Cell endogenous antioxidant composition and application

Abstract

The invention belongs to the field of daily chemicals, and discloses a cell endogenous antioxidant composition which is composed of a centella asiatica extract and tocopherol glucoside, and the mass ratio of the centella asiatica extract to the tocopherol glucoside is 1: (0.005-0.1). The composition is composed of a centella asiatica extract and tocopherol glucoside, and the centella asiatica extract and the tocopherol glucoside have better cell endogenous antioxidant synergy. Meanwhile, the invention further provides application of the composition.

Description

Technical Field

[0001] This invention relates to the field of daily chemical products, and more particularly to an endogenous antioxidant composition and its application. Background Technology

[0002] Centella asiatica extract has significant antioxidant effects. Its core components include asiaticoside and hydroxyasiaticoside, which protect skin cell health by inhibiting the generation of free radicals and clearing oxidative stress damage.

[0003] In this field, in order to further improve the antioxidant properties of Centella asiatica extract, the following literature discloses products that can be combined with Centella asiatica to prepare products with good antioxidant properties, or further processing of Centella asiatica to improve its antioxidant properties.

[0004] For example, patent application CN115813964A, which is entitled "An extraction process and application of Centella asiatica extract with antioxidant properties", improves the antioxidant properties of Centella asiatica products through extraction process.

[0005] Publication number KR20220141514A discloses a compound product of Centella asiatica and soy protein with enhanced antioxidant, anti-inflammatory and / or skin-whitening effects, a composition containing the same, and a method for preparing the same. The compound product is prepared by extracting Centella asiatica and soy protein together to obtain a composition with good antioxidant properties.

[0006] In this field, antioxidant performance is viewed from multiple perspectives, such as scavenging free radicals to resist oxidation, and ROS inhibition rate detection to determine its antioxidant performance.

[0007] Therefore, the technical problem to be solved in this case is: how to improve the performance of Centella asiatica extract from the perspective of endogenous antioxidants in cells. Summary of the Invention

[0008] The purpose of this invention is to provide an endogenous antioxidant composition comprising Centella asiatica extract and tocopherol glucoside, which have a good synergistic effect of endogenous antioxidant activity.

[0009] Furthermore, the present invention also provides applications of the composition.

[0010] To achieve the above objectives, this application discloses an endogenous antioxidant composition composed of Centella asiatica extract and tocopherol glucoside, wherein the mass ratio of Centella asiatica extract to tocopherol glucoside is 1:0.005~0.1.

[0011] The above composition has demonstrated good antioxidant properties from multiple perspectives, including keratinocyte assay, simulated oxidative stress test, and ROS inhibition rate test. These antioxidant properties are endogenous antioxidant properties.

[0012] The endogenous antioxidant capacity described in this invention refers to the body's natural antioxidant mechanism, composed of antioxidant enzymes and substances, which neutralize free radicals, repair damaged cells, and protect tissues from oxidative damage. The body develops a complex oxidative stress response system in response to reactive oxygen species (ROS) damage. When exposed to ROS, the body can induce a series of protective proteins to alleviate cellular damage; the Nrf2-ARE pathway is the most important endogenous antioxidant stress pathway discovered to date.

[0013] To combat ROS and oxidative damage, the body has an antioxidant system that can be divided into three levels: 1. Inhibiting ROS production, mainly composed of endogenous antioxidant enzymes such as SOD and CAT; 2. Scavenging ROS, mainly endogenous and exogenous small molecule antioxidants; 3. Repairing ROS damage, including various cleavage / degradation and repair enzyme systems.

[0014] The synergistic mechanism by which Centella asiatica extract and tocopherol glucoside produce the aforementioned antioxidant properties lies in:

[0015] The asiaticus extract is rich in asiatic acid, asiaticoside, and asiaticoside, all of which have certain transdermal absorption capabilities and are non-toxic. During the inflammatory phase, asiatic acid reduces the release of pro-inflammatory mediators such as TNF-α, IL-1β, and IL-6 by inhibiting the activity of NF-κB and COX-2, thereby alleviating the inflammatory response. Ayticoside can reduce the expression of pro-inflammatory cytokines IL-1β and TLR2, and promote the secretion of AQP3, LOR, and IVL from HaCaT keratinocytes and the secretion of hyaluronic acid from human skin fibroblasts, demonstrating significant therapeutic potential for atopic dermatitis. Asiaticoside slows down photoaging by inhibiting the ROS and TGF-β1 / Smad signaling pathways. asiaticoside can significantly enhance superoxide dismutase activity and endothelial growth factor expression, optimize microcirculation, and improve skin vitality. Simultaneously, it inhibits tyrosinase mRNA translation and the binding of melanocyte-induced transcription factor (MITF) to DNA, reducing melanin production and the expression of melanin-synthesizing enzyme genes, effectively combating pigmentation. In terms of photoaging protection, asiaticoside can activate the Nrf2-HO-1 signaling axis, effectively repairing UV-induced DNA double-strand breaks by reducing DNA oxidative damage and regulating miRNA expression profiles. Regarding skin barrier function, it can significantly upregulate the expression of aquaporin 3, naupliin, naupliin, and hyaluronic acid synthase in HaCaT keratinocytes, promoting skin hydration.

[0016] Tocopherol glycosides are a class of compounds in which tocopherol (vitamin E) is linked to sugars (glucosides, mannose, etc.) via glycosidic bonds. These compounds differ from tocopherol in that they possess an additional hydrophilic sugar group. Therefore, they differ from tocopherol in both physicochemical properties and physiological activities. For example, tocopherol is an easily oxidized, pale yellow, viscous liquid that is insoluble in water but soluble in oil, while tocopherol glucosides are pale yellow or grayish-white solids with some water solubility and relatively stable properties. They possess anti-allergic and anti-inflammatory physiological activities not found in vitamin E. However, they also have some correlation; for instance, tocopherol glycosides can be considered prodrugs of tocopherol, and after entering the body, they are converted to tocopherol under the action of enzymes. The antioxidant activity of tocopherol is based on the fact that the benzene ring containing the phenolic hydroxyl group in the tocopherol structure can interact with various free radicals. However, due to the presence of a 16-carbon chain hydrocarbon group in its structure, its mobility in biological membranes is limited, and it cannot effectively quench free radicals generated in the aqueous phase. Tocopherol glucoside, on the other hand, has the advantages of being safe, having good antioxidant properties, and being highly water-soluble. Numerous animal studies have shown that tocopherol glucoside has good anti-radiation effects and can effectively prevent radiation-induced DNA damage. This anti-radiation ability is attributed to the ability of tocopherol glucoside to quench free radicals in tissues. Vitamin E (tocopherol) reduces oxidative damage by scavenging free radicals and decreasing the activation of inflammatory factors (such as the NF-κB pathway). It also enhances the endogenous antioxidant system (such as glutathione and SOD enzymes) and improves the cell's stress resistance. The nuclear factor-erythrocyte lineage-2-related factor 2 / antioxidant response element (Nrf2 / ARE) signaling pathway is a crucial pathway for regulating redox balance. It controls the expression of various downstream antioxidant enzymes, thereby scavenging ROS, neutralizing peroxidation products, and maintaining redox homeostasis. It plays a vital role in protecting the skin from UV-induced oxidative damage. Therefore, natural antioxidants targeting the Nrf2 / ARE signaling pathway are potential raw materials for combating skin photoaging. Polyphenols, vitamins, and carotenoids, among other active substances, have good antioxidant effects and are commonly used raw materials for combating skin photoaging. Tocopherol can enhance skin's antioxidant effect and inhibit oxidative damage by regulating the expression of related factors in the redox pathway. Since tocopherol glucoside is converted to tocopherol through enzymatic hydrolysis after entering the human body, it is speculated that tocopherol glucoside also has similar effects, but with a longer-lasting effect.

[0017] Children's skin barrier function is not fully developed, so they recover relatively slowly from sunburn. Sunburned skin is more prone to redness, dryness, itching, and other discomforts. Even if acute inflammation is relieved through first aid after sun damage occurs, the skin still needs deep repair to return to a healthy state. In addition to basic hydration and moisturizing, targeted anti-inflammatory, antioxidant, and free radical scavenging care is crucial.

[0018] Centella asiatica extract has antioxidant, anti-inflammatory, and skin-repair-promoting effects, helping children's skin resist external stimuli and relieve dryness and redness. It is especially suitable for children with sensitive skin or weak skin barriers. Centella asiatica is widely used in children's skin care products, and its safety has been verified by the market. Tocopherol glucoside can reduce skin inflammation by inhibiting the release of inflammatory mediators, while promoting skin cell repair and regeneration. It helps improve skin problems caused by oxidative stress, such as redness, sensitivity, and dullness. The two have a synergistic effect, addressing children's skin photodamage from an endogenous antioxidant perspective.

[0019] In the above-mentioned endogenous antioxidant composition, the mass ratio of Centella asiatica extract to tocopherol glucoside is 1:0.02~0.1.

[0020] Furthermore, the present invention also discloses the use of the composition described above in the preparation of cosmetics.

[0021] Finally, the present invention also discloses a cosmetic comprising the composition described above;

[0022] Preferably, the content of the composition in the cosmetic is 0.05~1wt%.

[0023] The present invention has the following advantages and effects compared with the prior art:

[0024] Compared with existing technologies, this application presents a composition for emergency treatment and sustainable stabilization of sun damage in children. Centella asiatica quickly combats inflammation and resolves skin redness, while tocopherol glucoside reduces skin inflammation. After entering the body, it is converted into tocopherol under the action of enzymes, providing slow-release and continuous deep repair, and restoring the skin barrier function. Attached Figure Description

[0025] Figure 1 Fluorescence micrograph of the negative control group in an in vitro keratinocyte reactive oxygen species (ROS) inhibition test;

[0026] Figure 2 Fluorescence micrograph of the positive control group in an in vitro keratinocyte reactive oxygen species (ROS) inhibition test;

[0027] Figure 3 A fluorescence micrograph of sample 2 from the in vitro keratinocyte reactive oxygen species (ROS) inhibition test. Detailed Implementation

[0028] The present invention will now be clearly and completely described in conjunction with embodiments thereof. It should be noted that, in this description, unless specific conditions are specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0029] The raw material information used in this invention is shown in Table 1;

[0030] Table 1. Explanation of Raw Material Sources

[0031] Serial Number Product Name Active ingredient INCI Active ingredient content supplier 1 Ji'anshu PRO Centella Asiatica flower / leaf / stem extracts 100% Draco Natural Products Inc. 2 JSH-TGS-4 Tocopherol glucoside 100% Jiangsu Jianersheng Biotechnology Co., Ltd. 3 Vitamin E Tocopherol (Vitamin E) 100% DSM Vitamins (Shanghai) Co., Ltd.

[0032] The raw materials used in subsequent experiments are all shown above.

[0033] The relevant test formulations for this invention are shown in Table 2;

[0034] Table 2 Composition Ratios

[0035] Centella Asiatica extract Tocopherol glucoside Vitamin E Sample 1 1 0.005 0 Sample 2 1 0.02 0 Sample 3 1 0.05 0 Sample 4 1 0.1 0 Sample 5 1 0 0 Sample 6 0 1 0 Sample 7 1 0 0.02 Sample 8 0 0 1

[0036] Efficacy test:

[0037] I. In vitro keratinocyte reactive oxygen species (ROS) inhibition assay:

[0038] 1. Experimental objective and principle:

[0039] Reactive oxygen species (ROS) damage genetic material and proteins, generate lipofuscin, and destroy collagen in organisms, ultimately leading to skin aging and sagging. UVB, through its high energy, directly damages the DNA, proteins, and lipids of HaCaT cells, or activates intracellular photosensitizing substances to trigger photochemical reactions, thereby producing ROS. Simultaneously, UVB significantly increases intracellular ROS levels by disrupting mitochondrial electron transport chain function and activating NADPH oxidase. HaCaT cells, as an epidermal keratinocyte model, are often used to simulate skin oxidative stress. During oxidative stress, ROS can activate the Nrf2 / KEAP1 / ARE pathway. ROS modifies the KEAP1 protein, altering its conformation and preventing the degradation of Nrf2. Nrf2 translocates into the nucleus, binds to antioxidant response elements (AREs), and initiates the expression of antioxidant genes such as SOD, CAT, and GPX, enhancing ROS scavenging capacity.

[0040] 2. Test Indicators:

[0041] The criteria for determining the ROS inhibition rate of cells are as follows: if the ROS inhibition rate of the test sample cells is positive and the fluorescence intensity value is significantly different from that of the negative control (P<0.05), it indicates that the test sample has antioxidant efficacy at this concentration.

[0042] Test methods

[0043] (1) Sample preparation

[0044] Sample group: Dilute the sample with pure water to a 4% solution (i.e., the effective ingredient content is 4wt%), then filter it through a 0.22 μm filter, and collect the filtrate as the sample mother solution.

[0045] (2) Experimental grouping

[0046] Negative control group (NC): Cells (+), UVB (+), fluorescent probe (+), sample (-);

[0047] Positive control group (PC): Cells (+), UVB (+), fluorescent probe (+), Vitamin E (+);

[0048] Sample group (S): Cells (+) UVB (+) Fluorescent probe (+) Sample (+)

[0049] Nude cell group: Cells (-) UVB (-) Fluorescent probe (+) Sample (-)

[0050] (3) Experimental operation steps

[0051] Cell viability assay: Cells were seeded into 96-well plates. After 24 h, the culture medium was discarded, and basal culture medium containing different concentrations of the test samples was added. After 24 h, the OD490nm was measured by MTT assay, and the effect of the test samples on cell viability was analyzed by t-test.

[0052] ROS inhibition rate determination: After the experimental groups were administered drugs, the well plates were placed in a CO2 incubator for 24 h. After incubation, except for the blank group (BC) and the bare cell wells, the remaining wells were induced with ROS using stimulants and then loaded with fluorescent probes. After CO2 incubation, the wells were photographed under an inverted fluorescence microscope with a FITC channel, an incident light wavelength of 525 nm, an excitation light wavelength of 488 nm, and a 20x objective lens, with the same exposure time.

[0053] (4) Calculation formula

[0054]

[0055] In the formula:

[0056] T—the average of three fluorescence intensities of the sample group;

[0057] C—The average of three fluorescence intensities in the negative control group;

[0058] C0—The average of three fluorescence intensities in the naked cell group.

[0059] The results of the ROS inhibition rate test are shown in Table 3 and Figures 1 to 3 As shown, where Figure 1 Fluorescence micrograph of the negative control group in an in vitro keratinocyte reactive oxygen species (ROS) inhibition test; Figure 2 Fluorescence micrograph of the positive control group in an in vitro keratinocyte reactive oxygen species (ROS) inhibition test; Figure 3 A fluorescence micrograph of sample 2 from the in vitro keratinocyte reactive oxygen species (ROS) inhibition test.

[0060] Table 3. Results of ROS inhibition rate test of dilution solutions of samples 1-8

[0061] sample ROS inhibition rate negative control group 0.00±0.85 Positive control group 69.10±0.87 Sample 1 43.15±0.77 Sample 2 63.30±0.76 Sample 3 64.10±0.68 Sample 4 64.45±0.86 Sample 5 38.50±0.74 Sample 6 22.26±0.80 Sample 7 53.20±0.82 Sample 8 50.35±0.67

[0062] Results analysis:

[0063] 1. From the perspective of material ratio, when the ratio of Centella asiatica extract and tocopherol glucoside is close to or exceeds 1:0.02, the performance does not change significantly. From the perspective of cost, the optimal ratio is 1:0.02.

[0064] 2. Combining the data from samples 5 and 6, it can be seen that when Centella asiatica extract is used alone, its ROS inhibition rate is moderate, while that of tocopherol glucoside is slightly worse. When it is combined with a very small amount of tocopherol glucoside, as seen in sample 1, the performance improved from 38.50 to 43.15. When the ratio was further adjusted to 1:0.02, the performance showed a significant leap, indicating that the two have a synergistic effect in terms of ROS inhibition rate.

[0065] 3. Combining the data from samples 6 and 8, it can be seen that although tocopherol glucoside and vitamin E are both tocopherol and their derivatives, vitamin E alone has a more significant effect; when used in combination with Centella asiatica extract, the latter also showed synergy, but the former had better synergy.

[0066] II. Moisturizing Efficacy Test

[0067] Volunteer Recruitment: Participants with healthy skin and no history of cosmetic allergies were selected, aged 20-35, including 22 males and 28 females. The participants were randomly divided into 5 groups of 10 each.

[0068] Test method: Room temperature 25℃±1℃; humidity 50%±5%. Before the test, volunteers cleaned the flexor surface of their forearms with a uniform mild cleanser for the same amount of time. After sitting quietly in a constant environment for 30 minutes, a 5cm×5cm area of ​​skin on the forearm was selected as the test area, and the sample was gently applied to the test site.

[0069] The test formula is referenced in Table 4; Table 4 shows the formula for the skin lotion.

[0070] Table 4 Skin Lotion Formula Table

[0071]

[0072] The preparation method for skin lotion is as follows:

[0073] 1. Stir and heat the A phase raw material to 80-85℃, and stir until homogenized and completely dispersed.

[0074] 2. Heat the B phase raw material to 80-85℃, then add it to the A phase, homogenize and cool it down.

[0075] 3. Pre-dissolve the C-phase raw material separately.

[0076] 4. Pre-dissolve the D-phase raw material separately.

[0077] 5. Cool down to 40-45℃, add C-phase and D-phase raw materials and stir evenly.

[0078] Test items:

[0079] 1. Skin moisture content test

[0080] Skin moisture content determination: The skin moisture content of each sample was measured before use and at 1 hour, 2 hours, 4 hours and 7 days after use (D7) using a skin moisture meter. The average value was taken from three measurements.

[0081] Parameter description: Moisture content of the stratum corneum of the skin; the higher the measured value, the higher the moisture content of the stratum corneum of the skin.

[0082] Results: Before and after using the product, the moisture content of the stratum corneum of the skin on the sample side was significantly improved after use compared with before use (P<0.05), indicating that the test product has moisturizing effect.

[0083] The products shown here are the skin care lotions listed in Table 4. Subsequent tables will use the sample number added in Table 4 to represent products containing that sample, and the same applies thereafter.

[0084] Skin moisture content determination: The skin moisture content of each sample was measured before use and at 1h, 2h, 4h and D7 after use using a skin moisture meter. The average value was taken for three measurements, and the results are shown in Table 5.

[0085] Table 5. Skin moisture test results of samples 2 and 5-8

[0086] sample Sample 2 Sample 5 Sample 6 Sample 7 Sample 8 Before use 29.88±1.85 30.08±2.05 29.52±1.87 30.32±1.90 29.98±2.22 After using for 1 hour 78.32±1.80 70.32±1.95 63.12±1.90 75.17±1.98 75.45±2.05 After using for 2 hours 70.24±2.03 56.01±1.97 60.24±2.01 65.45±1.96 65.20±1.92 After 4 hours of use 61.20±1.95 42.04±1.87 52.65±2.10 52.75±2.13 48.18±2.00 After using 7D 44.30±2.12 35.10±1.98 38.25±2.05 41.02±1.85 33.08±2.08

[0087] Based on the test data of samples 2 and 5-8, sample 5 showed the worst short-term effect in increasing skin hydration and also the worst long-term moisturizing effect. Although sample 6 had a better long-term moisturizing effect than sample 5, it was weak in increasing skin moisture content in the short term. This is likely because Centella asiatica extract contains some low-molecular-weight polysaccharides, which can provide immediate moisturizing. Compared with sample 2, samples 7 and 8 had weaker immediate and long-term moisturizing effects. Sample 2 was the best in both immediate and long-term moisturizing. The above experiments show that Centella asiatica extract can effectively improve immediate moisturizing effects, while tocopherol glucoside also has a significant effect on long-term moisturizing and water retention. The combination of the two can improve skin hydration from different dimensions and play a synergistic role.

[0088] 2. Transdermal water loss test

[0089] Transepidermal water loss rate: The transepidermal water loss rate was measured continuously for 30 seconds using a transepidermal water meter, and the average value of the last 20 seconds was recorded.

[0090] Parameter description: Reflects skin barrier function; the smaller the measured value, the better the skin barrier function.

[0091] Results: Before and after the trial of the product, the transepidermal water loss rate of the sample skin was significantly improved after the trial compared with before the trial (P<0.05), indicating that the test substance has the effect of improving the skin barrier.

[0092] The skin moisture loss of each sample was measured before use and at 1 hour, 2 hours, 4 hours, and 7 days (D7) after use using a transdermal water loss meter. The results are shown in Table 6.

[0093] Table 6 Results of transdermal water loss test of compositions of samples 2 and 5-8

[0094] sample Sample 2 Sample 5 Sample 6 Sample 7 Sample 8 Before use 11.92 10.85 13.01 10.92 10.55 After using for 1 hour 7.12 7.20 9.46 6.57 6.32 After using for 2 hours 7.92 8.45 10.43 7.32 8.38 After 4 hours of use 8.02 8.75 10.46 7.47 8.47 After using 7D 8.04 8.78 10.51 7.82 8.58

[0095] Based on the transdermal water loss test data, Sample 2 showed the best improvement in transdermal water loss rate in both short and long time periods, with more significant improvement in skin barrier repair. Furthermore, the change in transdermal water loss rate was not significant over time, which may be due to the sustained-release effect of tocopherol glucoside on the skin.

[0096] III. Skin Repair Effect Test

[0097] Forty individuals with severe skin redness due to work or living environment were randomly divided into 8 groups of 5 participants each. A 28-day test was conducted. During the test, a blank sample (shown as blank emulsion without the added composition) was applied to the left cheek, and the product (shown in Table 4) was applied to the right cheek. Skin redness was tested on days 0, 7, 14, and 28. The test results are shown in Table 7.

[0098] Table 7. Skin redness test results of samples 2 and 5-8

[0099]

[0100] Note: The lower the skin redness test value, the less red.

[0101] The test data from the skin repair test showed that Sample 2 had the best redness-reducing effect on both day 7 and day 28, while Sample 8 had the worst redness-reducing effect. Although Sample 5 also had a good redness-reducing effect on day 7, its effect on day 28 was not as good as it became over time. Sample 6 required a longer period of use to maintain its redness-reducing effect. In summary, Centella asiatica extract and tocopherol glucoside have good soothing and redness-reducing effects, possibly due to the immediate soothing and redness-reducing effect of Centella asiatica extract, while tocopherol glucoside reduces skin inflammation by inhibiting the release of inflammatory mediators and promotes skin cell repair and regeneration. The two work synergistically to improve skin problems caused by oxidative stress.

Claims

1. A cellular endogenous antioxidant composition, characterized in that, It is composed of Centella asiatica extract and tocopherol glucoside, wherein the mass ratio of Centella asiatica extract to tocopherol glucoside is 1:0.005~0.

1.

2. The endogenous antioxidant composition according to claim 1, characterized in that, The mass ratio of Centella asiatica extract to tocopherol glucoside is 1:0.02~0.

1.

3. Use of the composition as described in claim 1 or 2 to prepare cosmetics.

4. A cosmetic product, characterized in that, Contains the composition as described in claim 1 or 2.

5. The cosmetic product according to claim 4, characterized in that, The content of the composition in the cosmetic product is 0.05~1wt%.

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