CN106798729B - Preparation method and application of nano lipid microbubble - Google Patents
Preparation method and application of nano lipid microbubble Download PDFInfo
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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)
- A61K36/87—Vitaceae or Ampelidaceae (Vine or Grape family), e.g. wine grapes, muscadine or peppervine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The invention provides a preparation method of nano lipid microbubbles, which comprises the steps of extracting grape juice, filtering, extracting concentrated solution, purifying the nano lipid microbubbles and storing the nano lipid microbubbles. The nano lipid microbubble prepared by the invention has the advantage of stable structure and special functions, and can be used for manufacturing medicines and health-care products for protecting intestinal tracts and livers independently or in a compatible way or as an additive component. The preparation method adopts a filtering method, does not use expensive centrifugal equipment, greatly reduces the cost, has simple preparation steps, greatly increases the output, is convenient for realizing industrial production, and simultaneously enables the nano lipid microvesicle to be really put into application.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to a preparation method and application of a nano lipid microbubble.
Background
The preparation of the medicines for protecting the intestinal tract, the liver and the like mainly comprises the preparation of chemical compounds serving as raw materials, Chinese herbal medicine formulas and the like. The preparation of the medicines with the purposes by adopting the plant nano lipid microvesicle as a raw material is not reported at present. The lipid microbubble (1iposome) is an artificial membrane, is a micro vesicle formed by lipid bilayer layers, and has the diameter of 25-1000 nm. The lipid microvesicles prepared by the traditional method have no biological function, but only serve as drug carriers and transgenic carriers, and other drugs or bioactive substances need to be coated on the lipid microvesicles to ensure that the lipid microvesicles have special biological functions.
In addition, most of the existing preparation of vesicles with active functions (similar to lipid microbubble structure) adopts a centrifugation method to prepare from animal cell culture supernatant or body fluid, and has the disadvantages of high instrument price, slow preparation speed, low yield, difficulty in realizing industrial production and difficulty in really forming and applying the nano microbubbles.
Disclosure of Invention
The invention aims to provide a preparation method of nano lipid microbubbles, which does not adopt expensive centrifugal equipment, is easy to prepare, has simple steps and greatly reduces the cost, and is convenient for realizing industrial production. The prepared nanometer lipid microbubble has stable structure, pure nature, no toxic or side effect, easy absorption and high activity, and can be used alone or in combination or as an additive component for liver and intestinal tract protection medicines and health products.
In order to achieve the purpose, the technical scheme of the invention is as follows: a method of preparing a nano lipid microbubble, the method comprising the steps of:
(1) extracting grape juice: cleaning grape raw materials and squeezing juice;
(2) and (3) filtering: filtering the juice obtained in the step (1) to remove residues, and collecting filtered liquid;
(3) extracting a concentrated solution: extracting the concentrated solution enriched with the nano lipid microvesicle from the filtrate obtained in the step (2) by microfiltration and ultrafiltration;
(4) purification of the nano-lipid microbubbles: diluting the concentrated solution enriched with the nano-lipid microvesicles obtained in the step (3), and then obtaining the concentrated solution of the nano-lipid microvesicles without impurities through ultrafiltration;
(5) and (4) carrying out freeze-drying treatment on the nano lipid microbubble concentrated solution obtained in the step (4) to obtain a nano lipid microbubble finished product.
Wherein the nano lipid microvesicles have a lipid membrane structure.
Wherein the grape raw material is at least one of red grape, purple grape and black grape.
Wherein the step (2) specifically comprises: filtering the juice obtained in the step (1) by using a filtering material with 5-12500 meshes to remove residues, and collecting the filtered liquid.
Wherein the step (3) is specifically as follows: filtering the filtrate obtained in the step (2) by adopting a microfiltration membrane with the diameter of 0.1-0.5 mu m, and collecting the microfiltration liquid; and ultrafiltering the microfiltered liquid by adopting an ultrafiltration membrane with the diameter of 0.001-0.05 mu m, removing the filtrate, and collecting the concentrated solution rich in the nano lipid microbubbles.
Wherein, the extraction, the filtration and the extraction of the concentrated solution of the grape juice are all carried out in the environment of 0-37 ℃.
The invention also provides an application of the nano lipid microbubble, and the nano lipid microbubble is applied to medicines and health care products for protecting intestinal tracts and livers independently or in a compatible way or as an additive component.
The filtration of the invention can adopt at least one filtration material with mesh diameter, and the filtration is carried out in sequence according to the mesh diameter from large to small.
In the invention, the nano lipid microvesicle, the grape-derived nano lipid microvesicle, the grape nano lipid microvesicle and the grape-derived active ingredient nano lipid microvesicle all refer to the nano lipid microvesicle prepared by the invention.
The lipid microbubble prepared from grapes has strong biological functions, and has the advantages of improving the bioavailability and lightening toxic and side effects when the medicine is in a nanometer size (1-100nm) and the like, so that the absorption efficiency of the lipid microbubble is higher.
In order to identify the grape-derived nano lipid microvesicles, the samples are observed by a transmission electron microscope, and the results show that the samples have a microvesicle structure of less than 100 nanometers. In order to identify the biological function of the grape-derived nano lipid microbubble, the invention carries out an in vivo experiment of the nano lipid microbubble for protecting the alcoholic liver of the acute mouse and an in vivo experiment of the nano lipid microbubble for protecting the DSS-induced acute enteritis mouse. The results show that the grape nano lipid microbubble prepared by the method can obviously reduce the glutamic-oxaloacetic transaminase (AST), the glutamic-pyruvic transaminase (ALT) and the AST/ALT ratio of an acute alcoholic liver model mouse, and can inhibit the damage of the liver and protect the liver. The nano lipid microbubble can also promote the repair of large intestine and small intestine intestinal tissues and prolong the survival time of mice.
The grape is used as a raw material, and the prepared plant nano lipid microbubble is a membrane structure microbubble with the diameter of 1-100nm, has the advantages of stable structure, pure nature, no toxic or side effect, easy absorption and high activity, has a strong function of protecting the liver and the intestinal tract, and can be used in liver and intestinal tract protection medicines and health care products independently or in a compatible manner or as an additive component.
The preparation method adopts a filtering method, does not use expensive centrifugal equipment, greatly reduces the cost, has simple preparation steps, greatly increases the output which is measured by kg and greatly exceeds the limit that the nanometer microvesicles prepared from animal cell culture supernatant and body fluid by adopting the centrifugal equipment can only produce a few grams, is convenient for realizing industrial production, and simultaneously enables the nanometer lipid microvesicles to be really put into application.
Drawings
Fig. 1 is a particle structure diagram of the grape-derived nanolipid microvesicle of the present invention.
FIG. 2 shows the results of liver function index evaluation of mice protected by grape-derived nanolipid microvesicles.
FIG. 3 shows the pathological evaluation results of mouse liver protected by grape-derived nanolipid microvesicle.
Fig. 4 is the result of evaluating the effect of the grape-derived nano-lipid microvesicles prepared in example 1 on the treatment of an intestinal injury model.
Fig. 5 is the result of evaluating the effect of the grape-derived nano-lipid microvesicles prepared in example 1 on the treatment of an intestinal injury model.
Fig. 6 is the result of evaluating the effect of the grape-derived nano-lipid microvesicles prepared in example 1 on the treatment of an intestinal injury model.
Fig. 7 is the result of evaluating the effect of the grape-derived nano-lipid microvesicles prepared in example 1 on the treatment of an intestinal injury model.
Detailed Description
The technical solution of the present invention will be further described with reference to the following specific examples, but the present invention is not limited to these examples.
The invention is described in detail below with the aid of non-limiting examples. It will be apparent to those skilled in the art that certain changes and modifications may be made in the invention without departing from the spirit and principles of the invention. It is to be understood that such changes and modifications in parallel are intended to be included within the scope of the appended claims.
Example 1
Preparation of grape-derived active ingredient nanometer lipid microbubble.
(1) Squeezing juice from the red grapes cleaned by clear water by adopting a squeezing type squeezer at the temperature of 0-37 ℃;
(2) filtering the squeezed juice by a 5-12000 mesh filter screen (operating at 0-37 ℃), and collecting filtered liquid;
(3) extracting a concentrated solution: carrying out ultrasonic treatment on the filtered liquid, then filtering by adopting a microfiltration membrane of 0.1-0.5 mu m (micrometer), and collecting the microfiltered liquid; ultrafiltering the microfiltered liquid by adopting an ultrafiltration membrane of 0.001-0.05 mu m, removing the filtrate, and collecting the concentrated solution rich in the nano lipid microbubbles;
(4) purification of the nano-lipid microbubbles: diluting the concentrated liquid enriched with lipid microbubbles with Phosphate Buffered Saline (PBS) or normal saline, and then performing ultrafiltration to obtain a nano lipid microbubble concentrated liquid without impurities;
(5) preservation of the nano-lipid microbubbles: and (3) carrying out freeze-drying treatment on the nano lipid microbubble concentrated solution or directly freezing and storing.
The structure of the nano lipid microbubble particles is observed by an electron microscope to identify the structure and biological functions of the nano lipid microbubble prepared by the invention, and the result shows that the nano particles prepared by the method have the nano microbubble (1-100nm) with the membrane structure, as shown in figure 1, the sample particles have the bubble structure of 1-100 nm.
The output of the nano lipid microbubble prepared by the method is measured by kg, which greatly exceeds the limit that the nano microbubble prepared by centrifugation can only produce a few grams in the prior art, so that the nano lipid microbubble can really realize industrialized production and can be really put into application.
Example 2
The grape-derived nanolipid microvesicles prepared in example 1 were used for evaluation of liver-protecting effect: the mouse is gavaged with 50% alcohol to construct an acute mouse alcoholic liver model, and the drug is administrated in a gavage mode every day. After 7 days, the mouse is killed by taking pathological sections of the liver of the mouse to observe the liver injury of the mouse.
In vivo experiments of mice prove that after the mice are fed with alcohol, the liver is damaged, the glutamic-oxaloacetic transaminase (AST) and the glutamic-pyruvic transaminase (ALT) are obviously increased, the AST/ALT ratio is also obviously increased, and the AST, ALT and AST/ALT ratio of the mice fed with alcohol are obviously reduced (figure 2). As shown in figure 2, the liver function indexes of the model mouse of alcoholic liver after being fed with the grape-derived nano lipid microbubbles, namely, the ratios of glutamic-oxaloacetic transaminase (AST) to glutamic-pyruvic transaminase (ALT) to AST/ALT are obviously reduced, which shows that the medicine can effectively improve the liver function and effectively protect the liver.
FIG. 3 is the pathological evaluation result of mouse liver protection by grape-derived nano lipid microbubbles, and the pathological result shows that the stem cells are enlarged and have fuzzy outlines after the mouse is fed with alcohol, and the liver cell damage is reduced after the mouse is fed with the grape-derived nano lipid microbubbles. It provides a new way for clinically treating liver diseases.
Example 3
Evaluation of the effect of the grape-derived active ingredient nano-membrane vesicles prepared in example 1 on treatment of intestinal injury model: (1) adding 3% Dextran Sodium Sulfate (DSS) into the mouse drinking water to construct a mouse acute enteritis model; (2) administration is carried out in a gastric lavage mode every day; (3) a part of mice are sacrificed for 7 days, large intestines and small intestines are taken out, the length of the large intestines is measured, and pathological sections are used for observing the damage of the large intestines and the small intestines of the intestines; (4) another portion of the mice were observed for survival time.
The grape-derived nano lipid microbubble can obviously relieve intestinal injury in a DSS-induced acute Ulcerative Colitis (UC) model mouse. C57BL/6 mice freely drink 3% DSS solution to establish an acute UC model, the experimental group (group C) is administrated by intragastric administration every day, each group is supplemented with 200 mug of grape nano lipid microvesicle every day, the control group (group B) is intragastric administered with solvent (PBS) with the same volume, and the survival time of the mice is observed. Fig. 4 is the result of evaluating the effect of the nano lipid microvesicles for treating intestinal injury model. The result shows that the survival time of the enteritis mice is obviously prolonged after the mice are fed with the grape-derived nano lipid microvesicles.
After 7 days, the mice were sacrificed, the large and small intestines were removed, the length of the large intestine was measured, and the damage of the tissue was observed by HE staining of a cryosection. The control group mice had a shortened large intestine (B), the experimental group had a reduced shortened large intestine (C), and the group A was normal mice given no DSS and only gastric lavage solvent (PBS). Fig. 5 is the result of evaluating the effect of grape-derived nano-lipid microvesicles for treating intestinal injury model. Therefore, the shortening degree of the large intestine of the enteritis mice is reduced after the mice are fed with the grape-derived nano lipid microvesicles.
Fig. 6 is the results of pathological section of grape-derived nano-lipid microvesicles used to treat intestinal injury model. The intestinal mucosa of the large intestine of the mice in the control group is seriously damaged, the intestinal crypt structure is damaged (B), the intestinal mucosa damage of the mice in the experimental group is reduced after the administration, the mice still have the complete crypt structure (C), and the group A is normal mice which are not administered with DSS and only are perfused with gastric lavage solvent (PBS). The pathological tissue section is combined with the HE staining evaluation result to show that the structure of the large intestine of the enteritis mouse is obviously reduced after the rat is fed with the grape-derived nano lipid microvesicle.
Fig. 7 is the results of pathological section of grape-derived nano-lipid microvesicles used to treat intestinal injury model. The intestinal mucosa of the mice in the control group is seriously damaged, the intestinal mucosa is thinned, the intestinal crypt structure is damaged (B), the intestinal mucosa damage of the mice in the experimental group is relieved after administration, the mice still have the complete crypt structure (C), and the group A is normal mice which are not administered with DSS and only infused with gastric lavage solvent (PBS). The pathological tissue section is combined with the HE staining evaluation result to show that the structure of the small intestine of the enteritis mouse is obviously reduced after the rat is fed with the grape-derived nano lipid microvesicle.
The traditional Chinese medicine composition can effectively improve liver functions, protect livers, promote repair of intestinal injury of enteritis model mice and prolong survival time of the mice, and provides a new way for clinically treating liver and intestinal diseases.
The method for preparing the grape nano lipid microbubble has the advantages that:
(1) the prepared grape nano lipid microbubble has stable structure. The nano lipid microbubble prepared by the method has a lipid membrane structure and a stable structure.
(2) The nano lipid microbubble can protect the liver and the intestinal tract, and can be used as an effective treatment medicine or health-care product for liver and intestinal tract diseases.
(3) The preparation method adopts a filtering method, does not use expensive centrifugal equipment, greatly reduces the cost, has simple preparation steps, greatly increases the output, is convenient for realizing industrial production, and simultaneously enables the nano lipid microvesicle to be really put into application.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the inventive concept of the present invention, and these changes and modifications are all within the scope of the present invention.
Claims (4)
1. A method for preparing a nano lipid microbubble, which comprises the following steps:
(1) extracting grape juice: cleaning grape raw materials and squeezing juice;
(2) and (3) filtering: filtering the juice obtained in the step (1) to remove residues, and collecting filtered liquid;
(3) extracting a concentrated solution: extracting the concentrated solution enriched with the nano lipid microvesicle from the filtrate obtained in the step (2) by microfiltration and ultrafiltration;
(4) purification of the nano-lipid microbubbles: diluting the concentrated solution enriched with the nano-lipid microvesicles obtained in the step (3), and then removing impurities through ultrafiltration to obtain a nano-lipid microvesicle concentrated solution;
(5) preservation of the nano-lipid microbubbles: freeze-drying the nano lipid microbubble concentrated solution obtained in the step (4) or directly freezing and storing to obtain a nano lipid microbubble finished product;
the nano lipid microbubble has a lipid membrane structure and the diameter is 1-100 nm;
the step (2) is specifically as follows: filtering the juice obtained in the step (1) by using a filtering material of 5-12500 meshes to remove residues, and collecting the filtered liquid;
the step (3) is specifically as follows: carrying out ultrasonic treatment on the filtrate obtained in the step (2), then filtering by adopting a microfiltration membrane with the diameter of 0.1-0.5 mu m, and collecting the microfiltration liquid; and ultrafiltering the microfiltered liquid by adopting an ultrafiltration membrane with the diameter of 0.001-0.05 mu m, removing the filtrate, and collecting the concentrated solution rich in the nano lipid microbubbles.
2. The nanolipid microbubble of claim 1, wherein the grape material is at least one of red grapes, purple grapes, and black grapes.
3. The method of claim 1, wherein the steps of extracting the juice, filtering and extracting the concentrated solution are performed at 0-37 ℃.
4. Use of the nanolipid microvesicles of any one of claims 1 to 3 in the preparation of a medicament and health product for protecting the intestine and liver.
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| WO2013070324A1 (en) * | 2011-11-07 | 2013-05-16 | University Of Louisville Research Foundation, Inc. | Edible plant-derived microvesicle compositions for diagnosis and treatment of disease |
| US20170035700A1 (en) * | 2014-04-11 | 2017-02-09 | University Of Louisville Research Foundation, Inc. | Coated edible plant-derived microvesicle compositions and methods for using the same |
| US9717733B2 (en) * | 2014-08-13 | 2017-08-01 | University Of Louisville Research Foundation, Inc. | Edible plant-derived microvesicle compositions including conjugated therapeutic agents and methods for using the same |
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|---|---|---|---|---|
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