Composition for inhibiting melanin production and its uses

Abstract

This invention provides a composition for suppressing melanin production and its use. [Solution] The composition contains platelets and / or growth factors isolated from blood and has the ability to suppress tyrosinase activity and alleviate inflammation. Furthermore, the composition of the present invention can be used in the manufacture of formulations for improving skin pigmentation or alleviating melasma.

Description

[Technical Field]

[0001] The present invention relates to compositions related to the inhibition of melanin production, and more particularly to compositions containing platelets and / or growth factors separated from blood, and formulations containing such compositions. [Background technology]

[0002] The skin is the largest organ in the human body and is divided into the epidermis (composed of the stratum corneum and basal layer), the dermis, and the subcutaneous tissue. Skin color is influenced by various pigments, including melanin, carotene, and hemoglobin. Melanin is mainly produced by melanocytes, which are distributed in the basal layer.

[0003] Tyrosinase is an oxidase and the rate-limiting enzyme that controls melanin production. When melanocytes are stimulated, the tyrosinase activity within them is activated, initiating a series of chemical reactions that ultimately produce melanin. Furthermore, when melanosomes containing large amounts of melanin are released into keratinocytes, it causes skin darkening and the formation of various types of spots (e.g., nevi, melasma). Therefore, suppressing the production and activity of tyrosinase is considered a key step in reducing melanin production. However, if melanin cannot act, vitiligo occurs, leading to further skin pathologies. [Overview of the project] [Problems that the invention aims to solve]

[0004] Currently, commercially available whitening ingredients are primarily added to cosmetics or skincare products to suppress melanin production and prevent pigmentation. If these are not added according to usage concentration limits, they can cause allergies or apoptosis (programmed cell death) or death of melanocytes (melanocytes). Therefore, there is still a need for compositions or formulations that can reduce melanin production without causing apoptosis or death of melanocytes. [Means for solving the problem]

[0005] The object of the present invention is to provide a composition for inhibiting melanin production that contains platelets and / or growth factors separated from blood.

[0006] In one embodiment, the composition contains platelets or growth factors separated from blood, and preferably contains platelets separated from plasma.

[0007] Preferably, the composition is 2.5 × 10 7 ~10×10 9 It contains [number] platelets.

[0008] Preferably, the composition is 2.5 × 10 7 ~10×10 8 It contains [number] platelets.

[0009] Preferably, the composition is 5 × 10 7 ~10×10 8 It contains [number] platelets.

[0010] In another embodiment, the composition contains platelet-derived growth factor BB (PDGF-BB) and / or transforming growth factor β (TGF-β).

[0011] Preferably, the concentration of platelet-derived growth factor BB (PDGF-BB) in the composition is 0.1 ng / mL to 5 ng / mL.

[0012] Preferably, the concentration of platelet-derived growth factor BB (PDGF-BB) in the composition is 0.1 ng / mL to 3 ng / mL.

[0013] Preferably, the concentration of platelet-derived growth factor BB (PDGF-BB) in the composition is 0.25 ng / mL to 2 ng / mL.

[0014] Preferably, the concentration of platelet-derived growth factor BB (PDGF-BB) in the composition is 0.25 ng / mL to 1 ng / mL.

[0015] Preferably, the concentration of the transforming growth factor β (TGF-β) in the composition is 0.5 ng / mL to 50 ng / mL.

[0016] Preferably, the concentration of the transforming growth factor β (TGF-β) in the composition is 0.5 ng / mL to 30 ng / mL.

[0017] Preferably, the concentration of the transforming growth factor β (TGF-β) in the composition is 1 ng / mL to 20 ng / mL.

[0018] Preferably, the concentration of the transforming growth factor β (TGF-β) in the composition is 1 ng / mL to 10 ng / mL.

[0019] In yet another embodiment, the melanin production inhibitory composition of the present invention is in the form of freeze-dried, dried powder.

[0020] Preferably, 4 × 10 per gram of the dry powder of the composition. 8 ~1.5×10 11It contains individual platelets.

[0021] Preferably, per 1 gram of the dry powder of the composition, it contains 4×10 8 ~1.5×10 10 individual platelets.

[0022] Preferably, per 1 gram of the dry powder of the composition, it contains 8×10 8 ~1.5×10 10 individual platelets.

[0023] Preferably, per 1 gram of the dry powder of the composition, it contains the PDGF-BB concentration of 15~850 ng.

[0024] Preferably, per 1 gram of the dry powder of the composition, it has the PDGF-BB concentration of 15~500 ng.

[0025] Preferably, per 1 gram of the dry powder of the composition, it contains the PDGF-BB concentration of 35~350 ng.

[0026] Preferably, per 1 gram of the dry powder of the composition, it contains the PDGF-BB concentration of 35~200 ng.

[0027] Preferably, per 1 gram of the dry powder of the composition, it contains the TGF-β concentration of 75~8,500 ng.

[0028] Preferably, per 1 gram of the dry powder of the composition, it contains the TGF-β concentration of 75~5,000 ng.

[0029] Preferably, per 1 gram of the dry powder of the composition, it contains the TGF-β concentration of 150~3,500 ng.

[0030] Preferably, per 1 gram of the dry powder of the composition, it contains the TGF-β concentration of 150~2,000 ng.

[0031] Another object of the present invention is to provide the use of a melanin production inhibitory composition for producing a formulation for improving skin pigmentation in an individual.

[0032] In one embodiment, the pigmentation is caused by inflammation of cells.

[0033] In one embodiment, the pigmentation is induced by tyrosinase.

[0034] Another object of the present invention is to provide the use of a melanin production inhibitory composition for producing a formulation to alleviate melasma in an individual.

[0035] The melanin production inhibitory composition of the present invention is 1 × 10 7 , 1.25 × 10 7 , 1.5×10 7 , 1.75 × 10 7 , 2×10 7 , 2.25×10 7 , 2.5×10 7 , 2.75×10 7 , 3 x 10 7 , 3.25×10 7 , 3.5×10 7 , 3.75×10 7 , 4×10 7 , 4.25×10 7 , 4.5×10 7 , 4.75×10 7 , 5×10 7 , 5.25×10 7 , 5.5×10 7 , 5.75×10 7 , 6×10 7 , 6.25×10 7 , 6.5×10 7 , 6.75×10 7 , 7×10 7 , 7.25×10 7 , 7.5×10 7 , 7.75×10 7 , 8×10 7 , 8.25×10 7 , 8.5×10 7 , 8.75×107 , 9×10 7 , 9.25×10 7 , 9.5×10 7 , 9.75×10 7 , 1 x 10 8 , 1.25 × 10 8 , 1.5×10 8 , 2×10 8 , 3 x 10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1 x 10 9 , 2×10 9 , 3 x 10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , or 1 × 10 10 It contains [number] platelets.

[0036] In the present invention, the melanin production inhibitory composition contains a growth factor isolated from blood, the growth factor being obtained by centrifuging a whole blood sample (e.g., whole blood from a non-human mammal or human) to obtain platelet-rich plasma (PRP), adding an activator as needed (e.g., calcium chloride (CaCl2), thrombin, collagen, adenosine diphosphate (ADP), etc.), and then centrifuging again.

[0037] In the present invention, the concentration of PDGF-BB is preferably 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5 ng / mL.

[0038] In the present invention, the concentration of TGF-β is preferably 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6 .4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9 The values ​​are 0.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 50 ng / mL.

[0039] The composition of the present invention can be made into an injectable or topically applied formulation, and the formulation can use a suitable skin-applied base or injectable carrier, or an aqueous solution, a non-aqueous solvent, a suspension, an emulsion, or a lyophilized formulation, and is sterilized according to a known method.

[0040] The compositions of the present invention can be used to form formulations with pharmaceutically acceptable solvents, carriers, or excipients. Examples of such solvents include, but are not limited to, water, physiological saline, sterile water, deionized water, and glycerol.

[0041] The composition of the present invention may further contain additional ingredients such as antibacterial agents or preservatives. [Brief explanation of the drawing]

[0042] [Figure 1] This figure shows the effect of the concentration of platelet-derived growth factor BB (PDGF-BB) in the composition of the present invention on melanocyte proliferation. [Figure 2] This figure shows the effect of the concentration of platelet-derived growth factor BB (PDGF-BB) in the composition of the present invention on melanin production. [Figure 3] This figure shows the results of suppressing melanin production by the concentration of platelet-derived growth factor BB (PDGF-BB) in the composition of the present invention. [Figure 4] This figure shows the results of suppressing melanin production by varying the concentration of transforming growth factor β (TGF-β) in the composition of the present invention. [Figure 5] This figure shows the results of suppressing melanin production with different platelet counts in the composition of the present invention. [Figure 6] This figure shows the results of suppressing inflammation of melanocytes with different platelet counts and different whitening ingredients in the compositions of the present invention. [Figure 7] This figure shows the results of inhibiting tyrosinase activity with different platelet counts and different whitening ingredients in the compositions of the present invention. [Modes for carrying out the invention]

[0043] The detailed description of the embodiments is intended to illustrate preferred embodiments of the present invention and should be understood not as intended to limit the scope of the invention thereto.

[0044] 1. Preparation of dried growth factor powder containing platelet-derived growth factor BB (PDGF-BB)

[0045] The growth factor of the present invention is isolated from whole blood of a human or non-human mammal.

[0046] 250 ml (mL) of whole blood was placed in a blood bag containing an anticoagulant and centrifuged at 300-1,500 × g for 3-10 minutes to obtain platelet-rich plasma (PRP), also known as plasma layer solution.

[0047] The aforementioned PRP was treated with calcium chloride (CaCl2) activator, followed by centrifugation again, and the supernatant was removed to completely remove proteins from the plasma (PLASMA).

[0048] An equal volume of sterile water for injection was added to the removed supernatant to obtain growth factors released by platelet activation, and then 1 mL was dispensed per vial into sample vials. Subsequently, freeze-drying was performed to obtain dried growth factor powder.

[0049] 2-1. Preparation of a culture medium specifically for melanocytes containing dried platelet-derived growth factor BB (PDGF-BB) powder

[0050] Dry powder medium was prepared by dissolving the growth factor dry powder in 1 mL of melanocyte-specific basal cell medium (containing a melanocyte-specific growth factor kit). The growth factor dry powder contains 15 to 850 ng of PDGF-BB per gram. The melanocyte-specific basal cell medium and the dry powder medium were mixed in predetermined proportions and analyzed and confirmed by enzyme-linked immunosorbent assay (ELISA) to obtain melanocyte-specific media containing PDGF-BB dry powder medium with final PDGF-BB concentrations of 0 nanograms (0 ng / mL), 0.12, 0.25, 0.5, 1.0, and 2.0 ng / mL per milliliter.

[0051] 2-2. Effects of platelet-derived growth factor BB (PDGF-BB) on melanocytes (MELANOCYTE)

[0052] A 96-well plate was divided into untreated wells (control wells, i.e., 0 ng / mL PDGF-BB) and growth factor wells containing 0.12, 0.25, 0.5, 1.0, and 2.0 ng / mL PDGF-BB, with three replicates per well.

[0053] Human epidermal melanocytes (HUMAN EPIDERMAL MELANOCYTES, HEM; obtained from ATCC, product number PCS-200-012) were cultured in a basal cell medium for melanocytes (obtained from ATCC, product number PCS-200-030) containing a melanocyte-specific growth factor kit (obtained from ATCC, product number PCS-200-041), and then prepared by replacing the medium with fresh medium.

[0054] HEM cells were added to untreated wells (control group wells) and wells containing 0.12, 0.25, 0.5, 1.0, and 2.0 ng / mL of PDGF-BB growth factor, ensuring that each well contained the same number of HEM cells. The cells were then cultured for 24 hours in a 37°C, 5% carbon dioxide (CO2) incubator. After removing the old medium (used medium), a melanocyte-specific medium containing the corresponding dried growth factor powder for each group was added to each well, and the culture was continued for a further 48 hours.

[0055] Cells in untreated wells (control group wells) and growth factor wells containing 0.12, 0.25, 0.5, 1.0, and 2.0 ng / mL of PDGF-BB were each mixed with 100 microliters (μL) of CCK-8 reagent (brand name: Dojin Chemical Laboratories, model number: CK04-01), and the mixtures were incubated at 37°C in a 5% carbon dioxide (CO2) incubator for 1 to 3 hours. After the reaction was complete, the cell culture supernatant (SUSPENSION) from each well was aspirated and transferred to a microcentrifuge tube, and analyzed at 450 nm absorbance using a multifunction microplate spectrophotometer (model number: VARIOSKAN LUX®, brand name: THERMO SCIENTIFIC®). Regression curves between absorbance and cell number were constructed according to the CCK-8 reagent product manual, and the absorbance was converted to the number of viable cells.

[0056] The experimental results are shown in Table 1 and Figure 1. Compared to the control group's melanocyte count, PDGF-BB effectively promoted the normal proliferation of melanocytes.

[0057] [Table 1]

[0058] 2-3. The effect of platelet-derived growth factor BB (PDGF-BB) on melanin production by melanocytes (MELANOCYTE).

[0059] A 96-well plate was divided into untreated wells (control group, 0 ng / mL) and growth factor wells containing PDGF-BB at concentrations of 0.12, 0.25, 0.5, 1.0, and 2.0 ng / mL, with three replicates per well.

[0060] Human epidermal melanocytes (HEM; obtained from ATCC, product number PCS-200-012) were cultured in melanocyte-specific basal cell medium (obtained from ATCC, product number PCS-200-030) with a melanocyte-specific growth factor kit (obtained from ATCC, product number PCS-200-041), and then the medium was replaced with fresh medium to prepare the sample.

[0061] HEM cells were added to untreated wells (control wells, 0 ng / mL PDGF-BB) and to growth factor wells containing 0.12, 0.25, 0.5, 1.0, and 2.0 ng / mL PDGF-BB, respectively, ensuring that each well contained the same number of HEM cells. The cells were then cultured for 24 hours in a 37°C, 5% carbon dioxide (CO2) incubator. After removing the old medium (used medium), a melanocyte-specific medium containing the corresponding dried growth factor powder for each group was added to each well, and the culture was continued for a further 48 hours.

[0062] Cells in untreated wells (control wells, 0 ng / mL) and growth factor wells containing PDGF-BB at concentrations of 0.12, 0.25, 0.5, 1.0, and 2.0 ng / mL were each mixed with 500 μL of TrypLE reagent (brand name: GIBCO, model number: A1285901) and incubated at 37°C in a 5% carbon dioxide (CARBON DIOXIDE, CO2) incubator for 3–5 minutes. After the reaction was complete, 500 μL of basal cell medium specifically for melanocytes was added to stop the reaction of the TrypLE reagent.

[0063] The cell solution from each well was transferred to a microcentrifuge tube and centrifuged using a microcentrifuge (brand name: KUBOTA, model number: 3520) at 1,000-3,000 rpm for 10-30 minutes; preferably 2,000 rpm for 20 minutes). The supernatant was removed to obtain a cell pellet. 110 μL of 1N NaOH (brand name: SIGMA, model number: S5881) was added to each tube and reacted for 10-20 minutes. After the reaction was complete, each mixture was transferred to a microcentrifuge tube and analyzed at 475 nm using a multifunction microplate spectrophotometer (model number: VARIOSKAN LUX (trademark), brand name: THERMO SCIENTIFIC (trademark)) to obtain the absorbance of melanin.

[0064] The experimental results are shown in Table 2 and Figure 2. Compared to the melanin levels of the control group, it was shown that under the influence of PDGF-BB, the amount of melanin (MELANIN) increased synchronously with the proliferation of melanocytes (MELANOCYTE).

[0065] [Table 2]

[0066] 2-4. Inhibitory effect of platelet-derived growth factor BB (PDGF-BB) on melanin production.

[0067] Based on the experimental results described above, the melanin production inhibition rate per melanocyte (MELANOCYTE) was calculated. The calculation formula is as follows: Melanin production inhibition rate (%) = (1 - amount of melanin per unit cell (test group)) ÷ amount of melanin per unit cell (control group) × 100%

[0068] The experimental results are shown in Table 3 and Figure 3. Using the amount of melanin in the control group as a baseline, the calculations showed that under the action of PDGF-BB, the total amount of melanin (MELANIN) increased in sync with the proliferation of melanocytes (MELANOCYTE). However, it was found that PDGF-BB actually suppressed melanin production in individual melanocytes. When the PDGF-BB concentration reached 0.12 ng / mL, the melanin production inhibition rate reached 7%. When the PDGF-BB concentration was 0.25 ng / mL or higher, the melanin production inhibition rate reached 10% or more. Furthermore, in the range of PDGF-BB concentration from 0.25 ng / mL to 1 ng / mL, the melanin production inhibition rate reached 14% or higher.

[0069] [Table 3]

[0070] 3. Preparation of a dried growth factor powder containing transforming growth factor β (TGF-β)

[0071] The growth factor of the present invention is isolated from whole blood of a human or non-human mammal.

[0072] 250 ml (mL) of whole blood was placed in a blood bag containing an anticoagulant, and centrifugation was performed at 300-1,500 × g for 3-10 minutes to obtain platelet-rich plasma (PRP), also known as plasma layer solution.

[0073] A thrombin activator was added to the aforementioned PRP, and the mixture was centrifuged again. The supernatant was then removed to completely remove proteins from the plasma.

[0074] An equal volume of sterile water for injection was added to the removed supernatant to obtain growth factors released by platelet activation, and then 1 mL was dispensed per vial into sample vials. Subsequently, the growth factor powder was obtained by freeze-drying.

[0075] 4-1. Preparation of a culture medium specifically for melanocytes containing Transforming Growth Factor β (TGF-β) dried powder

[0076] Dry powder medium was prepared by dissolving the growth factor dry powder in 1 mL of melanocyte-specific basal cell medium (containing a melanocyte-specific growth factor kit). The growth factor dry powder contains 75 to 8,500 ng of TGF-β per gram. The melanocyte-specific basal cell medium and the dry powder medium were mixed in predetermined proportions and analyzed and confirmed by enzyme-linked immunosorbent assay (ELISA) to obtain melanocyte-specific media containing TGF-β dry powder medium with final TGF-β concentrations of 0 nanograms (0 ng / mL), 0.5, 1.0, 5.0, 10.0, and 20.0 ng / mL per milliliter.

[0077] 4-2. Inhibitory effect of transforming growth factor β (TGF-β) on melanocyte (MELANOCYTE) production

[0078] The 96-well plate was divided into untreated wells (control wells, i.e., 0 ng / mL TGF-β) and growth factor wells containing 0.5, 1.0, 5.0, 10.0, and 20.0 ng / mL TGF-β, with three replicates per well.

[0079] Human epidermal melanocytes (HUMAN EPIDERMAL MELANOCYTES, HEM; obtained from ATCC, product number PCS-200-012) were cultured in a basal cell medium for melanocytes (obtained from ATCC, product number PCS-200-030) containing a melanocyte-specific growth factor kit (obtained from ATCC, product number PCS-200-041), and then prepared by replacing the medium with fresh medium.

[0080] HEM cells were added to untreated wells (control group wells) and wells containing 0.5, 1.0, 5.0, 10.0, and 20.0 ng / mL of TGF-β growth factor, ensuring that each well contained the same number of HEM cells. The cells were then cultured for 24 hours in a 37°C, 5% carbon dioxide (CO2) incubator. After removing the old medium (used medium), a melanocyte-specific medium containing TGF-β dry powder medium corresponding to each group was added to each well, and the culture was continued for a further 48 hours.

[0081] Cells in untreated wells (control wells, i.e., 0 ng / mL TGF-β) and growth factor wells containing 0.5, 1.0, 5.0, 10.0, and 20.0 ng / mL TGF-β were each mixed with 500 μL of TrypLE reagent (brand name: GIBCO, model number: A1285901) and incubated at 37°C in a 5% carbon dioxide (CARBON DIOXIDE, CO2) incubator for 3–5 minutes. After the reaction was complete, 500 μL of basal cell medium specifically for melanocytes was added to stop the reaction of the TrypLE reagent.

[0082] The cell solution from each well was transferred to a microcentrifuge tube and centrifuged using a microcentrifuge (brand name: KUBOTA, model number: 3520) at 1,000-3,000 rpm (preferably 2,000 rpm for 20 minutes). The supernatant was removed to obtain a cell pellet. 110 μL of 1N NaOH (brand name: SIGMA, model number: S5881) was added to each tube and allowed to react for 10-20 minutes. After the reaction was complete, each mixture was transferred to a microcentrifuge tube and analyzed at 475 nm using a multifunction microplate spectrophotometer (model number: VARIOSKAN LUX (trademark), brand name: THERMO SCIENTIFIC (trademark)) to obtain the absorbance of melanin.

[0083] Based on the experimental results described above, the formula used to calculate the melanin production inhibition rate per melanocyte (MELANOCYTE) is as follows: Melanin production inhibition rate (%) = (1 - amount of melanin per unit cell (test group)) ÷ amount of melanin per unit cell (control group) × 100%

[0084] The experimental results are shown in Table 4 and Figure 4. Using the amount of melanin in the control group as a baseline, the calculations showed that when the TGF-β concentration reached 0.5 ng / mL, the melanin production inhibition rate reached 7%; when the TGF-β concentration reached 1.0 ng / mL or higher, the melanin production inhibition rate reached 10% or higher; and furthermore, in the range of TGF-β concentration from 1.0 ng / mL to 10 ng / mL, the melanin production inhibition rate reached 14% or higher.

[0085] [Table 4]

[0086] 5. Preparation of dried platelet powder

[0087] The platelets of this invention are isolated from whole blood of a human or non-human mammal.

[0088] 250 ml (mL) of whole blood was placed in a blood bag containing an anticoagulant and centrifuged at 300-1,500 × g for 3-10 minutes to obtain platelet-rich plasma (PRP), also known as plasma layer solution.

[0089] The plasma was centrifuged again until the platelets gathered at the bottom, and the supernatant was removed to completely remove proteins from the plasma. An equal volume of sterile water for injection was added to obtain purified platelets, which were then dispensed into sample vials at a rate of 1 mL per vial. Subsequently, platelet-dried powder was obtained by lyophilization.

[0090] 6-1. Preparation of a culture medium specifically for melanocytes containing dried platelet powder

[0091] Platelet-free dried powder was dissolved in 1 mL of basal cell medium specifically for melanocytes (containing a melanocyte-specific growth factor kit) to prepare platelet-free dried powder medium (DRY POWDER MEDIUM). The platelet-free dried powder was 4 × 10⁶ per gram. 8 ~1.5×10 11 It contains 10 platelets. Melanocyte-specific basal cell medium and the platelet dry powder medium are mixed in a predetermined ratio, and the final platelet concentration is 1.25 × 10⁶ (no platelets, control group) 7 pieces, 2.5×10 7 individual platelets, 5 x 10 7 individual platelets, 10 x 10 7 10 platelets, and 15 × 10 7 A melanocyte-specific culture medium containing platelet-dried powder medium was obtained.

[0092] 6-2. Inhibitory effect of platelets on melanocyte (MELANOCYTE) production

[0093] In this invention, the effect of platelet count on melanin production was further investigated.

[0094] A 96-well plate was prepared with untreated wells (control wells, i.e., 0 platelets) and 1.25 × 10⁶ 7 pieces, 2.5×10 7 pieces, 5×10 7 pieces, 10×10 7 pieces, and 15 × 10 7 The platelets were divided into treatment wells, and three replicates were performed in each well.

[0095] HEM cells were placed in untreated wells (control group wells) and 1.25 × 10⁶ wells. 7 pieces, 2.5×10 7 pieces, 5×10 7 pieces, 10×10 7 pieces, and 15 × 10 7 The HEM cells were added to treatment wells containing the same number of platelets, ensuring that each well contained the same number of HEM cells, and incubated for 24 hours in a 37°C, 5% carbon dioxide (CO2) incubator. After removing the old medium (used medium), a melanocyte-specific medium containing dried platelet powder corresponding to each group was added to each well, and incubation was continued for a further 48 hours.

[0096] 500 μL of TrypLE reagent (brand name: GIBCO, model number: A1285901) was added to the cells in each well, and the incubators were placed in a 37°C, 5% carbon dioxide (CO2) incubator for 3-5 minutes. After the reaction was complete, 500 μL of basal cell medium specifically for melanocytes was added to stop the reaction of the TrypLE reagent.

[0097] Transfer the cell lysate in each well to a microcentrifuge tube, and centrifuge it using a microcentrifuge (brand name: KUBOTA, model number: 3520) at 1,000 - 3,000 rpm for 10 - 30 minutes (preferably 2,000 rpm for 20 minutes). Remove the supernatant to obtain a cell pellet (PELLET). Add 110 μL of 1N NaOH (brand name: SIGMA, model number: S5881) to each tube and let it act for 10 - 20 minutes. After the reaction is completed, transfer each mixture to a microcentrifuge tube and analyze it using a multifunctional microplate spectrophotometer (model number: VARIOSKAN LUX (trademark), brand name: THERMO SCIENTIFIC (trademark)) at an absorbance value of 475 nm to obtain the absorbance of melanin. [[ID=...]]

[0098] [[ID=...]]Based on the above experimental results, the melanin production inhibition rate per melanocyte (MELANOCYTE) unit was calculated. The calculation formula is as follows. Melanin production inhibition rate (%) = (1 - melanin amount per unit cell (test group)) ÷ melanin amount per unit cell (control group) × 100%

[0099] The experimental results are shown in Table 5 and Figure 5. Based on the amount of melanin in the control group, as a result of the calculation, when the platelet concentration is in the range of 2.5×10 7 ~15×10 7 the melanin production inhibition rate reached 8% or more. When the platelet concentration is in the range of 2.5×10 7 ~10×10 7 the melanin production inhibition rate reached 12% or more. Furthermore, when the platelet concentration is within the range of 5×10 7 ~10×10 7 the melanin production inhibition rate reached 16% or more.

[0100]

Table 5

[0101] 7-1. Preparation of a melanocyte - specific medium containing platelet dry powder

[0102] Using 1 mL of a basal medium dedicated to melanocytes (containing a melanocyte growth factor kit), the dried platelet powder was dissolved to prepare a dried platelet powder medium (DRY POWDER MEDIUM). The basal cell medium for melanocytes and the dried platelet powder medium were mixed at a predetermined ratio so that the final platelet concentrations were platelets-free (control group), 1.25×10 7 , 2.5×10 7 , 5×10 7 , and 10×10 7 platelets, and a melanocyte-dedicated medium containing the dried platelet powder medium was obtained.

[0103] 7-2. Preparation of a melanocyte-dedicated medium containing different whitening components

[0104] Using the basal cell medium for melanocytes, arbutin (ARBUTIN; brand name: SIGMA, model number: SI-A4256) was prepared, and the final concentration of arbutin (ARBUTIN) was set to 50 μg / mL. Using the basal cell medium for melanocytes, glutathione (GLUTATHIONE; brand name: SIGMA, model number: SI-G6013) was prepared, and the final concentration of glutathione (GLUTATHIONE) was set to 100 μg / mL. Using the basal cell medium for melanocytes, tranexamic acid (TRANEXAMIC ACID; brand name: SHOU YUAN, Taiwan FDA drug manufacturing license number: 027356) was prepared, and the final concentration of tranexamic acid (TRANEXAMIC ACID) was set to 2 mg / mL.

[0105] 7-3. Effects of platelets and different whitening components on melanocyte (MELANOCYTE) inflammation

[0106] A 96-well plate was used for control group wells (i.e., 0 platelets), as well as 1.25×10 7 , 2.5×10 7 , 5×10 7 , and 10×10 7The samples were divided into wells containing individual platelets, wells treated with arbutin, wells treated with glutathione, and wells treated with tranexamic acid, with three replicates per well.

[0107] HEM cells were added to each well, ensuring that each well contained the same number of HEM cells, and the cells were incubated at 37°C in a 5% carbon dioxide (CO2) incubator for 24 hours. After removing the old medium (used medium), the melanocyte-specific medium prepared in steps 7-1 and 7-2 corresponding to each group was added to each well, and incubation was continued for a further 48 hours.

[0108] The cell culture supernatant (SUSPENSION) from each well was transferred to a microcentrifuge tube, and the tumor necrosis factor-α (TNF-α) concentration in the cell culture medium was measured using a human TNF-α ELISA kit (brand name: ABCAM, product number: AB181421). Subsequently, the absorbance was analyzed at 450 nm using a multifunction microplate spectrophotometer (model number: VARIOSKAN LUX (trademark), brand name: THERMO SCIENTIFIC (trademark)). Following the instructions for the TNF-α ELISA kit, a regression curve between absorbance and TNF-α was obtained, and the degree of inflammation suppression in melanocytes was evaluated by converting the absorbance to TNF-α concentration, and the inflammation suppression rate was calculated. The calculation formula is as follows. Inflammation suppression rate (%) = 1-(TNF-α concentration (test group) ÷ TNF-α concentration (control group)) × 100%

[0109] The experimental results are shown in Table 6 and Figure 6. Using the TNF-α concentration of the control group as a baseline, the calculations revealed that each platelet group possesses the ability to alleviate inflammation. Furthermore, the anti-inflammatory effect of platelets was found to be significantly superior to that of common commercially available skin-whitening ingredients.

[0110] [Table 6]

[0111] 8-1. Preparation of a culture medium specifically for melanocytes containing dried platelet powder

[0112] Platelet dry powder was dissolved in 1 mL of melanocyte-specific basal cell medium (containing a melanocyte-specific growth factor kit) to prepare platelet dry powder medium (DRY POWDER MEDIUM). The melanocyte-specific basal cell medium and the platelet dry powder medium were mixed in a predetermined ratio, and the final platelet concentration was 1.25 × 10⁶ for the platelet-free group (control group) and 1.25 × 10⁶ for the platelet-free group. 7 pieces, 2.5×10 7 pieces, 5×10 7 pieces, and 10 × 10 7 A melanocyte-specific culture medium containing platelet-dried powder medium was obtained.

[0113] 8-2. Preparation of a culture medium specifically for melanin cells containing different whitening ingredients

[0114] Arbutin (ARBUTIN; brand name: SIGMA, model number: SI-A4256) was prepared using a basal cell medium specifically for melanocytes, and the final concentration of arbutin (ARBUTIN) was 50 μg / mL. Glutathione (GLUTATHIONE; brand name: SIGMA, model number: SI-G6013) was prepared using a basal cell medium specifically for melanocytes, and the final concentration of glutathione (GLUTATHIONE) was 100 μg / mL. Tranexamic acid (TRANEXAMIC ACID; brand name: SHOU YUAN, Taiwan FDA drug manufacturing license number 027356) was prepared using a basal cell medium specifically for melanocytes, and the final concentration of tranexamic acid (TRANEXAMIC ACID) was 2 mg / mL.

[0115] 8-3. Effects of platelets and different skin-whitening ingredients on tyrosinase activity

[0116] A 96-well plate was prepared, with a control group well (i.e., 0 platelets) and a 1.25 × 10⁶ well. 7 pieces, 2.5×10 7 pieces, 5×10 7 pieces, and 10 × 10 7 The samples were divided into wells containing individual platelets, wells treated with arbutin, wells treated with glutathione, and wells treated with tranexamic acid, with three replicates per well.

[0117] HEM cells were added to each well, ensuring that each well contained the same number of HEM cells, and the cells were incubated at 37°C in a 5% carbon dioxide (CO2) incubator for 24 hours. After removing the old medium (used medium), the melanocyte-specific medium prepared in steps 8-1 and 8-2 corresponding to each group was added to each well, and incubation was continued for a further 48 hours.

[0118] The cell culture supernatant (SUSPENSION) from each well was transferred to a microcentrifuge tube. The tyrosinase concentration in the cell culture medium was measured using a human tyrosinase ELISA kit (brand name: ANTIBODIES, product number: A312872). Subsequently, the absorbance was analyzed at 450 nm using a multifunction microplate spectrophotometer (model number: VARIOSKAN LUX®, brand name: THERMO SCIENTIFIC®). Following the instructions for the tyrosinase ELISA kit, a regression curve between absorbance and tyrosinase was obtained, and the degree of tyrosinase activity inhibition was evaluated by converting the absorbance to tyrosinase concentration, and the tyrosinase inhibition rate was calculated. The calculation formula is as follows. Tyrosinase inhibition rate (%) = 1 - (Tyrosinase concentration (test group) ÷ Tyrosinase concentration (control group)) × 100%

[0119] The experimental results are shown in Table 7 and Figure 7. Based on calculations using the tyrosinase concentration of the control group as a baseline, it was found that each platelet group had the ability to suppress tyrosinase activity. Furthermore, the tyrosinase activity-inhibiting effect of platelets was found to be significantly superior to that of common commercially available skin-whitening ingredients.

[0120] [Table 7]

Claims

1. A composition for inhibiting melanin production, comprising platelets and / or growth factors separated from blood.

2. The composition according to claim 1, wherein the composition contains platelets separated from plasma.

3. The above composition is 2.5 × 10 ７ ~10 x 10 ９ The composition according to claim 2, comprising [number] platelets.

4. The above composition is 2.5 × 10 ７ ~10 x 10 ８ The composition according to claim 3, comprising [number] platelets.

5. The above composition is 5 × 10 ７ ~10 x 10 ８ The composition according to claim 2, comprising [number] platelets.

6. The composition according to claim 1, wherein the growth factor is selected from platelet-derived growth factor BB (PDGF-BB) or transforming growth factor β (TGF-β).

7. The composition according to claim 6, wherein the concentration of PDGF-BB in the composition is 0.1 ng / mL to 5 ng / mL.

8. The composition according to claim 7, wherein the concentration of PDGF-BB in the composition is 0.1 ng / mL to 3 ng / mL.

9. The composition according to claim 8, wherein the concentration of PDGF-BB in the composition is 0.25 ng / mL to 2 ng / mL.

10. The composition according to claim 6, wherein the concentration of TGF-β in the composition is 0.5 ng / mL to 50 ng / mL.

11. The composition according to claim 10, wherein the concentration of TGF-β in the composition is 0.5 ng / mL to 30 ng / mL.

12. The composition according to claim 11, wherein the concentration of TGF-β in the composition is 1 ng / mL to 20 ng / mL.

13. The composition according to claim 1, wherein the composition is a dried powder formed by freeze-drying.

14. In the above composition, per gram of the dry powder, 4 × 10 ８ ~1.5 x 10 １１ The composition according to claim 13, comprising [number] platelets.

15. The composition according to claim 13, wherein the composition contains 15 ng to 850 ng of PDGF-BB per gram of the dried powder.

16. The composition according to claim 13, wherein the composition contains 75 ng to 8,500 ng of TGF-β per gram of the dried powder.

17. Use of the composition according to any one of claims 1 to 16 for producing a formulation for improving skin pigmentation in an individual.

18. The use according to claim 17, wherein the skin pigmentation includes melasma.

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