Composition for inhibiting TRPV1 activation and preparation method and application thereof
By combining the root extract of Glycyrrhiza inflata and Glycyrrhiza chalcone B through a specific preparation process, the inhibitory effect of TRPV1 is enhanced, which solves the problem of poor efficacy of existing TRPV1 antagonists and achieves highly efficient inhibition of TRPV1 and improvement of skin condition.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUANGZHOU HUANYA COSMETIC SCI & TECH CO LTD
- Filing Date
- 2026-01-15
- Publication Date
- 2026-05-12
AI Technical Summary
Existing TRPV1 antagonists, such as AMG9810, have failed to achieve the expected inhibitory effect. There is a lack of more effective TRPV1 inhibitory ingredients in sensitive skin care products, making it difficult to effectively relieve skin discomfort symptoms.
A combination of Glycyrrhiza inflata root extract and glycyrrhizin B was prepared by enzymatic hydrolysis and alcohol extraction to increase the content of total flavonoids and glycyrrhizin B, and to synergistically inhibit TRPV1 activation, including inhibiting TRPV1 receptor expression and calcium ion influx.
It significantly inhibits TRPV1 activation, reduces skin nerve sensitivity, and relieves discomfort symptoms such as burning, stinging, and itching in sensitive skin, broadening the range of skincare product choices and providing safer and more suitable solutions for sensitive skin.
Smart Images
Figure CN122005377A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of cosmetic technology, and specifically relates to a composition for inhibiting TRPV1 activation, its preparation method, and its application. Background Technology
[0002] Sensitive skin is generally considered to be a condition characterized by intolerance to changes in the external environment and / or the use of skincare products. People with sensitive skin often cannot tolerate regular skincare products, so when choosing skincare products, they should prioritize those with gentler and safer ingredients to avoid skin discomfort.
[0003] The causes of sensitive skin are complex, resulting from the combined effects of multiple factors, and its pathogenesis is not yet fully understood. However, current research generally suggests that its formation is closely related to factors such as abnormalities in the skin's sensory nervous system, impaired barrier function, inflammatory responses, external stimuli, psychological stress, and skin microecological dysbiosis, with these factors exhibiting mutually reinforcing and influencing relationships. Among these factors, sensitive skin is particularly closely associated with sensory nerve hyperresponsiveness, and related studies indicate that sensitive skin is often accompanied by excessive activation of transient potential capsaicin channel-1 (TRPV1) (Reference: Kueper T, et al. Exp Dermatol. 2010).
[0004] TRPV1 is a calcium-permeable, non-selective cation channel receptor and one of the main sensors for pain and heat, while also participating in the transmission of pruritus. TRPV1 can be activated by various stimuli such as pH, capsaicin, ultraviolet radiation, and heat damage (>42°C), inducing pain sensations through keratinocyte transmission. After activation, TRPV1 can induce the release of neuropeptides (such as substance P) in the local skin, increasing calcium ion influx and triggering neurogenic inflammation. Furthermore, its activation can cause mast cells to secrete endothelin-1, which further activates immune cells, leading to the release of inflammatory mediators (such as IL-1, IL-6, and TNF-α) and upregulation of VEGF, causing vasodilation and exacerbating skin inflammation. Therefore, the TRPV1 channel can serve as a therapeutic target for relieving inflammatory symptoms and pain. While the traditional TRPV1 antagonist AMG9810 (CAS: 545395-94-6) possesses some TRPV1 inhibitory activity, its efficacy has not yet met expectations. Therefore, developing more effective TRPV1 inhibitory products has become a core requirement and important direction for the innovation and upgrading of skincare products for sensitive skin. Summary of the Invention
[0005] The present invention aims to at least solve one of the technical problems existing in the prior art. Therefore, the object of the present invention is to provide a composition for inhibiting TRPV1 activation, its preparation method, and its application; the composition of the present invention can effectively inhibit TRPV1 activation, including effectively reducing TRPV1 expression and inhibiting intracellular calcium ion signaling, with comprehensive effects, low effective concentration, and its inhibitory effect is superior to that of traditional TRPV1 antagonists (AMG9810).
[0006] In a first aspect, the present invention provides the use of a composition in the preparation of a product that inhibits TRPV1 activation, the composition comprising a root extract of Glycyrrhiza inflata and glycyrrhizin B, wherein the root extract of Glycyrrhiza inflata is prepared by enzymatic hydrolysis and alcohol extraction of Glycyrrhiza inflata root.
[0007] In some embodiments of the present invention, the effective concentration of the extract of Glycyrrhiza uralensis root is ≥0.001wt%.
[0008] In some embodiments of the present invention, the effective concentration of the licorice root extract is 0.001wt%-2wt%. This effective concentration can be any point value or any two-point range value between 0.001wt%-2wt%, for example, it can be 0.001wt%, 0.01wt%, 0.05wt%, 0.1wt%, 0.2wt%, 1wt%, 2wt%.
[0009] In some embodiments of the present invention, the effective concentration of glycyrrhizin B is ≥5 μM.
[0010] In some embodiments of the present invention, the effective concentration of glycyrrhizin B is 5 μM-40 μM. This effective concentration can be any point value or any two points within the range of 5 μM-40 μM, such as 5 μM, 10 μM, 20 μM, or 40 μM.
[0011] In some embodiments of the present invention, the enzymatic hydrolysis uses a complex enzyme consisting of a mixture of cellulase and hemicellulase in a mass ratio of (1-2):1.
[0012] In some embodiments of the present invention, the pH of the enzymatic hydrolysis is 5-6.5.
[0013] In some embodiments of the present invention, the enzymatic hydrolysis temperature is 45-55°C.
[0014] In some embodiments of the present invention, the enzymatic hydrolysis time is 1-2 hours.
[0015] In some embodiments of the present invention, the alcohol extraction uses an ethanol solution with a volume concentration of 70%-90%.
[0016] In some embodiments of the present invention, the temperature of the alcohol extraction is 45-55°C.
[0017] In some embodiments of the present invention, the alcohol extraction time is 30-50 min.
[0018] In some embodiments of the present invention, the alcohol extraction process is assisted by ultrasonic extraction, and the frequency of the ultrasonic extraction is 5-6 kHz.
[0019] In some embodiments of the present invention, the preparation method of the licorice root extract specifically includes the following steps: 1) Mix the root of Glycyrrhiza uralensis inflata, cellulase, hemicellulase, water and pH adjuster, enzymatically hydrolyze, filter, and obtain the residue of Glycyrrhiza uralensis inflata. 2) Mix the residue of licorice root with ethanol solution and then extract with ethanol. The extraction process is assisted by ultrasonic extraction. After filtration, licorice root extract is obtained. 3) The extract of Glycyrrhiza inflata root was concentrated under reduced pressure to remove ethanol, thus obtaining Glycyrrhiza inflata root extract.
[0020] In some embodiments of the present invention, the inhibition of TRPV1 activation includes inhibiting the expression level of TRPV1 and / or inhibiting the influx of calcium ions after TRPV1 activation.
[0021] In some embodiments of the present invention, the TRPV1 activation is TRPV1 activation induced by an irritant, including capsaicin and / or phenolic compounds.
[0022] In some embodiments of the present invention, the phenolic compound includes phenoxyethanol or resorcinol.
[0023] In some embodiments of the present invention, the extract of Glycyrrhiza uralensis root contains total flavonoids and glycyrrhizin B, wherein the content of total flavonoids is greater than 200 mg / g and the content of glycyrrhizin B is greater than 2 mg / g.
[0024] In some embodiments of the present invention, the total flavonoids in the root extract of Glycyrrhiza inflata are 210-230 mg / g.
[0025] In some embodiments of the present invention, the content of glycyrrhizin B in the root extract of Glycyrrhiza inflata is 2.5-3 mg / g.
[0026] In a second aspect, the present invention provides a product for inhibiting TRPV1 activation, the product comprising licorice root extract and glycyrrhizin B, wherein the licorice root extract is prepared by enzymatic hydrolysis and alcohol extraction of licorice root.
[0027] In some embodiments of the present invention, the product includes cosmetics or topical skin preparations.
[0028] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The composition provided by the present invention, through the combination of licorice root extract and glycyrrhizin B, has synergistic effect, low effective concentration, and can effectively inhibit TRPV1 activation. Specifically, it can inhibit the expression of TRPV1 receptor and inhibit the influx of calcium ions after TRPV1 activation. In particular, it has a significant inhibitory effect on TRPV1 activation induced by irritating components such as capsaicin and phenolic compounds, which is superior to traditional TRPV1 antagonists (AMG9810).
[0029] (2) The licorice root extract in the composition of the present invention is prepared by a specific extraction process, which can effectively increase the content of total flavonoids and glycyrrhizin B, thereby further enhancing its inhibitory effect on TRPV1.
[0030] (3) The application of the composition of the present invention is expected to reduce the sensitivity of skin nerves at the target level and specifically relieve the common discomfort symptoms of sensitive skin such as burning, stinging, itching and redness. In particular, for sensitive skin people who are intolerant to some ingredients, it will effectively broaden the range of skin care products they can choose and provide a safer and more adaptable solution for sensitive skin care.
[0031] (4) Applying the composition of the present invention to cosmetic products can effectively improve the skin condition of people with sensitive skin. Attached Figure Description
[0032] Figure 1 The results show that phenoxyethanol induces TRPV1 expression. Figure 2 The results show the effects of different inducers on intracellular calcium flux. Detailed Implementation
[0033] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.
[0034] Unless otherwise specified, the raw materials, reagents, and apparatus used in the following examples can be obtained from conventional commercial sources or by existing known methods.
[0035] Example 1 This embodiment provides an extract of Glycyrrhiza uralensis root, the preparation method of which includes the following steps: 1) Mix 500g of licorice root with swelling fruit, 50g of cellulase, 50g of hemicellulase and 4000g of water, adjust the pH to 5.8 with citric acid, stir at 100rpm for 1.5h at 50℃, filter to obtain licorice root residue with swelling fruit. 2) Mix the licorice root residue obtained in step 1) with 2500g of 80% ethanol solution (V / V), extract at 50℃ and ultrasonic frequency of 5kHz for 40min, filter, and obtain licorice root extract. 3) The extract of Glycyrrhiza inflata root obtained in step 2) was concentrated under reduced pressure at 50°C to remove ethanol, thus obtaining Glycyrrhiza inflata root extract.
[0036] Example 2 This embodiment provides a composition consisting of the root extract of Glycyrrhiza uralensis from Example 1 and glycyrrhizin B (CAS: 58749-23-8).
[0037] Comparative Example 1 This embodiment provides an extract of Glycyrrhiza uralensis root, the preparation method of which includes the following steps: 500g of Glycyrrhiza inflata root and 5000g of 80% ethanol solution (V / V) were mixed and extracted by reflux at 80℃ for 2h. After filtration, the filtrate was concentrated under reduced pressure at 50℃ to remove ethanol, thus obtaining Glycyrrhiza inflata root extract.
[0038] Comparative Example 2 This embodiment provides a natural flavonoid compound, glycyrrhizin B (CAS: 58749-23-8).
[0039] Comparative Example 3 This embodiment provides a natural flavonoid compound, glycyrrhizin A (CAS: 58749-22-7).
[0040] Comparative Example 4 This embodiment provides a natural flavonoid compound, glycyrrhizin C (CAS: 144506-14-9).
[0041] Comparative Example 5 This comparative example provides a natural flavonoid compound, 4-hydroxychalcone (CAS: 2657-25-2).
[0042] Comparative Example 6 This comparative example provides a composition consisting of the root extract of Glycyrrhiza uralensis from Example 1 and glycyrrhizin A (CAS: 58749-22-7).
[0043] Comparative Example 7 This comparative example provides a composition consisting of the root extract of Glycyrrhiza uralensis from Example 1 and glycyrrhizin C (CAS: 144506-14-9).
[0044] Comparative Example 8 This comparative example provides a composition consisting of the root extract of Glycyrrhiza uralensis from Example 1 and 4-hydroxychalcone (CAS: 2657-25-2).
[0045] Comparative Example 9 This comparative example provides a TRPV1 antagonist, AMG9810 (CAS: 545395-94-6).
[0046] Experimental Example 1: Determination of Total Flavonoids and Glycyrrhizin Chalcone Monomer Content 1.1 Experimental Principle (1) Determination of total flavonoid content: Flavonoids are important secondary metabolites in plants and have strong antioxidant activity. The phenolic hydroxyl groups in flavonoid molecules can react with aluminum salts (such as aluminum nitrate) to form yellow complexes, which have a maximum absorption peak at a wavelength of 510-520 nm. The total flavonoid content can be quantified by absorbance (using rutin as a standard). Therefore, the total flavonoid content in the root extract of Glycyrrhiza uralensis can be determined by this spectrophotometric method.
[0047] (2) Determination of glycyrrhizin B monomer content: The different flavonoids are separated by the retention difference on the C18 column. The separated components flow out of the column in sequence and enter the detector. The detection signal is converted into a chromatogram. The content of the target substance in the sample can be calculated by measuring the peak area or peak height of the chromatographic peak of the target substance and comparing it with a standard of known concentration.
[0048] 1.2 Experimental Methods This test example tested the total flavonoid content and glycyrrhizin B monomer content in the root extract of Glycyrrhiza inflata from Example 1 and the root extract of Glycyrrhiza inflata from Comparative Example 1 (hereinafter referred to as "samples").
[0049] (1) Determination of total flavonoid content Adjust the total flavonoid concentration of the sample solution to the determination range. Accurately pipette 1.0 g into a dry, clean test tube, add 0.2 mL of 5% sodium nitrite solution, shake well, and let stand for 5 min. Add 0.2 mL of 10% aluminum nitrate solution, shake well, and let stand for 6 min. Add 1.0 mL of 5% sodium hydroxide solution, and add solvent to the mark. Shake well. Quickly measure the absorbance of each tube at a wavelength of 510 nm. Calculate the total flavonoid concentration of the sample solution based on the absorbance value according to the standard curve of rutin solution. The total flavonoid content in the sample is expressed as rutin.
[0050] (2) Determination of glycyrrhizin B monomer content Sample preparation: Take 1.0 g of sample and place it in a 10 mL volumetric flask. Add chromatographic grade methanol to the mark, shake well, and filter through a 0.45 μm filter membrane to obtain the test sample.
[0051] Chromatographic conditions: An Agilent Poroshell 120 EC-C18 column (150 mm × 4.6 mm, 2.7 µm) was used; gradient elution was performed using 0.05% phosphoric acid (A)-methanol (B) as the mobile phase (0 min, 10% B; 3 min, 25% B; 12 min, 30% B; 20 min, 40% B; 40 min, 90% B; 50 min, 95% B); flow rate was 1.0 mL / min; detection wavelength was 270 nm; column temperature was 30 °C.
[0052] 1.3 Experimental Results The experimental results are shown in Table 1.
[0053] Table 1. Effects of different preparation methods on the flavonoid content in Glycyrrhiza uralensis root extract.
[0054] As shown in Table 1, Example 1, through enzymatic hydrolysis and ultrasonic alcohol extraction, increased the content of natural flavonoids in the root of *Glycyrrhiza inflata*, with significantly higher contents of total flavonoids and glycyrrhizin B compared to Comparative Example 1. This result indicates that this process can yield an extract of *Glycyrrhiza inflata* root rich in glycyrrhizin B.
[0055] Experiment Example 2: Inhibition of TRPV1 receptor gene expression in keratinocytes 2.1 Preparation of sample solution Using DMEM complete culture medium as a solvent, sample solutions with mass concentrations of 0.05% and 0.01% were prepared according to Example 1 and tested.
[0056] Using DMEM complete medium as a solvent, two groups of sample solutions with different concentrations were prepared in Example 2, including 0.05% glycyrrhiza uralensis root extract and 10 μM glycyrrhiza chalcone B; and 0.01% glycyrrhiza uralensis root extract and 5 μM glycyrrhiza chalcone B; and the results were tested.
[0057] Using DMEM complete culture medium as a solvent, Comparative Example 1 was prepared into sample solutions with mass concentrations of 0.05% and 0.01% for testing.
[0058] Using DMEM complete medium as solvent, Comparative Example 2 was prepared into sample solutions with molar concentrations of 10 μM and 5 μM, respectively, for testing.
[0059] Using DMEM complete medium as solvent, Comparative Example 3 was prepared into sample solutions with molar concentrations of 10 μM and 5 μM, respectively, for testing.
[0060] Using DMEM complete medium as solvent, Comparative Example 4 was prepared into sample solutions with molar concentrations of 10 μM and 5 μM, respectively, for testing.
[0061] Using DMEM complete medium as solvent, Comparative Example 5 was prepared into sample solutions with molar concentrations of 10 μM and 5 μM, respectively, for testing.
[0062] Using DMEM complete medium as solvent, Comparative Example 6 was prepared into two groups of sample solutions with different concentrations, including 0.05% glycyrrhiza uralensis root extract and 10 μM glycyrrhiza chalcone A; and 0.01% glycyrrhiza uralensis root extract and 5 μM glycyrrhiza chalcone A; and the results were tested.
[0063] Using DMEM complete medium as solvent, Comparative Example 7 was prepared into two groups of sample solutions with different concentrations, including 0.05% glycyrrhiza uralensis root extract and 10 μM glycyrrhiza chalcone C; and 0.01% glycyrrhiza uralensis root extract and 5 μM glycyrrhiza chalcone C; and the results were tested.
[0064] Using DMEM complete medium as solvent, Comparative Example 8 was prepared into two groups of sample solutions with different concentrations, including 0.05% glycyrrhiza uralensis root extract and 10 μM 4-hydroxychalcone; and 0.01% glycyrrhiza uralensis root extract and 5 μM 4-hydroxychalcone; and the results were tested.
[0065] 2.2 Experimental Principle TRPV1 is an ion channel receptor expressed on the cell membrane and one of the main sensors for pain and heat in the skin, also associated with the transmission of pruritus. TRPV1 can be activated by various stimuli, including pH, capsaicin, ultraviolet radiation, heat damage (>42℃), and certain irritating chemicals. Skin keratinocytes express TRPV1, and activation leads to increased calcium ion influx, further transmitting pain and inducing exacerbated skin-related inflammatory responses. Therefore, immortalized human keratinocytes (HaCaT) can be used to detect the effects of different components on TRPV1 receptor expression. Upregulation of TRPV1 expression reflects, to some extent, increased cellular sensitivity to potential stimuli.
[0066] 2.3 Experimental Methods (1) Cell selection and culture To further improve the sensitivity of in vitro detection of TRPV1 receptor expression levels, this experiment used a TRPV1-overexpressing HaCaT cell line. This cell line had a fragment containing the human TRPV1 mRNA sequence inserted into the lentiviral vector pCDH-CMV-MCS-EF1-Puro. After transfecting HaCaT cells, resistance selection was performed, and tolerant cells were selected for expansion culture to construct a stable TRPV1-overexpressing cell line. Untransfected cells served as blank control cells. See reference: Zhou LD, et al. J Cosmet Dermatol. 2023 Apr;22(4):1369-1376. The cell line was cultured in DMEM complete medium (containing 10% FBS + antibiotics) in a carbon dioxide incubator at 37°C and 5% CO2, and passaged every 2-3 days.
[0067] (2) Cell treatment Different concentrations of the examples and comparative examples were applied to HaCaT cell lines overexpressing TRPV1 for 48 h to detect the effect of different components on TRPV1 expression. At the same time, a model was established using 0.03% phenoxyethanol (a phenolic preservative) to induce increased TRPV1 expression. Different concentrations of the examples and comparative examples were then added, and after 48 h of treatment, the inhibitory effect of different components on TRPV1 activation induced by phenoxyethanol was detected.
[0068] (3) RNA extraction Cell lysis was performed by adding cell lysis solution to each well of a 12-well plate. Chloroform (1:5 volume ratio) was added to the lysis buffer, and the mixture was shaken thoroughly. After standing at room temperature for 5 minutes, the plate was centrifuged at 12000 rpm for 15 minutes at 4°C. The colorless supernatant was transferred to a new 1.5 mL centrifuge tube, and an equal volume of isopropanol was added. The mixture was incubated at room temperature for 10 minutes, and then centrifuged at 12000 rpm for 10 minutes at 4°C. The supernatant was discarded. 500 µL of 75% ethanol was added, and the plate was centrifuged at 7500 rpm for 5 minutes at 4°C. The supernatant was discarded, and the plate was allowed to air dry at room temperature for 1-2 minutes. Finally, an appropriate amount of DEPC water was added to dissolve the RNA precipitate.
[0069] (4) cDNA synthesis and RT-PCR The reverse transcription kit and quantitative real-time PCR kit used in this experiment were purchased from Nanjing Novizan Biotechnology Co., Ltd. The procedures were followed according to the kit instructions, and the expression of relevant genes was calculated using a relative quantitative method.
[0070] 2.4 Experimental Results The results are as follows Figure 1 As shown in Tables 2 and 3.
[0071] Table 2. Inhibitory effects of different components on TRPV1 expression
[0072] As shown in Table 2, Example 1 effectively inhibited TRPV1 expression, with a significantly stronger inhibitory effect compared to Comparative Example 1 (conventional ethanol extraction). This indicates that the present invention, through specific enzymatic hydrolysis and ethanol extraction processes to increase the content of glycyrrhizin B, exhibits a stronger inhibitory effect on TRPV1. Meanwhile, Comparative Examples 2 (glycyrrhizin B), 3 (glycyrrhizin A), and 4 (glycyrrhizin C) showed certain inhibitory effects on TRPV1 expression at concentrations of 5–10 μM. Combining Example 1 with glycyrrhizin B (Example 2) significantly enhanced the inhibitory effect on TRPV1, demonstrating a synergistic inhibitory effect compared to the individual components. However, replacing the monomeric component glycyrrhizin B in Example 2 with glycyrrhizin A, glycyrrhizin C, and 4-hydroxychalcone significantly weakened the effect, indicating that Example 2 possesses unique components, and not all combinations of chalcone monomers can exhibit a synergistic inhibitory effect on TRPV1.
[0073] Table 3. Inhibitory effects of different components on phenoxyethanol-induced TRPV1 expression
[0074] Phenoxyethanol is a preservative containing a phenolic structure. Figure 1 The results of phenoxyethanol-induced TRPV1 expression are presented (data are expressed as mean ± standard error; the blank group consisted of normal HaCaT cells, the control group consisted of TRPV1-overexpressing HaCaT cells, and the phenoxyethanol-treated group consisted of HaCaT cells overexpressing TRPV1 treated with 0.03% phenoxyethanol for 48 h; one-way ANOVA analysis was performed, **** represents p < 0.0001). Figure 1 It can be seen that phenoxyethanol can significantly upregulate TRPV1 expression. Combined with the results in Table 3, it can be seen that even with phenoxyethanol-induced upregulation of TRPV1 expression, Example 1 can also inhibit TRPV1 expression, with a significantly better effect than Comparative Example 1. Example 2 shows an even better inhibitory effect, superior to Example 1, Comparative Example 1, and Comparative Example 2. Similarly, after replacing the components in Example 2, the effects of Comparative Examples 6 to 8 are significantly weaker than those of Example 2.
[0075] In summary, Example 1, by using this specific extraction process to increase the content of glycyrrhizin B, exhibited a significant inhibitory effect on TRPV1 expression, although the effective concentration was relatively low. However, the combination of glycyrrhizin B with glycyrrhizin significantly enhanced the inhibitory effect on TRPV1 expression, demonstrating synergistic effects. Furthermore, this synergy is not unique to combinations of Glycyrrhiza uralensis root extract with other structurally similar chalcone components; Example 2 exhibits a certain degree of component uniqueness.
[0076] Experimental Example 3: Intracellular Calcium Flow Inhibition Test in Keratinocytes 3.1 Preparation of sample solution Using DMEM complete culture medium as a solvent, sample solutions with mass concentrations of 2%, 1%, 0.2%, 0.1%, and 0.001% were prepared according to Example 1 and tested.
[0077] Using DMEM complete medium as a solvent, five groups of sample solutions of different concentrations were prepared according to Example 2, including: 0.5% glycyrrhiza uralensis root extract and 10 μM glycyrrhiza chalcone B; 0.001% glycyrrhiza uralensis root extract and 10 μM glycyrrhiza chalcone B; 1% glycyrrhiza uralensis root extract and 20 μM glycyrrhiza chalcone B; 0.2% glycyrrhiza uralensis root extract and 40 μM glycyrrhiza chalcone B; and 0.1% glycyrrhiza uralensis root extract and 20 μM glycyrrhiza chalcone B.
[0078] Using DMEM complete culture medium as solvent, Comparative Example 1 was prepared into sample solutions with mass concentrations of 1%, 0.2%, 0.1%, and 0.001% for testing.
[0079] Using DMEM complete medium as solvent, Comparative Example 2 was prepared into sample solutions with molar concentrations of 40 μM, 20 μM, and 10 μM, respectively, for testing.
[0080] Using DMEM complete medium as solvent, Comparative Example 3 was prepared into sample solutions with molar concentrations of 40 μM, 20 μM, and 10 μM for testing.
[0081] Using DMEM complete medium as solvent, Comparative Example 6 was prepared into three groups of sample solutions with different concentrations: 1% glycyrrhiza uralensis root extract and 20 μM glycyrrhiza chalcone A; 0.2% glycyrrhiza uralensis root extract and 40 μM glycyrrhiza chalcone A; and 0.1% glycyrrhiza uralensis root extract and 20 μM glycyrrhiza chalcone A.
[0082] Using DMEM complete medium as solvent, Comparative Example 9 was prepared into sample solutions with a molar concentration of 10 μM for testing.
[0083] 3.2 Experimental Principle TRPV1, as a calcium-permeable ion channel receptor, is characterized by its high permeability to calcium ions. When TRPV1 is activated, the channel protein conformation changes, the channel pores open, allowing extracellular calcium ions to rapidly flow into the cell down their concentration gradient, directly leading to a sharp increase in intracellular calcium ion concentration. On one hand, increased calcium flow can activate voltage-gated sodium channels at nerve endings, triggering action potentials and transmitting signals such as pain, burning, and itching to the central nervous system. On the other hand, increased calcium flow stimulates keratinocytes and mast cells to release inflammatory factors, neuropeptides (such as substance P), histamine, endothelin-1, etc., inducing an inflammatory response. Therefore, intracellular calcium ions can be labeled using the calcium ion fluorescent probe Calbryte 520, and the intensity of the fluorescence signal can be analyzed using a fluorescence microscope to reflect changes in intracellular calcium ion concentration. Higher intracellular calcium ion concentration indicates a higher degree of TRPV1 activation.
[0084] 3.3 Experimental Methods (1) Cell selection and culture Same as 2.3(1) in Experimental Example 2.
[0085] (2) Cell treatment Cells were seeded in 96-well plates with black borders. On the second day, different concentrations of the control and experimental samples were added and incubated for 10 min. Following the probe instructions, calcium ion dye Calbryte 520 was added and stained for 30 min. Cells were washed three times with HHBS buffer. After adding the inducer, fluorescence images were acquired and analyzed.
[0086] 3.4 Experimental Results The results are as follows Figure 2 As shown in Tables 4 and 5.
[0087] Table 4. Results of different components on the inhibition of capsaicin-induced calcium flow.
[0088] Figure 2 The effects of different inducers on intracellular calcium flux are presented (data are expressed as mean ± standard error; the control group represents TRPV1-overexpressing HaCaT cells; capsaicin and resorcinol were used to treat HaCaT cells overexpressing the disease at 5 μM and 300 μM, respectively; one-way ANOVA analysis was performed; * represents p < 0.05; **** represents p < 0.0001). Figure 2The results showed that both capsaicin and resorcinol significantly induced an increase in intracellular calcium flux in keratinocytes. Capsaicin increased intracellular calcium flux signal by 286.1%, while resorcinol increased it by 53.7%. Table 4 shows that Example 1, compared to Comparative Example 1 (conventional ethanol extraction), exhibited a stronger inhibitory effect on capsaicin-induced calcium flux increase, indicating that the content of glycyrrhizin B is concentration-dependent on the activation of TRPV1 induced by capsaicin. This invention, by increasing the content of glycyrrhizin B through a specific extraction process, demonstrated a stronger inhibitory effect. Meanwhile, the inhibitory effect of Comparative Example 2 (glycyrrhizin B) was weaker than that of Comparative Example 3 (glycyrrhizin A) and Comparative Example 9 (the known potent TRPV1 antagonist AMG9810). However, the combination of Example 1 and glycyrrhizin B (Example 2) significantly enhanced the effect on capsaicin-induced calcium flux increase, demonstrating superiority over Example 1, Comparative Examples 1-3, and Comparative Example 9, and exhibiting a synergistic inhibitory effect compared to single components at the same concentration.
[0089] Table 5. Results of different components inhibiting resorcinol-induced calcium flow.
[0090] As shown in Table 5, Example 1 exhibited a better inhibitory effect on resorcinol-induced intracellular calcium influx, outperforming Comparative Example 1. This indicates that the content of glycyrrhizin B is concentration-dependent on the TRPV1 activation induced by resorcinol. The present invention, by increasing the content of glycyrrhizin B through a specific extraction process, demonstrated a stronger inhibitory effect. Comparative Example 2 (glycyrrhizin B) showed a certain inhibitory effect on the increase in calcium influx induced by resorcinol, while Comparative Example 3 (glycyrrhizin A) showed a poor effect on this type of stimulating component. Example 2 significantly enhanced the effect on the increase in calcium influx induced by resorcinol, significantly outperforming Example 1, Comparative Examples 1-3, and Comparative Example 9. Furthermore, compared with single components at the same concentration, it exhibited a synergistic inhibitory effect. In contrast, the inhibitory effect of combining the root extract of *Glycyrrhiza uralensis* with glycyrrhizin A (Comparative Example 6) was significantly weakened.
[0091] Based on the above results, Examples 1 and 2 can inhibit the increase of intracellular calcium flow in keratinocytes, especially showing significant inhibitory effects on intracellular calcium flow caused by capsaicin and phenols. This suggests that the above-mentioned ingredients may be able to alleviate skin irritation and improve skin irritation caused by phenols, capsaicin and other ingredients by adding small amounts in practical applications.
[0092] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.
Claims
1. The use of a composition in the preparation of a product that inhibits TRPV1 activation, characterized in that, The composition includes Glycyrrhiza inflata root extract and glycyrrhizin B, wherein the Glycyrrhiza inflata root extract is prepared by enzymatic hydrolysis and alcohol extraction of Glycyrrhiza inflata root.
2. The application according to claim 1, characterized in that, The effective concentration of the extract of Glycyrrhiza uralensis root is ≥0.001wt%.
3. The application according to claim 1, characterized in that, The effective concentration of glycyrrhizin B is ≥5 μM.
4. The application according to claim 1, characterized in that, The enzymatic hydrolysis uses a complex enzyme consisting of cellulase and hemicellulase mixed in a mass ratio of (1-2):
1.
5. The application according to claim 1, characterized in that, The pH of the enzymatic hydrolysis is 5-6.5; and / or the temperature of the enzymatic hydrolysis is 45-55℃; and / or the time of the enzymatic hydrolysis is 1-2 hours.
6. The application according to claim 1, characterized in that, The alcohol extraction uses an ethanol solution with a volume concentration of 70%-90%; and / or, the alcohol extraction temperature is 45-55℃; and / or, the alcohol extraction time is 30-50 min.
7. The application according to claim 1, characterized in that, The alcohol extraction process is assisted by ultrasonic extraction, and the frequency of the ultrasonic extraction is 5-6 kHz.
8. The application according to claim 1, characterized in that, The inhibition of TRPV1 activation includes inhibiting the expression level of TRPV1 and / or inhibiting the influx of calcium ions after TRPV1 activation.
9. The application according to claim 1, characterized in that, The extract of Glycyrrhiza uralensis root contains total flavonoids and glycyrrhizin B, wherein the content of total flavonoids is greater than 200 mg / g and the content of glycyrrhizin B is greater than 2 mg / g.
10. A product for inhibiting TRPV1 activation, characterized in that, The product includes Glycyrrhiza inflata root extract and Glycyrrhiza chalcone B, wherein the Glycyrrhiza inflata root extract is prepared by enzymatic hydrolysis and alcohol extraction of Glycyrrhiza inflata root.