European pine bud extract with the function of regulating calcium ions in skin cells, its preparation method and application

Abstract

The present invention relates to the technical field of daily-use skin care products, and particularly relates to a Pinus sylvestris L. bud extract having the effect of regulating calcium ions in skin cells, a preparation method thereof, and an application thereof. The present invention provides a preparation method of a Pinus sylvestris L. bud extract, which comprises enzymatically hydrolyzing Pinus sylvestris L. buds, water and a mixed enzyme, and then inactivating the enzyme to obtain the Pinus sylvestris L. bud extract. The Pinus sylvestris L. bud extract prepared by the present invention can increase PMCA protein and does not depend on TRP series proteins. Therefore, no matter what kind of calcium ion influx is generated due to the activation of any TRP protein, the Pinus sylvestris L. bud extract can expel calcium ions in the cells out of the cells and reduce the calcium ion concentration in the cells. When applied in cosmetics, it has the effects of anti-inflammation, soothing and repairing the barrier.

Description

Technical Field

[0001] The present invention relates to the technical field of daily-use skin care products, and particularly relates to a Pinus sylvestris bud extract having an effect of regulating calcium ions in skin cells, a preparation method thereof, and an application thereof. Background Art

[0002] Calcium ions play a very important role in the physiological activities of the skin. As a unique second messenger, calcium ions play an important role in regulating biological processes such as cell proliferation, survival, apoptosis, and immune responses. The free calcium ions in cells generally maintain a very low concentration. If cells are in a high calcium concentration state for a long time, multiple inflammatory pathways will be activated, including the NLRP3 inflammasome, the JAK-STAT pathway, the NF-κB pathway, etc., leading to skin inflammation.

[0003] On the other hand, calcium ions have a dual regulatory effect on the keratinocytes of the skin. Low concentrations of calcium ions can promote the proliferation of keratinocytes and provide sufficient keratinocytes; high concentrations of calcium ions, on the contrary, will promote the differentiation of cells into the stratum corneum, making the stratum corneum more firm.

[0004] On the cell membrane of skin cells, there are channel proteins that control the entry and exit of calcium ions. Among them, the TRP series proteins and the ORAI series proteins can allow calcium ions to enter the cell from outside the cell. After these channel proteins are activated, the intracellular calcium ion concentration will increase. There is also a PMCA protein on the cell membrane that can pump the calcium ions in the cell out of the cell. The increase in the number of such channel proteins can accelerate the pumping of calcium ions in the cell to the outside of the cell and reduce the intracellular calcium ion concentration.

[0005] Therefore, there are many cosmetic raw materials that achieve a soothing and anti-inflammatory effect by regulating the calcium ion concentration in skin cells. However, most of these types of cosmetic raw materials produce effects by inhibiting the TRP series proteins. Since the TRP series includes multiple proteins from TRPV1 to TRPV7, and TRP inhibitors often can only inhibit one of these proteins and have no inhibitory effect on other proteins. Therefore, after using cosmetic raw materials with TRP inhibitory effects, it is not possible to well inhibit the entry of calcium ions into the cell interior. How to develop cosmetic raw materials with the effect of regulating calcium ions is a new research and development direction for those skilled in the art.

[0006] At present, many cosmetic raw materials on the market are derived from pine trees, but the specific varieties and extraction parts are not very clear, so the efficacy is unstable. Moreover, the common processes for plant extracts on the market involve high-temperature boiling, high-temperature pulverization, organic solvent extraction, etc. High-temperature environments often damage the active ingredients in plants. Organic solvent extraction can extract the components in plants that are insoluble in water, and these components often have higher biological activities. However, organic solvents are likely to cause environmental pollution, and the residues of organic solvents in cosmetic raw materials can cause harm to the skin. If organic solvents are not used and only water is used as the extraction medium, it is impossible to extract and dissolve the active ingredients in plants well.

[0007] Therefore, how to provide a method that can effectively extract the active ingredients in plants and is environmentally friendly and pollution-free is a technical problem that those skilled in the art urgently need to solve. Summary of the Invention

[0008] The purpose of the present invention is to provide a Pinus sylvestris bud extract with the effect of regulating calcium ions in skin cells, its preparation method and application. The present invention first discovers that the Pinus sylvestris bud extract has the effect of regulating calcium ions in skin cells, so when applied in cosmetics, it has the effects of anti-inflammation, soothing and repairing the barrier.

[0009] The purpose of the present invention is to provide a cosmetic raw material that can increase the protein expression of PMCA in skin cells, so that when the skin is stimulated, the calcium ion concentration inside the skin cells can be reduced, achieving the purpose of anti-inflammation, soothing and repairing the skin barrier.

[0010] In order to achieve the above-mentioned invention purposes, the present invention provides the following technical solutions:

[0011] The present invention provides a preparation method of a Pinus sylvestris bud extract, which is obtained by enzymatically hydrolyzing and inactivating enzymes a mixture of Pinus sylvestris buds, water and a mixed enzyme; the mixed enzyme is: cellulase, hemicellulase, pectinase, protease, SQ-SO28 extraction enzyme, SQ-IF27 extraction enzyme and SQ-ID6 extraction enzyme.

[0012] Pinus sylvestris is distributed in China, Europe and North America. We selected the Pinus sylvestris variety, Pinus sylvestris, which grows in the Nouvelle-Aquitaine region in the southwestern part of France, and further selected the buds of Pinus sylvestris for extraction.

[0013] Preferably, the mass ratio of the Pinus sylvestris buds, water and the mixed enzyme is 1-2:8-9:0.1-0.5.

[0014] Preferably, the mass ratio of the cellulase, hemicellulase, pectinase, protease, SQ-SO28 extraction enzyme, SQ-IF27 extraction enzyme, and SQ-ID6 extraction enzyme is: 1-2:1-2:1-2:1-2:1-1.5:1-1.5:1-1.5;

[0015] Preferably, the enzyme activities of the cellulase, hemicellulase, pectinase, protease, SQ-SO28 extraction enzyme, SQ-IF27 extraction enzyme, and SQ-ID6 extraction enzyme are: cellulase 2000-4000 U / mL, hemicellulase 2000-4000 U / mL, pectinase 1000-1500 U / mL, protease 1000-1500 U / mL, SQ-SO28 extraction enzyme 500-800 U / mL, SQ-IF27 extraction enzyme 500-800 U / mL, and SQ-ID6 extraction enzyme 500-800 U / mL.

[0016] Preferably, the temperature of the enzymatic hydrolysis is 25-30 °C; the time of the enzymatic hydrolysis is 3-6 h.

[0017] Preferably, the temperature for inactivating the enzyme is 50-60 °C, and the time for inactivating the enzyme is 30-60 min.

[0018] Preferably, after inactivating the enzyme, a filtration step is further included; the filtration method is: sequentially filtering with microporous membranes having diameters of 0.5 μm and 0.22 μm.

[0019] The present invention also provides a Pinus sylvestris L. bud extract prepared by the preparation method of the Pinus sylvestris L. bud extract described above.

[0020] The present invention also provides the application of the Pinus sylvestris L. bud extract in the preparation of skin care products.

[0021] Preferably, the skin care product has the effects of anti-inflammation and / or maintaining the calcium ion balance in cells.

[0022] Preferably, the skin care product is an eye cream, lotion, sunscreen product, concealer product, lotion or essence.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The Pinus sylvestris L. bud extract prepared by the present invention can increase the PMCA protein and does not depend on the TRP series of proteins. Therefore, no matter what causes the calcium ion influx due to the activation of any TRP protein, the Pinus sylvestris L. bud extract can expel the calcium ions in the cells out of the cells and reduce the calcium ion concentration in the cells. When applied in cosmetics, it has the effects of anti-inflammation, soothing, and repairing the barrier.

[0025] The present invention uses a low-temperature composite enzyme extraction process in the extraction process of European red pine buds. The composite enzyme can destroy the cell wall of plant cells under low temperature conditions to release active ingredients and avoid high temperature damage to the active ingredients. It can also ionize and glycosylate components that are difficult to dissolve in water and can be dissolved in water, so pure water can be used for extraction without the need for organic solvents.

[0026] The cellulase, hemicellulase and pectinase used in the present invention can all be used to decompose the cell walls of plant cells and release the active ingredients in the cells. Due to the characteristics of the cells of the European red pine buds, if only one or two of the three enzymes are used, the cell walls are not completely destroyed. Then the extract after filtration is transparent and clear, indicating that the soluble matter has not been released. And there is a large amount of residue on the microporous membrane of the first filtration. Therefore, these three enzymes need to be combined together to decompose the cell walls.

[0027] The active ingredients in the cells of European red pine buds, such as polyphenols, flavonoids, and polysaccharides, will bind to proteins in the cells to form complexes. If protease is not used, on the one hand, the active ingredients cannot be separated from the protein complex and cannot be released and dissolved; on the other hand, the final extract will be prone to precipitation. The present invention has been tested and if protease is not used, the color of the filtered extract is lighter, indicating that there are fewer active substances. After standing for 2 hours, flocculent precipitation begins to appear in the extract. And there is more residue on the microporous membrane of the second filtration. Therefore, it is necessary to add protease to the complex enzyme combination to promote the release of active ingredients.

[0028] Many of the active ingredients in the cells of European red pine buds are not water-soluble. Therefore, the present invention selects three kinds of extraction enzymes, SQ-SO28 extraction enzyme, SQ-IF27 extraction enzyme, and SQ-ID6 extraction enzyme. These three extraction enzymes can utilize the acid radical ions, sodium ions, potassium ions, and glucose in the plant cells to ionize and glycosylate the insoluble polyphenols and flavonoid components to form a state that can be dissolved in water. Therefore, an extract with stable properties can be obtained without the need for an organic solvent.

[0029] The present invention uses the combination of the above enzymes to extract European red pine buds, and the obtained extract has excellent biological activity and can be used as a cosmetic raw material to increase the content of PMCA protein in skin cells.

[0030] In the present invention, butanediol is added as a stabilizer after obtaining the refined extract, which can inhibit the growth of microorganisms in the extract during the subsequent transportation and storage of the extract, and can maintain the stability of the extract in high and low temperature environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can also be obtained based on the provided accompanying drawings.

[0032] Figure 1 Immunofluorescence staining diagrams of different groups for Example 4; among them, the left diagrams are all: nuclear staining, showing blue fluorescence, the middle diagrams are all: PMCA protein staining, showing red fluorescence, and the right diagrams are all: merged images.

[0033] Figure 2 Results diagrams of gene expression levels of different groups for Example 4.

[0034] Figure 3 Immunofluorescence staining diagram for Example 5; among them, in the left diagram: nuclear blue fluorescence, calcium ion green fluorescence; the right diagram is a partial enlarged view of the left diagram.

[0035] Figure 4 Calcium ion concentration curve for Example 5.

[0036] Figure 5 Immunofluorescence staining for Example 6, the left diagrams are all: NF-κB protein showing red fluorescence, the middle diagrams are all: nuclei showing blue fluorescence, and the right diagrams are all: merged images.

[0037] Figure 6 ELISA detection diagram for Example 6. Detailed implementation manners

[0038] The present invention provides a preparation method of a Pinus sylvestris var. mongolica bud extract. The Pinus sylvestris var. mongolica buds, water, and a mixed enzyme are mixed and enzymolyzed, and then the enzyme is inactivated to obtain the Pinus sylvestris var. mongolica bud extract; the mixed enzyme is: cellulase, hemicellulase, pectinase, protease, SQ-SO28 extraction enzyme, SQ-IF27 extraction enzyme, and SQ-ID6 extraction enzyme.

[0039] In the present invention, the mass ratio of the Pinus sylvestris var. mongolica buds, water, and the mixed enzyme is 1-2:8-9:0.1-0.5; preferably 1.5:8.5:0.2-0.4; more preferably 1.5:8.5:0.3.

[0040] In the present invention, the mass ratio of the cellulase, hemicellulase, pectinase, protease, SQ-SO28 extraction enzyme, SQ-IF27 extraction enzyme and SQ-ID6 extraction enzyme is: 1-2:1-2:1-2:1-2:1-1.5:1-1.5:1-1.5; preferably 1.5:1.5:1.5:1.5:1.1-1.4:1.1-1.4:1.1-1.4; more preferably 1.5:1.5:1.5:1.5:1.3:1.3:1.3.

[0041] In the present invention, the enzyme activities of the cellulase, hemicellulase, pectinase, protease, SQ-SO28 extraction enzyme, SQ-IF27 extraction enzyme and SQ-ID6 extraction enzyme are: cellulase 2000-4000 U / mL, hemicellulase 2000-4000 U / mL, pectinase 1000-1500 U / mL, protease 1000-1500 U / mL, SQ-SO28 extraction enzyme 500-800 U / mL, SQ-IF27 extraction enzyme 500-800 U / mL, SQ-ID6 extraction enzyme 500-800 U / mL; preferably cellulase 2400-3800 U / mL, hemicellulase 2400-3800 U / mL, pectinase 1100-1400 U / mL, protease 1100-1400 U / mL, SQ-SO28 extraction enzyme 600-700 U / mL, SQ-IF27 extraction enzyme 600-700 U / mL, SQ-ID6 extraction enzyme 600-700 U / mL; more preferably cellulase 2800-3200 U / mL, hemicellulase 2800-3200 U / mL, pectinase 1200-1300 U / mL, protease 1200-1300 U / mL, SQ-SO28 extraction enzyme 650 U / mL, SQ-IF27 extraction enzyme 650 U / mL, SQ-ID6 extraction enzyme 650 U / mL; even more preferably cellulase 3000 U / mL, hemicellulase 3000 U / mL, pectinase 1250 U / mL, protease 1250 U / mL, SQ-SO28 extraction enzyme 650 U / mL, SQ-IF27 extraction enzyme 650 U / mL, SQ-ID6 extraction enzyme 650 U / mL.

[0042] In the present invention, the cellulase, hemicellulase, pectinase, protease are derived from: Novozyme Corp.; the SQ-SO28 extraction enzyme, SQ-IF27 extraction enzyme and SQ-ID6 extraction enzyme are derived from: Proteus SA.

[0043] In the present invention, the temperature of the enzymatic hydrolysis is 25-30 °C; preferably 26-29 °C; more preferably 27-28 °C; even more preferably 28 °C.

[0044] In the present invention, the enzymatic hydrolysis time is 3 to 6 h; preferably 4 to 5 h; more preferably 5 h.

[0045] In the present invention, the temperature for inactivating the enzyme is 50 to 60 °C; preferably 52 to 58 °C; more preferably 54 to 56 °C; most preferably 55 °C.

[0046] In the present invention, the time for inactivating the enzyme is 30 to 60 min; preferably 35 to 55 min; more preferably 40 to 50 min; most preferably 45 min.

[0047] In the present invention, after inactivating the enzyme, a filtration step is further included; the filtration method is: sequentially filtering using microporous membranes with diameters of 0.5 μm and 0.22 μm.

[0048] The present invention also provides a Pinus sylvestris L. bud extract prepared by the preparation method of the Pinus sylvestris L. bud extract described above.

[0049] The present invention also provides the application of the Pinus sylvestris L. bud extract in the preparation of skin care products.

[0050] In the present invention, the skin care product has the efficacy of anti-inflammation and / or maintaining the calcium ion balance in cells.

[0051] In the present invention, the skin care product is an eye cream, lotion, sunscreen product, concealer product, lotion or essence; preferably an essence.

[0052] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention.

[0053] In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well-known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are conventional means well-known to those skilled in the art.

[0054] Example 1:

[0055] A preparation method of a Pinus sylvestris L. bud extract is as follows:

[0056] (1) Collect Pinus sylvestris L. buds, wash them with clear water, dry them until there is no moisture on the surface, package them with PE plastic bags, evacuate them, and store them in a cold storage at 4 °C for later use.

[0057] (2) Take out the buds of Pinus sylvestris var. mongolica Litv., put them into a reactor for extraction. Then add a compound enzyme, including: cellulase, hemicellulase, pectinase, protease, SQ-SO28 extraction enzyme, SQ-IF27 extraction enzyme, SQ-ID6 extraction enzyme.

[0058] The mass ratio of the buds of Pinus sylvestris var. mongolica Litv., water and the mixed enzyme is 1:8:0.1;

[0059] The mass ratio of cellulase, hemicellulase, pectinase, protease, SQ-SO28 extraction enzyme, SQ-IF27 extraction enzyme and SQ-ID6 extraction enzyme is: 1:1:1:1:1:1:1;

[0060] The enzyme activities of cellulase, hemicellulase, pectinase, protease, SQ-SO28 extraction enzyme, SQ-IF27 extraction enzyme and SQ-ID6 extraction enzyme are: cellulase 2000 U / mL, hemicellulase 2000 U / mL, pectinase 1000 U / mL, protease 1000 U / mL, SQ-SO28 extraction enzyme 500 U / mL, SQ-IF27 extraction enzyme 500 U / mL, SQ-ID6 extraction enzyme 500 U / mL;

[0061] Control the temperature at 25 °C, keep warm for 3 h, continuously stir the mixture in the reactor to obtain a crude extract.

[0062] (3) Heat the crude extract in step (2) to 50 °C and keep warm for 30 min to inactivate the compound enzyme.

[0063] (4) Transfer the crude extract in step (3) to a sealed container and perform two high-pressure filtrations using a microporous membrane. The diameter of the microporous membrane for the first filtration is 0.5 μm, and the diameter of the microporous membrane for the second filtration is 0.22 μm. After filtration, a refined extract is obtained.

[0064] (5) Keep the refined extract in step (4) in a sealed container and add butanediol with the same mass as the refined extract through a suction pump.

[0065] (6) Transfer the refined extract obtained in step (5) to a sterile room, perform sub-packaging and sealing in the sterile room to finally obtain the extract of the buds of Pinus sylvestris var. mongolica Litv.

[0066] Example 2:

[0067] A preparation method of the extract of the buds of Pinus sylvestris var. mongolica Litv. is as follows:

[0068] (1) Collect the buds of Pinus sylvestris var. mongolica Litv., wash them with clear water, dry them until there is no moisture on the surface, package them with PE plastic bags, evacuate them, and store them in a cold storage at 4 °C for later use.

[0069] (2) Take out the buds of Pinus sylvestris var. mongolica Litv., put them into a reactor for extraction. Then add a composite enzyme, including: cellulase, hemicellulase, pectinase, protease, SQ-SO28 extraction enzyme, SQ-IF27 extraction enzyme, SQ-ID6 extraction enzyme.

[0070] The mass ratio of the buds of Pinus sylvestris var. mongolica Litv., water and the mixed enzyme is 2:9:0.5;

[0071] The mass ratio of cellulase, hemicellulase, pectinase, protease, SQ-SO28 extraction enzyme, SQ-IF27 extraction enzyme and SQ-ID6 extraction enzyme is: 2:2:2:2:1.5:1.5:1.5;

[0072] The enzyme activities of cellulase, hemicellulase, pectinase, protease, SQ-SO28 extraction enzyme, SQ-IF27 extraction enzyme and SQ-ID6 extraction enzyme are: cellulase 4000 U / mL, hemicellulase 4000 U / mL, pectinase 1500 U / mL, protease 1500 U / mL, SQ-SO28 extraction enzyme 800 U / mL, SQ-IF27 extraction enzyme 800 U / mL, SQ-ID6 extraction enzyme 800 U / mL;

[0073] Control the temperature at 30 °C, keep warm for 6 h, continuously stir the mixture in the reactor to obtain a crude extract.

[0074] (3) Heat the crude extract in step (2) to 60 °C and keep warm for 60 min to inactivate the composite enzyme.

[0075] (4) Transfer the crude extract in step (3) to a sealed container and perform two high-pressure filtrations using a microporous membrane. The diameter of the microporous membrane for the first filtration is 0.5 μm, and the diameter of the microporous membrane for the second filtration is 0.22 μm. After filtration, a refined extract is obtained.

[0076] (5) Keep the refined extract in step (4) in a sealed container and add butanediol with the same mass as the refined extract through a suction pump.

[0077] (6) Transfer the extract obtained in step (5) to a sterile room, perform sub-packaging and sealing in the sterile room, and finally obtain the extract of the buds of Pinus sylvestris var. mongolica Litv.

[0078] Example 3:

[0079] A preparation method of the extract of the buds of Pinus sylvestris var. mongolica Litv. is as follows:

[0080] (1) Collect the buds of Pinus sylvestris var. mongolica Litv., wash them with clear water, dry them until there is no moisture on the surface, package them with a PE plastic bag, evacuate the air, and store them in a cold storage at 4 °C for later use.

[0081] (2) Take out the buds of Pinus sylvestris var. mongolica Litv. and put them into a reactor for extraction. Then add a compound enzyme, including: cellulase, hemicellulase, pectinase, protease, SQ-SO28 extraction enzyme, SQ-IF27 extraction enzyme, and SQ-ID6 extraction enzyme.

[0082] The mass ratio of the buds of Pinus sylvestris var. mongolica Litv., water and the mixed enzyme is 1.5:8.5:0.3;

[0083] The mass ratio of cellulase, hemicellulase, pectinase, protease, SQ-SO28 extraction enzyme, SQ-IF27 extraction enzyme, and SQ-ID6 extraction enzyme is: 1.5:1.5:1.5:1.5:1.3:1.3:1.3;

[0084] The enzyme activities of cellulase, hemicellulase, pectinase, protease, SQ-SO28 extraction enzyme, SQ-IF27 extraction enzyme, and SQ-ID6 extraction enzyme are: cellulase 3000 U / mL, hemicellulase 3000 U / mL, pectinase 1250 U / mL, protease 1250 U / mL, SQ-SO28 extraction enzyme 650 U / mL, SQ-IF27 extraction enzyme 650 U / mL, SQ-ID6 extraction enzyme 650 U / mL;

[0085] Control the temperature at 28 °C, keep warm for 5 h, continuously stir the mixture in the reactor to obtain a crude extract.

[0086] (3) Heat the crude extract in step (2) to 55 °C and keep warm for 45 min to inactivate the compound enzyme.

[0087] (4) Transfer the crude extract in step (3) to a sealed container and perform two high-pressure filtrations using a microporous membrane. The diameter of the microporous membrane for the first filtration is 0.5 μm, and the diameter of the microporous membrane for the second filtration is 0.22 μm. After filtration, a refined extract is obtained.

[0088] (5) Keep the refined extract in step (4) in a sealed container and add butanediol with the same mass as the refined extract through a suction pump.

[0089] (6) Transfer the extract obtained in step (5) to a sterile room, perform sub-packaging and sealing in the sterile room, and finally obtain the extract of the buds of Pinus sylvestris var. mongolica Litv.

[0090] Example 4: The extract of the buds of Pinus sylvestris var. mongolica Litv. increases the expression of PMCA

[0091] 4.1. Test purpose:

[0092] Detect the gene and protein expression levels of PMCA in human skin epidermal cells HaCaT cells.

[0093] The calcium ion concentration inside the cell is lower than that outside the cell. The efflux of calcium ions from the cell is against the concentration gradient and is an energy-consuming active transport. For every molecule of ATP hydrolyzed by PMCA, one calcium ion bound to it can be transported from the cytoplasm to the extracellular space. The increase in PMCA can promote the efflux of calcium ions from the cell interior, achieving the effect of maintaining the stability of intracellular calcium ions.

[0094] 4.2, Materials and Equipment:

[0095] Human skin epidermal cells (human immortalized keratinocyte cell line HaCaT) were purchased from CLS Cell Lines Service GmbH, Germany.

[0096] Keratinocyte Growth Medium (KGM), fetal bovine serum, high-glucose DMEM medium, and cell culture-related reagents were purchased from Gibco.

[0097] Reverse transcription kits, basic chemical reagents, lysis buffer, and related proteases were from Thermo Fisher Scientific, Hyclone, TaKaRa, and SIGMA Aldrich.

[0098] Immunofluorescence staining and PMCA-related antibodies were from Abcam, USA.

[0099] RT-PCR equipment was from Thermo Fisher Scientific.

[0100] Fluorescence confocal microscopy imaging system was from Leica.

[0101] 4.3, Test Methods:

[0102] Cell Culture:

[0103] Human skin epidermal cells HaCaT were cultured. After cell resuscitation and 7 days of in vitro culture to form a confluent monolayer, and confirmed by microscopy to have the morphology of keratinocytes. Passage culture was performed, the cell number was adjusted, and they were inoculated into culture well plates for testing.

[0104] The test group was added with the active ingredient Pinus sylvestris L. bud extract (prepared by the method of Example 3, the same below) at concentrations of 0.2% (active substance 0.2%) and 0.6% (active substance 0.6%) according to the mass fraction of the culture medium. The control group was not added with the active ingredient. The cells were incubated for 24 hours, washed, and then subjected to subsequent tests. RT-PCR was used to determine the gene expression level, and cell immunofluorescence staining was used to determine the protein expression level.

[0105] The method of RT-PCR was: For every 1×10 6Total RNA was extracted from the cells by adding 1 mL of Trizol reagent. After measuring the concentration, it was reverse transcribed into cDNA using a reverse transcription kit. The gene expression level of PMCA was calculated based on the Ct value.

[0106] The method of immunofluorescence staining was as follows: The cells were treated with standard procedures, including cell fixation, permeabilization with Triton, and blocking with BSA. After washing with PBS, incubation with the primary antibody and then the secondary antibody was performed in sequence. After washing, DAPI staining was carried out and the slides were mounted. Fluorescence microscopy was used to take pictures after washing. The fluorescence intensity was calculated to obtain the PMCA protein expression level.

[0107] 4.4. Test results:

[0108] The results are as Figure 1 and Figure 2 : Compared with the control group, in the cells with 0.2% Pinus sylvestris bud extract added (active substance 0.2%), the expression of PMCA protein increased by 84%, and in the cells with 0.6% Pinus sylvestris bud extract added (active substance 0.6%), the expression of PMCA protein increased by 152%. In this experiment, all data had p < 0.01. Therefore, Pinus sylvestris bud extract increased the gene expression and protein expression of PMCA in human skin epidermal cells HaCaT cells. It can promote the faster excretion of calcium ions from the cells, which is beneficial to maintaining the free calcium ion concentration in the cytoplasm at an extremely low level.

[0109] Example 5: Pinus sylvestris bud extract maintains intracellular calcium ion balance

[0110] 5.1. Test purpose:

[0111] To detect the changes in calcium ions in human skin epidermal cells HaCaT cells.

[0112] The calcium ion concentration in the cells is finely regulated. It is about 10 - 100 nmol / L inside the cells and about 1 - 2 mmol / L outside the cells. When keratinocytes are stimulated, calcium channels, such as TRP channels, will open, and calcium ions will flow into the cells to form calcium ion peaks, which will further activate the inflammatory pathway and skin nerve cells, resulting in a stinging sensation on the skin. The increase in PMCA can promote the excretion of calcium ions from inside the cells, achieving the effect of maintaining the stability of intracellular calcium ions.

[0113] 5.2. Materials and equipment:

[0114] Human skin epidermal cells (human immortalized keratinocyte cell line HaCaT) were purchased from CLS Cell Lines Service GmbH, Germany.

[0115] Keratinocyte Growth Medium (KGM), fetal bovine serum, high-glucose DMEM medium, and cell culture-related reagents were purchased from Gibco.

[0116] Dihydrocapsaicin, lysis buffer, and related proteases were from ThermoFisher Scientific, Hyclone, TaKaRa, and SIGMA Aldrich.

[0117] Calcium ion fluorescence detection Fluo 4-AM kit was from ThermoFisher Scientific.

[0118] Fluorescence confocal microscopy imaging system was from Leica.

[0119] 5.3. Test method:

[0120] Cell culture: Human skin epidermal cells HaCaT were cultured in vitro for 7 days after cell resuscitation until they grew into a confluent monolayer. After confirmation by microscopy, they had the morphology of keratinocytes. They were subcultured, the cell number was adjusted, and they were inoculated into culture wells for testing.

[0121] In the 0.5% active ingredient group, the active ingredient Pinus sylvestris L. bud extract (prepared by the method of Example 3, the same below) was added at a concentration of 0.5% according to the mass fraction of the culture medium. The control group did not add the active ingredient, and the cells were incubated for 24 hours. The culture medium was changed, and the active substance was washed away for subsequent testing.

[0122] Calcium ion fluorescence detection: In each cell culture well, according to the specified amount in the kit instructions, Fluo 4-AM fluorescent reagent was added and incubated at 37°C for 30 minutes, and then the fluorescent reagent was washed away. Buffer was added, DAPI was added for staining, and after incubation at room temperature for 30 minutes, it was washed away. The stimulant dihydrocapsaicin was added, and image recording was performed using a fluorescence confocal microscopy imaging system.

[0123] 5.4. Test results are as Figure 3 and Figure 4 : The cell nucleus shows blue fluorescence, and calcium ions show green fluorescence. The right figure is a partial magnification of the left figure (cropped 60 seconds after the addition of the stimulant). Compared with the control group, in the cells with 0.5% Pinus sylvestris L. bud extract added, the green fluorescence was significantly reduced, indicating that the calcium ion concentration remained at a very low level.

[0124] The fluorescence intensity of calcium ions was recorded by video, and the data was recorded as a data chart using analysis software. Within dozens of seconds after the addition of the stimulant, the calcium ion concentration in the control group cells increased rapidly, forming a calcium ion peak. In the cells treated with the active substance, the increase rate of calcium ions was very slow. The peak value of the calcium ion concentration also decreased a lot.

[0125] Therefore, the extract of Pinus sylvestris buds increases the expression of PMCA in human skin epidermal HaCaT cells, which can promote the faster excretion of calcium ions from cells. Under the stimulation of dihydrocapsaicin, the TRP receptors of cells are activated, and calcium ions rapidly flow into the cells, forming a calcium ion peak. In cells treated with the active substance, calcium ions can be quickly excreted from the cells, and the rising speed of calcium ions is very slow, and almost no calcium ion peak is generated.

[0126] Example 6: The extract of Pinus sylvestris buds inhibits the expression of inflammation-related factors

[0127] 6.1. Test purpose:

[0128] To detect the expression levels of inflammation-related factors in human skin epidermal HaCaT cells.

[0129] The calcium ion concentration in cells is finely regulated, and the calcium ion concentration inside the cells is lower than that outside the cells. When keratinocytes are stimulated, calcium ions will flow into the cells. If the cells are in a high calcium ion state for a long time, it will further activate multiple inflammatory pathways, including the NF-κB inflammatory pathway.

[0130] 6.2. Materials and equipment:

[0131] Human skin epidermal cells (human immortalized keratinocyte cell line HaCaT), purchased from CLS Cell Lines Service GmbH, Germany.

[0132] Keratinocyte growth medium (KGM), fetal bovine serum, DMEM high-glucose medium, and cell culture-related reagents, purchased from Gibco.

[0133] Serine protease, basic chemical reagents, lysis buffer, related proteases, from Thermo Fisher Scientific, Hyclone, TaKaRa, SIGMA Aldrich.

[0134] Immunofluorescence staining, related antibodies corresponding to each inflammatory factor, from Abcam, USA.

[0135] ELISA microplate reader, from Thermo Fisher Scientific.

[0136] Fluorescence confocal microscope imaging system, from Leica.

[0137] 6.3. Test method:

[0138] Cell culture: Human skin epidermal cells HaCaT were resuscitated and cultured in vitro for 7 days to grow into a confluent monolayer. After confirmation under a microscope, they had the morphology of keratinocytes. Passage culture was performed, the cell number was adjusted, and they were inoculated into culture well plates for testing.

[0139] In the test group, the active ingredient Pinus sylvestris L. bud extract (prepared by the method of Example 3, the same below) was added at concentrations of 0.2% and 0.6% by mass fraction of the culture medium. The control group was not added with the active ingredient. The cells were incubated for 24 hours, washed, and then subjected to subsequent tests.

[0140] The specific test method was as follows: Serine protease at 100 ng / mL was added and cultured for 1 hour. After washing, the cells were further cultured for 6 hours to induce the cells to enter an inflammatory state.

[0141] Then, the protein expression level of NF-κB was determined by immunofluorescence staining of cells. ELISA was used to detect the protein expression levels of phosphorylated IKK, IL-1β, IL-6, IL-8, NLRP3, and ASC.

[0142] The specific method was immunofluorescence staining: The cells were treated with standard procedures, including cell fixation, permeabilization with Triton, and blocking with BSA. After washing with PBS, incubation with the primary antibody and then the secondary antibody was performed in sequence. After washing, DAPI staining was carried out and the slides were sealed. After washing, fluorescence microscopy was used to take pictures. The fluorescence intensity was calculated using the corresponding software to obtain the protein expression level of NF-κB.

[0143] ELISA detection: The cells were treated with standard procedures, including adding lysis buffer after cell suspension, lysing and extracting with an ultrasonic extractor, and collecting the supernatant after centrifugation. Incubation with the primary antibody and then the secondary antibody was performed in sequence. After washing, HRP working solution and TMB substrate solution were added in sequence, and the reaction was stopped by adding the stop solution. Detection was carried out with an enzyme-linked immunosorbent assay (ELISA) reader to obtain the expression levels of the corresponding proteins.

[0144] 6.4 Test results:

[0145] The results were as Figure 5 and Figure 6 : Immunofluorescence staining: Stimulation with serine protease would increase the intracellular calcium ion concentration. When the cells were in a high calcium ion state for a long time, the inflammatory signaling pathway would be activated. From Figure 5It can be seen that, compared with the blank group, the stimulation of serine protease increased the NF-κB protein in the control group by 375% (p < 0.01). Compared with the control group, in the cells with 0.2% Pinus sylvestris bud extract added (active substance 0.2%), the expression of NF-κB protein decreased by 49%, and in the cells with 0.6% Pinus sylvestris bud extract added (active substance 0.6%), the expression of NF-κB protein decreased by 64% (all data in this experiment were p < 0.01).

[0146] ELISA detection: The stimulation of serine protease will increase the calcium ion concentration in cells. When cells are in a high calcium ion state for a long time, the inflammatory signaling pathway will be activated. From Figure 6 It can be seen that, compared with the control group, in the cells with 0.2% and 0.6% active substances (Pinus sylvestris bud extract) added, the protein expression levels of various inflammatory factors decreased to varying degrees. In this experiment, taking the data of the control group as the standard, the reduction ratios of various indicators of different groups relative to the control group were calculated, and all data were p < 0.01.

[0147] Test conclusion: Serine protease can activate the PAR2 pathway of human skin epidermal cells HaCaT cells, and further increase the calcium ion concentration in cells through multiple signaling pathways. When cells are in a high calcium ion state for a long time, it will activate calcineurin, calmodulin kinase II CaMKII, protein kinase PKCa, calcium ion-dependent protein kinase PNCK, and further activate the NF-κB inflammatory pathway and other inflammatory signaling pathways. Through this experiment, we observed that, compared with the blank group, the stimulation of serine protease increased the NF-κB protein in the control group by 375% (p < 0.01).

[0148] The Pinus sylvestris bud extract increased the expression of PMCA in HaCaT cells. It can promote the faster excretion of calcium ions from cells. Even under the stimulation of serine protease, the cells treated with the active substance can maintain a low calcium ion concentration in cells and reduce various different inflammatory factors.

[0149] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a European red pine bud extract, characterized in that: The European red pine bud extract is obtained by mixing European red pine buds, water and mixed enzymes for enzymolysis and inactivating the enzymes; the mixed enzymes are: cellulase, hemicellulase, pectinase, protease, SQ-SO28 extraction enzyme, SQ-IF27 extraction enzyme and SQ-ID6 extraction enzyme; The mass ratio of the cellulase, hemicellulase, pectinase, protease, SQ-SO28 extracting enzyme, SQ-IF27 extracting enzyme and SQ-ID6 extracting enzyme is: 1-2:1-2:1-2:1-2:1-1.5:1-1.5:1-1.5; The cellulase, hemicellulase, pectinase and protease are from Novozyme Corp.; SQ-SO28 extracting enzyme, SQ-IF27 extracting enzyme and SQ-ID6 extracting enzyme are from Proteus SA; The mass ratio of the European red pine buds, water and mixed enzyme is 1-2:8-9:0.1-0.

5.

2. The method for preparing a pine bud extract according to claim 1, characterized in that: The enzymatic activities of the cellulase, hemicellulase, pectinase, protease, SQ-SO28 extracting enzyme, SQ-IF27 extracting enzyme and SQ-ID6 extracting enzyme are as follows: cellulase 2000-4000 U / mL, hemicellulase 2000-4000 U / mL, pectinase 1000-1500 U / mL, protease 1000-1500 U / mL, SQ-SO28 extracting enzyme 500-800 U / mL, SQ-IF27 extracting enzyme 500-800 U / mL, SQ-ID6 extracting enzyme 500-800 U / mL.

3. The method for preparing a pine bud extract according to claim 1, characterized in that: The temperature of the enzymolysis is 25-30° C.; the time of the enzymolysis is 3-6 hours.

4. The method for preparing a pine bud extract according to claim 1, characterized in that: The enzyme inactivation temperature is 50-60° C., and the enzyme inactivation time is 30-60 min.

5. The method for preparing a Scots pine bud extract according to claim 1 or 4, characterized in that: After the enzyme is inactivated, a filtering step is also included; the filtering method is: filtering is performed using microporous membranes with diameters of 0.5 μm and 0.22 μm in sequence.

6. The European red pine bud extract obtained by the preparation method of the European red pine bud extract according to any one of claims 1 to 5.

7. Use of the European red pine bud extract according to claim 6 in preparing skin care products.

8. The use according to claim 7, characterized in that: The skin care product has the effects of anti-inflammation and / or maintaining the balance of calcium ions in cells.

9. The use according to claim 7 or 8, characterized in that: The skin care product is eye cream, lotion, sunscreen product, concealer product, toner or essence.

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