CN117323284A - Cosmetic composition comprising fermented velvet flower callus culture extract and mixture of pomegranate-derived exosomes - Google Patents
Cosmetic composition comprising fermented velvet flower callus culture extract and mixture of pomegranate-derived exosomes Download PDFInfo
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- CN117323284A CN117323284A CN202310757895.2A CN202310757895A CN117323284A CN 117323284 A CN117323284 A CN 117323284A CN 202310757895 A CN202310757895 A CN 202310757895A CN 117323284 A CN117323284 A CN 117323284A
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- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
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Abstract
The present invention relates to a composition comprising a mixture of a fermented chenopodium faberi callus culture extract and a pomegranate-derived exosome, and provides a cosmetic composition having more excellent skin moisturizing, anti-inflammatory, anti-aging and wrinkle-improving effects than the use of the fermented chenopodium faberi callus culture extract or the pomegranate-derived exosome alone.
Description
Technical Field
The present invention relates to a cosmetic composition comprising a fermented edelweiss callus culture extract and a mixture of exosomes derived from punica granatum, which has effects of moisturizing skin, anti-inflammatory, anti-aging or improving wrinkles.
Background
Anti-inflammatory, whitening, skin moisturizing and anti-wrinkle are always a focus of attention as a beautiful and anti-aging mark. With this concern, products for whitening, moisturizing and anti-aging skin are actively being introduced in the cosmetic market, and as consumer interest increases, demand is increasing, and related cosmetic industries are growing into high added value industries.
Recently, according to many studies, many materials have been developed which can maximize not only an anti-inflammatory effect and an effect of promoting skin moisturizing ability but also an anti-wrinkle effect by changing the activity of a collagen synthesis-related element that generates wrinkles, and the demands of research and consumers have increased.
Endoplasmic reticulum means a microcapsule having a membrane structure with a diameter of about 20nm to 5 μm, and is classified into exosomes (exosomes), exosomes (ectosomes), microbubbles (microvesicles), microparticles (microparticles), and the like according to its size and composition. Wherein exosomes are substances secreted outside cells in all living cells such as animal cells, microorganisms, plants, body fluids, etc., and are substances having a size of 50nm to 200nm as a means of intercellular communication. Exosomes consist of phospholipid bilayers resembling cell membranes, which efficiently and easily transmit corresponding signals inside the cell by transmitting proteins to the target cells, and which can complex signals because a variety of secondary metabolites and substances related to genetic information can be delivered at once. But has the disadvantages of high manufacturing cost and poor price competitiveness.
In addition, in the development of conventional cosmetic materials, plant cells cultured from velvet flowers in plant resources, adventitious root cultures, or extracts thereof are used, and fermentation processes are also performed. However, unlike these prior studies, studies on efficacy differences based on the mixing ratio of exosomes are very little.
Thus, in the present invention, it was confirmed that in the case of preparing a complex by mixing a fermented chenopodium faberi callus culture extract and a pomegranate-derived exosome, wrinkle improvement, skin moisturizing, anti-inflammatory effects can be promoted and enhanced as compared to the case of using the fermented chenopodium faberi callus culture extract or the pomegranate-derived exosome alone, thereby completing the present invention.
Prior art literature
Patent literature
Korean laid-open patent publication No. 10-2016-0043551
Korean patent publication No. 10-1842700
Disclosure of Invention
Problems to be solved by the invention
The present invention is directed to a cosmetic composition comprising a mixture of a fermented chenopodium faberi callus culture extract and an exosome derived from punica granatum as active ingredients, and having effects of improving skin moisture, anti-inflammatory, anti-aging and improving wrinkles.
Solution for solving the problem
In order to achieve the above-described object, the present inventors have confirmed that when a fermented velvet flower callus culture extract and a pomegranate-derived exosome are used in a mixture in a certain ratio, the results thereof show that the effects of improving wrinkles, skin moisturizing, and anti-inflammatory effects and the like can be improved, and that the effects of improving wrinkles, skin moisturizing, and anti-inflammatory effects are improved as compared to the use of a velvet flower callus culture extract or a pomegranate-derived exosome alone, thereby completing the present invention.
The present invention provides a cosmetic composition comprising a mixture of a velvet flower (Leontopodium alpinum) callus culture extract and an exosome derived from pomegranate pulp as an active ingredient.
The cosmetic composition of the present invention contains a natural extract as an active ingredient, not only has excellent biosafety, but also minimizes skin irritation. In particular, the plant cell culture extract of the velvet flower and the exosome derived from the pomegranate act on the skin at the same time, so that the synergistic effect of the plant cell culture extract and the exosome derived from the pomegranate can obviously enhance the functions of anti-inflammation, anti-aging, wrinkle improvement, skin moisture preservation and the like.
The above-mentioned mixture of fermented edelweiss callus culture extract and pomegranate-derived exosomes can be mixed in an amount of 0.1 to 10 weight% relative to the total weight of the cosmetic composition.
In addition, the above mixture can be obtained by mixing the fermented velvet flower callus culture extract and the pomegranate-derived exosomes in a weight ratio of 1:9 to 9:1, and in a specific embodiment, the present inventors confirmed that the fermented velvet flower callus culture extract and the pomegranate-derived exosomes in a weight ratio of 9:1, 7:3, 5:5, 3:7 are more excellent in wrinkle improvement, skin moisturization and anti-inflammatory effects in the case where the fermented velvet flower callus culture extract contained in the mixture is identical to or more than the pomegranate pulp-derived exosomes, and tested.
In the above-mentioned extract of the callus of the velvet flower, the term "callus" is a plant cell or an adventitious root culture, and may refer to a plant cell mass formed by division of the velvet flower plant cell.
The above-mentioned velvet flower callus culture extract may be a fermented extract obtained by inoculating a lactobacillus (lactobacillus sp.) strain.
The fermentation refers to a process of decomposing an organic substance by using enzymes possessed by a microorganism itself. The fermented metabolic substances contain various amino acids, organic acids and antioxidant substances beneficial to skin, so that skin metabolism is promoted, and skin texture is elastic and smooth. In addition, the particles become smaller through the fermentation process, so that the absorption rate is good, and the skin problem or allergic side effect can be relieved.
The above fermented extract can be prepared by the following means: the extract is naturally dried or dried by using a device such as a rotary vacuum concentrator or a freeze dryer, and the culture or extract is diluted in a solvent, preferably purified water, at a certain concentration, and then inoculated with a microorganism for fermentation, that is, lactobacillus sp, and fermented.
The lactobacillus strain may be lactobacillus pentosus (Lactobacillus pentosus), lactobacillus brevis (Lactobacillus brevis), lactobacillus plantarum (Lactobacillus plantarum), lactobacillus casei (Lactobacillus casei) or lactobacillus acidophilus (Lactobacillus acidophilus), but is not limited thereto.
The cosmetic composition of the present invention can be used for moisturizing skin, anti-inflammatory, anti-aging or improving skin wrinkles.
The term "skin moisturization" as used above means all the actions of maintaining the skin tissue steady state by properly adjusting the moisture loss (moisture evaporation) or the like of the skin. The skin moisturizing effect may be accompanied by various additional skin improving effects such as a keratolytic effect and a skin irritation reducing effect.
The term "anti-inflammatory" refers to the effect of inhibiting inflammation, and it is known that the regulation of inflammatory response is very complex, which enhances the recovery system in the organism and reduces damage. However, if the inflammatory reaction continues due to repeated tissue injury or regeneration, excessive Reactive Oxygen Species (ROS) are generated in the cells associated with inflammation, and permanent gene deformation is caused. That is, ROS are closely related to inflammatory responses that regulate various cellular actions in the body.
The above "improvement of wrinkles" refers to a phenomenon of inhibiting or preventing the skin from generating wrinkles, or relieving wrinkles that have been generated.
In addition, the present invention provides a method for preparing a mixture composed of a fermented chenopodium faberi callus culture extract and an exosome derived from punica granatum, comprising the steps of: preparing a velvet flower callus culture extract; fermenting the velvet flower callus culture extract; preparing exosomes by using pomegranate pulp; and mixing the fermented velvet flower callus culture extract and exosomes isolated from pomegranate pulp in a certain ratio to prepare a mixture.
In the step of separating the exosomes from the above-mentioned pomegranate pulp, after pulverizing the portion of the pomegranate pulp, distilled water is used as a solvent, and heat-treated at a temperature of 60 to 70 ℃ for 2 to 3 hours, thereby performing hot water extraction, and then cooling and filtering to obtain exosomes. Specifically, the above-mentioned pomegranate pulp-derived exosomes can be obtained by the following steps: step (a), adding distilled water into the pomegranate pulp raw material and crushing; step (b), centrifugally separating the crushed material, and filtering supernatant to separate and remove dead cells and foreign matters; step (c) of subjecting the filtered supernatant to ultra-high speed centrifugation to form a primary particle layer comprising exosomes; a step (d) of subjecting the supernatant, which has been subjected to the centrifugation in the step (c), to centrifugation again to form a secondary particle layer containing exosomes; and (e) separating exosomes from the pomegranate pulp by re-suspending the primary and secondary particle layers in purified water. More specifically, the above-mentioned pomegranate pulp-derived exosomes can be obtained by the following steps: step (a), adding distilled water with the weight ratio of 20 times to the pomegranate pulp raw material, crushing, and stabilizing the crushed material for 24 hours at the temperature of 4 ℃; step (b), after centrifugally separating the crushed material by using a centrifugal separator, taking only supernatant fluid of the crushed liquid to separate and remove dead cells and foreign matters; step (c) of filtering the supernatant to remove residues; step (d), centrifugally separating the supernatant by using a super-high speed centrifugal separator, and precipitating exosomes to form a primary particle layer; step (e), after the supernatant is subjected to centrifugal separation again by using a super-high speed centrifugal separator, precipitating additional exosomes to form a secondary particle layer; step (f), repeating the steps (d) and (e) 2 to 3 times to isolate high purity exosomes; and (g) separating exosomes from the pomegranate pulp by re-suspending the particle layer formed in the above step in purified water.
The mixture of the velvet flower callus culture extract and the pomegranate-derived exosomes prepared by the above method may be formulated into various forms of cosmetic compositions by conventional methods.
The cosmetic composition may be in the form of a lotion, astringent, nourishing lotion, nourishing cream, massage cream, eye essence, cleansing cream, facial mask, powder, body cream, body essence, body wash, hair dye, shampoo, conditioner, hair setting agent, hair tonic, ointment, gel, cream, patch, spray, powder, skin adhesive type, etc., but is not limited thereto.
In addition, for each dosage form, in addition to the above-described essential components, one of ordinary skill can appropriately select and combine other components according to the kind of other dosage forms, the purpose of use, and the like.
The above cosmetic composition can be provided in all dosage forms suitable for topical application. For example, it can be provided in the form of a solution, oil-in-water emulsion, water-in-oil emulsion, suspension, solid, gel, powder, paste, microneedle, foam, or aerosol composition. The composition of such dosage forms may be prepared according to methods conventional in the art.
The cosmetic composition of the present specification may further comprise functional additives other than the compounds of the present specification and ingredients contained in conventional cosmetic compositions. The functional additive may comprise a component selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymeric peptides, polymeric polysaccharides, sphingolipids, and seaweed extracts. The cosmetic composition according to the present specification may contain other ingredients that bring about synergistic effects on the main effects within a range that does not impair the main effects. Also, the cosmetic composition according to the present specification may further comprise a humectant, an emollient, a surfactant, an ultraviolet absorber, a preservative, a sterilizing agent, an antioxidant, a pH adjuster, an organic pigment, and an inorganic pigment, a perfume, a coolant, or an antiperspirant. The blending amount of the above components can be easily selected by a person of ordinary skill within the range that does not impair the object and effect of the present specification, and may be 0.001 to 10 weight percent, specifically 0.01 to 3 weight percent, based on the total weight of the composition.
Effects of the invention
Embodiments of the present invention can provide a cosmetic composition having excellent skin moisturizing, anti-inflammatory, anti-aging and wrinkle-improving effects by preparing a fermented chenopodium faberi callus culture extract and a pomegranate-derived exosome mixture.
The fermented edentum callus culture extract and the pomegranate-derived exosome mixture provided by the present invention have excellent skin moisturizing effects in addition to excellent wrinkle-improving and anti-inflammatory effects, compared with the use of the fermented edentum callus culture extract or the pomegranate-derived exosome alone, and thus, the effect as a cosmetic composition is excellent when it is applied as an active ingredient to a cosmetic substrate.
Drawings
Fig. 1 is a graph evaluating the moisturizing and continuous moisturizing ability of formulation example 2 and comparative formulation examples 1 to 3 according to the present invention.
Fig. 2 is a graph evaluating the moisturizing and continuous moisturizing ability of dosage form examples 1 to 6 according to the present invention.
Fig. 3 is a graph evaluating the moisturizing and continuous moisturizing ability of formulation example 8 and comparative formulation examples 4 to 6 according to the present invention.
Fig. 4 is a graph evaluating the moisturizing and sustained moisturizing ability of dosage form examples 7 to 12 according to the present invention.
Fig. 5 is a graph evaluating the amount of improved percutaneous moisture loss according to formulation example 2 of the present invention and comparative formulation examples 1 to 3.
Fig. 6 is a graph evaluating the improved amount of percutaneous moisture loss according to dosage form examples 1 to 6 of the present invention.
Fig. 7 is a graph evaluating the amount of improved percutaneous moisture loss according to formulation example 8 of the present invention and comparative formulation examples 4 to 6.
Fig. 8 is a graph evaluating the improved amount of percutaneous moisture loss according to dosage form examples 7 to 12 of the present invention.
FIG. 9 is a graph showing changes in activity of Nitrogen Oxides (NO) which are inflammatory-related factors of examples 1 to 4 and comparative examples 1 and 2 of the present invention.
FIG. 10 is a graph showing changes in the inhibitory activity of TNF-. Alpha.production as an inflammatory factor of examples 1 to 4 and comparative examples 1 and 2 of the present invention.
FIG. 11 is a graph showing changes in the activity of procollagen type 1 (pro-collagen type 1) which is a skin elasticity-related protein of examples 1 to 4 and comparative examples 1 and 2 of the present invention.
FIG. 12 is a graph showing changes in the activity of MMP-1, which is a skin elasticity-related protein of examples 1 to 4 and comparative examples 1 and 2 of the present invention.
Detailed Description
Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the present invention is not limited to the embodiments described herein, but may be embodied in other forms. The embodiments described herein are provided to make the disclosure more thorough and complete, and to fully convey the concept of the invention to those skilled in the art. In the following description of the present invention, a detailed description thereof will be omitted in case it is determined that the detailed description about the related known functions or compositions unnecessarily confuses the gist of the present invention.
Comparative example 1: preparation of fermented velvet flower callus culture extract
100g of dried velvet flower callus powder was obtained by washing, dehydrating and drying plant cells cultured from germinated seedlings of velvet flower stems, flowers or leaves, velvet flower seeds, and added to 1L of purified water, stirred at 50 ℃ for 8 hours, and subjected to primary extraction, and hot water extraction at 98 ℃ for 10 minutes. After filtering the extract with a filter, the extract was concentrated under reduced pressure at a temperature of 30 ℃ and freeze-dried, whereby a solid was obtained. The obtained extract of the callus of the velvet flower is mixed with purified water, and then inoculated with lactobacillus plantarum (Lactobacillus plantarum) as a fermentation strain, and fermented at 15-25 ℃ for 3 days. After the fermentation process is finished, filtering by a filter, and adding 1, 2-hexanediol to obtain a fermentation extract. Table 1 below shows the ingredients used to prepare the fermented chenopodium falciferum callus culture extract according to the present invention.
TABLE 1
| Composition of the components | Comparative example 1 (weight percent) |
| Velvet flower callus culture extract | 0.1 |
| Lactic acid bacterium fermented product | 0.1 |
| 1, 2-hexanediol | 2 |
| Purified water | Allowance of |
| Sum up | 100 |
Comparative example 2: preparation of Granati-derived exosomes-
In order to separate exosomes from the pomegranate pulp, purified water in an amount of 20 times by weight is added to the portion of the pomegranate pulp and soaked, and then crushed by a crusher. Thereafter, in order to increase the purity of exosomes, the pulverized pomegranate is centrifugally separated at 3000 to 4000xg for 10 minutes using a centrifugal separator, and only the supernatant is taken except for the pellet layer to separate and remove the residues. For the supernatant obtained in the present process, centrifugal separation was performed at 10000 to 15000xg for 20 to 30 minutes using an ultra high speed centrifugal separator (ultra high speed centrifugal), and in order to further improve the purity of the obtained supernatant, further improvement of the purity was attempted by filtering the supernatant and separating and removing the residue. The exosomes present in the high-purity supernatant were centrifuged at 100000 to 150000xg by means of an ultra-high-speed centrifugal separator for 2 to 4 hours, and then the exosomes were precipitated in the pellet layer. Exosomes were prepared by suspending exosomes as the final particle layer in sterile distilled water again, dispersing them.
< examples 1 to 4: preparation of mixture of fermented velvet flower callus culture extract and pomegranate exosomes >
The final mixture was prepared by mixing the fermented chenopodium falciferum callus culture extract obtained in the above comparative examples 1 and 2 with the pomegranate-derived exosomes. Representation 2 shows the ingredients used to prepare the fermented chenopodium falciferum callus culture extract and the pomegranate-derived exosome mixture according to the invention.
TABLE 2
| Comparative example 1 (weight percent) | Comparative example 2 (weight percent) | |
| Example 1 | 9 | 1 |
| Example 2 | 7 | 3 |
| Example 3 | 5 | 5 |
| Example 4 | 3 | 7 |
< dosage form examples 1 to 6 and comparative dosage form examples 1 to 3: preparation of essence >
Disodium edetate, glycerol, propylene glycol, carbomer were dispersed in purified water, and an emulsifying system dissolved in an aqueous phase heated to 70 to 75 ℃ was added, followed by stirring with an emulsifying disperser (AGI MIXER) for 5 minutes. After tromethamine was added at a temperature of 60 to 65 ℃, the mixture was neutralized by stirring with an emulsifying disperser for 3 minutes. 1, 2-hexanediol and ethylhexyl glycerol were added at 45℃and cooled to 30℃after stirring for 3 minutes. Thereafter, cosmetic materials (formulation examples 1 to 6) comprising the fermented chenopodium faberi callus culture extract and the pomegranate-derived exosome mixture of examples 1 to 3, and cosmetic materials (comparative formulation examples 1, 2) comprising the fermented chenopodium faberi callus culture extract and the pomegranate-derived exosome, respectively, of comparative examples 1,2 were added, and stirred and defoamed for 3 minutes, thereby preparing a concentrate formulation. Table 3 below shows the ingredients used to prepare the serum with or without all of examples 1 to 3 and comparative examples 1,2 according to the present invention.
TABLE 3 Table 3
< dosage form examples 7 to 12 and comparative dosage form examples 4 to 6: preparation of cream-
The transparent emulsion system was prepared by heating ethyl cetyl hexanoate, cetostearyl alcohol, glycerol stearate to 75 ℃ to 80 ℃ and dissolving. Disodium edetate, glycerol, propylene glycol, cetostearyl olive oil ester, sorbitan olive oil ester, carbomer are dispersed in purified water, and after adding an emulsifying system dissolved in an aqueous phase heated to 70 to 75 ℃, emulsified for 5 minutes with a high-speed stirrer at 3500 to 5000 rpm. After the tromethamine was added at a temperature of 60 to 65 ℃, the mixture was neutralized by stirring at 3000 to 3500rpm using a high speed stirrer for 3 minutes. 1, 2-hexanediol and ethylhexyl glycerol were added at 45℃and stirred for 3 minutes, and then cooled to 30 ℃. Thereafter, cosmetic materials (formulation examples 7 to 12) comprising the fermented chenopodium faberi callus culture extracts of examples 1 to 3 and the pomegranate exosome mixture, and cosmetic materials (comparative formulation examples 4, 5) comprising the fermented chenopodium faberi callus culture extracts and the pomegranate-derived exosome of comparative examples 1,2, respectively, were added, and deaerated with stirring for 3 minutes, thereby preparing a cream. Table 4 below shows the ingredients used to prepare creams that contained or did not contain all of the examples 1 to 3 and comparative examples 1,2 according to the present invention.
TABLE 4 Table 4
Experimental example 1 evaluation of Patch test
After the formulation examples 1 to 12 according to the present invention and comparative formulation examples 1 to 6 were applied to the skin, whether the test product had primary irritation to the human skin was evaluated by visual evaluation, and a skin patch test was performed for objective verification. Experiments were performed in 10 healthy adults as subjects. After 25mg of the sample was applied in the chamber of the patch test tester (IQ Ultra), it was applied in a closed state to the test site, the inner side of the lower part of the arm, for 24 hours. After removing the patch, skin reactions after 1 hour, 24 hours, and 48 hours were observed. The results of the skin irritation test for humans were evaluated according to the criteria of the international contact dermatitis study group (The International Contact Dermatitis Research Group, ICDRG). As shown in table 5 below, it was confirmed by evaluation that neither formulation examples 1 to 12 nor comparative formulation examples 1 to 6 caused skin irritation, and that they could be safely applied to the skin.
TABLE 5
Experimental example 2 evaluation of moisturizing ability and continuous moisturizing ability
In the preparation phase, in order to make the measurement conditions of the subjects the same, the test site was kept in a clean and dry state, and after keeping the skin stable for at least 30 minutes in a place where the constant temperature and humidity (22±2 ℃ r.h.40% to 60%) was maintained. In the measurement phase, 2mg/cm of the sample was applied to a selected test site (5 cm. Times.4 cm) on the forearm using a micropipette (micropipette) 2 Is a test product of (a). The measurement was performed 3 times before, after and 3 hours after the use of the product, and the average value was obtained using 3 values. Skin moisture measurements were performed using an Afford Luo Di moisture detector MC-1000 (Aphrodite moisture checker MC-1000) and the results are shown in Table 6 below and FIGS. 1-4.
TABLE 6
| Sample of | Moisture retention immediately after application (%) | Moisture retention after 3 hours of application (%) |
| Formulation example 1 | 127.2 | 70.4 |
| Formulation example 2 | 139.0 | 80.3 |
| Formulation example 3 | 119.8 | 56.7 |
| Formulation example 4 | 116.7 | 52.8 |
| Formulation example 5 | 122.2 | 60.6 |
| Formulation example 6 | 112.0 | 50.1 |
| Formulation example 7 | 121.6 | 53.5 |
| Formulation example 8 | 138.1 | 65.0 |
| Formulation example 9 | 95.1 | 56.9 |
| Dosage form example 10 | 104.1 | 50.5 |
| Formulation example 11 | 121.1 | 59.5 |
| Formulation example 12 | 80.9 | 23.5 |
| Comparative formulation example 1 | 109.6 | 47.4 |
| Comparative formulation example 2 | 90.5 | 34.0 |
| Comparative formulation example 3 | 61.9 | 7.2 |
| Comparative formulation example 4 | 84.4 | 38.6 |
| Comparative formulation example 5 | 53.7 | 15.5 |
| Comparative formulation example 6 | 30.6 | 4.0 |
As is clear from table 6 and fig. 1 to 4, the moisture retention immediately after coating and the moisture retention after coating both increased and continued as shown in the dosage form examples 1 to 12 containing the fermented velvet flower callus culture extract and the pomegranate-derived exosome mixture of the present invention, compared to the comparative dosage form examples 1,2, 4 and 5 containing the fermented velvet flower callus culture extract and the pomegranate-derived exosome alone. In particular, the formulations 2 and 8 containing 10% of example 2 were excellent in moisturizing ability immediately after coating and moisturizing ability after coating for 3 hours. Therefore, it is more preferable that the mixed fermented velvet flower callus culture extract and the pomegranate-derived exosomes are contained in the fermented velvet flower callus culture extract and the pomegranate-derived exosomes, respectively, and the moisturizing effect is most excellent when the ratio of the fermented velvet flower callus culture extract to the pomegranate-derived exosomes is 7:3.
Experimental example 3 evaluation of percutaneous moisture loss amount
In the preparation phase, in order to make the measurement conditions of the subjects the same, the test site was kept in a clean and dry state, and after keeping the skin stable for at least 30 minutes in a place where the constant temperature and humidity (22±2 ℃ r.h.40% to 60%) was maintained. In the measurement phase, 2mg/cm of the sample was applied to a selected test site (5 cm. Times.4 cm) on the forearm using a micropipette (micropipette) 2 Is a test product of (a). The measurement was performed 3 times in total for the product before use, immediately after application, and 3 hours after use, and the average value was determined from 3 values. Using the percutaneous water loss rate testerTM 300) and the results are shown in table 7 below and fig. 5 to 8.
TABLE 7
As is clear from table 7 and fig. 5 to 8, the percutaneous moisture loss amount immediately after and after 3 hours of application, which is shown in the dosage form examples 1 to 12 containing the fermented chenopodium falciferum callus culture extract and the pomegranate-derived exosome mixture of the present invention, was improved as compared with the comparative dosage form examples 1,2, 4 and 5 containing the fermented chenopodium falciferum callus culture extract and the pomegranate-derived exosome alone. In particular, the dosage forms 2 and 8 of example 2 containing 10% showed the most excellent effect of improving the amount of the percutaneous moisture loss immediately after the application and after the application for 3 hours. Therefore, it is more preferable that the mixed fermented velvet flower callus culture extract and the pomegranate-derived exosome are compared with the fermented velvet flower callus culture extract and the pomegranate-derived exosome which are contained separately, and the percutaneous moisture loss improvement effect is most excellent when the ratio of the fermented velvet flower callus culture extract to the pomegranate-derived exosome is 7:3.
Experimental example 4 cytotoxicity evaluation
In a 96-well plate (well plate) at 6X 10 3 Normal Human Fibroblasts (NHF) were seeded at an amount of 1.5X10 cells/well 4 Cell/well/amount of seeded human immortalized keratinocytes (HaCaT) at 1×10 5 Cell/well amounts after inoculation of mouse mononuclear macrophage leukemia cells (RAW 264.7), they were cultured under cell culture conditions. After 24 hours, the culture medium was removed and washed with Phosphate Buffered Saline (PBS), fibroblast Basal Medium (FBM) without supplement (supplement) was used for NHF, while Darbert Modified Eagle Medium (DMEM) without Fetal Bovine Serum (FBS) was used for HaCaT, RAW264.7 cells, and the cells were starved. The next day, a concentration of the test substance was treated and incubated for 24 hours. Water-soluble tetrazolium salt reagent WST-1 diluted 10-fold in culture medium was added to each well at 100. Mu.l, and after 2 hours of incubation, absorbance was measured at 450 nm.
For cytotoxicity testing, the test was performed at a concentration of the test substance of at least 0.1% and at most 10%. As a result, the results of the treatments of comparative examples 1 and 2 and examples 1 to 4 with NHF, haCaT, RAW 264.7.264.7, respectively, showed a cell viability of 90% or more at the highest concentration of 10%.
The concentration of the efficacy test was selected to be non-cytotoxic and the concentration dependence of efficacy was confirmed and is shown in table 8.
TABLE 8
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Experimental example 5 evaluation of NO production inhibitory Capacity
In a 96-well plate (well plate) at 5X 10 4 Cell/well amounts after inoculation with RAW264.7, culture was performed under cell culture conditions. After 24 hours, the culture medium was removed and washed with PBS, and the cells were starved using DMEM medium without FBS. The next day, a concentration of test substance was treated with 5. Mu.g/ml lipopolysaccharide (Lipop olysaccaride, LPS) and incubated. After 24 hours, the same amount of cell culture solution and griess reagent (griess reagent) were added and mixed, and then reacted at room temperature for 15 minutes. Absorbance was measured at 560nm, and the amount of Nitrogen Oxides (NO) as an inflammatory mediator was determined using a standard curve obtained from Sodium nitrite (Sodium nit rite). The amount of final NO was converted to the amount of NO in each certain protein and compared with a negative control group, and is shown in fig. 9.
From the experimental results, it was confirmed that NO inhibitory activity was excellent in the case of the mixed-fermented chenopodium falciferum callus culture extract and the pomegranate-derived exosomes. In particular, the NO production-inhibiting activity was most excellent when the mixing ratio of the fermented velvet flower callus culture extract and the pomegranate-derived exosomes was 7:3 (example 2). Therefore, the effect of inhibiting NO generation was more excellent when the fermented velvet flower callus culture extract and the pomegranate-derived exosomes were used in combination than when the fermented velvet flower callus culture extract and the pomegranate-derived exosomes were used alone, and the effects due to the optimum mixing ratio were confirmed, and are shown in table 9.
TABLE 9
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Experimental example 6 evaluation of TNF-alpha production inhibitory Activity ]
In Dulbecco 'modified Eagle' medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, streptomycin and penicillin at about 37℃with 5% CO 2 Macrophage RAW264.7 was cultured and 1×10 5 Cell/well amounts were seeded in 96-well plates.
Next, the compositions prepared according to examples 1 to 4 and comparative examples 1 and 2 were treated in accordance with the concentrations, stimulated with LPS (5. Mu.g/mL) and cultured for 24 hours, and then cell culture broth was recovered and the amount of TNF-. Alpha.was measured using an enzyme-linked immunosorbent assay kit (ELISA kit) (R & D System Inc. Minneapolis, minnesota). The amount of TNF- α was quantified using the TNF- α standard curve for each concentration contained in the Kit (Kit).
In addition, indomethacin (20. Mu.g/mL) was used as a positive control group by treatment instead of the sample, and was used in comparison with the TNF-. Alpha.inhibitory activity of the sample.
From the experimental results, it was confirmed that the LPS-treated group induced production of TNF-. Alpha. 929.3pg/mL, which was increased by about 4.1-fold in statistical significance when compared to the negative control group (226.3 pg/mL). In examples 2 to 4, production of TNF-. Alpha.was demonstrated in 579.0pg/mL, 644.38pg/mL and 811.23pg/mL, and in particular, it was confirmed that example 2 showed an inhibitory activity having a statistically significant meaning of about 37.7% as compared with the LPS control group. Furthermore, the mixed fermented raw material of the velvet flower callus culture extract and the pomegranate-derived exosomes appears to increase TNF- α inhibitory activity in a concentration-dependent manner. It was finally confirmed that example 2 has the most excellent TNF-. Alpha.inhibitory activity among examples 2 to 4.
Therefore, it was confirmed that the effect of suppressing TNF- α production was more excellent when the fermented velvet flower callus culture extract and the pomegranate-derived exosomes were used in combination than when the fermented velvet flower callus culture extract (comparative example 1) and the pomegranate-derived exosomes were used alone (comparative example 2), and the mixing ratio was very important, and this is shown in table 10.
Table 10
Experimental example 7 evaluation of Type 1 Procollagen (Procolagen Type 1) production ability >)
At 6X 10 in 96-well plates 3 Cell/well amounts after NHF inoculation, culture under cell culture conditions. After 24 hours, the culture was removed and washed with PBS, and the cells were starved using FBM medium without supplement (supplement). The next day, a concentration of the test substance was treated and incubated for 24 hours. After experiments with type 1 procollagen Elisa reagent, the absorbance was measured at 450 nm. The amount of final Procollagen (protocol) was converted to the amount of Procollagen per certain protein and compared to a negative control group and is shown in fig. 11.
From the experimental results, it was confirmed that when the fermented chenopodium falciferum callus culture extract and the pomegranate-derived exosomes were used in combination, the type 1 procollagen activity was increased, resulting in more excellent collagen activity, compared to the case where the fermented chenopodium falciferum callus culture extract and the pomegranate-derived exosomes were used alone at the same concentration and under the same conditions. In particular, in the case where the mixing ratio of the fermented velvet flower callus culture extract and the pomegranate-derived exosomes is 7:3 (example 2), the type 1 procollagen activity is greatly increased. Therefore, it was confirmed that the effect of improving wrinkles when the fermented velvet flower callus culture extract and the pomegranate-derived exosomes were used in combination was more excellent than when the fermented velvet flower callus culture extract and the pomegranate-derived exosomes were used alone, and the mixing ratio was very important, and it is shown in table 11.
TABLE 11
Experimental example 8 evaluation of inhibition of production of matrix Metalloprotease 1 (MMP-1)
At 2.5X10 in 96 well plates 5 Cell/well mass after seeding HaCaT cells, culture was performed under cell culture conditions. After 24 hours, the medium was removed and washed with PBS, and the cells were starved using DMEM medium (serum free) without FBS. The next day, culture was performed by irradiation of medium-wave Ultraviolet (UVB).
At 6X 10 in 96-well plate 3 Cell/well amounts after NHF inoculation, culture under cell culture conditions. After 24 hours, cells were starved using FBM medium without supplement (supplement). The following day, human fibroblasts were treated with the culture medium of UVB-stimulated HaCaT along with the sample and cultured. After incubation for 24 hours, absorbance was measured at 450nm after experiments with MMP-1Elisa reagent. The final MMP-1 amounts were converted to MMP-1 amounts in each of the defined proteins and compared to the negative control group and are shown in FIG. 12.
From the experimental results, it was confirmed that MMP-1 inhibitory activity was more excellent when the fermented velvet flower callus culture extract and the pomegranate-derived exosomes were used in combination, as compared to when the fermented velvet flower callus culture extract and the pomegranate-derived exosomes were used alone, at the same concentration and under the same conditions. In particular, when the mixing ratio of the fermented velvet flower callus culture extract and the pomegranate-derived exosomes was 7:3 (example 2), the increase in the MMP-1 inhibitory activity was shown to be large. Therefore, it was confirmed that the effect of improving wrinkles when the fermented velvet flower callus culture extract and the pomegranate-derived exosomes were used in combination was more excellent than when the fermented velvet flower callus culture extract and the pomegranate-derived exosomes were used alone, and the mixing ratio was very important, and this is shown in table 12.
Table 12
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While the present invention has been described with reference to one embodiment, those skilled in the art will recognize that various modifications and changes can be made to the present invention without departing from the spirit and scope of the invention as set forth in the appended claims. Therefore, when the technical features of the claims of the present invention are substantially included in the modified embodiment, it should be considered as being included in the technical scope of the present invention as well.
Claims (8)
1. A cosmetic composition characterized in that,
comprises a mixture of a velvet flower callus culture extract and an exosome derived from punica granatum as an active ingredient.
2. A cosmetic composition according to claim 1, characterized in that,
comprising from 0.1 to 10.0 weight percent of said mixture, relative to the total weight of the composition.
3. A cosmetic composition according to claim 1, characterized in that,
in the mixture, the velvet flower callus culture extract and the pomegranate-derived exosomes are mixed in a weight ratio of 1:9 to 9:1.
4. A cosmetic composition according to claim 1, characterized in that,
the velvet flower callus culture extract is a fermentation extract obtained by inoculating a lactobacillus strain.
5. A cosmetic composition according to claim 1, characterized in that,
the pomegranate source exosome is prepared by the following method:
step (a), adding distilled water to the pomegranate pulp raw material and pulverizing to prepare a pulverized product of the pomegranate pulp raw material;
step (b), after centrifugal separation of the crushed material, filtering supernatant to separate and remove dead cells and foreign matters;
step (c) of subjecting the filtered supernatant to ultra-high speed centrifugation to form a primary particle layer comprising exosomes;
a step (d) of subjecting the supernatant, which has been subjected to the centrifugation in the step (c), to centrifugation again to form a secondary particle layer containing exosomes; and
and (e) separating exosomes from the pomegranate pulp by re-suspending the primary and secondary particle layers in purified water.
6. A cosmetic composition according to claim 5, characterized in that,
repeating the steps (c) and (d) 2 to 3 times to isolate the exosomes of high purity.
7. The cosmetic composition according to any one of claim 1 to 6, characterized in that,
the composition is formulated into one or more of emollient water, nourishing lotion, moisturizing cream, nourishing cream, massage cream, essence, ampoule, gel, eye cream, cleansing cream, facial cleanser, cleansing water, facial mask, spray, and powder.
8. The cosmetic composition according to any one of claim 1 to 6, characterized in that,
the composition is used for moisturizing skin, anti-inflammatory, anti-aging or improving skin wrinkles.
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| KR10-2022-0080851 | 2022-06-30 | ||
| KR1020220080851A KR20240002803A (en) | 2022-06-30 | 2022-06-30 | A cosmetic composition comprising mixture of fermented Leontopodium alpinum callus culture extract and pomegranate-derived exosome |
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| KR101740097B1 (en) | 2014-10-13 | 2017-05-29 | 주식회사 바이오에프디엔씨 | Anti-inflammation and Anti-aging composition for skin external application comprising Leontopodium alpinum Cell Culture Extract and Methods for preparing the Same |
| KR101842700B1 (en) | 2017-03-27 | 2018-03-28 | 주식회사 아미코스메틱 | A cosmetic composition comprising leontopodium alpinum cell culture extract and natural complex extract |
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