阅读说明：本技术 从艳山姜叶中提取二氢醉胡椒素的方法 (Method for extracting dihydromethysticin from alpinia japonica leaves ) 是由 张嫩玲 游华林 沈祥春 于 2021-09-24 设计创作，主要内容包括：本发明公开了从艳山姜叶中提取二氢醉胡椒素的方法,属于天然产物提取技术领域。本发明所述二氢醉胡椒素的提取方法主要包括醇提、萃取、结晶以及重结晶等步骤。本发明的二氢醉胡椒素的提取方法,操作简便、快速,提取分离过程中主要采用重结晶法,工艺方法简单环保,且由该方法获得的二氢醉胡椒素,其纯度高于90％。此外,通过本发明所述的提取方法,可使二氢醉胡椒素的提取率达到0.88％,远高于现有技术中的其它各种提取方法。(The invention discloses a method for extracting dihydromethysticin from alpinia japonica leaves, belonging to the technical field of natural product extraction. The extraction method of the dihydromethysticin mainly comprises the steps of alcohol extraction, crystallization, recrystallization and the like. The extraction method of the dihydromethysticin is simple and rapid to operate, a recrystallization method is mainly adopted in the extraction and separation process, the process method is simple and environment-friendly, and the purity of the dihydromethysticin obtained by the method is higher than 90%. In addition, the extraction method of the invention can lead the extraction rate of the dihydromethysticin to reach 0.88 percent, which is much higher than other extraction methods in the prior art.)

1. The method for extracting dihydromethysticin from the alpinia japonica leaves is characterized by comprising the following steps:

drying and crushing the alpinia japonica leaves, performing reflux extraction on the crushed alpinia japonica leaves, filtering and removing slag to obtain an extracting solution, concentrating the extracting solution to a certain volume under reduced pressure, removing pigment to obtain a concentrated solution after the pigment is removed, and recovering a solvent in the concentrated solution to obtain an extract A; adding an extracting agent into the extract A for extraction, collecting extract liquor, and concentrating to obtain an extract B; adding a mixed solution of ethyl acetate and methanol or a mixed solution of chloroform and methanol into the extract B until the extract B is completely dissolved, standing for crystallization, filtering to obtain a crude crystal A, and concentrating the filtrate to obtain an extract C; adding a mixed solution of dichloromethane and methanol into the extract C until the extract C is completely dissolved, standing for crystallization, and filtering to obtain coarse crystals B; and combining the crude crystals A and B, and recrystallizing to obtain the dihydromethysticin.

2. The extraction method according to claim 1, wherein the solvent used for reflux extraction is one or more of ethanol, methanol or chloroform.

3. The extraction method according to claim 1, wherein the conditions of the reflux extraction are: the extraction temperature is 76-85 ℃, and the extraction time is 110-150 min.

4. The extraction method according to claim 1, wherein the pigment is removed with activated carbon.

5. The extraction method of claim 1, wherein the extractant is one of ethyl acetate or chloroform.

6. The extraction method according to claim 1, wherein in the mixed solution of ethyl acetate and methanol, the volume ratio of ethyl acetate to methanol is 9: 1-1: 1; in the mixed solution of chloroform and methanol, the volume ratio of chloroform to methanol is 1: 1-7: 3.

7. The extraction method according to claim 1, wherein the crystals are washed with methanol during the filtration to obtain the crude crystals A and B.

8. The extraction method according to claim 1, wherein the volume ratio of dichloromethane to methanol in the mixed solution of dichloromethane and methanol is 6:4 to 9: 1.

9. The extraction method according to claim 1, wherein the recrystallization is performed using chloroform or methanol.

10. The extraction method according to claim 9, wherein the recrystallization with chloroform or methanol is performed under conditions in which chloroform or methanol is added to the crude crystals, the crude crystals are dissolved in an environment of 40 to 50 ℃, and then the recrystallization is performed at room temperature.

Technical Field

The invention belongs to the technical field of natural product extraction, and particularly relates to a method for extracting dihydromethysticin from alpinia japonica leaves.

Background

Alpinia zerumbet (Pers.) burtt. et Smith is a perennial herb of Alpinia genus of zingiberaceae family, and has a wide resource distribution. The alpinia zerumbet is pungent, astringent and warm, has the effects of promoting qi circulation, relieving pain, warming middle energizer, eliminating dampness, strengthening spleen and warming stomach, and is used for treating symptoms such as abdominal psychroalgia, dyspepsia, chest and abdomen distending pain, phlegm-dampness stagnation, vomiting, diarrhea, cough and the like.

Modern pharmacological research shows that the alpinia zerumbet has good effects on the cardiovascular system, the digestive system, the blood system and the like. The chemical components contained in the alpinia zerumbet medicinal material provide a certain medicinal material basis for the pharmacological activity of the alpinia zerumbet medicinal material, at present, researchers concentrate on the research on the chemical components and the pharmacological activity of medicinal parts in the alpinia zerumbet, and the research shows that the alpinia zerumbet medicinal material mainly contains compounds such as volatile oils, flavonoids, diterpenoids, organic acids and the like, and more than 100 chemical components are determined at present and comprise dihydromethysticine.

Dihydromethysticin (DDK) with molecular formula of C₁₄H₁₄O₃Has neuroprotective, anti-obesity, antiviral, and anti-cell proliferation effects. The structural formula of dihydromethysticin is as follows:

at present, the method for separating dihydromethysticin from the alpinia zerumbet leaves is developed based on the activity of the dihydromethysticin, and the obtained amount is small, so that no suitable extraction and separation method for obtaining a large amount of the compound exists. In addition, the existing method for separating the dihydromethysticin is mainly a column chromatography method, and the method is complicated, time-consuming and material-consuming, and is not beneficial to production and development. Therefore, optimization of the extraction method of dihydromethysticin is an urgent problem to be solved.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a method for extracting and separating dihydromethysticin from alpinia japonica leaves, which is simple, convenient, rapid and environment-friendly, and the purity of the separated compound is high.

The technical scheme of the invention is as follows:

the method for extracting dihydromethysticin from the alpinia japonica leaves comprises the following steps:

drying and crushing the alpinia japonica leaves, performing reflux extraction on the crushed alpinia japonica leaves, filtering and removing slag to obtain an extracting solution, concentrating the extracting solution to a certain volume under reduced pressure, removing pigment to obtain a concentrated solution after the pigment is removed, and recovering a solvent in the concentrated solution to obtain an extract A. And adding an extracting agent into the extract A for extraction, collecting extract liquor, and concentrating to obtain an extract B. And adding a mixed solution of ethyl acetate and methanol or a mixed solution of chloroform and methanol into the extract B until the extract B is completely dissolved, standing for crystallization, filtering to obtain a crude crystal A, and concentrating the filtrate to obtain an extract C. And adding a mixed solution of dichloromethane and methanol into the extract C until the extract C is completely dissolved, standing for crystallization, and filtering to obtain coarse crystals B. And combining the crude crystals A and B, and recrystallizing to obtain the dihydromethysticin.

In the above extraction method, the solvent used for reflux extraction is selected from one or more of ethanol, methanol or chloroform. Preferably, the reflux extraction solvent is selected from ethanol, and further preferably 95% ethanol.

In the extraction method, the material-to-liquid ratio (g/mL) of the Zingiber zerumbet Hance leaves to the reflux extraction solvent is 1: 15-25. Preferably, the feed-to-liquid ratio is 1: 20.

In the extraction method, the reflux extraction conditions are as follows: the extraction temperature is 76-85 ℃, and the extraction time is 110-150 min. Preferably, the extraction temperature is 80 ℃ and the extraction time is 120 min.

In the extraction method, the reflux extraction times are 1-3.

In the above extraction method, the extract is concentrated under reduced pressure to 1/5 of the total volume before removing pigment.

In the above extraction method, activated carbon is used to remove pigment. The adding amount of the active carbon is based on the standard that the pigment in the extracting solution can be fully removed; preferably, the addition amount of the activated carbon is 1-3%. The method for removing the active carbon comprises the following steps: after adding activated carbon to fully adsorb the pigment, filtering to remove activated carbon residue.

In the extraction method, the extractant is one of ethyl acetate or chloroform.

In the extraction method, the addition amount of the extracting agent is 2 times of the volume of the extract A based on the fact that the extract A can be fully extracted in the extraction process of the extract A. The selection of the extraction times is also based on the realization of the full extraction of the extract A, and can be selected to be 1-3 times.

In the extraction method, the volume ratio of ethyl acetate to methanol in the mixed solution of ethyl acetate and methanol is 9: 1-1: 1. Preferably, the volume ratio of ethyl acetate to methanol is 7: 3.

In the extraction method, the volume ratio of chloroform to methanol in the mixed solution of chloroform and methanol is 1: 1-7: 3. Preferably, the volume ratio of chloroform to methanol is 6: 4.

In the extraction method, standing and crystallizing can be realized by standing for 3-5 days, or volatilizing the volume of the mixed solution to 1/5 of the original volume of the mixed solution. The final result of standing crystallization is the precipitation of a large amount of colorless needle crystals.

In the extraction method, in the filtering process of obtaining the coarse crystal A and the coarse crystal B, the filtering is selected from reduced pressure suction filtration, and the vacuum degree is 0.08 MPa. Preferably, the crystals are washed with methanol during filtration.

In the extraction method, the volume ratio of dichloromethane to methanol in the mixed solution of dichloromethane and methanol is 6: 4-9: 1. Preferably, the volume ratio of dichloromethane to methanol is 8: 2.

In the above extraction method, when the crystallization of the crude crystal B is difficult, a small amount of the crude crystal A may be added as a seed crystal.

In the above extraction method, chloroform is used for recrystallization. And adopting chloroform recrystallization conditions to add chloroform into the crude crystal, placing the crude crystal in an environment of 40-50 ℃ until the crude crystal is dissolved, and then recrystallizing at room temperature. In the present invention, the purpose of recrystallization can be achieved by using methanol, but methanol is less volatile at room temperature than chloroform, and therefore, in a specific embodiment, it is preferable to perform recrystallization using chloroform.

The invention has the beneficial effects that:

the extraction method of the dihydromethysticin is simple and rapid to operate, a recrystallization method is mainly adopted in the extraction and separation process, the process method is simple and environment-friendly, and the purity of the dihydromethysticin obtained by the method is higher than 90%. In addition, the extraction method of the invention can lead the extraction rate of the dihydromethysticin to reach 0.88 percent, which is much higher than other extraction methods in the prior art.

Drawings

FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of dihydromethysticin;

FIG. 2 is a nuclear magnetic resonance carbon spectrum of dihydromethysticin.

Detailed Description

Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified. The present invention will be described in further detail with reference to the following data in conjunction with specific examples. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.

Example 1

Extracting dihydromethysticin from the alpinia japonica leaves as follows:

drying and crushing 2kg of zingiber zerumbet leaves (taken from interlink county of Zhenfeng county, Guizhou), performing reflux extraction on the crushed zingiber zerumbet leaves by adopting 20L of 95% ethanol (the material-liquid ratio is 1:20) (the extraction temperature is 80 ℃, the extraction time is 2 hours), performing reflux extraction for 2 times, filtering to remove medicine residues after the reflux extraction is finished, obtaining an extracting solution, performing reduced pressure concentration on the extracting solution until the extracting solution is 1/5 of the total volume, adding 20g of activated carbon to remove pigments, obtaining a concentrated solution after the pigments are removed, performing reduced pressure concentration on the concentrated solution, and recovering ethanol to obtain an extract A84 g. And extracting the extract A with ethyl acetate for 3 times, wherein the dosage of the ethyl acetate in each extraction is 5000mL, collecting and combining the extraction liquid for 3 times, and concentrating under reduced pressure to obtain 49g of extract B. Adding 150mL of mixed solution of ethyl acetate and methanol (the volume ratio of ethyl acetate to methanol is 7:3) into the extract B, completely dissolving the extract B, standing for 5 days, and precipitating needle crystals; and (3) carrying out reduced pressure suction filtration (the vacuum degree is 0.08MPa) on the solution system after crystallization, cleaning and crystallizing with a small amount of methanol in the suction filtration process to obtain crude crystals A13.7g, and then carrying out reduced pressure concentration on the filtrate obtained by suction filtration to obtain an extract C34.3 g. Adding 100mL of mixed solution of dichloromethane and methanol (the volume ratio of dichloromethane to methanol is 8:2) into the extract C, standing for 5 days to precipitate needle crystals, performing reduced pressure suction filtration (the vacuum degree is 0.08MPa) on the solution system after crystallization, and cleaning the crystals with a small amount of methanol in the suction filtration process to obtain 6.3g of coarse crystals B. And (3) combining the crude crystals A and B, and recrystallizing with chloroform (the recrystallization step is that the combined crude crystals are stored in a 250mL conical flask, 15mL of chloroform is added under the condition of 45 ℃ water bath until the crude crystals are completely dissolved, the chloroform solution is kept still and volatilized at the room temperature of 25 ℃, and transparent needle crystals are taken from the wall of the conical flask), so that 17.6g of dihydromethypiperine with the purity of 93 percent is obtained.

Fig. 1 and fig. 2 show the nmr hydrogen spectrum and carbon spectrum of dihydromethysticin, respectively, and the data of the spectra are as follows:

¹H-NMR(400MHz,CD₃OD)δ:7.32-7.10(5H,m,H-2',3',4',5',6'),5.93(1H,d,J＝2.2Hz,H-5),5.52(1H,d,J＝2.2Hz,H-3),3.82(3H,s,4-OCH₃),2.97(2H,t,J＝7.6Hz,H-7),2.80(2H,t,J＝7.6Hz,H-8)；¹³C-NMR(100MHz,DMSO-d₆)δ:164.8(C-2),86.9(C-3),172.5(C-4),100.6(C-5),166.4(C-6),34.8(C-7),32.4(C-8),139.9(C-1'),128.1(C-2'),128.2(C-3',5'),126.1(C-4'),55.5(4-OCH₃)。

example 2

Drying and crushing 2kg of Zingiber zerumbet leaves (taken from interlink village in Zhenfeng county, Guizhou province), performing reflux extraction on the crushed Zingiber zerumbet leaves by adopting 20L of 95% ethanol (the material-liquid ratio is 1:20) (the extraction temperature is 80 ℃, the extraction time is 2 hours), performing reflux extraction for 3 times, filtering to remove medicine residues after the reflux extraction is finished, obtaining an extracting solution, performing reduced pressure concentration on the extracting solution until the total volume is 1/5, adding 20g of activated carbon to remove pigments, obtaining a concentrated solution after the pigments are removed, performing reduced pressure concentration on the concentrated solution, and recovering ethanol to obtain an extract A93 g. And (3) extracting the extract A with chloroform for 3 times, wherein the dosage of the chloroform in each extraction is 5000mL, collecting and combining the extraction liquid for 3 times, and concentrating under reduced pressure to obtain 45g of extract B. Adding 150mL of mixed solution of chloroform and methanol (the volume ratio of chloroform to methanol is 6:4) into the extract B, completely dissolving the extract B, standing for 5 days, and precipitating needle crystals; and (3) carrying out reduced pressure suction filtration (the vacuum degree is 0.08MPa) on the solution system after crystallization, cleaning and crystallizing with a small amount of methanol in the suction filtration process to obtain 10.4g of coarse crystals A, and then carrying out reduced pressure concentration on the filtrate obtained by suction filtration to obtain 32.3g of extract C. Adding 100mL of mixed solution of dichloromethane and methanol (the volume ratio of dichloromethane to methanol is 8:2) into the extract C, standing for 5 days to precipitate needle crystals, performing reduced pressure suction filtration (the vacuum degree is 0.08MPa) on the solution system after crystallization, and cleaning the crystals with a small amount of methanol in the suction filtration process to obtain 2.9g of coarse crystals B. Combining the crude crystals A and B, and recrystallizing with chloroform (the recrystallization step is that the combined crude crystals are stored in a 250mL conical flask, 15mL of chloroform is added under the condition of 45 ℃ water bath until the crude crystals are completely dissolved, the chloroform solution is kept still and volatilized at the room temperature of 25 ℃, and transparent needle crystals are taken from the wall of the conical flask), so that 11.4g of dihydromethypiperine with the purity of 94 percent is obtained.

Example 3

Drying and crushing 2kg of zingiber zerumbet leaves (taken from interlink village in Zhenfeng county, Guizhou province), performing reflux extraction on the crushed zingiber zerumbet leaves by adopting 20L of methanol (the material-liquid ratio is 1:20) (the extraction temperature is 70 ℃, the extraction time is 2 hours), performing reflux extraction for 3 times, filtering and removing medicine residues after the reflux extraction is finished to obtain an extracting solution, performing reduced pressure concentration on the extracting solution to 1/5 of the total volume, adding 20g of activated carbon to remove pigments to obtain a concentrated solution after the pigments are removed, performing reduced pressure concentration on the concentrated solution, and recovering the methanol to obtain an extract A71 g. And (3) extracting the extract A with chloroform for 3 times, wherein the dosage of the chloroform in each extraction is 5000mL, collecting and combining the extraction liquid for 3 times, and concentrating under reduced pressure to obtain 36g of extract B. Adding 150mL of mixed solution of chloroform and methanol (the volume ratio of chloroform to methanol is 6:4) into the extract B, standing for 5 days, and precipitating needle crystals; and (3) carrying out reduced pressure suction filtration (the vacuum degree is 0.08MPa) on the solution system after crystallization, cleaning and crystallizing with a small amount of methanol in the suction filtration process to obtain 7.9g of coarse crystals A, and then carrying out reduced pressure concentration on the filtrate obtained by suction filtration to obtain 27.1g of extract C. Adding 100mL of mixed solution of dichloromethane and methanol (the volume ratio of dichloromethane to methanol is 8:2) into the extract C, standing for 5 days to precipitate needle crystals, performing reduced pressure suction filtration (the vacuum degree is 0.08MPa) on the solution system after crystallization, and cleaning the crystals with a small amount of methanol in the suction filtration process to obtain 1.1g of coarse crystals B. And (3) combining the crude crystals A and B, and recrystallizing with chloroform (the recrystallization step is that the combined crude crystals are stored in a 250mL conical flask, 15mL of chloroform is added under the condition of 45 ℃ water bath until the crude crystals are completely dissolved, the chloroform solution is kept still and volatilized at the room temperature of 25 ℃, and transparent needle crystals are taken from the wall of the conical flask to obtain 8.1g of dihydromethypiperine with the purity of 94%.

Example 4

Drying and crushing 2kg of zingiber zerumbet leaves (taken from interlink village in Zhenfeng county, Guizhou province), performing reflux extraction on the crushed zingiber zerumbet leaves by adopting 20L of chloroform (the material-liquid ratio is 1:20) (the extraction temperature is 60 ℃, the extraction time is 1h), performing reflux extraction for 3 times, filtering and removing dregs after the reflux extraction is finished to obtain an extracting solution, performing reduced pressure concentration on the extracting solution to 1/5 of the total volume, adding 20g of activated carbon to remove pigments to obtain a concentrated solution after the pigments are removed, performing reduced pressure concentration on the concentrated solution, and recovering chloroform to obtain 48g of extract.

In this example, chloroform was used to reflux-extract the alpinia zerumbet leaves, and the results showed that the total extract amount was much less than in examples 1-3, and therefore it was difficult to obtain dihydromethysticin with a higher extraction rate.

Example 5

Drying and crushing 2kg of zingiber zerumbet leaves (taken from interlink village in Zhenfeng county of Guizhou province), performing reflux extraction on the crushed zingiber zerumbet leaves by adopting 20L of 95% ethanol (the material-liquid ratio is 1:20) (the extraction temperature is 80 ℃, the extraction time is 2 hours), performing reflux extraction for 2 times, filtering to remove medicine residues after the reflux extraction is finished, obtaining an extracting solution, and concentrating the extracting solution under reduced pressure to obtain 107g of extract A. And extracting the extract A with ethyl acetate for 3 times, wherein the dosage of the ethyl acetate in each extraction is 5000mL, collecting and combining the extraction liquid for 3 times, and concentrating under reduced pressure to obtain 61g of extract B. And (3) performing silica gel column chromatography (the particle size of the silica gel is 200-300 meshes) on the extract B, sequentially eluting petroleum ether-ethyl acetate (95:5)2L, petroleum ether-ethyl acetate (9:1)3L, petroleum ether-ethyl acetate (8:2)4L, petroleum ether-ethyl acetate (7:3)4L, petroleum ether-ethyl acetate (6:4)2L, petroleum ether-ethyl acetate (1:1)2L, petroleum ether-ethyl acetate (2:3)2L and methanol 3L, collecting the effluent, performing TLC (thin layer chromatography) detection on the effluent in the eluting process, determining that the dihydromethystichopin flows out from the eluate of the petroleum ether-ethyl acetate (8:2), continuously performing TLC detection, collecting the effluent containing the dihydromethystichopin, and performing reduced pressure concentration to obtain 13g of a crude fraction containing the dihydromethystichopin. The crude stream is subjected to silica gel column chromatography (the silica gel particle size is 200-300 meshes), eluted by petroleum ether and ethyl acetate (90:10), and subjected to TLC detection and combination to obtain 8g of dihydromethypiperine with the purity of 85%.

The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.