Mitochondrial anti-aging liposome and its preparation method and application

Abstract

The invention discloses a mitochondrial anti-aging liposome and its preparation method and application, specifically related to the field of cosmetic technology, it is intended to solve the problems such as poor stability of the functional components acting on "mitochondria" in the prior art, unobvious anti-aging effect, etc., it includes active ingredients and nanocarrier raw materials, the nanocarrier raw materials are wrapped in the periphery of the active ingredients, wherein the active ingredients include 1 to 5 parts of ribose, 0.1 to 3 parts of decarboxylated carnosine hydrochloride, 0.01 to 0.5 parts of collagen and 0.01 to 0.05 parts of palmitoyl tripeptide-1 by mass fractions; the nanocarrier raw materials include 1 to 10 parts of phospholipids, 1 to 40 parts of emulsifiers, 10 to 60 parts of polyols and 30 to 80 parts of water by mass fractions. The present invention can act on mitochondria, thereby activating cell autophagy, promoting the regeneration and repair of skin cells, and achieving significant anti-aging effects.

Description

Technical Field

[0001] The invention relates to a mitochondrial anti-aging liposome and a preparation method and application thereof, belonging to the technical field of cosmetics. Background Art

[0002] Mitochondria are known as the "engines" of human cells and are the bioenergetics center of eukaryotic cells. As the organelle that plays a primary role in energy generation and oxidative stress, they not only provide more than 90% of the energy source for cells, but also can deeply regulate energy metabolism.

[0003] As we age, the function of mitochondria gradually declines, which is manifested as reduced energy production and increased oxidative stress. These changes lead to a decline in cell function, which in turn leads to the decline of tissue and organ function, and ultimately manifests as aging. Therefore, the decline of mitochondrial function is considered an important part of the aging mechanism.

[0004] In recent years, studies have discovered some molecules and mechanisms related to mitochondrial DNA repair. These molecules include repair enzymes, DNA polymerases, and mitochondrial DNA repair proteins, which help maintain the integrity and stability of mitochondrial DNA by recognizing and repairing mtDNA damage. Therefore, by enhancing the ability to repair mitochondrial DNA, manifestations associated with aging can be reversed. Among them, autophagy is the process by which cells clean up damaged mitochondria and recycle cellular components, which helps maintain the youthful state of cells and mitochondria. Some active ingredients can promote the expression of autophagy-related proteins, help remove dysfunctional mitochondria, and slow down cell aging.

[0005] However, many active ingredients that act on "mitochondria" have poor stability. Direct application in cosmetic products can easily lead to their oxidative denaturation or inactivation, ultimately greatly reducing their anti-aging effects.

[0006] The quality of many anti-aging cosmetics currently available is uneven. Most cosmetics containing anti-aging ingredients have the following problems: the active ingredients are unstable and easily degraded and inactivated. The active ingredients in most anti-aging skin care products on the market are generally polyphenols, vitamins or peptides, which are sensitive to enzymes, light, heat, pH value, etc. If they are added directly to the formula without nano-encapsulation treatment, it is easy to cause the unstable active ingredients to become inactivated.

[0007] Moreover, since the stratum corneum of the skin has a strong keratinocyte barrier function, many active molecules, such as active peptides, are difficult to penetrate into the stratum corneum, which affects the skin care effect at the targeted site, and the anti-aging effect is minimal, and the anti-aging mechanism is single. In addition, due to the complexity of the skin aging mechanism, the synergistic effect of active substances with multiple mechanisms of action is required to effectively exert the anti-aging effect of the skin. However, the anti-aging skin care products on the market currently mainly solve the skin aging problem from the antioxidant mechanism, with a single mechanism of action, which ultimately leads to unsatisfactory anti-aging effects. Summary of the invention

[0008] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a mitochondrial anti-aging liposome and its preparation method and application, which can act on mitochondria to activate cell autophagy, promote the regeneration and repair of skin cells, and achieve significant anti-aging effects.

[0009] To achieve the above object, the present invention is implemented by adopting the following technical solutions:

[0010] In a first aspect, the present invention provides a mitochondrial anti-aging liposome, comprising an active ingredient and a nanocarrier raw material, wherein the active ingredient comprises, by weight, 1 to 5 parts of ribose, 0.1 to 3 parts of decarboxylated carnosine hydrochloride, 0.01 to 0.5 parts of collagen, and 0.01 to 0.05 parts of palmitoyl tripeptide-1; and the nanocarrier raw material comprises, by weight, 1 to 10 parts of phospholipids, 1 to 40 parts of emulsifiers, 10 to 60 parts of polyols, and 30 to 80 parts of water.

[0011] Furthermore, the phospholipid is one or more of soybean lecithin, hydrogenated lecithin, egg yolk lecithin, hydrogenated soybean lecithin, and hydrogenated egg yolk lecithin.

[0012] Furthermore, the emulsifier is one or more of polyoxyethylene castor oil emulsifiers, polyoxyethylene hydrogenated castor oil emulsifiers, polyglycerol emulsifiers, poloxamer, coconut glucoside, triglycerides, pyrrolidones, polyglyceryl esters, Tween 80, Tween 20, Tween 60, PEG-20 hydrogenated castor oil, PEG-40 hydrogenated castor oil, and PEG-60 hydrogenated castor oil.

[0013] Furthermore, the polyol is one or more of glycerol, propylene glycol, 1,3-butylene glycol, 1,3-propanediol, 1,2-pentanediol, ethoxydiglycol, 1,2-hexanediol, dipropylene glycol, isopropanol, polyethylene glycol-200, PPG-10 sorbitol, and octyldodecanol.

[0014] Furthermore, the nanocarrier raw material also includes 0.1-2 parts of an antioxidant, and the antioxidant is one or more of vitamin E, sodium bisulfite, rutin, butylated hydroxytoluene, tert-butylhydroquinone, and butylated hydroxyanisole.

[0015] In a second aspect, the present invention provides a method for preparing the mitochondrial anti-aging liposomes described in any one of the above items, comprising:

[0016] Evenly mix palmitoyl tripeptide-1, ribose, emulsifier, polyol and phospholipid to obtain a mixed solution A;

[0017] Mix decarboxylated carnosine hydrochloride, collagen and water to obtain a mixed solution B;

[0018] Pour the mixed solution A into the mixed solution B and mix evenly to obtain the mixed solution C;

[0019] The mixed solution C is nano-processed to obtain mitochondrial anti-aging liposomes.

[0020] Furthermore, when the nanocarrier raw material further includes 0.1 to 2 parts of an antioxidant, the antioxidant is also mixed into the mixed solution B.

[0021] Furthermore, the mixing operation is carried out at 40-65°C.

[0022] Furthermore, the nano-processing includes homogenizing the mixed solution C using a high-pressure homogenizer.

[0023] In a third aspect, the present invention also provides the use of the mitochondrial anti-aging liposomes described in the above technical solution in the preparation of cosmetics.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The mitochondrial anti-aging liposomes provided by the present invention are rationally matched with a variety of anti-aging functional ingredients, combined with nano-carrier technology, and the four scientifically matched anti-aging functional ingredients, ribose, decarboxylated carnosine hydrochloride, collagen and palmitoyl tripeptide-1, are packaged and delivered by nano-carriers through novel nano-technology. The mitochondrial anti-aging liposomes obtained by nano-encapsulation have good stability and water dispersibility, increase the solubility of the anti-aging functional ingredients, and also improve the irritation of the functional ingredients, so that the anti-aging functional ingredients can reach a sufficient concentration in the product to exert the corresponding functional effects;

[0026] The present invention effectively improves the solubility of each anti-aging functional component while providing a stable storage space for it, thereby avoiding unnecessary degradation or inactivation of the functional components before storage and use, and is beneficial to increasing the concentration of functional substances in anti-aging products;

[0027] The present invention comprehensively considers multiple anti-aging mechanisms, targets mitochondria, thereby activating cell autophagy, promoting the regeneration and repair of skin cells, and can achieve significant anti-aging effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a schematic diagram of the chicken embryo chorioallantoic membrane irritation test results of the mitochondrial anti-aging liposomes prepared in Examples 1 to 3 of the present invention after being diluted 10 times;

[0029] Figure 2 It is a schematic diagram of the in vitro skin penetration test results of free RhoB and RhoB nanocarriers of the present invention;

[0030] Figure 3 This is a laser confocal observation of the uptake behavior of HaCaT cells on the free RhoB and RhoB nanocarriers of the present invention;

[0031] Figure 4 This is a laser confocal observation of the uptake behavior of HSF cells on the free RhoB and RhoB nanocarriers of the present invention;

[0032] Figure 5 Schematic diagram of HaCaT cell proliferation rate detection after the mitochondrial anti-aging liposomes prepared in Example 1 and Comparative Example 7 were added to 100 μL DMEM complete medium at concentrations of 200, 400 and 800 mg / L respectively and a blank control group after HaCaT cells were cultured for 24 hours in the present invention;

[0033] Figure 6 The figure is a schematic diagram of detecting the proliferation rate of HSF cells after the HSF cells are cultured for 24 hours, wherein the mitochondrial anti-aging liposomes prepared in Example 1 and Comparative Example 7 are added to 100 μL DMEM complete medium at concentrations of 200, 400 and 800 mg / L respectively, and the blank control group is cultured for 48 hours;

[0034] Figure 7 The figure is a schematic diagram of the ROS fluorescence intensity detection results of the HSF cells in the present invention after being cultured for 24 hours and then added to the experimental group, model group and blank control group for further culture for 48 hours, wherein the experimental group is respectively added with 400 mg / L of the mitochondrial anti-aging liposomes prepared in Example 1 and Comparative Examples 1 to 7 and DMEM medium containing 0.6 mmol / L H2O2, the model group is added with DMEM medium containing 0.6 mmol / L H2O2, and the blank control group is added with DMEM medium;

[0035] Figure 8It is a schematic diagram of β-galactosidase staining after HSF cells are cultured for 24 hours and then added to the experimental group (Example 1, Comparative Example 7) and the control group in a CO2 incubator for 48 hours, wherein the experimental group is the mitochondrial anti-aging liposomes prepared by Example 1 and Comparative Example 7 at a concentration of 400 mg / L respectively;

[0036] Fig. 9 This is a schematic diagram of the fluorescence intensity detection results of the MDC staining method after HSF cells were cultured for 24 hours and then added to the experimental group and the blank control group and continued to be cultured for 48 hours, wherein the experimental group was added with 400 mg / L concentration of mitochondrial anti-aging liposomes prepared in Example 1 and Comparative Examples 1 to 7 in DMEM culture medium. DETAILED DESCRIPTION

[0037] The present invention will be further described below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the protection scope of the present invention.

[0038] Example 1

[0039] The embodiment of the present invention provides a method for preparing mitochondrial anti-aging liposomes, comprising the following steps:

[0040] 0.02 parts of palmitoyl tripeptide-1, 2 parts of ribose, 5 parts of lecithin, 12 parts of 1,2-pentanediol, 3 parts of PEG-40 hydrogenated castor oil, 10 parts of glycerin, and 10 parts of ethoxydiglycol were stirred at 55° C. to form a uniform and clear mixed solution A.

[0041] 1 part of decarboxylated carnosine hydrochloride, 0.1 part of collagen, 48 parts of water and 0.5 parts of sodium bisulfite were stirred at 55°C to form a uniform and clear mixed solution B.

[0042] Pour the mixed solution A into the mixed solution B and mix them, stirring at 55°C to form a uniform mixed solution C.

[0043] After the mixed solution C was homogenized in a high-pressure homogenizer at 850 bar, mitochondrial anti-aging liposomes were obtained, and the average particle size was 22.96 nm, and the polymer dispersity index (PDI) was 0.162.

[0044] Example 2

[0045] 0.05 parts of palmitoyl tripeptide-1, 3 parts of ribose, 3 parts of hydrogenated lecithin, 5 parts of 1,2-pentanediol, 5 parts of PEG-40 hydrogenated castor oil, 2 parts of Tween 80, 8 parts of 1,3-propylene glycol, and 12 parts of butylene glycol were stirred at 55° C. to form a uniform and clear mixed solution A.

[0046] 0.5 parts of decarboxylated carnosine hydrochloride, 0.1 parts of collagen, 50 parts of water and 0.5 parts of butylated hydroxytoluene were stirred at 55° C. to form a uniform and clear mixed solution B.

[0047] After the mixed solution C was homogenized in a high-pressure homogenizer at 850 bar, mitochondrial anti-aging liposomes were obtained, and the average particle size was 24.23 nm, and the polymer dispersity index (PDI) was 0.145.

[0048] Example 3

[0049] 0.02 parts of palmitoyl tripeptide-1, 2 parts of ribose, 4 parts of soybean lecithin, 6 parts of 1,2-pentanediol, 3 parts of PEG-60 hydrogenated castor oil, 1 part of coconut glucoside, 10 parts of glycerin, and 8 parts of butylene glycol were stirred at 55° C. to form a uniform and clear mixed solution A.

[0050] 1.5 parts of decarboxylated carnosine hydrochloride, 0.15 parts of collagen and 52 parts of water were stirred at 55°C to form a uniform and clear mixed solution B.

[0051] After the mixed solution C was homogenized in a high-pressure homogenizer at 850 bar, mitochondrial anti-aging liposomes were obtained, and the average particle size was 26.16 nm, and the polymer dispersity index (PDI) was 0.176.

[0052] Comparative Example

[0053] Using Example 1 as a control, the active substance therein was replaced with an equal amount of a single active ingredient, and the same method was used to prepare nanocomposites containing different single active ingredients.

[0054] Comparative Example 1:

[0055] The only difference from Example 1 is that the active ingredient contains only 3.12 parts of ribose.

[0056] Comparative Example 2:

[0057] The only difference from Example 1 is that the active fraction contains only 3.12 parts of decarboxylated carnosine hydrochloride.

[0058] Comparative Example 3:

[0059] The only difference from Example 1 is that the active fraction contains only 3.12 parts of collagen.

[0060] Comparative Example 4:

[0061] The only difference from Example 1 is that the active ingredient contains only 3.12 parts of palmitoyl tripeptide-1.

[0062] Comparative Example 5:

[0063] The only difference from Example 1 is that the active ingredient contains only 0.02 parts of palmitoyl tripeptide-1, 1 part of decarboxylated carnosine hydrochloride and 0.1 part of collagen.

[0064] Comparative Example 6:

[0065] The only difference from Example 1 is that the active ingredient contains only 2 parts of ribose.

[0066] Comparative Example 7:

[0067] The active ingredients ribose, decarboxylated carnosine hydrochloride, collagen, and palmitoyl tripeptide-1 were mixed with water to obtain a free composition having the same active ingredient content as that of Example 1.

[0068] The performance of the mitochondrial anti-aging liposomes obtained in Examples 1 to 3 and Comparative Examples 1 to 7 is analyzed below:

[0069] The average particle size of the mitochondrial anti-aging liposomes obtained in Examples 1 to 3 and Comparative Examples 1 to 6 was within 100 nm, and the polymer dispersibility index (PDI) was 0.1 to 0.4. The obtained mitochondrial anti-aging liposomes were placed in a sealed container at -20°C, room temperature, 4°C, and 45°C for 3 months, respectively, and no stratification or precipitation occurred, and the particle size did not change significantly, indicating that the obtained mitochondrial anti-aging liposomes had good stability.

[0070] Performance test 1: Chicken embryo allantoic membrane irritation test

[0071] The mitochondrial anti-aging liposomes prepared in Examples 1 to 3 were diluted 10 times, 0.2 mL was taken out, and dripped on the surface of the chorioallantoic membrane. The changes in the CAM blood vessels were observed within 5 minutes, and the initial time of congestion, bleeding and coagulation of the CAM blood vessels was recorded, and the stimulation score IS was calculated.

[0072] The results are as follows Figure 1 As shown, the results of Examples 1 to 3 are similar. When the mitochondrial anti-aging liposomes diluted 10 times were in contact with the chicken embryo chorioallantoic membrane for 300 s, there was no capillary bleeding, vascular melting, or coagulation, and the reaction integral was 0.07, indicating that the mitochondrial anti-aging liposomes described in Examples 1-3 after being diluted 10 times are safe and non-irritating.

[0073] Performance Test 2: Patch Test Experiment

[0074] 30 subjects were selected, and 10% of the mitochondrial anti-aging liposomes described in Examples 1-3 and a blank control were applied to the curved side of the subjects' forearms for 24 hours. After the spot tester was removed, the skin reaction was observed after a 30-minute interval after the indentation disappeared. The skin reaction was observed again 24 hours and 48 hours after the spot tester was removed.

[0075] The results showed that none of the 30 subjects had light red spots, erythema, edematous erythema, significant redness and swelling, infiltration or papules, and papules or blisters, etc., indicating that the mitochondrial anti-aging liposomes described in Examples 1-3 are non-irritating to human skin.

[0076] Performance Test 3: In vitro skin penetration test

[0077] Preparation of free RhoB and RhoB nanocarriers: Rhodamine B (RhoB) was added to the aqueous phase to replace the active ingredient as a fluorescent marker, and Rhodamine B nanocarriers (RhoB nanocarriers) were prepared by the method of preparing mitochondrial anti-aging liposomes, and an equal concentration of free Rhodamine B solution was prepared as a control sample (free RhoB).

[0078] The skin permeation experiment of ex vivo pig skin was carried out by vertical Franz diffusion cell method. The skin was fixed between the receiving chamber and the supply chamber, and the experiment was divided into two groups, namely the free RhoB group and the RhoB nanocarrier group. 0.5g of RhoB nanocarrier compound essence (5% RhoB nanocarrier) and free RhoB compound essence (consistent with the RhoB concentration in RhoB nanocarrier) were taken in the supply chamber, PBS was used as the receiving solution, and the mixture was stirred and diffused at 37°C. After 2h and 4h, the residual sample on the skin was gently wiped off, the skin in the target area was removed, and the skin was rinsed again. After thorough cleaning, the residual moisture was wiped off. The sample was frozen and sectioned, and the sections were observed by laser confocal microscopy, and representative areas were selected for photography.

[0079] Results Figure 2 , free RhoB was mostly concentrated in the stratum corneum at 2 hours and failed to penetrate the stratum corneum barrier. RhoB nanocarriers had penetrated the stratum corneum barrier at 2 hours and entered the active epidermis of the skin. As time went on, the fluorescence penetration depth of the RhoB nanocarrier skin increased at 4 hours, reaching a skin depth of 254.4μm, indicating that it had reached the dermis of the skin. The results showed that within the same period of time, the fluorescence intensity and penetration depth of the RhoB nanocarrier in the skin were significantly stronger than those of free RhoB, indicating that the nanocarrier can effectively deliver the encapsulated active ingredients to the deep tissues of the skin and reach the dermis, the target tissue for skin anti-aging effects.

[0080] Performance Test 4: Observation of Cell Uptake Behavior by Laser Confocal Microscopy

[0081] HaCaT cells in the logarithmic growth phase were cultured at 3.0×10 5 HSF cells were expressed at 1.6×10 per dish. 5The cells were inoculated in 35 mm confocal dishes and cultured for 24 h, and then incubated for 2 h and 4 h respectively with DMEM medium containing free RhoB and RhoB nanocarriers. After incubation, the medium was discarded and the cells were washed three times with PBS solution, fixed with 4% paraformaldehyde and stained with DAPI solution for 15 min each, and observed and photographed under a 60x objective lens using a laser confocal microscope.

[0082] Results Figure 3 and Figure 4 , from left to right in the figure are DAPI (a dye) fluorescence, RhoB fluorescence, and the fluorescence of DAPI and RhoB together. As the incubation time increases, the fluorescence intensity in HaCaT cells and HSF cells is significantly enhanced. After 2 hours of incubation, the intracellular fluorescence intensity of the free RhoB solution group is weak, while the fluorescence intensity of the RhoB nanocarrier group is significantly stronger than that of free RhoB. After 4 hours of incubation, the fluorescence intensity of the RhoB nanocarrier group is further enhanced. The experimental results show that compared with the free RhoB solution, the RhoB nanocarrier can be more absorbed by HaCaT cells and HSF cells, indicating that the nanocarrier can effectively deliver the encapsulated active ingredients into the skin target cells to exert their effects.

[0083] Performance Test 5: Cell Proliferation Experiment

[0084] HaCaT cells in logarithmic growth phase were collected and counted at 8×10 3 The density of HSF cells was 4×10 3 The density of cells / well was 100 μL per well, and the adaptive culture was continued for 24 hours. 100 μL of DMEM complete medium containing Example 1 (the concentrations of the mitochondrial anti-aging liposomes described in Example 1 were 200, 400 and 800 mg / L, respectively) and DMEM complete medium of Comparative Example 7 (the concentration of the mitochondrial anti-aging liposomes described in Example 1 was consistent) was added to each well, and 100 μL of DMEM complete medium was added to the control group, with 3 replicates per group. The culture was continued for 48 hours, and the cell proliferation rate was measured by CCK-8 method.

[0085] Results Figure 5 (Compared with the control group, * P <0.05) and Figure 6 (Compared with the control group, * P <0.05, ** P <0.01; Compared with Comparative Example 7, # P<0.05), compared with the control group, when the concentration of the mitochondrial anti-aging liposomes described in Example 1 was 400 mg / L, it had a proliferation-promoting effect on HaCaT cells (P<0.05). Compared with the control group, when the concentration of the mitochondrial anti-aging liposomes described in Example 1 was 400 mg / L and 800 mg / L, it had a proliferation-promoting effect on HSF cells (P<0.01 or P<0.05); compared with comparative example 7, when the concentration of the mitochondrial anti-aging liposomes described in Example 1 was 400 mg / L and 800 mg / L, the proliferation effect on HSF cells was significantly improved (P<0.05).

[0086] The above experimental results show that the mitochondrial anti-aging liposomes described in Example 1 can promote the proliferation of HaCaT cells and HSF cells, which may be related to the improvement of mitochondrial function because mitochondria are the main source of energy supply during cell proliferation.

[0087] Performance Test 6: ROS Fluorescence Intensity Detection

[0088] HSF cells were cultured at 4 × 10 4 The cells were inoculated into a 24-well plate at a density of 500 μL per well. After culturing for 24 h, the supernatant was discarded. The model group was added with DMEM medium containing 0.6 mmol / L H2O2, and the experimental group was added with DMEM complete medium containing 0.6 mmol / L H2O2 and 400 mg / L of the mitochondrial anti-aging liposomes described in Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5, Comparative Example 6, Comparative Example 7 and Example 1. A control group without H2O2 was set up. After continuing to culture for 24 h, DMEM containing 20 μM DCFH-DA was added and continued to incubate for 20 min. The cells were washed with PBS three times, and the cells were collected and the fluorescence intensity was detected by flow cytometry.

[0089] Results Figure 7 (Compared with the model group, ** P <0.01; compared with Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, ## P <0.01; Compared with Comparative Example 7, && P<0.01), the average fluorescence intensity of ROS in the model group HSF cells after injury by 0.6 mmol / L H2O2 was significantly higher than that in the control group. Compared with the model group, Comparative Examples 1, 2, 3, 4, 5, 6, 7 and Example 1 can significantly reduce the fluorescence intensity of ROS (P<0.01). Compared with Comparative Examples 1, 2, 3 and 4, Example 1 can significantly reduce the fluorescence intensity of ROS (P<0.01), indicating that the antioxidant effect of the nanocomposite of ribose and decarboxylated carnosine hydrochloride, collagen and palmitoyl tripeptide-1 is better than that of the nanocomposite of a single raw material. Compared with the model group, the ROS fluorescence intensity reduction rate of the nanocomposition described in Comparative Example 5 was 14.4%, the ROS fluorescence intensity reduction rate of the nanocomposition described in Comparative Example 6 was 16.8%, and the ROS fluorescence intensity reduction rate of the mitochondrial anti-aging liposome described in Example 1 was 38.6%. The ROS fluorescence intensity reduction rate of the mitochondrial anti-aging liposome described in Example 1 was greater than the sum of the ROS fluorescence intensity reduction rates of the nanocompositions described in Comparative Examples 5 and 6, further indicating that the compounding of ribose with decarboxylated carnosine hydrochloride, collagen, and palmitoyl tripeptide-1 has a synergistic effect. Compared with the free composition (Comparative Example 7), Example 1 can significantly reduce the ROS fluorescence intensity (P<0.01).

[0090] The above experimental results prove that the combination of ribose and decarboxylated carnosine hydrochloride, collagen, and palmitoyl tripeptide-1 can significantly promote the effect of antioxidant ingredients, have a synergistic effect, and the antioxidant effect of the encapsulated nanocomposite is greatly enhanced than that of the free ingredients.

[0091] Performance Test 7: β-Galactosidase Staining

[0092] HSF cells in logarithmic growth phase were cultured at 4×10 4 The cells were inoculated in a 24-well plate at a density of 500 μL per well, and the cells were cultured for 24 h. The experimental group was added with 400 mg / L of the mitochondrial anti-aging liposomes described in Comparative Example 7 and Example 1, and the cells used were 15th generation HSF cells. Incubated in a CO2 incubator for 48 h, and the cells in each group were washed 3 times with PBS according to the instructions of the kit. 1 mL of β-galactosidase staining fixative was added to each well, fixed at room temperature for 15 min, washed 3 times with PBS, 1 mL of β-galactosidase staining working solution was added, incubated at 37°C overnight, and the staining results were observed under a microscope.

[0093] Results Figure 8, after 15 generations of cells were stained with β-galactosidase, the cells were dark blue, indicating that the cells were obviously senescent. After the cells were treated with Comparative Example 7 and Example 1, the blue color became lighter. Compared with Comparative Example 7, the color of the mitochondrial anti-aging liposome cells described in Example 1 was lighter, indicating that after the HSF cells were treated with the mitochondrial anti-aging liposomes described in Example 1, the cell senescence state was weakened, and it had an anti-aging effect.

[0094] Performance Test 8: MDC staining method to detect cell autophagy expression

[0095] HSF cells were cultured at 8 × 10 4 The cells were inoculated into 6-well plates at a density of 2 mL per well. After 24 h of culture, the supernatant was discarded. The experimental group was added with DMEM complete medium containing mitochondrial anti-aging liposomes described in Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5, Comparative Example 6, Comparative Example 7 and Example 1 at a concentration of 400 mg / L. After 48 h of continuous culture, the cells were rinsed once with PBS, incubated at 37°C in a cell culture incubator for 4 h, rinsed once with PBS, 1 mL of 50 μmol / L MDC was added to each well, incubated at room temperature in the dark for 30 min, rinsed 3 times with PBS, and the cells were collected and the fluorescence intensity was detected by flow cytometry.

[0096] Results Fig. 9 (Compared with the control group, ** P <0.01; Compared with Comparative Example 1 and Comparative Example 6, ## P <0.01; Compared with Comparative Example 7, && P<0.01), compared with the control group, the relative fluorescence intensity of MDC in Comparative Example 1, Comparative Example 6, Comparative Example 7 and Example 1 increased significantly (P<0.01), indicating that ribose has the effect of activating autophagy in HSF cells. Compared with the control group, there was no significant difference in the change of the relative fluorescence intensity of MDC in Comparative Example 2, Comparative Example 3, Comparative Example 4 and Comparative Example 5 (P>0.05), indicating that decarboxylated carnosine hydrochloride, collagen and palmitoyl tripeptide-1 do not have the effect of activating autophagy in HSF cells. Compared with Comparative Example 1 and Comparative Example 6, the relative fluorescence intensity of the mitochondrial anti-aging liposome MDC described in Example 1 was significantly increased (P<0.01), indicating that the nanocomposition (Example 1) of ribose and four raw materials of decarboxylated carnosine hydrochloride, collagen, and palmitoyl tripeptide-1 was better than the nanocomposition containing ribose alone (Comparative Example 1 and Comparative Example 6) in promoting the formation of autophagosomes, while decarboxylated carnosine hydrochloride, collagen, and palmitoyl tripeptide-1 had no effect on promoting the formation of autophagosomes, suggesting that the compounding of ribose with four raw materials of decarboxylated carnosine hydrochloride, collagen, and palmitoyl tripeptide-1 had a synergistic effect. Compared with the free composition (Comparative Example 7), the relative fluorescence intensity of the mitochondrial anti-aging liposome MDC described in Example 1 was significantly increased (P<0.01).

[0097] The experimental results show that the combination of ribose with decarboxylated carnosine hydrochloride, collagen, and palmitoyl tripeptide-1 can significantly promote the formation of autophagosomes, have a synergistic effect, and the effect of the encapsulated nanocomposite in promoting cell autophagy is better than that of the free components at the same dose.

[0098] Comprehensively analyzing all performance tests, it can be seen that the mitochondrial anti-aging liposomes provided by the present invention are safe and non-irritating to human skin, and can be used as cosmetic ingredients. The nanocarrier can effectively deliver the encapsulated active ingredients to the deep tissue of the skin, reaching the target tissue for skin anti-aging effects - the dermis, so that the active ingredients can enter the target cells of the skin to exert their effects.

[0099] The combination of ribose and decarboxylated carnosine hydrochloride, collagen, and palmitoyl tripeptide-1 can significantly promote the effect of antioxidant ingredients, have a synergistic effect, and the antioxidant effect of the encapsulated nanocomposite is greatly enhanced than that of the free ingredients.

[0100] Mitochondrial anti-aging liposomes can promote cell proliferation and cell autophagy, slow down cell aging, and have antioxidant properties, which can protect mitochondria from oxidative damage and maintain their normal functions.

[0101] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A mitochondrial anti-aging liposome, characterized in that: The invention comprises an active ingredient and a nanocarrier raw material, wherein the nanocarrier raw material is wrapped around the periphery of the active ingredient, wherein the active ingredient is composed of 1 to 5 parts of ribose, 0.1 to 3 parts of decarboxylated carnosine hydrochloride, 0.01 to 0.5 parts of collagen and 0.01 to 0.05 parts of palmitoyl tripeptide-1 in parts by weight; and the nanocarrier raw material comprises 1 to 10 parts of phospholipids, 1 to 40 parts of emulsifiers, 10 to 60 parts of polyols and 30 to 80 parts of water in parts by weight; The preparation method of the mitochondrial anti-aging liposome comprises: Evenly mix palmitoyl tripeptide-1, ribose, emulsifier, polyol and phospholipid to obtain a mixed solution A; Mix decarboxylated carnosine hydrochloride, collagen and water to obtain a mixed solution B; Pour the mixed solution A into the mixed solution B and mix evenly to obtain the mixed solution C; The mixed solution C is nano-processed to obtain mitochondrial anti-aging liposomes.

2. The mitochondrial anti-aging liposome according to claim 1, characterized in that: The phospholipids are one or more of soybean lecithin, hydrogenated lecithin, egg yolk lecithin, hydrogenated soybean lecithin, and hydrogenated egg yolk lecithin.

3. The mitochondrial anti-aging liposome according to claim 1, characterized in that: The emulsifier is one or more of polyoxyethylene castor oil emulsifiers, polyoxyethylene hydrogenated castor oil emulsifiers, polyglycerol emulsifiers, poloxamer, coconut glucoside, triglycerides, pyrrolidones, Tween 80, Tween 20, Tween 60, PEG-20 hydrogenated castor oil, PEG-40 hydrogenated castor oil, and PEG-60 hydrogenated castor oil.

4. The mitochondrial anti-aging liposome according to claim 1, characterized in that: The polyol is one or more of glycerol, propylene glycol, 1,3-butylene glycol, 1,3-propanediol, 1,2-pentanediol, ethoxydiglycol, 1,2-hexanediol, dipropylene glycol, isopropanol, polyethylene glycol-200, PPG-10 sorbitol, and octyldodecanol.

5. The mitochondrial anti-aging liposome according to claim 1, characterized in that: The nanocarrier raw material also includes 0.1-2 parts of antioxidant, and the antioxidant is one or more of vitamin E, sodium bisulfite, rutin, butylated hydroxytoluene, tert-butylhydroquinone, and butylated hydroxyanisole.

6. The mitochondrial anti-aging liposome according to claim 1, characterized in that: When the nanocarrier raw material further includes 0.1-2 parts of antioxidant, the antioxidant is also mixed into the mixed solution B.

7. The mitochondrial anti-aging liposome according to claim 1, characterized in that: The mixing operations are all carried out at 40-65°C.

8. The mitochondrial anti-aging liposome according to claim 1, characterized in that: The nano-processing includes homogenizing the mixed solution C using a high-pressure homogenizer.

9. Use of the mitochondrial anti-aging liposome according to any one of claims 1 to 8 in the preparation of cosmetics.

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