CN114601859B - Blueberry extract, preparation method thereof and application of blueberry extract in preparation of medicines with blood lipid reducing and/or weight losing effects - Google Patents
Blueberry extract, preparation method thereof and application of blueberry extract in preparation of medicines with blood lipid reducing and/or weight losing effects Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 32
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- 239000008280 blood Substances 0.000 title claims abstract description 26
- 230000001603 reducing effect Effects 0.000 title claims abstract description 26
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- 150000002632 lipids Chemical class 0.000 title description 14
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- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 66
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- 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)
- A61K36/45—Ericaceae or Vacciniaceae (Heath or Blueberry family), e.g. blueberry, cranberry or bilberry
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation or decoction
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- A61K2236/50—Methods involving additional extraction steps
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Abstract
The invention relates to the technical field of biological medicines, and particularly discloses a blueberry extract and a preparation method thereof and application of the blueberry extract in preparation of medicines with blood fat reducing and/or weight losing effects. The preparation method of the blueberry extract comprises the following steps: (1) Taking fresh blueberry fruits, and then adding water at 0-5 ℃ for crushing to obtain crushed mixed solution; (2) Carrying out solid-liquid separation on the crushed mixed liquid, and taking the liquid to obtain blueberry crushed liquid; (3) Adding ethyl acetate into the blueberry crushing liquid for extraction; (4) And eluting and separating the ethyl acetate extract by adopting a preparative liquid chromatograph, concentrating and drying the eluent to obtain the blueberry extract. Research shows that the blueberry extract prepared by the method has excellent blood fat reducing and weight losing effects; therefore, the blueberry extract provided by the invention can be further used as an effective component for developing medicines with the effects of reducing blood fat and/or losing weight.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a blueberry extract and a preparation method thereof and application thereof in preparing medicines with blood fat reducing and/or weight losing effects.
Background
Obesity, a degree of significant overweight, and excessive fat layer thickness, especially excessive triglyceride accumulation, results. With obvious changes of life quality and life style of people, the incidence rate of obesity is high, the growing situation is more obvious, and the obesity is caused by excessive fat accumulation in a body due to excessive food intake or change of body metabolism, and is a disease promoting factor of most metabolic chronic diseases. It is found that obese people are more prone to suffering from hyperlipidemia, hypertension and other diseases. In addition to lifestyle changes, diet control, exercise, drugs, surgery, etc., clinical attempts have been made to use various drugs for the treatment of obesity (e.g., thyroid extract, 2-nitrophenol, and amphetamine), but the efficacy and safety are not ideal.
Blueberry is also called cowberry fruit and is rich in nutrition, and is called as fruit queen; the fruit has attractive color and unique flavor; it also has vision improving, autoimmune function enhancing, anticancer, memory improving, antioxidant, and antiaging effects. In addition, the blueberry has a certain blood lipid reducing effect although reported in the prior art; however, the prior art is not fully studied on the hypolipidemic effect or the slimming effect of blueberries, and further research on the substance basis of the hypolipidemic effect or the slimming effect is still needed by the person skilled in the art. If an effective part or a monomer compound with excellent blood fat reducing or weight losing effects can be developed from blueberries, the application value is important.
Disclosure of Invention
In view of the above, the invention firstly provides a preparation method of a blueberry extract, and researches show that the blueberry extract prepared by the method has an excellent blood lipid reducing effect.
The technical scheme of the invention is as follows:
the preparation method of the blueberry extract is characterized by comprising the following steps of:
(1) Taking fresh blueberry fruits, and then adding water at 0-5 ℃ for crushing to obtain crushed mixed liquid;
(2) Carrying out solid-liquid separation on the crushed mixed liquid, and taking the liquid to obtain blueberry crushed liquid;
(3) Adding ethyl acetate into the blueberry crushed liquid for extraction to obtain ethyl acetate extract, concentrating and drying the ethyl acetate extract to obtain ethyl acetate extract;
(4) And eluting and separating the ethyl acetate extract by adopting a preparative liquid chromatograph, concentrating and drying the eluent to obtain the blueberry extract.
The inventor shows in the research that the blueberry extract prepared by extracting the blueberry crushed liquid with ethyl acetate and then using a preparative liquid chromatograph has excellent blood fat reducing and weight losing effects.
Preferably, the water in the step (1) is water at 1-3 ℃.
Preferably, the dosage ratio of the fresh blueberry fruits to the water in the step (1) is 1 kg:6-12L.
Most preferably, the dosage ratio of the fresh blueberry fruit to the water in the step (1) is 1kg:8L.
Preferably, in the step (2), the concrete method for carrying out solid-liquid separation on the crushed mixed liquid is as follows: centrifuging the crushed mixed solution, filtering the supernatant, and taking the filtrate to obtain the blueberry crushed solution.
Preferably, in the step (3), the volume ratio of the blueberry crushing liquid to the ethyl acetate is 1:0.5-3;
further preferably, in the step (3), the volume ratio of the blueberry breaking solution to the ethyl acetate is 1:1-2.
Preferably, the specific conditions of the preparative liquid chromatograph in step (4) are: the chromatographic column adopts a reversed phase preparation type chromatographic column; the detection wavelength is 300-330 nm; the column temperature is 20-30 ℃; and (3) taking an aqueous solution containing 0.05-0.2% by volume of trifluoroacetic acid as a mobile phase A, and taking a methanol solution containing 0.05-0.2% by volume of trifluoroacetic acid as a mobile phase B, and performing gradient elution.
Further preferably, the specific conditions of the preparative liquid chromatograph in step (4) are: the chromatographic column adopts C18 reversed phase preparation type chromatographic column; the detection wavelength is 315nm; the column temperature was 25 ℃; the gradient elution was carried out with an aqueous solution containing 0.1 vol% trifluoroacetic acid as mobile phase A and a methanol solution containing 0.1 vol% trifluoroacetic acid as mobile phase B.
Preferably, the specific conditions of the gradient elution include: 0-3 min, wherein the volume change of the mobile phase B is 0-8%; 3-11 min, wherein the volume change of the mobile phase B is 8-22%; 11-19 min, wherein the volume of the mobile phase B is 22% and is kept unchanged; collecting eluent eluted in a time period of 15.9-18.3 min, concentrating and drying to obtain the blueberry extract.
The inventor needs to emphasize that the method of the invention is adopted to extract the blueberry crushed liquid by ethyl acetate and then prepare the blueberry extract by a preparative liquid chromatograph; the gradient elution condition of the preparative liquid chromatograph plays a decisive role in preparing blueberry extract with excellent blood fat reducing and weight losing effects; the blueberry extracts prepared under different gradient elution conditions have quite different blood fat reducing and weight losing effects; the inventor has shown through a great deal of experimental study that the blueberry extract prepared under the gradient elution condition of the preparation type liquid chromatograph has the functions of reducing blood fat and losing weight far higher than blueberry extracts prepared under other gradient elution conditions; the existing research shows that only the blueberry extract prepared under the gradient elution condition of the preparation type liquid chromatograph provided by the invention has excellent blood fat reducing and weight losing effects.
The invention also provides a blueberry extract prepared by the preparation method.
Preferably, the blueberry extract is applied to the preparation of medicines with the effects of reducing blood fat and/or losing weight.
The beneficial effects are that: the invention provides a brand-new preparation method of a blueberry extract; firstly, extracting blueberry crushed liquid by using ethyl acetate, and then preparing blueberry extract by using a preparation type liquid chromatograph; research shows that the blueberry extract prepared under the gradient elution condition of the preparation type liquid chromatograph has excellent blood fat reducing and weight losing effects; therefore, the blueberry extract provided by the invention can be further used as an effective component for developing medicines with the effects of reducing blood fat and/or losing weight.
Detailed Description
The technical scheme of the present invention will be clearly and completely described in the following examples. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 preparation of blueberry extract
(1) Taking fresh blueberry fruits, and then adding water at the temperature of 2 ℃ for crushing to obtain crushed mixed liquid; wherein the dosage ratio of the fresh blueberry fruits to the water is 1kg:8L;
(2) Centrifuging the crushed mixed solution, filtering the supernatant, and taking filtrate to obtain the blueberry crushed solution;
(3) Adding ethyl acetate into the blueberry crushed liquid for extraction to obtain ethyl acetate extract, concentrating and drying the ethyl acetate extract to obtain ethyl acetate extract; wherein, the volume ratio of the blueberry crushing liquid to the ethyl acetate is 1:1;
(4) Eluting and separating the ethyl acetate extract by adopting a preparative liquid chromatograph, concentrating and drying the eluent to obtain a blueberry extract;
the specific conditions of the preparative liquid chromatograph in the step (4) are as follows: the chromatographic column adopts C18 reversed phase preparation type chromatographic column; the detection wavelength is 315nm; the column temperature was 25 ℃; performing gradient elution by taking an aqueous solution containing 0.1 volume percent of trifluoroacetic acid as a mobile phase A and taking a methanol solution containing 0.1 volume percent of trifluoroacetic acid as a mobile phase B;
specific conditions of the gradient elution include: 0-3 min, wherein the volume change of the mobile phase B is 0-8%; 3-11 min, wherein the volume change of the mobile phase B is 8-22%; 11-19 min, wherein the volume of the mobile phase B is 22% and is kept unchanged; collecting eluent eluted in a time period of 15.9-18.3 min, concentrating and drying to obtain the blueberry extract.
Comparative example 1 preparation of blueberry extract
(1) Taking fresh blueberry fruits, and then adding water at the temperature of 2 ℃ for crushing to obtain crushed mixed liquid; wherein the dosage ratio of the fresh blueberry fruits to the water is 1kg:8L;
(2) Centrifuging the crushed mixed solution, filtering the supernatant, and taking filtrate to obtain the blueberry crushed solution;
(3) Adding ethyl acetate into the blueberry crushed liquid for extraction to obtain ethyl acetate extract, concentrating and drying the ethyl acetate extract to obtain an ethyl acetate extract, namely the blueberry extract; the volume ratio of the blueberry crushing liquid to the ethyl acetate is 1:1.
The difference between comparative example 1 and example 1 is that only the blueberry crushed solution is extracted by ethyl acetate, and then the ethyl acetate extract is taken to obtain the blueberry extract; in example 1, the ethyl acetate extract was further concentrated and enriched with the active ingredient by preparative liquid chromatograph to obtain blueberry extract.
Comparative example 2 preparation of blueberry extract
(1) Taking fresh blueberry fruits, and then adding water at the temperature of 2 ℃ for crushing to obtain crushed mixed liquid; wherein the dosage ratio of the fresh blueberry fruits to the water is 1kg:8L;
(2) Centrifuging the crushed mixed solution, filtering the supernatant, and taking filtrate to obtain the blueberry crushed solution;
(3) Adding ethyl acetate into the blueberry crushed liquid for extraction to obtain ethyl acetate extract, concentrating and drying the ethyl acetate extract to obtain ethyl acetate extract; wherein, the volume ratio of the blueberry crushing liquid to the ethyl acetate is 1:1;
(4) Eluting and separating the ethyl acetate extract by adopting a preparative liquid chromatograph, concentrating and drying the eluent to obtain a blueberry extract;
the specific conditions of the preparative liquid chromatograph in the step (4) are as follows: the chromatographic column adopts C18 reversed phase preparation type chromatographic column; the detection wavelength is 315nm; the column temperature was 25 ℃; performing gradient elution by taking an aqueous solution containing 0.1 volume percent of trifluoroacetic acid as a mobile phase A and taking a methanol solution containing 0.1 volume percent of trifluoroacetic acid as a mobile phase B;
specific conditions of the gradient elution include: 0-11 min, wherein the volume change of the mobile phase B is 0-22%; 11-19 min, wherein the volume of the mobile phase B is 22% and is kept unchanged; collecting eluent eluted in a time period of 15.9-18.3 min, concentrating and drying to obtain the blueberry extract.
Comparative example 2 differs from example 1 in that the gradient elution conditions of the preparative liquid chromatograph are different; the gradient elution conditions for comparative example 2 were: 0-11 min, wherein the volume change of the mobile phase B is 0-22%; 11-19 min, wherein the volume of the mobile phase B is 22% and is kept unchanged; and collecting the eluent eluted in the time period of 15.9-18.3 min. Whereas the gradient elution conditions for example 1 were: 0-3 min, wherein the volume change of the mobile phase B is 0-8%; 3-11 min, wherein the volume change of the mobile phase B is 8-22%; 11-19 min, wherein the volume of the mobile phase B is 22% and is kept unchanged; and collecting the eluent eluted in the time period of 15.9-18.3 min.
Comparative example 3 preparation of blueberry extract
(1) Taking fresh blueberry fruits, and then adding water at the temperature of 2 ℃ for crushing to obtain crushed mixed liquid; wherein the dosage ratio of the fresh blueberry fruits to the water is 1kg:8L;
(2) Centrifuging the crushed mixed solution, filtering the supernatant, and taking filtrate to obtain the blueberry crushed solution;
(3) Adding ethyl acetate into the blueberry crushed liquid for extraction to obtain ethyl acetate extract, concentrating and drying the ethyl acetate extract to obtain ethyl acetate extract; wherein, the volume ratio of the blueberry crushing liquid to the ethyl acetate is 1:1;
(4) Eluting and separating the ethyl acetate extract by adopting a preparative liquid chromatograph, concentrating and drying the eluent to obtain a blueberry extract;
the specific conditions of the preparative liquid chromatograph in the step (4) are as follows: the chromatographic column adopts C18 reversed phase preparation type chromatographic column; the detection wavelength is 315nm; the column temperature was 25 ℃; performing gradient elution by taking an aqueous solution containing 0.1 volume percent of trifluoroacetic acid as a mobile phase A and taking a methanol solution containing 0.1 volume percent of trifluoroacetic acid as a mobile phase B;
specific conditions of the gradient elution include: 0-3 min, wherein the volume change of the mobile phase B is 0-15%; 3-11 min, wherein the volume change of the mobile phase B is 15-25%; 11-19 min, wherein the volume of the mobile phase B is 25% and is kept unchanged; collecting eluent eluted in a time period of 15.9-18.3 min, concentrating and drying to obtain the blueberry extract.
Comparative example 3 differs from example 1 in that the gradient elution conditions of the preparative liquid chromatograph are different; the gradient elution conditions for comparative example 3 were: 0-3 min, wherein the volume change of the mobile phase B is 0-15%; 3-11 min, wherein the volume change of the mobile phase B is 15-25%; 11-19 min, wherein the volume of the mobile phase B is 25% and is kept unchanged; and collecting the eluent eluted in the time period of 15.9-18.3 min. Whereas the gradient elution conditions for example 1 were: 0-3 min, wherein the volume change of the mobile phase B is 0-8%; 3-11 min, wherein the volume change of the mobile phase B is 8-22%; 11-19 min, wherein the volume of the mobile phase B is 22% and is kept unchanged; and collecting the eluent eluted in the time period of 15.9-18.3 min.
Experimental example 1 evaluation of lipid-lowering and weight-reducing Activity
The method for constructing the high-fat obesity model mouse model comprises the following steps: SPF grade 6 week old 16-20 g male C57BL/6 male mice are taken; after 5 days of adaptive feeding, mice were fed a high-fat obesity model feed (TP 23300 type 60% high-fat obesity model feed) to induce obesity models, and the obesity model feed was fed for week 4 (day 28) to construct high-fat obesity model mice.
The high fat and high sugar obese model mice were randomly divided into 5 groups (10 animals per group) which were respectively a model group (no administration), an experimental group 1 (blueberry extraction prepared by administration example 1), a control group 1 (blueberry extraction prepared by administration comparison 1), a control group 2 (blueberry extraction prepared by administration comparison 2), a control group 3 (blueberry extraction prepared by administration comparison 3), and fed with the high fat obese model feed continuously. Another 10 normal mice were fed normal feed as a blank control. Dissolving blueberry extract to be tested in water according to the ratio of 2.0 mg/mL, and carrying out gastric lavage administration on the blueberry extract according to the administration dosage of 150 mg/(kg.d) of the experimental group and the control group; the blank and model groups were filled with an equal amount of water. After 8 weeks of continuous administration, all mice were fasted with 12 h, anesthetized with isoflurane, and serum was isolated by centrifugation at 4000 rpm through orbital vein Cong Caixie, and the serum was assayed for triglyceride, total cholesterol, low density lipoprotein sterol, and high density lipoprotein sterol according to the kit instructions.
The experimental results are shown in Table 1.
TABLE 1 Experimental results of lipid-lowering and weight-losing Activity of blueberry extract of the invention
* Compared with the obesity model group,p ＜ 0.05， ** compared with the obesity model group,p ＜ 0.01； # compared with the blank group,p ＜ 0.05， ## in contrast to the blank group,p ＜ 0.01。
as can be seen from the experimental data in table 1, the serum lipid levels of triglyceride, total cholesterol, low density lipoprotein sterol, etc. in the mice of the model group are significantly higher than those in the blank group; this indicates that the high fat obesity model mouse model was successfully constructed.
As can be seen from the experimental data in table 1, the serum lipid levels of triglyceride, total cholesterol, and low-density lipoprotein in the mice of the experimental group 1 are far lower than those of the mice of the control group 1; and the lipid levels of triglyceride, total cholesterol, low-density lipoprotein sterol and the like in the serum of the mice in the experimental group 1 tend to be close to those of the blank group; this illustrates: the blueberry extract prepared by extracting the blueberry crushing liquid with ethyl acetate and then using a preparative liquid chromatograph has far better blood lipid reducing and weight losing effects than the blueberry extract obtained by only extracting with ethyl acetate, and has very excellent blood lipid reducing and weight losing effects.
As can be seen from the experimental data in table 1, the improvement of the lipid levels of triglyceride, total cholesterol, and low-density lipoprotein sterol in the serum of the mice in the control group 2 is not significant compared with the control group 1; the serum lipid levels of triglyceride, total cholesterol, and low-density lipoprotein sterol in the control group 3 mice were not improved as compared with the control group 1; and the serum lipid levels of triglyceride, total cholesterol, low density lipoprotein sterol and the like in the control group 2 and 3 mice are far higher than those in the experimental group 1. This illustrates: the method is characterized in that blueberry crushed liquid is extracted by ethyl acetate and then a blueberry extract is prepared by a preparative liquid chromatograph; the gradient elution condition of the preparative liquid chromatograph plays a decisive role in preparing blueberry extract with excellent blood fat reducing and weight losing effects; the gradient elution conditions are improperly selected, so that the blood lipid reducing and weight losing effects of the prepared blueberry extract can not be effectively improved, and the blood lipid reducing and weight losing effects of the blueberry extract can be possibly reduced; the experimental result shows that the blueberry extract prepared under the gradient elution condition of the preparation type liquid chromatograph has the functions of reducing blood fat and losing weight which are far higher than blueberry extracts prepared under other gradient elution conditions; and only the blueberry extract prepared under the gradient elution condition of the preparation type liquid chromatograph provided by the invention has excellent blood fat reducing and weight losing effects, and the blueberry extract prepared under the gradient elution condition of other preparation type liquid chromatographs does not show excellent blood fat reducing and weight losing effects.
Finally, it is noted that the above-mentioned preferred embodiments are only intended to illustrate rather than limit the invention, and that, although the invention has been described in detail by means of the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (8)
1. The application of the blueberry extract in preparing the medicament with the blood fat reducing effect is characterized in that the preparation method of the blueberry extract comprises the following steps:
(1) Taking fresh blueberry fruits, and then adding water at 0-5 ℃ for crushing to obtain crushed mixed solution;
(2) Carrying out solid-liquid separation on the crushed mixed liquid, and taking the liquid to obtain blueberry crushed liquid;
(3) Adding ethyl acetate into the blueberry crushed liquid for extraction to obtain ethyl acetate extract, concentrating and drying the ethyl acetate extract to obtain ethyl acetate extract;
(4) Eluting and separating the ethyl acetate extract by adopting a preparative liquid chromatograph, concentrating and drying the eluent to obtain a blueberry extract;
the specific conditions of the preparative liquid chromatograph in the step (4) are as follows: the chromatographic column adopts a reversed phase preparation type chromatographic column; the detection wavelength is 300-330 nm; the column temperature is 20-30 ℃; taking an aqueous solution containing 0.05-0.2 volume percent of trifluoroacetic acid as a mobile phase A, and taking a methanol solution containing 0.05-0.2 volume percent of trifluoroacetic acid as a mobile phase B for gradient elution;
specific conditions of the gradient elution include: 0 to 3min, the volume change of the mobile phase B is 0 to 8 percent; 3-11 min, the volume change of the mobile phase B is 8-22%; the volume of the mobile phase B is 22% and is kept unchanged after 11-19 min; collecting the eluent eluted in the time period of 15.9-18.3 min, concentrating and drying to obtain the blueberry extract.
2. The use according to claim 1, wherein the water in step (1) is water at 1-3 ℃.
3. The use according to claim 1, wherein the ratio of fresh blueberry fruit to water in step (1) is 1 kg:6-12L.
4. Use according to claim 3, characterized in that the ratio of fresh blueberry to water in step (1) is 1kg:8l.
5. The use according to claim 1, wherein the specific method of solid-liquid separation of the crushed mixture in step (2) is: centrifuging the crushed mixed solution, filtering the supernatant, and taking the filtrate to obtain the blueberry crushed solution.
6. The use according to claim 1, wherein the volume ratio of the blueberry breaking liquid to the ethyl acetate in step (3) is 1:0.5-3.
7. The use according to claim 6, wherein the volume ratio of the blueberry breaking liquid to the ethyl acetate in step (3) is 1:1-2.
8. The use according to claim 1, wherein the specific conditions of the preparative liquid chromatograph in step (4) are: the chromatographic column adopts C18 reversed phase preparation type chromatographic column; the detection wavelength is 315nm; the column temperature was 25 ℃; the gradient elution was carried out with an aqueous solution containing 0.1 vol% trifluoroacetic acid as mobile phase A and a methanol solution containing 0.1 vol% trifluoroacetic acid as mobile phase B.
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