APPLICATION OF ACTIVE INGREDIENT WGX50 IN ZANTHOXYLUM BUNGEANUM EXTRACT

Abstract

An application method of the WGX50 of active ingredient in Zanthoxylum bungeanum extract is provided, after testing the cytotoxicity of keratinocytes and melanocytes with different concentrations of WGX50, conducting a moisturizing gene test based on keratinocytes, a whitening gene test based on melanocytes and an anti-inflammatory test based on UVB stimulation of keratinocytes. First discovers that WGX50 has the efficacys of moisturizing, whitening and anti-inflammation, and the discovery of the new efficacies of WGX50 provides a potential scheme for preparing multifunctional cosmetics, opens up new ideas for preparing cosmetics, and contributes to the realization of higher commercial value.

Claims

1. An application method of (E)-N-[2-(3,4-dimethoxyphenyl) ethyl]-3-phenylacrylamide (WGX50) of active ingredient in Zanthoxylum bungeanum extract, wherein the WGX50 of active ingredient in Zanthoxylum bungeanum extract is applied for one or more selected from the group consisting of moisturizing, whitening and anti-inflammation.

2. The application method of WGX50 of active ingredient in Zanthoxylum bungeanum extract according to claim 1, wherein the WGX50 of active ingredient is applied in promoting expression of AQP3 gene in keratinocytes, and the WGX50 of active ingredient has an efficacy of the moisturizing.

3. The application method of WGX50 of active ingredient in Zanthoxylum bungeanum extract according to claim 1, wherein the WGX 50 of active ingredient is applied in inhibiting expressions of MC1R, MITF, TYR and TYP-1 genes, and the WGX50 of active ingredient has an efficacy of the whitening.

4. The application method of WGX50 of active ingredient in Zanthoxylum bungeanum extract according to claim 1, wherein the WGX50 of active ingredient is applied in decreasing secretions of TNFα and IL-1α, and the WGX50 of active ingredient has an efficacy of the anti-inflammation.

5. The application method of WGX50 of active ingredient in Zanthoxylum bungeanum extract according to claim 1, wherein the WGX50 of active ingredient has the following structure: ##STR00002##

Description

BRIEF DESCRIPTION OF THE FIGURES

[0020] FIG. 1 is a diagram showing the cell viability change trend of keratinocytes of the invention.

[0021] FIG. 2 is a diagram showing the cell morphology of the keratinocytes of the invention.

[0022] FIG. 3 is a diagram showing the cell viability change trend of the melanocytes of the invention.

[0023] FIG. 4 is a diagram showing the change trend of moisturizing genes in keratinocytes of the invention.

[0024] FIG. 5 is a diagram showing the melanocyte whitening gene change trend of the invention.

[0025] FIG. 6 is a diagram showing the TNFα content change trend of the invention.

[0026] FIG. 7 is a diagram showing the IL-1α content change trend of the invention.

DESCRIPTION OF THE INVENTION

[0027] The invention will be described in detail with reference to the drawings and specific embodiments below.

[0028] The cells used in this test are keratinocytes and melanocytes, and all cells are produced by Guangdong Biocell Biotechnology Co., Ltd, in China. WGX50 powder is prepared according to the method disclosed in the Chinese patent application No. 201110439656.X (corresponding to Chinese patent publication No. CN103130674A) entitled “Chemical Synthesis Method of Potential Medicine gx50, gx51, gx52, and gx180 for the preventing and treating the Alzheimer's disease”.

[0029] The reagents used are PBS (Boster), MTT(Sigma), DMSO(Sigma), DMEM (Guangdong Biocell), NBS (Sijiqing), Medium-254(Gibco), HMGS(Gibco), KC2500 (Guangdong Biocell), Dexamethasone (Sigma), RNAiso Plus (TaKaRa), reverse transcription kit (TaKaRa), fluorescent dye (TaKaRa), TNFα kit (Abcam) and IL1-α kit (Abcam).

[0030] The main equipment used includes CO.sub.2 incubator (Thermo), clean bench (Airtech), inverted microscope (Olympus), microoscillator (Kylin-Bell), enzyme-labeled instrument (BioTek) and flow cytometer (Beckman).

[0031] Embodiment 1, cytotoxicity test based on keratinocytes. Set up 8 concentration gradients, and set up 3 repeating holes under each concentration. At the same time, solvent control hole, zero adjustment hole (KC2500) and positive control hole (PC, 4% Dimethyl sulfoxide, DMSO) are set up in the experiment. After incubation for 24 hours, the cell viability is detected by MTT method, and the maximum safe concentration of cell administration is screened. The MTT test results are shown in Table 1, in which the GraphPad Prism Program software is used for mapping, and T-test statistical analysis is used among all groups. P-value<0.05 indicates significant difference, and P-value<0.01 indicates extremely significant difference. The change trend of cell viability is shown in FIG. 1. According to the results of MTT assay, the concentration near the inflection point of cell vitality is selected, and after incubation and culture for 24 hours, the cell morphology is observed under the inverted microscope and photographed (20×), and the morphological detection results are shown in FIG. 2.

TABLE-US-00001 TABLE 1 Toxicity test results of keratinocytes Concentration gradient (μM) {circle around (1)} {circle around (2)} {circle around (3)} {circle around (4)} {circle around (5)} {circle around (6)} {circle around (7)} {circle around (8)} PC Control 0.5 1 2.5 5 10 25 50 100 4% DMSO / Cell Mean 106.81% 93.72% 93.60% 93.01% 84.00% 66.77% 57.35% 50.36% 30.27%  100% vitality SD  3.29%  1.96%  0.89%  1.29%  6.36%  5.71%  6.02%  4.62%  2.09% 1.88% P-value 0.036 0.016 0.006 0.006 0.014 0.001 0.000 0.000 0.000 /

[0032] According to MTT and morphological results, WGX50 shows no cytotoxicity to keratinocytes in the concentration range of 5 μM.

[0033] Embodiment 2, cytotoxicity test based on melanocytes. Set up 8 concentration gradients, and set up 3 repeating holes under each concentration. At the same time, solvent control well, Medium-254 well and positive control well (PC, 8% DMSO) are set up in the experiment. After incubation for 24 hours, the cell viability is detected by MTT method, and the maximum safe concentration of cell administration is screened. The results of MTT assay are shown in Table 2, and the change trend of cell viability is shown in FIG. 3.

TABLE-US-00002 TABLE 2 Toxicity test results of melanocytes Concentration gradient (μM) {circle around (1)} {circle around (2)} {circle around (3)} {circle around (4)} {circle around (5)} {circle around (6)} {circle around (7)} {circle around (8)} PC Control 0.5 1 2.5 5 10 25 50 100 8% DMSO / Cell Mean 105.78% 99.02% 96.68% 95.56% 99.51% 99.51% 108.86% 96.62% 24.23% 100.00% vitality SD  3.33%  1.07%  4.62%  2.62%  4.82%  3.79%  7.78%  3.15%  6.00%  5.68% P-value 0.196 0.783 0.476 0.394 0.914 0.907 0.186 0.121 0.000 /

[0034] According to the results of MTT, WGX50 shows no cytotoxicity to melanocytes in the concentration range of 100 μM.

[0035] Embodiment 3, moisturizing gene test based on keratinocytes. The experimental design of moisturizing gene test based on keratinocytes is shown in Table 3.

TABLE-US-00003 TABLE 3 Experimental design of moisturizing gene test Sample information Detection Systems Experi- Sample Incu- ment Sample concen- Detection Detection Detection bation grouping name tration Indicator model method time Blank BC / AQP3, Keratino- qRT-PCR 24 h control CD44, cytes group Filaggrin, Sample WGX50 5 μM claudin-1, group HAS2

[0036] AQP3 aquaporin in the detection index, located on the cell membrane, has the function of transporting water and plays an important role in the process of skin moisturizing.

[0037] Gene detection process: after incubation for 24 hours, the cells are sampled, and RNA extraction, reverse transcription and fluorescence quantitative PCR detection are carried out according to the kit instruction, and the results are calculated by 2.sup.−ΔΔΔCT method. When statistical analysis is carried out by T-Test method, significance is represented by *, P-value<0.05 is represented by *, and P-value<0.01 is represented by * *.

[0038] The detection results are shown in Table 4, and the trend of gene change is shown in FIG. 4.

TABLE-US-00004 TABLE 4 Test results of moisturizing gene BC (Blank Increased control group) WGX50 ratio Sample P- P- (compared name Mean SD value Mean SD value to BC) AQP3 1.004 0.099 / 1.345 0.127  0.005**   34.20% Filaggrin 1.029 0.287 / 1.101 0.076 0.646    7.20% Claudin-1 1.007 0.139 / 1.182 0.249 0.267   17.50% HAS2 1.046 0.354 / 0.898 0.289 0.543 −14.70% CD44 1.004 0.101 / 0.785 0.092 0.101 −21.90%

[0039] Compared with the blank control group, WGX50 significantly promotes the expression of AQP3 gene in keratinocytes (P-value<0.01).

[0040] Embodiment 4, whitening gene test based on melanocytes. The experimental design of whitening gene test based on melanocytes is shown in Table 5.

TABLE-US-00005 TABLE 5 Experimental design of whitening gene test Sample information Detection Systems Experi- Sample Incu- ment Sample concen- Detection Detection Detection bation grouping name tration Indicator model method time Blank BC / MC1R, Melano- qRT-PCR 24 h control MITF, cytes group TYR, Sample WGX50 100 μM TRP-1 group

[0041] Gene detection process: after 24 hours of incubation, the cells are sampled, and RNA extraction, reverse transcription and fluorescence quantitative PCR detection are carried out according to the kit instructions, and the results are calculated by 2.sup.−ΔΔΔCT method. When statistical analysis is carried out by T-Test method, significance is represented by *, P-value<0.05 is represented by *, and P-value<0.01 is represented by * *.

[0042] The detection results are shown in Table 6, and the trend of gene change is shown in FIG. 5.

TABLE-US-00006 TABLE 6 Results of whitening gene detection BC (Blank Increased control group) WGX50 ratio Sample P- P- (compared name Mean SD value Mean SD value to BC) MC1R 1.001 0.043 / 0.528 0.079 0.000**  47.2% MITF 1.01  0.16  / 0.432 0.029 0.000**  57.8% TYR 1.006 0.127 / 0.488 0.122 0.000**  51.8% TRP-1 1.005 0.118 / 0.304 0.022 0.000** 70.10%

[0043] Compared with the blank control group, WGX50 significantly inhibits the expression of MC1R, MITF, TYR and TYP-1 genes in melanocytes (P-value<0.01).

[0044] Embodiment 5, anti-inflammatory test of keratinocytes stimulated by UVB. The experimental design of anti-inflammatory test of keratinocytes stimulated by UVB is shown in Table 7.

TABLE-US-00007 TABLE 7 Experimental design of anti-inflammatory test Sample information Detection Systems Experiment Sample Sample Detection Detection Detection Incubation grouping name concentration Stimulus Indicator model method time Blank BC-O DMSO (0.1%) / TNFα, Keratinocytes ELISA 24 h control IL1-α (oil soluble) Solvent NC-O UVB control (300 ml/cm.sup.2) (oil soluble) Positive PC 100 μg/mL control (Dexamethasone) Sample WGX50 5 μM group

[0045] TNFα and IL1-α expression detection process: after 24 hours of incubation, the cell culture supernatant is collected, and the detection and analysis are performed according to the operating instructions of the TNFα and IL1-α kit. When using the T-Test method for statistical analysis, the NC group is compared with the BC group, and the significance is indicated by #, P-value<0.05 is indicated as #, P-value<0.01 is indicated as ##; sample group and positive control group are compared with NC group, and significance is represented by *, P-value<0.05 is represented by *, P-value<0.01 is represented by * *.

[0046] The results of TNFα measurement are shown in Table 8, and the change trend is shown in FIG. 6.

TABLE-US-00008 TABLE 8 Detection results of TNFα Average Decreased ratio concentration (compared Sample name (pg/mL) SD P-value to NC) BC 3.13 0.95 / / (Blank control group) NC 130.46 2.85 0.00 / (Solvent control group) PC 8.34 0.41 0.00** 94% (Positive control group) WGX50 18.97 1.04 0.00** 85%

[0047] Compared with the blank control group, the TNFα secretion in the positive control group is significantly increased (P-value<0.01), indicating that the UVB stimulation conditions in this experiment are effective. Compared with the solvent control group, the secretion of TNFα decreases significantly (P-value<0.01) at the concentration of 100 μg/mL dexamethasone, which shows that this experiment is effective.

[0048] Compared with the positive control group, the secretion of WGX50 TNFα decreases significantly (P-value<0.01).

[0049] The detection results of IL-1α a are shown in Table 9 and the change trend is shown in FIG. 7.

TABLE-US-00009 TABLE 9 Detection results of IL-1α Average Decreased ratio concentration (compared Sample name (pg/mL) SD P-value to NC) BC 53.86 10.05 / / (Blank control group) NC 132.52 18.75 0.00## / (Solvent control group) PC 93.97  1.62 0.02*  29% (Positive control group) WGX50 43.65  0.59 0.00** 67%

[0050] Compared with the blank control group, the IL-1α secretion in the positive control group significantly increases (P-value<0.05), indicating that the UVB stimulation conditions in this experiment are effective. Compared with the solvent control group, the secretion of IL-1α decreases significantly (P-Value<0.05) at the concentration of dexamethasone of 100 μg/mL, which indicates that this experiment is effective.

[0051] Compared with the positive control group, the secretion of WGX50 IL-1α decreases significantly (P-Value<0.01).