Three monoterpenoid indole alkaloid compounds extracted from gossypium hirsutum as well as preparation method and application of three monoterpenoid indole alkaloid compounds

Abstract

The invention relates to three monoterpenoid indole alkaloid compounds extracted from gossypium hirsutum as well as a preparation method and application thereof, can effectively solve the medicinal problem of gossypium hirsutum, and adopts the technical scheme that the structural formula of the three monoterpenoid indole alkaloid compounds extracted from gossypium hirsutum is as follows: # imgabs0 #. The preparation method is simple and easy to implement, and the three new compounds extracted from the gossypium hirsutum can inhibit lipopolysaccharide induced N9 microglial cell inflammation damage, have a remarkable neuroinflammation resisting effect, have the prospect of being further developed into drugs for preventing and treating neurodegenerative diseases and are innovations on the aspect of compounds extracted from the gossypium hirsutum.

Description

1. Technical Field

[0001] The invention relates to the field of medical technology, in particular to three monoterpene indole alkaloid compounds extracted from Cotton Tree Cotton Tree, and a preparation method and application thereof. 2. Background Technology

[0002] Neurodegenerative diseases are a disease state in which the structure and function of cellular neurons in the brain and spinal cord are gradually lost. They include Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, different types of spinocerebellar ataxia, etc., which seriously threaten human health and quality of life. Its pathogenesis is still unclear. Existing therapeutic drugs can only improve symptoms to a certain extent, but cannot fundamentally prevent or reverse the progression of the disease. Therefore, it is urgent to find new therapeutic drugs. Studies have shown that neuroinflammation is a key driving factor in the development of neurodegenerative diseases, and it runs through the entire pathogenesis of neurodegenerative diseases. It is the direct cause of neuronal damage or apoptosis. There is great hope for the treatment of neurodegenerative diseases by intervening in neuroinflammation. Therefore, finding compounds with anti-neuroinflammatory effects is of great significance for the research and development of therapeutic drugs for neurodegenerative diseases.

[0003] Alstonia mairei Lévl. is a plant of the genus Alstonia in the family Apocynaceae. It is endemic to my country and is distributed in Yunnan, Guizhou, Sichuan and other places. Its roots and leaves can be used for medicinal purposes, and have the effects of clearing away heat and detoxification, relieving pain and hemostasis, draining pus and promoting tissue regeneration. It can be used externally to treat traumatic bleeding and sores. Modern chemical and pharmacological studies have shown that Alstonia mairei Lévl. contains chemical components such as monoterpene indole alkaloids, flavonoids, triterpenes, and coumarins, and has multiple biological activities such as anti-inflammatory, anti-tumor, acetylcholinesterase inhibition, antioxidant, and neuroprotective. However, there are no reports on the extraction of compounds with anti-neuroinflammatory effects from Alstonia mairei Lévl. III. Summary of the invention

[0004] In view of the above situation, in order to solve the defects of the prior art, the purpose of the present invention is to provide three monoterpene indole alkaloid compounds extracted from Cotton Tree, and their preparation method and application, which can effectively solve the medicinal problem of Cotton Tree.

[0005] The technical solution provided by the present invention is that the structural formulas of the three monoterpene indole alkaloid compounds extracted from Cotton Tree are as follows:

[0006]

[0007] The method for extracting three monoterpene indole alkaloid compounds of the present invention comprises the following steps:

[0008] 1) crushing the dried cottonwood branches and leaves, extracting by immersion with methanol or ethanol with a volume fraction of 70-95% or heating under reflux, concentrating and drying the extract under reduced pressure, dispersing the obtained alcohol extract in a hydrochloric acid aqueous solution, adjusting the pH to 2-3, adding an equal volume of ethyl acetate or dichloromethane or chloroform for extraction, taking the acid water layer, adjusting the pH to 9-11 with ammonia water, and then adding an equal volume of ethyl acetate or dichloromethane or chloroform for extraction, the obtained organic phase extract is the total alkaloids, the total alkaloids are crudely separated by alumina column chromatography, eluted with a dichloromethane-methanol solvent system at a gradient of 100:0-0:100, collecting the eluate, and combining the same fractions by thin layer identification to obtain 14 components FA-FN;

[0009] 2) separating the component FA obtained in step 1) by alumina column chromatography, eluting with a petroleum ether-acetone solvent system at a gradient of 100:0 to 0:100, collecting the eluate, and combining the same fractions by thin layer chromatography to obtain 16 components FA1 to FA16;

[0010] 3) The component FA6 obtained in step 2) was subjected to gel column chromatography, and eluted isocratically with a mixed solvent of petroleum ether: dichloromethane: methanol = 5:5:1 as the eluent, and the same fractions were combined after thin layer analysis to obtain four components FA6-1 to FA6-4;

[0011] 4) The component FA6-1 obtained in step 3) was separated and purified by reverse phase preparative high performance liquid chromatography, the mobile phase was an acetonitrile-water system, the elution conditions were 0 to 40 min, 30% to 100% acetonitrile, and the main peak FA6-1-1 with a retention time of 13.6 min was collected to obtain compound 1, i.e., Alstonmairine A;

[0012] 5) separating the component FE obtained in step 1) by ODS column chromatography, eluting with a methanol-water solvent system at a gradient of 20:80-100:0, collecting the eluate, and combining the same fractions by thin layer chromatography to obtain 9 components FE1-FE9;

[0013] 6) The component FE6 obtained in step 5) was subjected to sephadex LH-20 column chromatography, eluted isocratically with methanol as solvent, and the same fractions were combined after thin layer analysis to obtain 7 components FE6-1 to FE6-7;

[0014] 7) Purifying the component FE6-2 obtained in step 6) by reverse phase preparative high performance liquid separation, using an acetonitrile-water system as the mobile phase, eluting under the conditions of 0 to 35 min, 16% to 60% acetonitrile, collecting the main peak FE6-2-2 with a retention time of 22.1 min, and obtaining compound 2, i.e., Alstonmairine B;

[0015] 8) The component FE5 obtained in step 5) was separated by alumina column chromatography, and eluted with a petroleum ether-acetone solvent system at a gradient of 100:5 to 0:100, and the eluate was collected and the same fractions were combined by thin layer chromatography to obtain 12 components FE5-1 to FE5-12;

[0016] 9) The component FE5-9 obtained in step 8) was separated and purified by reverse phase preparative high performance liquid separation, the mobile phase was acetonitrile-water system, the elution condition was 35% acetonitrile isocratic elution, and the main peak FE5-9-2 with a retention time of 12.3 min was collected to obtain compound 3, namely Alstonmairine C.

[0017] The invention discloses an application of three monoterpene indole alkaloid compounds extracted from cottonwood trees in the preparation of anti-neuroinflammatory drugs.

[0018] Application of compounds 1, 2 and 3 prepared by the extraction method of the present invention in the preparation of anti-neuroinflammatory drugs.

[0019] The raw materials for the preparation of the invention are abundant and easily available, and the preparation method is simple and easy. The three new compounds extracted from cotton wool can inhibit lipopolysaccharide (LPS)-induced inflammatory damage to N9 microglia, have significant anti-neuroinflammatory effects, and have the prospect of being further developed into preventive and therapeutic drugs for neurodegenerative diseases. This is an innovation in the compounds extracted from cotton wool. IV. Description of the drawings

[0020] Figure 1 This is the HRESIMS spectrum of compound 1 of the present invention.

[0021] Figure 2 The compound 1 of the present invention 1 H NMR (500 MHz, CDCl 3 ) spectrum.

[0022] Figure 3 The compound 1 of the present invention 13 C NMR (125 MHz, CDCl 3 ) spectrum.

[0023] Figure 4 This is the HRESIMS spectrum of compound 2 of the present invention.

[0024] Figure 5 The compound 2 of the present invention 1 H NMR (500MHz, CD 3 OD) spectrum.

[0025] Figure 6 The compound 2 of the present invention 13 C NMR (125MHz, CD3 OD) spectrum.

[0026] Figure 7 This is the HRESIMS spectrum of compound 3 of the present invention.

[0027] Figure 8 is the compound 3 of the present invention 1 H NMR (500MHz, CD 3 OD) spectrum.

[0028] Fig. 9 is the compound 3 of the present invention 13 C NMR (125MHz, CD 3 OD) spectrum. V. Specific implementation methods

[0029] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples.

[0030] Example 1

[0031] The present invention, when implemented, specifically comprises the following steps:

[0032] 1) 7.8 kg of dried cottonwood branches and leaves were crushed, and refluxed with 8 times the amount of 90% ethanol for 3 times, each time for 2 hours. The extracts were combined and concentrated under reduced pressure to dryness. The obtained alcohol extract was dispersed in a 1% hydrochloric acid aqueous solution, the pH was adjusted to 2-3, and an equal volume of ethyl acetate was added for extraction 3-5 times. The acidic water layer was taken, the pH was adjusted to 10 with ammonia water, and then an equal volume of ethyl acetate was added for extraction 3-5 times. The obtained organic phase extract was the total alkaloids. The total alkaloids were crudely separated by alumina column chromatography, and the dichloromethane-methanol solvent system was used for gradient elution at 100:0, 100:1, 100:2, 100:3, 100:5, 100:8, 10:1, 5:1, 3:1, 2:1, 1:1, and 0:100. The eluent was collected, and the same fractions were combined by thin layer identification to obtain 14 components FA-FN;

[0033] 2) separating the component FA obtained in step 1) by alumina column chromatography, eluting with a petroleum ether-acetone solvent system at a gradient ratio of 100:0, 100:1, 80:1, 60:1, 50:1, 40:1, 30:1, 25:1, 20:1, 15:1, 10:1, 0:100, collecting the eluate, and combining the same fractions by thin layer chromatography to obtain 16 components FA1-FA16;

[0034] 3) The component FA6 obtained in step 2) was subjected to gel column chromatography, and eluted isocratically with a mixed solvent of petroleum ether: dichloromethane: methanol = 5:5:1 as the eluent, and the same fractions were combined after thin layer analysis to obtain four components FA6-1 to FA6-4;

[0035] 4) The component FA6-1 obtained in step 3) was separated and purified by reverse phase preparative high performance liquid chromatography, the chromatographic column specification model was 10×250 mm, the particle size was 5 μm, the C-18 Fisher Wharton chromatographic column, the mobile phase was an acetonitrile-water system, the elution conditions were 0 to 40 min, 30% to 100% acetonitrile, the flow rate was 3 mL / min, and the main peak FA6-1-1 with a retention time of 13.6 min was collected to obtain compound 1, i.e., Alstonmairine A;

[0036] 5) separating the component FE obtained in step 1) by ODS column chromatography, eluting with a methanol-water solvent system at a gradient ratio of 20:80, 40:60, 60:40, 80:20, and 100:0, collecting the eluate, and combining the same fractions by thin layer chromatography to obtain 9 components FE1 to FE9;

[0037] 6) The component FE6 obtained in step 5) was subjected to sephadex LH-20 column chromatography, eluted isocratically with methanol as solvent, and the same fractions were combined after thin layer analysis to obtain 7 components FE6-1 to FE6-7;

[0038] 7) The component FE6-2 obtained in step 6) was separated and purified by reverse phase preparative high performance liquid phase separation, the chromatographic column specification model was 10×250 mm, the particle size was 5 μm, the C-18 Fisher Wharton chromatographic column, the mobile phase was acetonitrile-water system, the elution conditions were 0 to 35 min, 16% to 60% acetonitrile, the flow rate was 3 mL / min, and the main peak FE6-2-2 with a retention time of 22.1 min was collected to obtain compound 2, namely Alstonmairine B;

[0039] 8) The component FE5 obtained in step 5) was separated by alumina column chromatography, and eluted with a petroleum ether-acetone solvent system at a gradient ratio of 100:5, 100:8, 100:10, 6:1, 3:1, and 0:100, and the eluate was collected and the same fractions were combined by thin layer chromatography to obtain 12 components FE5-1 to FE5-12;

[0040] 9) The component FE5-9 obtained in step 8) was separated and purified by reverse phase preparative high performance liquid separation, the chromatographic column specification model was 10×250 mm, the particle size was 5 μm, the C-18 Fisher Wharton chromatographic column, the mobile phase was acetonitrile-water system, the elution condition was 35% acetonitrile isocratic elution, the flow rate was 3 mL / min, and the main peak FE5-9-2 with a retention time of 12.3 min was collected to obtain compound 3, i.e., Alstonmairine C.

[0041] The three new compounds (Alstonmairine AC) extracted by the present invention are derived from the branches and leaves of Alstonia mairei Levl., a plant of the genus Alstonia in the Apocynaceae family. The biological activity study shows that the three compounds can inhibit LPS-induced N9 microglial inflammatory damage and thus play a neuroprotective role. They can be used for the prevention and treatment of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, different types of spinocerebellar ataxia, etc. The relevant test data are as follows:

[0042] 1. Structural confirmation

[0043] The present invention determines the chemical structures of compounds 1-3 by analyzing the physical and chemical properties of three new compounds extracted from Cotton Tree. The chemical structures of compounds 1-3 are determined by combining multiple spectral methods such as UV, IR, NMR, MS, and computational chemistry methods such as ECD calculation.

[0044] Alstonmairine A(1): Pale yellow amorphous powder. UV(MeOH)λ max (logε):209(3.00),221(2.95)nm; IR(KBr)ν max :3406,2923,2854,1744,1675,1458,1295,1239,1035cm -1 ;HR-ESI-MS m / z 369.1799[M+H] + (calcd.for C 21 H 25 N 2 O 4 ,369.1809); 1 H NMR and 13 The CNMR data are shown in Tables 1 and 2.

[0045] Alstonmairine B(2): Pale yellow amorphous powder. UV(MeOH)λ max (logε):203(1.23),218(1.17)nm; IR(KBr)ν max :2924,1737,1614,1457,1057cm -1 ; HR-ESI-MS m / z 393.1772 [M+Na] + (calcd.for C 21 H 26 N 2 O 4 Na,393.1785); 1H NMR and 13 C NMR data are shown in Tables 1 and 2.

[0046] Alstonmairine C(3): Pale yellow amorphous powder, UV(MeOH)λ max (logε):203(1.18),218(1.22)nm; IR(KBr)ν max :3392,3292,2927,2853,1725,1617,1455,1375,755cm -1 ;HR-ESI-MS m / z 393.1774[M+Na] + (calcd.for C 21 H 26 N 2 O 4 Na,393.1785); 1 HNMR and 13 The CNMR data are shown in Tables 1 and 2.

[0047] Table 1. Compounds 1-3 1 H NMR data (δ H ,J in Hz)

[0048]

[0049] Table 2. Compounds 1-3 13 C NMR data

[0050]

[0051] II. Study on the Anti-neuroinflammatory Activity of Compounds 1-3

[0052] 1. Screening of safe concentrations of compounds 1-3 for N9 cells based on MTT assay

[0053] N9 cells were placed at 37°C with 5% CO 2 Culture in an incubator until the logarithmic growth phase, and the cell density is 3×10 4 / mL, 200μL per well was inoculated in a 96-well plate, and after 24h, it was divided into a normal group (CON) and different concentrations of drug groups (10, 50, 100μM). After 24h of culture, 20μL of MTT solution (5mg / mL) was added to each well, and the culture was continued for 4h. The culture medium was carefully aspirated, and 150μL of DMSO was added to each well. The blue-purple crystals in the well were completely dissolved by shaking for 10min. The absorbance value (OD) of each well was measured by an enzyme-labeled instrument at 490nm to calculate the cell viability.

[0054] The experimental results are shown in Table 3. Compared with the CON group, compound 1-3 had no effect on cell viability at a concentration of 50 μM; when the concentration was 100 μM, cell viability was significantly reduced (P<0.01), so the maximum safe concentration for subsequent experiments was set to 50 μM.

[0055] Table 3. Effects of different concentrations of compounds 1-3 on N9 cells ( n=4)

[0056]

[0057] Note: Compared with the normal group, && P<0.01

[0058] 2. MTT assay was used to detect the effects of different concentrations of compounds 1-3 on LPS-induced N9 cell injury

[0059] N9 cells were placed at 37°C with 5% CO 2 Culture in an incubator until the logarithmic growth phase, and the cell density is 3×10 4 / mL, 200μL per well was inoculated in a 96-well plate, and after 24h, it was divided into a normal group (CON), a model group (LPS, 5μg / mL) and different concentrations of drug groups (0.1, 1, 10, 50μM + LPS, 5μg / mL). After 24h of culture, 20μL of MTT solution (5mg / mL) was added to each well, and the culture was continued for 4h. The culture medium was carefully aspirated, and 150μL of DMSO was added to each well. The blue-purple crystals in the well were completely dissolved by shaking for 10min. The absorbance value (OD) of each well was measured by an enzyme-labeled instrument at 490nm to calculate the cell viability.

[0060] The experimental results are shown in Tables 4-6. Compared with the CON group, the cell viability of the model group (M) was significantly reduced (P<0.01); compared with the M group, when the concentrations of compounds 1-3 were 10μM and 50μM, the cell viability was significantly increased (P<0.01); when the concentration of compounds 2 and 3 was 1μM, the cell viability was increased to a certain extent (P<0.05).

[0061] The results showed that compounds 1-3 had a very significant inhibitory effect on LPS-induced N9 microglial inflammatory damage at concentrations of 10 μM and 50 μM, and can be used to prepare anti-neuroinflammatory drugs. They have good application prospects in the research and development of drugs for neurodegenerative diseases related to neuroinflammation.

[0062] Table 4 Effects of different concentrations of compound 1 on LPS-induced N9 cell injury ( n=4)

[0063]

[0064]

[0065] Note: CON means normal group, M means model group. Compared with CON group, ## P<0.01.

[0066] Compared with group M, ** P<0.01.

[0067] Table 5 Effects of different concentrations of compound 2 on LPS-induced N9 cell injury ( n=4)

[0068]

[0069] Note: CON means normal group, M means model group. Compared with CON group, ## P<0.01.

[0070] Compared with group M, * P<0.05, ** P<0.01.

[0071] Table 6 Effects of different concentrations of compound 3 on LPS-induced N9 cell injury ( n=4)

[0072]

[0073] Note: CON means normal group, M means model group. Compared with CON group, ## P<0.01.

[0074] Compared with group M, * P<0.05, ** P<0.01.

[0075] Conclusion

[0076] In summary, the three novel monoterpene indole alkaloid compounds (Alstonmairine AC) extracted by the present invention can inhibit LPS-induced N9 microglial inflammatory damage and thus play a neuroprotective role, and have the prospect of being further developed into preventive and therapeutic drugs for neurodegenerative diseases, and can be used for the prevention and treatment of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, different types of spinocerebellar ataxia, etc., and have practical clinical significance and promotion and application value, and huge economic and social benefits.

[0077] It should be pointed out that the above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the profession can make changes or modify the technical contents disclosed above into equivalent embodiments with equivalent changes without departing from the scope of the technical solution of the present invention, which all fall within the protection scope of the present invention.

Claims

1. A compound of three monoterpene indole alkaloids extracted from Cotton Tree, characterized in that: The structural formulas of the three monoterpene indole alkaloid compounds are shown below:

2. The method for extracting three monoterpene indole alkaloid compounds from Cotton Tree of Claim 1, characterized in that: The following steps are involved: 1) crushing the dried cottonwood branches and leaves, extracting by immersion with methanol or ethanol with a volume fraction of 70-95% or heating under reflux, concentrating and drying the extract under reduced pressure, dispersing the obtained alcohol extract in a hydrochloric acid aqueous solution, adjusting the pH to 2-3, adding an equal volume of ethyl acetate or dichloromethane or chloroform for extraction, taking the acid water layer, adjusting the pH to 9-11 with ammonia water, and then adding an equal volume of ethyl acetate or dichloromethane or chloroform for extraction, the obtained organic phase extract is the total alkaloids, the total alkaloids are crudely separated by alumina column chromatography, eluted with a dichloromethane-methanol solvent system at a gradient of 100:0-0:100, collecting the eluate, and combining the same fractions by thin layer identification to obtain 14 components FA-FN; 2) separating the component FA obtained in step 1) by alumina column chromatography, eluting with a petroleum ether-acetone solvent system at a gradient of 100:0 to 0:100, collecting the eluate, and combining the same fractions by thin layer chromatography to obtain 16 components FA1 to FA16; 3) The component FA6 obtained in step 2) was subjected to gel column chromatography, and eluted isocratically with a mixed solvent of petroleum ether: dichloromethane: methanol = 5:5:1 as the eluent, and the same fractions were combined after thin layer analysis to obtain four components FA6-1 to FA6-4; 4) The component FA6-1 obtained in step 3) was separated and purified by reverse phase preparative high performance liquid chromatography, the mobile phase was an acetonitrile-water system, the elution conditions were 0 to 40 min, 30% to 100% acetonitrile, and the main peak FA6-1-1 with a retention time of 13.6 min was collected to obtain compound 1, i.e., Alstonmairine A; 5) separating the component FE obtained in step 1) by ODS column chromatography, eluting with a methanol-water solvent system at a gradient of 20:80-100:0, collecting the eluate, and combining the same fractions by thin layer chromatography to obtain 9 components FE1-FE9; 6) The component FE6 obtained in step 5) was subjected to sephadex LH-20 column chromatography, eluted isocratically with methanol as solvent, and the same fractions were combined after thin layer analysis to obtain 7 components FE6-1 to FE6-7; 7) Purifying the component FE6-2 obtained in step 6) by reverse phase preparative high performance liquid separation, using an acetonitrile-water system as the mobile phase, eluting under the conditions of 0 to 35 min, 16% to 60% acetonitrile, collecting the main peak FE6-2-2 with a retention time of 22.1 min, and obtaining compound 2, i.e., Alstonmairine B; 8) The component FE5 obtained in step 5) was separated by alumina column chromatography, and eluted with a petroleum ether-acetone solvent system at a gradient of 100:5 to 0:100, and the eluate was collected and the same fractions were combined by thin layer chromatography to obtain 12 components FE5-1 to FE5-12; 9) The component FE5-9 obtained in step 8) was separated and purified by reverse phase preparative high performance liquid separation, the mobile phase was acetonitrile-water system, the elution condition was 35% acetonitrile isocratic elution, and the main peak FE5-9-2 with a retention time of 12.3 min was collected to obtain compound 3, namely Alstonmairine C.

3. The method for extracting three monoterpene indole alkaloid compounds from Cotton Tree according to claim 2, characterized in that: The following steps are involved: 1) 7.8 kg of dried cottonwood branches and leaves were crushed, and refluxed with 8 times the amount of 90% ethanol for 3 times, each time for 2 hours. The extracts were combined and concentrated under reduced pressure to dryness. The obtained alcohol extract was dispersed in a 1% hydrochloric acid aqueous solution, the pH was adjusted to 2-3, and an equal volume of ethyl acetate was added for extraction 3-5 times. The acidic water layer was taken, the pH was adjusted to 10 with ammonia water, and then an equal volume of ethyl acetate was added for extraction 3-5 times. The obtained organic phase extract was the total alkaloids. The total alkaloids were crudely separated by alumina column chromatography, and the dichloromethane-methanol solvent system was used for gradient elution at 100:0, 100:1, 100:2, 100:3, 100:5, 100:8, 10:1, 5:1, 3:1, 2:1, 1:1, and 0:

100. The eluent was collected, and the same fractions were combined by thin layer identification to obtain 14 components FA-FN; 2) separating the component FA obtained in step 1) by alumina column chromatography, eluting with a petroleum ether-acetone solvent system at a gradient ratio of 100:0, 100:1, 80:1, 60:1, 50:1, 40:1, 30:1, 25:1, 20:1, 15:1, 10:1, 0:100, collecting the eluate, and combining the same fractions by thin layer chromatography to obtain 16 components FA1-FA16; 3) The component FA6 obtained in step 2) was subjected to gel column chromatography, and eluted isocratically with a mixed solvent of petroleum ether: dichloromethane: methanol = 5:5:1 as the eluent, and the same fractions were combined after thin layer analysis to obtain four components FA6-1 to FA6-4; 4) The component FA6-1 obtained in step 3) was separated and purified by reverse phase preparative high performance liquid chromatography, the chromatographic column specification model was 10×250 mm, the particle size was 5 μm, the C-18 Fisher Wharton chromatographic column, the mobile phase was an acetonitrile-water system, the elution conditions were 0 to 40 min, 30% to 100% acetonitrile, the flow rate was 3 mL / min, and the main peak FA6-1-1 with a retention time of 13.6 min was collected to obtain compound 1, i.e., Alstonmairine A; 5) separating the component FE obtained in step 1) by ODS column chromatography, eluting with a methanol-water solvent system at a gradient ratio of 20:80, 40:60, 60:40, 80:20, and 100:0, collecting the eluate, and combining the same fractions by thin layer chromatography to obtain 9 components FE1 to FE9; 6) The component FE6 obtained in step 5) was subjected to sephadex LH-20 column chromatography, eluted isocratically with methanol as solvent, and the same fractions were combined after thin layer analysis to obtain 7 components FE6-1 to FE6-7; 7) The component FE6-2 obtained in step 6) was separated and purified by reverse phase preparative high performance liquid phase separation, the chromatographic column specification model was 10×250 mm, the particle size was 5 μm, the C-18 Fisher Wharton chromatographic column, the mobile phase was acetonitrile-water system, the elution conditions were 0 to 35 min, 16% to 60% acetonitrile, the flow rate was 3 mL / min, and the main peak FE6-2-2 with a retention time of 22.1 min was collected to obtain compound 2, namely Alstonmairine B; 8) The component FE5 obtained in step 5) was separated by alumina column chromatography, and eluted with a petroleum ether-acetone solvent system at a gradient ratio of 100:5, 100:8, 100:10, 6:1, 3:1, and 0:100, and the eluate was collected and the same fractions were combined by thin layer chromatography to obtain 12 components FE5-1 to FE5-12; 9) The component FE5-9 obtained in step 8) was separated and purified by reverse phase preparative high performance liquid separation, the chromatographic column specification model was 10×250 mm, the particle size was 5 μm, the C-18 Fisher Wharton chromatographic column, the mobile phase was acetonitrile-water system, the elution condition was 35% acetonitrile isocratic elution, the flow rate was 3 mL / min, and the main peak FE5-9-2 with a retention time of 12.3 min was collected to obtain compound 3, i.e., Alstonmairine C.

4. Use of the three monoterpene indole alkaloid compounds extracted from Cotton-horn Gossypium according to claim 1 in the preparation of anti-neuroinflammatory drugs.

5. Use of compounds 1, 2, and 3 prepared by the extraction method according to claim 2 or 3 in the preparation of anti-neuroinflammatory drugs.

Images