CN112870265A - Apple peel extract with fatty acid synthase inhibiting activity and application thereof - Google Patents
Apple peel extract with fatty acid synthase inhibiting activity and application thereof Download PDFInfo
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- CN112870265A CN112870265A CN202110108415.0A CN202110108415A CN112870265A CN 112870265 A CN112870265 A CN 112870265A CN 202110108415 A CN202110108415 A CN 202110108415A CN 112870265 A CN112870265 A CN 112870265A
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- extract
- apple peel
- organic solvent
- fatty acid
- acid synthase
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Abstract
The invention discloses a preparation method of an apple peel extract, and belongs to the technical field of biology. The preparation method comprises the following steps: s1, crushing, drying and degreasing the apple peel to obtain degreased apple peel; s2, adding an organic solvent-water mixed solvent into the degreased apple peel obtained in the step S1 for ultrasonic extraction, and collecting and combining extracting solutions to obtain a crude extracting solution; s3, concentrating the crude extract obtained in the step S2 under reduced pressure until the organic solvent is volatilized, drying to obtain an apple peel organic solvent extract, S4, dissolving the organic solvent extract obtained in the step S3 in an ethanol-water mixed solvent, performing chromatographic separation by using ethanol-water as a mobile phase through simulated moving bed chromatography, collecting an effluent, concentrating under reduced pressure, and drying to obtain the apple peel extract. The apple peel extract with the fatty acid synthase inhibition activity can be widely applied to the fields of pharmacy, health-care food, cosmetics and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an apple peel extract with fatty acid synthase inhibitory activity and application thereof.
Background
Higher animals' body fat is more synthesized by themselves in the body, on the one hand, by direct intake. Fatty Acid Synthase (FAS) is an important enzyme in the synthesis of fat in biological cells. Studies of fatty acid synthase in poultry by Tianxi et al found that there is a very high positive correlation between the fat level in the abdominal cavity of poultry and the activity of fatty acid synthase, and proposed that controlling FAS activity is an effective method for controlling the fat level of animals (Tianxi, 1994. regulation of body fat level and fatty acid synthase activity in animals. chemistry of life, 14, (1): 184; Tianweixi et al, 1996. relationship between body fat level and liver fatty acid synthase activity in layers of different growth stages. J. Biochem. 12(2): 234. 42. 236; Mei Li et al, 1999. fans influencing the levels of chicken synthase complex activity in fool. biochem Mol. Int,47(1): 63-69). Results of studies on mouse fatty acid synthase by louftus et al, university of hopkins, in 2000, demonstrated that inhibition of fatty acid synthase resulted in accumulation of malonyl-coa, which inhibited the expression of food-related hypothalamic neuropeptide Y, resulting in decreased appetite and weight loss without affecting the mobility of mice. Loftus et al indicate that fatty acid synthase can be a potential therapeutic target for weight control (Loftus. T.M.et. al.,2000.Science,288(5475): 2379-. In addition, many studies have found that the activity and expression of FAS is shown to be extremely low in almost all non-diseased adult tissues, while its activity is significantly increased and expression is significantly up-regulated in many types of cancer tissues, such as rectal cancer, breast cancer, prostate cancer, endometrial cancer, ovarian cancer, lung cancer, liver cancer, and the like. Meanwhile, it has been found that cerulenin, an inhibitor of FAS, can kill cancer cells and inhibit the growth of xenograft tumors, and other FAS inhibitors such as cerulenin derivative C75, antiobesity drug orlistat, EGCG in green tea and other flavonoids of natural origin such as luteolin, quercetin, kaempferol and the like and antibiotic triclosan have also been confirmed to inhibit the growth of tumor cells by inducing apoptosis. The above results indicate that fatty acid synthase may be a potential dual target for the treatment of obesity and tumors. Therefore, the development and development of inhibitors of fatty acid synthase would have great practical significance and potential for development.
The apple is a plant of Malus of Maloideae of Rosaceae, and its tree is deciduous tree. The apple has high nutritive value, is rich in minerals and vitamins, has rich calcium content, is beneficial to metabolizing excessive salt in the body, and has the effects of promoting the production of body fluid to quench thirst, clearing heat and relieving restlessness, invigorating stomach and promoting digestion and the like. CN105498274B discloses a two-aqueous phase system and application thereof in separating flavone from Hongxing apple peel. CN103204895B discloses a method for separating ursolic acid from apple peel. CN103431216B discloses a goat fattening pellet feed containing apple peel and a preparation method thereof. CN1166662C discloses a production process for extracting apple polyphenol by using apple peel residues. The apple peel products or preparations obtained in the invention patents are based on the traditional knowledge and efficacy of apple peel, and the used methods are conventional extraction and separation methods, so that more valuable substances in the apple peel cannot be fully extracted, analyzed and utilized.
Disclosure of Invention
In order to solve at least one of the above technical problems, the technical solution adopted by the present invention is as follows:
the invention provides a preparation method of an apple peel extract, which comprises the following steps:
s1, crushing, drying and degreasing the apple peel to obtain degreased apple peel;
s2, adding an organic solvent-water mixed solvent into the degreased apple peel obtained in the step S1 for ultrasonic extraction, and collecting and combining extracting solutions to obtain a crude extracting solution;
s3, concentrating the crude extract obtained in the step S2 under reduced pressure until the organic solvent is volatilized, and drying to obtain the apple peel organic solvent extract.
S4, dissolving the organic solvent extract obtained in the step S3 in an ethanol-water mixed solvent, performing chromatographic separation by simulated moving bed chromatography with ethanol-water as a mobile phase, collecting an effluent, concentrating under reduced pressure, and drying to obtain the apple peel extract.
The preparation method of the invention is most key to the preparation of the apple peel extract by degreasing, ultrasonic extraction and simulated moving bed chromatographic separation. Wherein the degreasing operation is to further remove fatty acid components in the apple peel, the ultrasonic extraction is to extract the effective components with maximum efficiency under the condition of relatively low temperature, and the simulated moving bed chromatography can enrich the effective components. Unlike conventional extraction methods, the present method takes full account of the effect of temperature on the chemical components of the extract, and therefore all operations are performed at temperatures not higher than 60 ℃.
In some embodiments of the present invention, in the step S1, the degreasing is a degreasing by reflux extraction with petroleum ether or cyclohexane.
In some embodiments of the invention, the reflux extraction degreasing with petroleum ether or cyclohexane is that apple peel is mixed according to a feed-liquid ratio of 1 g: 8 ml-1 g: adding 12ml of petroleum ether or cyclohexane, heating and refluxing for 2-3 times in water bath at the temperature of 30-60 ℃, wherein each time lasts for 10-30 min, and filtering and removing the petroleum ether or cyclohexane to obtain the degreased apple peel.
In some embodiments of the present invention, the organic solvent-water mixed solvent in step S2, wherein the type of the organic solvent refers to any one of formic acid, acetic acid, ethanol, n-propanol, isopropanol and acetone, and the volume percentage of the organic solvent is 30% to 75%;
the material-liquid ratio of the degreased apple peel to the organic solvent-water mixed solvent in the step S2 is 1 g: 8 ml-1 g: 15 ml;
in step S2, the ultrasonic extraction conditions are: ultrasonic extraction is carried out for 1 to 3 times at the temperature of between 20 and 30 ℃, and each time lasts for 10 to 30 min.
In some embodiments of the present invention, the vacuum concentration in the step S3 is vacuum concentration at a temperature of 30 ℃ to 60 ℃ and a pressure of 0.01 to 0.1 MPa.
In some embodiments of the present invention, in step S4, the simulated moving bed is composed of an eluent pump, a raw material pump, an extraction liquid pump, 4-12 chromatographic columns, a multi-channel rotary valve and a computer control system, the extract obtained in step 3 is dissolved by an eluent, and then injected into a raw material port between zones 2 and 3 of the simulated moving bed for separation, the eluent is injected from an inlet of zone 1, the weakly retained component is extracted from an outlet of zone 3, the target component solution is extracted from an extraction port of zone 2 by the extraction liquid pump, and the apple peel extract is obtained after concentration and drying.
In a second aspect, the present invention provides an apple peel extract produced by the method of any one of the first aspects of the present invention.
In a third aspect, the present invention provides a fatty acid synthase inhibitor, the active ingredient of which is the apple peel extract according to the second aspect of the present invention.
Use of an apple peel extract according to the second aspect of the invention or a fatty acid synthase inhibitor according to the third aspect of the invention in the manufacture of a formulation having anti-obesity or anti-cancer properties.
In some embodiments of the invention, the formulation is a pharmaceutical, nutraceutical, or functional cosmetic.
The invention has the advantages of
Compared with the prior art, the invention has the following beneficial effects:
(1) the present invention discloses for the first time that an extract having fatty acid synthase inhibitory activity can be obtained from the apple peel of an edible plant by the method of the present invention and is a natural, plant-derived, non-toxic inhibitor of fatty acid synthase.
(2) Experiments prove that the extract prepared by the invention can obviously inhibit the activity of fatty acid synthase, and the activity of the extract is superior to that of currently known fatty acid synthase inhibitors, such as cerulenin, EGCG, resveratrol and the like. (comparison of the half inhibitory concentrations of the extracts prepared according to the invention with several currently known inhibitors for the inhibitory activity of fatty acid synthase, see FIG. 1).
(3) Because the strength of the activity of the fatty acid synthase directly influences the amount of fat synthesized in biological cells and the capability of tumor cells for synthesizing fatty acid, the activity of the fatty acid synthase is inhibited, so that the process of synthesizing fat in the biological cells can be weakened, and the fat content can be controlled or even reduced; on the other hand, the fatty acid synthesis ability of the tumor cells is reduced, so that the growth of the tumor cells can be inhibited. Therefore, the extract of the invention is used as an active ingredient to prepare various dosage forms of anti-cancer drugs or weight-losing drugs, has the characteristics of obvious efficacy and activity, clear action mechanism, definite target spot and the like, and meets the requirement of pharmaceutical preparation development. (the extract prepared by the invention has obvious inhibition effect on the cell viability of breast cancer cell strains MDA-MB-231 and MCF-7 with high fatty acid synthase expression, see figure 2).
(4) Since apple is a common fruit, apple peel is edible. The extract prepared from the apple peel can be used as a raw material to prepare health-care food, functional cosmetics, common food and the like with the function of resisting cancer or losing weight, and meets the national requirements of the restriction and regulation on the raw materials of the health-care food and the common food, so that the apple peel extract with the function of inhibiting the activity of fatty acid synthase has wider application and development prospects.
Drawings
FIG. 1 is a graph showing the results of comparing the inhibitory activity of fatty acid synthase activity of three extracts prepared in examples 1 to 3 of the present invention with that of three control inhibitors. Wherein, 1 is the extract A of the invention in example 1, 2 is the extract B of the invention in example 2, 3 is the extract C of the invention in example 3, 4 is cerulenin, 5 is EGCG, and 6 is resveratrol; the unit of ordinate is microgram/ml.
FIG. 2 shows the effect of three extracts prepared in examples 1-3 on the activity of MDA-MB-231 and MCF-7 cells of human breast cancer. The vertical axis represents the relative activity of MDA-MB-231 or MCF-7 cells, and the horizontal axes 1-4 represent: DMSO-treated groups, extract A, extract B, extract C (sample concentrations were all 10. mu.g/ml).
Figure 3 shows a schematic diagram of the simulated moving bed chromatography principle.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments.
Examples
The following examples are used herein to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and the disclosures and references cited herein and the materials to which they refer are incorporated by reference.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
The experimental procedures in the following examples are conventional unless otherwise specified. The instruments used in the following examples are, unless otherwise specified, laboratory-standard instruments; the test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
In the examples described below, the fatty acid synthase activity measurement method used is a conventional method for measuring the activity of an enzyme in tumor cells in vitro. That is, after the Cancer cells are disrupted, they are assayed by a standard assay using acetyl coenzyme A and malonyl coenzyme A, NADPH as substrates (Tian W.X.et al, 1985.J.biol.chem.,260: 11375. 11387; Li P., et al, 2014.Mol Cancer 13: 138). The specific method is that cancer cells are inoculated on a cell culture dish (with the diameter of 10cm) at the density of 70-80%, and after the cells adhere to the wall overnight, culture media with different drug concentrations are added. After 24h drug treatment, the adherent cells were washed twice with pre-cooled PBS and transferred to centrifuge tubes with a cell scraper on ice. Cells were sonicated until clear. Collecting the crushed liquid, centrifuging at 13000rpm for 20min at 4 ℃, taking the supernatant, and measuring the enzyme activity. FAS was monitored continuously at 37 ℃ for 120 seconds at 340nm in the UV range using the method reported previously. Wherein 500. mu.L of the assay system contains 25mM KH2PO4-K2HPO4The total volume of the buffer for viability assay (pH 7), 0.25mM EDTA, 0.25mM DTT, 30 μ M acetyl-coa, 100 μ M malonyl-coa, and 350 μ M NADPH was made up to 500 μ L with the supernatant. And (3) absorbing by taking malonyl coenzyme A which is not added as a background, and finally determining the total protein concentration by using a BCA method, wherein each sample is repeatedly determined for more than three times. The viability of FAS was calculated as nmol NADPH consumption per mg total protein per min. All experiments were repeated three times.
The degree of inhibition of Fatty Acid Synthase (FAS) activity is expressed in terms of relative activity, and is calculated as: the fatty acid synthase activity value was determined as a control by adding only the corresponding extraction solvent to the substrate, and this value was represented by A0 and was set to 100%. The test group added with the fatty acid synthase inhibitor extract liquid has the fatty acid synthase activity value of 50% of A0, and the concentration of the inhibitor is IC50 value.
Example 1 extract A having fatty acid synthase inhibitory Activity
This example provides an extract a having fatty acid synthase inhibitory activity, which is prepared by a method comprising the steps of:
(1) crushing and drying apple peel, and then carrying out reflux extraction and degreasing by using petroleum ether, wherein the specific operation is that the apple peel is prepared by mixing the following raw materials according to a material-liquid ratio of 1 g: adding 12ml of petroleum ether, heating and refluxing for 3 times in water bath at 50 ℃ for 15min each time, and filtering and removing the petroleum ether solution to obtain degreased apple peel;
(2) adding the degreased apple peel obtained in the step (1) into a mixture according to a material-liquid ratio of 1 g: adding 30% acetic acid-water solution into 12ml, performing ultrasonic extraction at 20 deg.C for 10min for 3 times, collecting the extractive solutions, and mixing to obtain crude extractive solution;
(3) and (3) concentrating the crude extract obtained in the step (2) under reduced pressure at the temperature of 50 ℃ and the pressure of 0.05MPa until no organic solvent smell exists, continuously concentrating under reduced pressure until the solvent is volatilized, and drying to obtain the apple peel organic solvent extract.
(4) And (4) carrying out chromatographic separation on the organic solvent extract obtained in the step (3) on a simulated moving bed. The simulated moving bed consists of an eluent pump, a raw material pump, an extraction liquid pump, 4-12 chromatographic columns, a multi-channel rotary valve and a computer control system. And (4) after the extract obtained in the step (3) is dissolved by the eluent, injecting the dissolved extract into a raw material port between a zone 2 and a zone 3 of the simulated moving bed for separation, injecting the eluent from an inlet of the zone 1, leading out the weakly retained component from an outlet of the zone 3, pumping the target component solution from an extraction port of the zone 2 by an extract pump, and concentrating and drying the solution to obtain the target extract A.
An appropriate amount of extract A was dissolved in DMSO as a test sample solution, and the test was carried out by the above-mentioned standard method for measuring the fatty acid synthase activity, and the results showed that: the half inhibitory concentration (IC50) of extract A against fatty acid synthase was 1.94. mu.g/mL, indicating that extract A strongly inhibits the activity of fatty acid synthase and that the required effective dose is low.
Example 2 extract B having fatty acid synthase inhibitory Activity
This example provides an extract B having fatty acid synthase inhibitory activity, which is prepared by a method comprising the steps of:
(1) crushing and drying apple peel, and then carrying out reflux extraction and degreasing by using petroleum ether, wherein the specific operation is that the apple peel is prepared by mixing the following raw materials according to a material-liquid ratio of 1 g: adding 10ml of petroleum ether, heating and refluxing for 2 times in water bath at 45 ℃ for 30min each time, and filtering and removing the petroleum ether solution to obtain degreased apple peel;
(2) adding the degreased apple peel obtained in the step (1) into a mixture according to a material-liquid ratio of 1 g: adding 60% acetone-water solution into 10ml, performing ultrasonic extraction at 27 deg.C for 20min for 2 times, collecting the extractive solutions, and mixing to obtain crude extractive solution;
(3) and (3) concentrating the crude extract obtained in the step (2) under reduced pressure at the temperature of 55 ℃ and the pressure of 0.01MPa until no acetone smell exists, continuing concentrating under reduced pressure until the solvent is volatilized, and drying to obtain the organic solvent extract.
(4) And (4) carrying out chromatographic separation on the organic solvent extract obtained in the step (3) on a simulated moving bed. The simulated moving bed consists of an eluent pump, a raw material pump, an extraction liquid pump, 4-12 chromatographic columns, a multi-channel rotary valve and a computer control system. And (4) after the extract obtained in the step (3) is dissolved by the eluent, injecting the dissolved extract into a raw material port between a zone 2 and a zone 3 of the simulated moving bed for separation, injecting the eluent from an inlet of the zone 1, leading out the weakly retained component from an outlet of the zone 3, pumping the target component solution from an extraction port of the zone 2 by an extract pump, and concentrating and drying the solution to obtain a target extract B.
An appropriate amount of extract B was taken as a test sample solution in DMSO, and the test was performed using the above-described standard method for measuring fatty acid synthase activity, and the results showed that: the half inhibitory concentration (IC50) of extract B against fatty acid synthase was 2.19. mu.g/mL, indicating that extract B has a strong inhibitory effect on the activity of fatty acid synthase and that the required effective dose is low.
Example 3 extract C having fatty acid synthase inhibitory Activity
This example provides an extract C having fatty acid synthase inhibitory activity, which is prepared by a method comprising the steps of:
(1) crushing and drying apple peel, and then refluxing and extracting and degreasing by using cyclohexane, wherein the specific operation is that the apple peel is prepared by mixing the following raw materials in a ratio of 1 g: adding cyclohexane into 8ml of the mixture, heating and refluxing for 2 times in water bath at the temperature of 60 ℃, wherein each time is 20min, and filtering and removing cyclohexane solution to obtain degreased apple peel;
(2) adding the degreased apple peel obtained in the step (1) into a mixture according to a material-liquid ratio of 1 g: adding 75% ethanol-water solution into 8ml, performing ultrasonic extraction at 30 deg.C for 30min for 1 time, collecting the extractive solution, and mixing to obtain crude extractive solution;
(3) and (3) concentrating the crude extract obtained in the step (2) under reduced pressure at the temperature of 30 ℃ and the pressure of 0.1MPa until no ethanol smell exists, continuing concentrating under reduced pressure until the solvent is volatilized, and drying to obtain the organic solvent extract.
(4) And (4) carrying out chromatographic separation on the organic solvent extract obtained in the step (3) on a simulated moving bed. The simulated moving bed consists of an eluent pump, a raw material pump, an extraction liquid pump, 4-12 chromatographic columns, a multi-channel rotary valve and a computer control system. And (4) after the extract obtained in the step (3) is dissolved by the eluent, injecting the dissolved extract into a raw material port between a zone 2 and a zone 3 of the simulated moving bed for separation, injecting the eluent from an inlet of the zone 1, leading out the weakly retained component from an outlet of the zone 3, pumping the target component solution from an extraction port of the zone 2 by an extract pump, and concentrating and drying the solution to obtain a target extract C.
An appropriate amount of extract C was dissolved in DMSO as a test sample solution, and the test was performed using the above-described standard method for measuring fatty acid synthase activity, and the results showed that: the half inhibitory concentration (IC50) of extract C against fatty acid synthase was 1.82. mu.g/mL, indicating that extract C has a strong inhibitory effect on the activity of fatty acid synthase and requires a low effective dose.
Example 4 inhibitory Activity of the extracts of examples 1 to 3 on fatty acid synthase
The half inhibitory concentrations of the three currently known inhibitors for the fatty acid synthase inhibitory activity were compared, and the fatty acid synthase activity assay was performed as described above, using the conventional method for measuring the activity of the enzyme in tumor cells in vitro. The three controls were not pre-treated before activity measurements, but were dissolved in DMSO, and the concentrations used in the measurements varied according to their activity.
Control 1: cerulenin.
Cerulenin is a widely used inhibitor of natural Fatty Acid Synthase (FAS). Cerulenin is produced by the fungus Cephalosporium caeruleueus. Cerulenin for use in the present invention is available from Sigma-Aldrich, Cat. 219557.
Control 2: EGCG.
EGCG (epigallocatechin gallate), is the main component of green tea polyphenols, and is a catechin monomer separated from tea leaves. EGCG for use in the present invention is available from Sigma-Aldrich under catalog number 93894.
Control 3: resveratrol.
Resveratrol is present in many plants, especially in grape skins in high amounts. Resveratrol used in the present invention was purchased from Sigma-Aldrich and was available under the catalog number 34092.
The results of comparing the extracts A, B and C having fatty acid synthase inhibitory activity prepared in examples 1 to 3 with the half inhibitory concentrations of the above-mentioned three currently known inhibitors for fatty acid synthase inhibitory activity are shown in FIG. 1. Wherein 1 is the extract A of example 1, 2 is the extract B of example 2, 3 is the extract C of example 3, 4 is cerulenin, 5 is EGCG, 6 is resveratrol; the unit of ordinate is microgram/ml.
As can be seen from figure 1, the extract A, B, C of the invention has obvious inhibition on the activity of FAS, and the effect is stronger than that of the accepted FAS inhibitors of Cerulenin, EGCG and resveratrol.
Example 5 Effect of the extracts of examples 1-3 on the Activity of breast cancer cells MDA-MB-231 and MCF-7
The cell viability was determined by the CCK-8 method. Inoculating the cells to a 96-well plate, making the cell density to be 70% -80% after the cells are attached overnight, removing the culture medium containing the serum, adding the serum-free culture medium containing different drug concentrations, and arranging 6 multiple wells for each concentration of the drug. After the cells are treated with the drug for 24 hours, the culture medium is aspirated, 100 mu L of fresh serum-free culture medium and 10 mu L of CCK-8 solution are added into each well, incubation is carried out for 1-2 hours at 37 ℃, and then the light absorption value under 450nm is detected by using an enzyme-linked immunosorbent assay. Data are the average of 6 replicates, and all experiments were repeated three more times. Relative viability of cells in the apple peel-treated group was calculated as 100% based on the viability of cells in the DMSO-treated group.
The effect of each extract on the viability of breast cancer cells MDA-MB-231 and MCF-7 is shown in FIG. 2. The results show that extracts A, B and C can significantly reduce MDA-MB-231 and MCF-7 cell viability (p < 0.05).
The above results indicate that the apple peel extract of the present invention can be used in the preparation of drugs, health foods or functional cosmetics with weight-loss or anticancer effects.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (10)
1. The preparation method of the apple peel extract is characterized by comprising the following steps:
s1, crushing, drying and degreasing the apple peel to obtain degreased apple peel;
s2, adding an organic solvent-water mixed solvent into the degreased apple peel obtained in the step S1 for ultrasonic extraction, and collecting and combining extracting solutions to obtain a crude extracting solution;
s3, concentrating the crude extract obtained in the step S2 under reduced pressure until the organic solvent is volatilized, and drying to obtain the apple peel organic solvent extract.
S4, dissolving the organic solvent extract obtained in the step S3 in an ethanol-water mixed solvent, performing chromatographic separation by simulated moving bed chromatography with ethanol-water as a mobile phase, collecting an effluent, concentrating under reduced pressure, and drying to obtain the apple peel extract.
2. The method according to claim 1, wherein in the step S1, the degreasing is a degreasing by reflux extraction with petroleum ether or cyclohexane.
3. The production method according to claim 3, characterized in that: the reflux extraction and degreasing by using petroleum ether or cyclohexane is to prepare the apple peel by mixing the following raw materials according to a material-liquid ratio of 1 g: 8 ml-1 g: adding 12ml of petroleum ether or cyclohexane, heating and refluxing for 2-3 times in water bath at the temperature of 30-60 ℃, wherein each time lasts for 10-30 min, and filtering and removing the petroleum ether or cyclohexane to obtain the degreased apple peel.
4. The method according to claim 1, wherein the organic solvent-water mixed solvent in step S2 is any one of formic acid, acetic acid, ethanol, n-propanol, isopropanol, and acetone, and the volume percentage of the organic solvent is 30% to 75%; the material-liquid ratio of the degreased apple peel to the organic solvent-water mixed solvent in the step S2 is 1 g: 8 ml-1 g: 15 ml;
in step S2, the ultrasonic extraction conditions are: ultrasonic extraction is carried out for 1 to 3 times at the temperature of between 20 and 30 ℃, and each time lasts for 10 to 30 min.
5. The method according to claim 1, wherein the vacuum concentration in step S3 is performed under a vacuum at a temperature of 30 ℃ to 60 ℃ and a pressure of 0.01MPa to 0.1 MPa.
6. The preparation method according to claim 5, wherein in step S4, the simulated moving bed comprises an eluent pump, a raw material pump, an extract pump, 4-12 chromatographic columns, a multi-channel rotary valve and a computer control system, the extract obtained in step 3 is dissolved by the eluent, injected into a raw material port between a zone 2 and a zone 3 of the simulated moving bed for separation, the eluent is injected from a zone 1 inlet, the weak retention component is led out from a zone 3 outlet, the target component solution is extracted from a zone 2 extraction port by the extract pump, and the apple peel extract is obtained after concentration and drying.
7. Apple peel extract prepared by the process of any one of claims 1 to 6.
8. A fatty acid synthase inhibitor, the active ingredient of which is the apple peel extract of claim 7.
9. Use of the apple peel extract of claim 7 or the fatty acid synthase inhibitor of claim 8 for the preparation of a formulation with slimming or anticancer function.
10. Use according to claim 9, wherein the preparation is a medicament, a nutraceutical or a functional cosmetic.
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| CN114315955A (en) * | 2021-12-28 | 2022-04-12 | 吉林大学 | Protein purification device and method based on electric membrane filtration technology |
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