and hot topic for scientists both domestically and internationally. Studies have shown that alkaloids in natural medicines have anti-tumor effects. Therefore, this invention uses human non-small cell lung cancer A549 and normal human lung epithelial cells BEAS-2B to screen for the anti-lung cancer activity of alismalkaloid A extracted and isolated from Alisma plantago-aquatica. Experimental results show that alismalkaloid A can significantly reduce cell viability and Bcl2 expression, induce apoptosis and mitochondrial membrane potential depolarization in A549 lung cancer cells, and significantly increase the expression of Bax, Caspase3, and Caspase9, without affecting normal human lung epithelial cells BEAS-2B, suggesting that it may induce apoptosis in A549 cells through the mitochondrial apoptosis pathway. However, how to extract active compound compounds from Alisma plantago-aquatica for the treatment of lung cancer and realize their application in the preparation of anti-lung cancer drugs has not been publicly reported to date. Summary of the Invention

[0003] In view of the above situation and to overcome the defects of the prior art, the purpose of this invention is to provide a method for extracting alismoalkaloid A from Alisma plantago-aquatica and its application, which can effectively solve the problem of extracting alismoalkaloid from Alisma plantago-aquatica and realize its application in the preparation of drugs for treating lung cancer.

[0004] The technical solution provided by this invention is a method for extracting the compound alismoalkaloid A from Alisma plantago-aquatica, wherein the chemical structural formula of the compound alismoalkaloid A is as follows:

[0005]

[0006] The preparation method is as follows: Take 40-50 kg of dried and pulverized Alisma plantago-aquatica, and soak it in 4-6 times its weight-volume of 50% acetone at room temperature for 22-26 hours. (Weight-volume refers to kg for solids and L for liquids). Extract the extract three times using a flash extractor, each time for 30 seconds. Filter the extract, combine the filtrates, and concentrate under reduced pressure to obtain 9.5-10.2 kg of extract. Dissolve the extract in 15-17 L of water, and extract it successively with equal volumes of petroleum ether, dichloromethane, ethyl acetate, and n-butanol 5-7 times each to obtain the petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction, n-butanol fraction, and water fraction, respectively. Concentrate and dry each fraction. The dichloromethane fraction (278.4-282.4 g) was dissolved in methanol, mixed with 200-300 mesh silica gel, and packed into a column. Gradient elution was performed using dichloromethane:methanol ratios of 100:0, 80:1, 30:1, 15:1, 8:1, 5:1, 3:1, 2:1, and 1:1 (v / v), at a flow rate of 10 mL / min. Elution was performed every 200 mL, with the volume of mobile phase used for each gradient determined by anisaldehyde-concentrated sulfuric acid thin-layer chromatography. Elution was completed in 5-6 days. The dichloromethane:methanol fraction (2:1) was combined, labeled fraction C-8, concentrated, and dried. Fraction C-8 was dissolved in methanol and purified by Toyopearl chromatography. An HW-40C column was used as the eluting agent, with 50% methanol (v / v) at a flow rate of 0.6 mL / min and a mobile phase volume of 750-850 mL. The fractions were identified by anisaldehyde-concentrated sulfuric acid thin-layer chromatography. Fractions of 61-120 mL were combined and labeled as fraction C-8-2. Fraction C-8-2 was dissolved in methanol and analyzed using a Toyopearl chromatography system. An HW-40C column was used for elution with 45% methanol (v / v) at a flow rate of 0.6 mL / min. The mobile phase volume was 450-550 mL. Anisaldehyde-concentrated sulfuric acid thin-layer chromatography was used for identification. Fractions of 81-135 mL were combined and labeled as fraction C-8-2-3. Fraction C-8-2-3 was dissolved in methanol, packed into 200-300 mesh silica gel, and eluted with dichloromethane:methanol at a v / v ratio of 30:1 at a flow rate of 5 mL / min. Anisaldehyde-concentrated sulfuric acid thin-layer chromatography was used for identification. Fractions of 44-130 mL were combined and labeled as fraction C-8-2-3-3. Fraction C-8-2-3-3 was separated by semi-preparative HPLC and loaded onto a YMC-Pack (250×10 mm, 5 μm particle size, 12 nm pore size). An ODS-AA column was used, with a mobile phase of acetonitrile:trifluoroacetic acid aqueous solution at a volume ratio of 21:79 (3 / 10,000), a flow rate of 3 mL / min, and a collection retention time t. R The fraction with a flow rate of 31.0–32.5 min was concentrated and dried to obtain the compound alisma alkaloid A.

[0007] The compound alisma alkaloid A prepared by this method can significantly reduce cell viability and Bcl2 expression, induce apoptosis and mitochondrial membrane potential depolarization in A549 lung cancer cells, and significantly increase the expression of Bax, Caspase3, and Caspase9. It has no effect on normal human lung epithelial cells BEAS-2B, and has the effect of inducing apoptosis in A549 cells through the mitochondrial apoptosis pathway, thus realizing the application of alisma alkaloid A in the preparation of drugs for treating lung cancer.

[0008] The method of this invention is scientific, reasonable, novel, unique, and easy to operate. It can effectively extract the compound alismoalkaloid A from Alisma plantago-aquatica. This compound can induce apoptosis in A549 cells through the mitochondrial apoptosis pathway, realizing the application of alismoalkaloid A in the preparation of drugs for treating lung cancer. This expands the medicinal and commercial value of Alisma plantago-aquatica and has great economic and social benefits. Attached Figure Description

[0009] Figure 1 This is a chemical structural diagram of the compound alisma alkaloid A of this invention.

[0010] Figure 2 The compound of this invention is alismoalkaloid A. 1 H-NMR (500MHz, CD3OD) spectrum.

[0011] Figure 3 The compound of this invention is alismoalkaloid A. 13 C-NMR (125MHz, CD3OD) spectrum.

[0012] Figure 4 This is the DEPT 135 spectrum of alismoalkaloid A, a compound of this invention.

[0013] Figure 5 The compound of this invention is alismoalkaloid A. 1 H- 1 H COSY spectrum.

[0014] Figure 6 This is the HSQC spectrum of the compound Alisma alkaloid A of this invention.

[0015] Figure 7This is the HMBC spectrum of the compound Alisma alkaloid A of this invention.

[0016] Figure 8 This is the HR-ESI-MS spectrum of alismoalkaloid A, a compound of this invention.

[0017] Figure 9 This is the UV spectrum of alisma alkaloid A, a compound of this invention.

[0018] Figure 10 The image shows the IR spectrum of alisma alkaloid A, a compound of this invention.

[0019] Figure 11 The graph shows the effect of the compound Alisma alkaloid A (as described in this invention) on human non-small cell lung cancer A549 cells (A549 cells in logarithmic growth phase were used at a concentration of 2 × 10⁻⁶ cells). 4 cells / mL or 5×10 4 Cells / mL were seeded at 200 μL or 3 mL per well in 96 or 6-well plates. After 24 h, the cells were divided into a normal control group (Con) and alismalkaloid A (0.1, 1, 10, 20, 50 μM). A: A549 cell viability was measured by MTT assay 24 h after drug treatment; B: BEAS-2B cytotoxicity was measured by MTT assay 24 h after drug treatment; C: Cell index was observed 24 h after drug treatment using a label-free real-time monitoring system; D: A549 cell apoptosis level was measured by flow cytometry; E: A549 cell JC-1 level was measured by flow cytometry; F: Apoptosis level quantification; G: JC-1 level quantification; H: Fluorescence imaging of Bax, Bcl2, Caspase3, and Caspase9; I: Bax quantification; J: Bcl2 quantification; K: Caspase3 quantification; L: Caspase9 quantification. Compared with the Con group, * P<0.05, ** P<0.01. Con: normal control group; 10, 20 μM were different doses of alismalkaloid A. Detailed Implementation

[0020] The specific implementation of the present invention will be described in detail below with reference to examples and specific circumstances.

[0021] In specific implementation, the preparation method of the present invention is given by the following examples.

[0022] Example 1

[0023] This invention discloses a method for extracting alisma alkaloid A from Alisma plantago-aquatica. 45 kg of dried and pulverized Alisma plantago-aquatica is soaked in 5 times its weight-volume of 50% acetone at room temperature for 24 hours. The extract is then subjected to flash extraction three times, each time for 30 seconds. The extracts are filtered, combined, and concentrated under reduced pressure to obtain 9.8 kg of extract. The extract is dissolved in 16 L of water and extracted six times each with equal volumes of petroleum ether, dichloromethane, ethyl acetate, and n-butanol, yielding the petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction, n-butanol fraction, and water fraction, respectively. Each fraction is concentrated and dried. The dichloromethane fraction (280.6 g) is then extracted. Dissolve in methanol, mix with 200-300 mesh silica gel and pack onto a column. Elute using gradient ratios of dichloromethane:methanol = 100:0, 80:1, 30:1, 15:1, 8:1, 5:1, 3:1, 2:1, and 1:1 (v / v), at a flow rate of 10 mL / min. Check every 200 mL. The volume of mobile phase used for each gradient is determined by anisaldehyde-concentrated sulfuric acid thin-layer chromatography. Elution is completed after 6 days. Combine the fractions of dichloromethane:methanol = 2:1, label them fraction C-8, and concentrate and dry. Dissolve fraction C-8 in methanol and elute with 50% methanol (v / v) onto a Toyopearl HW-40C column at a flow rate of 0.6 mL / min. Use 800 mL of mobile phase and check with anisaldehyde-concentrated sulfuric acid thin-layer chromatography. Combine the fractions of 61-120 mL, label them fraction C-8-2. Dissolve fraction C-8-2 in methanol and elute using a Toyopearl column. An HW-40C column was used for elution with 45% methanol (v / v) at a flow rate of 0.6 mL / min and a mobile phase volume of 500 mL. Anisaldehyde-sulfuric acid thin-layer chromatography was used for identification. Fractions of 81-135 mL were combined and labeled as fraction C-8-2-3. Fraction C-8-2-3 was dissolved in methanol, packed into 200-300 mesh silica gel, and eluted with dichloromethane:methanol at a v / v ratio of 30:1 at a flow rate of 5 mL / min. Anisaldehyde-sulfuric acid thin-layer chromatography was used for identification. Fractions of 44-130 mL were combined and labeled as fraction C-8-2-3-3. Fraction C-8-2-3-3 was separated by semi-preparative HPLC and loaded onto a YMC-Pack (250×10 mm, 5 μm particle size, 12 nm pore size). An ODS-AA column was used, with a mobile phase of acetonitrile:trifluoroacetic acid aqueous solution at a volume ratio of 21:79 (3 / 10,000, mass concentration), a flow rate of 3 mL / min, and a collection retention time t. R The fraction with a flow rate of 31.0–32.5 min was concentrated and dried to obtain the compound alisma alkaloid A.

[0024] Example 2

[0025] This invention discloses a method for extracting alisma alkaloid A from Alisma plantago-aquatica. 42 kg of dried and pulverized Alisma plantago-aquatica is soaked in 4 times its weight-volume of 50% acetone at room temperature for 25 hours. The extract is then subjected to flash extraction three times, each time for 30 seconds. The extracts are filtered, combined, and concentrated under reduced pressure to obtain 9.8 kg of extract. The extract is dissolved in 15.5 L of water and extracted five times each with equal volumes of petroleum ether, dichloromethane, ethyl acetate, and n-butanol, yielding the petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction, n-butanol fraction, and water fraction, respectively. Each fraction is concentrated and dried. The dichloromethane fraction (278.6 g) is then extracted. Dissolve in methanol, mix with 200-300 mesh silica gel and pack onto a column. Elute using gradient ratios of dichloromethane:methanol = 100:0, 80:1, 30:1, 15:1, 8:1, 5:1, 3:1, 2:1, and 1:1 (v / v), at a flow rate of 10 mL / min. Check every 200 mL. The volume of mobile phase used for each gradient is determined by anisaldehyde-concentrated sulfuric acid thin-layer chromatography. Elute for 5 days. Combine the fractions of dichloromethane:methanol = 2:1, label as fraction C-8, and concentrate and dry. Dissolve fraction C-8 in methanol and elute with 50% methanol (v / v) on a Toyopearl HW-40C column at a flow rate of 0.6 mL / min. Use 750 mL of mobile phase and check with anisaldehyde-concentrated sulfuric acid thin-layer chromatography. Combine the fractions of 61-120 mL, label as fraction C-8-2. Dissolve fraction C-8-2 in methanol and elute using a Toyopearl column. An HW-40C column was used for elution with 45% methanol (v / v) at a flow rate of 0.6 mL / min and a mobile phase volume of 460 mL. Anisaldehyde-sulfuric acid thin-layer chromatography was used for identification. Fractions of 81-135 mL were combined and labeled as fraction C-8-2-3. Fraction C-8-2-3 was dissolved in methanol, packed into 200-300 mesh silica gel, and eluted with dichloromethane:methanol at a v / v ratio of 30:1 at a flow rate of 5 mL / min. Anisaldehyde-sulfuric acid thin-layer chromatography was used for identification. Fractions of 44-130 mL were combined and labeled as fraction C-8-2-3-3. Fraction C-8-2-3-3 was separated by semi-preparative HPLC and loaded onto a YMC-Pack (250×10 mm, 5 μm particle size, 12 nm pore size). An ODS-AA column was used, with a mobile phase of acetonitrile:trifluoroacetic acid aqueous solution at a volume ratio of 21:79 (3 / 10,000), a flow rate of 3 mL / min, and a collection retention time t. R The fraction with a flow rate of 31.0–32.5 min was concentrated and dried to obtain the compound alisma alkaloid A.

[0026] Example 3

[0027] This invention discloses a method for extracting alisma alkaloid A from Alisma plantago-aquatica. 49 kg of dried and pulverized Alisma plantago-aquatica is soaked in 6 times its weight-volume of 50% acetone at room temperature for 23 hours. The extract is then subjected to flash extraction three times, each time for 30 seconds. The extracts are filtered, combined, and concentrated under reduced pressure to obtain 10.1 kg of extract. The extract is dissolved in 17 L of water and extracted seven times each with equal volumes of petroleum ether, dichloromethane, ethyl acetate, and n-butanol, yielding the petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction, n-butanol fraction, and water fraction, respectively. Each fraction is concentrated and dried. The dichloromethane fraction (282.4 g) is then extracted. Dissolve in methanol, mix with 200-300 mesh silica gel and pack onto a column. Elute using gradient ratios of dichloromethane:methanol = 100:0, 80:1, 30:1, 15:1, 8:1, 5:1, 3:1, 2:1, and 1:1 (v / v), at a flow rate of 10 mL / min. Check every 200 mL. The volume of mobile phase used for each gradient is determined by anisaldehyde-concentrated sulfuric acid thin-layer chromatography. Elute for 6 days. Combine the fractions of dichloromethane:methanol = 2:1, label as fraction C-8, and concentrate and dry. Dissolve fraction C-8 in methanol and elute with 50% methanol (v / v) onto a Toyopearl HW-40C column at a flow rate of 0.6 mL / min. Use 850 mL of mobile phase and check with anisaldehyde-concentrated sulfuric acid thin-layer chromatography. Combine the fractions of 61-120 mL, label as fraction C-8-2. Dissolve fraction C-8-2 in methanol and elute using a Toyopearl column. An HW-40C column was used for elution with 45% methanol (v / v) at a flow rate of 0.6 mL / min and a mobile phase volume of 550 mL. Anisaldehyde-sulfuric acid thin-layer chromatography was used for identification. Fractions of 81-135 mL were combined and labeled as fraction C-8-2-3. Fraction C-8-2-3 was dissolved in methanol, packed into 200-300 mesh silica gel, and eluted with dichloromethane:methanol at a v / v ratio of 30:1 at a flow rate of 5 mL / min. Anisaldehyde-sulfuric acid thin-layer chromatography was used for identification. Fractions of 44-130 mL were combined and labeled as fraction C-8-2-3-3. Fraction C-8-2-3-3 was separated by semi-preparative HPLC and loaded onto a YMC-Pack (250×10 mm, 5 μm particle size, 12 nm pore size). An ODS-AA column was used, with a mobile phase of acetonitrile:trifluoroacetic acid aqueous solution at a volume ratio of 21:79 (3 / 10,000), a flow rate of 3 mL / min, and a collection retention time t. R The fraction with a flow rate of 31.0–32.5 min was concentrated and dried to obtain the compound alisma alkaloid A.

[0028] This compound, alisma alkaloid A, possesses anti-lung cancer activity and can be effectively used in the preparation of anti-lung cancer drugs. The application of alisma alkaloid A in anti-lung cancer drugs has been demonstrated through structural identification and experiments, showing excellent results. Relevant information is as follows:

[0029] 1. Instruments and reagents

[0030] 1.1 Experimental Apparatus

[0031] Table 1 Instruments

[0032]

[0033]

[0034] 1.2 Experimental Reagents

[0035] Table 2 Reagents

[0036]

[0037]

[0038] 1.3 Experimental Materials

[0039] Column chromatography packing material: Toyopearl HW-40C (TOSOH Corporation, Japan); thin layer chromatography silica gel (particle size range 10-40 μm) (Qingdao Ocean Chemical Plant); column chromatography silica gel H (100-200 mesh, 200-300 mesh, Qingdao Ocean Chemical).

[0040] The Oriental Ze Xie was harvested in June 2022 from Jiyang Town, Fujian Province. It was identified by Professor Chen Suiqing of Henan University of Traditional Chinese Medicine 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.

[0041] 2. Structural identification

[0042] The compound in Example 1 of this invention is a colorless solid, composed of... Figure 2-10 The HR-ESI-MS results show a quasi-molecular ion peak at m / z 481.2295 [M+Na]. + ,(calcd.For C 40 H 38 O 16 Na481.23O9), its molecular formula was determined to be C 25 H 34 O6N2;UV(MeOH)λ max:206(2.40), 280(0.45); IR(KBr)ν max cm -1 :3394,1638,1517,1453,1275,1199,1203,1151,1127,1035cm -1 The chemical structural formula is:

[0043]

[0044] The relevant data is shown in Table 1:

[0045] Table 1. NMR data (in CD3OD) of compound salviamarinic acid A

[0046]

[0047]

[0048] The structures of the other examples were identified, and the results showed that they were compounds with the same chemical structure, namely alismoalkaloid A.

[0049] 3. Activity Experiment

[0050] 3.1 Cell Culture

[0051] Thaw frozen A549 cells in a 37°C water bath until they reach an ice-water balance. Immediately centrifuge at 1000 rpm for 5 min, discard the supernatant, and transfer the cells to a culture dish containing 10% FBS in Ham'F-12K medium (penicillin and streptomycin, both 100 kU / L). Incubate at 37°C in a 5% CO2 incubator until the cells reach 80-90% confluence. Passage the cells. Replace the medium with fresh medium every 24 hours.

[0052] Thaw frozen BEAS-2B cells in a 37°C water bath until they reach an ice-water balance. Immediately centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and transfer the cells to a culture dish containing 10% FBS in DMEM high-glucose medium (penicillin and streptomycin, both 100 kU / L). Incubate at 37°C in a 5% CO2 incubator until the cells reach 80-90% confluence. Passage the cells. Replace the medium with fresh medium every 24 hours.

[0053] 3.2 Experimental study on the effect of alismalkaloid A on A549 cells based on MTT assay

[0054] A549 cells were cultured at 37°C in a 5% CO2 incubator until the logarithmic growth phase, and then cultured at a cell density of 2 × 10⁻⁶ cells / year. 4Cells were seeded at a density of 200 μL / well in 96-well plates. After 24 h, the plates were divided into a normal control group (Con) and alismalkaloid A (0.1, 1, 10, 20, and 50 μM). After 24 h of culture, 20 μL of MTT solution (5 mg / mL) was added to each well, and the plates were cultured for another 4 h. The culture medium was carefully aspirated, and 150 μL of DMSO was added to each well. The plates were shaken for 10 min to completely dissolve the blue-purple crystals. The OD value of each well was measured at 490 nm using a microplate reader to calculate cell viability.

[0055] 3.3 Experimental study on the effect of alismalkaloid A on BEAS-2B cells based on MTT assay

[0056] BEAS-2B cells were cultured at 37°C in a 5% CO2 incubator until the logarithmic growth phase, with a cell density of 2.5 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of 200 μL / mL in 96-well plates. After 24 h, the plates were divided into a normal control group (Con) and alismalkaloid A groups (0.1, 1, 10, 20, and 50 μM). After 24 h of incubation, 20 μL of MTT solution (5 mg / mL) was added to each well, and the plates were incubated for another 4 h. The culture medium was carefully aspirated, and 150 μL of DMSO was added to each well. The plates were shaken for 10 min to completely dissolve the blue-purple crystals. The absorbance (OD) of each well was measured at 490 nm using a microplate reader, and cell viability was calculated.

[0057] 3.4 Annexin V-PE / 7-AAD method for detecting apoptosis levels

[0058] A549 cells were fed at a rate of 5 × 10⁻⁶ 4 Cells were seeded at a density of 3 mL / well in 6-well plates. After 24 hours of adhesion, the plates were divided into a normal control group (Con) and alismalkaloid A (10 μM and 20 μM). Cell samples were obtained after 24 hours of culture. The apoptosis level of A549 cells was analyzed by flow cytometry according to the apoptosis detection kit.

[0059] 3.5 Detection of mitochondrial membrane potential levels

[0060] A549 cells were fed at a rate of 5 × 10⁻⁶ 4 Cells were seeded at a density of 3 mL / well in 6-well plates. After 24 hours of adhesion, the cells were divided into a normal control group (Con) and alismalkaloid A (10 μM, 20 μM). Cell samples were obtained after 24 hours of culture. The mitochondrial depolarization level of A549 cells was detected according to the JC-1 kit, and the JC-1 level of A549 cells was analyzed by flow cytometry.

[0061] 3.6 Experiment using a high-content imaging analysis system to detect the expression of mitochondrial apoptosis-related proteins

[0062] A549 cells in logarithmic growth phase were harvested at a rate of 2 × 10⁻⁶. 4 200 μL of alimons per mL were seeded into each well of a 96-well plate. After 24 h, the plates were divided into a normal control group (Con) and alismalkaloid A (10 and 20 μM). After 24 h of treatment, the supernatant was collected, fixed with formaldehyde, permeabilized with Triton, and blocked with bovine serum albumin for 1.5 h. Then, the plates were incubated overnight with primary antibodies (Bcl2, Bax, Caspase3, Caspase9, and β-actin, all dissolved in bovine serum albumin (BSA) at a dilution of 1:1000). Secondary antibody (dissolved in BSA at a dilution of 1:1000) was added and incubated for 1 h. The plates were then washed 4 times with PBST for 5 min each time, and once with PBS for 5 min each time. The results were analyzed using a high-content imaging system, and the fluorescence values ​​were normalized and statistically analyzed as protein expression levels.

[0063] 3.7 Statistical Analysis

[0064] Experimental data are expressed as mean ± standard deviation (x̄ ± sd) and statistical analysis was performed using SPSS 26.0. One-way ANOVA was used for comparisons between groups. P < 0.05 indicated a statistically significant difference, and P < 0.01 indicated a highly statistically significant difference.

[0065] 4. Conclusion

[0066] The anticancer activity of alismalkaloid A was screened using human non-small cell lung cancer A549 cells. The results showed that alismalkaloid A significantly reduced cell viability and Bcl2 expression (P<0.05 or P<0.01); and induced apoptosis and mitochondrial depolarization in lung cancer cells, while significantly increasing the expression of Bax, Caspase3, and Caspase9 (P<0.01) (see [link to study]). Figure 11 It is non-toxic to normal human lung epithelial cells BEAS-2B, suggesting that it can induce apoptosis in lung cancer cells and has significant anti-cancer activity.

[0067] With the further development and application of traditional Chinese medicine (TCM), the anti-tumor effects of its active ingredients have gained clinical recognition and attracted widespread attention. Due to their low toxicity and therapeutic efficacy, TCM active ingredients exert their anti-cancer effects through mechanisms such as inhibiting cancer cell proliferation and inducing cancer cell apoptosis. Clinical research experiments have shown that Alisma plantago-aquatica contains various chemical components with broad biological activities. Its traditional pharmacological effects are mainly focused on lowering blood lipids, blood pressure, blood sugar, promoting diuresis, relieving constipation, and inhibiting atherosclerosis; however, research reports on its anti-cancer effects are relatively few. Lung cancer is one of the diseases with the highest incidence and mortality rates worldwide. Based on its pathological type, lung cancer can be divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC is the most common, accounting for more than 85% of all lung cancers, seriously threatening human health. Experiments have shown that drug-mediated tumor cell apoptosis is an important approach to cancer treatment, and the alkaloid components in natural medicines have the characteristics of high efficiency and low toxicity in anti-tumor effects. The mitochondrial apoptosis pathway is the most important signaling pathway for cell apoptosis caused by tumor resistance to anticancer drugs. In this pathway, Bax and Bcl-2 work together to change the permeability of the mitochondrial membrane, while Caspase9 and Caspase3 are responsible for initiating and executing apoptosis, respectively. This invention utilizes 50% hydrated acetone to disrupt the tissue and extract a novel alkaloid compound, alismalkaloid A, from the dichloromethane fraction of *Alisma plantago-aquatica*. An in vitro NSCLC model was established using human non-small cell lung cancer (NSCLC) A549 cells for anticancer activity screening. Experimental results showed that alismalkaloid A significantly reduced cell viability and Bcl2 expression, induced lung cancer cell apoptosis and mitochondrial depolarization, and significantly increased the expression of Bax, Caspase3, and Caspase9. It showed no toxicity to normal human lung epithelial cells BEAS-2B, suggesting that it may induce lung cancer cell apoptosis by improving the mitochondrial apoptosis pathway, exhibiting significant anticancer activity. This invention effectively solves the problem of obtaining alismalkaloid A from *Alisma plantago-aquatica* for the preparation of anti-lung cancer drugs, expanding the medicinal value of *Alisma plantago-aquatica* and bringing significant economic and social benefits.

Claims

1. A method for extracting compound Alisma alkaloid A from Alisma plantago-aquatica, characterized in that, The chemical structural formula of the compound, alisma alkaloid A, is as follows: ； The preparation method is as follows: Take 40-50 kg of dried and pulverized Alisma plantago-aquatica, and soak it in 4-6 times its weight and volume of 50% acetone at room temperature for 22-26 hours. (Weight and volume refer to kg for solids and L for liquids). Extract the extract three times using a flash extractor, each time for 30 seconds. Filter the extract, combine the filtrates, and concentrate under reduced pressure to obtain 9.5-10.2 kg of extract. Dissolve the extract in 15-17 L of water, and extract it 5-7 times each with equal volumes of petroleum ether, dichloromethane, ethyl acetate, and n-butanol, respectively, to obtain the petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction, n-butanol fraction, and water fraction. After concentration and drying, the dichloromethane fraction was dissolved in methanol, mixed with 200-300 mesh silica gel, and packed into a column. Gradient elution was performed at dichloromethane:methanol ratios of 100:0, 80:1, 30:1, 15:1, 8:1, 5:1, 3:1, 2:1, and 1:1 (v / v), at a flow rate of 10 mL / min. Elution was performed every 200 mL, with the volume of each mobile phase determined by anisaldehyde-concentrated sulfuric acid thin-layer chromatography. Elution was completed in 5-6 days. The dichloromethane:methanol = 2:1 fraction was combined, labeled as fraction C-8, and concentrated and dried. Fraction C-8 was dissolved in methanol and purified by Toyopearl chromatography. An HW-40C column was used as the elution medium, with 50% methanol at a flow rate of 0.6 mL / min and a mobile phase volume of 750-850 mL. The fractions were identified by anisaldehyde-concentrated sulfuric acid thin-layer chromatography, and 61-120 mL fractions were combined and labeled as fraction C-8-2. Fraction C-8-2 was dissolved in methanol and purified by Toyopearl HW-40C chromatography. The chromatographic column was eluted with 45% methanol (v / v) at a flow rate of 0.6 mL / min, using 450-550 mL of mobile phase. Anisaldehyde-concentrated sulfuric acid thin-layer chromatography was used for identification. Fractions of 81-135 mL were combined and labeled as fraction C-8-2-3. Fraction C-8-2-3 was dissolved in methanol, packed into a column with 200-300 mesh silica gel, and eluted with dichloromethane:methanol at a v / v ratio of 30:1 at a flow rate of 5 mL / min. Anisaldehyde-concentrated sulfuric acid thin-layer chromatography was used for identification. Fractions of 44-130 mL were combined and labeled as fraction C-8-2-3-3. Fraction C-8-2-3-3 was separated by semi-preparative HPLC and loaded onto a YMC-Pack (250×10 mm, 5 μm particle size, 12 nm pore size). An ODS-A column was used, with a mobile phase of acetonitrile:trifluoroacetic acid aqueous solution at a volume ratio of 21:79 (3 / 10,000), a flow rate of 3 mL / min, and a collection retention time t. R The fraction was concentrated and dried over a period of 31.0–32.5 min to obtain the compound alismoalkaloid A.

2. The method for extracting compound Alisma alkaloid A from Alisma plantago-aquatica according to claim 1, characterized in that, 45 kg of dried and pulverized Alisma plantago-aquatica was soaked in 50% acetone (5 times the weight volume of Alisma plantago-aquatica) at room temperature for 24 hours. The extract was then subjected to flash extraction three times for 30 seconds each time. After filtration, the filtrates were combined and concentrated under reduced pressure to obtain 9.8 kg of extract. The extract was dissolved in 16 L of water and extracted six times each with equal volumes of petroleum ether, dichloromethane, ethyl acetate, and n-butanol to obtain the petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction, n-butanol fraction, and water fraction, respectively. Each fraction was concentrated and dried. The dichloromethane fraction was dissolved in methanol. The sample was packed into a column using 00-300 mesh silica gel. Gradient elution was performed using dichloromethane:methanol ratios of 100:0, 80:1, 30:1, 15:1, 8:1, 5:1, 3:1, 2:1, and 1:1 (v / v), at a flow rate of 10 mL / min. Analysis was performed every 200 mL. The volume of mobile phase used for each gradient was determined by anisaldehyde-concentrated sulfuric acid thin-layer chromatography. Elution was completed after 6 days. The fraction with dichloromethane:methanol = 2:1 was combined and labeled as fraction C-8, then concentrated and dried. Fraction C-8 was dissolved in methanol and eluted using a Toyopearl HW-40C column with 50% methanol (v / v) at a flow rate of 0.6 mL / min and a mobile phase volume of 800 mL. Analysis was performed by anisaldehyde-concentrated sulfuric acid thin-layer chromatography. The fractions of 61-120 mL were combined and labeled as fraction C-8-2. Fraction C-8-2 was dissolved in methanol and eluted using a Toyopearl column. An HW-40C column was used for elution with 45% methanol (v / v) at a flow rate of 0.6 mL / min and a mobile phase volume of 500 mL. Anisaldehyde-sulfuric acid thin-layer chromatography was used for identification. Fractions of 81-135 mL were combined and labeled as fraction C-8-2-3. Fraction C-8-2-3 was dissolved in methanol, packed into 200-300 mesh silica gel, and eluted with dichloromethane:methanol at a v / v ratio of 30:1 at a flow rate of 5 mL / min. Anisaldehyde-sulfuric acid thin-layer chromatography was used for identification. Fractions of 44-130 mL were combined and labeled as fraction C-8-2-3-3. Fraction C-8-2-3-3 was separated by semi-preparative HPLC and loaded onto a YMC-Pack (250×10 mm, 5 μm particle size, 12 nm pore size). An ODS-A column was used, with a mobile phase of acetonitrile:trifluoroacetic acid aqueous solution at a volume ratio of 21:79 (3 / 10,000, v / v), a flow rate of 3 mL / min, and a collection retention time t. R The fraction was concentrated and dried over a period of 31.0 to 32.5 minutes to obtain the compound alismoalkaloid A.

3. The method for extracting compound Alisma alkaloid A from Alisma plantago-aquatica according to claim 1, characterized in that, 42 kg of dried and pulverized Alisma plantago-aquatica was soaked in 4 times its weight and volume of 50% acetone at room temperature for 25 h. The extract was then subjected to flash extraction three times for 30 s each time. The extracts were filtered, and the filtrates were combined and concentrated under reduced pressure to obtain 9.8 kg of extract. The extract was dissolved in 15.5 L of water and extracted five times each with equal volumes of petroleum ether, dichloromethane, ethyl acetate, and n-butanol, yielding the petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction, n-butanol fraction, and water fraction, respectively. Each fraction was concentrated and dried. The dichloromethane fraction was dissolved in methanol. Pack the sample onto a column using 200-300 mesh silica gel. Perform gradient elution with dichloromethane:methanol ratios of 100:0, 80:1, 30:1, 15:1, 8:1, 5:1, 3:1, 2:1, and 1:1 (v / v), at a flow rate of 10 mL / min. Check every 200 mL. The volume of mobile phase used for each gradient is determined by anisaldehyde-concentrated sulfuric acid thin-layer chromatography. After 5 days of elution, combine the dichloromethane:methanol 2:1 fraction, label it fraction C-8, and concentrate and dry it. Dissolve fraction C-8 in methanol and pass it through a Toyopearl HW-40C column, eluting with 50% methanol at a flow rate of 0.6 mL / min using 750 mL of mobile phase. Check with anisaldehyde-concentrated sulfuric acid thin-layer chromatography. Combine the fractions of 61-120 mL, labeling them fraction C-8-2. Dissolve fraction C-8-2 in methanol and pass it through a Toyopearl HW-40C column. The chromatographic column was eluted with 45% methanol (v / v) at a flow rate of 0.6 mL / min using 460 mL of mobile phase. Anisaldehyde-concentrated sulfuric acid thin-layer chromatography was used for identification. Fractions of 81-135 mL were combined and labeled as fraction C-8-2-3. Fraction C-8-2-3 was dissolved in methanol, packed into a column with 200-300 mesh silica gel, and eluted with dichloromethane:methanol at a v / v ratio of 30:1 at a flow rate of 5 mL / min. Anisaldehyde-concentrated sulfuric acid thin-layer chromatography was used for identification. Fractions of 44-130 mL were combined and labeled as fraction C-8-2-3-3. Fraction C-8-2-3-3 was separated by semi-preparative HPLC and loaded onto a YMC-Pack (250 × 10 mm, 5 μm particle size, 12 nm pore size). An ODS-A column was used, with a mobile phase of acetonitrile:trifluoroacetic acid aqueous solution at a volume ratio of 21:79 (3 / 10,000), a flow rate of 3 mL / min, and a collection retention time t. R The fraction was concentrated and dried over a period of 31.0–32.5 min to obtain the compound alismoalkaloid A.

4. The method for extracting compound Alisma alkaloid A from Alisma plantago-aquatica according to claim 1, characterized in that, 49 kg of dried and pulverized Alisma plantago-aquatica was soaked in 50% acetone (6 times its weight volume) at room temperature for 23 h. The extract was then subjected to flash extraction three times for 30 s each time. After filtration, the filtrates were combined and concentrated under reduced pressure to obtain 10.1 kg of extract. The extract was dissolved in 17 L of water and extracted seven times each with equal volumes of petroleum ether, dichloromethane, ethyl acetate, and n-butanol to obtain the petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction, n-butanol fraction, and water fraction, respectively. Each fraction was concentrated and dried. The dichloromethane fraction was dissolved in methanol, mixed with 200-300 mesh silica gel, and packed into a column. Gradient elution was performed at dichloromethane:methanol ratios of 100:0, 80:1, 30:1, 15:1, 8:1, 5:1, 3:1, 2:1, and 1:1 (v / v), at a flow rate of 10 mL / min. The volume of mobile phase used in each gradient was determined by anisaldehyde-concentrated sulfuric acid thin-layer chromatography. After elution for 6 days, the fractions containing dichloromethane and methanol in a 2:1 ratio were combined and labeled as fraction C-8. The fractions were then concentrated and dried. Fraction C-8 was dissolved in methanol and passed through a Toyopearl HW-40C column, eluted with 50% methanol at a flow rate of 0.6 mL / min and a mobile phase volume of 850 mL. Identification was performed using anisaldehyde-concentrated sulfuric acid thin-layer chromatography. Fractions of 61-120 mL were combined and labeled as fraction C-8-2. Fraction C-8-2 was dissolved in methanol and passed through a Toyopearl HW-40C column. The chromatographic column was eluted with 45% methanol (v / v) at a flow rate of 0.6 mL / min using 550 mL of mobile phase. Anisaldehyde-sulfuric acid thin-layer chromatography was used for identification. Fractions of 81-135 mL were combined and labeled as fraction C-8-2-3. Fraction C-8-2-3 was dissolved in methanol, packed into a column with 200-300 mesh silica gel, and eluted with dichloromethane:methanol at a v / v ratio of 30:1 at a flow rate of 5 mL / min. Anisaldehyde-sulfuric acid thin-layer chromatography was used for identification. Fractions of 44-130 mL were combined and labeled as fraction C-8-2-3-3. Fraction C-8-2-3-3 was separated by semi-preparative HPLC and loaded onto a YMC-Pack (250 × 10 mm, 5 μm particle size, 12 nm pore size). An ODS-A column was used, with a mobile phase of acetonitrile:trifluoroacetic acid aqueous solution at a volume ratio of 21:79 (3 / 10,000), a flow rate of 3 mL / min, and a collection retention time t. R The fraction was concentrated and dried over a period of 31.0–32.5 min to obtain the compound alismoalkaloid A.

5. The use of alisma alkaloid A extracted by the method according to any one of claims 1-4 in the preparation of a drug for treating non-small cell lung cancer.

Images