Alkaloid components in pleurotus nebroseus and extraction and separation method and application thereof

Abstract

The present application relates to the technical field of separation and purification of Leucopaxillus giganteus, and is a Leucopaxillus giganteus alkaloid component, an extraction and separation method thereof and application. The Leucopaxillus giganteus alkaloid component is disclosed for the first time in the present application. The Leucopaxillus giganteus alkaloid component has a certain inhibitory effect on human HGC-27 (gastric cancer cell) cells, so that the Leucopaxillus giganteus alkaloid component can be used for preparing a drug for preventing gastric cancer or / and an anti-gastric cancer drug.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of Pleurotus tuoliensis separation and purification, and is a Pleurotus tuoliensis alkaloid component and an extraction and separation method and application thereof. BACKGROUND

[0002] Pleurotus tuoliensis belongs to Enmycophyta, Basidiomycetes, Agaricales, Pleurotaceae and Pleurotus, and is a higher fungus. The fruiting body thereof is generally large and pure white. The cap is 5 cm to 15 cm or larger in diameter and 3 cm to 4 cm in thickness. The initial stage is nearly spherical, and is sometimes lateral or eccentric. The surface is nearly smooth or like velvet. Pleurotus tuoliensis is only produced in Xinjiang Moli, Qinghe, Tuoli, Taicheng and Alatay in China, which are deserts and gobi in harsh climate. Related chemical researches show that polysaccharide as a main active ingredient of Pleurotus tuoliensis is widely studied, but other active ingredients are less studied. Alkaloid components as one of main effective components of Pleurotus tuoliensis are found to have effects of resisting stomach cancer and enhancing immunity.

[0003] Malignant tumors seriously threaten human life and health. Since many new anticancer drugs are expensive, long-term application is easy to cause drug resistance and side effects, so it is the key to tumor treatment to find anticancer drugs with high efficiency and low toxicity. Pleurotus tuoliensis is a fungus with medicinal and edible properties, and contains various active alkaloid compounds.

[0004] Therefore, it is of great significance to develop and utilize alkaloid component compounds of Pleurotus tuoliensis, further explore the potential medicinal value thereof, and determine and characterize the structure and physical and chemical properties of the monomer compounds, so as to develop and utilize Pleurotus tuoliensis. SUMMARY

[0005] The present application provides a Pleurotus tuoliensis alkaloid component and an extraction and separation method and application thereof, overcomes the deficiencies of the prior art, and discloses the Pleurotus tuoliensis alkaloid component for the first time, which can be used for preparing a drug for preventing stomach cancer or resisting stomach cancer.

[0006] One of the technical solutions of the present application is achieved by the following measures: a Pleurotus tuoliensis alkaloid component, the chemical structural formula of which is:

[0007] .

[0008] Tuoliensines D is a short name of the Pleurotus tuoliensis alkaloid component.

[0009] The following is a further optimization or / and improvement of one of the above technical solutions of the invention:

[0010] The alkaloid component in the above-mentioned Volvariella volvacea is obtained according to the following steps:

[0011] In the first step, the fruiting body of Volvariella volvacea is crushed and added with ethanol, and after being soaked at room temperature for 3 to 4 hours, it is extracted by heating reflux for 3 times, and the reflux extraction liquid of each time is combined and recovered under reduced pressure and concentrated to obtain Volvariella volvacea extract;

[0012] In the second step, the Volvariella volvacea extract is dispersed with water, and sequentially extracted with petroleum ether, dichloromethane and ethyl acetate to obtain petroleum ether part, dichloromethane part and ethyl acetate part extract;

[0013] In the third step, the ethyl acetate part extract is separated by silica gel column chromatography gradient elution to obtain 6 fractions, namely Fr1 to Fr6, wherein the silica gel column chromatography gradient eluent comprises petroleum ether and ethyl acetate;

[0014] In the fourth step, the third fraction Fr3 obtained from the 6 fractions is further purified and separated by high performance liquid chromatography gradient elution, and the eluate is collected, and the target product, the alkaloid component in Volvariella volvacea, is obtained at 9.66 minutes, wherein the high performance liquid chromatography eluent is a mixture of methanol and water.

[0015] In the first step, 8 to 12 ml of ethanol with a mass fraction of 95% is added per 1 g of Volvariella volvacea fruiting body.

[0016] In the first step, the heating reflux operation temperature is 50 to 60 DEG C, and the reflux time of each time is 1 to 3 hours.

[0017] In the third step, the volume ratio of petroleum ether and ethyl acetate is 5:1, 2:1, 1.5:1, 1:1, 1:5 and 1:0 in turn.

[0018] In the fourth step, the volume ratio of methanol and water is 15:85.

[0019] The second technical solution of the present application is realized by the following measures: a method for extracting and separating the alkaloid component in Volvariella volvacea, which is carried out according to the following steps:

[0020] In the first step, the fruiting body of Volvariella volvacea is crushed and added with ethanol, and after being soaked at room temperature for 3 to 4 hours, it is extracted by heating reflux for 3 times, and the reflux extraction liquid of each time is combined and recovered under reduced pressure and concentrated to obtain Volvariella volvacea extract;

[0021] In the second step, the Volvariella volvacea extract is dispersed with water, and sequentially extracted with petroleum ether, dichloromethane and ethyl acetate to obtain petroleum ether part, dichloromethane part and ethyl acetate part extract;

[0022] The third step involves separating the ethyl acetate fraction using silica gel column chromatography gradient elution to obtain six fractions, namely Fr1 to Fr6, wherein the silica gel column chromatography gradient elution solution includes petroleum ether and ethyl acetate.

[0023] The fourth step involves further purifying and separating the third fraction (Fr3) from the six fractions obtained by high-performance liquid chromatography (HPLC) gradient elution, and collecting the eluent. The alkaloids in the target product, Pleurotus eryngii, are obtained at 9.66 minutes. The HPLC eluent is a mixture of methanol and water.

[0024] The third technical solution of the present invention is achieved through the following measures: the application of alkaloid components in Pleurotus eryngii as a preparation of a drug for preventing gastric cancer.

[0025] The fourth technical solution of the present invention is achieved through the following measures: the application of alkaloid components in Pleurotus eryngii as a preparation of an anti-gastric cancer drug.

[0026] This invention discloses for the first time the alkaloid components in Pleurotus eryngii and conducts in vitro anticancer pharmacodynamic experiments on HGC-27 cells (human gastric cancer cells). The experiments clearly show that the compound has a strong inhibitory effect on HGC-27 cells, thus enabling the compound to be used in the preparation of drugs for the prevention of gastric cancer and / or the preparation of anti-gastric cancer drugs. Attached Figure Description

[0027] Appendix Figure 1 This is a chemical structure diagram of the alkaloid components in Pleurotus eryngii as described in this invention.

[0028] Appendix Figure 2 The alkaloid components in Pleurotus eryngii as described in this invention 1 H-NMR spectrum.

[0029] Appendix Figure 3 The alkaloid components in Pleurotus eryngii as described in this invention 13 C-APT spectrum.

[0030] Appendix Figure 4 The alkaloid components in Pleurotus eryngii as described in this invention 1 H- 1 H COSY spectrum.

[0031] Appendix Figure 5 The alkaloid components in Pleurotus eryngii as described in this invention 1 H- 13 C HSQC spectrum.

[0032] Appendix Figure 6 The alkaloid components in Pleurotus eryngii as described in this invention 1 H- 13 C HMBC spectrum.

[0033] Appendix Figure 7 The alkaloid components in Pleurotus eryngii as described in this invention 1 H- 1 H ROESY spectrum. Detailed Implementation

[0034] This invention is not limited to the following embodiments, and specific implementation methods can be determined according to the technical solution of this invention and the actual situation. Unless otherwise specified, all chemical reagents and chemical products mentioned in this invention are chemical reagents and chemical products that are known and commonly used in the prior art; room temperature in this invention generally refers to a temperature of 15°C to 25°C, and is generally defined as 25°C.

[0035] The present invention will be further described below with reference to embodiments:

[0036] Example 1: The chemical structural formula of the alkaloid components in this white oyster mushroom is as follows:

[0037] .

[0038] Tuoliensines D is an abbreviation for the alkaloid components in Pleurotus eryngii.

[0039] The alkaloid components in the white lingzhi mushroom described in this invention were subjected to 1H NMR spectroscopy. 1 H-NMR, carbon nuclear magnetic resonance (NMR) 13 C-APT) and two-dimensional nuclear magnetic resonance spectra (C-APT) 1 H- 1 H COSY、 1 H- 13 C HSQC, 1 H- 13 C HMBC, 1 H- 1 H ROESY) analysis revealed alkaloid components in Pleurotus eryngii. 1 H-NMR spectrum as shown Figure 2 As shown, the alkaloid D of Pyrrosia lingua 13 C-APT spectrum as shown Figure 3 As shown, the alkaloid components in Pleurotus eryngii 1 H- 1 H COSY spectrum as shown Figure 4 As shown, the alkaloid components in Pleurotus eryngii 1 H- 13 C HSQC spectrum as shown Figure 5 As shown, the alkaloid components in Pleurotus eryngii 1 H- 13 C HMBC spectrum as follows Figure 6 As shown, the alkaloid components in Pleurotus eryngii1 H- 1 H ROESY spectrum as shown Figure 7 As shown, for one of them Figure 2 and Figure 3 Perform spectrum analysis and... Figure 2 and Figure 3 Each peak is assigned a location. Figure 2 and Figure 3 The peak assignments are shown in Table 1. From the data in Table 1, the chemical structural formulas of the alkaloid components in the *Pleurotus eryngii* described in this invention are as follows: Figure 1 As shown, it is readily soluble in methanol.

[0040] Example 2: As an optimization of the above example, the following steps were followed:

[0041] The first step is to crush the fruiting bodies of Pleurotus eryngii and add ethanol, soak them at room temperature for 3 to 4 hours, then heat and reflux to extract three times. Combine the reflux extracts from each extraction, and recover and concentrate them under reduced pressure to obtain Pleurotus eryngii extract.

[0042] The second step is to disperse the extract of Pleurotus eryngii in water and extract it sequentially with petroleum ether, dichloromethane and ethyl acetate to obtain petroleum ether fraction, dichloromethane fraction and ethyl acetate fraction extract.

[0043] The third step involves separating the ethyl acetate fraction using silica gel column chromatography gradient elution to obtain six fractions, namely Fr1 to Fr6, wherein the silica gel column chromatography gradient elution solution includes petroleum ether and ethyl acetate.

[0044] The fourth step involves further purifying and separating the third fraction (Fr3) from the six fractions obtained by high-performance liquid chromatography (HPLC) gradient elution, and collecting the eluent. The alkaloids in the target product, Pleurotus eryngii, are obtained at 9.66 minutes. The HPLC eluent is a mixture of methanol and water.

[0045] In this invention, the chromatographic conditions for high performance liquid chromatography are: YMC-Pack ODS-A, 25℃, 250 mm × 10 mm, 5 μm, 15% methanol.

[0046] Example 3: As an optimization of the above example, in the first step, 8 ml to 12 ml of 95% ethanol is added for every 1 g of Pleurotus eryngii fruiting body.

[0047] Example 4: As an optimization of the above example, in the first step, the temperature of the heating reflux operation is 50°C to 60°C, and the reflux time is 1 hour to 3 hours each time.

[0048] Example 5: As an optimization of the above example, in the third step, 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.

[0049] Example 6: As an optimization of the above-mentioned examples, in the fourth step, the volume ratio of methanol and water is 15:85.

[0050] Example 7: The extraction and separation method of the alkaloid component in the white mushroom is characterized in that the following steps are performed:

[0051] In the first step, the fruiting body of the white mushroom is crushed and added with ethanol, and after being soaked at room temperature for 3 to 4 hours, it is extracted by heating reflux for 3 times. The reflux extraction liquid of each time is combined and recovered and concentrated under reduced pressure to obtain a white mushroom extract;

[0052] In the second step, the white mushroom extract is dispersed with water, and sequentially extracted with petroleum ether, dichloromethane and ethyl acetate to obtain petroleum ether, dichloromethane and ethyl acetate extract;

[0053] In the third step, the ethyl acetate extract is separated by gradient elution with a silica gel column to obtain 6 fractions, namely Fr1 to Fr6, wherein the gradient eluent of the silica gel column chromatography includes petroleum ether and ethyl acetate;

[0054] In the fourth step, the third fraction Fr3 obtained from the 6 fractions is further purified and separated by gradient elution with high performance liquid chromatography, and the eluate is collected to obtain the target product, the alkaloid component in the white mushroom, at 9.66 minutes, wherein the high performance liquid chromatography eluent is a mixture of methanol and water.

[0055] Example 8: The alkaloid component in the white mushroom is used for preparing a drug for preventing gastric cancer.

[0056] Example 9: The alkaloid component in the white mushroom is used for preparing an anti-gastric cancer drug.

[0057] Example 10: The alkaloid component in the white mushroom is obtained by the following method:

[0058] In the first step, the fruiting body of the white mushroom is crushed and added with 95% ethanol, and after being soaked at room temperature for 3 hours, it is extracted by heating reflux for 3 times at 50°C, each for 2 hours. The reflux extraction liquid of each time is combined and recovered and concentrated under reduced pressure to obtain a white mushroom extract;

[0059] In the second step, the white mushroom extract is dispersed with water, and sequentially extracted with petroleum ether, dichloromethane and ethyl acetate to obtain petroleum ether, dichloromethane and ethyl acetate extract;

[0060] In the third step, the ethyl acetate extract is separated by gradient elution with a silica gel column to obtain 6 fractions, namely Fr1 to Fr6, wherein the volume ratio of the gradient eluent of the silica gel column chromatography, petroleum ether and ethyl acetate, is 5:1, 2:1, 1.5:1, 1:1, 1:5 and 1:0, respectively;

[0061] In the fourth step, the third fraction Fr3 of the six fractions was purified and separated by high performance liquid chromatography gradient elution, and the eluent was collected. The alkaloids in the target product, Pleurotus eryngii, were obtained at 9.66 minutes. The volume ratio of methanol to water in the high performance liquid chromatography eluent was 15:85.

[0062] The alkaloids in the white lingzhi mushroom prepared in Example 10 of this invention were subjected to in vitro anti-gastric cancer pharmacodynamic experiments using the MTT colorimetric method.

[0063] Using alkaloids from Pleurotus eryngii as the experimental group and cisplatin as the control group, a blank group was also established. HGC-27 (human gastric cancer cells) were used as the experimental subjects in all three groups. The culture medium was diluted and inoculated at 4 × 10⁻⁶. 5 The culture medium was seeded at a density of 100 μL per well in 96-well plates. After normal incubation for 24 hours, the corresponding drugs were added to each group to achieve final drug concentrations of 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), for a total of 5 concentrations, with 3 replicates per concentration. After 48 hours of incubation, 10 μL of MTT was added to each well for staining. After another 4 hours of incubation, the original culture medium was discarded, and 150 μL of DMSO was added to each well. The plates were shaken at low speed for 10 min to fully dissolve the crystals. The optical density was measured at 570 nm using an ELISA reader, and the 50% inhibitory concentration (IC50) was calculated based on the optical density. 50 (μg / mL), IC50 for optical density value calculation 50 The calculation method is a known existing technique. The IC50 values ​​of the experimental and control groups for HGC-27 cells were compared. 50 As shown in Table 2, the data in Table 2 can be seen that the alkaloids in the white lingzhi mushroom described in this invention have a certain inhibitory effect on HGC-27 cells.

[0064] In summary, this invention discloses for the first time the alkaloid components in Pleurotus eryngii. The alkaloid components in Pleurotus eryngii described in this invention have a certain inhibitory effect on HGC-27 cells, thereby enabling the alkaloid components in Pleurotus eryngii of this invention to be used in the preparation of drugs for the prevention of gastric cancer and / or the preparation of anti-gastric cancer drugs.

[0065] The above technical features constitute various embodiments of the present invention, which have strong adaptability and implementation effect. Unnecessary technical features can be added or removed according to actual needs to meet the needs of different situations.

[0066]

Claims

1. A method for extracting and separating alkaloid components from Clitocybe maxima, characterized in that The following steps are taken: In the first step, the fruiting body of the white fungus is crushed and added with ethanol, and after being soaked at room temperature for 3 to 4 hours, it is extracted by heating reflux for 3 times. The reflux extraction liquid of each time is combined and recovered and concentrated under reduced pressure to obtain a white fungus extract; In the second step, the white fungus extract is dispersed with water, and sequentially extracted with petroleum ether, dichloromethane and ethyl acetate to obtain a petroleum ether part, a dichloromethane part and an ethyl acetate part extract; In the third step, the ethyl acetate part extract is separated by silica gel column chromatography gradient elution to obtain 6 fractions, namely Fr1 to Fr6, wherein the silica gel column chromatography gradient elution liquid comprises petroleum ether and ethyl acetate; In the fourth step, the third fraction Fr3 obtained in the third step is further purified and separated by high performance liquid chromatography gradient elution, and the eluate is collected to obtain the target product white fungus alkaloid component at 9.66 minutes, wherein the high performance liquid chromatography eluent is a mixture of methanol and water, and the chemical structural formula of the target product white fungus alkaloid component is: 。 2. The method according to claim 1, wherein the method comprises the following steps: 1) extracting the alkaloids from the white- gilled mushrooms; 2) separating the alkaloids from the extract; and 3) purifying the alkaloids. In the first step, 8 to 12 ml of ethanol with a mass fraction of 95% is added to 1 g of the fruiting body of the white fungus.

3. The method according to claim 1 or 2, characterized in that In the first step, the heating reflux operation temperature is 50 to 60°C, and the reflux time is 1 to 3 hours each time.

4. The method according to claim 3, wherein the method comprises the following steps: 1) extracting the alkaloids from the white spirit mushroom; 2) separating the alkaloids from the extract; and 3) purifying the alkaloids. In the third step, 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.

5. The method according to claim 1 or 2 or 4, characterized in that In the fourth step, the volume ratio of methanol to water is 15:

85.

6. The white fungus alkaloid component obtained by the extraction and separation method according to any one of claims 1 to 5 as a preparation for preventing gastric cancer.

7. The white fungus alkaloid component obtained by the extraction and separation method according to any one of claims 1 to 5 as a preparation for resisting gastric cancer.

Images