Indole alkaloid in pleurotus nebrodensis and preparation method and application thereof

Abstract

The invention relates to the technical field of pleurotus nebrodensis separation and purification, in particular to indole alkaloid in pleurotus nebrodensis and a preparation method and application thereof.The indole alkaloid in pleurotus nebrodensis is prepared by the steps that pleurotus nebrodensis sporocarp is used as a purification and separation object, and dichloromethane part extract is obtained through reflux extraction and extraction; and purifying and separating the dichloromethane part extract by adopting silica gel column chromatography, gel column chromatography and high performance liquid chromatography to obtain the indole alkaloids in the pleurotus nebrodensis. The invention discloses the indole alkaloid in the pleurotus nebrodensis for the first time, and the indole alkaloid in the pleurotus nebrodensis disclosed by the invention has a certain inhibition effect on HGC-27 cells, so that the indole alkaloid in the pleurotus nebrodensis can be applied to preparation of drugs for preventing and resisting gastric cancer.

Description

Technical Field

[0001] The invention relates to the technical field of separation and purification of Pleurotus nebrodensis, and is an indole alkaloid in Pleurotus nebrodensis and a preparation method thereof, and also comprises application of the indole alkaloid in Pleurotus nebrodensis in preparing a drug for preventing gastric cancer or / and resisting gastric cancer. Background Art

[0002] Bailing mushroom, Chinese name Bailing Pleurotus tuoliensis, also known as Tianshan Bailingzhi, Asafetida mushroom, Asafetida mushroom, etc., belongs to the phylum Basidiomycetes, class Agaricaceae, order Agaricaceae, genus Pleurotus, and is a higher fungus. 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 flat spherical in the early stage, mostly lateral, sometimes eccentric, and the surface is nearly smooth or velvety. It is parasitic or saprophytic in the wild on the hypertrophic rhizome of the traditional Chinese medicine Asafetida. In my country, it is only produced in the desert Gobi with harsh climates such as Mulei, Qinghe, Toli, Tacheng, and Altay in Xinjiang. Relevant chemical studies have shown that polysaccharides as the main active ingredient of Bailing mushroom have been widely studied, but other active ingredients have been less studied. Alkaloid components, as one of the main active ingredients of Bailing mushroom, have been found to have multiple effects such as anti-tumor and immunity enhancement. 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.

[0003] Patent document with publication number CN112142667A discloses an alkaloid compound, a preparation method and use thereof, which uses Radix Bletillae Sinensis as an extraction raw material to extract two bisbenzylisoquinoline alkaloids and four morphinane-type alkaloids, and systematically evaluates their application in the prevention or treatment of neurodegenerative diseases and cancer drugs.

[0004] Jia Yuemei et al. (Study on the chemical components of Pleurotus eryngii and its anti-cervical cancer cell activity [J]. Natural Product Research and Development, 2023 (35) 5, 2088-2093) used ODS, silica gel column chromatography, semi-preparative high performance liquid chromatography and other methods to separate and purify it, combined with modern spectral technology and physical and chemical properties to identify the compound structure, and isolated 11 compounds from Pleurotus eryngii extract, which were identified as sickle pyrone A (1), 5-hydroxydihydrosickle pyrone C (2), 3-methyl ether sickle pyrone (3), 2-acetonyl-3-methyl-7-me-thoxynaphthazarin (4), (-)-cyclonerodiol (5), 2-isopropanol-3-methyl-7-methoxy-naphthazar in(6), 1,2-dihydroxymentholactone(7), 6,7-dihydroxy-3,6-dimethyl-2-isovaleroyl-4,5,6,7-tetrahydrobenzofuran(8), dihydro-3-exopleurolide(9), 5-hydroxymethylfurfural(10), 6,7-dihydroxy-3,6-dimethyl-2-isovaleroyl-4,5,6,7-tetrahydrobenzofuran(11). All compounds were isolated from Pleurotus eryngii for the first time. The obtained compounds were screened for their anti-cervical cancer cell Hela activity. Except for compounds 5, 9 and 10, which had weaker activities, the other compounds showed strong anti-cervical cancer cell activity. Among them, compound 3 had the strongest activity, IC 50 It is 3.78 μmol / L, which is worthy of further development. Summary of the invention

[0005] The present invention provides an indole alkaloid in Pleurotus eryngii and a preparation method and application thereof. The present invention discloses for the first time the indole alkaloid in Pleurotus eryngii and discloses for the first time the application of the indole alkaloid in Pleurotus eryngii in the preparation of a gastric cancer prevention drug and a gastric cancer resistance drug, thereby providing a new drug for the prevention and treatment of gastric cancer.

[0006] One of the technical solutions of the present invention is achieved by the following measures: an indole alkaloid (i.e. Tuoliensines B) in Pleurotus eryngii has a chemical structural formula of: .

[0007] The following is a further optimization and / or improvement of one of the above-mentioned technical solutions: The indole alkaloids in Pleurotus nebrodensis are prepared according to the following steps: The first step is to crush the Pleurotus eryngii fruiting body and add ethanol, and heat and reflux extract at 50° C. to 60° C. for 3 times, each time for 1 hour to 3 hours, and combine the reflux extracts of each time, recover and concentrate under reduced pressure to obtain Pleurotus eryngii extract; The second step is to disperse the total extract of Pleurotus nebrodensis into a suspension with water, extract it with petroleum ether and dichloromethane in sequence, and concentrate the extract to obtain a petroleum ether extract and a dichloromethane extract; The third step is to obtain 6 fractions by gradient elution separation of the dichloromethane extract by silica gel column chromatography, wherein the silica gel column chromatography gradient eluent includes petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 50:1, 20:1, 10:1, 5:1, 2:1, and 1:1, respectively; In the fourth step, the sixth fraction Fr6 was separated by gel column chromatography, and then purified and separated by high performance liquid chromatography gradient elution, and the eluate was collected to obtain indole alkaloids in Pleurotus nebrodensis at 17.2 minutes.

[0008] The gel column chromatography may adopt Sephadex LH-20 gel column chromatography.

[0009] In the first step, 8 ml to 12 ml of 95% ethanol is added to every 1 g of Pleurotus nebrodensis fruiting body.

[0010] In the fourth step, the HPLC eluent is a mixture of methanol and water, wherein the volume ratio of methanol to water is 50:50.

[0011] The second technical solution of the present invention is achieved by the following measures: A method for preparing indole alkaloids from Pleurotus eryngii, comprising the following steps: The first step is to crush the Pleurotus eryngii fruiting body and add ethanol, and heat and reflux extract at 50° C. to 60° C. for 3 times, each time for 1 hour to 3 hours, and combine the reflux extracts of each time, recover and concentrate under reduced pressure to obtain Pleurotus eryngii extract; The second step is to disperse the total extract of Pleurotus nebrodensis into a suspension with water, extract it with petroleum ether and dichloromethane in sequence, and concentrate the extract to obtain a petroleum ether extract and a dichloromethane extract; The third step is to obtain 6 fractions by gradient elution separation of the dichloromethane extract by silica gel column chromatography, wherein the silica gel column chromatography gradient eluent includes petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 50:1, 20:1, 10:1, 5:1, 2:1, and 1:1, respectively; In the fourth step, the sixth fraction Fr6 was separated by Sephadex LH-20 gel column chromatography and then purified by high performance liquid chromatography gradient elution, and the eluate was collected to obtain indole alkaloids in Pleurotus nebrodensis at 17.2 minutes.

[0012] The following is a further optimization and / or improvement of the second technical solution of the above invention: In the first step, 8 ml to 12 ml of 95% ethanol is added to every 1 g of Pleurotus nebrodensis fruiting body.

[0013] In the fourth step, the HPLC eluent is a mixture of methanol and water, wherein the volume ratio of methanol to water is 50:50.

[0014] The third technical solution of the present invention is achieved through the following measures: the use of the indole alkaloids in Pleurotus eryngii described in one of the technical solutions as a drug for the prevention of gastric cancer or / and resistance to gastric cancer.

[0015] The fourth technical solution of the present invention is achieved by the following measures: the use of the indole alkaloids in Pleurotus eryngii described in one of the technical solutions as a drug for the prevention of human gastric cancer or / and resistance to human gastric cancer.

[0016] The present invention discloses for the first time indole alkaloids in Pleurotus eryngii. The indole alkaloids in Pleurotus eryngii described in the present invention have a certain inhibitory effect on HGC-27 cells, so that the indole alkaloids in Pleurotus eryngii described in the present invention can be used for preparing gastric cancer prevention drugs and gastric cancer resistance drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Attached Figure 1 The figure is the chemical structure diagram of Tuoliensines B described in the present invention.

[0018] Attached Figure 2 Tuoliensines B of the present invention 1 H-NMR spectrum.

[0019] Attached Figure 3 Tuoliensines B of the present invention 13 C-APT spectrum.

[0020] Attached Figure 4 Tuoliensines B of the present invention 1 H- 1 H COSY spectrum.

[0021] Attached Figure 5 Tuoliensines B of the present invention 1 H- 13 C HSQC spectrum.

[0022] Attached Figure 6 Tuoliensines B of the present invention 1 H- 13 C HMBC spectrum.

[0023] Attached Figure 7 Tuoliensines B of the present invention 1 H- 1 H ROESY spectrum. DETAILED DESCRIPTION

[0024] The present invention is not limited by the following embodiments, and specific implementation methods can be determined based on the technical solution of the present invention and actual conditions.

[0025] Unless otherwise specified, the various chemical reagents and chemicals mentioned in the present invention are all chemical reagents and chemicals commonly known in the prior art; 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 unless otherwise specified, for example, hydrochloric acid solution is an aqueous hydrochloric acid 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.

[0026] The present invention will be further described below in conjunction with embodiments: Example 1: A Tuoliensines B, whose chemical structural formula is: .

[0027] Example 2: The Tuoliensines B was prepared according to the following steps: The first step is to crush the Pleurotus eryngii fruiting body and add ethanol, and heat and reflux extract at 50° C. to 60° C. for 3 times, each time for 1 hour to 3 hours, and combine the reflux extracts of each time, recover and concentrate under reduced pressure to obtain Pleurotus eryngii extract; The second step is to disperse the total extract of Pleurotus nebrodensis into a suspension with water, extract it with petroleum ether and dichloromethane in sequence, and concentrate the extract to obtain a petroleum ether extract and a dichloromethane extract; The third step is to obtain 6 fractions (i.e., Fr1-Fr6) by gradient elution of the extract of the dichloromethane part by silica gel column chromatography, wherein the gradient eluent of the silica gel column chromatography includes petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 50:1, 20:1, 10:1, 5:1, 2:1, and 1:1, respectively; In the fourth step, the sixth fraction Fr6 was separated by Sephadex LH-20 gel column chromatography and then purified by high performance liquid chromatography gradient elution, and the eluate was collected to obtain Tuoliensines B at 17.2 minutes.

[0028] Example 3: As an optimization of the above Example 2, in the first step, 8 ml to 12 ml of 95% ethanol is added per 1 g of Pleurotus nebrodensis fruiting body.

[0029] Example 4: As an optimization of the above-mentioned Example 2, in the fourth step, the HPLC eluent is a mixture of methanol and water, wherein the volume ratio of methanol to water is 50:50.

[0030] Example 5: Use of the indole alkaloids in Pleurotus nebrodensis described in the above examples as a drug for the prevention and / or resistance of gastric cancer.

[0031] Example 6: Use of the indole alkaloids in Pleurotus nebrodensis described in the above examples as a drug for the prevention and / or resistance of human gastric cancer.

[0032] Example 7: The Tuoliensines B was prepared according to the following steps: The first step is to crush the Pleurotus eryngii fruiting body and add 95% ethanol, add 8ml to 12ml of 95% ethanol to every 1g of Pleurotus eryngii fruiting body, heat and reflux for extraction 3 times at 50°C to 60°C, each time for 1 hour to 3 hours, combine the reflux extracts of each time, recover under reduced pressure, and concentrate to obtain Pleurotus eryngii extract; the second step is to disperse the total extract of Pleurotus eryngii into a suspension with water, extract with petroleum ether and dichloromethane in turn, and concentrate the extract to obtain a petroleum ether extract and a dichloromethane extract; the third step is to take the dichloromethane extract and separate it by gradient elution with silica gel column chromatography to obtain 6 fractions (i.e., Fr1-Fr6), wherein the silica gel column chromatography gradient eluent includes petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 50:1, 20:1, 10:1, 5:1, 2:1, and 1:1 in turn; the fourth step is to separate the sixth fraction Fr6 by Sephadex LH-20 Gel column chromatography separation, and then purification and separation after high performance liquid chromatography gradient elution, and collection of the eluate, Tuoliensines B was obtained at 17.2 minutes; Wherein, in the fourth step, when Sephadex LH-20 gel column chromatography is used for separation, methanol is used as the eluent; the eluent of the high performance liquid chromatography is a mixture of methanol and water, and the volume ratio of methanol to water is 50:50.

[0033] The chemical structural formula of Tuoliensines B obtained in Example 7 is as follows Figure 1 shown.

[0034] Example 8: The Tuoliensines B was prepared according to the following steps: The first step is to crush the Pleurotus eryngii fruiting body and add 95% ethanol, add 8 ml of 95% ethanol to each 1g of Pleurotus eryngii fruiting body, heat and reflux for extraction 3 times at 60°C, each time for 1 hour, combine the reflux extracts of each time, recover under reduced pressure, and concentrate to obtain Pleurotus eryngii extract; the second step is to disperse the total extract of Pleurotus eryngii into a suspension with water, extract it with petroleum ether and dichloromethane in turn, and concentrate the extract to obtain a petroleum ether extract and a dichloromethane extract; the third step is to take the dichloromethane extract and separate it by gradient elution with silica gel column chromatography to obtain 6 fractions (i.e., Fr1-Fr6), wherein the silica gel column chromatography gradient eluent includes petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 50:1, 20:1, 10:1, 5:1, 2:1, and 1:1 in turn; the fourth step is to separate the sixth fraction Fr6 by Sephadex LH-20 gel column chromatography, and then by high performance liquid chromatography ((YMC-Pack ODS-A, 25°C, 250 mm×10 mm, 5μm)) was used for gradient elution and the eluate was collected to obtain Tuoliensines B at 17.2 minutes; Wherein, in the fourth step, when Sephadex LH-20 gel column chromatography is used for separation, methanol is used as the eluent, and the high performance liquid chromatography eluent is a mixture of methanol and water, and the volume ratio of methanol to water is 50:50.

[0035] The Tuoliensines B described in Example 8 was subjected to H NMR spectroscopy ( 1 H-NMR), carbon nuclear magnetic resonance spectroscopy ( 13 C-APT) and NMR 2D spectra ( 1 H- 1 H COSY, 1 H- 13 C HSQC, 1 H- 13 C HMBC, 1 H- 1 H ROESY) analysis.

[0036] Tuoliensines B described in Example 8 1 H-NMR spectrum Figure 2 As shown, the Tuoliensines B described in Example 8 13 C-APT spectrum Figure 3 As shown, the Tuoliensines B described in Example 8 1 H- 1 H COSY spectrum Figure 4 As shown, the Tuoliensines B described in Example 8 1 H-13 C HSQC spectrum is shown in Figure 5 As shown, the Tuoliensines B described in Example 8 1 H- 13 C HMBC spectrum Figure 6 As shown, the Tuoliensines B described in Example 8 1 H- 1 HROESY spectrum 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 assignments are shown in Table 1. From the data in Table 1, it can be seen that the chemical structure of Tuoliensines B described in Example 8 is as follows Figure 1 As shown, it is easily soluble in methanol.

[0037] The Tuoliensines B described in 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.

[0038] Experimental methods: Tuoliensines B 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 5 The 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 minutes 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), optical density value was used to calculate IC 50 The calculation method of is a known technology.

[0039] Experimental results: IC of experimental group and control group on HGC-27 cells 50 As shown in Table 2.

[0040] It can be seen from the data in Table 2 that Tuoliensines B described in the present invention has a certain inhibitory effect on HGC-27 cells.

[0041] In summary, the present invention discloses Tuoliensines B for the first time. The Tuoliensines B of the present invention has a certain inhibitory effect on HGC-27 cells, so that the Tuoliensines B of the present invention can be used for preparing gastric cancer prevention drugs and anti-gastric cancer drugs.

[0042] The above technical features respectively constitute various 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.

[0043]

Claims

1. An indole alkaloid in Pleurotus nebrodensis, characterized in that: Its chemical structure is: 。 2. The indole alkaloids in Pleurotus nebrodensis according to claim 1, characterized in that: Prepared according to the following steps: The first step is to crush the Pleurotus eryngii fruiting body and add ethanol, heat and reflux to extract, combine the reflux extracts of each time, recover and concentrate under reduced pressure to obtain Pleurotus eryngii extract; The second step is to disperse the total extract of Pleurotus nebrodensis into a suspension with water, extract it with petroleum ether and dichloromethane in sequence, and concentrate the extract to obtain a petroleum ether extract and a dichloromethane extract; The third step is to obtain 6 fractions by gradient elution separation of the dichloromethane extract by silica gel column chromatography, wherein the silica gel column chromatography gradient eluent includes petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 50:1, 20:1, 10:1, 5:1, 2:1, and 1:1, respectively; In the fourth step, the sixth fraction was separated by gel column chromatography, and then purified and separated by high performance liquid chromatography gradient elution, and the eluate was collected to obtain indole alkaloids in Pleurotus nebrodensis at 17.2 minutes.

3. The indole alkaloids in Pleurotus nebrodensis according to claim 2, characterized in that: In the first step, 8 ml to 12 ml of 95% ethanol is added per 1 g of Pleurotus nebrodensis fruiting body.

4. The indole alkaloids in Pleurotus nebrodensis according to claim 2 or 3, characterized in that: In the first step, the temperature of the heating reflux extraction is 50° C. to 60° C., and the heating reflux extraction is performed at least 3 times, each time for 1 hour to 3 hours.

5. The indole alkaloids in Pleurotus nebrodensis according to claim 2 or 3, characterized in that: In the fourth step, the HPLC eluent is a mixture of methanol and water, wherein the volume ratio of methanol to water is 50:

50.

6. The indole alkaloids in Pleurotus nebrodensis according to claim 4, characterized in that: In the fourth step, the HPLC eluent is a mixture of methanol and water, wherein the volume ratio of methanol to water is 50:

50.

7. A method for preparing indole alkaloids from Pleurotus nebrodensis according to claim 1, 3, 4, 5 or 6, characterized in that: Follow the steps below: The first step is to crush the Pleurotus eryngii fruiting body and add ethanol, heat and reflux to extract, combine the reflux extracts of each time, recover and concentrate under reduced pressure to obtain Pleurotus eryngii extract; The second step is to disperse the total extract of Pleurotus nebrodensis into a suspension with water, extract it with petroleum ether and dichloromethane in sequence, and concentrate the extract to obtain a petroleum ether extract and a dichloromethane extract; The third step is to obtain 6 fractions by gradient elution separation of the dichloromethane extract by silica gel column chromatography, wherein the silica gel column chromatography gradient eluent includes petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 50:1, 20:1, 10:1, 5:1, 2:1, and 1:1, respectively; In the fourth step, the sixth fraction was separated by gel column chromatography, and then purified and separated by high performance liquid chromatography gradient elution, and the eluate was collected to obtain indole alkaloids in Pleurotus nebrodensis at 17.2 minutes.

8. Use of the indole alkaloids in Pleurotus nebrodensis according to any one of claims 1 to 6 as a drug for preventing or / and resisting gastric cancer.

9. Use of the indole alkaloids in Pleurotus nebrodensis according to any one of claims 1 to 6 as a drug for preventing or / and resisting human gastric cancer.

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