CN108143751B - Preparation method and application of male silkworm moth exosome - Google Patents
Preparation method and application of male silkworm moth exosome Download PDFInfo
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
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- A61Q19/00—Preparations for care of the skin
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
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- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/84—Products or compounds obtained by lyophilisation, freeze-drying
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Abstract
The invention discloses a preparation method and application of male silkworm moth exosomes, and belongs to the technical field of biology. The invention adopts healthy male silkworm moth living body to prepare homogenate, performs differential centrifugal separation and low-temperature ultra-speed purification to obtain high-purity exosome, has good curative effect in animal skin injury experiments, can prepare exosome into freeze-dried powder, is stored for a long time, forms different preparation products by matching with different auxiliary materials, and has excellent market prospect and clinical value.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an exosome with biological activity extracted from healthy living male silkworm moths and application thereof.
Background
Male Bombycis mori is used by emperors as a precious tonic for tonifying kidney, resisting aging, prolonging life and prolonging life in Tang and Song times; the Ming Dynasty famous medicien Li Shizhen refers to male silkworm moth as Shenchong Guobao in Ben Cao gang mu, which shows that the male silkworm moth has obvious effects of nourishing liver and kidney, strengthening yang, resisting aging, preventing senile dementia, preventing male prostatic hyperplasia, regulating female endocrine dyscrasia, etc. The kidney-tonifying and yang-strengthening product of the male silkworm moth can be seen in the market, but the product for treating wound, ulcer, scald and other symptoms is not seen, and the reason is that the effective components for treating the wound are damaged and lost when the male silkworm moth dry body is prepared.
In recent years, research shows that exosomes are vesicular bodies actively secreted by cells, have the diameter of 40-100nm and the density of 1.10-1.18g/ml, and are widely present in living animals, plants and microorganisms. The exosome selectively wraps various bioactive substances such as lipid, protein, mRNA and miRNA, the membrane surface carries certain surface markers of a host, the bioactive substances can be absorbed by target cells through the modes of ligand-receptor combination and the like, and the exosome has the definite functions of reducing the tissue damage degree, promoting the damaged tissue morphology and functional repair, and may have the potential biological functions of regulating the body immunity, regulating the cell growth and differentiation and the like.
At present, a plurality of methods for extracting exosome are adopted, such as 1. ultracentrifugation (differential centrifugation): low-speed centrifugation and high-speed centrifugation are alternately carried out to separate vesicle particles with similar sizes. The super-separation method is popular because of simple operation and more vesicles. 2. Density gradient centrifugation: under the action of ultracentrifugal force, the sucrose solution is made to form a density layer continuously distributed from low to high, and the zone separation method is used. Exosomes in the sample will be enriched at a density range of 1.13-1.19g/ml by density gradient centrifugation. But the procedure is cumbersome and time consuming. 3. Magnetic bead immunization: the surface of the exosome is provided with a specific marker (such as CD63 and CD9 protein), and the exosome can be adsorbed and separated by using magnetic beads coated with anti-marker antibodies to be combined with exosome vesicles after incubation. The magnetic bead method has the advantages of high specificity, simple and convenient operation, no influence on the complete form of the exosome and the like, but has low efficiency, the bioactivity of the exosome is easily influenced by pH and salt concentration, is not beneficial to downstream experiments, and is difficult to widely popularize. 4. The kit extraction method comprises the following steps: various commercial exosome extraction kits are available on the market, some of which filter out impurity components by specially designed filters, some of which separate and purify by Size Exclusion Chromatography (SEC), and some of which precipitate exosomes by compound precipitation. The kit does not need special equipment, and the extraction efficiency and the purification effect are gradually improved with the continuous updating and updating of products, but the kit aiming at the extraction of the insect exosome is lacked at present. Ultracentrifugation is therefore the most common exosome purification means suitable for industrial production.
Disclosure of Invention
The invention aims to provide a preparation method and application of male silkworm moth exosomes. Mixing with different adjuvants to obtain different preparations.
After a lot of experiments and diligent efforts, the inventor obtains the following technical scheme: a preparation method of male silkworm moth exosomes comprises the following steps:
(1) soaking healthy male silkworm moth living bodies in normal saline or PBS, crushing the living bodies by a tissue homogenizer to prepare homogenate, centrifuging the homogenate for 30min at 500-1000g, collecting supernatant, and using a precipitate for other purposes;
(2) taking 10000-;
(3) centrifuging 100000g of the supernatant in the step (2) for 100min, discarding the supernatant, and collecting the precipitate;
(4) adding PBS into the precipitate obtained in the step (3), mixing again, centrifuging for 120min at 120000g, removing the supernatant, collecting the precipitate, and suspending the precipitate with PBS;
(5) adding mannitol into the PBS suspension in the step (4) until the final concentration is 10%, uniformly mixing, and filtering by using a filter membrane;
(6) subpackaging the filtrate obtained in the step (5) and placing the subpackaged filtrate in a freezing bin of a freeze dryer, cooling to-40 ℃ at the speed of 1-2 ℃/min, and maintaining at-40 ℃ for 2 hours; then starting vacuum, maintaining the vacuum degree of the dryer at 160-50 ℃ to 60 ℃ below zero, controlling the temperature of the condenser at 50 ℃ below zero to 60 ℃ below zero, gradually increasing the temperature of the shelf from 40 ℃ below zero to 6 ℃ for 20-24h, then entering an analysis drying stage, increasing the temperature of the shelf from 6 ℃ to 35 ℃ and maintaining the vacuum degree at 220-250 μ bar for 4-6h, when the temperature of the product is within 1-2 ℃ of the temperature of the shelf, closing the large butterfly valve for 60s, ensuring that the pressure in the drying box does not obviously increase, ending the freeze-drying process, and then pressing the cover to obtain the male silkworm moth exosome freeze-dried powder.
An application of male silkworm moth exosome, wherein the male silkworm moth exosome freeze-dried powder can be applied to skin care products.
An application of exosome of male Bombycis Mori is provided, and the skin care product containing exosome can be any one of water agent, paste, cream, pellicle and spray.
An application of exosome of male Bombycis Mori comprises adding humectant, antiseptic, whitening agent, thickener or/and emulsifier into skin care product containing exosome.
An application of the male silkworm moth exosome for repairing the damaged skin, preventing scar, beautifying and whitening skin is disclosed.
Compared with the prior art, the invention has the advantages that:
the exosome provided by the invention is extracted from living male silkworm moths, high-purity exosome is obtained by differential centrifugal separation and low-temperature ultra-speed purification, and the prepared freeze-dried powder can be stored in a refrigerator for a long time and has excellent market prospect and clinical value after being prepared into various skin care products or pharmaceutical preparations.
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FIG. 1 is an electron microscope image of male silkworm moth exosomes.
Detailed description of the preferred embodiment
The following are specific embodiments of the present invention and further description of the technical solutions of the present invention, but the present invention is not limited to the scope of the embodiments, and all changes or equivalent substitutions that do not depart from the spirit of the present invention are included in the scope of the present invention.
Example 1 extraction of Male Bombycis mori exosomes and preparation of lyophilized powder
Taking healthy living male silkworm moths, soaking the male silkworm moths in normal saline, smashing the male silkworm moths into homogenate by a tissue homogenizer, and carrying out the following steps:
(1) homogenizing 3000g, centrifuging for 30min, collecting supernatant, and precipitating for other use;
(2) centrifuging 10000g of the supernatant in the step (1) for 100min, collecting the supernatant, and precipitating part for other use;
(3) centrifuging 100000g of the supernatant in the step (2) for 100min, discarding the supernatant, and collecting the precipitate;
(4) adding the precipitate in the step (3) into PBS for remixing, centrifuging for 120min at 120000g, removing the supernatant, and collecting the precipitate; suspending the precipitate with PBS;
(5) adding mannitol into the PBS suspension in the step (4) until the final concentration is 10%, uniformly mixing, and filtering with a filter membrane with the aperture of 0.22 mu m;
(6) subpackaging the filtrate obtained in the step (5) and placing the subpackaged filtrate in a freezing bin of a freeze dryer, cooling to-40 ℃ at the speed of 1-2 ℃/min, and maintaining at-40 ℃ for 2 hours; then starting vacuum to make the dryer obtain necessary vacuum degree, establishing vacuum drying pressure condition, raising temperature of shelf and providing energy required for sublimation of ice crystal for product. The vacuum degree of the drying box is maintained at 200 mu bar for 160 plus materials, the temperature of the condenser is controlled at minus 50 ℃ to minus 60 ℃, the temperature of the shelf is gradually increased from minus 40 ℃ to 6 ℃ for 20-24h, then the analysis drying stage is carried out, the temperature of the shelf is increased from 6 ℃ to 35 ℃, the vacuum degree is maintained at 250 mu bar for 4-6h, when the temperature of the product is within the range of 1-2 ℃ relative to the temperature of the shelf, the pressure in the drying box is not obviously increased within 60s after the large butterfly valve is closed, the freeze-drying process is finished, and then the gland is carried out to obtain the male silkworm exosome freeze-dried powder.
And (4) taking out a small amount of the exosome suspension obtained in the step (4) and observing the exosome suspension by using an electron microscope, wherein the vesicle particle size is 60-120nm as shown in a figure 1.
Example 2: preparation of drug containing male silkworm moth exosome for treating injury in aqueous solution form
The main components (w/v) of the water agent are as follows: 1-5% of glycerol, 1-10% of propylene glycol and a thickening agent; 1-2% of Tween and 0.1% of ethyl p-hydroxybenzoate, and 60ml of PBS buffer solution are mixed, 10-50 μ g of male silkworm moth exosome freeze-dried powder is added, 5ml of 0.01M phosphate buffer solution is added, PBS is supplemented to 100ml of final volume after mixing, 0.45 μ M of filter membrane is used for filtering, sterilizing and insoluble substances, and the preparation process of the product containing male silkworm moth exosomes in the form of an aqueous preparation is completed.
The prepared male silkworm moth exosome-containing aqueous solution can be used as a spray, a skin lotion and the like, and can be sprayed and coated.
Example 3: preparation of emulsion-type product containing male silkworm moth exosome
The emulsified product can be prepared by different methods such as oil-in-water or water-in-oil, such as 10% glycerol, 2% Tween 80, 2% cholesterol, 5% lecithin, 0.2% ethylparaben and 70ml PBS buffer solution, heating, homogenizing and emulsifying, cooling to 37-45 deg.C, adding 10-50 μ g male Bombycis Mori exosome lyophilized powder, adding 5ml 0.01M phosphate buffer solution, mixing, adding water to 100ml final volume, homogenizing, filtering with 0.45 μ M filter membrane for sterilization and insoluble substance, and completing the preparation process of male Bombycis Mori exosome-containing product in the form of emulsion preparation.
Example 4: preparation of product in form of cream or ointment containing male silkworm moth exosome
The cream ointment can be prepared by the following formulation as an example. 5% of glycerol, 10% of vaseline, 2% of lanolin, 1% of tween, 5% of PEG400, 2% of glycerol monostearate, 0.2% of ethylparaben and 60ml of PBS buffer solution are mixed, heated, homogenized and emulsified, then 10-50 mu g of male silkworm moth exosome freeze-dried powder is added, 5ml of 0.01M phosphate buffer solution is added, water is supplemented to 100ml of final volume after mixing, and after homogenization, filtration sterilization and insoluble substances are carried out by a 0.45 mu M filter membrane, thus completing the preparation process of the product containing the male silkworm moth exosome in the form of cream.
Example 5: combined use of male silkworm moth exosomes and antibiotics
5% glycerin, 10% vaseline, 2% lanolin, 1% tween, 5% PEG400, 2% glyceryl monostearate, 0.2% ethylparaben, 0.3% silver sulfadiazine and 60ml PBS buffer solution are mixed, heated, homogenized and emulsified, then 1-10 μ g of male silkworm moth exosome freeze-dried powder is added, 5ml of 0.01M phosphate buffer solution is added, kojic acid, glabridin, Vc, Ve and arbutin are added, PBS is added to the final volume of 100ml after mixing, and after homogenization, filtration sterilization and insoluble substances are carried out by a 0.45 μ M filter membrane, thus completing the product containing male silkworm moth exosome.
Claims (4)
1. A preparation method of male silkworm moth exosomes is characterized by comprising the following steps: the method comprises the following steps:
(1) soaking living body of male silkworm moth in physiological saline or PBS, crushing the living body into homogenate by a tissue homogenizer, centrifuging for 30min after homogenizing, collecting supernatant, and using the precipitate as other component, wherein the centrifugal force is 3000 g;
(2) centrifuging the supernatant in the step (1) for 100min, collecting the supernatant, and using the precipitate for other purposes, wherein the centrifugal force is 10000 g;
(3) centrifuging the supernatant in the step (2) for 100min, discarding the supernatant, and collecting the precipitate, wherein the centrifugal force is 100000 g;
(4) adding PBS into the precipitate obtained in the step (3), mixing again, centrifuging for 120min, removing supernatant, collecting the precipitate, and suspending the precipitate with PBS, wherein the centrifugal force is 120000 g;
(5) adding mannitol into the PBS suspension in the step (4) until the final concentration is 10%, uniformly mixing, and filtering by using a filter membrane;
(6) subpackaging the filtrate obtained in the step (5) and placing the subpackaged filtrate in a freezing bin of a freeze dryer, cooling to-40 ℃ at the speed of 1-2 ℃/min, and maintaining at-40 ℃ for 2 hours; and then starting vacuum, maintaining the vacuum degree of the dryer at 160-plus-200 mu bar, controlling the temperature of the condenser at-50 ℃ to-60 ℃, gradually increasing the temperature of the shelf from-40 ℃ to 6 ℃ for 20-24h, then entering an analysis drying stage, increasing the temperature of the shelf from 6 ℃ to 35 ℃, maintaining the vacuum degree at 220-plus-250 mu bar for 4-6h, closing the large butterfly valve for 60s when the temperature of the product is within 1-2 ℃ of the temperature of the shelf, and ending the freeze-drying process to obtain the male silkworm exosome freeze-dried powder.
2. The use of the exosome prepared by the preparation method of claim 1, which is characterized in that: application of lyophilized powder of male Bombycis Mori exosome in preparing skin care product is provided.
3. The use of an exosome according to claim 2, wherein: the skin care product is any one of aqua, ointment, cream, film and spray.
4. The use of an exosome according to claim 2, wherein: the skin care product can be added with humectant, antiseptic, whitening agent, thickener or/and emulsifier.
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| JP2014230521A (en) * | 2013-05-29 | 2014-12-11 | 国立大学法人 東京医科歯科大学 | Composition for reprogramming of cell |
| WO2015009325A1 (en) * | 2013-07-18 | 2015-01-22 | Al-Qahtani Ahmed H | Skin cream |
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| CN105483081B (en) * | 2015-11-13 | 2019-09-20 | 中国人民解放军第二军医大学 | MiRNA145-5p modifies excretion body and its preparation and application of umbilical cord mesenchymal stem cells secretion |
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| JP2014230521A (en) * | 2013-05-29 | 2014-12-11 | 国立大学法人 東京医科歯科大学 | Composition for reprogramming of cell |
| WO2015009325A1 (en) * | 2013-07-18 | 2015-01-22 | Al-Qahtani Ahmed H | Skin cream |
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