Alkaloid component in pleurotus nebrodensis as well as preparation method and application of alkaloid component

Abstract

The invention relates to the technical field of ferula asafetida endophytic fungus strain separation and purification, in particular to an alkaloid component in pleurotus nebrodensis as well as a preparation method and application thereof, and the alkaloid component in pleurotus nebrodensis, namely an alkaloid compound in pleurotus nebrodensis, is prepared by the following steps: crushing pleurotus nebrodensis sporocarp, adding methanol, heating, refluxing and extracting; dispersing the pleurotus nebrodensis total extract into a suspension with water, and extracting to obtain a petroleum ether part and ethyl acetate part extract; and carrying out gradient elution on the ethyl acetate part extract by silica gel column chromatography, and separating and purifying the third fraction by high performance liquid chromatography to obtain the alkaloid compounds in pleurotus nebrodensis. The invention discloses the alkaloid component in the pleurotus nebrodensis, namely the pleurotus nebrodensis alkali C for the first time, and the pleurotus nebrodensis alkali C has a certain inhibition effect on HGC-27 cells, so that the alkaloid component in the pleurotus nebrodensis, namely the pleurotus nebrodensis alkali C, can be applied to the preparation of tumor prevention drugs and antitumor drugs.

Description

Technical Field

[0001] The invention relates to the technical field of separation and purification of Pleurotus nebrodensis, and is an alkaloid component in Pleurotus nebrodensis and a preparation method thereof and an application thereof in preparing tumor prevention and anti-tumor drugs. The alkaloid component in Pleurotus nebrodensis is an alkaloid compound in Pleurotus nebrodensis, which is recorded as Pleurotus nebrodensis C. Background Art

[0002] Bailing mushroom, Chinese name Bailing Pleurotus tuoliensis, also known as Tianshan Bailingzhi, Asafetida mushroom, Asafetida mushroom, etc., belongs to the Basidiomycetes, Agaricaceae, Pleurotus 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 at the beginning, mostly lateral, sometimes eccentric, and the surface is nearly smooth or velvety. In the wild, it parasitizes or saprophytes 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, Tuoli, 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 there are relatively few studies on other active ingredients. 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. Summary of the invention

[0003] The present invention provides an alkaloid component in Pleurotus nebrodensis and a preparation method thereof and an application thereof in preparing tumor prevention and anti-tumor drugs, which overcomes the deficiencies of the above-mentioned prior art and discloses for the first time an alkaloid component in Pleurotus nebrodensis (pleurotus nebrodensis alkaloid C), so that the alkaloid component can be used in preparing tumor prevention and anti-tumor drugs.

[0004] One of the technical solutions of the present invention is achieved by the following measures: an alkaloid compound in Pleurotus nebrodensis (also known as Pleurotus nebrodensis alkaloid C) has a chemical structural formula of: .

[0005] The following is a further optimization and / or improvement of one of the above-mentioned technical solutions: The alkaloid compounds in the above-mentioned Pleurotus nebrodensis are prepared according to the following steps: The first step is to crush the Pleurotus eryngii fruiting body and add methanol, heat and reflux to extract, and concentrate the extract 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 respectively, 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 perform gradient elution on a silica gel column to obtain six fractions in sequence after separation; The fourth step is to separate and purify the third fraction by high performance liquid chromatography to obtain alkaloid compounds in Pleurotus nebrodensis.

[0006] In the first step, the heating reflux extraction is: heating reflux extraction at 50° C. to 60° C. for 3 times, each time for 1 hour to 3 hours, and combining the extracts from each time.

[0007] In the first step, 8 ml to 12 ml of methanol is added per 1 g of Pleurotus nebrodensis fruiting body.

[0008] In the third step, the eluent for gradient elution is a mixture of dichloromethane and methanol, and the volume ratios of dichloromethane and methanol for gradient elution are 5:1, 2:1, 1.5:1, 1:1, 1:5, and 1:0, respectively.

[0009] In the fourth step, the eluent for HPLC separation is a mixture of methanol and water, the volume ratio of methanol to water is 15:85, and the chromatographic column is YMC-Pack ODS-A, 250 mm×10 mm 5 μm.

[0010] The second technical solution of the present invention is achieved by the following measures: A method for preparing an alkaloid compound (Pleurotus eryngii C) in Pleurotus eryngii is carried out according to the following steps: The first step is to crush the Pleurotus eryngii fruiting body and add methanol, heat and reflux to extract, and concentrate the extract 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 respectively, 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 perform gradient elution on a silica gel column to obtain six fractions in sequence after separation; The fourth step is to separate and purify the third fraction by high performance liquid chromatography to obtain alkaloid compounds in Pleurotus nebrodensis.

[0011] The third technical solution of the present invention is achieved through the following measures: an application of alkaloid compounds in Pleurotus nebrodensis as a drug for preparing tumor prevention.

[0012] The fourth technical solution of the present invention is achieved through the following measures: an application of alkaloid compounds in Pleurotus nebrodensis as a preparation of anti-tumor drugs.

[0013] The present invention discloses for the first time an alkaloid component (pleurotus eryngii alkaloid C) in Pleurotus eryngii, which has a certain inhibitory effect on HGC-27 cells, so that the alkaloid component in Pleurotus eryngii of the present invention can be used for preparing tumor prevention drugs and anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Attached Figure 1 The alkaloid components in Pleurotus eryngii of the present invention 1 H-NMR spectrum.

[0015] Attached Figure 2 The alkaloid components in Pleurotus eryngii of the present invention 13 C-APT spectrum.

[0016] Attached Figure 3 The alkaloid components in Pleurotus eryngii of the present invention 1 H- 1 H COSY spectrum.

[0017] Attached Figure 4 The alkaloid components in Pleurotus eryngii of the present invention 1 H- 13 C HSQC spectrum.

[0018] Attached Figure 5 The alkaloid components in Pleurotus eryngii of the present invention 1 H- 13 C HMBC spectrum.

[0019] Attached Figure 6 The alkaloid components in Pleurotus eryngii of the present invention 1 H- 1 H ROESY spectrum. DETAILED DESCRIPTION

[0020] 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 known and commonly used chemical reagents and chemicals in the prior art; unless otherwise specified, the percentages in the present invention are all mass percentages; the normal temperature and room temperature in the present invention generally refer to the temperature of 15°C to 25°C, and are generally defined as 25°C.

[0021] The present invention will be further described below in conjunction with embodiments: Example 1: The alkaloid compound in Pleurotus nebrodensis has the chemical structural formula: .

[0022] The alkaloid compound in Pleurotus eryngii of the present invention (i.e., the alkaloid component in Pleurotus eryngii) is named as: 4-amino-3-methylfuro[3,4 -c ]pyridazin-7 (5 H )-one; recorded as Tuoliensines C or Tuoliensines C.

[0023] The alkaloid component (Bai Ling Mushroom alkaloid C) in the present invention is 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 HROESY) analysis. 1 H-NMR spectrum Figure 1 As shown, 13 C-APT spectrum Figure 2 As shown, 1 H- 1 H COSY spectrum Figure 3 As shown, 1 H- 13 C HSQC spectrum is shown in Figure 4 As shown, 1 H- 13 C HMBC spectrum Figure 5 As shown, 1 H- 1 H ROESY spectrum is shown in Figure 6 As shown. Figure 1 and Figure 2 Perform graph analysis and Figure 1 and Figure 2 Each peak was assigned, and the peak assignment is shown in Table 1.

[0024] From the data in Table 1, it can be seen that the chemical structural formula of the alkaloid component (Bai Ling Mossine C) in the Pleurotus eryngii of the present invention is as follows, and it is easily soluble in methanol. .

[0025] Example 2: As an optimization of the above example, the alkaloid compound (Pleurotus eryngii alkaloid C) in Pleurotus eryngii was prepared according to the following steps: The first step is to crush the Pleurotus eryngii fruiting body and add methanol, heat and reflux to extract, and concentrate the extract 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 respectively, 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 perform gradient elution on a silica gel column to obtain six fractions in sequence after separation; In the fourth step, the third fraction is separated and purified by high performance liquid chromatography to obtain the alkaloid compounds in Pleurotus nebrodensis (Pleurotus nebrodensis alkaloids C).

[0026] Example 3: As an optimization of the above example, in the first step, the heating reflux extraction is: heating reflux extraction at 50°C to 60°C for 3 times, each time for 1 hour to 3 hours, and combining the extracts from each time.

[0027] Example 4: As an optimization of the above example, in the first step, 8 ml to 12 ml of methanol is added to every 1 g of Pleurotus nebrodensis fruiting body.

[0028] Example 5: As an optimization of the above example, in the third step, the eluent for gradient elution is a mixture of dichloromethane and methanol, and the volume ratios of dichloromethane and methanol for gradient elution are 5:1, 2:1, 1.5:1, 1:1, 1:5, and 1:0, respectively.

[0029] Example 6: As an optimization of the above example, in the fourth step, the eluent for HPLC separation is a mixture of methanol and water, the volume ratio of methanol to water is 15:85, and the chromatographic column is YMC-Pack ODS-A, 250mm×10mm 5μm. At a temperature of 25°C, after HPLC elution and separation, the alkaloid compound (Bai Ling Mushroom alkaloid C) in Pleurotus nebrodensis was obtained at 49.9 minutes.

[0030] Example 7: Application of the alkaloid compound (pleurotus eryngii alkaloid C) in Pleurotus nebrodensis as a drug for preparing tumor prevention.

[0031] Example 8: Application of the alkaloid compound (pleurotus eryngii alkaloid C) in Pleurotus nebrodensis as an anti-tumor drug.

[0032] Example 9: The alkaloid compound (Pleurotus eryngii alkaloid C) in Pleurotus eryngii is obtained according to the following method: The first step is to crush the Pleurotus eryngii fruiting body and add methanol, heat and reflux extraction at 55°C for 3 times, each time for 2.5 hours, combine the reflux extracts of each time and recover and concentrate under reduced pressure to obtain the Pleurotus eryngii total extract, and add 10 ml of methanol to every 1 g of Pleurotus eryngii fruiting body.

[0033] 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 gradient elution separation of the ethyl acetate extract using silica gel column chromatography, 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 in Fr1-Fr6 was purified and separated by high performance liquid chromatography, and the eluate was collected; the high performance liquid chromatography eluent was a mixture of methanol and water, the volume ratio of methanol to water was 15:85, the chromatographic column was YMC-Pack ODS-A, 250mm×10mm 5μm, the temperature was 25°C, and the alkaloid compound in Pleurotus nebrodensis (Pleurotus nebrodensis C) was obtained at 49.9 minutes.

[0034] Example 10: The alkaloid compound (Pleurotus eryngii alkaloid C) in Pleurotus eryngii is obtained according to the following method: The first step is to crush the Pleurotus eryngii fruiting body and add methanol, heat and reflux extraction at 60°C for 3 times, each time for 2 hours, combine the reflux extracts of each time and recover and concentrate under reduced pressure to obtain the total extract of Pleurotus eryngii, and add 9 ml of methanol to every 1 g of Pleurotus eryngii fruiting body.

[0035] 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 gradient elution separation of the ethyl acetate extract using silica gel column chromatography, 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 in Fr1-Fr6 was purified and separated by HPLC gradient elution, and the eluate was collected; the HPLC eluent was a mixture of methanol and water, the volume ratio of methanol to water was 15:85, the chromatographic column was YMC-Pack ODS-A, 250mm×10mm 5μm, the temperature was 25°C, and the alkaloid compound (pleurotus eryngii alkaloid C) in Pleurotus nebrodensis was obtained at 49.9 minutes.

[0036] Example 11: The alkaloid compound (pleurotus nebrodensis alkaloid C) in the present invention was subjected to an in vitro antitumor pharmacodynamics experiment, and the in vitro antitumor pharmacodynamics experiment adopted the MTT colorimetric method.

[0037] The alkaloid compound (Bai Ling Mushroom C) in Pleurotus eryngii was used as the experimental group, Cisplatin (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 density of the cells was inoculated in a 96-well plate, with 100 μL per well. After normal culture in the incubator for 24 hours, the corresponding drugs were added to each group, so that 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 culture for 48 hours, 10 μL of MTT was added to each well for staining. After further culture for four hours, the original culture medium was discarded, 150 μL of DMSO was added to each well, and the plates were shaken at a low speed 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 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 pleurotus eryngii alkaloid C described in the present invention has a certain inhibitory effect on HGC-27 cells.

[0038] In summary, the present invention discloses for the first time an alkaloid component pleurotus eryngii, pleurotus eryngii alkaloid component C in Pleurotus eryngii. The alkaloid component pleurotus eryngii C in the present invention has a certain inhibitory effect on HGC-27 cells, so that the alkaloid component in Pleurotus eryngii of the present invention can be used for preparing tumor prevention drugs and anti-tumor drugs.

[0039] 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.

Claims

1. An alkaloid compound in Pleurotus eryngii, characterized in that The chemical structure is: 。 2. The alkaloid compound 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 methanol, heat and reflux to extract, and concentrate the extract 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 respectively, 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 perform gradient elution on a silica gel column to obtain six fractions in sequence after separation; The fourth step is to separate and purify the third fraction by high performance liquid chromatography to obtain alkaloid compounds in Pleurotus nebrodensis.

3. The alkaloid compound in Pleurotus nebrodensis according to claim 2, characterized in that In the first step, the heating reflux extraction is: heating reflux extraction at 50° C. to 60° C. for 3 times, each time for 1 hour to 3 hours, and combining the extracts from each time.

4. The alkaloid compound in Pleurotus nebrodensis according to claim 2 or 3, characterized in that In the first step, 8 ml to 12 ml of methanol was added per 1 g of Pleurotus nebrodensis fruiting body.

5. The alkaloid compound in Pleurotus nebrodensis according to any one of claims 2 to 4, characterized in that In the third step, the eluent for gradient elution is a mixture of dichloromethane and methanol, and the volume ratios of dichloromethane and methanol for gradient elution are 5:1, 2:1, 1.5:1, 1:1, 1:5, and 1:0, respectively.

6. The alkaloid compound in Pleurotus nebrodensis according to any one of claims 2 to 5, characterized in that In the fourth step, the eluent for HPLC separation is a mixture of methanol and water, the volume ratio of methanol to water is 15:85, and the chromatographic column is YMC-Pack ODS-A, 250 mm×10 mm 5 μm.

7. A method for preparing alkaloid compounds from Pleurotus nebrodensis according to any one of claims 1 to 6, characterized in that Follow these steps: The first step is to crush the Pleurotus eryngii fruiting body and add methanol, heat and reflux to extract, and concentrate the extract 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 respectively, 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 perform gradient elution on a silica gel column to obtain six fractions in sequence after separation; The fourth step is to separate and purify the third fraction by high performance liquid chromatography to obtain alkaloid compounds in Pleurotus nebrodensis.

8. Use of the alkaloid compound in Pleurotus nebrodensis according to any one of claims 1 to 6 as a drug for preparing tumor prevention.

9. Use of the alkaloid compound in Pleurotus nebrodensis according to any one of claims 1 to 6 as an anti-tumor drug.

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