Aaptamine alkaloid and preparation method and application thereof

Abstract

The application provides an aaptamine alkaloid and a preparation method and application thereof. The aaptamine alkaloid has a structure shown in formula I. The aaptamine alkaloid can significantly inhibit LPS-induced BV2 cell NO production, and inhibit expression of inflammatory factors IL-6, IL-1beta and TNF-alpha, and has an anti-neuroinflammatory effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to aaptamine alkaloid and a preparation method and application thereof. BACKGROUND

[0002] Marine drugs are drugs developed by using modern scientific methods and technologies with marine organisms as drug sources. At present, more than 40,000 marine natural products have been found in the world, more than 50% of which have various biological activities and strong drug-like and drug-making properties. Marine sponges, as marine invertebrates, are important resources for the research of marine drug source compounds. There are more than 20 species of sponges in the genus Aaptos in the South China Sea, and the sponges in this genus can metabolize aaptamine alkaloids with a unique benzo[de][1,6]naphthyridine skeleton. Such alkaloids show good biological activities in many aspects such as antibacterial, antitumor, antiviral, enzyme inhibitor, and antifouling (Larghi, E. L.; Bohn, M. L.; Kaufman, T. S. Aaptamine and related products. Their isolation, chemical syntheses, and biological activity. Tetrahedron. 2009, 65, 4257-4282.).

[0003] Among the aaptamine homologues, there is a class of triazatetracyclo-aaptamine (TTA) alkaloids, the structural formula of which is shown in 1-12 as follows:

[0004]

[0005] Such alkaloids were obtained early from the Indonesian sponge A. suberitoides (Pham, C.; Hartmann, R. P., Stork, B., Wesselborg, S., Lin, W., Proksch, P. Aaptamine derivatives from the indonesian sponge Aaptos suberitoides. J. Nat. prod. 2013, 76, 103-106.). Later, a series of TTA tetranortriterpenoid aaptamine alkaloids were obtained from the South China Sea sponge Aaptos ([1] Yu, H. B.; Sun, F.; Ma, G. Y.; Gan, J. H.; Hu, W. Z.; Han, B. N.; Jiao, W. H.; Lin, H. W. Cytotoxic aaptamine derivatives from the South China Sea sponge Aaptos aaptos. J. Nat. Prod. 2014, 77, 2124-2129. [2] Gan, J. H.; Hu, W. Z.; Yu H. B.; Yang, F.; Gao, M. X.; Shi, H. J.; Kang, Y. F.; Han, B. N. Three new aaptamine derivatives from the South China Sea sponge Aaptos aaptos. J. Asian Nat. Prod. Res. 2015, 17, 1231-1238. [3] Yu, H. B.; Yang, F.; Sun, F. Li, J.; Jiao, W. H.; Gan, J. H.; Hu, W. Z.; Lin, H. W. Aaptamine derivatives with antifungal and anti-HIV-1 activities from the South China Sea sponge Aaptos aaptos. Mar. Drugs. 2014, 12, 6003-6013.). The diazacyclic structure in TTA alkaloids is formed by the condensation of C-9 nitrogen substitution structure with NH-1 via Schiff base ring. It is speculated that there should be an intermediate of C-9 nitrogen substitution and non-cyclization. So far, there has been no report on C-9 nitrogen substitution aaptamine monomer alkaloids. Given that aaptamine alkaloids generally have good biological activity, in order to enrich the types of marine drugs, more such natural products still need to be developed. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application aims to provide a kind of aaptamine alkaloid and its preparation method and application.A kind of aaptamine alkaloid with the structure shown in formula I is isolated from sponges Aaptossuberitoides for the first time in the present application, and it is found that it has anti-neuritis effect, provides a new lead compound for the research and development of anti-neuritis drugs, and enriches the types of marine drugs.

[0007] To achieve this purpose, the technical scheme adopted by the present application is as follows:

[0008] In a first aspect, the present application provides a kind of aaptamine alkaloid or its pharmaceutically acceptable salt, the aaptamine alkaloid has the structure shown in formula I:

[0009]

[0010] Wherein, R1 is hydroxyl or methoxy;

[0011] R2 is hydrogen atom, methyl, Representative group of connecting bond.

[0012] In some embodiments of the present application, the aaptamine alkaloid is selected from compound II-compound VI;

[0013]

[0014] Wherein, the chemical name of compound II is N-(8-hydroxy-1H-benzo [de] [1, 6] naphthyridin-9-yl) acetamide, molecular formula C 13 H 11 N3O2.

[0015] The chemical name of compound III is N-(8-methoxy-1H-benzo [de] [1, 6] naphthyridin-9-yl) acetamide, molecular formula C 14 H 13 N3O2.

[0016] The chemical name of compound IV is (S)-2-hydroxy-N-(8-hydroxy-1H-benzo [de] [1, 6] naphthyridin-9-yl) propanamide, molecular formula C 14 H 13 N3O3.

[0017] The chemical name of compound V is N-(8-methoxy-1H-benzo[de][1,6]naphthyridin-9- yl)formamide, molecular formula C 13 H 11 N3O2.

[0018] The chemical name of compound VI is 4-hydroxy-N-(8-methoxy-1H-benzo[de][1,6]naphthyridin-9- yl)benzamide, molecular formula C 19 H 15 N3O3.

[0019] In some embodiments of the present application, the pharmaceutically acceptable salt is a salt of the aaptamine alkaloid with an organic acid, an inorganic acid or a base.

[0020] In some embodiments of the present application, the inorganic acid is selected from one or more of hydrochloric acid, sulfuric acid, phosphoric acid, diphosphoric acid, hydrobromic acid and nitric acid.

[0021] In some embodiments of the present application, the organic acid is selected from one or more of acetic acid, maleic acid, fumaric acid, tartaric acid, succinic acid, lactic acid, p-toluenesulfonic acid, salicylic acid and oxalic acid.

[0022] In some embodiments of the present application, the base is selected from one or more of potassium hydroxide, sodium hydroxide, calcium hydroxide, lithium hydroxide, aqueous ammonia, sodium carbonate and sodium bicarbonate.

[0023] In a second aspect, the present application provides a method for preparing an aaptamine alkaloid or a pharmaceutically acceptable salt thereof according to the first aspect, the method comprising the following steps:

[0024] S1, sequentially extracting, concentrating, desalting, re-dispersing and extracting the sponge Aaptos suberitoides to obtain a total extract;

[0025] S2, directionally separating the total extract by a chromatographic method to obtain a trace alkaloid component containing compounds II to VI, and obtaining compounds II to VI after separation and purification;

[0026] S3, optionally, preparing the compounds II to VI into a pharmaceutically acceptable salt.

[0027] It should be noted that the "optionally" in step S3 means that step S3 can be performed or not performed, and those skilled in the art can easily understand that when a pharmaceutically acceptable salt of compounds II to VI is needed, step S3 is performed.

[0028] In some embodiments of the present application, step S1 comprises:

[0029] The sponge Aaptos suberitoides is soaked in an ethanol-water mixed solvent to obtain a first extract;

[0030] The soaked sponge Aaptos suberitoides is crushed and mixed with ethanol, and then subjected to ultrasonic treatment to obtain a second extract;

[0031] The first extract and the second extract are combined to obtain a total extract;

[0032] The total extract is concentrated under reduced pressure to obtain a first extract powder; the first extract powder is desalted with methanol, and then concentrated under reduced pressure to obtain a second extract powder;

[0033] The second extract powder is dispersed in water, and then extracted with ethyl acetate to separate an ethyl acetate phase and an aqueous phase;

[0034] The aqueous phase is extracted with n-butanol to separate an n-butanol phase, which is the total extract.

[0035] In some embodiments of the present application, in the soaking step, the volume percentage of ethanol in the ethanol-water mixed solvent is 95-98%; for example, it can be 95%, 95.5%, 96%, 96.5%, 97%, 97.5% or 98%, etc. However, the present application is not limited to the listed values, and other unlisted values within this range are also applicable.

[0036] In some embodiments of the present application, in the soaking step, the amount of the ethanol-water mixed solution used is 1-5 L per kilogram of sponge Aaptos suberitoides.

[0037] In the present application, the temperature and time of the soaking step are not particularly limited and can be routinely selected by those skilled in the art. For example, the soaking temperature can be 20-25℃, such as 20℃, 21℃, 22℃, 23℃, 24℃ or 25℃, etc.; the soaking time can be 24-72 h, such as 24 h, 26 h, 30 h, 32 h, 35 h, 38 h, 40 h, 45 h, 50 h, 55 h, 60 h, 65 h, 70 h or 72 h, etc.

[0038] In some embodiments of the present application, the ultrasonic treatment is performed 1-5 times (e.g., 1 time, 2 times, 3 times, 4 times, 5 times), each time for 0.5-3 hours (e.g., 0.5 hours, 0.8 hours, 1 hour, 1.2 hours, 1.5 hours, 1.8 hours, 2 hours, 2.2 hours, 2.5 hours, 2.8 hours, or 3 hours, etc.), and the amount of ethanol used in each ultrasonic treatment is 1-5 liters per kilogram of Aaptos suberitoides sponges.

[0039] In the present application, the conditions for the reduced-pressure concentration are not particularly limited and can be routinely selected by those skilled in the art. For example, the temperature during the reduced-pressure concentration can be 35-37°C, and the pressure can be 100-120 Pa.

[0040] In some embodiments of the present application, each of the extraction with ethyl acetate and the extraction with n-butanol is independently performed 1-5 times (e.g., 1 time, 2 times, 3 times, 4 times, 5 times), and the amount of the ethyl acetate and the n-butanol used in each extraction is independently 1-3 times (e.g., 1 time, 2 times, 3 times) the volume of the solution to be extracted.

[0041] In some embodiments of the present application, in step S2, the operation of directing separation of the total extract by chromatography to obtain the trace alkaloid component containing compounds II-VI comprises:

[0042] The total extract is subjected to silica gel column chromatography, isocratically eluted with an eluent composed of dichloromethane and methanol in a volume ratio of 5-15:1 (e.g., 5:1, 6:1, 8:1, 10:1, 12:1, 13:1, or 15:1, etc.), and sequentially obtains components B1-B6 according to the elution order;

[0043] Components B3-B6 are respectively subjected to silica gel column chromatography, isocratically eluted with an eluent composed of dichloromethane and methanol in a volume ratio of 5-15:1 (e.g., 5:1, 6:1, 8:1, 10:1, 12:1, 13:1, or 15:1, etc.), the eluates are concentrated and combined, and analyzed by thin layer chromatography (TLC), wherein the main spot (yellow, 254 nm dark spot, 365 nm fluorescence) is aaptamine monomer, and components other than the aaptamine monomer are stained with modified bismuth potassium iodine, and the components with orange-red color are combined to obtain the trace alkaloid component containing compounds II-VI.

[0044] In some embodiments of the present application, in step S2, the operation of separation and purification comprises:

[0045] The trace alkaloid components containing compound II-compound VI are subjected to silica gel column chromatography, isocratically eluted with eluent consisting of dichloromethane and methanol in a volume ratio of 5-15:1 (for example, it can be 5:1, 6:1, 8:1, 10:1, 12:1, 13:1 or 15:1, etc.), and sequentially obtained are components BH1-BH7 in the order of elution;

[0046] The component BH1 is subjected to hydroxypropyl dextran gel column chromatography, isocratically eluted with methanol aqueous solution in a volume percentage of 40-60% (for example, it can be 40%, 42%, 43%, 45%, 46%, 48%, 50%, 52%, 53%, 55%, 56%, 58% or 60%, etc.) as eluent, and sequentially obtained are components BH1-1-BH1-8 in the order of elution;

[0047] The component BH6 is subjected to octadecyl silica gel (ODS) column chromatography, and sequentially gradient eluted with methanol aqueous solution in a volume percentage of 8-15% (for example, it can be 8%, 10%, 12%, 15%, etc.), 18-25% (for example, it can be 18%, 20%, 22%, 25%, etc.), 28-35% (for example, it can be 28%, 30%, 32%, 35%, etc.), 38-45% (for example, it can be 38%, 40%, 42%, 45%, etc.), 48-55% (for example, it can be 48%, 50%, 52%, 55%, etc.), 58-65% (for example, it can be 58%, 60%, 62%, 65%, etc.), 68-75% (for example, it can be 68%, 70%, 72%, 75%, etc.) as eluent, and sequentially obtained are components BH6-1-BH6-15 in the order of elution;

[0048] The component BH1-2 is purified by liquid chromatography to obtain compound IV;

[0049] The component BH6-2 is purified by liquid chromatography to obtain compound II;

[0050] The component BH6-4 is separated by liquid chromatography, and sequentially obtained are component BH6-4-1 and component BH6-4-2 in the order of sequence; the component BH6-4-1 is purified by liquid chromatography to obtain compound III, and the component BH6-4-2 is purified by liquid chromatography to obtain compound V;

[0051] The component BH6-13 is purified by liquid chromatography to obtain compound VI.

[0052] In some embodiments of the present application, the conditions of the liquid chromatography include:

[0053] The chromatographic column is a C18 silica gel chromatographic column;

[0054] and / or, the mobile phase is composed of acetonitrile aqueous solution and trifluoroacetic acid (TFA), the volume ratio of which is 1000:(0.2-0.6) (for example, it can be 1000:0.2, 1000:0.3, 1000:0.4, 1000:0.5 or 1000:0.6, etc.), and the volume percentage of acetonitrile in the acetonitrile aqueous solution is 5-18% (for example, it can be 5%, 6%, 8%, 10%, 12%, 13%, 15%, 16% or 18%, etc.);

[0055] and / or, the elution mode is isocratic elution.

[0056] In a third aspect, the present application provides a use of the aaptamine alkaloid or the pharmaceutically acceptable salt thereof according to the first aspect in the preparation of an anti-neurogenic drug.

[0057] In a fourth aspect, the present application provides an anti-neurogenic drug, which comprises the aaptamine alkaloid or the pharmaceutically acceptable salt thereof according to the first aspect, and a pharmaceutically acceptable carrier or excipient.

[0058] Compared with the prior art, the present application has the following beneficial effects:

[0059] The present application firstly isolates a class of aaptamine alkaloids with the structure shown in Formula I from the sponge Aaptos suberitoides, and researches find that the aaptamine alkaloids can significantly inhibit the NO production of LPS-induced BV2 cells, and inhibit the expression of inflammatory factors IL-6, IL-1β and TNF-α, and have an anti-neurogenic effect. The present application expands the structural subtypes of aaptamine alkaloids, and provides a scientific basis for developing aaptamine alkaloids as a new type of anti-neurogenic lead compound. BRIEF DESCRIPTION OF DRAWINGS

[0060] Figure 1A a cell viability data graph of LPS-induced BV2 cells treated by compound II;

[0061] Figure 1B a cell viability data graph of LPS-induced BV2 cells treated by compound III;

[0062] Figure 1C a cell viability data graph of LPS-induced BV2 cells treated by compound IV;

[0063] Figure 1D a cell viability data graph of LPS-induced BV2 cells treated by compound V;

[0064] Figure 1E a cell viability data graph of LPS-induced BV2 cells treated by compound VI;

[0065] Figure 2A Graph of NO inhibition rate of compound II on LPS-induced BV2 cells;

[0066] Figure 2B Graph of NO inhibition rate of compound III on LPS-induced BV2 cells;

[0067] Figure 2C Graph of NO inhibition rate of compound IV on LPS-induced BV2 cells;

[0068] Figure 2D Graph of NO inhibition rate of compound V on LPS-induced BV2 cells;

[0069] Figure 2E Graph of NO inhibition rate of compound VI on LPS-induced BV2 cells;

[0070] Figure 3A Graph of TNF-α expression of LPS-induced BV2 cells treated by compound VI;

[0071] Figure 3B Graph of IL-6 expression of LPS-induced BV2 cells treated by compound VI;

[0072] Figure 3C Graph of IL-1β expression of LPS-induced BV2 cells treated by compound VI. DETAILED DESCRIPTION

[0073] The technical solutions of the present application will be further described below in combination with the drawings and through specific embodiments. It should be understood by those skilled in the art that the specific embodiments are only used to help understand the present application, and should not be regarded as specific limitations to the present application.

[0074] In the embodiments of the present application, some of the materials used are as follows:

[0075] Sponge Aaptos suberitoides: collected from the South China Sea;

[0076] Silica gel column chromatography filler: HG / T 2354-2010, purchased from Qingdao Marine Chemical Industry;

[0077] ODS column chromatography filler: reversed-phase C8 silica gel / octyl silica gel 300A, purchased from Fuji, Japan;

[0078] Hydroxypropyl dextran gel column chromatography filler: sephadex LH-20;

[0079] Liquid chromatography column: Grace Prevail C18 (filler particle size 5 μm, length 250 mm, diameter 4.6 mm).

[0080] Example 1

[0081] This example is used to illustrate the discovery process of aaptamine alkaloid compounds of the present application:

[0082] S1, Preparation of total extract:

[0083] Sponges Aaptos suberitoides (1.5 kg) were soaked in a mixture of ethanol and water (95% ethanol by volume, 3 L) at room temperature for 36 h, and a first extract was obtained by separation;

[0084] The soaked sponges Aaptos suberitoides were homogenized and then ultrasonically treated in ethanol for 2 times, each time with 2 L of ethanol added, and each time for 1 h. The ultrasonically treated solutions were combined to obtain a second extract;

[0085] The first extract and the second extract were combined to obtain a total extract;

[0086] The total extract was concentrated under reduced pressure (40°C, 110 Pa) to obtain a first extract powder. The first extract powder was desalted with anhydrous methanol (400 mL), and then concentrated under reduced pressure (40°C, 110 Pa) to obtain a second extract powder;

[0087] The second extract powder was suspended in water (400 mL), and then extracted with ethyl acetate 3 times, each time with 800 mL of ethyl acetate. The ethyl acetate phase and the water phase were separated;

[0088] The water phase was extracted with n-butanol 3 times, each time with 800 mL of n-butanol. The n-butanol phase was separated.

[0089] S2, Separation and purification:

[0090] The n-butanol phase (35.43 g) was subjected to silica gel column chromatography, and eluted with an eluent consisting of dichloromethane and methanol in a volume ratio of 10:1. Fractions B1-B6 were obtained in order of elution;

[0091] The components B3-B6 were subjected to silica gel column chromatography, isocratic elution was performed using eluent consisting of dichloromethane and methanol in a volume ratio of 8:1, the eluate was concentrated and combined, and TLC analysis was performed using a developing agent consisting of a mixture of dichloromethane and methanol in a volume ratio of 10:1, wherein the main spot (yellow, 254 nm dark spot, 365 nm fluorescence) was aaptamine monomer, and components other than the aaptamine monomer were stained with modified bismuth potassium iodine, and the components showing orange-red color were combined to obtain trace alkaloid components (480 mg, denoted as BH components) containing compounds II-VI;

[0092] The BH components were subjected to silica gel column chromatography, isocratic elution was performed using eluent consisting of dichloromethane and methanol in a volume ratio of 8:1, and components BH1-BH7 were obtained in the order of elution;

[0093] The component BH1 (32 mg) was subjected to hydroxypropyl dextran gel column chromatography, isocratic elution was performed using methanol aqueous solution with a methanol volume percentage of 50% as eluent, and components BH1-1-BH1-8 were obtained in the order of elution;

[0094] The component BH6 (79 mg) was subjected to ODS column chromatography, gradient elution was performed using methanol aqueous solution with a methanol volume percentage of 10%, 20%, 30%, 40%, 50%, 60%, and 70% as eluent in sequence (whether to replace the next mobile phase was selected according to the elution of the spot), and components BH6-1-BH6-15 were obtained in the order of elution;

[0095] The component BH1-2 (6.3 mg) was purified by liquid chromatography (mobile phase: 5 vol% acetonitrile aqueous solution: TFA in a volume ratio of 1000:0.4, flow rate 1 mL / min, isocratic elution) to obtain compound IV (2.1 mg, retention time R t = 30 min);

[0096] The component BH6-2 (6.6 mg) was purified by liquid chromatography (mobile phase: 10 vol% acetonitrile aqueous solution: TFA in a volume ratio of 1000:0.4, flow rate 1 mL / min, isocratic elution) to obtain compound II (3.1 mg, R t = 32 min);

[0097] The component BH6-4 (11.2 mg) was separated by liquid chromatography (mobile phase: 13 vol% acetonitrile aqueous solution: TFA in a volume ratio of 1000:0.4, flow rate 1 mL / min, isocratic elution) to obtain component BH6-4-1 (R t = 11 min) and component BH6-4-2 (R t=16min); Component BH6-4-1 (3.0 mg) was purified by liquid chromatography (mobile phase: 13 vol% acetonitrile aqueous solution: TFA volume ratio = 1000:0.4, flow rate 1 mL / min, isocratic elution) to obtain compound III (1.3 mg, R). t =36 min), the component BH6-4-2 (6.1 mg) was purified by liquid chromatography (mobile phase: 10 vol% acetonitrile aqueous solution: TFA volume ratio = 1000:0.4, flow rate 1 mL / min, isocratic elution) to obtain compound V (5.3 mg, R). t =34min);

[0098] The component BH6-13 (4.7 mg) was purified by liquid chromatography to obtain compound VI (3.7 mg, R). t =36min).

[0099] Structural assessment:

[0100] use 1 H NMR, 13 Compounds II through VI were characterized by C NMR, and the NMR data are shown in Tables 1 and 2. Their structural formulas are as follows:

[0101]

[0102] Table 1. Compounds II to VI 1 H NMR data (DMSO-d6, J in Hz)

[0103]

[0104]

[0105] In Table 1, 'a' represents... 1 H NMR (600MHz), b represents 1 H NMR (500MHz).

[0106] Table 2. Compounds II to VI 13 C10 NMR data (DMSO-d6)

[0107]

[0108]

[0109] In Table 2, 'a' represents... 13 C NMR (150MHz), b represents 13 C NMR (125MHz).

[0110] Example 2: In vitro anti-neuritis activity experiment

[0111] S1, cytotoxicity screening

[0112] The cytotoxicity was measured by CCK8 method, and the specific steps were as follows: after counting BV2 cells, the cells were inoculated in a 96-well plate using serum-free DMEM medium, the cell density was 8x10 3 The compound was prepared into a 10mM stock solution with DMSO (dimethyl sulfoxide), and before use, it was diluted into 6 concentration gradients with serum-free DMEM medium containing 1 μg / mL LPS (lipopolysaccharide), which were 1, 2, 5, 10, 20, and 40 μM, respectively. After the cells adhered, the original culture medium was discarded, 100 μL of the prepared compound solution was added to each well of the experimental group; 100 μL of serum-free DMEM medium containing 1‰ DMSO and 1 μg / mL LPS was added to each well of the negative control group; 100 μL of pure serum-free DMEM medium was added to each well of the blank control group, and there was no cell in the well, 3 replicate wells were set for each concentration in each group. After 24h of action, 10 μL of CCK8 was added to each well, and after 1h of incubation at 37℃, the absorbance OD value of each group was detected by an enzyme-labeled instrument at 450nm. The cell viability calculation formula was as follows:

[0113] Cell viability (%) = (OD 实验组 - OD 空白组 ) / (OD 阴性组 - OD 空白组 ) x 100%.

[0114] The experimental results of compound II to compound VI are shown in Figures 1A-1E respectively. As can be seen from the figure, there is no significant difference in cell viability between the experimental group, the negative control group and the blank control group, indicating that the compounds II to VI of the present application have no obvious cytotoxicity.

[0115] S2, NO inhibitory activity

[0116] After counting BV-2 cells, the cells were resuspended with serum-free DMEM high-glucose medium, inoculated in a 96-well plate, and 100 μL of cell suspension was added to each well, the cell density was 8x10 3The compound was prepared into 10 mM stock solution with DMSO, and diluted into 6 concentration gradients of 1, 2, 5, 10, 20 and 40 μM with serum-free DMEM high glucose culture medium containing 1 μg / mL LPS before use. After the cells adhered, the original culture medium was discarded, 100 μL of the prepared compound solution was added to each well of the experimental group; 100 μL of serum-free DMEM high glucose culture medium containing 1‰ DMSO and 1 μg / mL LPS was added to each well of the negative control group; 100 μL of serum-free DMEM high glucose culture medium containing 40 μM nitric oxide synthase inhibitor LNMMA and 1 μg / mL LPS was added to each well of the positive drug group, 3 replicate wells were set for each concentration in each group, and the plate was placed in a 37°C incubator. After 24 h of action, the supernatant was taken for detection of NO content.

[0117] Griess method was used to detect the content of NO: Biyun Tian S0021M nitric oxide detection kit was used, and the operation was carried out according to the instructions. Griess Reagent I and II were taken out and restored to room temperature. NaNO2 standard was diluted with DMEM medium to prepare standard solutions with NaNO2 concentrations of 0, 1, 2, 5, 10, 20, 40, 60 and 100 μM. The standard solutions and supernatants of each group were added to the 96-well plate at 50 μL / well. Room temperature Griess Reagent I was added to each well at 50 μL / well, and the reaction was avoided for 5 min. Room temperature Griess Reagent II was added to each well at 50 μL / well, and the absorbance OD value was measured at 540 nm by a microplate reader. The OD values of each well of the NaNO2 standard solution and the corresponding concentrations were used to draw a standard curve. The NO concentration was calculated according to the standard curve and the OD values of other wells. The NO inhibition rate was calculated according to the following formula:

[0118] Inhibition rate (%) = (1-NO concentration 实验组 / NO concentration 阴性组 ) x 100%.

[0119] The experimental results of compounds II to VI are shown in Figures 2A-2E respectively. As can be seen from the figure, compounds II to VI can all inhibit the production of NO induced by LPS, and all have significant differences at a concentration of 10-40 μM. Meanwhile, the comparison of the data of each group shows that the inhibitory activity of compound VI is the most significant.

[0120] S3, ELISA detection of inflammatory factors

[0121] After the BV-2 cells were counted, the cells were resuspended with serum-free DMEM medium, and the cells were inoculated in a 6-well plate at a cell density of 5-6 x 10 5Cells / well, starved culture for 24 h. Compound VI was prepared as a 10 mM stock solution with DMSO, and diluted to four concentration gradients (4, 8, 20, and 40 μM) with serum-free DMEM medium before use. After starvation culture, 500 μL of the prepared compound solution was added to each well of the experimental group; 500 μL of serum-free DMEM medium containing 2‰ DMSO was added to each well of the negative control and model groups. One h after administration, 500 μL of serum-free DMEM medium was added to each well of the negative control group, and 500 μL of serum-free DMEM medium containing 2 μg / mL LPS was added to each well of the experimental and model groups. The final concentrations of the compound administered were 1, 2, 5, and 10 μM, and the LPS concentration was 500 ng / mL. After incubation at 37°C for 24 h, the supernatant was collected by centrifugation, and the levels of inflammatory factors (TNF-α, IL-6, and IL-1β) in the cell supernatant were detected by ELISA.

[0122] The inhibitory effects of compound VI on the expression of LPS-induced inflammatory factors TNF-α, IL-6, and IL-1β in BV2 cells are as follows: Figures 3A-3C As shown in the figure, compared with the negative control group, the expression levels of inflammatory factors IL-6, IL-1β, and TNF-α in LPS-induced BV2 cells in the model group were significantly increased. However, compared with the model group, the expression levels of inflammatory factors IL-6, IL-1β, and TNF-α in LPS-induced BV2 cells decreased after treatment with compound VI in the experimental group, and this decrease increased with increasing concentration of compound VI. This indicates that compound VI can inhibit the expression of inflammatory factors IL-6, IL-1β, and TNF-α in LPS-induced BV2 cells and has an anti-neuritis effect.

[0123] The above description is merely a specific embodiment of this disclosure, enabling those skilled in the art to understand or implement it. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this disclosure. Therefore, this disclosure is not to be limited to the embodiments described herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An aaptamine alkaloid or a pharmaceutically acceptable salt thereof, characterized in that, The aaptamine alkaloid has a structure shown in Formula I: wherein R1 is hydroxyl or methoxyl; R2is a hydrogen atom, a methyl group, - represents the bond of the radical.

2. The aaptamine alkaloid or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The aaptamine alkaloid is selected from compounds II-VI; 3. The aaptamine alkaloid or a pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that, The pharmaceutically acceptable salt is a salt of the aaptamine alkaloid with an organic acid, an inorganic acid or a base.

4. The aaptamine alkaloid or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that, The inorganic acid is selected from one or more of hydrochloric acid, sulfuric acid, phosphoric acid, diphosphoric acid, hydrobromic acid and nitric acid.

5. The aaptamine alkaloid or a pharmaceutically acceptable salt thereof according to claim 3, wherein The organic acid is selected from one or more of acetic acid, maleic acid, fumaric acid, tartaric acid, succinic acid, lactic acid, p-toluenesulfonic acid, salicylic acid and oxalic acid.

6. The aaptamine alkaloid or a pharmaceutically acceptable salt thereof according to claim 3, wherein The base is selected from one or more of potassium hydroxide, sodium hydroxide, calcium hydroxide, lithium hydroxide, ammonia, sodium carbonate and sodium bicarbonate.

7. A process for the preparation of aaptamine alkaloid or a pharmaceutically acceptable salt thereof as claimed in claim 2, wherein, The preparation method comprises the following steps: S1. Extracting, concentrating, desalting, re-dispersing and extracting sponge Aaptos suberitoides in sequence to obtain a total extract; S2. Directionally separating the total extract by a chromatographic method to obtain a trace alkaloid component containing compounds II-VI through enrichment, and obtaining compounds II-VI after separation and purification; S3. Optionally, preparing the compounds II-VI into pharmaceutically acceptable salts.

8. The preparation method according to claim 7, characterized in that, Step S1 comprises: Soaking sponge Aaptos suberitoides with an ethanol water mixture to obtain a first extract; Crushing the soaked sponge Aaptos suberitoides, mixing with ethanol and performing ultrasonic treatment to obtain a second extract; Combining the first extract and the second extract to obtain a total extract; Concentrating the total extract under reduced pressure to obtain a first extract powder, desalting the first extract powder with methanol and concentrating under reduced pressure to obtain a second extract powder; Dispersing the second extract powder with water and then extracting with ethyl acetate to separate to obtain an ethyl acetate phase and an aqueous phase; Extracting the aqueous phase with n-butanol to separate to obtain an n-butanol phase, which is the total extract.

9. The production method according to claim 8, characterized by, In the soaking step, the volume percentage of ethanol in the ethanol water mixture is 95-98%, and the amount of the ethanol water mixture used is 1-5 L per kilogram of sponge Aaptos suberitoides.

10. The preparation method according to claim 8, characterized in that, The ultrasonic treatment is performed 1-5 times, each time for 0.5-3 h, and the amount of ethanol used in each ultrasonic treatment is 1-5 L per kilogram of sponge Aaptos suberitoides.

11. The preparation method according to claim 8, characterized in that, Each of the extraction with ethyl acetate and the extraction with n-butanol is independently performed 1-5 times, and the amount of ethyl acetate and n-butanol used in each extraction is independently 1-3 times the volume of the solution to be extracted.

12. The method of claim 7, wherein, In step S2, the operation of directionally separating the total extract by a chromatographic method to obtain a trace alkaloid component containing compounds II-VI through enrichment comprises: Performing silica gel column chromatography on the total extract, isocratically eluting with an eluent composed of dichloromethane and methanol in a volume ratio of 5-15:1, and sequentially obtaining components B1-B6 according to the elution order; The components B3-B6 are subjected to silica gel column chromatography, isocratic elution is performed with eluent consisting of dichloromethane and methanol in a volume ratio of 5-15:1, the eluate is concentrated and combined, thin layer chromatography is used for analysis, the main spot is aaptamine monomer, components other than the aaptamine monomer are stained with modified bismuth potassium iodine, the components with orange-red color are combined to obtain the trace alkaloid component containing compounds II-VI.

13. The production method according to claim 7 or 12, characterized by, In step S2, the separating and purifying operation comprises: The trace alkaloid component containing compounds II-VI is subjected to silica gel column chromatography, isocratic elution is performed with eluent consisting of dichloromethane and methanol in a volume ratio of 5-15:1, and components BH1-BH7 are sequentially obtained in elution order; Component BH1 is subjected to hydroxypropyl dextran gel column chromatography, isocratic elution is performed with methanol aqueous solution with a methanol volume percentage of 40-60% as eluent, and components BH1-1-BH1-8 are sequentially obtained in elution order; Component BH6 is subjected to n-octadecyl silica gel column chromatography, gradient elution is performed with methanol aqueous solution with a methanol volume percentage of 8-15%, 18-25%, 28-35%, 38-45%, 48-55%, 58-65%, and 68-75% as eluent in sequence, and components BH6-1-BH6-15 are sequentially obtained in elution order; Component BH1-2 is subjected to liquid chromatography to obtain compound IV; Component BH6-2 is subjected to liquid chromatography to obtain compound II; Component BH6-4 is subjected to liquid chromatography to sequentially obtain components BH6-4-1 and BH6-4-2 in sequence; component BH6-4-1 is subjected to liquid chromatography to obtain compound III, and component BH6-4-2 is subjected to liquid chromatography to obtain compound V; Component BH6-13 is subjected to liquid chromatography to obtain compound VI.

14. The method of claim 13, wherein, The conditions of the liquid chromatography comprise: The chromatographic column is a C18 silica gel chromatographic column; and / or, the mobile phase consists of acetonitrile aqueous solution and trifluoroacetic acid in a volume ratio of 1000:(0.2-0.6), and the acetonitrile in the acetonitrile aqueous solution has a volume percentage of 5-18%; and / or, the elution mode is isocratic elution.

15. Use of the aaptamine alkaloid or a pharmaceutically acceptable salt thereof according to any one of claims 1-3 in the preparation of an anti-neuritis drug.

16. An anti-neuritic agent, characterized by comprising the compound of claim 1. The aaptamine alkaloid or a pharmaceutically acceptable salt thereof according to any one of claims 1-3, and a pharmaceutically acceptable carrier or excipient.

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