CN111759869A - Sea-buckthorn extract and multi-step extraction method and application thereof - Google Patents
Sea-buckthorn extract and multi-step extraction method and application thereof Download PDFInfo
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- CN111759869A CN111759869A CN202010778999.8A CN202010778999A CN111759869A CN 111759869 A CN111759869 A CN 111759869A CN 202010778999 A CN202010778999 A CN 202010778999A CN 111759869 A CN111759869 A CN 111759869A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
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Abstract
The invention discloses a sea-buckthorn extract and a multi-step extraction method and application thereof, wherein sea-buckthorn and ethanol are mixed and ultrasonically extracted, the ethanol is removed to obtain a pre-extract, the pre-extract is extracted by water and petroleum ether, and the rest part after the petroleum ether extraction is extracted by ethyl acetate; the method has the advantages that the sea buckthorn extracts obtained by extracting different solvents have good inhibition effect on the activity of alpha-glucosidase, the full extraction and utilization of active substances of sea buckthorn are realized, the inhibition capability is superior to that of the existing alpha-glucosidase inhibitor acarbose, and the sea buckthorn extracts can be used for preparing alpha-glucosidase inhibitors and products for preventing and/or treating diseases with alpha-glucosidase as a target point, and are safe and efficient.
Description
Technical Field
The invention relates to the field of extraction of effective substances in sea-buckthorn, in particular to a sea-buckthorn extract, a multi-step extraction method and application thereof.
Background
Seabuckthorn (the name of Latin, Hippophae rhamnoides Linn.) is a deciduous shrub of the genus Hippophae of the family Elaeagnaceae, and is drought-resistant and sand-resistant, and can survive in saline-alkali lands, thus being widely used for water and soil conservation. Sea buckthorn is planted in great amount in northwest China for desert greening.
The sea buckthorn fruit is rich in nutrition, and contains active substances such as various vitamins, flavonoids, triterpenoids, oil, fatty acid, phenols, volatile oils, trace elements, phospholipids, 5-hydroxytryptamine and the like, and various amino acids and proteins required by a human body according to measurement.
In addition, the current sea-buckthorn is widely applied to the production of sea-buckthorn fruit juice and sea-buckthorn raw pulp, and in the production process, a large amount of sea-buckthorn pomace is produced after the sea-buckthorn is squeezed, and the sea-buckthorn pomace is usually discarded as waste or produced into dry powder to be used as feed, so that the price is very low. If the utilization value of the seabuckthorn fruit residues can be developed more, the method has great economic significance.
Disclosure of Invention
The invention mainly solves the technical problem of providing a seabuckthorn extract and an extraction method thereof, which can extract an extract with active value from seabuckthorn pomace.
Alpha-glucosidase plays an important role in the absorption process of food, and must be bound thereto for the food to be digested and absorbed. The sugar-reducing mechanism of the alpha-glucosidase inhibitor is that the speed of decomposing starch into glucose is slowed down by inhibiting the alpha-glucosidase on the intestinal mucosa, and the absorption of glucose by the small intestine is reduced and delayed, so that the blood sugar is reduced, and the effect on postprandial hyperglycemia is obvious.
Experiments show that the sea buckthorn extracts obtained by extracting with different solvents have a good inhibition effect on the activity of alpha-glucosidase, the inhibition capability of the sea buckthorn extracts is superior to that of acarbose serving as the existing alpha-glucosidase inhibitor, and the sea buckthorn extracts can be used for preparing the alpha-glucosidase inhibitor, wherein the inhibition effect of the sea buckthorn extracts obtained by extracting with ethyl acetate is optimal.
The alpha-glucosidase inhibitor does not stimulate the secretion of insulin, and the single use of the medicine does not usually cause hypoglycemia, so that the alpha-glucosidase inhibitor can help reduce the fluctuation of blood sugar, can obviously reduce the probability of cardiovascular diseases of diabetics, and has the most obvious effect of improving myocardial infarction. Is one of the few oral hypoglycemic drugs which can interfere with impaired glucose tolerance. The alpha-glucosidase inhibitor has been reported to have definite curative effect on diabetes, and also has certain curative effect on obesity, chronic hepatitis B, AIDS and tumor.
Based on the experimental results of the invention, the invention can provide the following technical scheme:
a method for extracting seabuckthorn fruit extract A comprises the following steps: mixing fructus Hippophae pomace with ethanol, extracting, removing ethanol to obtain pre-extract, extracting the pre-extract with water and petroleum ether, mixing petroleum ether phases, and removing solvent to obtain extract A.
The extraction method of the sea-buckthorn pomace and ethanol mixed extraction is not limited, and any extraction method available in the field can be applied to the method, such as ultrasonic extraction, reflux extraction, ultrahigh pressure extraction, microwave extraction and the like.
In a specific embodiment of the invention, the sea buckthorn pomace and ethanol are mixed and extracted by ultrasonic extraction.
In the present invention, the "extraction of the pre-extract with water and petroleum ether" means that the extraction is performed using a solvent system of water and petroleum ether. If the material to be extracted contains a proper amount of water, only petroleum ether can be added to form a water and petroleum ether solvent system for extraction; if the material to be extracted contains no water or has extremely low water content, petroleum ether and proper amount of water can be added to form a water and petroleum ether solvent system for extraction; and so on.
The invention also provides an extraction method of the seabuckthorn extract B, and by adopting the method, after the pre-extract is extracted by using water and petroleum ether, the residual water phase is A1, the water phase A1 is extracted by ethyl acetate, and the ethyl acetate phase is combined to remove the solvent to obtain the extract B.
The invention also provides an extraction method of the seabuckthorn extract C, the method is adopted, after ethyl acetate is used for extracting the water phase A1, the residual water phase is B1, the water phase B1 is extracted by n-butanol, and the n-butanol phase is combined to remove the solvent to obtain the extract C.
And concentrating the residual water phase after n-butanol extraction under reduced pressure to obtain water fraction D.
In a specific embodiment of the present invention, the sea buckthorn pomace: the feed-liquid ratio of ethanol is 1 g: 1-15 mL, preferably 1 g: 10 mL;
further, the temperature during ultrasonic treatment is 30-80 ℃, and preferably 45 ℃;
further, the ultrasonic time is 1-5 h, preferably 1-3 h, and more preferably 2 h;
further, the ethanol concentration was 95%.
Further, the ultrasonic power is 200W, and the frequency is 40 KHz.
In a particular embodiment of the invention, the pre-extract: the proportion of water is 1 g: 2-15 mL; further 1 g: 3-10 mL; further 1 g: 9.5-10.5 mL;
pre-extraction: the proportion of petroleum ether is 1 g: 2-15 mL; further 1 g: 3-10 mL; further 1 g: 9.5-10.5 mL;
further, water: volume of petroleum ether is 1: 0.5 to 1.5, preferably 1: 1;
further, the pre-extract is dissolved in water and then mixed with petroleum ether for extraction.
In a particular embodiment of the invention, the pre-extract: the proportion of ethyl acetate was 1 g: 2-10 mL; further 1 g: 3-9 mL; further 1 g: 8-9 mL.
Further, the pre-extract: the volume ratio of n-butanol is 1 g: 1-10 mL, further 1 g: 2-8 mL; further 1 g: 7-8 mL.
The invention also provides the seabuckthorn extract obtained by the method.
The invention also provides the application of the seabuckthorn extract in preparing products for preventing and/or treating one or more of impaired glucose tolerance, diabetes, obesity, chronic hepatitis B, AIDS and tumors.
Impaired glucose tolerance, i.e., pre-diabetes, mainly includes Impaired Fasting Glucose (IFG) and Impaired Glucose Tolerance (IGT). Can effectively improve impaired glucose regulation, prevent and/or treat impaired glucose tolerance and/or diabetes, and simultaneously prevent or improve diabetic complications, and can be used for preparing related products.
The invention also provides application of the seabuckthorn extract in preparation of alpha-glycosidase inhibition.
The invention also provides a product which comprises the seabuckthorn extract.
In the present invention, the products include, but are not limited to, pharmaceuticals, nutraceuticals, foods, and the like.
The invention has the beneficial effects that:
(1) the extraction method has simple steps, is simple, convenient and quick, can efficiently obtain the seabuckthorn extract, and realizes the full extraction and utilization of effective substances of the seabuckthorn by a multi-step extraction mode.
(2) The different sea buckthorn extracts obtained by extracting with different solvents are proved by experiments to be capable of inhibiting the activity of the alpha-glucosidase, have better effect than acarbose, can be used for preparing alpha-glucosidase inhibitor products, and can also be used for preparing products for preventing and/or treating diseases taking the alpha-glucosidase as a target point, such as products for treating diseases of impaired glucose tolerance, diabetes, hypertension, obesity, chronic hepatitis B, AIDS, tumors and the like, thereby realizing waste utilization and changing waste into valuable.
Detailed Description
The technical solutions of the present invention are described clearly and completely below, and it is obvious that the described embodiments are some, not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
(1) Accurately weighing 1kg of dried fructus Hippophae residue, placing in a container, adding 10L of ethanol (95%), performing ultrasonic extraction at 45 deg.C for 2 hr with ultrasonic power of 200W and frequency of 40KHz, filtering to obtain filtrate, removing solvent by rotary evaporation, and concentrating under reduced pressure to obtain 405.36g of pre-extract.
(2) 405.36g of the pre-extract is dissolved by 4000mL of water, 4000mL of petroleum ether is added for extraction, organic phases are combined, and 28.24g of petroleum ether part of the sea buckthorn extract is obtained after the solvent is removed by rotary evaporation.
(3) Adding 3500mL of ethyl acetate into the part left after the petroleum ether extraction in the step (2) for extraction, combining organic phases, and removing the solvent by rotary evaporation to obtain 76.57g of ethyl acetate part of the seabuckthorn extract.
(4) Adding 3000mL of n-butanol into the part left after the ethyl acetate extraction in the step (3) for extraction, combining organic phases, and removing the solvent by rotary evaporation to obtain 95.12g of n-butanol part of the seabuckthorn extract, wherein the rest part after the n-butanol extraction is 134.56g of water part.
Example 2
(1) Accurately weighing 1kg of dried fructus Hippophae residue, placing in a container, adding 15L of ethanol (95%), performing ultrasonic extraction at 80 deg.C for 1 hr with ultrasonic power of 200W and frequency of 40KHz, filtering to obtain filtrate, removing solvent by rotary evaporation, and concentrating under reduced pressure to obtain 332.54g of pre-extract.
(2) 332.54g of the pre-extract is dissolved by 2000mL of water, 2000mL of petroleum ether is added for extraction, organic phases are combined, and 13.04g of petroleum ether part of the sea buckthorn extract is obtained after the solvent is removed by rotary evaporation.
(3) Adding 1000mL of ethyl acetate into the part left after the petroleum ether extraction in the step (2) for extraction, combining organic phases, and removing the solvent by rotary evaporation to obtain 57.56g of ethyl acetate part of the seabuckthorn extract.
(4) Adding 800mL of n-butanol into the part left after the ethyl acetate extraction in the step (3) for extraction, combining organic phases, and removing the solvent by rotary evaporation to obtain 75.74g of n-butanol part of the seabuckthorn extract, wherein the rest part after the n-butanol extraction is 108.05g of water part.
Example 3
(1) Accurately weighing 1kg of dried fructus Hippophae pomace, placing in a container, adding 5000mL of ethanol (95%), performing ultrasonic extraction at 30 deg.C for 3 hr with ultrasonic power of 200W and frequency of 40KHz, filtering to obtain filtrate, removing solvent by rotary evaporation, and concentrating under reduced pressure to obtain 270.32g of pre-extract.
(2) 270.32g of the pre-extract is dissolved by 1000mL of water, 1000mL of petroleum ether is added for extraction, organic phases are combined, and 11.02g of petroleum ether part of the sea buckthorn extract is obtained after the solvent is removed by rotary evaporation.
(3) Adding 800mL of ethyl acetate into the part left after the petroleum ether extraction in the step (2) for extraction, combining organic phases, and removing the solvent by rotary evaporation to obtain 49.02g of ethyl acetate part of the seabuckthorn extract.
(4) Adding 600mL of n-butanol into the rest part of the extract obtained in the step (3) after the ethyl acetate extraction for extraction, combining organic phases, and removing the solvent by rotary evaporation to obtain 67.54g of n-butanol part of the seabuckthorn extract, wherein the rest part of the extract obtained after the n-butanol extraction is 92.10g of water part.
Test example 1
1. Preparation of reagents
(1) Preparation of liquid A: NaH2PO4·2H2Weighing 15.603g, metering to 500mL, storing in a brown bottle at 4 ℃ for later use.
(2) Preparing liquid B: na (Na)2HPO4·12H2Weighing 35.822g, metering to 500mL, storing in a brown bottle at 4 ℃ for later use.
(3) Preparation of 0.1M phosphate buffer: 51mL of the solution A and 49mL of the solution B are measured, 100mL of water is added, and the mixture is mixed uniformly to obtain a phosphate buffer solution with the pH value of 6.8, and the phosphate buffer solution is stored in a brown bottle at 4 ℃ for later use.
(4) Yeast α -glucosidase: the enzyme solution was diluted to 20U/mL with 100U/mL of the enzyme stock solution using a phosphate buffer solution (pH 6.8), frozen for use, and diluted to 1U/mL with a phosphate buffer solution (pH 6.8) before use.
(5) Substrate pNPG preparation: 0.3766g of pNPG is precisely weighed, added into a proper amount of sodium phosphate buffer solution for dissolution, and then the volume is determined to 50mL to prepare 25mmol/L mother liquor, and the mother liquor is prepared into 0.5mmol/L with the sodium phosphate buffer solution before use for standby.
(6) Preparing an acarbose inhibitor: 13.9mg of acarbose is precisely weighed, and the volume is adjusted to 1mL by DMSO to prepare 13.9mg/mL for later use.
(7)0.1mol/L of Na2CO3Preparation: weighing 1.06g of Na2CO3Adding a proper amount of distilled water into a beaker for dissolving, fixing the volume to 100mL, and storing at 4 ℃ for later use.
2. Preparation of inhibitors
Accurately weighing 100mg of sea buckthorn extract into a 10mL volumetric flask, dissolving with a small amount of DMSO, fixing the volume to scale, diluting with DMSO to 4 times the concentration of 2.5mg/mL as mother liquor, and diluting to appropriate concentration when in use to obtain a series of sea buckthorn extract inhibitors with different concentrations. When the glycosidase inhibition effect is not good, the mother liquor with larger concentration can be reconfigured to obtain the IC50 value of the test object for the subsequent analysis of the alpha-glycosidase inhibition effect.
3. Experiment of inhibition effect of sea buckthorn extract on alpha-glucosidase activity
The inhibition of the α -glucosidase activity by the different extracts extracted in example 1 was determined separately, as is conventional in the art for determining the inhibition of α -glucosidase activity, as follows:
the test process comprises the following steps: performing tests on a 96-well plate according to the steps shown in Table 1, wherein each group comprises 3 parallel groups, uniformly mixing the reagent to be tested, the phosphate buffer solution and the enzyme solution, preserving the temperature in a constant temperature oscillator at 37 ℃ for 10min, taking out the mixture after the test is finished, adding 50 mu L of 0.5mmol/L pNPG solution, fully and uniformly mixing the mixture, performing water bath reaction at 37 ℃ for 20min, and adding 50 mu L of 0.1mol/L Na after the test is finished2CO3The reaction was stopped in solution at 405nmThe absorbance values are measured at lower part of the time, and the inhibition rate and the IC50 value of each sample α -glucosidase can be calculated according to a formula.
The formula:
wherein Ac is blank absorbance, ABAs is the absorbance of the control group, As is the absorbance of the sample group, ASBThe absorbance values are for sample blanks.
The IC50 values were calculated and the results are shown in Table 1.
TABLE 1 test procedure and method (Unit: μ L)
TABLE 2 IC50 values for alpha-glycosidase at different sites
| Petroleum ether site | Ethyl acetate fraction | N-butanol fraction | Water part | Acarbose | |
| IC50(mg/mL) | 0.401 | 0.053 | 0.471 | 6.824 | 1.273 |
Acarbose is an alpha-glucosidase inhibitor, which can inhibit the activity of various alpha-glucosidase such as maltase, isomaltase, glucoamylase and sucrase, so that the rate of starch decomposition into oligosaccharides such as maltose (disaccharide), maltotriose and dextrin (oligosaccharide) and further glucose decomposition is reduced, and the rate of sucrose decomposition into glucose and fructose is reduced, thereby causing the absorption of glucose in intestinal tract to be slowed down, relieving postprandial hyperglycemia and achieving the effect of reducing blood sugar. Can reduce fasting blood glucose and glycosylated hemoglobin after long-term administration.
The experimental results show that different sea buckthorn extracts obtained by extraction with different solvents can effectively inhibit the activity of alpha-glucosidase, and have better effect than acarbose, wherein the extract extracted with ethyl acetate has the strongest inhibition effect, the half-inhibition concentration of the extract on the alpha-glucosidase is far lower than the half-inhibition concentration of the acarbose on the alpha-glucosidase, the effect is obvious, and the sea buckthorn extracts can be used for preparing alpha-glucosidase inhibitors and medicines for treating diseases taking the alpha-glucosidase as a target point, such as medicines for treating diseases of impaired glucose tolerance, diabetes, hypertension, obesity, chronic hepatitis B, AIDS, tumors and the like.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. The extraction method of the seabuckthorn extract A is characterized by comprising the following steps: mixing fructus Hippophae pomace with ethanol, extracting, removing ethanol to obtain pre-extract, extracting the pre-extract with water and petroleum ether, mixing petroleum ether phases, and removing solvent to obtain extract A.
2. A method for extracting a seabuckthorn extract B is characterized by comprising the following steps: the aqueous phase remaining after extraction by the process of claim 1 is A1, aqueous phase A1 is extracted with ethyl acetate, and the ethyl acetate phases are combined to remove the solvent to give extract B.
3. A method for extracting seabuckthorn fruit extract C is characterized by comprising the following steps: the aqueous phase A1 was extracted with ethyl acetate according to the method of claim 2, the remaining aqueous phase B1 was extracted with n-butanol, and the n-butanol phases were combined to remove the solvent to give extract C.
4. The extraction method according to any one of claims 1 to 3, wherein the ratio of sea buckthorn pomace: the feed-liquid ratio of ethanol is 1 g: 1-15 mL, preferably 1 g: 10 mL;
further, the temperature during ultrasonic treatment is 30-80 ℃, and preferably 45 ℃;
further, the ultrasonic time is 1-5 h, preferably 1-3 h, and more preferably 2 h;
further, the ethanol concentration is 95%;
further, the ultrasonic power is 200W, and the frequency is 40 KHz.
5. The extraction process according to any one of claims 1 to 3, characterized in that the pre-extract: the proportion of water is 1 g: 2-15 mL; further 1 g: 3-10 mL; further 1 g: 9.5-10.5 mL;
pre-extraction: the proportion of petroleum ether is 1 g: 2-15 mL; further 1 g: 3-10 mL; further 1 g: 9.5-10.5 mL;
further, water: volume of petroleum ether is 1: 0.5 to 1.5, preferably 1: 1;
further, the pre-extract is dissolved in water and then mixed with petroleum ether for extraction.
6. Extraction process according to claim 2 or 3, characterized in that the pre-extract: the proportion of ethyl acetate was 1 g: 2-10 mL; further 1 g: 3-9 mL; further 1 g: 8-9 mL;
further, the pre-extract: the volume ratio of n-butanol is 1 g: 1-10 mL, further 1 g: 2-8 mL; further 1 g: 7-8 mL.
7. A sea buckthorn extract characterized by being obtained by extraction according to the method of any one of claims 1 to 6.
8. The use of the hippophae rhamnoides extract of claim 7 in the preparation of a product for preventing and/or treating one or more of impaired glucose tolerance, diabetes, obesity, chronic hepatitis b, aids, and tumors.
9. Use of the hippophae rhamnoides extract of claim 7 for the preparation of alpha-glucosidase inhibition.
10. A product comprising one or more of the hippophae rhamnoides extracts of claim 7.
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