Glycosides from endophytic fungi of xanthoceras sorbifolia bge. and preparation method and application thereof

Abstract

The present application relates to glycosides in endophytic fungi of Malania oleifera, and a preparation method and application thereof, wherein the glycosides have structures shown in compounds AN12 to AN18.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of fungal secondary metabolites, and particularly relates to glycosides from Ceriops tagal endophytic fungi and a preparation method and application thereof. BACKGROUND

[0002] Ceriops tagal belongs to the family Rhizophoraceae and the genus Ceriops, and is one of the most representative mangrove plants. In China and India, the plant has been used as a medicinal plant for a long time to stop bleeding, relieve constipation and itching, and treat sores, frostbite and malaria, etc. The chemical components of the plant mainly include diterpenes, triterpenes, polyphenols and steroids, etc. In addition, the endophytic fungi of Ceriops tagal have also attracted widespread attention, and terpenoids, alkaloids, isocoumarins, chromones, furan derivatives, quinones, phenols and steroids, etc. have been isolated from the secondary metabolites thereof. The applicant previously obtained a series of benzopyranone derivatives from the fungus Colletotrichum gloeosporioides of Ceriops tagal (Chinese patent application number: CN 201910639955.4), and in the present application, a series of anti-tumor active glycosides were obtained by analyzing different fractions of the same extract. SUMMARY

[0003] The present application provides a glycoside compound from Ceriops tagal endophytic fungi, a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof, characterized in that the glycoside compound has the structure shown in compounds AN12 to AN18:

[0004]

[0005]

[0006] Another embodiment of the present application provides a method for simultaneously preparing the above glycoside compounds AN12 to AN18, characterized in that it comprises the following steps:

[0007] (1) preparing a seed culture medium, inoculating the fungus Colletotrichum gloeosporioides into the seed culture medium, and culturing at 28°C for 3 days to obtain a seed culture solution;

[0008] (2) inoculating the seed culture solution obtained in step (1) into a fermentation culture medium, and culturing at 28°C for 28-30 days to obtain a fermentation product;

[0009] (3) separating the fermentation broth and the mycelium in the fermentation product obtained in step (2), wherein the fermentation broth is extracted with an equal volume of ethyl acetate for 3-4 times, and the combined extract is concentrated under reduced pressure to obtain an extract.

[0010] (4) The extract obtained in step (3) is subjected to vacuum silica gel column chromatography, and eluted with petroleum ether-ethyl acetate in a gradient of 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, 0:100, and two column volumes are collected for each gradient. The fractions obtained in the gradient elution of 30:70, 20:80 and 10:90 are combined, concentrated, and subjected to Sephadex LH-20 gel column chromatography with chloroform:methanol = 1:1 as the eluent. The resulting product is further subjected to high performance liquid chromatography (HPLC) with an Agilent C18 column (9.4 x 250 mm, 7 μm) at a flow rate of 2 mL / min and a mobile phase of CH3OH:H2O = 40:60 to 30:70 to obtain compounds AN12 to AN18.

[0011] wherein the ratio of the eluent or the mobile phase is volume ratio; the seed culture medium contains 1.5-3.0% of glucose, 0.1-0.5% of yeast extract, 0.1-0.5% of protein peptone, 0.11-0.6% of crude sea salt, and an appropriate amount of water; the fermentation culture medium contains 1.6-3.5% of glucose, 0.1-0.5% of yeast extract, 0.1-0.5% of protein peptone, 0.11-0.6% of crude sea salt, and an appropriate amount of water; the above percentages are weight percentages; and the seed culture medium and the fermentation culture medium are sterilized at 120°C for 25-30 minutes.

[0012] Another embodiment of the present application provides use of the above-mentioned compounds AN12 to AN18, stereoisomers, tautomers or pharmaceutically acceptable salts thereof in the preparation of an antitumor drug.

[0013] Another embodiment of the present application provides use of the above-mentioned compounds AN12 to AN18, stereoisomers, tautomers or pharmaceutically acceptable salts thereof in the preparation of an antitumor drug.

[0014] Another embodiment of the present application provides use of the above-mentioned compounds AN12 to AN18, stereoisomers, tautomers or pharmaceutically acceptable salts thereof in the preparation of an antitumor drug.

[0015] The present application provides a pharmaceutical composition characterized by using the above-mentioned compounds AN12 to AN18, stereoisomers, tautomers or pharmaceutically acceptable salts thereof as an effective ingredient.

[0016] The above-mentioned pharmaceutical composition provided by the present application can further comprise other anti-tumor drugs; and can also comprise a pharmaceutically acceptable excipient (preferably a pharmaceutically acceptable carrier, diluent or excipient). The dosage form of the above-mentioned pharmaceutical composition can be a solid preparation, a semi-solid preparation or a liquid preparation.

[0017] The term "pharmaceutically acceptable salt" in the present application refers to a non-toxic addition salt of an inorganic or organic acid and / or base, which can be referred to "Salt selection for basic drugs", Int. J. Pharm. (1986), 33, 201-217.

[0018] The "Colletotrichum gloeosporioides" fungus described in the present application is collected from the roots of the mangrove plant Xylocarpus granatum in Dongzhaigang Mangrove Nature Reserve in Haikou City by the inventors, and after isolation and purification, the strain is identified as Colletotrichum by morphology and molecular biology. The sequence of the ITS region of the fungus has been submitted to NCBI (GenBank accession No. MF508974). The "Colletotrichum gloeosporioides" fungus described in the present application has been disclosed in the inventors' previous research papers "The Journal of Antibiotics, volume 72, pages 513-517, (2019) and Chem. Biodiversity 2020, 17, e1900547"; and also disclosed in the applicant's previous Chinese patent application No. CN201910639955.4, and has been preserved in the Key Laboratory of Chemicals of Tropical Medicinal Resources of the Ministry of Education (formerly known as the Key Laboratory of Chemicals of Tropical Medicinal Plants of the Ministry of Education) of the applicant. The public can obtain the "Colletotrichum gloeosporioides" fungus described in the present application according to the method recorded in the above-mentioned research papers of the inventors, or purchase it from the Key Laboratory of Chemicals of Tropical Medicinal Resources of the Ministry of Education of the applicant. The applicant promises that the Key Laboratory of Chemicals of Tropical Medicinal Resources of the Ministry of Education can provide the "Colletotrichum gloeosporioides" fungus described in the present application to the public for 20 years from the filing date of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is the structure of compound AN12 and COSY, HMBC correlation diagram;

[0020] Figure 2 is the structure of compound AN13 and COSY, HMBC correlation diagram;

[0021] Figure 3is the structure of compound AN 14 and COSY, HMBC correlation charts;

[0022] Figure 4 is the structure of compound AN 15 and COSY, HMBC correlation charts;

[0023] Figure 5 is the structure of compound AN 16 and COSY, HMBC correlation charts;

[0024] Figure 6 is the structure of compound AN 17 and COSY, HMBC correlation charts;

[0025] Figure 7 is the structure of compound AN 18 and COSY, HMBC correlation charts;

[0026] Figure 8 is the DEPT spectrum of AN 12;

[0027] Figure 9 is the COSY spectrum of AN 12;

[0028] Figure 10 is the HSQC spectrum of AN 12;

[0029] Figure 11 is the HMBC spectrum of AN 12;

[0030] Figure 12 is the NOESY spectrum of AN 12;

[0031] Figure 13 is the DEPT spectrum of AN 13;

[0032] Figure 14 is the COSY spectrum of AN 13;

[0033] Figure 15 is the HSQC spectrum of AN 13;

[0034] Figure 16 is the DEPT spectrum of AN 14;

[0035] Figure 17 is the COSY spectrum of AN 14;

[0036] Figure 18 is the HSQC spectrum of AN 14;

[0037] Figure 19 is the DEPT spectrum of AN 15;

[0038] Figure 20 is the COSY spectrum of AN 15;

[0039] Figure 21is a HSQC spectrum of AN 15;

[0040] Figure 22 is a DEPT spectrum of AN 16

[0041] Figure 23 is a COSY spectrum of AN 16;

[0042] Figure 24 is a HSQC spectrum of AN 16;

[0043] Figure 25 is a DEPT spectrum of AN 17

[0044] Figure 26 is a COSY spectrum of AN 17;

[0045] Figure 27 is a HSQC spectrum of AN 17;

[0046] Figure 28 is a DEPT spectrum of AN 18

[0047] Figure 29 is a COSY spectrum of AN 18;

[0048] Figure 30 is a HSQC spectrum of AN 18. DETAILED DESCRIPTION

[0049] For further understanding of the present application, the following examples are provided to make it more specific. However, these examples are only for better understanding of the present application and do not limit the scope or the principle of the present application, and the embodiments of the present application are not limited to the following.

[0050] Example 1

[0051] (1) Seed culture of Colletotrichum gloeosporioides

[0052] Prepare seed culture medium: glucose 20 g, peptone 2 g, yeast extract 2 g, crude sea salt 2.5 g, water 1.0 L, evenly distribute in two 1000 mL conical flasks, and sterilize at 120°C for 25 minutes.

[0053] Inoculate the strain of Colletotrichum gloeosporioides into the prepared seed culture medium, and incubate at 28°C for 3 days to obtain seed culture solution;

[0054] (2) Fermentation of Colletotrichum gloeosporioides

[0055] Preparation of fermentation medium: glucose 1.1 kg, peptone 100 g, yeast extract 100 g, sea salt 125 g, water 50 L, evenly divided into 100 1000 mL conical flasks, 120 ℃ for 25-30 min.

[0056] Take an appropriate amount of seed culture solution (8 mL / flask) obtained in step (1) into the conical flask containing the fermentation medium, and incubate at 28 ℃ for 30 days to obtain the fermentation product.

[0057] (3) Preparation of extract

[0058] Separate the fermentation broth and the bacterial cells in the fermentation product obtained in step (2), extract the fermentation broth with an equal volume of ethyl acetate for 3 times, combine the extract and concentrate under reduced pressure to obtain the extract (20.0 g);

[0059] (4) Extraction and separation of compounds AN12-AN18

[0060] The extract obtained in step (3) is subjected to reduced pressure silica gel column chromatography, and eluted with petroleum ether-ethyl acetate in a gradient of 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, the fractions eluted in the gradients of 30:70, 20:80 and 10:90 are combined, concentrated, and subjected to Sephadex LH-20 gel column chromatography with chloroform:methanol = 1:1 as the eluent, and then subjected to high performance liquid chromatography (HPLC) preparation with an Agilent C18, 9.4×250 mm, 7 μm column, a flow rate of 2 mL / min, and a mobile phase of CH3OH:H2O = 40:60 to 30:70, to finally obtain compound AN12 (12.8 mg), AN13 (13.4 mg), AN14 (16.1 mg), AN15 (9.2 mg), AN16 (10.3 mg), AN17 (11.2 mg), and AN18 (10.8 mg).

[0061]

[0062] The structure analysis and related structure confirmation data of compounds AN12-AN18 are as follows:

[0063] Compound AN12, brown oil, has absorption under ultraviolet light at 254 nm, and high resolution mass spectrometry HR-ESI-MS (m / z 259.0811, [M+H] + , calculated as C 11 H 15 O7 + , 259.0812) indicates that the molecular formula is C 11 H 14O7, 5 degrees of unsaturation. 1 HNMR spectra (Table 1) showed one methylene (δ H 3.58, 2H), three aromatic hydrogens (δ H 6.75, 1H; 6.34, 1H; 6.34, 1H), four methine (δ H 5.73, 1H; 4.25, 1H; 4.22, 1H; 4.12, 1H). 13 CNMR and DEPT spectra showed 11 carbon signals, including three quaternary carbons (δ C 152.2, 152.2, 131.9), one methylene carbon (δ C 63.3), four methine carbons (δ C 103.3, 88.5, 74.2, 72.1). It was very clear from the carbon spectrum that this molecule was a phenolic glycoside. 1 H- 1 H COSY showed three aromatic hydrogens formed a coupling system, suggesting that the aglycone was a 1,2,3-trisubstituted benzene. The chemical shifts of two aromatic carbons and aromatic hydrogens were identical, suggesting that there was a symmetry factor in the trisubstitution. The carbon chemical shifts of the sugar moiety were initially judged to be α-D-ribofuranoside. After consulting the literature, they were found to be identical to the carbon chemical shifts of the sugar moiety of the known compound daldiniside A. Therefore, the sugar moiety of AN12 was determined to be D-ribofuranoside. In the NOESY spectrum, the correlation peaks of H-1'(δ H 5.73) and H-2'(δ H 4.22) were found, from which it was determined that the configuration of the glycosidic bond was α. Thus, the structure of compound AN12 was determined to be 2,6-dihydroxyphenyl-1-O-α-D-ribofuranoside.

[0064] Table 1. H NMR and 1 H NMR and 13 C NMR data

[0065]

[0066] [a] Recorded in CH3OD.

[0067] Compound AN13, brown oil, had an absorption under UV light at 254 nm, and high resolution mass spectrometry HR-ESI-MS (m / z 345.1180, [M+H] + , calculated as C 15 H 21 O9+ 345.1189) suggests the molecular formula is C 15 H 20 O9, with 6 degrees of unsaturation. 1 H NMR spectrum (Table 1) shows this compound has 3 methylene hydrogens (δ H 3.70, 1H; 3.64, 1H; 3.05, 2H; 1.69, 2H), 1 aromatic hydrogen (δ H 6.14), 4 methine hydrogens (δ H 5.64, 1H; 4.19, 1H; 4.12, 1H; 4.09, 1H), 1 methyl hydrogen (δ H 0.98, 3H). 13 C NMR and DEPT spectra show this compound has 15 carbon signals, of which 6 are quaternary carbons (δ C 208.4, 165.79, 165.79, 165.32, 165.32, 107.25), 3 methylene carbons (δ C 63.61, 47.53, 19.75), 5 methine carbons (δ C 101.92, 97.26, 88.41, 73.83, 71.56, 63.61), 1 methyl carbon (δ C 14.82). From the carbon spectrum, this compound is also a phenolic glycoside. 1 H- 1 H COSY shows 2 methylene hydrogens (δ H 3.05, 2H; 1.69, 2H) form a n-propyl coupling system. HMBC shows the ketone carbonyl (δ C 208.4) is connected to this n-propyl and the adjacent carbon (δ C 107.3) of the aromatic hydrogen. From the carbon spectral data, the sugar unit is also D-ribofuranose. HMBC shows this sugar unit is connected to C-2 (δ C 165.8) through a glycosidic bond. By testing NOESY and one-dimensional selective NOE, no cross peaks between H-1' and H-2' were found, so the relative configuration of the glycosidic bond is β. Thus, the structure of compound AN13 is identified as 2-O-(β-D-ribofuranosyl)-3,4,5-trihydroxyphenylpropyl ketone.

[0068] Compound AN14, white powder, has absorption under 254 nm UV light, high resolution mass spectrum HR-ESI-MS (m / z 345.1189, [M+H] + , calculated as C 15 H 21 O9 +345.1180) suggests the molecular formula of C 15 H 20 O9, with 6 degrees of unsaturation. 1 H NMR spectrum (Table 2) shows two methylene protons (δ H 3.62, 2H; 2.83, IH; 2.76, IH), two aromatic protons (δ H 7.36, IH; 6.41, IH). 13 C NMR and DEPT spectra show 15 carbon signals, of which 5 are quaternary carbons (δ C 199.9, 158.3, 153.9, 137.3, 109.2), 2 are methylene carbons (δ C 63.1, 44.2), 7 are methine carbons (δ C 130.9, 108.6, 104.4, 88.3, 75.6, 73.6, 71.5), and 1 is a methyl carbon (δ C 20.9). 1 H- 1 H COSY and coupling constants suggest two aromatic protons are ortho-coupled, indicating the presence of a 1,2,3,4-tetrasubstituted benzene ring in the molecule. From HMBC, the coupling system (δ 1 H- 1 H COSY also reveals another coupling system (δ H 4.71, 2.83, 2.76, 1.54). From HMBC, this coupling system is connected to the benzene ring through the ketone carbonyl (δ C 199.9) at low field. Similar to the previous compound, the chemical shifts at δ C 104.4, 88.3, 73.6, 71.5, 63.1 constitute the sugar unit of the glycoside, which is also D-ribofuranose. No correlation between the anomeric hydrogen H-1' and H-2' is observed by NOESY and one-dimensional selective NOE, indicating that the glycosidic linkage is in the β configuration. Thus, the structure of this compound is deduced to be 2-O-(β-D-ribofuranosyl)-3,6-dihydroxyphenyl 9-hydroxy-propyl ketone.

[0069] Table 2. H NMR and 1 H NMR and 13 C NMR data

[0070]

[0071] [a] Recorded in (CD3)2CO.

[0072] Compound AN15 is a brown oily substance that absorbs under 254 nm UV light. High-resolution mass spectrometry (HR-ESI-MS) was used to analyze it (m / z 329.1230, [M+H]). + Calculated as C 15 H 21 O8 + (329.1231) indicates the molecular formula is C 15 H 20 O8 has an unsaturation degree of 6. 1 The 1H NMR spectrum (Table 2) shows that this compound has 3 methylene groups (δ¹⁸). H 3.66, 2H; 3.07, 2H; 1.69, 2H), 2 aromatic hydrogens (δ H 6.14,2H). 13 C10 NMR and DEPT spectra showed that the compound had a total of 15 carbon signals, including 5 quaternary carbons (δ¹⁸ C⁻¹). C 206.94, 164.90, 164.90, 164.35, 106.28), 3 methylene carbons (δ C 62.97, 46.37, 18.64), 6 methine carbons (δ C 101.04, 96.64, 96.64, 88.08, 78.84, 73.00), 1 methyl group (δ C 14.18). 1 H- 1 H COSY indicates 2 methylene (δ) C 46.37, 18.64) and methyl (δ) C 14.18) forms an n-propyl group, which HMBC shows is formed through a carbonyl group (δ). C 206.94) and the C-1 (δ) of the benzene ring C (106.28) connected. 1 The 1H NMR spectrum showed that the compound contained two aromatic hydrogens, which presented a single peak with identical chemical shifts, indicating that they were two aromatic hydrogens located at the para position. This, combined with the presence of these two aromatic carbons... 13 The identical C10 NMR chemical shifts indicate that the compound possesses a certain degree of symmetry. Therefore, the two quaternary carbons with identical chemical shifts (δ¹⁸C) are... C 164.90) is classified as C-2 and C-6. Similar to the aforementioned compounds, the chemical shift is δ. C101.04, 88.08, 73.00, 70.84, 62.97 of the sugar unit of the glycoside, and also D-ribofuranose. By NOESY, it was found that the end group hydrogen H-1' had no correlation peak with H-2', judging that the glycosidic bond configuration was β configuration. Thus, the structure of compound AN15 was identified as 4-O-(β-D-ribofuranosyl)-2,6-dihydroxyphenyl n-propyl ketone.

[0073] Compound AN16, brown oil, had absorption under 254 nm UV light, high resolution mass spectrum HR-ESI-MS (m / z 327.1230, [M+H] + , calculated as C 16 H 23 O7 + , 327.1438) suggested that the molecular formula was C 16 H 22 O7, and the unsaturation degree was 6. By analyzing 1 H NMR and 13 C NMR spectra (Table 3), C-4 was connected with a methoxy group (δ C 55.07), and C-8 was connected with a glycosidic bond. From the chemical shift of the carbon spectrum and the coupling constant of the hydrogen spectrum, it could be judged that the sugar was still D-ribofuranose. By NOESY, it was found that the end group hydrogen H-1' had a correlation peak with H-2', judging that the glycosidic bond configuration was α configuration. Therefore, it was inferred that the structure of compound AN16 was 3,4-dihydro-2-methyl-4-methoxy-8-O-(α-D-ribofuranosyl)-2H-1-benzopyran.

[0074] Compound AN17, brown oil, had absorption under 254 nm UV light, high resolution mass spectrum HR-ESI-MS (m / z 353.1228, [M+H] + , calculated as C 17 H 21 O8 + , 353.1231) suggested that the molecular formula was C 17 H 20 O8, and the unsaturation degree was 8. 1 H NMR and 13 C NMR spectra showed that the compound and compound AN15 had similar mother nucleus structures. The main difference was that compound AN17 had only 2 hydrogen (δ H 6.70, 1H; 6.52, 1H) in the meta position on the benzene ring. From HMBC, it could be judged that the hydrogen with a chemical shift of δ H 6.70 belonged to H-5. The hydrogen with a chemical shift of δ C101.9, 88.2, 73.5, 71.0, 63.1 of the carbon atoms constitute the sugar unit of the glycoside and are also D-ribofuranose. The monosaccharide is attached to C-6 via a glycosidic bond as shown by HMBC. NOESY shows that H-1' is correlated with H-2', thus the configuration of the glycosidic bond is determined to be α. Thus, the structure of compound AN17 is identified as 8-hydroxy-2-propyl-6-O-(α-D-ribofuranosyl)-4H-chromone.

[0075] Table 3.1H NMR and13C NMR data of compounds AN16-AN17 1 H NMR and 13 C NMR data

[0076]

[0077]

[0078] [a] Recorded in CDCl3, [b] Recorded in CH3OD.

[0079] Compound AN18 is one of a pair of enantiomers 13 C NMR data are almost all in pairs). Brown oil, high resolution mass spectrum HR-ESI-MS (m / z 349.0898, [M+Na] + , calculated for C 15 H 18 O8Na + , 349.0899) suggests the molecular formula of C 15 H 18 O8, with 7 degrees of unsaturation. After literature search, one set of NMR data is identical to that of the known compound 7-O-(α-D-ribofuranosyl)-2,3-dihydro-5-hydroxy-2-methyl-chromen-4-one, thus the other set of NMR data is assigned to its enantiomer, with S configuration at C-2. Since the coupling constant of the anomeric hydrogen cannot be used to determine the configuration of the glycosidic bond, by testing the NOESY spectrum, it is found that 1'-H is correlated with 2'-H, thus the configuration of the glycosidic bond of this pair of enantiomers is determined to be α. Thus, the structure of compound AN18 is identified as (2S)-2,3-dihydro-5-hydroxy-2-methyl-7-O-(α-D-ribofuranosyl)-4H-benzopyran-4-one.

[0080] Table 4.1H NMR and13C NMR data of compound AN18 and its enantiomer1 H NMR and 13 C NMR data

[0081]

[0082]

[0083] [a] Recorded in CH3OD.

[0084] Example 2

[0085] (1) Seed culture of Colletotrichum gloeosporioides

[0086] Prepare seed culture medium (10.0 L): glucose 1.5% (by weight, the same below), yeast extract 0.5%, peptone 0.1%, crude sea salt 0.11%, the rest being water; evenly distribute in 16 1000 mL conical flasks, sterilize at 120°C for 25-30 minutes.

[0087] Inoculate Colletotrichum gloeosporioides into the prepared seed culture medium, and incubate at 28°C for 3 days to obtain seed culture solution;

[0088] (2) Fermentation of Colletotrichum gloeosporioides

[0089] Prepare fermentation medium (100 L): glucose 1.6% (by weight, the same below), yeast extract 0.5%, peptone 0.1%, crude sea salt 0.11%, the rest being water; evenly distribute in 200 1000 mL conical flasks, sterilize at 120°C for 30 minutes.

[0090] Take an appropriate amount of seed culture solution obtained in step (1) and inoculate into the conical flask containing fermentation medium, and incubate at 28°C for 28 days to obtain fermentation product.

[0091] (3) Preparation of extract

[0092] Separate the fermentation broth and mycelium in the fermentation product obtained in step (2), extract the fermentation broth with an equal volume of ethyl acetate 4 times, combine the extract and concentrate under reduced pressure to obtain the extract;

[0093] (4) Extraction and separation of compounds AN12-AN18

[0094] The extract obtained in step (3) was subjected to column chromatography on silica gel under reduced pressure, and eluted with petroleum ether-ethyl acetate in a gradient of 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100, and two column volumes were collected for each gradient. The fractions obtained in the gradient elution of 30:70, 20:80, and 10:90 were combined, concentrated, and then subjected to column chromatography on Sephadex LH-20 with chloroform-methanol (1:1) as the eluent. Finally, the compounds AN12 to AN18 were obtained by preparative HPLC on an Agilent C18 column (9.4 x 250 mm, 7 μm) at a flow rate of 2 mL / min with a mobile phase of CH3OH:H2O=40:60 to 30:70. (TLC detection was consistent with that in Example 1, and the structural confirmation data were consistent.)

[0095] Example 3

[0096] (1) Cultivation of the Colletotrichum gloeosporioides strain

[0097] Seed culture medium (1.0 L) was prepared with 3.0% (by weight, the same hereinafter) glucose, 0.1% yeast extract, 0.5% peptone, and 0.6% crude sea salt, and the rest was water. The medium was evenly divided into three 500 mL conical flasks and sterilized at 120°C for 25-30 minutes.

[0098] The Colletotrichum gloeosporioides strain was inoculated into the prepared seed culture medium, and the seed culture was obtained by incubation at 28°C for 3 days.

[0099] (2) Fermentation of the Colletotrichum gloeosporioides strain

[0100] Fermentation medium (10 L) was prepared with 3.5% (by weight, the same hereinafter) glucose, 0.1% yeast extract, 0.5% peptone, and 0.6% crude sea salt, and the rest was water. The medium was evenly divided into twenty 1000 mL conical flasks and sterilized at 120°C for 25 minutes.

[0101] An appropriate amount of the seed culture obtained in step (1) was inoculated into the conical flasks containing the fermentation medium, and the fermentation product was obtained by incubation at 28°C for 29 days.

[0102] (3) Preparation of the extract

[0103] The fermentation broth and the mycelium in the fermentation product obtained in step (2) were separated, and the fermentation broth was extracted with an equal volume of ethyl acetate three times. The combined extract was concentrated under reduced pressure to obtain the extract.

[0104] (4) Extraction and isolation of compounds AN12 to AN18

[0105] The extract obtained in step (3) was subjected to column chromatography on silica gel under reduced pressure, and eluted with petroleum ether-ethyl acetate in gradient of 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100, respectively, and two column volumes were collected for each gradient. The fractions obtained from the gradient elution of 30:70, 20:80 and 10:90 were combined, concentrated, and subjected to column chromatography on Sephadex LH-20 with chloroform-methanol (1:1) as the eluent. The compounds AN12 to AN18 were finally obtained by preparative HPLC on an Agilent C18 column (9.4 x 250 mm, 7 μm) at a flow rate of 2 mL / min with CH3OH:H2O (40:60 to 30:70) as the mobile phase (TLC detection was consistent with that of Example 1, and the structural confirmation data were consistent).

[0106] Example 4 Anti-tumor activity

[0107] The cytotoxic activity of compounds AN12 to AN18 against tumor cell line A549 was tested by MTT method. Tumor cells in exponential growth phase were taken, adherent cells were detached by adding 0.02% Trypsin-EDTA, and a single cell suspension was prepared using RPMI1640 culture medium containing 10% fetal bovine serum. After counting and adjusting the cell number, the cells were inoculated into 96-well plates and incubated in a 37°C carbon dioxide incubator for 24 h. The test compounds were set at 2.00, 5.00, 10.00, and 20.00 μg / mL, respectively, with 3 parallel samples for each group. The test samples were dissolved in DMSO and diluted with RPMI1640, and then added to the 96-well plates and incubated in a 37°C carbon dioxide incubator for 72 h. MTT was dissolved in serum-free RPMI1640, and 50 μL was added to each well. The plates were incubated in a 37°C carbon dioxide incubator for 4 h, removed, and the supernatant was aspirated. DMSO (150 μL) was added to each well to dissolve the formazan, and the absorbance was measured at 630 nm using an enzyme-labeled instrument. The corresponding inhibition percentage and IC 50 values of compounds AN12 to AN18 against tumor cell line A549 were all less than 20.00 μg / mL, and the detailed test results of AN16 are shown in Table 5. 50

[0108] Table 5 Inhibitory effect of compound AN16 on tumor cells A549

[0109] ​

Claims

1. A glycoside compound or a pharmaceutically acceptable salt thereof derived from an endophytic fungus of *Hylocereus undatus*, characterized in that... The glycosides have the structures shown in compounds AN12 to AN17: 、 、 、 、 、 。 2. A method for simultaneously preparing glycoside compounds AN12 to AN18, characterized in that... Includes the following steps: (1) Prepare seed culture medium by inoculating seeds of Colletotrichum gloeosporioides into the seed culture medium and culturing at 28℃ for 3 days to obtain seed culture solution. (2) Inoculate the seed culture medium obtained in step (1) into the fermentation medium and incubate at a constant temperature of 28℃ for 28-30 days to obtain the fermentation product; (3) Separate the fermentation broth and cell cells from the fermentation product obtained in step (2). The fermentation broth is extracted with an equal volume of ethyl acetate 3-4 times. The extracts are combined and concentrated under reduced pressure to obtain an extract. (4) The extract obtained in step (3) was subjected to vacuum silica gel column chromatography, using petroleum ether-ethyl acetate gradients of 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. The fractions obtained from the 30:70, 20:80, and 10:90 gradients were combined, concentrated, and then subjected to Sephadex LH-20 gel column chromatography with chloroform:methanol = 1:1 as the eluent. The mixture was then prepared by high performance liquid chromatography (HPLC) using an Agilent C18 column (9.4 × 250 mm, 7 μm), a flow rate of 2 mL / min, and a mobile phase of CH3OH:H2O = 40:60 to 30:

70. The final compounds AN12 to AN18 were obtained. The structures of compounds AN12 to AN18 are as follows: 、 、 、 、 、 、 。 3. The use of the glycoside compounds AN12 to AN18 or pharmaceutically acceptable salts thereof prepared according to claim 2 in the preparation of an antitumor drug, wherein the tumor cell line of the tumor is A549.

4. A pharmaceutical composition, characterized in that... The active ingredient is the glycoside compound AN12 to AN17 as described in claim 1 or a pharmaceutically acceptable salt thereof.

5. The pharmaceutical composition according to claim 4, characterized in that... It also includes other anti-tumor drugs.

6. The pharmaceutical composition according to any one of claims 4-5, characterized in that... It also contains pharmaceutically acceptable excipients.

7. The pharmaceutical composition according to claim 6, characterized in that... The pharmaceutically acceptable excipients are selected from pharmaceutically acceptable carriers, diluents, or excipients.

8. The pharmaceutical composition according to claim 7, characterized in that... The dosage form of the pharmaceutical composition is a solid dosage form, a semi-solid dosage form, or a liquid dosage form.

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