Application of cacumen biotae extract in activating Nrf2 protein in cells

Abstract

The invention discloses application of a cacumen biotae extract in activating Nrf2 protein in cells, and particularly relates to the field of cosmetics. Wherein the activation of the Nrf2 protein in the cell is realized by interrupting the Keap1-Nrf2 protein-protein interaction in the cell, and performing targeted combination with the Keap1 protein and releasing the Nrf2 protein. By activating the Nrf2 pathway, the Chinese arborvitae twig and leaf extract can simultaneously achieve anti-oxidation, anti-inflammatory, anti-aging and whitening effects, and meets the requirements of cosmetics for multi-target effects.

Description

Technical Field

[0001] This application relates to the field of cosmetics, and in particular to the application of Platycladus orientalis leaf extract in activating Nrf2 protein in cells. Background Technology

[0002] Oriental arborvitae (Platycladus orientalis), also known as Chinese arborvitae, is an evergreen tree belonging to the genus Platycladus in the family Cupressaceae. Its leaves (oriental arborvitae leaves), seeds (arborvitae kernels), and branches are all used in traditional Chinese medicine, boasting a long history of medicinal use and listed as a superior herb in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica). Traditional Chinese medicine considers oriental arborvitae leaves to be cold in nature and bitter in taste, entering the lung, liver, and large intestine meridians, and is commonly used to treat hair loss, bleeding due to blood heat, and skin inflammation.

[0003] Modern research indicates that Platycladus orientalis leaves have wide applications in skincare, pharmaceuticals, and daily chemical products. Studies have shown that the active ingredients in Platycladus orientalis can enhance the expression of antioxidant enzymes (such as superoxide dismutase) in cells, reducing oxidative damage caused by external stimuli. Furthermore, the active ingredients in Platycladus orientalis exhibit potential anti-aging and whitening value through their inhibition of tyrosinase. Therefore, the current mechanism of action of the active ingredients in Platycladus orientalis is through downstream enzymatic reactions in the antioxidant regulatory pathway. This mechanism leads to unclear inhibitory targets for the extracts, making it difficult to design synergistic effects with other cosmetic ingredients with known and well-defined targets, thus limiting the efficacy of Platycladus orientalis extracts. Summary of the Invention

[0004] The main purpose of this application is to provide an application of Platycladus orientalis leaf extract in activating Nrf2 protein in cells, aiming to solve the problem that the inhibitory target of the extract is not clear in the prior art, which limits the effect of Platycladus orientalis extract.

[0005] To achieve the above objectives, this application provides the use of Platycladus orientalis leaf extract in activating cellular Nrf2 protein.

[0006] Optionally, the Nrf2 protein in the cell is activated by disrupting the Keap1-Nrf2 protein-protein interaction in the cell and targeting and binding the Keap1 protein to release the Nrf2 protein.

[0007] Optionally, when using Platycladus orientalis leaf extract to activate Nrf2 protein in cells, the concentration of Platycladus orientalis leaf extract in the culture medium is 1-4 mg / ml.

[0008] Optionally, the preparation method of the Platycladus orientalis leaf extract includes: pulverizing Platycladus orientalis leaves to obtain Platycladus orientalis leaf powder; adding NaOH solution to the Platycladus orientalis leaf powder for soaking and extraction to obtain an extract; concentrating and freeze-drying the extract to obtain the Platycladus orientalis leaf extract.

[0009] To achieve the above objectives, this application also provides the use of Platycladus orientalis leaf extract in the preparation of products that activate cell Nrf2 protein.

[0010] Optionally, the product is a topical skin agent.

[0011] Compared with the prior art, the beneficial effects of this application are as follows: The application of the Platycladus orientalis leaf extract in activating Nrf2 protein in cells, as described in this invention, involves interrupting the Keap1-Nrf2PPI pathway and releasing Nrf2 protein by reacting with Keap1 protein, thereby increasing Nrf2 protein levels and activating the Nrf2 pathway. This activates the expression of downstream antioxidant enzymes (such as HO-1), exerting antioxidant effects and maintaining cell state. By activating the Nrf2 pathway, the Platycladus orientalis leaf extract can simultaneously achieve antioxidant, anti-inflammatory, anti-aging, and whitening effects, meeting the needs of cosmetics for multi-target efficacy. Based on this mechanism, it can synergistically interact with other cosmetic ingredients with known and well-defined targets, expanding the effects of the Platycladus orientalis extract. Attached Figure Description

[0012] Figure 1 This is a graph showing the cytotoxicity of Platycladus orientalis extract on HaCaT cells in Example 1 of this application. Figure 2 This is a diagram showing the cell thermal migration results of Keap1 and Nrf2 proteins in HaCaT cells by the Platycladus orientalis extract in Example 1 of this application. Figure 3 This is an immunoprecipitation verification diagram showing the disruption of Keap1-Nrf2 PPI in HaCaT cells by the Platycladus orientalis extract in Example 1 of this application; Figure 4 This is a diagram illustrating the effect of the Platycladus orientalis extract in Example 1 of this application on activating the levels of Nrf2 protein and downstream antioxidant proteins in HaCaT cells.

[0013] The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0015] It is worth noting that under normal conditions, Keap1 (Kelch-like epichlorohydrin-associated protein 1) and Nrf2 (nuclear transcription-associated factor 2) proteins in cells undergo a protein-protein interaction (PPI), maintaining the Nrf2 protein level in the cytoplasm at a low level through ubiquitination and proteasome degradation. Therefore, this application uses Platycladus orientalis extract to interrupt the Keap1-Nrf2 PPI, upregulate Nrf2 protein levels, limit cellular oxidative stress levels, and maintain skin redox balance to combat abnormal skin conditions, as detailed below.

[0016] The first embodiment of the present invention provides the application of Platycladus orientalis leaf extract in activating Nrf2 protein in cells. Specifically, activation of Nrf2 protein in cells is achieved by the Platycladus orientalis leaf extract disrupting the Keap1-Nrf2 PPI in cells and targeting and binding to Keap1 protein, thereby releasing Nrf2 protein.

[0017] In this embodiment, the active ingredient flavonoids (biflavonoids) in Platycladus orientalis leaves interrupt the Keap1-Nrf2 PPI and, along with the Keap1 protein, release the Nrf2 protein, thereby increasing the Nrf2 protein level and activating the Nrf2 pathway. This activates the expression of downstream antioxidant enzymes (such as HO-1), exerting antioxidant effects and maintaining cell state. By activating the Nrf2 pathway, the active ingredients of Platycladus orientalis can simultaneously achieve antioxidant, anti-inflammatory, anti-aging, and whitening effects, meeting the needs of cosmetics for multi-target efficacy.

[0018] For example, the culture medium contains 1-4 mg / ml of Platycladus orientalis leaf extract.

[0019] Furthermore, the preparation method of Platycladus orientalis leaf extract includes: Step 1: Crush the arborvitae leaves to obtain arborvitae leaf powder; Step 2: Add NaOH solution (pH=12.4) to the arborvitae leaf powder for soaking and extraction. Repeat 3-5 times to obtain the extract. Store the extract at 3-5℃ for 22-26 hours to allow solid impurities in the extract to settle completely. Step 3: Filter the supernatant after sedimentation to remove finer particulate impurities and collect the filtered clear liquid. Step 4: The filtered extract is transferred to a rotary evaporator for concentration. Specifically, the water bath temperature is set to 60-70℃, the system pressure is maintained between 0.007-0.012 MPa, the rotation speed of the flask is controlled at 200-300 rpm, and the duration is 4-6 hours. The purpose is to evaporate most of the solvent (water) to obtain a viscous product. The product is then freeze-dried at a temperature of -20℃ to -50℃ and a vacuum degree of 18-24 Pa for 70-74 hours. After freeze-drying, the target product is obtained—a reddish-brown, granular freeze-dried active ingredient solid, namely, the Platycladus orientalis leaf extract. For ease of cell experiments, this embodiment uses dimethyl sulfoxide (DMSO) for initial dissolution, followed by the addition of Tween 80 as a solubilizer. Finally, phosphate-buffered saline (PBS) is added to bring the total solution volume to 1 ml (1000 μL) to ensure a final concentration of 100 mg / ml for the Platycladus orientalis leaf extract.

[0020] A second embodiment of the present invention provides the application of Platycladus orientalis leaf extract in the preparation of a product that activates the Nrf2 protein in cells. The product is a topical skin agent, for example, a cosmetic ingredient with anti-aging and antioxidant effects.

[0021] Example 1: Extraction of Platycladus orientalis leaf extract The leaves of Platycladus orientalis were pulverized into powder using a pulverizer. 30 g of this powder was weighed and added to 150 ml of 0.025 mol / L NaOH solution (pH=12.4) to obtain a mixture. The mixture was then soaked and extracted at 4℃ for three consecutive times, each extraction lasting 24 h. After three extractions, all extracts were combined and stored at 4℃ for another 24 h to obtain the extract. The supernatant of the extract was aspirated and filtered through a 0.45 μM pore size filter membrane. The clarified filtrate was then transferred to a rotary evaporator for concentration. The water bath temperature was set to 60℃, the system pressure to 0.007 rpm, the rotation speed to 200 rpm, and the concentration time to 4 h, resulting in a viscous Platycladus orientalis extract. The viscous Platycladus orientalis extract adhering to the inner wall of the round-bottom flask of the rotary evaporator was scraped off with a spatula and lyophilized at -30℃ and a vacuum of 18 Pa for 72 h to obtain the active components of Platycladus orientalis.

[0022] Weigh 100 mg of the active ingredient of Platycladus orientalis, first dissolve it in 50 μL of dimethyl sulfoxide, then add 100 μL of Tween 80 as a solubilizer, and finally add phosphate buffer to make up the total volume of the solution to 1 ml, so as to obtain Platycladus orientalis extract with an active ingredient concentration of 100 mg / ml, which is used for subsequent cell experiments.

[0023] Example 2: Toxicity verification of Platycladus orientalis extract Step 1: Take well-grown HaCaT cells and seed them into 96-well clear flat-bottom plates, 5 × 10⁶ cells per well. 3 Cells were cultured in a complete culture medium consisting of high-glucose DMEM, 10% fetal bovine serum, and 1% penicillin and antibiotic-resistant cells, under the following conditions: 5% carbon dioxide and 37°C. Step 2: After 4 hours of adhesion, the culture medium was replaced with a medium containing different concentrations (10 mg / ml, 3 mg / ml, 1 mg / ml, 0.3 mg / ml, 0.1 mg / ml, 0.03 mg / ml, 0.01 mg / ml, 0.003 mg / ml, 0.001 mg / ml, 0 mg / ml) of Platycladus orientalis extract. Step 3: Incubate at 5% carbon dioxide and 37°C for 72 hours; Step 4: Replace the culture medium with 100 μL MTT (3-(4,5-dimethyl-2-thiazole)-2,5-diphenyltetrazolium bromide thiazole blue) reagent (1 mg / mL). After incubation for 4 h, replace with 100 μL LDMSO and measure using a 570 nm microplate reader.

[0024] Experimental results are as follows Figure 1 As shown in the experimental results, the 72hIC value of the active ingredients in Platycladus orientalis is [data missing]. 50 The concentration was 9.101 mg / mL, indicating that the cytotoxicity of the active ingredient in Platycladus orientalis is very low, proving its safety as a cosmetic raw material.

[0025] Example 3 uses a cell thermal migration assay to investigate the ability of Platycladus orientalis leaf extract to target Keap1 and Nrf2 in the cellular environment.

[0026] Step 1: Obtain well-grown human keratinocytes (HaCaT) at a volume of 5 × 10⁶ cells. 6 Add cell lysis buffer containing protease inhibitors and phosphatase inhibitors, and place on ice for lysis; Step 2: Incubate HaCaT cell lysate with 2 mg / ml of Platycladus orientalis extract and dimethyl sulfoxide at room temperature for 60 min. Step 3: The incubated lysate was aliquoted into 1.5 mL centrifuge tubes and heated individually at specified temperatures (25℃, 55℃, 60℃, 65℃, 70℃, 75℃). After centrifugation, the supernatant of the heated lysate was collected and then analyzed by Western blot (WB) using Keap1, Nrf2, and β-actin antibodies.

[0027] Experimental results are as follows Figure 2As shown in the figure, the experimental results indicate that the extract of Platycladus orientalis significantly stabilized the Keap1 protein, while having no significant effect on the thermal stability of the Nrf2 protein. This result demonstrates that the extract of Platycladus orientalis leaves can bind to the Keap1 protein even in the complex environment of cell lysates.

[0028] Example 4 Step 1: Obtain well-grown HaCaT cells, with a cell volume of 2 × 10⁶ cells. 6 Add arborvitae extract containing different concentrations (1 mg / ml, 2 mg / ml) and treat for 4 hours; Step 2: Rinse adherent cells 2-3 times with PBS buffer; then add an appropriate volume of IP lysis buffer to the cells in the culture plate / flask for 3-5 min. Use a cell scraper to scrape off the cells and reagents, collecting them into a 1.5 mL centrifuge tube. Incubate on ice for 30 min to ensure complete cell lysis. Centrifuge at 4℃ and 12000 rpm for 15 min, collect the supernatant, and use 1 / 10 of the supernatant as input; the remaining sample is used as immunoprecipitation cell lysis buffer. Step 3: Use the BCA protein concentration assay kit to determine the protein concentration in the immunoprecipitated cell lysate obtained in Step 2 to standardize the loading amount. Step 4: Take the supernatant of the immunoprecipitated cell lysate obtained in Step 2 into a 1.5 ml centrifuge tube, add 2.5 μL (1.5 μg) of Nrf2 antibody (add the same amount of ordinary IgG of the same species as the antibody used for precipitation as a negative control, negative control experiment), and then incubate at 4 °C for 2 h; Step 5: Add 20 μl of magnetic beads (mix thoroughly before use) to the centrifuge tube after incubation in step 4, gently tap it with your finger to mix, and incubate overnight at 4°C with shaking. Step 6: Place the centrifuge tube on a magnetic rack and let it stand for 5-10 seconds, then aspirate the supernatant (avoid contact with the magnetic beads as much as possible). Add 1 mL of washing buffer to the centrifuge tube, place it on a vertical shaker and wash for 3 minutes. Then, place the centrifuge tube on a magnetic rack and let it stand for 5-10 seconds, then aspirate the supernatant (avoid contact with the magnetic beads as much as possible). Repeat this process 3 times. Step 7: After the final wash, discard the supernatant, add an appropriate volume of 2×SDS (sodium dodecyl sulfate) loading buffer containing mercaptoethanol, boil in water for 10 min, and store the sample at -20℃ to obtain the IP sample.

[0029] Step 8: Perform Western blot analysis on the Input and IP samples using Keap1, Nrf2, and β-actin antibodies.

[0030] Experimental results are as follows Figure 3As shown in the figure, the experimental results indicate that the extract of Platycladus orientalis leaves can effectively disrupt the Keap1-Nrf2 protein-protein interaction in living cells at a concentration of 2 mg / ml.

[0031] Example 5 Step 1: Take well-grown HaCaT cells and use 2×10⁻⁶ cells. 6 Cells were seeded at a density in six-well plates; 0 mg / ml, 0.5 mg / ml, 1 mg / ml and 2 mg / ml of Platycladus orientalis leaf extract were added to the six-well plates and incubated for 12 hours before harvesting HaCaT cell lysates; Step 2: Prepare WB samples, separate HaCaT cell lysates using 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), and transfer them to a PVDF (polyvinylidene fluoride) membrane. Step 3: Incubate the PVDF membrane overnight with Keap1, Nrf2, HO-1, and β-actin antibodies. Wash the membrane three times with washing buffer. Add secondary antibody (1:1000) to the PVDF membrane, incubate for 2 hours, and then detect protein bands using an enhanced chemiluminescence reagent. Analyze the results using ImageLab.

[0032] Experimental results are as follows Figure 4 As shown in the results, the experimental results indicate that, at a concentration of 2 mg / ml, treatment of HaCaT cells with Platycladus orientalis leaf extract for 12 hours can activate intracellular Nrf2 protein and stimulate the expression of the downstream antioxidant protein HO-1 (heme oxygenase-1). This further demonstrates that Platycladus orientalis leaf extract can increase intracellular Nrf2 levels by interrupting Keap1-Nrf2 PPI, thereby activating the Nrf2 pathway and exerting antioxidant effects to maintain cell condition.

[0033] The above are merely preferred embodiments of this application and do not limit the patent scope of this application. Any equivalent structural or procedural transformations made using the content of this application's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of this application.

Claims

1. Application of Platycladus orientalis leaf extract in activating cellular Nrf2 protein.

2. The application of the Platycladus orientalis leaf extract according to claim 1 in activating Nrf2 protein in cells, characterized in that, The activation of the Nrf2 protein in the cell is achieved by disrupting the Keap1-Nrf2 protein-protein interaction in the cell and targeting and binding the Keap1 protein to release the Nrf2 protein.

3. The application of the Platycladus orientalis leaf extract according to claim 1 in activating Nrf2 protein in cells, characterized in that, When using Platycladus orientalis leaf extract to activate Nrf2 protein in cells, the concentration of Platycladus orientalis leaf extract in the culture medium is 1-4 mg / ml.

4. The application of the Platycladus orientalis leaf extract according to claim 1 in activating Nrf2 protein in cells, characterized in that, The preparation method of the Platycladus orientalis leaf extract includes: The leaves of the arborvitae were crushed to obtain arborvitae leaf powder; NaOH solution was added to the powdered arborvitae leaves for soaking and extraction to obtain an extract; The extract was concentrated and freeze-dried to obtain Platycladus orientalis leaf extract.

5. Application of Platycladus orientalis leaf extract in the preparation of products that activate cell Nrf2 protein.

6. The use of the Platycladus orientalis leaf extract according to claim 5 in the preparation of a product that activates cell Nrf2 protein, characterized in that, The product is a topical skin treatment.

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