CN113698377B - Method for extracting dihydromethysticin from alpinia japonica leaves - Google Patents
Method for extracting dihydromethysticin from alpinia japonica leaves Download PDFInfo
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- CN113698377B CN113698377B CN202111120433.7A CN202111120433A CN113698377B CN 113698377 B CN113698377 B CN 113698377B CN 202111120433 A CN202111120433 A CN 202111120433A CN 113698377 B CN113698377 B CN 113698377B
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- C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
- C07D309/34—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
- C07D309/36—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with oxygen atoms directly attached to ring carbon atoms
- C07D309/38—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with oxygen atoms directly attached to ring carbon atoms one oxygen atom in position 2 or 4, e.g. pyrones
Abstract
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.
Description
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 (per.) 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 the middle-jiao, eliminating dampness, strengthening the spleen and warming the stomach, and is used for treating symptoms such as abdominal psychroalgia, dyspepsia, chest and abdominal distending pain, phlegm-damp stagnation, vomiting and 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 C 14 H 14 O 3 It has neuroprotective, anti-obesity, antiviral, and anti-cell proliferation activities. The structural formula of dihydromethysticin is as follows:
at present, the method for separating the dihydromethysticin from the alpinia zeylanica leaves is developed based on the activity of the dihydromethysticin, and the obtained amount is small, so that a proper extraction and separation method for obtaining a large amount of the compound is not provided. 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 alpinia zeylanica 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 crystal A and the crude crystal B, and recrystallizing to obtain the dihydromethysticin.
In the above extraction method, the solvent used for reflux extraction is one or more selected from ethanol, methanol or chloroform. Preferably, the reflux extraction solvent is selected from ethanol, and further preferably 95% ethanol.
In the extraction method, the material-liquid ratio (g/mL) of the Zingiber zerumbet Hance leaves to the reflux extraction solvent is 1. Preferably, the feed-liquid ratio is 1.
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 120min.
In the above extraction method, the reflux extraction times are 1 to 3 times.
In the above extraction method, before removing pigment, the extractive solution is concentrated under reduced pressure to 1/5 of the total volume.
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 amount of activated carbon added is 1 to 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 to 3 times.
In the above extraction method, the volume ratio of ethyl acetate to methanol in the mixed solution of ethyl acetate and methanol is 9. Preferably, the volume ratio of ethyl acetate to methanol is 7.
In the above extraction method, the volume ratio of chloroform to methanol in the mixed solution of chloroform and methanol is 1. Preferably, the volume ratio of chloroform to methanol is 6.
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 0.08MPa. Preferably, the crystals are washed with methanol during filtration.
In the above extraction method, the volume ratio of dichloromethane to methanol in the mixed solution of dichloromethane and methanol is 6. Preferably, the volume ratio of dichloromethane to methanol is 8.
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. The condition of adopting chloroform for recrystallization is that chloroform is added into the coarse crystal and is put into the environment of 40-50 ℃ until the coarse crystal is dissolved, and then the recrystallization is carried out at room temperature. In the present invention, the purpose of recrystallization can also be achieved by using methanol, but methanol is less volatile at room temperature than chloroform, and therefore, in a specific embodiment, recrystallization is preferably performed by 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 the 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 alpinia zerumbet leaves as follows:
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 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 to 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 A84g. 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; and (3) carrying out reduced pressure suction filtration (the vacuum degree is 0.08 MPa) 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 extract C34.3 g. Adding 100mL of a mixed solution of dichloromethane and methanol (the volume ratio of dichloromethane to methanol is 8. 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:
1 H-NMR(400MHz,CD 3 OD)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-OCH 3 ),2.97(2H,t,J＝7.6Hz,H-7),2.80(2H,t,J＝7.6Hz,H-8)； 13 C-NMR(100MHz,DMSO-d 6 )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-OCH 3 )。
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 95% ethanol (the material-liquid ratio is 1: 20) (the extraction temperature is 80 ℃, the extraction time is 2 h), 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 to 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 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 a mixed solution of chloroform and methanol (the volume ratio of chloroform to methanol is 6; and (3) carrying out reduced pressure suction filtration (the vacuum degree is 0.08 MPa) 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 crystal 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 a mixed solution of dichloromethane and methanol (the volume ratio of dichloromethane to methanol is 8. 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 (the extraction temperature is 70 ℃ and the extraction time is 2 hours) by adopting 20L of methanol (the material-liquid ratio is 1. 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 a mixed solution of chloroform and methanol (the volume ratio of chloroform to methanol is 6; and (3) carrying out reduced pressure suction filtration (the vacuum degree is 0.08 MPa) on the solution system after crystallization, cleaning and crystallizing with a small amount of methanol in the suction filtration process to obtain crude crystals A7.9 g, and then carrying out reduced pressure concentration on the filtrate obtained by suction filtration to obtain extract C27.1 g. Adding 100mL of a mixed solution of dichloromethane and methanol (the volume ratio of dichloromethane to methanol is 8. 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.
In this example, the reflux extraction of alpinia zerumbet leaves with chloroform showed that the total extract amount was much less than in examples 1 to 3, and thus it was difficult to obtain dihydromethysticin at a high extraction rate.
Example 5
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 95% ethanol (the material-liquid ratio is 1 to 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. Subjecting the extract B to silica gel column chromatography (silica gel particle size 200-300 mesh), eluting with 2L of petroleum ether-ethyl acetate (95). The crude stream was subjected to silica gel column chromatography (silica gel particle size 200-300 mesh), eluted with petroleum ether: ethyl acetate (90: 10), and combined by TLC detection to give dihydromethypiperine 8g, 85% pure.
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.
Claims (5)
1. The method for extracting dihydromethysticin from alpinia zeylanica leaves is characterized by comprising the following steps:
drying and crushing the alpinia japonica leaves, and performing reflux extraction on the crushed alpinia japonica leaves by adopting 95% ethanol, wherein the extraction temperature is 76-85 ℃, and the extraction time is 110-150 min; filtering to remove residues to obtain an extracting solution, concentrating the extracting solution under reduced pressure to a certain volume, removing pigments to obtain a concentrated solution with pigments removed, and recovering a solvent in the concentrated solution to obtain an extract A; adding ethyl acetate or chloroform into the extract A for extraction, collecting the 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; mixing the coarse crystal A and the coarse crystal B, and recrystallizing to obtain dihydromethysticin;
in the mixed solution of ethyl acetate and methanol, the volume ratio of ethyl acetate to methanol is 7; in the mixed solution of chloroform and methanol, the volume ratio of chloroform to methanol is 6; in the mixed solution of dichloromethane and methanol, the volume ratio of dichloromethane to methanol is 8.
2. The extraction method according to claim 1, wherein the pigment is removed with activated carbon.
3. 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.
4. The extraction method according to claim 1, wherein the recrystallization is performed using chloroform or methanol.
5. The extraction method according to claim 4, wherein the recrystallization with chloroform or methanol is carried out 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 carried out at room temperature.
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Non-Patent Citations (5)
| Title |
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| 5,6-Dehydrokawain from Alpinia zerumbetpromotes osteoblastic MC3T3-E1 cell differentiation;Momochika Kumagai等;《Bioscience, Biotechnology, and Biochemistry》;20161231;第80卷(第7期);第1425-1432页 * |
| Dihydro-5,6-dehydrokavain (DDK) from Alpinia zerumbet:Its Isolation, Synthesis, and Characterization;Tran Dang Xuan等;《Molecules》;20150909;第20卷;第16308页倒数第1段 * |
| HIV-1 Integrase and Neuraminidase Inhibitors from Alpinia zerumbet;Atul Upadhyay等;《J.Agric.Food Chem.》;20110209;第59卷;第2858页 * |
| Syntheses and Biological Activities of Dihydro-5,6-dehydrokawain Derivatives;Shinkichi Tawata等;《Biosci.Biotech.Biochem.》;19961231;第60卷(第10期);第1643页右栏倒数第2段 * |
| 艳山姜叶的化学成分研究;田壁榕等;《中药材》;20181130;第41卷(第11期);第2567-2570页 * |
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