Indole diterpene alkaloid and application thereof as antibacterial agent

Abstract

The invention belongs to the field of marine natural products, and relates to indole diterpene alkaloid and application thereof as an antibacterial agent. The indole diterpenoid alkaloid compound 1 has a strong cell proliferation inhibition effect, and the IC50 value of the indole diterpenoid alkaloid compound 1 on a human gastric cancer cell strain SGC-7901 is 19.16 [mu] g / mL; and the IC50 value on a human hepatoma cell line HepG2 is 6.27 mu g / mL. The IC50 of the positive drug cis-platinum to SGC-7901 and the IC50 of the positive drug cis-platinum to HepG2 are 3.56 mu g / mL and 1.99 mu g / mL respectively. The structure of the compound 1 is shown in the specification.

Description

[0001] Based on the examination opinion issued by the State Intellectual Property Office (Document No. 2025111100285130) regarding the lack of unity of invention, the applicant filed this divisional application for Chinese Patent Application No. 202410737079.X. The original application was filed on June 6, 2024, with priority number 202310721381.1 and priority date of June 19, 2023. The original invention was entitled "An Indole Diterpene Alkaloid Derived from Marine Fungi and Its Application". Technical Field

[0002] This invention belongs to the field of marine natural products, specifically relating to an indole diterpenoid alkaloid and its application as an antibacterial agent. Background Technology

[0003] Mangrove ecosystems are widely considered one of the most productive ecosystems in the world. Due to their unique ecological environment, microorganisms have developed effective adaptation mechanisms, producing unique chemical substances and enormous biodiversity. Mangrove-related fungi are an important source of natural products. Secondary metabolites of mangrove fungi can be classified into four categories based on their chemical structure and biosynthetic pathways: ketones, terpenes, alkaloids, and other nitrogen-containing metabolites. Many of these metabolites possess pharmacological activities such as antibacterial, antifungal, and cytotoxic effects. This invention provides an indole diterpenoid alkaloid derived from marine fungi and its applications. Summary of the Invention

[0004] This invention provides a marine fungus N4-3, isolated from the rhizosphere soil of the mangrove plant *Avicennia marina*, which originates from the Dongzhaigang Mangrove Nature Reserve in Haikou, South China Sea. The marine fungus N4-3 of this invention is characterized by the following preservation information: Depository Institution: China General Microbiological Culture Collection Center (CGMCC); Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; Deposit Date: April 6, 2023; Accession Number: CGMCC No. 40554; Classification: *Penicillium* sp.

[0005] Another embodiment of the present invention provides an indole diterpenoid alkaloid, its tautomer, or a pharmaceutically acceptable salt thereof, characterized in that the indole diterpenoid alkaloid has the structures shown in compounds 1, 2, 3, 4, and 5:

[0006] , , , , .

[0007] Another embodiment of the present invention provides a method for preparing the above-mentioned indole diterpene alkaloids 1, 2, 3, 4 and / or 5, characterized by comprising the following steps:

[0008] (1) Marine fungus N4-3 was cultured in a culture medium to obtain a seed culture;

[0009] (2) The seed liquid obtained in step (1) is inoculated into the fermentation medium and fermented to obtain the fermentation product;

[0010] (3) Separate the fermentation broth and cells from the fermentation product obtained in step (2). Extract the fermentation broth with organic solvent A 2-4 times. Combine the extracts and concentrate under reduced pressure to obtain fermentation broth extract. Extract the cells with organic solvent B 2-4 times. Combine the extracts and concentrate under reduced pressure to obtain cell extract. Combine the fermentation broth extract and cell extract to obtain the crude extract.

[0011] (4) The crude extract obtained in step (3) was separated by chromatography to obtain indole diterpenoid alkaloid compounds 1, 2, 3, 4 and / or 5.

[0012] The strain culture conditions and fermentation conditions (such as culture medium selection, temperature, time, etc.) in the above preparation method of the present invention are all conventional experimental operations in the field. Those skilled in the art can make reasonable selections according to the experimental conditions. In order to facilitate understanding of the present invention, only one conventional operation method is listed below: The strain culture medium in step (1) is a commonly used strain culture medium in the field, preferably PDB culture medium, and the formula (per liter of water) is preferably: glucose 20 g / L, potato 200 g / L, sea salt 30 g / L; the strain culture in step (1) is preferably cultured on a shaker, the temperature is preferably room temperature to 30 ℃, the shaker speed is preferably 150-200 r / min, and the culture time is preferably 2-4 days. The fermentation culture medium in step (2) is a commonly used fermentation culture medium in the field, which may be the same as or different from the strain culture medium in step (1), preferably PDB culture medium, and the formula (per liter of water) is preferably: glucose 20 g / L, potato 150-300 g / L, sea salt 30 g / L; the inoculum amount in step (2) is preferably 1-5%, and the fermentation culture temperature is room temperature to 30 ℃. oC. The culture time is 28-30 days. The organic solvent A in step (3) is preferably one or more of ethyl acetate, dichloromethane, chloroform, or diethyl ether; the organic solvent B is preferably one or more of methanol, ethanol, THF, or acetone. The fermentation broth extract and the cell extract are combined and optionally dried to obtain a crude extract. The chromatographic separation in step (4) is preferably one or a combination of several of the following: reduced-pressure silica gel column chromatography, normal-phase silica gel column chromatography, reversed-phase silica gel column chromatography, gel column chromatography, and HPLC. The preferred method is as follows: The crude extract of marine fungus N4-3 is first subjected to vacuum silica gel column chromatography with a gradient elution using petroleum ether-ethyl acetate as the eluent. The elution gradients are 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100. Two column volumes are collected for each gradient, and the fractions are separated into five groups based on polarity. The eluents from gradients 50:50 and 40:60 are combined, concentrated, and then subjected to Sephadex LH-20 gel column chromatography with CH2Cl2 / MeOH as the eluent, v / v. Elution was performed using a 1:1 mixed solvent, eluting for 4-5 column volumes. The eluent was concentrated under reduced pressure and then subjected to reversed-phase column chromatography (ODS) with a mobile phase of MeOH:H₂O = 85:15 to obtain indole diterpenoid alkaloids 1 and 4. The eluents obtained from gradients of 30:70 and 20:80 were combined and concentrated, then subjected to normal-phase silica gel column chromatography with a mobile phase of ethyl acetate:petroleum ether = 1:8 to 1:3, eluting for 6-8 column volumes. This was followed by Sephadex LH-20 gel column chromatography with a 1:1 v / v mixed solvent of CH₂Cl₂ / MeOH, eluting for 4-5 column volumes. The eluent was concentrated under reduced pressure and then subjected to reversed-phase column chromatography (ODS) with a mobile phase of MeOH:H₂O = 90:10 to 85:15 to obtain indole diterpenoid alkaloids 2, 3, and 5. Compounds 1-5 were optionally purified by semi-preparative HPLC. All eluent or mobile phase ratios described in this invention are volume ratios.

[0013] Another embodiment of the present invention provides a pharmaceutical composition characterized in that the pharmaceutical composition uses one or more of the above-mentioned indole diterpenoid alkaloid compounds 1, 2, 3, 4, 5, their tautomers, or pharmaceutically acceptable salts thereof as active ingredients. The pharmaceutical composition further includes a pharmaceutically acceptable carrier, diluent, or excipient. The dosage form of the pharmaceutical composition is selected from solid dosage forms, liquid dosage forms, semi-solid dosage forms, etc. The pharmaceutical composition is used for the prevention and treatment of human pathogens, tumors, and other diseases.

[0014] Another embodiment of the present invention provides the use of one or more of the above-mentioned indole diterpenoid alkaloid compounds 1, 2, 3, 4, 5, their tautomers, or pharmaceutically acceptable salts thereof in the preparation of antibacterial or antitumor drugs. The antibacterial drugs are preferably targeted at diseases caused by infections such as Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Escherichia coli, and Vibrio parahaemolyticus. The antitumor drugs are preferably targeted at gastric cancer, liver cancer, etc.

[0015] Another embodiment of the present invention provides the use of the above-mentioned marine fungus N4-3 in the preparation of one or more of indole diterpenoid alkaloid compounds 1, 2, 3, 4, and 5. Attached Figure Description

[0016] Figure 1 It is compounds 1 and 2. 1 H- 1 H COSY, HMBC, ROESY, NOESY diagrams.

[0017] Figure 2 This is the HMBC correlation diagram for compound 3.

[0018] Figure 3 It is compound 1 1 1H NMR (400 MHz, CDCl3) image.

[0019] Figure 4 It is compound 1 13 C NMR (100 MHz, CDCl3) image.

[0020] Figure 5 This is the high-resolution mass spectrum of compound 1.

[0021] Figure 6 It is compound 2. 1 1H NMR (600 MHz, CDCl3) image.

[0022] Figure 7 It is compound 2. 13 C NMR (150 MHz, CDCl3) image.

[0023] Figure 8 This is the high-resolution mass spectrum of compound 2.

[0024] Figure 9 It is compound 3. 1 1H NMR (600 MHz, CDCl3) image.

[0025] Figure 10 It is compound 3. 13 C NMR (150 MHz, CDCl3) image.

[0026] Figure 11 This is the high-resolution mass spectrum of compound 3.

[0027] Figure 12 It is compound 4. 1 1H NMR (600 MHz, CDCl3) image.

[0028] Figure 13 This is the HSQC (CDCl3) diagram of compound 1.

[0029] Figure 14 It is compound 1 1 H– 1 H COSY (CDCl3) diagram.

[0030] Figure 15 This is the HMBC(CDCl3) diagram of compound 1.

[0031] Figure 16 This is the ROESY (CDCl3) diagram of compound 1.

[0032] Figure 17 This is the ECD diagram of compound 1.

[0033] Figure 18 This is the HSQC (CDCl3) diagram of compound 2.

[0034] Figure 19 It is compound 2. 1 H– 1 H COSY (CDCl3) diagram.

[0035] Figure 20 This is the HMBC (CDCl3) diagram of compound 2.

[0036] Figure 21 This is the ROESY (CDCl3) diagram of compound 2.

[0037] Figure 22 This is the ECD diagram of compound 2. Detailed Implementation

[0038] Example 1

[0039] (1) Cultivation of marine fungus N4-3 strain

[0040] The culture medium used for culturing marine fungus N4-3 was prepared by adding the following to every 1000 mL of water: 200 g of boiled potato juice, 20 g of glucose, and 30 g of coarse sea salt. The solution was dispensed into Erlenmeyer flasks and cultured at room temperature on a shaker at 180 rpm for 3 days to obtain the seed culture.

[0041] (2) Fermentation of marine fungus N4-3

[0042] The fermentation medium used for the fermentation culture of marine fungus N4-3 was prepared by adding the following to every 1000 mL of water: 200 g of boiled potato juice, 20 g of glucose, and 30 g of coarse sea salt. The medium was dispensed into 100 Erlenmeyer flasks (100 1L flasks, each containing 400 mL of PDB medium). The seed culture was inoculated into the medium in the Erlenmeyer flasks at an inoculum size of 1.0%. Fermentation was carried out at room temperature for 30 days to obtain the fermentation product.

[0043] (3) Preparation of crude extract of marine fungus N4-3

[0044] Take the fermentation product obtained after fermentation culture in step (2), separate the fermentation broth and the cells, extract the fermentation broth with ethyl acetate 3 times, concentrate the extract under reduced pressure to obtain fermentation broth extract; extract the cells with methanol 3 times, concentrate under reduced pressure to obtain cell extract; combine the fermentation broth extract and cell extract (optionally, further drying operation is adopted) to obtain the crude extract.

[0045] (4) Isolation and purification of compounds 1-5

[0046] The crude extract of marine fungus N4-3 obtained in step (3) was first subjected to vacuum silica gel column chromatography (preferably 100-200 mesh silica gel) with petroleum ether-ethyl acetate as the eluent for gradient elution. The elution gradients were 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100. Two column volumes were collected for each gradient, and the fractions were separated into five groups according to their polarity. The eluents obtained from gradients 50:50 and 40:60 were combined and concentrated, and then subjected to Sephadex LH-20 gel column chromatography with CH2Cl2 / MeOH as the eluent, v / v. Elution was performed using a 1:1 mixed solvent for 4–5 column volumes, followed by concentration under reduced pressure and then reversed-phase column chromatography (ODS) with a mobile phase of MeOH:H2O = 85:15 to obtain indole diterpenoid alkaloids 1 (45.2 mg) and 4 (12.3 mg). Elutions obtained from gradients of 30:70 and 20:80 were combined, concentrated, and then subjected to normal-phase silica gel column chromatography with a mobile phase of ethyl acetate:petroleum ether = 1:8 to 1:3 for 6–8 column volumes. This was followed by Sephadex LH-20 gel column chromatography with a 1:1 mixed solvent of CH2Cl2 / MeOH (v / v) for 4–5 column volumes, followed by concentration under reduced pressure and then reversed-phase column chromatography (ODS) with a mobile phase of MeOH:H2O = 90:10 to 85:15 to obtain indole diterpenoid alkaloids 2 (6.2 mg), 3 (4.5 mg), and 5 (28.5 mg).

[0047] The structural characterization data of compounds 1-5 are as follows. Based on one-dimensional and two-dimensional NMR, MS, and ECD data, the structures of compounds 1-5 can be determined as follows:

[0048]

[0049] Compound 1: [α] 20 D −215 (c = 0.10, MeOH). UV (MeOH) λmax (log ε) 367(0.5), 285 (2.3), 259 (1.6), 204 (1.3) nm. ECD (0.25 mM, MeOH) λmax (Δε) 373(−24.6), 324 (+26.5), 281 (−13.3), 251 (+18.3), 224 (+10.9) , 205 (−12.5) nm.IR (KBr) νmax 3329, 2975, 2934, 1658, 1456, 1375, 1258, 1167, 1132, 1010,868, 534, 458. 1 H NMR (CDCl3, 400 MHz) and 13 C NMR (CDCl3, 100 MHz) is shown in Table 1; HRESIMS m / z 604.3042 [M + Na] + (calcd for C 37 H 43 NNaO5 + , 604.3033).

[0050] Table 1. Compound 1 1 H and 13 C NMR data

[0051]

[0052] Compound 2: [α] 20 D +15 (c = 0.10, MeOH). UV (MeOH) λ max (log ε) 368 (0.3),282 (2.7), 215 (1.3) nm. ECD (0.25 mM, MeOH) λ max(Δε) 353 (−13.6), 315 (−7.8), 280 (−61.4), 235 (+26.2), 215 (−15.2) nm. 1 H NMR (CDCl3, 600 MHz) and 13 CNMR (CDCl3, 150 MHz) is shown in Table 2; HRESIMS m / z 618.3187, [M + Na] + , (calcd forC 38 H 45 NNaO5 + , 618.3190).

[0053] Table 2. Compound 2 1 H and 13 C NMR data

[0054]

[0055] Compound 3: [α] 20 D −43 (c = 0.10, MeOH). UV (MeOH) λ max (log ε) 309 (0.7),242 (2.2), nm. 1 H NMR (CDCl3, 600 MHz) and 13 C NMR (CDCl3, 150 MHz) is shown in Table 3; HRESIMS m / z 650.3120, [M + Na] + , (calcd for C 38 H 45 NNaO7 + , 650.3088).

[0056] Table 3 Compounds 1 H and 13 C NMR data

[0057]

[0058] Compound 4: High-resolution mass spectrometry yields [M + Na] + The signal, 620.2993 (calcd for C 37 H 43 NNaO6 + The value of 620.2983 indicates that the molecular weight of this compound is 583 and its molecular formula is C620.2983. 37 H 45NO5 has an unsaturation degree of 16. 1 H NMR data revealed that in δ H There is an NH signal at 7.75 (H-1), in the low field region δ H Eight monomethyl signals were given at 1.45 (H-32), 1.33 (H-37 / 38), 1.28 (H-36), 1.25 (H-39 / 40), 1.09 (H-35), and 1.06 (H-33), δ H 7.49 (H-20) and 7.03 (H-30) are its aromatic hydrogen signals, and two olefin signals δ are also given. H 6.05 (H-11), 5.7-5.75 (H-6), and 6 groups of methylene signals and 6 methine signals. Compared with compound 1, it has two additional methylene signals δ. H 2.84-2.75 (27), δ H 3.27 (H-22) lacks two sets of methine signals, presumably due to the replacement of the two double bonds at C-22 and C-27 at rings A and B by double bonds at C-23 and C-28. Through literature review and data comparison, the compound was ultimately identified as the known compound shearinine O. 1 H NMR (600 MHz, CDCl3) δ 7.75 (1H, d, J = 5.4 Hz, H-1), 7.49 (1H, d, J = 3.3 Hz, H-20), 7.03 (1H, d, J = 4.9 Hz, H-30), 6.05 (1H, s, H-11), 5.7-5.75 (1H, m, H-6), 4.24 (1H, s, H-9), 3.27 (2H, d, J = 16.9 Hz, H-22), 3.04 (1H, d, J = 11.2 Hz, H-5a), 2.84-2.75 (2H, m, H-27), 2.73 (1H, m,H-16), 2.50-2.30 (2H, m, H-5b / 17a), 2.19-1.92 (5H, m, H-14a / 14b / 15a / 15b / 17b), 1.45 (3H, s, H-32), 1.33 (6H, s, H-37 / 38), 1.28 (3H, s, H-36), 1.25 (6H, s, H-39 / 40), 1.09 (3H, s, H-35), 1.06 (3H, s, H-33).

[0059] Compound 5: High-resolution mass spectrometry gives [m / z 604.3048 (C37 H 43 NNaO5 + [Calcd. 604.3033] indicates that the molecular weight of this compound is 581, and the molecular formula is C. 37 H 43 NO5 has an unsaturation degree of 17. Further analysis is needed. 1 1H NMR data (Table 2-6) reveal the δ H There is an NH signal at 7.81 (brd, J = 6.4 Hz), in the high field region δ H 1.54 (H-37 / 38), 1.47 (H-39 / 40), 1.45 (H-36), 1.37 (H-32), 1.24 (H-33), and 1.19 (H-35) yielded eight monomethyl signals and two aromatic hydrogen signals δ. H 7.20 (H-20) and 7.43 (H-30), and also give three olefin signals δ H 5.84 (H-11), 6.38 (H-22), and 6.50 (H-27), as well as five chemically inequivalent methylene signals and two methine signals δ H 4.32 (H-9), 2.79 (H-16). 13 C10 NMR shows a total of 37 carbons, including eight methyl signal δ groups. C 16.2 (C-32), 28.3 (C-33), 23.2 (C-35), 28.9 (C-36), 31.5 (C-37 / 38), 30.9 (C-39 / 40), one unsaturated ketone group (δ C 197.2, C-10), 4 oxygen-bound carbon δ C 104.5 (C-7), 88.0 (C-9), 73.3 (C-24), 72.8 (C-26) and eight olefinic or aromatic carbon δ C 117.7 (C-11), 110.4 (C-20), 121.4 (C-22), 129.0 (C-27), 104.2 (C-30). The above spectral signals indicate that compound 5 is also an indole diterpenoid compound. After comparison with literature, it was identified as 22,23-dehydroshearinine A.

[0060] Compound 5 1 H and 13 C NMR data

[0061]

[0062] Example 2 Evaluation of the antifungal activity of compounds 1-5 against plant pathogenic fungi

[0063] The inhibitory activity of the crude extract (Agricultural Industry Standard NY / T 1156.2-2006) against rice sheath blight pathogen was determined using the mycelial growth rate method. The positive control was fluopyram.

[0064] The specific method is as follows: The test drugs (compounds 1-5) are diluted with solvent to prepare a series of concentration gradient stock solutions. Under aseptic conditions, pre-melted culture medium is quantitatively added to sterile conical flasks according to the test treatment. Drug solutions are quantitatively aspirated sequentially from low to high concentration and added to the aforementioned conical flasks, then thoroughly mixed. Equal volumes are then poured into three identical petri dishes to prepare drug-containing PDA plates of the corresponding concentrations. Under aseptic conditions, using a sterile punch with a 7mm diameter, mycelial cakes are punched from the edge of the colony and inoculated into the center of the drug-containing plate using an inoculator. An equal volume of solvent is added to the solvent control. The plates are incubated at 25℃. When the control is almost fully colonized, the colony diameter (cm) is measured using the cross-sectional method. The average value is calculated, and the inhibition rate is calculated. This process is repeated three times to obtain the final result.

[0065]

[0066] The results showed that compounds 1-5 exhibited inhibition rates of over 10% against rice sheath blight pathogens at a concentration of 200 mg / L. The positive control, fluopyram, showed a 100% inhibition rate against rice sheath blight pathogens at a concentration of 20 mg / L.

[0067] Example 3: Antibacterial activity test against human pathogenic bacteria

[0068] The antibacterial activity of the compounds was determined using the microdilution method. Nine bacterial strains were tested, including seven Gram-positive bacteria: Staphylococcus aureus (ATCC 43300, S. aureus ATCC 33591, S. aureus ATTCC 25923, S. aureus ATCC 29213), Enterococcus faecalis (ATCC 51299), Enterococcus faecium (ATCC 35667), and Bacillus subtilis (ATCC 19659) and two Gram-negative bacteria: Escherichia coli (ATCC 25922) and Vibrio parahaemolyticus (ATCC 17802). Vancomycin hydrochloride and oxacillin sodium were used as positive controls, DMSO was used as a negative control, and bacterial culture medium was used as a blank control. The culture was carried out at 28 °C for 24 h.

[0069] Prepare LB medium, aliquot it evenly into 250 mL Erlenmeyer flasks, and autoclave for later use. Remove the cryopreserved tubes of the bacterial strain to be tested from the -80℃ freezer, allow them to regain activity at room temperature, and use a pipette to add 200 µL of the bacterial suspension to the prepared medium. Incubate at 180 r / min and 37 ℃ for a period of time. Accurately weigh 1 mg of the test compound and place it in a 2 mL EP tube. Add a certain amount of DMSO to dissolve it completely, sonicating any insoluble parts to achieve a compound concentration of 10 μg / μL. Accurately weigh appropriate amounts of the positive control drugs vancomycin hydrochloride and oxacillin sodium, and dissolve them in DMSO to achieve a concentration of 2.5 μg / μL.

[0070] First, an initial screening for antibacterial activity was performed. The bacterial suspension was diluted in a clean bench, with 100 µL of the diluted suspension set aside. A 96-well plate was taken out, and 200 μL of blank LB medium was added to B1-D1 as a blank control. 198 μL of medium containing the bacterial suspension was added to E1-G1, along with 2 μL of DMSO as a negative control. Another 198 μL of medium containing the bacterial suspension was added to B2-G11, and 2 μL of the test sample or positive control was added to B2-G11 as the sample group. The final sample concentration was 100 μg / mL. The plate was sealed with sealing film to prevent subsequent contamination. Shaking was prohibited during incubation to prevent cross-contamination. One strain was used per plate, and three replicates were performed, with the average value taken. After the operation, the plate was placed in a constant temperature incubator and incubated at 37℃ for 24 h. The OD values ​​were then read using a microplate reader. 600 The inhibition rate was calculated, and samples with an inhibition rate greater than 50% were selected for microdilution testing.

[0071]

[0072] The blank control was prepared by adding 200 μL of blank LB medium to B1-D1. 198 μL of the culture medium containing bacteria was added to E1-G1, along with 2 μL of DMSO as a negative control. 198 µL of the culture medium containing bacteria was then added to A2-A4, along with 2 µL of the test sample or positive control, and thoroughly mixed. 100 µL of the culture medium containing bacteria was added to B2-H10. 100 µL of the culture medium from A2-A4 was then added to B2-B4, and thoroughly mixed. The culture was then down-diluted to the H-displacement. 100 μL of the H-displacement was aspirated and discarded. 100 µL of the culture medium was then added to complete the AH-displacement. The final test concentrations were 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL, 1.5625 μg / mL, and 0.78125 μg / mL. Each 96-well plate was used to test only one strain. The plates were sealed with sealing film and incubated at 37°C for 24 hours. The OD values ​​were then read using a microplate reader. 600 The value was calculated and the proliferation inhibition rate was determined using the software GraphPad Prism 8. 50 value.

[0073]

[0074] a Dates are expressed in IC 50 values. b O = oxacillin sodium. c V = Vancomycin HCl.

[0075] Example 4 Cytotoxic Activity Test

[0076] (1) Resuscitation of tumor cells

[0077] The cytotoxic activity of compound 1 was tested using the MTT assay, with human gastric cancer cell line SGC-7901 and liver cancer cell line HepG2 as test tumor cell lines, and cisplatin as a positive control.

[0078] First, thaw the cancer cells, centrifuge for 5 min and discard the supernatant, then incubate the cancer cells at 37°C in a 5% CO2 incubator. Observe the growth status of the cancer cells routinely, then add 2-3 mL of PBS solution and 0.1-1 mL of 0.25% trypsin containing EDTA for digestion. When the cells reach a round shape, add 3 mL of culture medium to prepare a cell suspension for passage culture.

[0079] (2) MTT experiment

[0080] Human gastric cancer cells SGC-7901 and hepatocellular carcinoma cells HepG2 were added to 96-well plates, with 100 μL of suspension added to each well. The plates were incubated for one day in a 5% CO2 incubator at 37 °C. The test compound and positive control were added to each well, along with 100 mL of culture medium to serve as the negative and blank control groups, respectively. After a short time, 20 μL of LTT was added to each well, and the plates were incubated for another 4 hours. Excess liquid was aspirated, and 100 μL of DMSO was added to dissolve the cells. The plates were gently shaken for about 10 minutes, and the absorbance was read at 490 nm. The formula for calculating the proliferation inhibition rate is as follows:

[0081]

[0082] The results showed that compound 1 had a strong inhibitory effect on cell proliferation, and its IC50 value against the human gastric cancer cell line SGC-7901 was significantly reduced. 50 The value was 19.16 μg / mL; the IC50 value for the human hepatocellular carcinoma cell line HepG2 was... 50 The value was 6.27 μg / mL. The IC50 value of the positive control drug cisplatin against SGC-7901 and HepG2 was... 50 The concentrations were 3.56 and 1.99 μg / mL, respectively.

Claims

1. An indole diterpene alkaloid, its tautomer, or a pharmaceutically acceptable salt thereof, characterized in that... The indole diterpenoid alkaloids have the structures shown in compounds 1, 2, and 3: 、 、 。 2. A pharmaceutical composition, characterized in that... The pharmaceutical composition uses one or more of the indole diterpenoid alkaloids 1, 2, and 3 as described in claim 1, their tautomers, or their pharmaceutically acceptable salts as the active ingredient.

3. The pharmaceutical composition according to claim 2, characterized in that... The pharmaceutical composition also includes a pharmaceutically acceptable carrier, diluent, or excipient.

4. The pharmaceutical composition according to any one of claims 2-3, characterized in that... The dosage form of the pharmaceutical composition is selected from solid dosage forms, liquid dosage forms, and semi-solid dosage forms.

5. The use of one or more of the indole diterpenoid alkaloids 4, 5, 1, 2, 3, their tautomers, or their pharmaceutically acceptable salts in plant protection, particularly in the control of agricultural pathogens, wherein the structures of indole diterpenoid alkaloids 4, 5 are as follows: 、 。 6. Use of one or more of the indole diterpenoid alkaloids 4, 5, 1, 2, 3, their tautomers, or pharmaceutically acceptable salts thereof, as described in claim 1, in the preparation of medicaments for the prevention and treatment of agricultural pathogens, human pathogens, and tumors, wherein the structures of indole diterpenoid alkaloids 4 and 5 are as follows: 、 。 7. Use of one or more of the indole diterpenoid alkaloids 4, 5, 1, 2, 3, their tautomers, or pharmaceutically acceptable salts thereof, as described in claim 1, in the preparation of antibacterial or antitumor drugs, wherein the structures of indole diterpenoid alkaloids 4 and 5 are as follows: 、 。 8. The application according to claim 7, characterized in that... The antibacterial drug targets diseases caused by Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Bacillus subtilis, Escherichia coli, and Vibrio parahaemolyticus infections.

9. The application according to claim 7, characterized in that... The anti-tumor drugs are targeted at stomach cancer and liver cancer.

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