CN111218419B - Bitter gourd exosome and extraction method and application thereof - Google Patents
Bitter gourd exosome and extraction method and application thereof Download PDFInfo
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- 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)
- A61K36/42—Cucurbitaceae (Cucumber family)
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- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/31—Extraction of the material involving untreated material, e.g. fruit juice or sap obtained from fresh plants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
Abstract
The invention discloses a bitter gourd exosome and an extraction method and application thereof, wherein the extraction method comprises the following steps: taking a proper amount of fresh Yunnan wild balsam pear, removing seeds, cleaning and airing; squeezing to obtain juice, centrifuging, collecting supernatant, and removing precipitate; ultracentrifuging the obtained supernatant, removing the supernatant, taking the precipitate, and suspending the precipitate in 1-2mL of phosphate buffer saline; the suspension was filtered through a 0.22 μm filter, the filtrate was centrifuged again in an ultracentrifuge, the supernatant was discarded, and the pellet was suspended in 1-2mL of phosphate buffered saline. The extracted Momordica charantia exosome can be used for preparing medicine for treating ischemic cerebral apoplexy. The extraction method is convenient and effective, the extraction purity is high, and the extracted bitter gourd exosomes have the protection effect on nerve injury after stroke.
Description
Technical Field
The invention relates to the field of medicines, and in particular relates to a bitter gourd exosome and an extraction method and application thereof.
Background
Ischemic cerebral apoplexy has the characteristics of high morbidity, high disability rate and high mortality. The cerebral apoplexy comprises hemorrhagic apoplexy and ischemic stroke, wherein the ischemic stroke accounts for about 87%, and the ischemic stroke is mainly caused by blood flow reduction caused by cerebral vessel blockage and cerebral blood supply insufficiency. The clinical method for treating cerebral apoplexy is to use t-PA for thrombolysis, but t-PA has a strict treatment time window of 4.5h, bleeding conversion (more serious injury) can be caused after the treatment time window is exceeded, and a safer and more effective treatment medicament is urgently needed clinically.
Exosomes (Exosomes) are extracellular vesicles of particle size 30-200nm released by cells into the environment, are butterfly-shaped, and consist of specific proteins, lipids and nucleic acid substances. The carried multiple miRNAs can be combined with specific receptors on the surface of a target cell membrane, regulate multiple signal pathways and regulate multiple physiological and pathological processes. Whether exosome can be extracted from bitter gourd or not and is used for treating ischemic stroke, and no report is found.
Disclosure of Invention
One of the purposes of the invention is to provide an extraction method of bitter gourd exosomes, which can extract exosomes from bitter gourd plants.
The second purpose of the invention is to provide the application of the balsam pear exosome extracted by the method in preparing the medicine for treating ischemic cerebral apoplexy.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows: an extraction method of bitter gourd exosomes comprises the following steps:
(1) taking a proper amount of fresh Yunnan wild balsam pear, removing seeds, cleaning and airing;
(2) air drying, squeezing to obtain juice, and continuously centrifuging at 4 deg.C for 10min at 1000 g; 3000g, 20 min; 10000g, 40 min; centrifuging, taking the supernatant, and discarding the precipitate;
(3) ultracentrifuging the obtained supernatant, centrifuging for 90min at 4 deg.C and 150000g in an ultrarefrigerated centrifuge, discarding the supernatant after centrifugation, collecting the precipitate, and suspending the precipitate in 1-2mL PBS buffer solution;
(4) the suspension was filtered through a 0.22 μm filter, the filtrate was again centrifuged at 150000g for 90min at 4 ℃ in an ultrafreeze centrifuge, the supernatant was discarded after centrifugation, and the pellet was suspended in 1-2mL of PBS buffer.
The invention also provides the bitter gourd exosome extracted by the method.
The observation of a transmission electron microscope shows that the bitter gourd exosome is a saucer-like membrane bubble structure with a clear membrane, the particle size is mainly concentrated at about 120nm, and three marker proteins CD54, CD63 and TSG101 exist in immunoblot detection.
The invention also provides application of the balsam pear exosome extracted by the method in preparing a medicament for treating cerebral apoplexy.
The brain protection effect of the balsam pear exosomes is verified by using a rat Middle Cerebral Artery Occlusion (MCAO) cerebral apoplexy model. Ischemia is embolized for 2 hours, bitter gourd exosomes (800ug/kg) are injected intravenously 30 minutes before revascularization, and the embolization is carried out 24 hours after reperfusion: TTC (2, 3, 5-triphenyltetrazolium chloride) staining for infarct size, mNSS nerve score, EB staining and EB quantification, and permeability of BBB (blood brain barrier).
The result shows that the balsam pear exosome can reduce the cerebral infarction area of MCAO rats, reduce the neurological score of the MCAO rats, reduce the penetration of EB and reduce the damage of blood brain barriers.
Compared with the prior art, the invention has the following beneficial effects:
1. the extraction method of the bitter gourd exosomes provided by the invention is convenient and effective, and has high impurity removal rate and high extraction purity.
2. The bitter gourd exosome extracted by the method can reduce the cerebral infarction area of an MCAO rat, can reduce the nerve score of the MCAO rat, can reduce the permeation of the EB of the MCAO rat, has a protection effect on the blood brain barrier of the MCAO rat, and shows that the bitter gourd exosome has a protection effect on nerve injury after cerebral apoplexy.
Drawings
FIG. 1 is a flow chart of the extraction of Momordica Charantia exosomes of example 1 of the present invention;
FIG. 2 is a transmission electron micrograph of an exosome of Momordica charantia extracted in example 1 of the present invention;
FIG. 3 is a graph showing the particle size distribution of the isolated exosomes of Momordica charantia of example 1 of the present invention;
FIG. 4 is a band diagram of the marker protein of the exosomes of Momordica charantia extracted in example 1 of the present invention;
FIG. 5 is a TTC staining pattern for detecting cerebral infarction;
FIG. 6 is a TTC staining quantitation assay;
FIG. 7 is a mNSS nerve score graph;
FIG. 8 is a diagram of brain slices from EB penetration testing of the blood brain barrier;
FIG. 9 is an EB quantitative analysis chart.
Detailed Description
The invention is described in further detail below with reference to the figures and specific examples.
Example 1: extraction method of bitter gourd exosomes
Referring to the extraction process shown in figure 1, removing seeds of fresh Yunnan wild fructus Momordicae Charantiae 500g, washing for three times, each time for 1-3min, and air drying;
air drying, squeezing juice of fructus Momordicae Charantiae with fresh juice extractor to obtain juice of about 200ml, continuously centrifuging fructus Momordicae Charantiae juice at 4 deg.C for 10min at 1000 g; 3000g, 20 min; 10000g, 40 min; centrifuging, taking the supernatant, and discarding the precipitate;
ultracentrifuging the obtained supernatant, centrifuging for 90min at 4 deg.C and 150000g in an ultrarefrigerated centrifuge, discarding the supernatant after centrifugation, collecting the precipitate, and suspending the precipitate in 1-2ml PBS buffer solution;
filtering the suspension with 0.22um filter membrane, centrifuging the filtrate again in ultracentrifuge at 4 deg.C and 150000g for 90min, collecting precipitate after centrifugation, discarding supernatant, and suspending the precipitate in 1-2ml PBS buffer solution.
Placing the extracted bitter gourd exosomes under a transmission electron microscope to observe the form, and the steps are as follows: dripping the balsam pear exosome solution obtained by fresh low-temperature ultracentrifugation on the clean surface of the sealing film; placing the copper mesh membrane surface on the droplets of the balsam pear exosomes, suspending for 10 minutes, and slowly sucking dry by using filter paper; transferring a copper net to a 3% glutaraldehyde solution drop, suspending for 5 minutes, and sucking dry by using filter paper; transferring the copper net to the DW liquid drop for 10 times, each time for 2 minutes, and drying by using filter paper each time; transferring the copper mesh onto 4% uranyl acetate droplets, carrying out suction drying by using filter paper for 10 minutes; after natural drying (30 min), the record was observed by TEM. As a result, as shown in FIG. 2, the cell structure was mostly a structure of a saucer-like film with a clear film of less than 200nm in an electron microscope field.
The particle size of the extracted bitter gourd exosomes is measured, the exosomes and phosphate buffer solution are diluted by 1:10 and mixed uniformly, 500 microliters of the exosomes are added into a special cuvette for detection, and the result is shown in fig. 3, wherein the sizes of the exosomes are mainly concentrated at about 120nm and belong to the range of the sizes (30-200nm) of the exosomes.
Three marker proteins CD54, CD63 and TSG101 of the exosome are measured by an immunoblotting method, and the marker protein bands of the exosome are clearly visible (as shown in figure 4).
Example 2: brain protection effect of balsam pear exosomes
1. Laboratory animal
Male Sprague-dawley (sd) rats, 250 ± 20g body weight, clean grade, provided by the experimental animals centre of xu state university of medical, laboratory animal production licenses: SCXK (Shanghai) 2018 and 0006.
2. Primary reagent
Bitter gourd exosomes: extracting according to the method described in the embodiment; TTC (2, 3, 5-Triphenyltetrazolium chloride; Shanghai Biotech), Evans blue (EB; Beijing YinuoKa Tech Co., Ltd.), trichloroacetic acid (Beijing YinuoKa Tech Co., Ltd.), CD54/ICAM-1 Antibody #4915(CST), TSG101 Antibody (proteintech), CD63 Antibody (proteintech)
3. Instrument for measuring the position of a moving object
Ultracentrifuge (Beckman Optima L-100XP), transmission electron microscope (Tecnai G2 Spirit Twin. Tecnai G2 Spirit Twin), Zeta potential detector (Nicomp380ZLS), small animal anesthesia machine (ABS-100), Tanon electrophoresis (EPS 300)
4. Establishment, grouping and administration of MCAO models
SD rats were randomly divided into: blank control group, MCAO + balsam pear exosome group, each group contains 5 individuals.
MCAO model preparation: after the SD rat is anesthetized by isoflurane inhalation, the right side of the median of the neck is vertically incised, peripheral tissues are separated, the left Common Carotid Artery (CCA), External Carotid Artery (ECA) and Internal Carotid Artery (ICA) of the rat are exposed, the CCA proximal end and the ECA are tightly tied by using suture lines, the ICA is clamped by using a arteriole clamp, a small opening is cut by holding an ophthalmic scissors at 45 degrees between the CCA ligation part and the upper end three-way opening, a standard suture bolt corresponding to the body weight is inserted from the cut in a small way, the arteriole clamp is loosened, the incoming direction is adjusted to avoid pterygopalatine arteries, the standard suture bolts enter the ICA until the starting part of the Middle Cerebral Artery (MCA), and ischemia is timed to start. According to the weight of the rat, the insertion depth of the thread plug is about 17mm, the internal carotid artery is tightly tied and fixed, and the skin is completely sutured. After 2 hours of ischemia, the rat was replated 24 hours after removing the plug.
The administration mode comprises the following steps: the Momordica Charantia exosomes were suspended in sterile PBS and given intravenously at a dose of 800ug/kg upon re-infusion.
5. Immunoblotting
1/4 volumes of 5 Xprotein loading buffer (loading buffer) were added to the exosome samples and the proteins were denatured by heating in a boiling water bath for 5 min. 20ug of the protein sample was loaded and separated by SDS-PAGE, and the protein was electroporated onto a PVDF membrane activated with methanol by wet or semi-dry transfer. Slowly shaking the PVDF membrane in a sealing solution, sealing at room temperature for more than 4h, adding the diluted primary antibody, and incubating overnight at 4 ℃; after Washing the membrane with 1 × Washing Buffer (5min × 5 times), the diluted secondary antibody was added and incubated at room temperature for 1h, and then the membrane with 1 × Washing Buffer (5min × 5 times). ECL fluorescence color development was used according to the kit protocol.
6. TTC dyeing
SD rats in each model group are subjected to ischemia for 2h, and are subjected to TTC staining after 24h of repeated perfusion.
Taking the brain by decapitation: the method comprises the steps of killing animals by a rat decapitation method under ether anesthesia, quickly taking brains, freezing the brains in a refrigerator at the temperature of-20 ℃ for 30min, taking out the brains, removing cerebellum, olfactory bulbs and lower brainstem, cutting off coronal brain slices with the thickness of about 2mm, putting the brain slices in 2% TTC dye liquor, incubating in an oven at the temperature of 37 ℃ for 25 min, turning the brain slices in the middle for 1-2 times, and fully coloring the brain slices. The brain slices were removed, washed 3 times with PBS solution, stored in 4% paraformaldehyde solution for storage by photography, and infarct volume was calculated using ImageJ software.
Percent infarct ═ 100% (non-infarcted hemisphere-area of non-infarcted hemisphere)/area of non-infarcted hemisphere%
7. Blood Brain Barrier (BBB) permeability measurement
Weighing 29 Evans Blue (EB), dissolving with 100ml sterile physiological saline, preparing 2% EB solution for later use, and grouping: rats were sacrificed after 24 hours of anesthesia following 2 hours of ischemia reperfusion, and 2% EB (3ml/kg) solution was injected intravenously 2 hours prior to sacrifice. After anesthetizing the rat, the rat is in a supine position, skin and subcutaneous tissues are cut off from the lower edge of the xiphoid process, the xiphoid process is exposed, the rat is clamped and lifted by a needle holder, ribs connected with two sternums at two sides are cut off, the rat enters a thoracic cavity, the heart is exposed, the epicardium is peeled by tweezers, a disposable infusion set needle connected with physiological saline is inserted into the left ventricle, after blood return is observed, the infusion set is opened, the right auricle is cut off by an ophthalmic scissors, and the physiological saline is infused into the whole body. After approximately 200ml of saline infusion, the outflow from the right auricle was observed to be clear and clear, the liver became white, and the infusion was complete. The rat was decapitated and brain tissue was removed. After the brain is frozen in a refrigerator at the temperature of 20 ℃ below zero for 30min, brain slices are taken out for photographing, and the permeation condition of EB is observed.
The brain was divided into ischemic and non-ischemic sides, weighed and stored in a-80 ℃ freezer for use, and the ischemic side brain tissue was homogenized with cold PBS, and the same volume of 50% trichloroacetic acid was added to continue the homogenization, followed by centrifugation at 15000rpm for 20 minutes to collect the supernatant. The supernatant was measured for absorbance at 620 nm. The EB content was measured by the gradient concentration of the EB standard curve.
8. mNSS neurological score
Scoring is done according to the following scoring criteria, with higher scores indicating more severe damage.
Exercise test
9. Results of the experiment
The TTC staining test results are shown in fig. 5 and 6. The results show that the infarct volume part of the rat after ischemia/reperfusion is white, the non-infarct volume part is red, the Control rat has no infarct part, the infarct area of the MCAO rat is about 55%, the cerebral infarct area of the MCAO rat model rat can be obviously reduced after the balsam pear exosome is given, and the balsam pear exosome can play a role in protecting the brain after ischemia/reperfusion.
The mNSS neurological score results of the rats are shown in FIG. 7. The higher the neurological score, the more serious the neurological damage, the neurological score of the MCAO rat is far higher than that of the Control group, and the administration of the balsam pear exosome can obviously reduce the neurological score of the MCAO rat, which indicates that the balsam pear exosome can improve the neurological function defect of the MCAO rat when the MCAO rat is subjected to 24h reperfusion.
The results of the blood-brain barrier permeability experiments are shown in fig. 8 and 9. The result shows that the brain of the rat in the Control group has no EB penetration, the brain of the rat in the MCAO group has obvious EB penetration, and the EB penetration condition of the rat in the MCAO group can be obviously reduced after the balsam pear exosome is administrated, so that the balsam pear exosome can protect the integrity of a blood brain barrier and play a role in protecting the brain after ischemia/reperfusion.
Claims (1)
1. The application of the balsam pear exosome in preparing the medicine for treating ischemic cerebral apoplexy is characterized in that the balsam pear exosome is extracted by the following steps:
(1) taking a proper amount of fresh Yunnan wild balsam pear, removing seeds, cleaning and airing;
(2) air drying, squeezing to obtain juice, and continuously centrifuging at 4 deg.C for 10min at 1000 g; 3000g, 20 min; 10000g, 40 min; centrifuging, taking the supernatant, and discarding the precipitate;
(3) ultracentrifuging the obtained supernatant, centrifuging for 90min at 4 deg.C and 150000g in an ultrarefrigerated centrifuge, discarding the supernatant after centrifugation, taking the precipitate, and suspending the precipitate in 1-2mL phosphate buffer solution;
(4) the suspension was filtered through a 0.22 μm filter, the filtrate was again centrifuged at 150000g for 90min at 4 ℃ in an ultrafreeze centrifuge, the supernatant was discarded after centrifugation, and the precipitate was taken and suspended in 1-2mL phosphate buffered saline.
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| CN113462632B (en) * | 2021-08-13 | 2023-08-29 | 徐州医科大学 | Balsam pear exosome, extraction method and application thereof in preparation of medicines for treating burns and scalds |
| CN114259511B (en) * | 2021-12-29 | 2023-04-25 | 徐州医科大学 | Application of balsam pear exosome in preparing medicament for protecting radiation heart injury |
| TR2022013409A2 (en) * | 2022-08-26 | 2022-09-21 | Aye Exocure Tibbi Ueruenler Sanayi Ve Ticaret Anonim Sirketi | EXOSOMES FROM KUDRET NARI (MOMORDICA CHARANTIA) |
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