Folium artemisiae argyi extract for regulating sensory nerve ion channel as well as preparation method and application of folium artemisiae argyi extract

Abstract

The invention relates to the field of cosmetics, in particular to a folium artemisiae argyi extract for regulating and controlling sensory nerve ion channels and a preparation method and application thereof, and the folium artemisiae argyi extract is used for preparing a preparation for inhibiting pruritus perceived by a body. The preparation method comprises the following steps: A, taking folium artemisiae argyi, and cleaning to obtain a folium artemisiae argyi raw material; b, adding the folium artemisiae argyi raw material into water, and heating and extracting to obtain a first crude extract; c, cooling and sieving the first crude extract to obtain a second crude extract; d, adding a preservative into the second crude extract to obtain a third crude extract; e, adding a pH regulator into the third crude extract, and regulating the pH to be acidic to obtain a fourth crude extract; and F, carrying out solid-liquid separation on the fourth crude extract to obtain the folium artemisiae argyi extract. The extract disclosed by the invention can be applied to a hair washing and caring product formula and has the effect of relieving skin itch.

Description

Technical Field

[0001] The invention relates to the field of cosmetics and plant extracts, and in particular to an wormwood leaf extract for regulating sensory nerve ion channels, and a preparation method and application thereof. Background Art

[0002] Mugwort leaf is a traditional Chinese herbal medicine and a widely used and recognized Chinese medicine with skin care properties in the cosmetics industry. The preparation process of its extract is mainly water extraction, alcohol extraction and other solvent extraction methods, and the extract is obtained after multiple steps of impurity removal. This extraction method generally has many process procedures, which affects the timeliness, and the organic solvents such as ethanol used are highly dangerous and environmentally unfriendly. Summary of the invention

[0003] The purpose of the present invention is to disclose an wormwood leaf extract for regulating sensory nerve ion channels, a preparation method and application thereof, simplify the wormwood leaf extraction process, and the obtained extract has good stability, has antioxidant properties, inhibits mast cell histamine degranulation activity, downregulates the expression of neuronal cell itching-related ion channel TRPV1, is highly mild, and is non-irritating to the eyes, and can be used as a raw material for the formulation of skin / scalp itching products.

[0004] The process of the invention contains no organic solvent, is environmentally friendly and has a low risk factor.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions: A method for preparing an wormwood leaf extract comprises the following steps: A. Take mugwort leaves, wash them, and obtain mugwort leaf raw materials; B. adding the wormwood leaf raw material into water, performing heating extraction, and obtaining a first crude extract; C. cooling and sieving the first crude extract to obtain a second crude extract; D. adding a preservative to the second crude extract to obtain a third crude extract; E. adding a pH regulator to the third crude extract to adjust the pH to acidic to obtain a fourth crude extract; F. Performing solid-liquid separation on the fourth crude extract to obtain an Artemisia argyi extract.

[0006] The purification process after extraction only uses centrifugation for treatment, which greatly simplifies the post-processing process of the extract. It is suitable for cosmetic applications and the prepared extract has the effects of inhibiting mast cell histamine degranulation activity and down-regulating the expression activity of neuronal cell ion channel proteins, and has a mild effect of relieving skin itching. At the same time, no organic solvents are used for extraction, which is environmentally friendly and has a low risk factor.

[0007] Preferably, in step A, the cleaning includes stirring and cleaning in deionized water; in step B, the extraction temperature of the heated extraction includes 93° C., and the extraction time includes 3 hours; in step C, the sieving screen includes 300 mesh; in step D, the preservative includes one or more of phenoxyethanol and sodium benzoate; in step E, the pH adjuster includes citric acid; in step F, the solid-liquid separation includes centrifugal separation.

[0008] Preferably, in step A, the stirring speed of the stirring and washing comprises 110 rmp / min, and the stirring time comprises 10 min; in step B, the stirring is continued during the heating extraction, and the stirring speed comprises 155 rmp / min; in step C, the cooling temperature comprises room temperature; in step D, in the preservative, the mass ratio of the phenoxyethanol to the sodium benzoate comprises 7~8:2~3; in step E, the pH of the fourth crude extract comprises 5.5; in step F, the separation speed of the centrifugal separation comprises 3000 rmp / min, and the centrifugation time comprises 30 min.

[0009] Preferably, in step B, the mass ratio of the wormwood raw material to water is 1:10~20; in step D, the amount of the preservative added is 1.2~3.3% of the mass of the second crude extract; in step F, the solid content of the wormwood extract is 1~3%.

[0010] Preferably, in step A, the cleaning is performed in a mixer; in step B, the heating extraction is performed in a steam generator; in step C, the cooling method includes water cooling; in step F, the solid-liquid separation is performed in a centrifuge.

[0011] A wormwood extract obtained by the above-mentioned wormwood extract preparation method.

[0012] An application of the above-mentioned wormwood leaf extract is used to prepare a preparation for inhibiting body-perceived itching.

[0013] Preferably, the invention is used for preparing a preparation for inhibiting histamine degranulation of mast cells.

[0014] Preferably, the method is used for preparing a preparation comprising one or more of regulating the expression of ion channel proteins in neuronal cells and regulating the flow of neural ions.

[0015] Preferably, the neuronal cell ion channel protein includes transient receptor potential vanilloid channel 1 (TRPV1); and the neuronal ions include calcium ions.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. Simplify the extraction process of mugwort leaf extract and improve its effectiveness.

[0017] 2. The water extraction method is environmentally friendly and has a low risk factor. It is non-irritating to chicken embryos under 100% concentration test conditions, indicating that the extract has no eye irritation and is milder than alcohol extraction.

[0018] 3. The prepared extract is highly mild and non-irritating, and has the effects of inhibiting mast cell histamine degranulation, downregulating the expression of neuronal cell ion channel protein TRPV1 and regulating the calcium flow of nerve cells, and targetedly regulating sensory nerve ion channels, with dual-target effects of relieving skin / scalp itching.

[0019] 4. The extract has the effect of scavenging DPPH free radicals, with a free radical scavenging rate of 52.63%, and has certain antioxidant properties.

[0020] 5. The extract can be used in the formulation of hair care products to relieve skin itching. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the extract without centrifugation.

[0022] Figure 2 Schematic diagram of the centrifuged extract after 3 months.

[0023] Figure 3 The relative activity of cells in each group.

[0024] Figure 4 This is a comparison chart of histamine differences in different groups.

[0025] Figure 5 This is a mildness test chart.

[0026] Figure 6 The expression levels of ion channel protein TRPV1 in human neuroblastoma cells SH-SY5Y in different groups.

[0027] Figure 7 Ca2+ in SH-SY5Y cells transfected with TRPV1 virus in different groups 2+ concentration.

[0028] Figure 8 This is the chicken embryo irritation picture of 100% concentration of Artemisia argyi water extract.

[0029] Fig. 9 This is the chicken embryo irritation picture of 50% concentration of Artemisia annua alcohol extract. DETAILED DESCRIPTION

[0030] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0031] Example 1 Preparation process of Artemisia argyi extract: 1. Weigh 46g of dry moxa leaves and place them in a beaker. Add deionized water and stir with a blender for 10 minutes (speed 110rmp / min) to wash them clean.

[0032] 2. Place the washed mugwort leaves in a transparent emulsifying pot, add deionized water (solid-liquid ratio = 1:15), heat up the steam generator, and extract at 93°C for 3 hours. The stirring speed during the extraction process is about 155rmp / min.

[0033] 3. Cool the extract to room temperature with tap water, filter with a 300-mesh sieve, and obtain 578 g (1:15 solid-liquid ratio) of extract.

[0034] 4. Add 1% preservative (phenoxyethanol: sodium benzoate = 0.8:0.2).

[0035] 5. Adjust the pH to 5.5 with citric acid.

[0036] 6. Centrifugal treatment (3000rmp / min, 30min) 6. Solid content: 1.69%.

[0037] Example 2 Preparation process of Artemisia argyi extract: 1. Weigh 46g of dry moxa leaves and place them in a beaker. Add deionized water and stir with a blender for 10 minutes (speed 110rmp / min) to wash them clean.

[0038] 2. Place the washed mugwort leaves in a transparent emulsifying pot, add deionized water (solid-liquid ratio = 1:10), heat up the steam generator, and extract at 93°C for 3 hours. The stirring speed during the extraction process is about 155rmp / min.

[0039] 3. Cool the extract to room temperature with tap water, filter with a 300-mesh sieve, and obtain 382 g (1:10 solid-liquid ratio) of extract.

[0040] 4. Add 1% preservative (phenoxyethanol: sodium benzoate = 0.7:0.3).

[0041] 5. Adjust the pH to 5.5 with citric acid.

[0042] 6. Centrifugal treatment (3000rmp / min, 30min) 6. Solid content: 2.12%.

[0043] Example 3 Preparation process of Artemisia argyi extract: 1. Weigh 46g of dry moxa leaves and place them in a beaker. Add deionized water and stir with a blender for 10 minutes (speed 110rmp / min) to wash them clean.

[0044] 2. Place the washed mugwort leaves in a transparent emulsifying pot, add deionized water (solid-liquid ratio = 1:20), heat up the steam generator, and extract at 93°C for 3 hours. The stirring speed during the extraction process is about 155rmp / min.

[0045] 3. Cool the extract to room temperature with tap water, filter with a 300-mesh sieve, and obtain 781 g (1:20 solid-liquid ratio) of extract.

[0046] 4. Add 1% preservative (phenoxyethanol: sodium benzoate = 0.7:0.3).

[0047] 5. Adjust the pH to 5.5 with citric acid.

[0048] 6. Centrifugal treatment (3000rmp / min, 30min) 6. Solid content: 1.09%.

[0049] Effect Example 1 Taking Example 1 as an example, the extract after completing step (5) of Example 1 was taken for observation. After being left overnight, Figure 1 shown.

[0050] At the same time, the extract after completing step (6) of Example 1 was taken for observation. After being placed under the same conditions for three months, Figure 2 shown.

[0051] from Figure 1 , Figure 2 From the comparison, it can be seen that the extract that has not been centrifuged will precipitate after being placed, while the extract that has been centrifuged will still not precipitate after being placed for up to 3 months, and has better stability.

[0052] Effect Example 2 Taking Example 1 as an example, the Artemisia argyi leaf extract obtained in Example 1 was used to conduct an antiseptic challenge experiment.

[0053] The theoretical inoculation amount of bacteria is 5.0*106 cfu / g; the theoretical inoculation amount of mold is 1.2*10 5 .

[0054] The results are shown in Table 1.

[0055] Table 1

[0056] The results show that the wormwood leaf extract obtained by the present invention can pass the antiseptic challenge test.

[0057] Effect Example 3 Taking Example 1 as an example, the activity of inhibiting mast cell histamine degranulation was performed.

[0058] Test steps: (1) Cell activity test: 1): Routine cell culture. Inoculate the cell suspension into a 96-well cell culture plate and culture for 18-24 hours until the cells are 80% confluent.

[0059] 2) Discard the original culture medium in the wells, add different concentrations of test samples to each well, and return to the incubator for incubation for 24±1h.

[0060] 3) Take out the culture plate, add 20 μL of MTT solution to each well, and incubate in the incubator for 3-4 hours. Remove the liquid in the wells, add 100 μL of DMSO to each well, place it on an oscillator for 10-15 minutes, and measure the absorbance at a wavelength of 570 nm on a microplate reader.

[0061] 4) Data analysis: The cell activity of the negative control group was taken as 100%, and the relative cell activity (Viability) of each group was calculated.

[0062]

[0063] (2) Histamine release from mouse mast cells (P815): 1) Collect P815 cells in the logarithmic phase, inoculate the cell suspension into a 24-well plate, and culture in an incubator for 18-24 hours; 2) Remove the original culture medium in the wells, add samples of different concentrations (three concentrations of samples were added to the test samples, ketotifen fumarate was added to the positive control group, and the negative control group and model group were added with the same amount of serum-free culture medium), three replicate wells in each group, and incubate for 2 hours; 3) Remove the original culture medium in the wells. According to the experimental groups, add serum-free culture medium containing C48 / 80 to the negative control group, add serum-free culture medium containing C48 / 80 and ketotifen fumarate to the positive control group, and add C48 / 80 and three concentrations of the test samples to the sample group. After the end of the administration, stimulate with C48 / 80 for 60 minutes and terminate the reaction in an ice bath.

[0064] 4) Collect the supernatant, store at -80℃, and measure histamine using ELISA kit.

[0065] 5) Data analysis: Data were expressed as mean ± standard deviation and analyzed using SPSS. If p < 0.05, the difference was considered statistically significant.

[0066] Experimental results: The relative activity of cells in each group Figure 3 1.00%, 0.50%, and 0.25% were selected as the test concentrations for subsequent efficacy experiments to obtain the difference in histamine between different groups, as shown in Figure 2. Figure 4 shown.

[0067] According to statistical analysis, the sample "Artemisia argyi extract" can significantly inhibit the release of P815 histamine at 1.00%, 0.50% and 0.25% (p<0.05), and the inhibition rates are 66.29%, 67.55% and 60.66% respectively, which has a certain antipruritic effect.

[0068] Effect Example 4 The wormwood extract obtained by the method of the present invention (taking Example 1 as an example) was tested for mildness. The results are as follows Figure 5 shown.

[0069] Zein Irritation Studies have shown that mugwort extract is non-irritating.

[0070] Effect Example 5 After TRPV1 protein extraction, according to the number of samples, 50 volumes of BCA reagent A plus 1 volume of BCA reagent B (50:1) are prepared with an appropriate amount of BCA working solution and mixed thoroughly. BCA working solution is stable at room temperature for 24 hours. Completely dissolve the protein standard, take 10 μL and dilute it to 100 μL with PBS to a final concentration of 0.5 mg / mL. Add 0, 1, 2, 4, 8, 12, 16, 20 μL of the standard to the standard wells of a 96-well plate, and add the solution used to dilute the standard to 20 μL. Add an appropriate volume of sample to the sample wells of a 96-well plate, and add the solution used to dilute the standard to 20 μL. Add 200 μL of BCA working solution to each well and place at 37°C for 30 minutes. Determine at A 562 and calculate the protein concentration based on the standard curve. The unit is mg / mL.

[0071] like Figure 6 , 1.290% and 0.645% of Artemisia argyi extract can significantly down-regulate the expression of ion channel protein TRPV1 in SH-SY5Y cells.

[0072] Effect Example 6 Collect SH-SY5Y cells transfected with TRPV1 virus, inoculate the cell suspension into 12-well plates, and culture in an incubator for 18-24 h; add Fluo-3AM calcium ion fluorescent probe and incubate at 37°C for 30 min; collect cells and transfer them to 1.5 ml tubes, centrifuge at 10,000 g for 1 min, and discard the supernatant; add 1 ml PHF buffer to wash the cells, pipette up and down to resuspend, wash twice more, and resuspend the cells in a final volume of 500 μL PHF buffer, and conduct the experiment within 1 hour.

[0073] like Figure 7 In the positive control group, histamine could induce Ca2+ in SH-SY5Y cells transfected with TRPV1 virus. 2+ Level fluctuation, different concentrations of Artemisia argyi extract in the sample group have the effect of restoring intracellular Ca 2+ The role of level.

[0074] Effect Example 7 The water extract of Artemisia argyi obtained by the method of the present invention (taking Example 1 as an example) was compared with the alcohol extract of Artemisia argyi obtained by conventional alcohol extraction, as follows: 1. Test Purpose Prediction of conjunctival eye irritation of test samples by CAM membrane model 2. The test steps are shown in Table 2.

[0075] Table 2

[0076] 3. Test parameters 1. Stimulus Rating (IS) Result judgment: IS<1: non-irritating; 1≤IS<5: mildly irritating; 5≤IS<9: moderately irritating; IS≥9: strongly irritating / corrosive.

[0077] 4. The test data are shown in Table 3 and Table 4.

[0078] Table 3 - Eye irritation score of CAM membrane of 100% concentration of Artemisia argyi water extract (scoring method: IS)

[0079] 1. Calculate the formal experimental results, IS=0; 2. The IS score of the positive control 1% SLS benchmark substance is 5.98, which is moderately irritating; 3. 100% Artemisia argyi extract 24AB53 is classified as non-irritating to eyes.

[0080] Table 4 - Eye irritation score of CAM membrane with 50% concentration of Artemisia argyi alcohol extract (scoring method: IS)

[0081] 1.1. Calculate the formal experimental results, IS=9.39; 2. The IS score of the positive control 1% SLS benchmark substance is 5.98, which is moderately irritating; 3. 50% Artemisia argyi extract (alcohol extraction) 24YE92 is classified as a strong irritant to eyes.

[0082] 5. Test pictures The Artemisia argyi alcohol extract of the test sample 1 acted on the CAM membrane for 5 minutes, and the chicken embryo irritation pictures were taken, such as Figure 4 shown.

[0083] The wormwood water extract of test sample 2 acted on the CAM membrane for 5 minutes, and the chicken embryo irritation pictures were taken, such as Figure 5 shown.

[0084] 6. Test Conclusion The comprehensive test results are summarized and shown in Table 5.

[0085] Table 5

[0086] Effect Example 8 The Artemisia argyi leaf extract obtained in Example 1 was used to prepare a dual-purpose cleaning product for shampooing and bathing, and an application test evaluation was carried out. The results are shown in Table 6.

[0087] Table 6

[0088] As can be seen from Table 6, the shampoo and bath dual-purpose cleaning product prepared using the Artemisia argyi extract of the present invention can effectively relieve itching and oily hair, and achieves excellent results.

[0089] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A method for preparing an wormwood extract, characterized in that: The following steps are involved: A. Take mugwort leaves, wash them, and obtain mugwort leaf raw materials; B. adding the wormwood leaf raw material into water, performing heating extraction, and obtaining a first crude extract; C. cooling and sieving the first crude extract to obtain a second crude extract; D. adding a preservative to the second crude extract to obtain a third crude extract; E. adding a pH regulator to the third crude extract to adjust the pH to acidic to obtain a fourth crude extract; F. Performing solid-liquid separation on the fourth crude extract to obtain an Artemisia argyi extract.

2. The method for preparing the wormwood extract according to claim 1, characterized in that: In step A, the cleaning includes stirring and cleaning in deionized water; in step B, the extraction temperature of the heated extraction includes 93° C., and the extraction time includes 3 hours; in step C, the sieving screen includes 300 mesh; in step D, the preservative includes one or more of phenoxyethanol and sodium benzoate; in step E, the pH adjuster includes citric acid; in step F, the solid-liquid separation includes centrifugal separation.

3. The method for preparing the wormwood extract according to claim 2, characterized in that: In step A, the stirring speed of the stirring and washing includes 110 rmp / min, and the stirring time includes 10 min; in step B, the stirring is continued during the heating extraction, and the stirring speed includes 155 rmp / min; in step C, the cooling temperature includes room temperature; in step D, in the preservative, the mass ratio of the phenoxyethanol to the sodium benzoate includes 7~8:2~3; in step E, the pH of the fourth crude extract includes 5.5; in step F, the separation speed of the centrifugal separation includes 3000 rmp / min, and the centrifugation time includes 30 min.

4. The method for preparing the wormwood extract according to claim 1, characterized in that: In step B, the mass ratio of the wormwood raw material to water is 1:10~20; in step D, the amount of the preservative added is 1.2~3.3% of the mass of the second crude extract; in step F, the solid content of the wormwood extract is 1~3%.

5. The method for preparing the wormwood extract according to claim 1, characterized in that: In step A, the cleaning is performed in a mixer; in step B, the heating extraction is performed in a steam generator; in step C, the cooling method includes water cooling; in step F, the solid-liquid separation is performed in a centrifuge.

6. An Artemisia argyi extract obtained according to the preparation method of Artemisia argyi extract according to claim 1.

7. An application of the wormwood extract according to claim 6, characterized in that: Used to prepare a preparation for suppressing body-perceived itching.

8. The use according to claim 7, characterized in that: Used to prepare preparations for inhibiting mast cell histamine degranulation.

9. The use according to claim 7, characterized in that: Used for preparing a preparation comprising one or more of regulating the expression of ion channel proteins in neuronal cells and regulating the flow of neural ions.

10. The use according to claim 9, characterized in that: The neuronal cell ion channel protein includes transient receptor potential vanilloid channel 1; the neural ions include calcium ions.

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