CN116286591A - Extraction method and application of broccoli exosome - Google Patents
Extraction method and application of broccoli exosome Download PDFInfo
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Abstract
The invention provides an extraction method and application of an exosome of broccoli, wherein the extraction method comprises the following steps: (1) Cutting the washed broccoli, uniformly mixing the broccoli with PBS buffer solution, pulping, filtering and taking clear liquid; (2) centrifuging the clear liquid obtained in the step 1 for a plurality of times; (3) Performing ultracentrifugation on the supernatant subjected to the centrifugation in the step 2 for a plurality of times, retaining the precipitate, and re-suspending the precipitate by using PBS buffer; (4) Adding the suspension in the step 3 into sucrose solutions with different concentrations, ultracentrifugating, and collecting and combining bands between sucrose solution layers with different concentrations; (5) Mixing the band obtained in the step 4 with PBS buffer solution, and ultracentrifugating to remove sucrose to obtain precipitate which is the broccoli exosome. Meanwhile, the invention also provides application of the broccoli exosome obtained by the extraction method in preparing a medicine for relieving constipation.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to an extraction method and application of an exosome of broccoli.
Background
Constipation is one of the most common gastrointestinal disorders, being a risk factor for colorectal cancer, irritable bowel syndrome and some other gastrointestinal disorders, with a incidence of between 2% and 30%, especially in elderly people. Clinical medication and surgical treatment of a small number of severe patients require various degrees of side effects and the possibility of recurrence, which are often difficult to ignore, such as cardiac arrhythmias, abdominal cramps, coronary contractions, and even myocardial infarction. And the market is lack of healthy and harmless methods for treating constipation.
The exosomes are extracellular vesicles which are secreted by intracellular multivesicular bodies and are rich in bioactive substances, the diameter is 30-200nm, the main formation mechanism is that secretory cell plasma membranes are invaginated to form early-stage endosomes, the early-stage endosomes are regulated and controlled by in-vivo sorting complexes to form the multivesicular bodies, and the multivesicular bodies are fused with cell membranes to form the exosomes. The bioactive substances in exosomes mainly comprise nucleic acids (mRNA, miRNA, incRNA and circrnas), proteins, lipids and other substances, and exosomes do not contain organelle structures such as mitochondria, endoplasmic reticulum and the like. Exosomes mainly play a role in cell communication, disease diagnosis, organism immune response and the like, and have become research hotspots in the field of life science.
At present, a great deal of research discovers that exosomes can have beneficial effects on the intestinal tract and the organism by regulating intestinal microorganisms. Therefore, it is necessary to find an exosome effective in relieving constipation, and by extracting and trying the effective components, a further research basis is provided for the edible and harmless exosome to intervene in intestinal flora, improve intestinal microenvironment and thereby relieve constipation-related symptoms.
Disclosure of Invention
Aiming at the existing problems, the invention provides an extraction method of the broccoli exosome and application thereof in relieving constipation, which not only solves the problems of the medicine for treating the symptoms without treating the root cause, having resistance and having harm to the body, but also provides a further research foundation for the food therapy of the exosome.
In order to achieve the above purpose, the invention provides a method for extracting an exosome of broccoli and an application thereof, which are characterized in that:
(1) Cleaning and cutting the broccoli raw material, uniformly mixing the broccoli raw material with PBS buffer solution, pulping, filtering, and taking filtrate; wherein the mass ratio of the PBS buffer solution to the broccoli is 1:4-1:2;
(2) Repeating the low-speed centrifugation of the filtrate obtained in the step 1 for a plurality of times, wherein the supernatant is taken after each centrifugation and centrifuged again;
(3) Performing ultracentrifugation on the supernatant obtained after repeated centrifugation in the step 2 for a plurality of times, taking a precipitate, and re-suspending the precipitate with PBS buffer solution to form a suspension;
(4) Adding the suspension obtained in the step 3 into sucrose solution with gradient concentration for ultracentrifugation, and collecting and combining strips among sucrose solution layers with different concentrations, wherein the strips are green strip-shaped suspension parts formed among sucrose solution with different concentrations after centrifugation;
(5) Mixing the strip obtained in the step 4 with PBS buffer solution, and ultracentrifugating to remove sucrose, wherein the obtained precipitate is broccoli exosome;
the ultracentrifugation condition is 100000g-150000g,90min-150min.
Further, the temperature of the PBS buffer solution is 4+/-1 ℃, and the temperature can protect exosomes to the greatest extent from being influenced by temperature factors in the pulping process of the step 1.
Further, the step 2 is repeated for 3 times, the first centrifugation condition is 500g-1500g and 5min-15min, the second centrifugation condition is 3000g-5000g and 30min-50min, and the third centrifugation condition is 8000g-12000g and 40min-80min.
Further, the first centrifugation condition in the step 2 is 1500g and 15min; the second centrifugation condition is 5000g and 50min; the third centrifugation condition is 12000g and 80min; the centrifugal condition has the best effect of removing large particles, fine fragments and partial impurities.
Further, the sucrose solutions in step 4 were 60%, 45%, 30%, 15% and 8%, respectively, and were sequentially added to the centrifuge tube in order of concentration from top to bottom.
Further, the ultracentrifugation conditions of step 3, step 4 and step 5 were 150000g, 120min; step 3, the exosome purification effect is best under the centrifugation condition; step 5 provides the best sucrose removal under this centrifugation condition.
Meanwhile, the invention also provides application of the broccoli exosome obtained by the extraction method in preparing a medicine for relieving constipation.
The beneficial effects of the invention are as follows:
1. the extraction process of the broccoli exosome is simple, the raw materials are easy to obtain, and the cost is low.
2. By optimizing the extraction process flow of the broccoli exosomes, the extraction effect of the broccoli exosomes is effectively improved.
3. The broccoli exosome has the regulation function on relieving constipation, and provides a research basis for exosome diet therapy.
4. The temperature of PBS buffer solution in extracting the broccoli exosomes is 4+/-1 ℃, so that the exosomes can be protected from being influenced by temperature factors in the pulping process to the greatest extent.
Drawings
FIG. 1 is a transmission electron microscope image of a broccoli exosome at 8000 Xmagnification;
FIG. 2 is a transmission electron micrograph of the broccoli exosome at 600000 x magnification;
FIG. 3 is a graph of the nano-flow concentration of the exosomes of broccoli;
FIG. 4 is a graph of particle size of the broccoli source exosomes;
FIG. 5 is a graph showing the change in body weight of various groups of mice in the mouse experiment with the broccoli exosomes;
FIG. 6 is a graph of the moisture content of the stool of each group of mice in a mouse experiment for the broccoli exosomes;
FIG. 7 is a graph of total fecal gram in 6h for each group of mice in the mouse experiment for the broccoli exosomes;
FIG. 8 is a graph showing total stool particle count for each group of mice in a 6h mouse experiment for broccoli exosomes;
FIG. 9 is a graph of the first stool time of groups of mice in a mouse experiment for the broccoli exosomes;
fig. 10 is a graph of the time to first-grain blue stool for each group of mice in the mouse experiment for the broccoli exosomes.
Detailed Description
In order that the above objects and advantages of the invention will be readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
1. Extraction of broccoli exosomes
1.1 specific extraction method
Example 1
(1) Cleaning and cutting the broccoli raw material, uniformly mixing the broccoli raw material with PBS buffer solution at the temperature of 3 ℃, beating the mixture into a paste, and filtering the paste to obtain filtrate; wherein the mass ratio of the PBS buffer solution to the broccoli is 1:2;
(2) Centrifuging the filtrate obtained in the step 1, and centrifuging the supernatant again for 3 times under the conditions of 500g and 5min respectively; 3000g, 30min;8000g, 40min;
(3) Performing ultracentrifugation of 100000g and 90min on the supernatant obtained after 3 times of centrifugation in the step 2, taking precipitate, and re-suspending with PBS buffer to form suspension;
(4) Sequentially adding sucrose solutions with the concentration of 8%, 15%, 30%, 45% and 60% into a centrifuge tube according to the sequence from the concentration to the bottom to form sucrose solutions with gradient concentration, adding the suspension obtained in the step (3) into the sucrose solution, performing ultracentrifugation for 100000g and 90min, and collecting and combining strips among sucrose solution layers with different concentrations;
(5) Mixing the band obtained in the step 4 with PBS buffer solution, performing ultracentrifugation of 100000g and 90min, and removing sucrose to obtain precipitate as the broccoli exosome.
Example 2
(1) Cleaning and cutting the broccoli raw material, uniformly mixing the broccoli raw material with PBS buffer solution at the temperature of 5 ℃, beating the mixture into a paste, and filtering the paste to obtain filtrate; wherein the mass ratio of the PBS buffer solution to the broccoli is 1:3;
(2) Centrifuging the filtrate obtained in the step 1, and centrifuging the supernatant again for 3 times under the conditions of 1000g and 10min respectively; 4000g, 40min;10000g, 60min;
(3) Performing ultracentrifugation of 120000g and 120min on the supernatant obtained after 3 times of centrifugation in the step 2, taking precipitate, and re-suspending with PBS buffer to form suspension;
(4) Sequentially adding sucrose solutions with the concentration of 8%, 15%, 30%, 45% and 60% into a centrifuge tube from top to bottom according to the concentration to form sucrose solutions with gradient concentration, adding the suspension obtained in the step 3 into the sucrose solution, performing ultracentrifugation for 120000g and 120min, and collecting and combining strips among sucrose solution layers with different concentrations;
(5) Mixing the band obtained in the step 4 with PBS buffer solution at 5 ℃, and performing ultracentrifugation for 120min at 120 g to remove sucrose, wherein the obtained precipitate is broccoli exosome.
Example 3
(1) Cleaning and cutting the broccoli raw material, uniformly mixing the broccoli raw material with PBS buffer solution at the temperature of 4 ℃, beating the mixture into a paste, and filtering the paste to obtain filtrate; wherein the mass ratio of the PBS buffer solution to the broccoli is 1:4;
(2) Centrifuging the filtrate obtained in the step 1, and centrifuging the supernatant again for 3 times, wherein the centrifugation conditions of 3 times are 15000g and 15min respectively; 5000g, 50min;12000g, 80min;
(3) Performing ultracentrifugation for 150000g and 150min on the supernatant obtained after 3 times of centrifugation in the step 2, taking precipitate, and re-suspending the precipitate with PBS buffer solution to form suspension;
(4) Sequentially adding sucrose solutions with the concentration of 8%, 15%, 30%, 45% and 60% into a centrifuge tube from top to bottom according to the concentration to form sucrose solutions with gradient concentration, adding the suspension obtained in the step 3 into the sucrose solutions, performing ultracentrifugation for 150000g and 150min, and collecting and combining strips among sucrose solution layers with different concentrations;
(5) Mixing the band obtained in the step 4 with PBS buffer solution, performing ultracentrifugation for 150min at 150000g, and removing sucrose to obtain precipitate as the broccoli source exosome.
1.2 measurement of the content of the exosomes of broccoli
The protein concentration of the broccoli exosome obtained in the above example was measured using BCA protein quantification kit (neoseime biosciences). The measurement principle is that in alkaline medium, protein can make Cu 2+ Reduction to Cu 1+ Then using a unique reagent containing BCA to colorimetrically detect Cu 1+ A purple complex with a strong absorbance at 562nm was formed to determine the content of broccoli exosomes. The above embodiment is not limited to urinaryThe results of the body content test are shown in the following table:
1.3 analysis of exocrine Condition
1) Transmission electron microscope observation
Taking out 20uL of the exosomes obtained in the above example 3, dropwise adding the exosomes onto a copper mesh, adsorbing for 5min, and sucking the floating liquid through filter paper; dripping 20uL of phosphotungstic acid on a copper mesh for 3min, and sucking floating liquid by filter paper; drying for several minutes at normal temperature; and (3) performing electron microscope detection imaging at 100kv to obtain a transmission electron microscope imaging result, as shown in fig. 1 and 2. Fig. 1 is a view of an imaging of an apocrine body shot at a magnification of 8000, and fig. 2 is a view of an imaging of an apocrine body shot at a magnification of 600000.
2) Exosome particle size concentration detection
The exosomes obtained in the example 3 are detected by adopting a nano-flow detector, a standard working curve of the scattered light intensity and the particle size of the particles is established by utilizing a silica standard sphere, and the scattered light intensity of the exosomes to be detected under the same condition can be converted into the particle size, so that the particle size and the concentration distribution of the exosomes to be detected are obtained. The method is applicable when the refractive index of the exosomes to be measured is the same as or similar to the refractive index of the silica particles. As shown in FIG. 3, the abscissa is the lateral astigmatism, which is related to the particle size, with larger values indicating larger particles; the ordinate is fluorescence intensity, so that whether the particles have green fluorescence or not and the intensity of the fluorescence can be detected, and no fluorescence can be judged when the fluorescence intensity is lower than 100; the exosomes extracted by the method are substances which are non-fluorescent, have concentrated particle sizes and are of the same type. As shown in FIG. 4, the abscissa is the size of the measured particles, the ordinate is the number of particles, the concentration of the obtained exosomes is 8.13E+10 particles/ml, and the particle size is mainly distributed at 50-90nm, and the average particle size is 72.42nm.
2. Application of broccoli exosome extracted by the method in preparation of medicine for relieving constipation
The broccoli exosomes used in this example were obtained in example 3 above, and the broccoli exosomes obtained in example 3 above were dispersed into three kinds of broccoli exosomes solutions of different densities of 17.5mg/kg d, 37.5mg/kg d, 75mg/kg d, and stored in a-80 ℃ refrigerator for use.
1) Establishment of mouse model
Effect on loperamide hydrochloride (Pylobutamide hydrochloride, LOP) induced Constipation model (establishment) mice weight change: the 40 SPF-class Balb/c mice were randomly divided into 5 groups after one week of adaptive feeding, including a blank group (NC), a model group (M), a low dose group (LE), a medium dose group (ME) and a high dose group (HE), 8 in each group. The five groups do not carry out any treatment on the aspects of diet drinking water, eat standard feed and drink water freely. The blank control group is respectively filled with the physiological saline once in the morning of 1d to 7d and the physiological saline once in the morning of 8d to 35d and the afternoon; the model group is respectively subjected to the injection of physiological saline for one time in the morning of 1d to 7d and the injection of physiological saline for one time in the morning of 8d to 35d and the injection of loperamide hydrochloride of 10mg/kg in the afternoon for molding; the low dose group is respectively molded by pouring 17.5.5mg/kg of broccoli source exosomes in the morning of 1 d-7 d and pouring 17.5mg/kg of broccoli source exosomes in the morning of 8 d-35 d and pouring 10mg/kg of loperamide hydrochloride in the afternoon; the medium dose group is respectively molded by filling 37.5mg/kg of broccoli source exosomes in the morning of 1 d-7 d and filling 37.5mg/kg of broccoli source exosomes in the morning of 8 d-35 d and filling 10mg/kg of loperamide hydrochloride in the afternoon; the high dose group is respectively molded by filling 75mg/kg of broccoli source exosomes in the morning for 1 d-7 d and filling 75mg/kg of broccoli source exosomes in the morning for 8 d-35 d and filling 10mg/kg of loperamide hydrochloride in the afternoon. Mice were kept under an environment of 12h light, 12h dark, and a circadian light variation cycle and a temperature (22.+ -. 2) DEG C for five weeks.
2) Weight measurement
The weights of the mice were weighed and recorded before the initiation of the molding of the mice and after each cycle, respectively, and the weight gain of each group of mice was normal within 1 to 35 days of the experiment, indicating that the experimental animals were not abnormal, and the results are shown in fig. 5.
2) Fecal moisture content measurement
On day 34, after the completion of the stomach lavage, the mice are singly put into a cage box filled with water absorbing paper, feces are collected, the feces are weighed to be wet weight, and the dry weight is obtained after freeze drying, wherein the water content of the feces is calculated as follows:
compared with the water content of the mouse excrement of the blank control group, the water content of the mouse excrement of the model group is obviously reduced (/ P < 0.0001), wherein the more the obvious effect is shown, which indicates that the LOP can obviously reduce the water content of the mouse excrement; compared with the model group, the water content of the feces of the mice in each administration group is obviously increased, and the medium and low doses are more obvious, which indicates that the dose of the broccoli source exosomes is more beneficial to relieving the reduction effect of LOP on the water content of the feces of the mice, as shown in figure 6.
3) Measurement of mouse defecation
On day 35, mice of each group fasted but normally drunk for 12h except for the blank group, mice of the other groups were perfused with LOP (10 mg/kg BW) for 30min and then perfused with 0.4% Evan blue (10 ml/kg BW) and started to time, each experimental mouse was fed individually, fed with free food and drinking water, and the first bowel movement time, the total 6h bowel movement grain number and the total 6h bowel movement gram number were observed and recorded. As shown in fig. 7, the model group mice showed significantly reduced 6h fecal gram compared to the placebo group mice 6h fecal gram (< 0.0001), indicating that LOP significantly reduced fecal weight; compared with the model group, the gram number of the 6h feces of the mice in each administration group is obviously increased, and the medium dosage is most obvious, which proves that the dosage of the broccoli source exosome is more beneficial to relieving the reducing effect of LOP on the gram number of the 6h feces of the mice. As shown in fig. 8, compared with the 6h fecal particle number of mice in the blank control group, the 6h fecal particle number of mice in the model group is significantly reduced (P < 0.01), which indicates that LOP can significantly reduce the fecal particle number of mice; compared with the model group, the number of 6h feces of mice in each administration group is obviously increased, and the medium dosage is most obvious, which proves that the dosage of the broccoli source exosome is more beneficial to relieving the reduction effect of LOP on the number of 6h feces of mice. As shown in fig. 9, the time of the first stool of the mice in the model group is significantly prolonged (P < 0.001) compared with the time of the first stool of the mice in the blank group, which indicates that the LOP can significantly prolong the time of the first stool of the mice; compared with the model group, the time of the first feces of the mice in each administration group is obviously shortened, and the low dosage is most obvious, which proves that the dosage of the broccoli source exosome is more beneficial to relieving the prolongation of the LOP on the time of the first feces of the mice. As shown in fig. 10, compared with the time of the first blue stool of the mice in the blank control group, the time of the first blue stool of the mice in the model group is significantly prolonged (P < 0.0001), which indicates that the LOP can significantly prolong the time of the first blue stool of the mice; compared with the model group, the time of the first stool of the mice in each administration group is obviously shortened, and the low dosage is most obvious, which proves that the dosage of the broccoli source exosome is more beneficial to relieving the prolongation of the time of the first stool of the mice by LOP.
In the mouse model experiment, the influence of the broccoli exosomes on the mouse body weight, the water content of the mouse feces and the defecation amount of the mouse are shown in the following table:
the results show that the mouse model can effectively relieve constipation after being added into exosomes, and the mouse model has a good constipation relieving effect.
Claims (7)
1. The extraction method of the broccoli exosome is characterized by comprising the following steps:
(1) Cleaning and cutting the broccoli raw material, uniformly mixing the broccoli raw material with PBS buffer solution, pulping, filtering, and taking filtrate; wherein the mass ratio of the PBS buffer solution to the broccoli is 1:4-1:2;
(2) Repeating the low-speed centrifugation of the filtrate obtained in the step 1 for a plurality of times, wherein the supernatant is taken after each centrifugation and centrifuged again;
(3) Performing ultracentrifugation on the supernatant obtained after repeated centrifugation in the step 2 for a plurality of times, taking a precipitate, and re-suspending the precipitate with PBS buffer solution to form a suspension;
(4) Adding the suspension obtained in the step 3 into sucrose solution with gradient concentration for ultracentrifugation, and collecting and combining strips among sucrose solution layers with different concentrations, wherein the strips are green strip-shaped suspension parts formed among the sucrose solution layers with different concentrations after centrifugation;
(5) Mixing the strip obtained in the step 4 with PBS buffer solution, and ultracentrifugating to remove sucrose, wherein the obtained precipitate is broccoli exosome;
the ultracentrifugation condition is 100000g-150000g,90min-150min.
2. The method for extracting an exosome of broccoli according to claim 1, wherein: the temperature of the PBS buffer was 4.+ -. 1 ℃.
3. The method for extracting an exosome of broccoli according to claim 2, wherein: the step 2 is repeated for 3 times, the first centrifugation condition is 500g-1500g and 5min-15min, the second centrifugation condition is 3000g-5000g and 30min-50min, and the third centrifugation condition is 8000g-12000g and 40min-80min.
4. A method for extracting an exosome of broccoli according to claim 3, wherein: the first centrifugation condition in the step 2 is 1500g and 15min; the second centrifugation condition is 5000g and 50min; the third centrifugation condition is 12000g and 80min.
5. The method for extracting an exosome of broccoli according to claim 4, wherein: the sucrose solutions in step 4 were 60%, 45%, 30%, 15% and 8% in concentration, respectively, and were sequentially added to the centrifuge tube in order of concentration from top to bottom.
6. The method for extracting an exosome of broccoli according to claim 5, wherein: the ultracentrifugation conditions of step 3, step 4 and step 5 were 150000g, 120min.
7. Use of the broccoli exosome obtained by the extraction method of any one of claims 1 to 6 for the preparation of a medicament for relieving constipation.
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