CN111303180A - Method for extracting eurycomanone from eurycoma longifolia - Google Patents
Method for extracting eurycomanone from eurycoma longifolia Download PDFInfo
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- 240000005739 Eurycoma longifolia Species 0.000 title claims abstract description 59
- UCUWZJWAQQRCOR-UHFFFAOYSA-N Pasakbumin A Natural products O1C(=O)C(O)C2(O)C(=C)C(O)C3(O)C4C5(C)C(O)C(=O)C=C(C)C5CC1C42CO3 UCUWZJWAQQRCOR-UHFFFAOYSA-N 0.000 title claims abstract description 30
- UCUWZJWAQQRCOR-OKNZMGBLSA-N eurycomanone Chemical compound O1C(=O)[C@H](O)[C@@]2(O)C(=C)[C@@H](O)[C@@]3(O)[C@@H]4[C@@]5(C)[C@H](O)C(=O)C=C(C)[C@@H]5C[C@@H]1[C@]42CO3 UCUWZJWAQQRCOR-OKNZMGBLSA-N 0.000 title claims abstract description 30
- 239000009947 eurycomanone Substances 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 87
- 238000001914 filtration Methods 0.000 claims abstract description 45
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 39
- 238000000605 extraction Methods 0.000 claims abstract description 36
- 108090000790 Enzymes Proteins 0.000 claims abstract description 31
- 102000004190 Enzymes Human genes 0.000 claims abstract description 31
- 239000000243 solution Substances 0.000 claims abstract description 24
- 238000010992 reflux Methods 0.000 claims abstract description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 17
- 239000011259 mixed solution Substances 0.000 claims abstract description 15
- 239000000706 filtrate Substances 0.000 claims abstract description 14
- 239000000047 product Substances 0.000 claims abstract description 12
- 229940088598 enzyme Drugs 0.000 claims description 29
- 239000000843 powder Substances 0.000 claims description 22
- 230000000694 effects Effects 0.000 claims description 19
- 239000002994 raw material Substances 0.000 claims description 14
- 238000002156 mixing Methods 0.000 claims description 13
- 108010059820 Polygalacturonase Proteins 0.000 claims description 11
- 239000007864 aqueous solution Substances 0.000 claims description 11
- 108010093305 exopolygalacturonase Proteins 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 11
- 108010059892 Cellulase Proteins 0.000 claims description 10
- 229940106157 cellulase Drugs 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 10
- 239000006228 supernatant Substances 0.000 claims description 10
- 238000000746 purification Methods 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 8
- 239000013078 crystal Substances 0.000 claims description 7
- 238000001291 vacuum drying Methods 0.000 claims description 7
- 238000001704 evaporation Methods 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 238000002137 ultrasound extraction Methods 0.000 claims description 5
- 238000000227 grinding Methods 0.000 claims description 2
- 230000007071 enzymatic hydrolysis Effects 0.000 claims 2
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims 2
- 150000001875 compounds Chemical class 0.000 abstract description 20
- 239000012046 mixed solvent Substances 0.000 abstract description 13
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 abstract description 7
- 241000196324 Embryophyta Species 0.000 abstract description 4
- LJQKCYFTNDAAPC-UHFFFAOYSA-N ethanol;ethyl acetate Chemical compound CCO.CCOC(C)=O LJQKCYFTNDAAPC-UHFFFAOYSA-N 0.000 abstract description 3
- 230000000052 comparative effect Effects 0.000 description 18
- 150000002576 ketones Chemical class 0.000 description 7
- 239000000126 substance Substances 0.000 description 6
- JNELGWHKGNBSMD-UHFFFAOYSA-N xanthone Chemical compound C1=CC=C2C(=O)C3=CC=CC=C3OC2=C1 JNELGWHKGNBSMD-UHFFFAOYSA-N 0.000 description 6
- 238000009210 therapy by ultrasound Methods 0.000 description 5
- 239000002904 solvent Substances 0.000 description 4
- 210000002421 cell wall Anatomy 0.000 description 3
- 229920002678 cellulose Polymers 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 241000208340 Araliaceae Species 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 2
- 235000003140 Panax quinquefolius Nutrition 0.000 description 2
- IOSXSVZRTUWBHC-LBTVDEKVSA-N Quassin Chemical compound CC([C@@H]1CC(=O)O[C@@H]([C@]21C)C1)=C(OC)C(=O)[C@@H]2[C@]2(C)[C@@H]1[C@H](C)C=C(OC)C2=O IOSXSVZRTUWBHC-LBTVDEKVSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 235000008434 ginseng Nutrition 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- IOSXSVZRTUWBHC-UHFFFAOYSA-N quassin Natural products C1C(C23C)OC(=O)CC3C(C)=C(OC)C(=O)C2C2(C)C1C(C)C=C(OC)C2=O IOSXSVZRTUWBHC-UHFFFAOYSA-N 0.000 description 2
- 238000000638 solvent extraction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 208000019901 Anxiety disease Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 235000003901 Crambe Nutrition 0.000 description 1
- 241000220246 Crambe <angiosperm> Species 0.000 description 1
- 244000042238 Eurycoma Species 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010057672 Male sexual dysfunction Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- BNRNXUUZRGQAQC-UHFFFAOYSA-N Sildenafil Natural products CCCC1=NN(C)C(C(N2)=O)=C1N=C2C(C(=CC=1)OCC)=CC=1S(=O)(=O)N1CCN(C)CC1 BNRNXUUZRGQAQC-UHFFFAOYSA-N 0.000 description 1
- 241001093962 Simaroubaceae Species 0.000 description 1
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- 230000036506 anxiety Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 201000004792 malaria Diseases 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- DEIYFTQMQPDXOT-UHFFFAOYSA-N sildenafil citrate Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O.CCCC1=NN(C)C(C(N2)=O)=C1N=C2C(C(=CC=1)OCC)=CC=1S(=O)(=O)N1CCN(C)CC1 DEIYFTQMQPDXOT-UHFFFAOYSA-N 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 229940094720 viagra Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
- C07D493/10—Spiro-condensed systems
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to the field of plant extraction, in particular to a method for extracting eurycomanone from Eurycoma longifolia, which comprises the following steps: s1, crushing and dispersing eurycoma longifolia root tubers into water, and adding a compound enzyme for enzymolysis; s2, adding an ethanol water solution into the filter residue obtained after the filtration in the step S1 for reflux extraction; s3, adding a mixed solution of ethanol and ethyl acetate into the filter residue obtained after the filtration in the step S2 for reflux extraction; and S4, combining the filtrates, and purifying to obtain the product. The invention firstly adopts the compound enzyme for enzymolysis to facilitate the release of the eurysanone, and then utilizes the ethanol-water mixed solvent and the ethanol-ethyl acetate mixed solvent for extraction in turn, thereby improving the extraction rate of the eurysanone, and the optimal extraction rate of the eurysanone reaches more than 97.8 percent and the purity is 99.7 percent.
Description
Technical Field
The invention relates to the field of plant extraction, and particularly relates to a method for extracting eurycomanone from eurycoma longifolia.
Technical Field
Eurycoma longifolia is a plant of Eurycoma longifolia of Simaroubaceae, and is widely distributed in southeast Asia countries such as Malaysia, Indonesia, Vietnam, etc. The ginseng is originally called as Malaysia ginseng, natural Viagra and the like, and is called as Malaysia sanda treasure together with cubilose and tinfoil. The Eurycoma longifolia has effects of resisting cancer, resisting malaria, improving male sexual dysfunction, improving human body function, resisting anxiety, resisting osteoporosis, relieving pain, resisting inflammation, resisting bacteria, resisting parasite, regulating immunity and treating diabetes. The main active component of the Eurycoma longifolia, namely the eurycoma longifolia compound, the widely-tassel ketone is the most representative one of the Eurycoma longifolia compounds, and researches show that the widely-tassel ketone has higher content in the Eurycoma longifolia, and the content reaches 1.64 per thousand.
For example, CN103408564B discloses a process for extracting eurycomanone from eurycoma longifolia plants, but the process is complicated, uses various organic solvents such as ethanol, petroleum ether, ethyl acetate, chloroform, etc., and is repeatedly used, the solvent consumption is large, and the product purity is not high.
Disclosure of Invention
The invention aims to solve the problems of various process steps and low purity of extracted products in the prior art for extracting the eurycomanone from the eurycoma longifolia, and provides a method for extracting the eurycomanone from the eurycoma longifolia.
The purpose of the invention is realized by the following technical scheme:
a method for extracting eurycomanone from Eurycoma longifolia, which comprises the following steps:
s1, according to a material-liquid ratio of 1 g: (20-25) mL, grinding and dispersing eurycoma longifolia root tubers into water; adding 10-15U of complex enzyme into per gram of Eurycoma longifolia root tuber powder, and performing enzymolysis at 38-45 deg.C for 2-3h, wherein the complex enzyme is pectase and cellulase according to activity unit ratio (3-5): 1, mixing;
s2, in the mixture subjected to enzymolysis in the step S1, mixing the raw materials according to a material-liquid ratio of 1 g: (10-20) mL, adding an ethanol aqueous solution, extracting for 1-2h under reflux, and then filtering, wherein the mass fraction of ethanol in the ethanol aqueous solution is 93% -97%;
s3, in the filter residue obtained after the filtration in the step S2, adding the raw materials in a ratio of 1 g: (10-20) mL, adding a mixed solution of ethanol and ethyl acetate, performing reflux extraction for 1-2h, and then filtering, wherein the mass fraction of ethanol in the mixed solution is 28% -32%;
and S4, combining the filtrates obtained by filtering in the steps S2 and S3, and purifying to obtain the product.
The plant cell wall mainly comprises fiber and polysaccharide, and the complex enzyme is adopted to treat the Eurycoma longifolia so as to break the cell wall, thereby facilitating the release of effective components. The Eurycoma longifolia contains a plurality of chemical components, and two compounds of the quassin and the alkaloid are taken as main components. The xanthone belongs to the group of quassin, and its molecular formula is C20H24O9. The Eurycoma longifolia contains many substances with similar structures and similar physical properties to those of eurycoma longifolia, for example, 27C species are found in Eurycoma longifolia20Class of substances, 22 kinds of C19And (e) a substance. In the extraction process, the substances interact with each other, and the broad-leaved ketone is difficult to be completely extracted at one time. The invention promotes the release of effective components through the enzymolysis of the complex enzyme, and then carries out fractional extraction through different mixed solvents (the mixed solvents of ethanol and water and the mixed solvents of ethanol and ethyl acetate are sequentially used for extraction) so as to overcome the condition that the eurysanone is combined with different molecules at different extraction stages, thereby more fully extracting the eurysanone in the eurycoma longifolia.
Preferably, the complex enzyme in the step S1 is prepared by mixing pectinase and cellulase according to an activity unit ratio of 4: 1.
Preferably, after the complex enzyme enzymolysis in the step S1 and before the step S2, the mixture after the enzymolysis is subjected to ultrasonic extraction. Under the action of the ultrasonic wave, the effective components in Eurycoma longifolia plant cells can enter the solvent more conveniently.
Preferably, the ultrasonic extraction conditions are: extracting at 45-50 deg.C under 50-100Hz for 30-40 min.
Preferably, the eurycoma longifolia root tuber is pulverized into powder of 10-30 meshes in the step S1.
Preferably, the purification in step S4 includes the following steps:
(1) centrifuging the combined filtrate obtained in the step S4 for 15-20min at the rotation speed of 20000-25000 r/min; filtering and keeping supernatant;
(2) evaporating and concentrating the supernatant obtained in the step (1) at 50-55 ℃ until the concentration of the concentrated solution is 30-degree Be' to obtain a concentrated solution;
(3) adding anhydrous ethanol into the concentrated solution, stirring until the concentration of the concentrated solution is 10 ° Be', crystallizing at 2-4 deg.C, washing the obtained crystal, and vacuum drying.
In the purification process of the eurysanone, the eurysanone is firstly degreased by centrifugation and then is further purified by recrystallization, so that the purity of the eurysanone is effectively improved.
Preferably, the washing in the step (2) is to wash the crystals with ice water at 1-2 ℃.
Preferably, the temperature of vacuum drying in the step (2) is 52-54 ℃, and the vacuum degree is-0.05 MPa.
Compared with the prior art, the invention has the following technical effects:
the invention provides a method for extracting eurycomanone from eurycoma longifolia.A complex enzyme is firstly adopted to carry out enzymolysis on the cell wall of eurycoma longifolia cells so as to release eurycomanone. And extracting the eurysanone in the eurycoma longifolia by using an ultrasonic extraction mode, and sequentially extracting by using an ethanol-water miscible solvent and an ethanol-ethyl acetate miscible solvent. Improves the extraction rate of the xanthone, the optimal extraction rate of the xanthone reaches more than 97.8 percent, and the purity is 99.7 percent.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below with reference to specific examples and comparative examples. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
The equipment used in the present examples, comparative examples and experimental examples were conventional experimental equipment, and the materials and reagents used were commercially available unless otherwise specified.
Example 1
A method for extracting eurycomanone from Eurycoma longifolia, which comprises the following steps:
s1, according to a material-liquid ratio of 1 g: 20mL, crushing Eurycoma longifolia root tuber into 10-mesh powder, dispersing the powder into water, adding 10U of compound enzyme into each gram of Eurycoma longifolia root powder, and performing enzymolysis for 2 hours at the temperature of 45 ℃, wherein the compound enzyme is pectinase and cellulase, and the ratio of activity units of the compound enzyme to the activity units of 5: 1, mixing;
s2, in the mixture subjected to enzymolysis in the step S1, mixing the raw materials according to a material-liquid ratio of 1 g: 20mL, adding an ethanol aqueous solution, performing reflux extraction for 1h, and then filtering, wherein the mass fraction of ethanol in the ethanol aqueous solution is 95%;
s3, in the filter residue obtained after the filtration in the step S2, adding the raw materials in a ratio of 1 g: 20mL, adding a mixed solution of ethanol and ethyl acetate, performing reflux extraction for 1h, and then filtering, wherein the mass fraction of ethanol in the mixed solution is 28%;
and S4, combining the filtrates obtained by filtering in the steps S2 and S3, and purifying to obtain the product.
The purification in the above step S4 includes the following steps:
(1) combining the filtrates obtained in the step S4, and centrifuging for 15min at the rotating speed of 20000 r/min; filtering and keeping supernatant;
(2) evaporating and concentrating the supernatant obtained in the step (1) at 50 ℃ until the concentration is 30-degree Be' to obtain a concentrated solution;
(3) adding anhydrous ethanol into the concentrated solution, stirring to obtain a solution with a concentration of 10 ° Be', crystallizing at 2-4 deg.C, washing the obtained crystal with 1-2 deg.C water, and vacuum drying at 52 deg.C under-0.05 MPa.
Example 2
A method for extracting eurycomanone from Eurycoma longifolia, which comprises the following steps:
s1, according to a material-liquid ratio of 1 g: 20mL, crushing Eurycoma longifolia root tuber into 10-mesh powder, dispersing the powder into water, adding 10U of compound enzyme into each gram of Eurycoma longifolia root powder, and performing enzymolysis for 2 hours at the temperature of 45 ℃, wherein the compound enzyme is pectinase and cellulase, and the ratio of activity units of the compound enzyme to the activity units of 5: 1, mixing;
s2, carrying out 50Hz ultrasonic treatment on the mixture subjected to the enzymolysis in the step S1 for 30min at the temperature of 50 ℃, and then filtering;
s3, in the filter residue obtained after the filtration in the step S2, adding the raw materials in a ratio of 1 g: 20mL, adding an ethanol aqueous solution, performing reflux extraction for 1h, and then filtering, wherein the mass fraction of ethanol in the ethanol aqueous solution is 95%;
s4, in the filter residue obtained after the filtration in the step S3, adding the raw materials in a ratio of 1 g: 20mL, adding a mixed solution of ethanol and ethyl acetate, performing reflux extraction for 1h, and then filtering, wherein the mass fraction of ethanol in the mixed solution is 28%;
and S5, combining the filtrates obtained by filtering in the steps S2, S3 and S4, and purifying to obtain the product.
The purification in the above step S5 includes the following steps:
(1) combining the filtrates obtained in the step S5, and centrifuging for 15min at the rotating speed of 20000 r/min; filtering and keeping supernatant;
(2) evaporating and concentrating the supernatant obtained in the step (1) at 50 ℃ until the concentration is 30-degree Be' to obtain a concentrated solution;
(3) adding anhydrous ethanol into the concentrated solution, stirring to obtain a solution with a concentration of 10 ° Be', crystallizing at 2-4 deg.C, washing the obtained crystal with 1-2 deg.C water, and vacuum drying at 52 deg.C under-0.05 MPa.
Example 3
A method for extracting eurycomanone from Eurycoma longifolia, which comprises the following steps:
s1, according to a material-liquid ratio of 1 g: 25mL, crushing eurycoma longifolia root tuber powder of no 30 meshes, dispersing the powder into water, adding 15U of complex enzyme into each gram of eurycoma longifolia root tuber powder, and performing enzymolysis for 3 hours at the temperature of 38 ℃, wherein the complex enzyme is pectinase and cellulase, and the ratio of the activity units of the complex enzyme to the activity units of 3: 1, mixing;
s2, carrying out 100Hz ultrasonic treatment on the mixture subjected to the enzymolysis in the step S1 for 40min at the temperature of 45 ℃, and then filtering;
s3, in the filter residue obtained after the filtration in the step S2, adding the raw materials in a ratio of 1 g: 10mL, adding ethanol water solution, extracting for 2h under reflux, and then filtering, wherein the mass fraction of ethanol in the ethanol water solution is 97%;
s4, in the filter residue obtained after the filtration in the step S3, adding the raw materials in a ratio of 1 g: 10mL, adding a mixed solution of ethanol and ethyl acetate, performing reflux extraction for 2h, and then filtering, wherein the mass fraction of the ethanol in the mixed solution is 32%;
and S5, combining the filtrates obtained by filtering in the steps S2, S3 and S4, and purifying to obtain the product.
The purification in the above step S5 includes the following steps:
(1) centrifuging the combined filtrate obtained in the step S5 for 20min at 25000 r/min; filtering and keeping supernatant;
(2) evaporating and concentrating the supernatant obtained in the step (1) at 55 ℃ to obtain a concentrated solution with the concentration of 30 DEG Be';
(3) adding anhydrous ethanol into the concentrated solution, stirring to obtain a solution with a concentration of 10 ° Be', crystallizing at 2-4 deg.C, washing the obtained crystal with 1-2 deg.C water, and vacuum drying at 54 deg.C under-0.05 MPa.
Comparative example 1
A method for extracting eurycomanone from Eurycoma longifolia, which comprises the following steps:
s1, according to a material-liquid ratio of 1 g: 22mL, crushing Eurycoma longifolia root tuber into 25-mesh powder, dispersing the powder into water, adding 12U of compound enzyme into each gram of Eurycoma longifolia root tuber powder, and performing enzymolysis for 2.5 hours at the temperature of 40 ℃, wherein the compound enzyme is pectinase and cellulase, and the ratio of activity units of the compound enzyme to the activity units of the pectinase to the cellulose is 4:1, mixing;
s2, carrying out 80Hz ultrasonic treatment on the solution mixture subjected to the enzymolysis in the step S1 for 35min at the temperature of 48 ℃, and then filtering;
s3, in the filter residue obtained after the filtration in the step S2, adding the raw materials in a ratio of 1 g: 15mL, adding a mixed solution of ethanol and ethyl acetate, performing reflux extraction for 1.5h, and then filtering, wherein the mass fraction of ethanol in the mixed solution is 30%;
and S4, combining the filtrates obtained by filtering in the steps S2 and S3, and purifying to obtain the product. The purification procedure was as in example 3.
Compared with example 3, the comparative example does not adopt a mixed solvent of ethanol and water for reflux extraction.
Comparative example 2
A method for extracting eurycomanone from Eurycoma longifolia, which comprises the following steps:
s1, according to a material-liquid ratio of 1 g: 22mL, crushing Eurycoma longifolia root tuber into 25-mesh powder, dispersing the powder into water, adding 12U of compound enzyme into each gram of Eurycoma longifolia root tuber powder, and performing enzymolysis for 2.5 hours at the temperature of 40 ℃, wherein the compound enzyme is pectinase and cellulase, and the ratio of activity units of the compound enzyme to the activity units of the pectinase to the cellulose is 4:1, mixing;
s2, carrying out 80Hz ultrasonic treatment on the mixture subjected to the enzymolysis in the step S1 for 35min at the temperature of 48 ℃, and then filtering;
s3, in the filter residue obtained after the filtration in the step S2, adding the raw materials in a ratio of 1 g: 15mL, adding an ethanol aqueous solution, extracting for 1.5h under reflux, and then filtering, wherein the mass fraction of ethanol in the ethanol aqueous solution is 95%;
and S4, combining the filtrates obtained by filtering in the steps S2 and S3, and purifying to obtain the product. The purification procedure was as in example 3.
Compared with example 3, the comparative example does not adopt the mixed solvent of ethanol and ethyl acetate for extraction.
Comparative example 3
A method for extracting eurycomanone from Eurycoma longifolia, which comprises the following steps:
s1, according to a material-liquid ratio of 1 g: 22mL, crushing Eurycoma longifolia root tuber into 25-mesh powder, dispersing the powder into water, adding 12U of compound enzyme into each gram of Eurycoma longifolia root tuber powder, and performing enzymolysis for 2.5 hours at the temperature of 40 ℃, wherein the compound enzyme is pectinase and cellulase, and the ratio of activity units of the compound enzyme to the activity units of the pectinase to the cellulose is 4:1, mixing;
s2, carrying out 80Hz ultrasonic treatment on the mixture subjected to the enzymolysis in the step S1 for 35min at the temperature of 48 ℃, and then filtering;
s3, in the filter residue obtained after the filtration in the step S2, adding the raw materials in a ratio of 1 g: 15mL, adding 60% ethanol water solution by mass fraction, refluxing and extracting for 1.5h, and then filtering;
s4, in the filter residue obtained after the filtration in the step S3, adding the raw materials in a ratio of 1 g: 15mL, adding a mixed solution of ethanol and ethyl acetate, performing reflux extraction for 1.5h, and then filtering, wherein the mass fraction of ethanol in the mixed solution is 50%;
and S5, combining the filtrates obtained by filtering in the steps S2, S3 and S4, and purifying to obtain the product. The purification procedure was as in example 3.
In contrast to example 3, this comparative example employed a mixed solvent of ethanol and water in a ratio out of the range of the present invention, and a mixed solvent of ethanol and ethyl acetate out of the range of the present invention.
Experimental example 1
Three batches of Eurycoma longifolia were purchased from Ningxia Hua Ke agricultural ecological development Co., Ltd, Fujian province Changshun biotechnology Co., Ltd, and Yunnan Guangzheng Biotechnology Co., Ltd. And (3) determining the content of the eurycomanone in each batch of eurycoma longifolia medicinal materials. Reference is made to the "HPLC method for determining the content of eurycomanone in Eurycoma longifolia", Fujian analytical test, 2019,28 (3). The test results are shown in table 1.
TABLE 1 content of broad-leaved ketones in the starting materials
The method for extracting the eurycomanone from the commercially available eurycomane crambe medicinal materials is carried out according to the methods of the examples and the comparative examples, the extraction rate of the eurycomanone is calculated, and the purity of the extracted eurycomanone is measured. Reference to the measurement method: determination of the content of eurycomanone in Eurycoma longifolia by HPLC, Fujian analysis test, 2019,28 (3). The results are shown in Table 2.
TABLE 2 broad-tasone extraction products table
| Group of | Example 1 | Example 1 | Example 2 | Example 3 | Comparative example 1 | Comparative example 2 | Comparative example 3 |
| Extraction rate | 96.8％ | 97.7％ | 97.2％ | 97.8％ | 82.1％ | 82.3％ | 90.2％ |
| Purity of | 99.5％ | 99.6％ | 98.9％ | 99.7％ | 98.3％ | 98.1％ | 87.6％ |
It can be seen from table 1 that the extraction process or the resulting extraction yield of the eurysanone is significantly higher for each group of examples than for each group of comparative examples. The comparative example 1 does not adopt ethanol-water mixed solvent extraction, the comparative example 2 does not adopt ethanol-ethyl acetate mixed solvent extraction, and a fractional extraction method is not adopted, so that molecules with similar structures to the broad-leaved ketone cannot be well separated in the extraction process, the extraction rate is reduced, and the fractional extraction has obvious improvement effect on the extraction of the broad-leaved ketone substance. The proportion of ethanol aqueous solution used in comparative example 3 is out of the range of the present invention, and although the extraction rate of comparative example 3 is 90% or more, the purity is lower than that of other examples and comparative examples. The extraction rate and purity of the broad-leaved ketone are synergistically improved by the mixed solvent of ethanol and water and the mixed solvent of ethanol and ethyl acetate.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (8)
1. A method for extracting eurycomanone from Eurycoma longifolia, which is characterized by comprising the following steps:
s1, according to a material-liquid ratio of 1 g: (20-25) mL, grinding and dispersing eurycoma longifolia root tubers into water; adding 10-15U of complex enzyme into per gram of Eurycoma longifolia root tuber powder, and performing enzymolysis at 38-45 deg.C for 2-3h, wherein the complex enzyme is pectase and cellulase according to activity unit ratio (3-5): 1, mixing;
s2, in the mixture subjected to enzymolysis in the step S1, mixing the raw materials according to a material-liquid ratio of 1 g: (10-20) mL, adding an ethanol aqueous solution, extracting for 1-2h under reflux, and then filtering, wherein the mass fraction of ethanol in the ethanol aqueous solution is 93% -97%;
s3, in the filter residue obtained after the filtration in the step S2, adding the raw materials in a ratio of 1 g: (10-20) mL, adding a mixed solution of ethanol and ethyl acetate, performing reflux extraction for 1-2h, and then filtering, wherein the mass fraction of ethanol in the mixed solution is 28% -32%;
and S4, combining the filtrates obtained by filtering in the steps S2 and S3, and purifying to obtain the product.
2. The method for extracting eurycomanone from eurycoma longifolia as claimed in claim 1, wherein the complex enzyme in step S1 is prepared by mixing pectinase and cellulase at an activity unit ratio of 4: 1.
3. The method for extracting eurycomanone from eurycomane longifolia according to claim 1, wherein after the enzymatic hydrolysis of the complex enzyme in step S1 and before step S2, the mixture after the enzymatic hydrolysis is subjected to ultrasonic extraction.
4. The method for extracting eurycomanone from eurycoma longifolia according to claim 3, wherein the ultrasonic extraction conditions are as follows: extracting at 45-50 deg.C under 50-100Hz for 30-40 min.
5. The method for extracting eurycomanone from eurycoma longifolia as claimed in claim 1, wherein the root tuber of eurycoma longifolia is pulverized into powder of 10-30 mesh in step S1.
6. The method for extracting eurycomanone from eurycoma longifolia as claimed in claim 1, wherein the purification in step S4 comprises the following steps:
(1) centrifuging the combined filtrate obtained in the step S4 for 15-20min at the rotation speed of 20000-25000 r/min; filtering and keeping supernatant;
(2) evaporating and concentrating the supernatant obtained in the step (1) at 50-55 ℃ until the concentration is 30 DEG Be' to obtain a concentrated solution;
(3) adding anhydrous ethanol into the concentrated solution, stirring to obtain a solution with a concentration of 10 ° Be', crystallizing at 2-4 deg.C, washing the obtained crystal, and vacuum drying.
7. The method for extracting eurycomanone from eurycoma longifolia according to claim 6, wherein the washing in step (3) is performed by washing the crystals with water at 1-2 ℃.
8. The method for extracting eurycomanone from eurycomane according to claim 6, wherein the temperature of vacuum drying in step (3) is 52-54 ℃ and the vacuum degree is-0.05 MPa.
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