Indole pleurotus nebrodensis alkali A, preparation method thereof and application of indole pleurotus nebrodensis alkali A in preparation of drugs for preventing tumors and resisting tumors

Abstract

The invention relates to the technical field of indole alkaloid compounds, in particular to indole pleurotus nebrodensis alkali A, a preparation method thereof and application of the indole pleurotus nebrodensis alkali A to preparation of drugs for preventing and resisting tumors. Pleurotus nebrodensis sporocarp is smashed, ethyl alcohol is added, heating reflux preparation and concentration are conducted, obtained pleurotus nebrodensis total extract is dispersed into suspension with water, and then the suspension is placed in a drying oven to be dried to obtain the indole pleurotus nebrodensis alkali A. The indole pleurotus nebrodensis alkali A is obtained. The method comprises the following steps: sequentially extracting with petroleum ether, dichloromethane and ethyl acetate, concentrating an extracting solution to obtain ethyl acetate part extract, carrying out gradient elution separation on the ethyl acetate part extract by silica gel column chromatography, carrying out gradient elution on the obtained third fraction by high performance liquid chromatography, purifying and separating, and collecting eluate to obtain the indole pleurotus nebrodensis alkali A at 16.3 minutes. The indole pleurotus nebrodensis alkali A is disclosed for the first time, and it is found through in-vitro antitumor pharmacodynamic experiments that the indole pleurotus nebrodensis alkali A has a certain inhibition effect on human HGC-27 gastric cancer cells, so that the indole pleurotus nebrodensis alkali A can be used for preparing drugs for preventing and resisting gastric cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of indole alkaloids, and in particular to indole pleurotus eryngii alkaloid A and its application in preparing drugs for preventing and treating gastric cancer, and is an indole pleurotus eryngii alkaloid A and its preparation method and its application in preparing drugs for preventing and treating tumors and treating tumors. Background Art

[0002] Pleurotus tuoliensis is a higher fungus belonging to the phylum Enmycophyta, class Basidiomycetes, order Agaricales, family Pleurotaceae, genus Pleurotus. Its fruiting body is generally large, pure white, with a cap diameter of 5cm to 15cm or larger, and a thickness of 3cm to 4cm. It is nearly oblate in the early stage, mostly lateral, sometimes eccentric, with a nearly smooth or velvety surface.

[0003] Relevant chemical studies have shown that polysaccharides, as the main active ingredient of Pleurotus eryngii, have been widely studied, but other active ingredients have been less studied. Alkaloid components, as one of the main effective ingredients of Pleurotus eryngii, have been found to have multiple effects such as anti-tumor and immunity enhancement.

[0004] The Chinese patent application document with authorization announcement number CN115433185B discloses a monoterpene indole alkaloid and its preparation method and application, which belongs to the technical field of preparation and identification of active ingredients of traditional Chinese medicine. By studying the anti-tuberculosis active substance basis of Lifeisan, it was found that its monoterpene indole alkaloids are its main active ingredients, and its effective parts are obtained; and alkaloid components are separated from Lifeisan, and 2 new alkaloid compounds are separated from the effective parts, and show significant anti-Bacillus smegmatis activity, providing a basis for the further development and utilization of Lifeisan.

[0005] The Chinese patent application document with the authorization announcement number CN113480538B discloses the use of a monoterpene indole alkaloid derivative in the preparation of a drug for preventing or treating cerebral ischemia-reperfusion injury. The derivative has a good protective effect on primary cortical neurons injured by cerebral ischemia-reperfusion and can be used to prepare a drug for preventing cerebral ischemia-reperfusion injury.

[0006] The Chinese patent application document with the authorization announcement number CN111333628B discloses an indole alkaloid. The indole alkaloid of the present invention is prepared from the Chinese medicinal herb Evodia rutaecarpa, has the activity of inhibiting α-glucosidase, and can be used to prepare a hypoglycemic drug.

[0007] At present, malignant tumors seriously threaten human life and health. Since many new anticancer drugs are expensive and long-term use can easily cause drug resistance and side effects, finding highly effective and low-toxic anticancer drugs is the key to tumor treatment. As a medicinal and edible fungus, Pleurotus nebrodensis contains a variety of highly active alkaloid compounds. Therefore, the development and utilization of the alkaloid components of Pleurotus nebrodensis, further exploring its potential medicinal value, and determining and characterizing the structure and physicochemical properties of its monomer compounds are of great significance to the development and utilization of Pleurotus nebrodensis. Summary of the invention

[0008] The present invention provides an indole pleurotus eryngii alkaloid A and a preparation method thereof and an application thereof in preparing a drug for preventing and treating tumors, which overcomes the deficiencies of the above-mentioned prior art and discloses for the first time an indole pleurotus eryngii alkaloid A which has a certain inhibitory effect on human HGC-27 gastric cancer cells, thereby enabling it to be used in the preparation of a drug for preventing and treating gastric cancer.

[0009] One of the technical solutions of the present invention is achieved by the following measures: an indole pleurotus eryngii alkaloid A, the chemical structure of which is .

[0010] The following is a further optimization and / or improvement of one of the above-mentioned technical solutions: The above was prepared according to the following method: The first step is to crush the Pleurotus eryngii fruiting bodies and add 95% by mass ethanol, heat and reflux to prepare, combine the preparation liquid of each reflux, recover under reduced pressure, and concentrate to obtain the Pleurotus eryngii total extract; The second step is to disperse the total extract of Pleurotus nebrodensis into a suspension with water, extract it with petroleum ether, dichloromethane and ethyl acetate in sequence, and concentrate the extract to obtain a petroleum ether extract, a dichloromethane extract and an ethyl acetate extract; The third step is to take the ethyl acetate extract and separate it by silica gel column chromatography with gradient elution to obtain 6 fractions, namely Fr1 to Fr6; In the fourth step, the third fraction Fr3 obtained by gradient elution of silica gel column chromatography was purified and separated by gradient elution of high performance liquid chromatography, and the eluate was collected to obtain indole pleurotus eryngii A at 16.3 minutes.

[0011] 8 mL to 12 mL of ethanol was added to every 1 g of Pleurotus nebrodensis fruiting body.

[0012] In the first step, the heating temperature is 50° C. to 60° C., the reflux preparation times are 3 times, and the reflux preparation time each time is 1 hour to 3 hours.

[0013] In the third step, the gradient eluent of the silica gel column chromatography includes petroleum ether and ethyl acetate, and the volume ratios of petroleum ether and ethyl acetate are 5:1, 2:1, 1.5:1, 1:1, 1:5 and 1:0, respectively.

[0014] In the fourth step, the eluent for the HPLC gradient elution is a mixture of methanol and water, wherein the volume ratio of methanol to water is 15:85.

[0015] The second technical solution of the present invention is achieved by the following measures: A method for preparing indole pleurotus eryngii A is carried out according to the following method: The first step is to crush the Pleurotus eryngii fruiting bodies and add 95% by mass ethanol, heat and reflux to prepare, combine the preparation liquid of each reflux, recover under reduced pressure, and concentrate to obtain the Pleurotus eryngii total extract; The second step is to disperse the total extract of Pleurotus nebrodensis into a suspension with water, extract it with petroleum ether, dichloromethane and ethyl acetate in sequence, and concentrate the extract to obtain a petroleum ether extract, a dichloromethane extract and an ethyl acetate extract; The third step is to take the ethyl acetate extract and separate it by silica gel column chromatography with gradient elution to obtain 6 fractions, namely Fr1 to Fr6; In the fourth step, the third fraction Fr3 obtained by gradient elution of silica gel column chromatography was purified and separated by gradient elution of high performance liquid chromatography, and the eluate was collected to obtain indole pleurotus eryngii A at 16.3 minutes.

[0016] The following is a further optimization and / or improvement of the second technical solution of the above invention: 8 mL to 12 mL of ethanol was added to every 1 g of Pleurotus nebrodensis fruiting body.

[0017] In the first step, the heating temperature is 50° C. to 60° C., the reflux preparation times are 3 times, and the reflux preparation time each time is 1 hour to 3 hours.

[0018] In the third step, the gradient eluent of the silica gel column chromatography includes petroleum ether and ethyl acetate, and the volume ratios of petroleum ether and ethyl acetate are 5:1, 2:1, 1.5:1, 1:1, 1:5 and 1:0, respectively.

[0019] In the fourth step, the eluent for the HPLC gradient elution is a mixture of methanol and water, wherein the volume ratio of methanol to water is 15:85.

[0020] The third technical solution of the present invention is achieved through the following measures: an application of indole pleurotus eryngii alkaloid A in the preparation of a drug for preventing gastric cancer.

[0021] The fourth technical solution of the present invention is achieved through the following measures: an application of indole pleurotus eryngii alkaloid A in the preparation of anti-gastric cancer drugs.

[0022] The present invention discloses for the first time the indole pleurotus eryngii alkaloid A, and through in vitro antitumor pharmacodynamics experiments, finds that it has a certain inhibitory effect on human HGC-27 gastric cancer cells, thereby enabling it to be used as a drug for preparing a gastric cancer prevention and anti-gastric cancer drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Attached Figure 1 The figure is the chemical structure diagram of the indole pleurotus eryngii A of the present invention.

[0024] Attached Figure 2 The indole pleurotus eryngii alkaloid A of the present invention 1 H-NMR spectrum.

[0025] Attached Figure 3 The indole pleurotus eryngii alkaloid A of the present invention 13 C-APT spectrum.

[0026] Attached Figure 4 The indole pleurotus eryngii alkaloid A of the present invention 1 H- 1 H COSY spectrum.

[0027] Attached Figure 5 The indole pleurotus eryngii alkaloid A of the present invention 1 H- 13 C HSQC spectrum.

[0028] Attached Figure 6 The indole pleurotus eryngii alkaloid A of the present invention 1 H- 13 C HMBC spectrum.

[0029] Attached Figure 7 The indole pleurotus eryngii alkaloid A of the present invention 1 H- 1 H ROESY spectrum. DETAILED DESCRIPTION

[0030] The present invention is not limited by the following embodiments, and the specific implementation method can be determined according to the technical scheme of the present invention and the actual situation. Unless otherwise specified, the various chemical reagents and chemicals mentioned in the present invention are all chemical reagents and chemicals known and used in the prior art; unless otherwise specified, the percentages in the present invention are all mass percentages; unless otherwise specified, the solutions in the present invention are all aqueous solutions with water as the solvent, for example, hydrochloric acid solution is a hydrochloric acid aqueous solution; the normal temperature and room temperature in the present invention generally refer to a temperature of 15°C to 25°C, and are generally defined as 25°C.

[0031] The present invention will be further described below in conjunction with embodiments: Example 1: The indole tuoliensine A (Tuoliensines A for short) has a chemical structural formula of .

[0032] The present invention discloses Tuoliensines A for the first time. The Tuoliensines A described in this embodiment was subjected to nuclear magnetic resonance hydrogen spectrum ( 1 H-NMR), carbon nuclear magnetic resonance spectroscopy ( 13 C-APT) and NMR 2D spectra ( 1 H- 1 H COSY, 1 H- 13 CHSQC, 1 H- 13 C HMBC, 1 H- 1 H ROESY) analysis, the Tuoliensines A described in this example 1 H-NMR spectrum Figure 2 As shown, the Tuoliensines A described in the examples 13 C-APT spectrum Figure 3 As shown, the Tuoliensines A described in the examples 1 H- 1 H COSY spectrum Figure 4 As shown, the Tuoliensines A described in the examples 1 H- 13 C HSQC spectrum is shown in Figure 5 As shown, the Tuoliensines A described in the examples 1 H- 13 C HMBC spectrum Figure 6 As shown, the Tuoliensines A described in the examples 1 H- 1 H ROESY spectrum is shown in Figure 7 As shown, Figure 2 and Figure 3 Perform graph analysis and Figure 2 and Figure 3 Each peak is assigned Figure 2 and Figure 3 The peak attributions are shown in Table 1.

[0033] From the data in Table 1, it can be seen that the chemical structure of Tuoliensines A described in this embodiment is as follows Figure 1 As shown, it is easily soluble in methanol.

[0034] Example 2: As an optimization of the above example, the following method was used to prepare: The first step is to crush the Pleurotus eryngii fruiting bodies and add 95% by mass ethanol, heat and reflux to prepare, combine the preparation liquid of each reflux, recover under reduced pressure, and concentrate to obtain the Pleurotus eryngii total extract; The second step is to disperse the total extract of Pleurotus nebrodensis into a suspension with water, extract it with petroleum ether, dichloromethane and ethyl acetate in sequence, and concentrate the extract to obtain a petroleum ether extract, a dichloromethane extract and an ethyl acetate extract; The third step is to take the ethyl acetate extract and separate it by silica gel column chromatography with gradient elution to obtain 6 fractions, namely Fr1 to Fr6; In the fourth step, the third fraction Fr3 obtained by gradient elution of silica gel column chromatography was purified and separated by gradient elution of high performance liquid chromatography, and the eluate was collected to obtain indole pleurotus eryngii A at 16.3 minutes.

[0035] Example 3: As an optimization of the above example, in the first step, 8 mL to 12 mL of ethanol is added to every 1 g of the aerial part of Hyssopus officinalis.

[0036] Example 4: As an optimization of the above example, in the first step, the heating temperature is 50°C to 60°C, the number of reflux preparations is 3 times, and the reflux preparation time for each time is 1 hour to 3 hours.

[0037] Example 5: As an optimization of the above example, in the third step, the silica gel column chromatography gradient eluent includes petroleum ether and ethyl acetate, and the volume ratios of petroleum ether and ethyl acetate are 5:1, 2:1, 1.5:1, 1:1, 1:5, and 1:0, respectively.

[0038] Example 6: As an optimization of the above example, in the fourth step, the eluent for the HPLC gradient elution is a mixture of methanol and water, wherein the volume ratio of methanol to water is 15:85.

[0039] Example 7: The preparation method of the indole pleurotus eryngii alkaloid A is carried out according to the following method: The first step is to crush the Pleurotus eryngii fruiting bodies and add 95% by mass ethanol, heat and reflux to prepare, combine the preparation liquid of each reflux, recover under reduced pressure, and concentrate to obtain the Pleurotus eryngii total extract; The second step is to disperse the total extract of Pleurotus nebrodensis into a suspension with water, extract it with petroleum ether, dichloromethane and ethyl acetate in sequence, and concentrate the extract to obtain a petroleum ether extract, a dichloromethane extract and an ethyl acetate extract; The third step is to take the ethyl acetate extract and separate it by silica gel column chromatography with gradient elution to obtain 6 fractions, namely Fr1 to Fr6; In the fourth step, the third fraction Fr3 obtained by gradient elution of silica gel column chromatography was purified and separated by gradient elution of high performance liquid chromatography, and the eluate was collected to obtain indole pleurotus eryngii A at 16.3 minutes.

[0040] Example 8: Use of the indole pleurotus eryngii A in the preparation of a drug for preventing gastric cancer.

[0041] Example 9: Use of the indole pleurotus eryngii A in the preparation of anti-gastric cancer drugs.

[0042] Example 10: The indole pleurotus eryngii alkaloid A was prepared according to the following method: The first step is to crush the Pleurotus eryngii fruiting body and add 95% by mass ethanol, heat and reflux for 3 times at 50° C. to 60° C., each time for 1 hour to 3 hours, combine the reflux preparation liquid each time, recover under reduced pressure, and concentrate to obtain the Pleurotus eryngii total extract; The second step is to disperse the total extract of Pleurotus nebrodensis into a suspension with water, extract it with petroleum ether, dichloromethane and ethyl acetate in sequence, and concentrate the extract to obtain a petroleum ether extract, a dichloromethane extract and an ethyl acetate extract; The third step is to obtain 6 fractions, namely Fr1-Fr6, by using silica gel column chromatography gradient elution to separate the extract of ethyl acetate, wherein the silica gel column chromatography gradient eluent comprises petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 5:1, 2:1, 1.5:1, 1:1, 1:5, and 1:0, respectively; In the fourth step, the third fraction Fr3 was purified and separated by high performance liquid chromatography (YMC-Pack ODS-A, 25°C, 250 mm×10mm, 5μm, 15% methanol) after gradient elution, and the eluate was collected to obtain indole tuoliensine A (i.e., Tuoliensines A) at 16.3 minutes.

[0043] The Tuoliensines A of the present invention was subjected to an in vitro anti-tumor pharmacodynamics experiment, and the in vitro anti-tumor pharmacodynamics experiment utilized the MTT colorimetric method.

[0044] Tuoliensines A was used as the experimental group, Cisplatin was used as the control group, and a blank group was set up. HGC-27 (human gastric cancer cell) cells were selected as the experimental objects in the experimental group, the control group and the blank group. After the culture medium was diluted, 4×10 5The cells were inoculated in a 96-well plate at a density of 100 μL per well. After 24 hours of normal culture in an incubator, the corresponding drugs were added to each group, and the final concentrations of the drugs in each group were 12.5 μg / mL (group 1), 25 μg / mL (group 2), 50 μg / mL (group 3), 100 μg / mL (group 4), and 200 μg / mL (group 5), respectively. There were 5 concentrations in total, and 3 replicates for each concentration. After 48 hours of culture, 10 μL of MTT was added to each well for staining. After another four hours of culture, the original culture medium was discarded, 150 μL of DMSO was added to each well, and the plates were shaken at a low speed on a shaker for 10 min to fully dissolve the crystals. The optical density value was detected at a wavelength of 570 nm using an enzyme-linked immunosorbent assay, and the 50% inhibitory concentration (IC) was calculated based on the optical density value. 50 , μg / mL), where the optical density value is used to calculate IC 50 The calculation method of IC of the experimental group and the control group for HGC-27 cells is known in the art. 50 As shown in Table 2. From the data in Table 2, it can be seen that the Tuoliensines A described in the present invention has a certain inhibitory effect on HGC-27 cells.

[0045] It can be seen from the data in Table 2 that the Tuoliensines A described in the present invention has a certain inhibitory effect on HGC-27 cells.

[0046] In summary, the present invention discloses for the first time the indole pleurotus eryngii alkaloid A, and through in vitro antitumor pharmacodynamics experiments, it is found that it has a certain inhibitory effect on human HGC-27 gastric cancer cells, thereby enabling it to be used as a drug for the preparation of gastric cancer prevention and anti-gastric cancer drugs.

[0047] The above technical features constitute the embodiments of the present invention, which have strong adaptability and implementation effect. Non-essential technical features can be added or reduced according to actual needs to meet the requirements of different situations.

Claims

1. An indole pleurotus eryngii alkaloid A, characterized in that: The chemical structure is 。 2. The indole pleurotus eryngii alkaloid A according to claim 1, characterized in that: Prepared according to the following method: The first step is to crush the Pleurotus eryngii fruiting bodies and add 95% by mass ethanol, heat and reflux to prepare, combine the preparation liquid of each reflux, recover under reduced pressure, and concentrate to obtain the Pleurotus eryngii total extract; The second step is to disperse the total extract of Pleurotus nebrodensis into a suspension with water, extract it with petroleum ether, dichloromethane and ethyl acetate in sequence, and concentrate the extract to obtain a petroleum ether extract, a dichloromethane extract and an ethyl acetate extract; The third step is to take the ethyl acetate extract and separate it by silica gel column chromatography with gradient elution to obtain 6 fractions, namely Fr1 to Fr6; In the fourth step, the third fraction Fr3 obtained by gradient elution of silica gel column chromatography was purified and separated by gradient elution of high performance liquid chromatography, and the eluate was collected to obtain indole pleurotus eryngii A at 16.3 minutes.

3. The indole pleurotus eryngii alkaloid A according to claim 2, characterized in that: In the first step, 8 mL to 12 mL of ethanol was added to every 1 g of Pleurotus nebrodensis fruiting body.

4. The indole pleurotus eryngii alkaloid A according to claim 1, characterized in that: In the first step, the heating temperature is 50° C. to 60° C., the reflux preparation times are 3 times, and the reflux preparation time each time is 1 hour to 3 hours.

5. The indole pleurotus eryngii alkaloid A according to claim 1, characterized in that: In the third step, the gradient eluent of the silica gel column chromatography includes petroleum ether and ethyl acetate, and the volume ratios of petroleum ether to ethyl acetate are 5:1, 2:1, 1.5:1, 1:1, 1:5, and 1:0, respectively.

6. The indole pleurotus eryngii alkaloid A according to claim 5, characterized in that: In the fourth step, the eluent for the HPLC gradient elution is a mixture of methanol and water, wherein the volume ratio of methanol to water is 15:

85.

7. A method for preparing the indole pleurotus eryngii A according to claim 1, characterized in that: Proceed as follows: The first step is to crush the Pleurotus eryngii fruiting bodies and add 95% by mass ethanol, heat and reflux to prepare, combine the preparation liquid of each reflux, recover under reduced pressure, and concentrate to obtain the Pleurotus eryngii total extract; The second step is to disperse the total extract of Pleurotus nebrodensis into a suspension with water, extract it with petroleum ether, dichloromethane and ethyl acetate in sequence, and concentrate the extract to obtain a petroleum ether extract, a dichloromethane extract and an ethyl acetate extract; The third step is to take the ethyl acetate extract and separate it by silica gel column chromatography with gradient elution to obtain 6 fractions, namely Fr1 to Fr6; In the fourth step, the third fraction Fr3 obtained by gradient elution of silica gel column chromatography was purified and separated by gradient elution of high performance liquid chromatography, and the eluate was collected to obtain indole pleurotus eryngii A at 16.3 minutes.

8. The method for preparing the indole pleurotus eryngii A according to claim 7, characterized in that: In the first step, 8 mL to 12 mL of ethanol is added to every 1 g of Pleurotus nebrodensis fruiting body; or / and, in the first step, the heating temperature is 50° C. to 60° C., the reflux preparation times are 3 times, and the reflux preparation time for each time is 1 hour to 3 hours; or / and, in the third step, the silica gel column chromatography gradient eluent includes petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 5:1, 2:1, 1.5:1, 1:1, 1:5, 1:0 respectively; or / and, in the fourth step, the eluent of the high performance liquid chromatography gradient elution is a mixture of methanol and water, wherein the volume ratio of methanol to water is 15:

85.

9. Use of the indole pleurotus eryngii alkaloid A according to any one of claims 1 to 6 in the preparation of a drug for preventing gastric cancer.

10. Use of the indole pleurotus eryngii A according to any one of claims 1 to 6 in the preparation of anti-gastric cancer drugs.

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