Extraction method and application of brassica napus exosome

Abstract

The application provides a method for extracting and application of broccoli exosome, and the extraction method is as follows: (1) cut the washed broccoli into pieces, mix with PBS buffer, beat pulp, filter, and take the supernatant; (2) centrifuge the supernatant obtained in step 1 for multiple times; (3) ultracentrifuge the supernatant obtained in step 2 after multiple centrifugations, reserve the precipitate, and resuspend with PBS buffer; (4) ultracentrifuge the suspension in step 3 in sucrose solutions with different concentrations, and collect and combine the bands between different concentration sucrose solution layers; (5) mix the bands obtained in step 4 with PBS buffer, ultracentrifuge, remove sucrose, and the obtained precipitate is broccoli exosome. Meanwhile, the application also provides application of the broccoli exosome obtained by the above extraction method in preparation of a medicine for relieving constipation.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a method for extracting broccoli exosomes and its application. Background Technology

[0002] Constipation is one of the most common gastrointestinal disorders and a risk factor for colorectal cancer, irritable bowel syndrome, and several other gastrointestinal diseases, with an incidence rate between 2% and 30%, especially among the elderly. Clinical drug treatments and surgical treatments required in a few severe cases can both cause varying degrees of side effects and the possibility of recurrence. These adverse reactions are often difficult to ignore, such as arrhythmias, abdominal cramps, coronary artery constriction, and even myocardial infarction. Currently, there is a lack of healthy and harmless treatments for constipation on the market.

[0003] Exosomes are extracellular vesicles rich in bioactive substances, secreted by intracellular multivesicular bodies. They range in diameter from 30 to 200 nm. Their main formation mechanism involves the invagination of the secretory cell membrane to form early endosomes. These early endosomes are then regulated by in vivo sorting complexes to form multivesicular bodies, which eventually fuse with the cell membrane to form exosomes. The bioactive substances in exosomes mainly include nucleic acids (mRNA, miRNA, incRNA, and circRNA), proteins, and lipids. Exosomes do not contain organelles such as mitochondria or endoplasmic reticulum. Exosomes play a crucial role in cell communication, disease diagnosis, and immune responses, and have become a research hotspot in the life sciences.

[0004] Numerous studies have found that exosomes can have beneficial effects on the gut and the body by regulating gut microbiota. Therefore, it is necessary to seek an effective exosome for relieving constipation. By extracting and testing its active ingredients, we can provide a further research basis for edible and harmless exosome intervention in the gut microbiota, improving the gut microenvironment, and thus alleviating constipation-related symptoms. Summary of the Invention

[0005] To address existing problems, this invention provides a method for extracting broccoli exosomes and their application in relieving constipation. This method not only solves the problems of constipation medications that only treat the symptoms but not the root cause, leading to resistance and harm to the body, but also provides a foundation for further research on exosome-based dietary therapy.

[0006] To achieve the above objectives, this invention provides a method for extracting broccoli exosomes and its application, which is characterized by:

[0007] (1) Wash and chop the broccoli raw material, mix it with PBS buffer, blend it into a paste, filter it, and take the filtrate; the mass ratio of PBS buffer to broccoli is 1:4-1:2.

[0008] (2) Repeat the low-speed centrifugation of the filtrate obtained in step 1 several times, and take the supernatant after each centrifugation and centrifuge it again.

[0009] (3) Centrifuge the supernatant obtained after repeating step 2 several times, take the precipitate, and resuspend it with PBS buffer to form a suspension.

[0010] (4) The suspension obtained in step 3 is added to a sucrose solution with a gradient concentration and centrifuged at high speed. The strips between the sucrose solution layers of different concentrations are collected and combined. The strips are the green strip-shaped suspensions formed between the sucrose solutions of different concentration gradients after centrifugation.

[0011] (5) Mix the band obtained in step 4 with PBS buffer, centrifuge at high speed to remove sucrose, and the resulting precipitate is broccoli exosomes;

[0012] The ultracentrifugation conditions are 100,000g-150,000g and 90min-150min.

[0013] Furthermore, the temperature of the PBS buffer is 4℃±1℃, which can maximize the protection of exosomes from the temperature factors during the pulping process in step 1.

[0014] Furthermore, step 2 is repeated 3 times. The first centrifugation conditions are 500g-1500g and 5min-15min, the second centrifugation conditions are 3000g-5000g and 30min-50min, and the third centrifugation conditions are 8000g-12000g and 40min-80min.

[0015] Furthermore, in step 2, the first centrifugation conditions are 1500g for 15min; the second centrifugation conditions are 5000g for 50min; and the third centrifugation conditions are 12000g for 80min. Under these centrifugation conditions, the removal effect on large particles, small fragments, and some impurities is the best.

[0016] Furthermore, in step 4, the concentrations of the sucrose solutions are 60%, 45%, 30%, 15%, and 8%, respectively, and they are added to the centrifuge tubes in order of increasing concentration.

[0017] Furthermore, the ultracentrifugation conditions for steps 3, 4, and 5 were 150,000 g and 120 min; under these centrifugation conditions, step 3 showed the best exosome purification effect; and under these centrifugation conditions, step 5 showed the best sucrose removal effect.

[0018] In addition, the present invention also provides the application of broccoli exosomes obtained by the above extraction method in the preparation of drugs for relieving constipation.

[0019] The beneficial effects of this invention are:

[0020] 1. Broccoli exosome extraction process is simple, raw materials are readily available, and cost is low.

[0021] 2. By optimizing the extraction process of broccoli exosomes, the extraction efficiency of broccoli exosomes was effectively improved.

[0022] 3. Broccoli exosomes have a regulatory effect on relieving constipation, providing a research basis for exosome-based dietary therapy.

[0023] 4. The temperature of the PBS buffer used for extracting broccoli exosomes is 4℃±1℃, which can protect the exosomes from the influence of temperature during the pulping process to the greatest extent. Attached Figure Description

[0024] Figure 1 Transmission electron microscope image of broccoli exosomes magnified 8000 times;

[0025] Figure 2 Transmission electron microscope image of broccoli exosomes magnified 600,000 times;

[0026] Figure 3 Nanoflow cytometry concentration of broccoli exosomes;

[0027] Figure 4 Particle size distribution of broccoli-derived exosomes;

[0028] Figure 5 The graph shows the changes in body weight of mice in different groups during the mouse experiment using broccoli exosomes.

[0029] Figure 6 Figure 1 shows the fecal water content of mice in different groups of mice in the mouse experiment using broccoli exosomes.

[0030] Figure 7 Figure 1: Total fecal weight of mice in each group within 6 hours in mouse experiments with broccoli exosomes;

[0031] Figure 8 The graph shows the total number of feces in each group of mice within 6 hours in the mouse experiment with broccoli exosomes.

[0032] Figure 9 The graph shows the time of the first feces in each group of mice in the mouse experiment with broccoli exosomes.

[0033] Figure 10 The graph shows the time of the first blue stool in each group of mice in the broccoli exosome experiment. Detailed Implementation

[0034] To make the above-mentioned objectives and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to examples.

[0035] 1. Extraction of broccoli exosomes

[0036] 1.1 Specific extraction method

[0037] Example 1

[0038] (1) Wash and chop the broccoli raw material, mix it with PBS buffer at 3°C ​​and blend it into a paste, filter it and take the filtrate; the mass ratio of PBS buffer to broccoli is 1:2.

[0039] (2) Centrifuge the filtrate obtained in step 1, take the supernatant and centrifuge again, repeat 3 times, the centrifugation conditions for the 3 times are 500g, 5min; 3000g, 30min; 8000g, 40min;

[0040] (3) After repeating the centrifugation in step 2 three times, the supernatant obtained was subjected to ultracentrifugation at 100,000g for 90min. The precipitate was collected and resuspended in PBS buffer to form a suspension.

[0041] (4) Add sucrose solutions with concentrations of 8%, 15%, 30%, 45% and 60% to centrifuge tubes in descending order of concentration to form sucrose solutions with gradient concentrations. Then add the suspension obtained in step (3) to the sucrose solution centrifuge and perform ultracentrifugation at 100,000g for 90min. Collect and combine the bands between the layers of sucrose solutions of different concentrations.

[0042] (5) Mix the band obtained in step 4 with PBS buffer and centrifuge at 100000g for 90min to remove sucrose. The resulting precipitate is broccoli exosomes.

[0043] Example 2

[0044] (1) Wash and chop the broccoli raw material, mix it with PBS buffer at 5℃ and blend it into a paste, filter it and take the filtrate; the mass ratio of PBS buffer to broccoli is 1:3.

[0045] (2) Centrifuge the filtrate obtained in step 1, take the supernatant and centrifuge again, repeat 3 times, the centrifugation conditions for the 3 times are 1000g, 10min; 4000g, 40min; 10000g, 60min;

[0046] (3) After repeating the centrifugation in step 2 three times, the supernatant obtained was subjected to ultracentrifugation at 120000g for 120min. The precipitate was collected and resuspended in PBS buffer to form a suspension.

[0047] (4) Add sucrose solutions with concentrations of 8%, 15%, 30%, 45% and 60% to centrifuge tubes in descending order of concentration to form sucrose solutions with gradient concentrations. Then add the suspension obtained in step 3 to the sucrose solution centrifuge and perform ultracentrifugation at 120000g for 120min. Collect and combine the bands between the layers of sucrose solutions of different concentrations.

[0048] (5) Mix the band obtained in step 4 with PBS buffer at 5°C, and centrifuge at 120000g for 120min to remove sucrose. The precipitate obtained is broccoli exosomes.

[0049] Example 3

[0050] (1) Wash and chop the broccoli raw material, mix it with PBS buffer at 4℃ and blend it into a paste, filter it and take the filtrate; the mass ratio of PBS buffer to broccoli is 1:4.

[0051] (2) Centrifuge the filtrate obtained in step 1, take the supernatant and centrifuge again, repeat 3 times, the centrifugation conditions for the 3 times are 15000g, 15min; 5000g, 50min; 12000g, 80min;

[0052] (3) After repeating the centrifugation in step 2 three times, the supernatant obtained was subjected to ultracentrifugation at 150000g for 150min. The precipitate was collected and resuspended in PBS buffer to form a suspension.

[0053] (4) Add sucrose solutions with concentrations of 8%, 15%, 30%, 45% and 60% to centrifuge tubes in descending order of concentration to form sucrose solutions with gradient concentrations. Then add the suspension obtained in step 3 to the sucrose solution and perform ultracentrifugation at 150,000g for 150min. Collect and combine the bands between the layers of sucrose solutions of different concentrations.

[0054] (5) Mix the band obtained in step 4 with PBS buffer and centrifuge at 150000g for 150min to remove sucrose. The resulting precipitate is broccoli-derived exosomes.

[0055] 1.2 Determination of Broccoli Exosome Content

[0056] The protein concentration of broccoli exosomes obtained in the above examples was determined using a BCA protein quantification kit (NewSemi Biotechnology Co., Ltd.). The measurement principle is that, in an alkaline medium, proteins can react with Cu... 2+ Reduced to Cu 1+ The reaction was then performed, and Cu was detected colorimetrically using a unique reagent containing BCA. 1+A purple complex with strong absorption at 562 nm was formed to determine the content of broccoli exosomes. The exosome content test results of the above embodiments are shown in the table below:

[0057]

[0058] 1.3 Exosome Characterization Analysis

[0059] 1) Transmission electron microscopy observation

[0060] Take 20 μL of the exosomes obtained in Example 3 above, drop them onto a copper grid for adsorption for 5 min, and then absorb the floating liquid with filter paper; take 20 μL of phosphotungstic acid, drop them onto a copper grid for adsorption for 3 min, and then absorb the floating liquid with filter paper; dry at room temperature for several minutes; perform electron microscopy at 100 kV to obtain transmission electron microscopy imaging results, such as... Figure 1 and Figure 2 As shown. Figure 1 This is a transmission electron microscope image of broccoli exosomes taken at 8000 magnification. Figure 2 This is a transmission electron microscope image of broccoli exosomes taken at a magnification of 600,000.

[0061] 2) Exosome particle size concentration detection

[0062] The exosomes obtained in Example 3 were detected using a nanoflow cytometer. A standard working curve was established using silica standard spheres to convert the scattering intensity of the exosomes under the same conditions into particle size, thus obtaining the particle size and concentration distribution of the exosomes. This method is applicable when the refractive index of the exosomes is the same as or similar to that of the silica particles. Figure 3 As shown, the horizontal axis represents lateral astigmatism, which is related to particle size; a larger value indicates a larger particle. The vertical axis represents fluorescence intensity, which can detect whether the particles exhibit green fluorescence and the intensity of the fluorescence; a value below 100 indicates no fluorescence. The exosomes extracted using the method of this invention are non-fluorescent and have a relatively concentrated particle size, belonging to a single class of substances. Figure 4 As shown, the horizontal axis represents the size of the measured particles, and the vertical axis represents the number of particles. The obtained exosome concentration is 8.13E+10 particles / mL, and the particle size is mainly distributed in the range of 50-90 nm, with an average particle size of 72.42 nm.

[0063] 2. Application of broccoli exosomes extracted using the above method in the preparation of drugs for relieving constipation.

[0064] The broccoli exosomes used in this embodiment were obtained in Example 3 above. The broccoli exosomes obtained in Example 3 were dispersed into three different densities of broccoli exosome solutions: 17.5 mg / kgd, 37.5 mg / kgd, and 75 mg / kgd, and stored in a -80°C refrigerator for later use.

[0065] 1) Establishment of mouse model

[0066] Effects of LOP (pylobutamide hydrochloride) on body weight changes in a constipation model in mice: Forty SPF-grade Balb / c mice were randomly divided into five groups after one week of acclimatization: blank control group (NC), model group (M), low-dose group (LE), medium-dose group (ME), and high-dose group (HE), with eight mice in each group. All five groups received standard feed and free access to water without any dietary or water treatment. The blank control group received saline by gavage once every morning from day 1 to 7, and saline by gavage once every morning and afternoon from day 8 to 35. The model group received saline by gavage once every morning from day 1 to 7, and saline by gavage once every morning and 10 mg / kg loperamide hydrochloride by gavage in the afternoon from day 8 to 35. The low-dose group received 17.5 mg / kg broccoli-derived exosomes by gavage once every morning from day 1 to 7, and 17.5 mg / kg broccoli-derived exosomes by gavage once every morning and 10 mg / kg loperamide hydrochloride by gavage in the afternoon from day 8 to 35. The model was established using loperamide hydrochloride at a dose of 10 mg / kg. The medium-dose group received 37.5 mg / kg of broccoli-derived exosomes by gavage every morning from day 1 to 7, and 37.5 mg / kg of broccoli-derived exosomes by gavage every morning and 10 mg / kg of loperamide hydrochloride by gavage every afternoon from day 8 to 35. The high-dose group received 75 mg / kg of broccoli-derived exosomes by gavage every morning from day 1 to 7, and 75 mg / kg of broccoli-derived exosomes by gavage every morning and 10 mg / kg of loperamide hydrochloride by gavage every afternoon from day 8 to 35. Mice were housed in an environment with a 12-hour light / 12-hour dark diurnal cycle and a temperature of (22±2)℃ for five consecutive weeks.

[0067] 2) Weight measurement

[0068] Mice were weighed and their weight recorded before the start of the mouse model and after each cycle. During days 1–35 of the experiment, the weight gain of mice in each group was normal, indicating that the experimental animals showed no abnormalities. The results are as follows: Figure 5 .

[0069] 2) Measurement of fecal moisture content

[0070] On day 34, after gavage, mice were placed individually in cages lined with absorbent paper, and their feces were collected and weighed to obtain the wet weight. After freeze-drying, the wet weight was obtained. The formula for calculating fecal moisture content is as follows:

[0071]

[0072] Compared with the blank control group, the fecal water content of mice in the model group was significantly reduced (****P<0.0001, where * indicates a significant effect, and more * indicate a more significant effect), indicating that LOP can significantly reduce the fecal water content of mice. Compared with the model group, the fecal water content of mice in each treatment group was significantly increased, and the increase was more pronounced at low and medium doses, indicating that this dose of broccoli-derived exosomes is more effective in alleviating the effect of LOP on reducing the fecal water content of mice. Figure 6 As shown.

[0073] 3) Measurement of mouse defecation

[0074] On day 35, mice in each group (except the control group) were fasted but allowed normal water intake for 12 hours. Mice in other groups were administered LOP (10 mg / kg BW) by gavage for 30 minutes, followed by 0.4% Evans blue (10 ml / kg BW). Timing was started immediately. Each mouse was individually housed with free access to food and water. The time of the first defecation, the time of the first blue stool, the total number of stool particles in 6 hours, and the total weight of stool in 6 hours were observed and recorded. Figure 7 As shown, compared with the blank control group, the fecal weight at 6 hours was significantly lower in the model group (P < 0.0001), indicating that LOP can significantly reduce the fecal weight excreted by mice. Compared with the model group, the fecal weight at 6 hours was significantly higher in all treatment groups, with the medium dose showing the most significant increase, indicating that this dose of broccoli-derived exosomes is more effective in alleviating the effect of LOP on reducing the fecal weight at 6 hours in mice. Figure 8 As shown, compared with the blank control group, the number of fecal particles in the model group was significantly reduced at 6 hours (**P < 0.01), indicating that LOP can significantly reduce the number of fecal particles excreted by mice. Compared with the model group, the number of fecal particles in each treatment group was significantly increased at 6 hours, with the medium dose showing the most significant increase, indicating that this dose of broccoli-derived exosomes is more effective in alleviating the reduction in the number of fecal particles in mice at 6 hours caused by LOP. Figure 9 As shown, compared with the blank control group, the time to first feces in the model group was significantly prolonged (***P<0.001), indicating that LOP can significantly prolong the time to first feces in mice. Compared with the model group, the time to first feces in each treatment group was significantly shortened, with the low dose showing the most significant shortening, indicating that this dose of broccoli-derived exosomes is more effective in alleviating the prolonging effect of LOP on the time to first feces in mice. Figure 10 As shown, compared with the blank control group, the time to first blue feces in the model group was significantly prolonged (P < 0.0001), indicating that LOP can significantly prolong the time to first blue feces in mice. Compared with the model group, the time to first blue feces in each treatment group was significantly shortened, with the low dose showing the most significant effect, indicating that this dose of broccoli-derived exosomes is more effective in alleviating the prolonging effect of LOP on the time to first feces in mice.

[0075] The effects of broccoli exosomes on mouse body weight, fecal water content, and fecal volume in mouse models are shown in the table below:

[0076]

[0077]

[0078]

[0079] The above results indicate that the addition of exosomes to the mouse model can effectively relieve constipation, demonstrating a good effect in relieving constipation.

Claims

1. Use of a broccoli exosome in the preparation of a medicine for relieving constipation. The extraction method of the broccoli exosome comprises the following steps: (1) The broccoli raw material is washed, chopped, mixed with PBS buffer, and then beaten into a slurry, filtered, and the filtrate is taken; the mass ratio of PBS buffer to broccoli is 1:4-1:2; (2) The filtrate obtained in step 1 is repeatedly centrifuged at low speed, and the supernatant is taken after each centrifugation and then centrifuged again; (3) The supernatant obtained after repeating the centrifugation in step 2 is ultracentrifuged, the precipitate is resuspended with PBS buffer to form a suspension; (4) The suspension obtained in step 3 is added to a sucrose solution with gradient concentration for ultracentrifugation, and the bands between different concentrations of sucrose solution layers are collected and combined, the bands being the green strip-shaped suspended part formed between different concentrations of sucrose solution layers after centrifugation; (5) The bands obtained in step 4 are mixed with PBS buffer and ultracentrifuged to remove sucrose, and the obtained precipitate is broccoli exosome; The ultracentrifugation condition is 100000g-150000g, 90min-150min.

2. The use of a Brassica oleracea exosome in the preparation of a medicine for relieving constipation according to claim 1, characterized in that: The temperature of the PBS buffer is 4℃±1℃.

3. The use of Brassica oleracea exosome in the preparation of a drug for relieving constipation according to claim 2, characterized in that: The repeated centrifugation in step 2 is 3 times, the first centrifugation condition is 500g-1500g, 5min-15min, the second centrifugation condition is 3000g-5000g, 30min-50min, and the third centrifugation condition is 8000g-12000g, 40min-80min.

4. The use of Brassica oleracea exosome in the preparation of a drug for relieving constipation according to claim 3, characterized in that: The first centrifugation condition in step 2 is 1500g, 15min; the second centrifugation condition is 5000g, 50min; and the third centrifugation condition is 12000g, 80min.

5. The use of Brassica oleracea exosome according to claim 4 for the preparation of a medicament for relieving constipation, characterized in that: The concentration of the sucrose solution in step 4 is 60%, 45%, 30%, 15% and 8% respectively, and is added to the centrifuge tube in order from high to low.

6. The use of a Brassica oleracea exosome according to claim 5 for the preparation of a medicament for the relief of constipation, characterized in that: The ultracentrifugation condition in step 3, step 4 and step 5 is 150000g, 120min.

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