Aaptamine alkaloid as well as preparation method and application thereof

Abstract

The invention provides aaptamine alkaloid as well as a preparation method and application of the aaptamine alkaloid. The aaptamine alkaloid has a structure as shown in a formula I which is described in the specification. The aaptamine alkaloid can significantly inhibit the generation of LPS-induced BV2 cell NO, and inhibit the expression of inflammatory factors IL-6, IL-1beta and TNF-alpha, so that the aaptamine alkaloid has an anti-neuritis effect. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to an aaptamine alkaloid and a preparation method and application thereof. Background Art

[0002] Marine drugs are drugs developed using modern scientific methods and technologies with marine organisms as the drug source. At present, more than 40,000 marine natural products have been discovered internationally, of which more than 50% have various biological activities and strong drug-like properties. Sponges are marine invertebrates and are an important resource for the study of marine drug-derived compounds. There are abundant sponge species in the South China Sea, among which there are about 20 species of sponges in the genus Aaptos. Sponges in this genus can metabolize aaptamine alkaloids with a unique benzo[de][1,6]naphthyridine skeleton. This type of alkaloids shows good biological activity in many aspects such as antibacterial, antitumor, antiviral, enzyme inhibitor, and antifouling (Larghi, EL; Bohn, ML; Kaufman, TSAaptamine and related products. Their isolation, chemical syntheses, and biological activity. Tetrahedron. 2009, 65, 4257-4282.).

[0003] Among the aaptamine homologues, there is a class called triazatetracyclo-aaptamine alkaloids (TTA), whose structural formulas are shown in the following 1 to 12:

[0004]

[0005] This type of alkaloid was first obtained 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 Aaptossuberitoides. J. Nat. prod. 2013, 76, 103-106.). Later, a series of TTA tetracyclic aaptamine alkaloids were obtained from the South China Sea sponge Aaptosaaptos ([1] Yu, HB; Sun, F.; Ma, GY; Gan, JH; Hu, WZ; Han, BN; Jiao, WH; Lin, HWCytotoxic aaptamine derivativesfrom the South China Sea sponge Aaptosaaptos. J. Nat. Prod. 2014, 77, 2124-2129. [2] Gan, JH; Hu, WZ; Yu HB; Yang, F.; Gao, MX; Shi, HJ; Kang, YF; Han, BNThree new aaptamine derivatives from the South China Sea sponge Aaptosaaptos. J. Asian Nat. Prod. Res. 2015, 17, 1231-1238. [3] Yu, HB; Yang, F.; Sun, F. Li, J.; Jiao, WH; Gan, JH; Hu, WZ; Lin, HW Aaptamine derivatives with antifungal and anti-HIV-1 activities from the South China Sea sponge Aaptosaaptos. Mar. Drugs. 2014, 12, 6003-6013.). The dinitrogen heterocyclic ring in the TTA alkaloid structure is formed by the C-9 nitrogen substituted structure and NH-1 through Schiff base cyclization. It is speculated that there should be an intermediate with C-9 nitrogen substitution and no cyclization. So far, there has been no report on C-9 nitrogen substituted aaptamine monomer alkaloids. Given that aaptamine alkaloids generally have good biological activities, more such natural products remain to be developed in order to enrich the types of marine drugs. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide an aaptamine alkaloid and a preparation method and application thereof. The present invention is the first to separate a class of aaptamine alkaloids having a structure shown in Formula I from the sponge Aaptos suberitoides, and studies have found that the aaptamine alkaloids have anti-neuritis effects, providing new lead compounds for the research and development of anti-neuritis drugs, and enriching the types of marine drugs.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides an aaptamine alkaloid or a pharmaceutically acceptable salt thereof, wherein the aaptamine alkaloid has a structure shown in Formula I:

[0009]

[0010] Among them, R 1 is a hydroxyl group or a methoxy group;

[0011] R 2 A hydrogen atom, a methyl group, Represents the connecting bond of the group.

[0012] In some embodiments of the present invention, the aaptamine alkaloid is selected from Compound II to Compound VI;

[0013]

[0014] Among them, the chemical name of compound II is N-(8-hydroxy-1H-benzo[de][1,6]naphthyridin-9-yl)acetamide, and the molecular formula is C 13 H 11 N 3 O 2 .

[0015] The chemical name of compound III is N-(8-methoxy-1H-benzo[de][1,6]naphthyridin-9-yl)acetamide, and its molecular formula is C 14 H 13 N 3 O 2 .

[0016] The chemical name of compound IV is (S)-2-hydroxy-N-(8-hydroxy-1H-benzo[de][1,6]naphthyridin-9-yl)propanamid e, and its molecular formula is C 14 H 13 N3 O 3 .

[0017] The chemical name of compound V is N-(8-methoxy-1H-benzo[de][1,6]naphthyridin-9-yl)formamide, and its molecular formula is C 13 H 11 N 3 O 2 .

[0018] The chemical name of compound VI is 4-hydroxy-N-(8-methoxy-1H-benzo[de][1,6]naphthyridin-9-yl)benzamide, and its molecular formula is C 19 H 15 N 3 O 3 .

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

[0020] In some embodiments of the present invention, 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 invention, 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 invention, the base is selected from one or more of potassium hydroxide, sodium hydroxide, calcium hydroxide, lithium hydroxide, ammonia water, sodium carbonate and sodium bicarbonate.

[0023] In a second aspect, the present invention provides a method for preparing the aaptamine alkaloid or a pharmaceutically acceptable salt thereof as described in the first aspect, the preparation method comprising the following steps:

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

[0025] S2, using a chromatographic method to directionally separate the total extract, enriching to obtain trace alkaloid components containing compounds II to VI, and separating and purifying to obtain compounds II to VI;

[0026] S3. Optionally, the Compound II to Compound VI are prepared into pharmaceutically acceptable salts.

[0027] It should be noted that the “optionally” in step S3 means that step S3 may or may not be performed. It is easy for those skilled in the art to understand that when it is necessary to obtain a pharmaceutically acceptable salt of compound II to compound VI, step S3 is performed.

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

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

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

[0031] combining the first extract and the second extract to obtain a total extract;

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

[0033] Dispersing the second extract with water, then extracting with ethyl acetate to separate into an ethyl acetate phase and an aqueous phase;

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

[0035] In some embodiments of the present invention, 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 invention is not limited to the listed values, and other values ​​not listed within the range are also applicable.

[0036] In some embodiments of the present invention, in the soaking step, the amount of the ethanol-water mixture is 1-5 L / kg sponge Aaptos suberitoides.

[0037] In the present invention, there is no particular limitation on the temperature and time of the soaking, and those skilled in the art can make conventional selections. For example, the soaking temperature can be 20-25°C, such as 20°C, 21°C, 22°C, 23°C, 24°C or 25°C, etc.; the soaking time can be 24-72h, such as 24h, 26h, 30h, 32h, 35h, 38h, 40h, 45h, 50h, 55h, 60h, 65h, 70h or 72h, etc.

[0038] In some embodiments of the present invention, the ultrasonic treatment is carried out 1-5 times (for example, 1 time, 2 times, 3 times, 4 times, or 5 times), the time of each ultrasonic treatment is 0.5-3 h (for example, 0.5 h, 0.8 h, 1 h, 1.2 h, 1.5 h, 1.8 h, 2 h, 2.2 h, 2.5 h, 2.8 h, or 3 h, etc.), and the amount of ethanol used in each ultrasonic treatment is 1-5 L / kg sponge Aaptos suberitoides.

[0039] In the present invention, there is no particular limitation on the conditions for the reduced pressure concentration, and those skilled in the art can make conventional selections. 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 invention, the extraction with ethyl acetate and the extraction with n-butanol are each independently performed 1-5 times (for example, 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 each independently 1-3 times (for example, 1 time, 2 times, 3 times) of the volume of the solution to be extracted.

[0041] In some embodiments of the present invention, in step S2, the operation of using a chromatographic method to directionally separate the total extract and enriching to obtain trace alkaloid components containing compounds II to VI includes:

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

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

[0044] In some embodiments of the present invention, in step S2, the separation and purification operations include:

[0045] The trace alkaloid fraction containing compounds II to VI is subjected to silica gel column chromatography, and isocratically eluted with an eluent consisting of dichloromethane and methanol in a volume ratio of 5-15:1 (for example, 5:1, 6:1, 8:1, 10:1, 12:1, 13:1 or 15:1, etc.), to obtain fractions BH1 to BH7 in order of elution;

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

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

[0048] Component BH1-2 was purified by liquid chromatography to obtain compound IV;

[0049] Component BH6-2 was purified by liquid chromatography to obtain compound II;

[0050] Component BH6-4 is separated by liquid chromatography to obtain component BH6-4-1 and component BH6-4-2 in order; component BH6-4-1 is purified by liquid chromatography to obtain compound III, and component BH6-4-2 is purified by liquid chromatography to obtain compound V;

[0051] Component BH6-13 was purified by liquid chromatography to give compound VI.

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

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

[0054] And / or, the mobile phase is composed of an acetonitrile aqueous solution and trifluoroacetic acid (TFA) in a volume ratio of 1000:(0.2-0.6) (for example, 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, 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 invention provides a use of the aaptamine alkaloid or a pharmaceutically acceptable salt thereof as described in the first aspect in the preparation of an anti-neuritis drug.

[0057] In a fourth aspect, the present invention provides an anti-neuritis drug, comprising the aaptamine alkaloid or a pharmaceutically acceptable salt thereof as described in the first aspect, and a pharmaceutically acceptable carrier or excipient.

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

[0059] The present invention is the first to isolate a class of aaptamine alkaloids having a structure shown in Formula I from the sponge Aaptos suberitoides, and studies have found that the aaptamine alkaloids can significantly inhibit LPS-induced NO production in BV2 cells, and inhibit the expression of inflammatory factors IL-6, IL-1β and TNF-α, and have anti-neuritis effects. The present invention expands the structural subtypes of aaptamine alkaloids and provides a scientific basis for the development of aaptamine alkaloids as new anti-neuritis lead compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1A This is a graph showing the cell viability data of LPS-induced BV2 cells treated with compound II;

[0061] Figure 1B This is a graph showing the cell viability data of LPS-induced BV2 cells treated with compound III;

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

[0063] Figure 1D This is a graph showing the cell viability data of LPS-induced BV2 cells treated with compound V;

[0064] Figure 1E This is a graph showing the cell viability data of LPS-induced BV2 cells treated with compound VI;

[0065] Figure 2A This is the data graph of the NO inhibition rate of compound II on LPS-induced BV2 cells;

[0066] Figure 2B This is the data graph of the NO inhibition rate of compound III on LPS-induced BV2 cells;

[0067] Figure 2C This is the data graph of the NO inhibition rate of compound IV on LPS-induced BV2 cells;

[0068] Figure 2D This is the data graph of the NO inhibition rate of compound V on LPS-induced BV2 cells;

[0069] Figure 2E This is the data graph of the NO inhibition rate of compound VI on LPS-induced BV2 cells;

[0070] Figure 3A The data graph is a graph showing the expression of TNF-α in BV2 cells induced by LPS treated with compound VI;

[0071] Figure 3B The data graph is a graph showing the expression of IL-6 in LPS-induced BV2 cells treated with compound VI;

[0072] Figure 3C The data graph shows the IL-1β expression level in LPS-induced BV2 cells treated with compound VI. DETAILED DESCRIPTION

[0073] The technical solution of the present invention is further described below with reference to the accompanying drawings and through specific implementations. It should be understood by those skilled in the art that the specific implementations are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0074] In the embodiments of the present invention, some of the materials used are from the following sources:

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

[0076] Silica gel column chromatography filler: HG / T2354-2010, purchased from Qingdao Ocean Chemical;

[0077] ODS column chromatography packing: reverse 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 the aaptamine alkaloid compound of the present invention:

[0082] S1. Preparation of total extract:

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

[0084] After the soaked sponge Aaptos suberitoides was homogenized, it was ultrasonically treated twice in ethanol, each time the amount of ethanol added was 2 L, each ultrasonic time was 1 hour, and the ultrasonic solutions were combined to obtain a second extract;

[0085] combining the first extract and the second extract to obtain a total extract;

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

[0087] The second extract was suspended in water (400 mL), and then extracted with ethyl acetate for 3 times, each time with 800 mL of ethyl acetate, and separated to obtain an ethyl acetate phase and an aqueous phase;

[0088] The aqueous phase was extracted with n-butanol for 3 times, with 800 mL of n-butanol used each time, and 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 isocratically eluted with an eluent consisting of dichloromethane and methanol in a volume ratio of 10:1, and components B1 to B6 were obtained in sequence according to the elution order;

[0091] Components B3 to B6 were subjected to silica gel column chromatography respectively, and isocratically eluted with an eluent consisting of dichloromethane and methanol in a volume ratio of 8:1. The eluates were concentrated and combined, and analyzed by TLC (developing solvent was a mixture of dichloromethane and methanol in a volume ratio of 10:1), wherein the main spot (yellow, dark spot at 254 nm, fluorescence at 365 nm) was aaptamine monomer, and the components other than the aaptamine monomer were stained with improved potassium bismuth iodide, and the components showing orange-red color were combined to obtain a trace alkaloid component containing compounds II to VI (480 mg, recorded as component BH);

[0092] The BH component was subjected to silica gel column chromatography, and was isocratically eluted with an eluent consisting of dichloromethane and methanol in a volume ratio of 8:1, and components BH1 to BH7 were obtained in sequence according to the elution order;

[0093] Component BH1 (32 mg) was subjected to hydroxypropyl dextran gel column chromatography, and an aqueous solution of methanol with a volume percentage of 50% was used as an eluent for isocratic elution to obtain components BH1-1 to BH1-8 in the order of elution;

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

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

[0096] Component BH6-2 (6.6 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 II (3.1 mg, R t =32min);

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

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

[0099] Structure identification:

[0100] use 1 H NMR, 13 C NMR was used to characterize Compounds II to VI. The NMR data are shown in Tables 1 and 2. Their structural formulas are determined as follows:

[0101]

[0102] Table 1 Compounds II to VI 1 H NMR data (DMSO-d 6 , J in Hz)

[0103]

[0104]

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

[0106] Table 2 Compounds II to VI 13 C NMR data (DMSO-d 6 )

[0107]

[0108]

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

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

[0111] S1. Cytotoxic activity screening

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

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

[0114] The experimental results corresponding to compounds II to VI are as follows: Figure 1A to Figure 1E As shown in the figure, it can be seen that there is no significant difference in cell viability among the experimental group, the negative control group and the blank control group, indicating that compounds II to VI of the present invention have no obvious cytotoxicity.

[0115] S2, NO inhibitory activity

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

[0117] Griess method for detecting NO content: Use the Bio-Tech S0021M nitric oxide detection kit according to the instructions, take out Griess Reagent I and II, restore them to room temperature, dilute NaNO with DMEM medium 2 Standard, prepared NaNO 2 The concentrations of standard solutions were 0, 1, 2, 5, 10, 20, 40, 60, and 100 μM, respectively. The standard solutions and supernatants of each group were added to a 96-well plate at 50 μL / well. Room temperature Griess Reagent I was added to each well at 50 μL / well. After 5 minutes of reaction in the dark, room temperature Griess Reagent II was added to each well at 50 μL / well. The absorbance OD value was measured at 540 nm by a microplate reader. According to NaNO 2 The OD value of each well of the standard solution and the corresponding concentration are used to make a standard curve, and the NO concentration is calculated based on the standard curve and the OD value of other wells. The NO inhibition rate calculation formula is as follows:

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

[0119] The experimental results corresponding to compounds II to VI are as follows: Figure 2A to Figure 2E As shown in the figure, it can be seen that compounds II to VI can inhibit the production of NO induced by LPS, and there are significant differences at concentrations of 10-40 μM. At the same time, the comparison of data from each group shows that the inhibitory activity of compound VI is the most significant.

[0120] S3. ELISA detection of inflammatory factors

[0121] After BV-2 cells were digested and counted, the cells were resuspended in serum-free DMEM medium and inoculated into 6-well plates. 1000 μL of cell suspension was added to each well, and the cell density was 5-6×10 5 cells / well, starved for 24h. Compound VI was prepared into a 10mM stock solution with DMSO and diluted into 4 concentration gradients with serum-free DMEM medium before use, namely 4, 8, 20, and 40μM. After starvation culture, 500μL of the prepared compound solution was added to each well of the experimental group; 500μL of serum-free DMEM medium containing 2‰ DMSO was added to each well of the negative control group and the model group; 1h after administration, 500μL of serum-free DMEM medium was added to the negative control group, and 500μL of serum-free DMEM medium containing 2μg / mL LPS was added to the experimental group and the model group. The final compound administration concentrations were 1, 2, 5, and 10μM, and LPS was 500ng / mL. After 24h of incubation at 37℃, the supernatant was centrifuged and the content of inflammatory factors (TNF-α, IL-6, and IL-1β) in the cell supernatant was detected by ELISA.

[0122] The inhibitory effects of compound VI on the expression of inflammatory factors TNF-α, IL-6, and IL-1β in BV2 cells induced by LPS were as follows: Figure 3A to Figure 3C As shown in the figure, compared with the negative control group, the expression of inflammatory factors IL-6, IL-1β and TNF-α in BV2 cells induced by LPS in the model group was significantly increased; compared with the model group, the expression of inflammatory factors IL-6, IL-1β and TNF-α in BV2 cells induced by LPS in the experimental group treated with compound VI decreased, and the decrease increased with the increase of compound VI concentration, indicating that compound VI can inhibit the expression of inflammatory factors IL-6, IL-1β and TNF-α in BV2 cells induced by LPS, and has anti-neuritis effect.

[0123] The above description is only a specific embodiment of the present disclosure, so that those skilled in the art can understand or implement the present disclosure. Various modifications to these embodiments will be 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 the present disclosure. Therefore, the present disclosure will not be limited to the embodiments described herein, but will conform to 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 methoxy; R2 is a hydrogen atom, a methyl group, * Represents the connecting bond of the group.

2. The aaptamine alkaloid or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The aaptamine alkaloid is selected from Compound II to Compound 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 formed by the aaptamine alkaloid and an organic acid, an inorganic acid or a base; Preferably, the inorganic acid is selected from one or more of hydrochloric acid, sulfuric acid, phosphoric acid, diphosphoric acid, hydrobromic acid and nitric acid; Preferably, 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; Preferably, the base is selected from one or more of potassium hydroxide, sodium hydroxide, calcium hydroxide, lithium hydroxide, aqueous ammonia, sodium carbonate and sodium bicarbonate.

4. A method for preparing the aaptamine alkaloid or a pharmaceutically acceptable salt thereof as claimed in claim 2 or 3, characterized in that: The preparation method comprises the following steps: S1, extracting, concentrating, desalting, re-dispersing and extracting the sponge Aaptos suberitoides in sequence to obtain a total extract; S2, using a chromatographic method to directionally separate the total extract, enriching to obtain trace alkaloid components containing compounds II to VI, and separating and purifying to obtain compounds II to VI; S3. Optionally, the Compound II to Compound VI are prepared into pharmaceutically acceptable salts.

5. The preparation method according to claim 4, characterized in that: Step S1 includes: The sponge Aaptos suberitoides is soaked in a mixture of ethanol and water to obtain a first extract; The soaked sponge Aaptos suberitoides was crushed, mixed with ethanol, and subjected to ultrasonic treatment to obtain a second extract; combining the first extract and the second extract to obtain a total extract; The total extract is concentrated under reduced pressure to obtain a first extract; the first extract is desalted with methanol and concentrated under reduced pressure to obtain a second extract; Dispersing the second extract with water, then extracting with ethyl acetate to separate into an ethyl acetate phase and an aqueous phase; Extracting the aqueous phase with n-butanol to separate and obtain an n-butanol phase, which is the total extract; Preferably, 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 is 1-5 L / kg sponge Aaptos suberitoides; Preferably, the ultrasonic treatment is performed 1-5 times, the time of each ultrasonic treatment is 0.5-3h, and the amount of ethanol used in each ultrasonic treatment is 1-5L / kg sponge Aaptos suberitoides; Preferably, the extraction with ethyl acetate and the extraction with n-butanol are each independently performed 1-5 times, and the amount of the ethyl acetate and the n-butanol used in each extraction is each independently 1-3 times the volume of the solution to be extracted.

6. The preparation method according to claim 4 or 5, characterized in that: In step S2, the operation of using a chromatographic method to directionally separate the total extract and enriching to obtain trace alkaloid components containing compounds II to VI includes: The total extract is subjected to silica gel column chromatography, and isocratically eluted with an eluent consisting of dichloromethane and methanol in a volume ratio of 5-15:1, to obtain components B1 to B6 in sequence according to the elution order; Components B3 to B6 are subjected to silica gel column chromatography respectively, and are isocratically eluted with an eluent consisting of dichloromethane and methanol in a volume ratio of 5-15:

1. The eluates are concentrated and combined, and analyzed by thin layer chromatography, wherein the main spot is aaptamine monomer. Components other than the aaptamine monomer are stained with improved potassium bismuth iodide, and the components showing orange-red color are combined to obtain the trace alkaloid component containing compounds II to VI.

7. The preparation method according to any one of claims 4 to 6, characterized in that: In step S2, the separation and purification operations include: The trace alkaloid fraction containing compounds II to VI is subjected to silica gel column chromatography, and isocratically eluted with an eluent consisting of dichloromethane and methanol in a volume ratio of 5-15:1, to obtain fractions BH1 to BH7 in sequence according to the elution order; The component BH1 is subjected to hydroxypropyl dextran gel column chromatography, and isocratic elution is performed using a methanol aqueous solution with a methanol volume percentage of 40-60% as an eluent, and components BH1-1 to BH1-8 are obtained in sequence according to the elution order; The component BH6 is subjected to n-octadecyl silica gel column chromatography, and a methanol aqueous solution with a methanol volume percentage of 8-15%, 18-25%, 28-35%, 38-45%, 48-55%, 58-65%, and 68-75% is used as an eluent for gradient elution, and components BH6-1 to BH6-15 are obtained in the order of elution; Component BH1-2 was purified by liquid chromatography to obtain compound IV; Component BH6-2 was purified by liquid chromatography to obtain compound II; Component BH6-4 is separated by liquid chromatography to obtain component BH6-4-1 and component BH6-4-2 in order; component BH6-4-1 is purified by liquid chromatography to obtain compound III, and component BH6-4-2 is purified by liquid chromatography to obtain compound V; Component BH6-13 was purified by liquid chromatography to give compound VI.

8. The preparation method according to claim 7, characterized in that: The conditions of the liquid chromatography include: The chromatographic column is a C18 silica gel column; And / or, the mobile phase consists of an acetonitrile aqueous solution and trifluoroacetic acid in a volume ratio of 1000:(0.2-0.6), wherein the volume percentage of acetonitrile in the acetonitrile aqueous solution is 5-18%; And / or, the elution mode is isocratic elution.

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

10. An anti-neuritis drug, characterized in that: The invention comprises the aaptamine alkaloid or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 3, and a pharmaceutically acceptable carrier or excipient.

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