CN112941010A - Plant callus efficacy filtrate and exosome extraction method - Google Patents
Plant callus efficacy filtrate and exosome extraction method Download PDFInfo
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Abstract
The invention provides a method for extracting plant callus efficacy filtrate and exosome and application thereof. The extraction method comprises the steps of adding plant callus into hank's liquid, homogenizing, filtering to obtain filtrate, centrifuging under different centrifugal adjustment for multiple times, and collecting supernatant C to obtain plant callus effect filtrate; and (3) filling the light green or light yellow glue left in the container into a transparent precipitate, adding PBS buffer solution or normal saline, dissolving and freeze-drying the transparent precipitate to obtain light yellow or light green or white dry powder, namely the plant callus exosome. The plant callus efficacy filtrate and the plant callus exosome are both used for healing skin wound, moisturizing skin and delaying skin aging. The purified exosome product of the plant tissue obtained by the invention has stable property, can be transported and stored at normal temperature, can reduce the inflammatory reaction of the skin, activate the regeneration of epidermis, enhance the proliferation and differentiation capacity of skin cells, promote the healing of wound surfaces, and resist and reverse photoaging.
Description
Technical Field
The invention relates to the technical field of plant extraction, in particular to a method for extracting plant callus exosomes rich in callus efficacy component body fluid and a purified exosome product thereof from edelweiss callus or coastal cress callus or aniseed callus and application thereof.
Background
The callus is a highly vacuolated and amorphous irregular loose tissue which is formed under the condition of in vitro plant culture and contains a large number of plant stem cells. The plant callus is rich in plant stem cells, has various functional components, and has higher concentration of the functional components and wider component spectrum compared with common plant extracts. Common callus includes edelweiss callus, cress callus and aniseed callus. Edelweiss callus has now been isolated and identified: the collagen. The callus of the coastal cress is rich in compounds such as triterpenoid saponins, alkaloids, iridoids, volatile oil and the like, has the characteristics of complex and various components and the like, and has the effects of resisting inflammation, bacteria and aging proved by pharmacological tests. The callus of the hypecoum vulgare has been separated into anisole, alpha-anisyl ketone, methyl piperonyl alcohol, anisaldehyde, vitamin C, etc. The antioxidant activity of the hypecoum aquifolium extract is higher than that of alpha-tocopherol and BHT under all concentrations, the hypecoum aquifolium extract can inhibit the peroxidation of linoleic acid, can inhibit the activity of tyrosinase, has the effect of whitening, and has the effects of resisting inflammation and oxidation, promoting the metabolic balance of collagen and promoting the regeneration of collagen through pharmacological tests.
At present, most of plant extract products on the market are essential oil, hydrolat, cell sap and the like, and the function utilization is limited. Although essential oil is an aromatic substance unique to plants, the essential oil has certain effect, but contains less effective protein, and particularly, the effect of the essential oil is not ideal due to the fact that water-soluble protein does. The plant hydrolat only contains a small amount of low-boiling point water-soluble substances except a large amount of water molecules, and most of active substances are abandoned. The plant cell sap is a plant product produced by collecting culture solution of a edelweiss cell line or a cell strain after in vitro culture. The disadvantages are that the content of effective substances is low, the cell line cell strain has the possibility of wireless proliferation, and the risk of tumorigenicity or tumor induction when the cell line cell strain is applied to a human body is not eliminated. In recent years, researches show that the plant can secrete a vesicular body with the diameter of 40-100nm, is named as an exosome, selectively wraps various bioactive substances such as lipid, protein, mRNA, miRNA and the like, and has the definite functions of reducing the degree of tissue damage and promoting the morphology and functional repair of damaged tissues.
In the existing plant exosome extraction scheme, due to the difference of plant osmotic pressure, the vesicle structure of exosome is broken possibly due to the difference of osmotic pressure in the process of extracting for hours by adopting PBS or normal saline, so that the extraction yield of exosome is reduced, and the exosome cannot exert efficacy; in addition, in the existing plant exosome extraction scheme, coarse filtration of filter residues is lacked, and due to the characteristic of rich fiber of plant tissues, the plant tissues which are not subjected to coarse filtration float in homogenate, and low-speed centrifugation does not have practical operability. In addition, in the existing plant exosome extraction scheme, excipients such as glycerol, propylene glycol, mannitol and the like are added in the final extraction step, so that osmotic pressure changes and the extracted exosomes are cracked, and the actual efficacy of the finally extracted exosomes is reduced. Meanwhile, the added excipient has certain sensitization and chemical activity, and the operation difficulty is increased in the subsequent preparation of pharmacy and cosmetics.
Skin injuries are common in daily life, are as small as skin pigmentation, roughness, acne and comedo, are as large as skin burn and scald, wound surfaces can be large or small, deep or shallow, and injured parts are different, so that troubles and pains are brought to patients; at present, researchers collect exosomes from natural edible plants, and although the exosomes can be used for human body beauty, the skin repair capacity is still needed to be researched.
Disclosure of Invention
The invention provides a method for extracting plant callus effect ingredient body fluid and exosome from edelweiss callus or coastal cress callus or aniseed callus and application of the extracted plant callus effect ingredient body fluid and exosome aiming at various defects in the prior art for extracting and applying the plant exosome.
In order to achieve the aim, the invention provides a method for extracting plant callus efficacy filtrate and exosome, which is characterized by comprising the following specific steps:
(1) adding the plant callus into hank's liquid according to the volume ratio of 1: 1-1: 5, homogenizing, filtering by using a 300-2500-mesh screen, taking filtrate, centrifuging for 10-30 minutes by using 300-500 g, and taking supernatant A; the plant callus is a edelweiss callus, a stringy cress callus or a hypsizygus marmoreus callus;
(2) centrifuging the supernatant A for 10-30 minutes at 2000-4000 g, and taking a supernatant B;
(3) centrifuging the supernatant B for 90-110 minutes by using a centrifugal force of 100000-120000 g, and separating out a supernatant C, wherein the supernatant C is plant callus efficacy filtrate;
(4) and (3) collecting the light green or light yellow cementing transparent precipitate left in the container after the supernatant C is separated in the step (3), adding PBS buffer solution or normal saline to dissolve the precipitate, transferring the dissolved precipitate into a-80 ℃ refrigerator or liquid nitrogen to be subjected to quick freezing for 10-20 min, transferring the dissolved precipitate into a freeze dryer to be dried for 24 hours, and performing vacuum packaging to obtain light yellow or light green or white dry powder, namely the plant callus exosomes.
The invention has the following excellent technical scheme: the plant callus in the step (1) is cultured in vitro to form embryo-shaped callus; the volume ratio of the plant callus to the hank's liquid in the step (1) is 1: 1; filtering the homogenate in the step (1) by a 2500-mesh screen, and centrifuging the homogenized filtered liquid at 400g for 20 minutes to obtain supernatant A.
The invention has the following excellent technical scheme: centrifuging the supernatant A in the step (2) at 3000g for 20 minutes; and (4) centrifuging the supernatant B in the step (3) for 100 minutes by using a 11000 centrifugal force.
The invention has the following excellent technical scheme: and (4) directly adding the precipitate into a centrifugal tube in the step (4) to dissolve, adding the physiological saline into 1/20-1/10 of the volume of the centrifugal tube, transferring the precipitate into a penicillin bottle, placing the penicillin bottle into a refrigerator with the temperature of-80 ℃ or liquid nitrogen for quick freezing for 10-20 min, and then transferring the penicillin bottle into a freeze dryer for drying for 24 hours at the temperature of-40 ℃ to-80 ℃ and under the pressure of less than 100 pa.
The invention provides application of plant callus efficacy filtrate prepared according to the extraction method, which is characterized by comprising the following steps: the plant callus efficacy filtrate is independently used as a medicine or a skin care product for healing skin wounds, moisturizing skin and delaying skin aging, or is added into a medicine or a skin care product for healing skin wounds, moisturizing skin and delaying skin aging.
The invention has the following excellent technical scheme: when the plant callus effect filtrate is independently used as a medicine or a skin care product for healing skin wound surfaces, moisturizing skin and delaying skin aging, the dilution concentration of the plant callus effect filtrate is 40-60%, and the plant callus effect filtrate is used for promoting skin absorption through a needle-free high-pressure injection device.
The invention has the following excellent technical scheme: adding the plant callus efficacy filtrate into normal saline and glycerol, and uniformly mixing to prepare moisturizing spray, wherein the mixing volume ratio of the plant callus efficacy filtrate, the normal saline and the glycerol is 8-12: 7-10: 0.8-1.2.
The invention provides an application of a plant callus exosome prepared according to the extraction method, which is characterized in that: the plant callus exosome is independently used as a medicine or a skin care product for healing skin wounds, moisturizing skin and delaying skin aging, or is added into the medicine or the skin care product for healing skin wounds, moisturizing skin and delaying skin aging.
The invention has the following excellent technical scheme: when the plant callus exosomes are independently used as medicines or skin care products for healing skin wounds, moisturizing skin and delaying skin aging, the dilution concentration of the plant callus exosomes is 8-12%, and the plant callus exosomes are promoted to be absorbed by the skin through a needle-free high-pressure injection device.
The invention has the following excellent technical scheme: the plant callus exosome is emulsified and uniformly stirred according to the following formula to prepare the skin-care moisturizing cream, wherein the weight parts of various substances in the formula are as follows in percentage by mass: 0.05-2.0 parts of plant callus secretion, 1-5 parts of isopropyl palmitate, 1-5 parts of pentaerythritol distearate, 1-5 parts of cetearyl alcohol, 1-5 parts of isomeric hexadecane, 2-8 parts of alkyl polyglycoside, 2-5 parts of phospholipid, 1-3 parts of polyquaternary ammonium salt, 1-3 parts of hyaluronic acid, 0.01-0.2 part of preservative, 0-0.03 part of essence and the balance of distilled water.
The invention has the beneficial effects that:
(1) the method adopts hank's liquid (balanced salt solution) to extract the plant exosomes, can better maintain the stable structure of the plant exosomes through experimental research, adds a coarse filtration step after homogenate, sets a low-speed centrifugation part into two steps, improves the actual operability of exosome extraction, re-suspends the exosomes by using normal saline in a final extraction step, adopts the steps of freeze-drying and vacuum packaging, obtains the purified exosome product of the edelweiss callus or the coastal cress callus or the hypecoum vulgaris callus, has stable property, can be transported and stored at normal temperature, and can keep the form of the exosomes after dissolution;
(2) the method can extract plant exosomes and callus efficacy filtrate, and the obtained callus purification exosome product and callus efficacy filtrate can reduce skin inflammatory reaction, activate epidermal regeneration, enhance the proliferation and differentiation capacity of skin cells, promote wound healing, resist and reverse photoaging, have good migration promotion effect on fibroblasts and keratinocytes, are new biomedical products, and can be used for preparing various daily chemical products;
(3) the plant callus exosomes can be absorbed by cells in ways of endocytosis, exocytosis and the like, so that the application range of the plant callus exosomes as a skin care product is enlarged, and the plant callus exosomes can be also absorbed by the skin by using a needle-free high-pressure injection device, so that a better treatment or beauty effect is achieved;
(4) the callus efficacy filtrate or callus purification exosome product obtained in the invention can be independently applied in the aspects of preparing skin care products for promoting facial rejuvenation and biological medicines for promoting wound healing, and can also be added into skin care products and biological dressings for further development.
Drawings
FIG. 1 is a schematic diagram showing three kinds of callus exosomes of edelweiss, Crassipes maritima and a callus efficacy filtrate of the edelweiss in the embodiment of the invention, wherein the number 1 is the snowflower callus exosomes, the number 2 is the snowflower callus efficacy filtrate, the number 3 is the Crassipes maritima callus exosomes, the number 4 is the coastal Crassipes callus efficacy filtrate, the number 5 is the Crassius maritima callus exosomes, and the number 6 is the Crassius maritima callus efficacy filtrate;
FIG. 2 is the observation image of the exosome of the edelweiss callus under a transmission electron microscope in the example;
FIG. 3 is the observation image of the exosomes of the snowflake callus under the scanning electron microscope in the example;
FIG. 4 is a transmission electron microscope observation image of the callus exosomes of Oenanthe stolonifera in the example;
FIG. 5 is a transmission electron microscope observation image of the callus exosomes of Oenanthe stolonifera in the example;
FIG. 6 is the observation image of the callus exosomes of sea fennel in the example under the scanning electron microscope;
FIG. 7 is an observation image of sea fennel callus exosomes under a scanning electron microscope in the example;
FIG. 8 is a graph comparing the effect of various concentrations of the snowflake callus exosome product (PPCEP) on fibroblast-promoted migration in the examples;
FIG. 9 is a graph comparing the effect of various concentrations of the edelweiss callus exosome product (PPCEP) on the promotion of migration of keratinocytes in the examples;
FIG. 10 is a graph comparing the effect of various concentrations of the chenopodium hybridum callus efficiency filtrate (PPCaF) on the promotion of migration of fibroblasts in the examples;
FIG. 11 is a graph comparing the effect of the various concentrations of the edelweiss callus efficiency filtrate (PPCaF) on the promotion of migration of keratinocytes in the examples;
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The present invention will be further described with reference to the following examples.
The first embodiment is as follows: adding the edelweiss callus into hank's solution according to the volume ratio of 1:1, homogenizing, filtering by a 2500-mesh screen, taking filtrate, centrifuging for 10 minutes at 500g, and taking supernatant A; centrifuging at 4000g for 30 min, and collecting supernatant B; centrifuging supernatant B at 120000g centrifugal force for 90 min, pouring supernatant C into a new container, wherein the yellowish supernatant C is herba Centellae callus effect filtrate, and is shown in figure 1 with number 2; pouring out faint yellow cementing transparent precipitate attached to the tube wall to obtain the precipitate of the edelweiss callus exosome, adding 2mL of physiological saline, redissolving the purified exosome, transferring the precipitate into a penicillin bottle, carrying out quick freezing in liquid nitrogen, transferring the precipitate into a freeze dryer, drying for 24 hours, and carrying out vacuum packaging to obtain faint yellow dry powder which is the edelweiss callus exosome, wherein the view is specifically shown as the figure 1 with the number of 1.
Example two: adding the callus of the coastal cress into hank's solution according to the volume ratio of 1:1, homogenizing, filtering by a 2500-mesh screen, taking filtrate, centrifuging for 10 minutes at 500g, and taking supernatant A; centrifuging at 4000g for 30 min, and collecting supernatant B; centrifuging supernatant B at 120000g centrifugal force for 90 min, pouring supernatant C into a new container, wherein the yellowish supernatant C is herba Oenanthes Javanicae callus effect filtrate, and is shown in figure 1 with number 4; pouring out the light yellow glue attached to the tube wall to obtain transparent sediment, namely the purified secretion body sediment of the callus of the coastal cress, adding 2mL of normal saline, dissolving the purified secretion body again, transferring the purified secretion body into a penicillin bottle, carrying out quick freezing in liquid nitrogen, transferring the purified secretion body into a freeze dryer to be dried for 24 hours, and carrying out vacuum packaging to obtain light yellow dry powder, namely the purified secretion body of the callus of the coastal cress, wherein the view is shown as the figure with the number 3 in the figure 1.
Example three: adding the calluses of the hypecoum vulgare into hank's solution according to the volume ratio of 1:1, homogenizing, filtering by a 2500-mesh screen, taking filtrate, centrifuging for 10 minutes at 500g, and taking supernatant A; centrifuging at 4000g for 30 min, and collecting supernatant B; centrifuging the supernatant B at 120000g centrifugal force for 90 min, pouring supernatant C into a new container, wherein the yellowish supernatant C is the herba Anisi Stellati callus efficacy filtrate, and is shown in figure 1 with number 6; pouring out faint yellow cementing transparent precipitate attached to the tube wall, namely sea fennel callus purified exosome precipitate, adding 2mL of physiological saline, redissolving the purified exosome, transferring into a penicillin bottle, carrying out flash freezing in liquid nitrogen, transferring into a freeze dryer, drying for 24 hours, and carrying out vacuum packaging to obtain faint yellow dry powder, namely the sea fennel callus exosome, wherein the view is shown as the figure with the number 5 in the figure 1.
The inventors of the present application observed the cedar flower callus exosomes prepared in the first example (detailed shown in fig. 2 and 3), the coastal celery callus purification exosomes prepared in the second example (detailed shown in fig. 4 and 5), and the anisetree callus exosomes prepared in the third example (detailed shown in fig. 6 and 7) by using a transmission electron microscope and a scanning electron microscope, and the prepared exosomes have complete structures and sizes between 80-200nm as can be seen from the attached drawings.
The inventor of the present application also conducted comparative experimental studies on the effect of different concentrations of the cedar callus exosome (PPCEP) and the cedar callus efficacy filtrate (PPCaF) on promoting migration of fibroblasts and keratinocytes, specifically as follows:
test one: in vitro scratch experiments were performed on skin cells using the snowflake callus exosomes (PPCEPs) prepared in example one. Preparing 5%, 10% and 20% PPCEP solution by using a blank DMEM medium, adding the solution into a 6-pore plate which is fully paved with cells, scratching the ground by using a 1ml gun head, interfering in vitro fibroblasts and keratinocytes by using PPCEP with different concentrations, and comparing the solution with the blank DMEM medium and the DMEM medium containing 10% bovine serum to observe the migration rate of the fibroblasts and the keratinocytes at different time points. The test results are shown in fig. 8 (migration promoting effect of PPCEP with different concentrations on fibroblasts) and fig. 9 (migration promoting effect of PPCEP with different concentrations on keratinocytes), and it can be seen from the comparison results that PPCEP with different concentrations has a better migration promoting effect on fibroblasts and keratinocytes, which indicates that PPCEP is helpful for healing skin wounds and improving skin cell activity. The PPCEP with 10% concentration has the strongest migration promoting ability to fibroblasts and keratinocytes.
And (2) test II: in vitro scratch experiments were performed on skin cells using the edelweiss callus efficiency filtrate (PPCaF) prepared in example one. Preparing 20%, 50% and 100% PPCEP solution by using a blank DMEM medium, adding the solution into a 6-well plate which is fully paved with cells, scratching the ground by using a 1ml gun head, interfering in vitro fibroblasts and keratinocytes by using PPCaF with different concentrations, and comparing the solution with the blank DMEM medium and the DMEM medium containing 10% bovine serum to observe the migration rate of the fibroblasts and the keratinocytes at different time points. The test results are shown in fig. 10 (migration promoting effect of PPCaF with different concentrations on fibroblasts) and fig. 11 (migration promoting effect of PPCaF with different concentrations on keratinocytes), which shows that PPCaF with different concentrations has a better migration promoting effect on fibroblasts and keratinocytes, although the migration promoting ability of PPCaF on skin cells is weaker than that of positive control calf serum, compared with blank control, a significant promoting effect is still exhibited, which indicates that PPCaF contributes to healing of skin wound and improves skin cell activity.
The inventors of the present application also conducted experimental studies on the application of the callus exosomes and callus efficacy filtrates prepared in the present application, which are specifically as follows:
example four: the application of the functional filtrate (PPCaF) of the snowflake callus prepared in the first embodiment to preparation of a skin-care moisturizing spray specifically comprises the steps of taking 50mL of the PPCaf prepared in the first embodiment, adding 45mL of physiological saline and 5mL of glycerin to adjust skin touch, uniformly mixing, filling into a spray bottle, and pressurizing with nitrogen to prepare the functional filtrate (PPCaF) of the snowflake callus moisturizing spray.
The results of a pilot study on the skin care moisturizing spray of the chenopodium hybridum callus efficacy filtrate (PPCaF) prepared in example four are as follows:
1. subject No. 1 before spraying with the chenopodium alpinum callus effect filtrate (PPCaF) for skin care and moisture retention had skin moisture of 23.5%, 66.4% immediately after application, and 33.1% after 6 hours, with difference values of 42.9% and 9.6%, respectively, and had significant water replenishing effect and good moisture retention effect.
2. Test subject No. 2 had skin moisture of 26.4% before skin-care moisturizing spray with the filtrate of the effect of the edelweiss callus (PPCaF), 56.2% immediately after use and 28.2% after 6 hours, the difference values were 32.4% and 6%, respectively, and the moisturizing effect was good and good.
3. Test subject No. 2 before spraying with the chenopodium hybridum callus efficacy filtrate (PPCaF) for skin care and moisture retention, the skin moisture was 22.9%, 64.3% immediately after use and 36.1% after 6 hours, the difference values were 41.4% and 13.2%, the moisturizing effect was significant, and the moisturizing effect was good.
The test results show that the edelweiss callus efficacy filtrate (PPCaF) is used for preparing the skin-care moisturizing spray and has good moisturizing and hydrating effects.
Example five: the edelweiss callus exosome (PPCEP) prepared in the first example is used for other skin-care moisturizing cream, and the weight parts of the substances in the formula are as follows: according to 100 parts by weight, taking 0.05 part of PPCEP, 1 part of isopropyl palmitate, 1 part of pentaerythritol distearate, 5 parts of cetearyl alcohol, 5 parts of isomeric hexadecane, 8 parts of alkyl polyglycoside, 5 parts of phospholipid, 513 parts of polyquaternium-513, 1 part of hyaluronic acid, 0.2 part of preservative, 0.03 part of essence and the balance of distilled water; the raw materials are emulsified and stirred uniformly to prepare the edelweiss callus purified exosome product (PPCEP) skin-care moisturizing cream.
And the skin-care moisturizing cream containing the snowflake callus exosomes (PPCEP) prepared in the fifth example is tried, and the experimental study is as follows:
1. subject No. 1 used a edelweiss callus purified exosome product (PPCEP) before skin-care moisturizing cream, the skin moisture was 22.0%, 54.2% immediately after use, and 48.1% after 6 hours, the difference values were 32.2% and 26.1%, respectively, the moisturizing effect was significant, and the moisturizing effect was significant.
2. Subject No. 2 used a edelweiss callus purified exosome product (PPCEP) before skin-care moisturizing cream, the skin moisture was 24.1%, 48.2% immediately after use, 32.1% after 6 hours, the difference was 24.1% and 8%, the moisturizing effect was good, and the moisturizing effect was better.
3. Subject No. 3 used a edelweiss callus purified exosome product (PPCEP) before skin-care moisturizing cream, the skin moisture was 29.2%, 44.3% immediately after use, and 41.4% after 6 hours, the difference values were 15.1% and 12.2%, respectively, the moisturizing effect was good, and the moisturizing effect was good.
The experiments prove that the edelweiss callus exosome (PPCEP) prepared in the invention has good water replenishing and moisturizing effects when applied to skin cream.
Claims (10)
1. A method for extracting plant callus efficacy filtrate and exosome is characterized by comprising the following specific steps:
(1) adding the plant callus into hank's liquid according to the volume ratio of 1: 1-1: 5, homogenizing, filtering by using a 300-2500-mesh screen, taking filtrate, centrifuging for 10-30 minutes by using 300-500 g, and taking supernatant A; the plant callus is a edelweiss callus, a stringy cress callus or a hypsizygus marmoreus callus;
(2) centrifuging the supernatant A for 10-30 minutes at 2000-4000 g, and taking a supernatant B;
(3) centrifuging the supernatant B for 90-110 minutes by using a centrifugal force of 100000-120000 g, and separating out a supernatant C, wherein the supernatant C is plant callus efficacy filtrate;
(4) and (3) collecting the light green or light yellow cementing transparent precipitate left in the container after the supernatant C is separated in the step (3), adding PBS buffer solution or normal saline to dissolve the precipitate, transferring the dissolved precipitate into a-80 ℃ refrigerator or liquid nitrogen to be subjected to quick freezing for 10-20 min, transferring the dissolved precipitate into a freeze dryer to be dried for 24 hours, and performing vacuum packaging to obtain light yellow or light green or white dry powder, namely the plant callus exosomes.
2. The method for extracting plant callus efficacy filtrate and exosomes according to claim 1, characterized by comprising the following specific steps: the plant callus in the step (1) is cultured in vitro to form embryo-shaped callus; the volume ratio of the plant callus to the hank's liquid in the step (1) is 1: 1; filtering the homogenate in the step (1) by a 2500-mesh screen, and centrifuging the homogenized filtered liquid at 400g for 20 minutes to obtain supernatant A.
3. The method for extracting plant callus efficacy filtrate and exosomes according to claim 1, characterized by comprising the following specific steps: centrifuging the supernatant A in the step (2) at 3000g for 20 minutes; and (4) centrifuging the supernatant B in the step (3) for 100 minutes by using a 11000 centrifugal force.
4. The method for extracting plant callus efficacy filtrate and exosomes according to claim 1, characterized by comprising the following specific steps: and (4) directly adding the precipitate into a centrifugal tube in the step (4) to dissolve, adding the physiological saline into 1/20-1/10 of the volume of the centrifugal tube, transferring the precipitate into a penicillin bottle, placing the penicillin bottle into a refrigerator with the temperature of-80 ℃ or liquid nitrogen for quick freezing for 10-20 min, and then transferring the penicillin bottle into a freeze dryer for drying for 24 hours at the temperature of-40 ℃ to-80 ℃ and under the pressure of less than 100 pa.
5. Use of a plant callus efficiency filtrate prepared according to the extraction method of any one of claims 1 to 3, characterized in that: the plant callus efficacy filtrate is independently used as a medicine or a skin care product for healing skin wounds, moisturizing skin and delaying skin aging, or is added into a medicine or a skin care product for healing skin wounds, moisturizing skin and delaying skin aging.
6. The use of a plant callus efficacy filtrate as claimed in claim 5, wherein: when the plant callus effect filtrate is independently used as a medicine or a skin care product for healing skin wound surfaces, moisturizing skin and delaying skin aging, the dilution concentration of the plant callus effect filtrate is 40-60%, and the plant callus effect filtrate is used for promoting skin absorption through a needle-free high-pressure injection device.
7. The use of a plant callus efficacy filtrate as claimed in claim 5, wherein: adding the plant callus efficacy filtrate into normal saline and glycerol, and uniformly mixing to prepare moisturizing spray, wherein the mixing volume ratio of the plant callus efficacy filtrate, the normal saline and the glycerol is 8-12: 7-10: 0.8-1.2.
8. Use of plant callus exosomes prepared according to the extraction method of any one of claims 1 to 4, characterized in that: the plant callus exosome is independently used as a medicine or a skin care product for healing skin wounds, moisturizing skin and delaying skin aging, or is added into the medicine or the skin care product for healing skin wounds, moisturizing skin and delaying skin aging.
9. Use of a plant callus exosome according to claim 8, wherein: when the plant callus exosomes are independently used as medicines or skin care products for healing skin wounds, moisturizing skin and delaying skin aging, the dilution concentration of the plant callus exosomes is 8-12%, and the plant callus exosomes are promoted to be absorbed by the skin through a needle-free high-pressure injection device.
10. The application of the plant callus exosome according to claim 8, wherein the plant callus exosome is emulsified and uniformly stirred according to the following formula to prepare a skin-care moisturizing cream, and the weight parts of various substances in the formula are as follows, and the skin-care moisturizing cream is prepared from the following components in percentage by mass: 0.05-2.0 parts of plant callus secretion, 1-5 parts of isopropyl palmitate, 1-5 parts of pentaerythritol distearate, 1-5 parts of cetearyl alcohol, 1-5 parts of isomeric hexadecane, 2-8 parts of alkyl polyglycoside, 2-5 parts of phospholipid, 1-3 parts of polyquaternary ammonium salt, 1-3 parts of hyaluronic acid, 0.01-0.2 part of preservative, 0-0.03 part of essence and the balance of distilled water.
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