CN117752564A - Application of rose-derived extracellular vesicle-like nanoparticles in preparation of product with whitening and antioxidation effects - Google Patents
Application of rose-derived extracellular vesicle-like nanoparticles in preparation of product with whitening and antioxidation effects Download PDFInfo
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Abstract
The invention relates to application of rose-derived extracellular vesicle-like nano particles in preparation of a product with whitening and antioxidation effects. The invention creatively extracts and purifies the rose-derived extracellular vesicles, researches the physiological effects of the extracellular vesicles, discovers that the extracellular vesicles can be fully absorbed by melanoma cells, remarkably reduces the melanin content in the melanoma cells, remarkably reduces the melanin deposition on the body surface of zebra fish embryos, has excellent antioxidant capacity, and clinical experiments prove that the extracellular vesicles can reduce skin pigment and lighten skin color. The application of the modified starch to the preparation of products with whitening and antioxidation effects has potential and provides a new strategy for skin whitening and skin aging resistance.
Description
Technical Field
The invention belongs to the technical field of extracellular vesicles, relates to application of rose-derived extracellular vesicle-like nanoparticles, and in particular relates to application of rose-derived extracellular vesicle-like nanoparticles in preparation of products with whitening and antioxidation effects.
Background
Skin is the first line of defense in the human body, it protects the body from harmful external environmental factors, and interference with its normal structure and function can lead to various skin abnormalities including inflammation, immune skin disorders and skin aging. Attention to the skin condition is not only for external beauty, but also reflects the overall health, vitality and nutritional status of the skin. The requirements of consumers on low irritation, safety, environmental protection and the like of the beauty skin care products are gradually improved, and natural plant skin care gradually enters the field of view of the public. They have the disadvantages of low affinity and low solubility to the skin, which prevent the development of plant skin care products. Thus, a botanical product with low irritation and safety is sought that has a positive effect on plant skin care development. The rose is used as a traditional Chinese medicine plant, has low cost and better safety.
Extracellular vesicles (extracellular vesicles, EVs) contain a variety of proteins, lipids, and nucleic acids that can perform important physiological functions by mediating cell-to-cell communication, with almost all types of eukaryotic and prokaryotic cells secreting EVs. Plant EVs are similar in morphology to mammalian EVs, but have less of a study in terms of composition and function. It has been found that plant EVs are components of the innate immune system of plants and have antifungal effects. In addition, plant EVs have cross-species regulation function, not only can regulate physiological functions of mammalian cells, but also can intervene and prevent disease processes, and play a role in treating diseases. These findings demonstrate that plant EVs as a novel natural product are likely to be good candidates for new drug development. Most of the traditional Chinese medicines are plants, the cost is low, the side effect is small, but EVs derived from the traditional Chinese medicines are rarely researched. It is not known whether it is possible to extract extracellular vesicles from roses, whether the extracted extracellular vesicles of rose origin have biological activity.
Therefore, it is of interest to provide a method for extracting extracellular vesicles from roses and to further investigate the function of the extracted extracellular vesicles of rose origin to explore their potential for cosmetic use.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide the application of the rose-derived extracellular vesicle-like nano-particles, in particular to the application of the rose-derived extracellular vesicle-like nano-particles in the preparation of products with whitening and antioxidation effects.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the invention provides an application of rose-derived extracellular vesicle-like nanoparticles in preparing a product with whitening and antioxidation effects.
The invention creatively extracts and purifies the rose-derived extracellular vesicles, researches the physiological effects of the extracellular vesicles, discovers that the extracellular vesicles can be fully absorbed by melanoma cells, remarkably reduces the melanin content in the melanoma cells, remarkably reduces the melanin deposition on the body surface of zebra fish embryos, has excellent antioxidant capacity, and clinical experiments prove that the extracellular vesicles can reduce skin pigment and lighten skin color. Has potential in preparing products with whitening and antioxidation effects, and provides a new strategy for skin whitening and skin aging resistance.
The rose-derived extracellular vesicle-like nanoparticle according to the present invention can be prepared by any method known to those skilled in the art, and more preferably, the extraction method, which gives a rose-derived extracellular vesicle-like nanoparticle having higher purity and more excellent whitening and antioxidation effects, is more preferable.
Preferably, the rose-derived extracellular vesicle-like nanoparticle is prepared by a preparation method comprising the steps of:
filtering the juice of the rose raw material, centrifuging the filtrate, filtering the supernatant, ultracentrifugating the filtrate, collecting the precipitate, and resuspending the precipitate.
Preferably, the filtrate centrifugation is performed at 2000-5000g (e.g., 2000g, 2500g, 3000g, 3500g, 4000g, 4500g, 5000g, etc.) at 0-8 ℃ (e.g., 0 ℃,1 ℃, 2 ℃, 3 ℃, 4 ℃, 5 ℃, 6 ℃, 7 ℃, 8 ℃, etc.) for 20-40min (e.g., 20min, 25min, 30min, 35min, 40min, etc.), the supernatant is collected, and further centrifugation is performed at 8000-15000g (e.g., 8000g, 9000g, 10000g, 11000g, 12000g, 13000g, 14000g, 15000g, etc.) for 20-40min (e.g., 20min, 25min, 30min, 35min, 40min, etc.).
Specific point values in the numerical ranges are selectable, and will not be described in detail here.
Preferably, the ultracentrifugation is performed at 100000-180000g (e.g., 100000g, 110000g, 120000g, 130000g, 140000g, 150000g, 160000g, 170000g, 180000g, etc.) at 0-8 ℃ (e.g., 0 ℃,1 ℃, 2 ℃, 3 ℃, 4 ℃, 5 ℃, 6 ℃, 7 ℃, 8 ℃, etc.) for 40-100min (e.g., 40min, 50min, 60min, 70min, 80min, 90min, 100min, etc.).
Specific point values in the numerical ranges are selectable, and will not be described in detail here.
Preferably, the preparation method further comprises filtration using a 0.22 μm filter membrane after the sediment is resuspended.
In the present invention, the product includes a cosmetic or pharmaceutical product.
In the invention, the product is a cosmetic, and the dosage form of the product comprises emulsion, water agent, oil agent or gel agent.
In the invention, the product is a medicine, and the dosage forms comprise oil, emulsion, ointment, paste, film coating agent, gel, aerosol, spray, solution and liniment.
Preferably, the product further comprises an auxiliary material.
Preferably, the auxiliary materials comprise any one or a combination of at least two of carriers, fillers, thickeners, binders, emulsifying agents, pH regulators, antioxidants, preservatives, solubilizers, cosolvents or solvents.
In a second aspect, the invention provides the use of rose-derived extracellular vesicle-like nanoparticles for the preparation of a formulation for reducing the amount of melanin in melanoma cells.
According to the research result of the invention, the rose-derived extracellular vesicle-like nano-particles can reduce the content of melanin in melanoma cells at the cellular level (in vitro level), namely, the rose-derived extracellular vesicle-like nano-particles can be prepared into a simple preparation for test, which is used for exploring the physiological metabolic process of the melanoma cells, and the preparation claimed by the invention is not used for eliminating the etiology or focus, namely, the application of the preparation for reducing the content of melanin in the melanoma cells with the non-therapeutic purpose.
Compared with the prior art, the invention has the following beneficial effects:
the invention creatively extracts and purifies the rose-derived extracellular vesicles, researches the physiological effects of the extracellular vesicles, discovers that the extracellular vesicles can be fully absorbed by melanoma cells, remarkably reduces the melanin content in the melanoma cells, remarkably reduces the melanin deposition on the body surface of zebra fish embryos, has excellent antioxidant capacity, and clinical experiments prove that the extracellular vesicles can reduce skin pigment and lighten skin color. The application of the modified starch to the preparation of products with whitening and antioxidation effects has potential and provides a new strategy for skin whitening and skin aging resistance.
Drawings
FIG. 1 is an electron microscopy characterization of rose-derived extracellular vesicles (R-EVP);
FIG. 2A is a morphological diagram of mouse melanoma cells;
FIG. 2B is a graph showing the results of the RTCA method for detecting proliferation of R-EVP on melanoma cells in mice;
FIG. 3A is a graph of fluorescence microscopy for detecting internalization uptake of R-EVP by murine melanoma cells;
FIG. 3B is a graph showing the statistical result of melanin content of a mouse melanoma cell detected by an enzyme-labeled instrument;
fig. 4A is a graph of melanin content in zebra fish photographed under a stereoscopic microscope;
FIG. 4B is a graph of the melanin content of zebra fish embryos analyzed using imageJ software;
FIG. 5 is a graph showing the results of evaluation of antioxidation of rose-derived extracellular vesicles (R-EVPs);
FIG. 6A is a diagram of the skin conditions before and after use of the hydroquinone cream set and the R-EVP set;
FIG. 6B is a graph of individual results and statistical results of skin brightness (L values) for the R-EVP group;
fig. 6C is a graph of individual results and statistics of skin brightness (L x values) for the hydroquinone cream group.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
Example 1
Extraction of rose-derived extracellular vesicles (R-EVP):
(1) The method comprises the steps of (1) taking 1000g of dried rosebuds, carrying out primary cleaning by using sterile water, squeezing, filtering, collecting supernatant, standing in a clean 5-liter beaker, and centrifuging for 30min at 3000 g;
(2) After collecting the supernatant, it was centrifuged and filtered with a 0.22 μm filter;
(3) Collecting the supernatant in a clean 50mL centrifuge tube, and centrifuging at 10000g for 30min at 4 ℃;
(4) The supernatant was collected using a 50mL centrifuge tube and filtered using a 0.22 μm membrane pump; collecting the liquid after suction filtration in a centrifuge tube special for an ultra-high speed centrifuge, and centrifuging at 135000g for 70 minutes at 4 ℃;
(5) The supernatant was discarded, and after re-suspending the pellet with pre-chilled 1 XPBS buffer, the pellet was filtered using a 0.22 μm disposable needle filter, and finally the R-EVP was collected with a sterilized EP tube and stored in a refrigerator at-80℃for further use.
Example 2
Characterization of rose-derived extracellular vesicles (R-EVP):
the particle size, purity and morphology of the R-EVP extracted in example 1 were characterized.
(1) The particle size distribution of R-EVP is analyzed by a nanoparticle tracing analyzer, and the average particle size is 60.52 +/-10.66 nm, so that the particle size of the particle meets the particle size range of extracellular vesicles reported in the literature. The concentration is 4.8X10 12 particles/mL.
(2) Detecting the purity of the R-EVP by using Triton X-100 membrane rupture experiment: after the R-EVP is dissolved by using Triton X-100 with different concentrations, the change of particle number is tested by a nanoflow detector, and the research result shows that the purity of the extracted R-EVP is as high as 90 percent.
(3) The morphology of R-EVP was observed under a transmission electron microscope as shown in fig. 1: the surface is concave, the typical cup-holder-shaped and disc-shaped vesicle structure is shown, the cell membrane structure is clear and visible, the membrane boundary is clear and sharp, the dyeing background is clean, and the contrast is obvious.
Example 3
Internalization uptake assay of rose-derived extracellular vesicles (R-EVP):
the sufficient internalization absorption of R-EVP by mouse melanoma cells is the basis of the precondition of the subsequent action of R-EVP on mouse melanoma cells, so that whether R-EVP can be internalized and absorbed by mouse melanoma cells is verified before the subsequent experiment is carried out.
(1) Culture of murine melanoma cells: mouse melanoma cells were purchased from Shanghai Redson Biotechnology Inc., and the morphology of the mouse melanoma cells is shown in FIG. 2A.
(2) The effect of R-EVP on proliferation of melanoma cells of mice was examined by RTCA: mouse melanoma cells were seeded at appropriate cell concentrations in 16-well plates. R-EVP with different concentrations is used for co-culturing with mouse melanoma cells for 72 hours, and the method is based on the principle of an electrical impedance sensor and is used for monitoring the growth and proliferation of the cells in real time. As a result, as shown in FIG. 2B, the concentration (1X 10) 6 、1×10 7 、1×10 8 ) Has neither an inhibitory nor proliferation-promoting effect on mouse melanoma cells.
(3) Fluorescence microscopy detects R-EVP uptake by murine melanoma cells internalization:
(3.1) Dil-R-EVP solution preparation: an appropriate amount of R-EVP was taken and supplemented to 1mL with 1 XPBS buffer, after mixing well 10. Mu.L of DiI dye was added and the mixture was marked at 37℃for 30 minutes in the absence of light. The pellet was resuspended in 1 XPBS buffer and centrifuged 3 times under equivalent centrifugation conditions to wash unlabeled R-EVP and excess DiI dye after centrifugation at 100,000Xg for 70 minutes at 4℃with removal of the supernatant. Finally, the pellet was resuspended in 100. Mu.L PBS buffer and collected in an EP tube, labeled DiI+R-EVP, and used immediately or buffered in a-80℃refrigerator.
(3.2) fluorescence microscopy of R-EVP uptake by murine melanoma cells internalization: the labeled R-EVP at different particle concentrations were co-cultured with mouse melanoma cells for 2h, 4h and 8h. The uptake of R-EVP by murine melanoma cells was then examined using fluorescence microscopy. As shown in fig. 3A, dil-R-EVP was internalized and absorbed by the murine melanoma cells and the degree of internalization increased with increasing Dil-R-EVP concentration and prolonged co-incubation time, with the internalized Dil-R-EVP being largely located within the cytoplasm of the murine melanoma cells.
(3.3) study of R-EVP on regulation of melanin in melanoma cells in mice:
(3.3.1) Nano FCM detection of particle content of R-EVP: particle concentration was 4.8X10 12 particles/mL. During the subsequent study, we measured the amount of R-EVP used in terms of particle concentration.
(3.3.2) detection of melanin content of mouse melanoma cells by enzyme-labeled instrument: the particle concentration of R-EVP is 1×10 5 、1×10 6 、1×10 7 And 0.2 microliter of arbutin as a whitening positive drug is respectively interfered for 72 hours, washed 3 times with PBS buffer solution, and 600 microliter of 1M NaOH is added. Then, the cells are fully cracked and shed by blowing in water bath at 80 ℃ for 1 hour, transferred into a 96-well plate and marked with the cells and the medicines. The absorbance was measured using a microplate reader, and the relative melanin content value of each group relative to the normal group was calculated, and as a result, as shown in fig. 3B, R-EVP was able to reduce the melanin content in the melanoma cells of mice.
The results prove that the R-EVP can be fully internalized and absorbed by the melanoma cells of the mice, has no toxic effect on the melanoma cells of the mice, and can reduce the melanin content in the melanoma cells of the mice.
Example 4
In vivo test for reduction of pigmentation by rose-derived extracellular vesicles (R-EVP):
(1) Preparing a solution: preparing a zebra fish photographing fixing solution, pouring 20g of methyl cellulose into boiling water, uniformly stirring, and then adding 30 microliters of 500-fold diluted MS-222 anesthetic.
(2) Culturing: the sexually mature female and male zebra fish are paired in a ratio of 1:1, naturally mated and spawned, healthy AB-series zebra fish embryos which are 6-8 hpf after fertilization (fertilized after hours) are selected and randomly placed on 6-hole cell plates, and 3ml of solution is added into each hole. Embryo culture solution containing organic solvent is added dropwise into each hole, then capped and sealed, and the mixture is placed in an incubator to culture zebra fish embryos at 29.3 ℃. The concentration of R-EVP was set to 1X 10 8 、5×10 8 、1×10 9 、5×10 9 、1×10 10 Five groups, namely, a positive control group of 1.0mg/mL arbutin and a blank control group.
(3) After 72 hours of exposure, the development of the melanin on the body surface of the zebra fish embryo is observed. After the zebra fish embryo is anesthetized, the zebra fish embryo is placed on a glass slide with a groove, the zebra fish embryo is fixed by using prepared methylcellulose gel, and a digital image of the living zebra fish embryo is photographed by a stereoscopic microscope at a total magnification of 40 x; then, the black pigment content of the zebra fish embryos was analyzed using ImageJ software. Representative 6 tails are selected for each exposure concentration for photographing and recording, and a picture of melanin content in the zebra fish body is photographed under a stereoscopic microscope, as shown in fig. 4A, and quantitative analysis is performed, and the result is shown in fig. 4B, so that R-EVP can reduce pigmentation in the zebra fish body.
Example 5
Evaluation test of antioxidant Activity of Rose derived extracellular vesicles (R-EVP):
DPPH radical scavenging test was performed on the rose-derived extracellular vesicles (R-EVP) extracted in example 1.
Preparing a solution: first, DPPH 1mg is dissolved in about 20mL of methanol, and the solution is subjected to ultrasonic treatment for 5min and is fully shaken to ensure that the upper part and the lower part are uniform. Taking 1mL of the DPPH solution, and measuring an absorbance value at 515 nm; sample solutions (1X 10 concentrations respectively) were reconstituted 10 、1×10 11 、1×10 12 particles/mL).
The specific method comprises the following steps: the sample addition systems for each group are shown in the following table, the reaction solution was shaken and then placed in a water bath at 37℃for 15 minutes, and then taken out, and the absorbance value of the solution was measured by an ultraviolet-visible spectrophotometer.
| Reagent(s) | C1/mL | C2/mL | C3/mL |
| DPPH solution | 2 | 2 | 0 |
| Sample solution | 0 | 2 | 2 |
| Anhydrous methanol | 2 | 0 | 2 |
The calculation formula of the DPPH free radical clearance is shown as follows:
DPPH radical scavenging = 1- (A2-A3)/a1×100%
Wherein A1, A2 and A3 are absorbance values of C1, C2 and C3, respectively.
As shown in FIG. 5, it is clear that the rose-derived extracellular vesicles (R-EVP) according to the present invention have excellent antioxidant effect.
Example 6
Clinical trials of rose-derived extracellular vesicles (R-EVP):
the clinical efficacy of the rose-derived extracellular vesicles (R-EVP) extracted in example 1 was studied and compared to the hydroquinone cream, a drug that has been demonstrated to improve skin pigmentation on the market.
The operation method comprises the following steps:
(1) Preparing a test sample: 1mL of rose-derived extracellular vesicles (R-EVP), 48mL of PBS solution, 0.5mL of glycerol and 0.5mL of azone are uniformly mixed.
(2) Volunteer selection criteria and grouping conditions: subjects with facial chloasma, freckle and various pigmentation phenomena, 10 people in each group, were 18-70 years old.
(3) Sample trial method: the samples were applied to the skin using the same procedure, once a day, and continuously.
(4) Detecting the index: skin brightness (L x value).
The skin conditions before and after use of the hydroquinone cream set and the R-EVP set are shown in fig. 6A; individual results and statistics of skin brightness (L values) for the R-EVP group are shown in fig. 6B, and individual results and statistics of skin brightness (L values) for the hydroquinone cream group are shown in fig. 6C (P <0.001 (student's t test) compared to the control group), thus both R-EVP and hydroquinone cream can improve skin pigmentation.
The applicant states that the technical solution of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above embodiments, i.e. it does not mean that the present invention must be implemented by the above embodiments. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Claims (10)
1. Application of rose-derived extracellular vesicle-like nanoparticles in preparation of products with whitening and antioxidation effects.
2. The use according to claim 1, wherein the rose-derived extracellular vesicle-like nanoparticle is prepared by a preparation method comprising the steps of:
filtering the juice of the rose raw material, centrifuging the filtrate, filtering the supernatant, ultracentrifugating the filtrate, collecting the precipitate, and resuspending the precipitate.
3. Use according to claim 2, characterized in that the centrifugation of the filtrate is carried out at 2000-5000g for 20-40min at 0-8 ℃, the supernatant is collected and then at 8000-15000g for 20-40min.
4. Use according to claim 2 or 3, characterized in that the ultracentrifugation is performed at 100000-180000g for 40-100min at 0-8 ℃.
5. The use according to any one of claims 2 to 4, wherein the preparation method further comprises filtration using a 0.22 μm filter membrane after the sediment is resuspended.
6. The use according to claim 1, wherein the product comprises a cosmetic or pharmaceutical product.
7. The use according to claim 6, wherein the product is a cosmetic product in a form comprising an emulsion, a water, an oil or a gel;
preferably, the product is a pharmaceutical product, and the dosage forms comprise oil, emulsion, paste, film coating agent, gel, aerosol, spray, solution and liniment.
8. The use according to claim 1, wherein the product further comprises an auxiliary material.
9. The use according to claim 8, wherein the adjuvant comprises any one or a combination of at least two of a carrier, a filler, a thickener, a binder, an emulsifier, a pH adjuster, an antioxidant, a preservative, a solubiliser, a co-solvent or a solvent.
10. Use of rose-derived extracellular vesicle-like nanoparticles for the preparation of a formulation for reducing the amount of melanin in melanoma cells.
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| KR20060101100A (en) * | 2005-03-19 | 2006-09-22 | 정세영 | Rose extract having skin whitening effect |
| CN113318018A (en) * | 2021-04-13 | 2021-08-31 | 广东省禾基生物科技有限公司 | Rose extract and extraction method and application thereof |
| WO2022065774A1 (en) * | 2020-09-22 | 2022-03-31 | 주식회사 바이오솔루션 | Composition for improving skin condition, comprising plant-derived extracellular vesicle and method for preparing same |
| KR20220153866A (en) * | 2021-05-12 | 2022-11-21 | 주식회사 바이오에프디엔씨 | External Composition Comprising Plant Extracellular vesicle of Rose for Improving Skin |
| CN116472018A (en) * | 2020-11-16 | 2023-07-21 | 韩国外泌体生技有限公司 | Whitening cosmetic composition comprising exosomes derived from rose stem cells as active ingredients |
| CN117064931A (en) * | 2023-09-27 | 2023-11-17 | 广州中医药大学第三附属医院(广州中医药大学第三临床医学院、广州中医药大学附属骨伤科医院、广东省中医骨伤研究院) | Application of honeysuckle-derived extracellular vesicle-like nano-particles in preparation of anti-influenza virus drugs |
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- 2023-12-15 CN CN202311731566.7A patent/CN117752564A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20060101100A (en) * | 2005-03-19 | 2006-09-22 | 정세영 | Rose extract having skin whitening effect |
| WO2022065774A1 (en) * | 2020-09-22 | 2022-03-31 | 주식회사 바이오솔루션 | Composition for improving skin condition, comprising plant-derived extracellular vesicle and method for preparing same |
| CN116472018A (en) * | 2020-11-16 | 2023-07-21 | 韩国外泌体生技有限公司 | Whitening cosmetic composition comprising exosomes derived from rose stem cells as active ingredients |
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| KR20220153866A (en) * | 2021-05-12 | 2022-11-21 | 주식회사 바이오에프디엔씨 | External Composition Comprising Plant Extracellular vesicle of Rose for Improving Skin |
| CN117064931A (en) * | 2023-09-27 | 2023-11-17 | 广州中医药大学第三附属医院(广州中医药大学第三临床医学院、广州中医药大学附属骨伤科医院、广东省中医骨伤研究院) | Application of honeysuckle-derived extracellular vesicle-like nano-particles in preparation of anti-influenza virus drugs |
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