Broussonetia kaempferi extract as well as preparation method and application thereof

Abstract

The invention relates to a flammulina velutipes extract as well as a preparation method and application thereof. The preparation method comprises the following steps: S1, carrying out low-temperature ultrasonic cold extraction on dried and crushed flammulina velutipes by using a first solvent, filtering, and drying filter residues for later use; s2, carrying out heating reflux extraction on the dried filter residue by using a second solvent, filtering, repeating the operation for 2-3 times, merging the filtrate, and concentrating the filtrate to obtain a first extracting solution; s3, macroporous resin serves as an adsorbent, water, a third solvent and a fourth solvent are adopted to elute the first extracting solution, the fourth solvent elution part is collected and concentrated, and a second extracting solution is obtained; s4, decolorizing the second extracting solution by adopting a decolorizing agent, heating, stirring and filtering to obtain a third extracting solution; s5, concentrating and drying the third extracting solution to obtain the flame tree flower extract. The prepared flammulina velutipes extract is rich in active ingredient catalpol 6-caffeic acid ester, and the extract can be used for skin barrier repair.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and more specifically, relates to an extract of Spathodea campanulata Beauv. flowers, a preparation method thereof, and an application thereof. Background Art

[0002] The flowers of Spathodea campanulata Beauv. are the dried flower buds of Spathodea Campanulata Beauv. belonging to the genus Spathodea of the family Bignoniaceae. According to relevant literature records, the flowers of Spathodea campanulata Beauv. have high medicinal value. Research shows that the flowers of Spathodea campanulata Beauv. are rich in chemical components such as iridoid glycosides, alkaloids, saponins, glycosides, flavonoids, triterpenes, and tannins, and have pharmacological effects such as antibacterial, antioxidant, anti-inflammatory, antimalarial, and anti-ultraviolet. However, in existing reports, no preparation process of an extract of Spathodea campanulata Beauv. flowers suitable for industrial production and relevant research on applying the extract of Spathodea campanulata Beauv. flowers to the field of skin barrier repair have been found.

[0003] In addition, the main processes currently used for the preparation of plant extracts are: ① extraction with inorganic / organic solvents combined with macroporous resin elution; ② extraction with inorganic / organic solvents combined with macroporous resin elution and decolorization with a decolorizing agent; ③ extraction with inorganic / organic solvents combined with enzymatic hydrolysis; ④ extraction with inorganic / organic solvents combined with alcohol precipitation, etc. Although the above-mentioned plant extract preparation processes can extract the target substances, they cannot well balance the removal of pigments, impurities, and lipophilic impurities in the target substances, and the enzymatic hydrolysis process among them has strict requirements for the control of operating parameters. The above factors will all affect the extraction effect and use performance of the extract.

[0004] Therefore, there is an urgent need to propose an extract of Spathodea campanulata Beauv. flowers, a preparation method thereof, and an application thereof to solve the technical problem that the current preparation process of the extract of Spathodea campanulata Beauv. flowers cannot efficiently balance the preparation of the extract of Spathodea campanulata Beauv. flowers and the removal of pigments and impurities in the extract of Spathodea campanulata Beauv. flowers, resulting in poor extraction effect and quality of the extract, affecting the use performance of the extract of Spathodea campanulata Beauv. flowers. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the main purpose of the present invention is to propose an extract of Spathodea campanulata Beauv. flowers, a preparation method thereof, and an application thereof to solve at least one of the technical problems that the current preparation process of the extract of Spathodea campanulata Beauv. flowers cannot efficiently balance the preparation of the extract of Spathodea campanulata Beauv. flowers and the removal of pigments and impurities in the extract of Spathodea campanulata Beauv. flowers, resulting in poor extraction effect and quality of the extract, not being suitable for industrial production, and affecting the use performance of the extract of Spathodea campanulata Beauv. flowers.

[0006] To achieve this purpose, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a flame tree flower extract, wherein the flame tree flower extract comprises an active ingredient, catalpol 6-caffeate, and the mass percentage of catalpol 6-caffeate in the flame tree flower extract is not less than 41%.

[0008] In a second aspect, the present invention provides a method for preparing a flame tree flower extract, the preparation method comprising the following steps:

[0009] Step S1: extracting the dried and crushed flame tree flower with a first solvent at low temperature and ultrasonically, filtering, and drying the filter residue for later use;

[0010] Step S2: extracting the dried filter residue by heating it with a second solvent under reflux, filtering it, repeating the operation 2-3 times, combining the filtrates, and concentrating the filtrates to obtain a first extract;

[0011] Step S3: using a macroporous resin as an adsorbent, using water, a third solvent, and a fourth solvent to elute the first extract, collecting the fourth solvent elution portion, and concentrating to obtain a second extract;

[0012] Step S4: Decolorizing the second extract with a decolorizing agent, wherein the decolorizing agent is compounded from activated carbon and silica gel, and the amount of the decolorizing agent added is 0.1%-1% by mass of the second extract, heating, stirring, and filtering to obtain a third extract;

[0013] Step S5: concentrating and drying the third extract to obtain the flame tree flower extract.

[0014] Preferably, the first solvent in step S1 is an ethanol aqueous solution, and the mass percentage content of ethanol is greater than 95%; the second solvent in step S2 is an ethanol aqueous solution, and the mass percentage content of ethanol is 35%-45%; the third solvent in step S3 is an ethanol aqueous solution, and the mass percentage content of ethanol is 15%-25%; the fourth solvent in step S3 is an ethanol aqueous solution, and the mass percentage content of ethanol is 40%-60%.

[0015] It should be noted that the flame tree flower is rich in anthocyanins and a small proportion of carotenoids. Anthocyanins give the flame tree flower a bright red color, while carotenoids provide the flame tree flower with yellow and orange colors. However, during the extraction process of the flame tree flower extract, excessive pigments in the extract are not conducive to its subsequent application.

[0016] In step S1, the dried and pulverized flame tree flowers are subjected to low-temperature ultrasonic cold extraction with a first solvent. Among them, ultrasonic low-temperature cold extraction combines the cavitation effect of ultrasonic waves and the characteristics of low-temperature extraction. Generally speaking, the formation conditions of the ultrasonic cavitation effect vary according to the types of plants to be extracted. In the present invention, for flame tree flowers, there is a specific ultrasonic power in the first solvent (ethanol aqueous solution) that generates the cavitation effect to release anthocyanins and carotenoids while destroying the cell structure of the flame tree flowers. Anthocyanins are easily soluble in the polar solvent ethanol aqueous solution, while carotenoids are fat-soluble pigments and are slightly soluble in the polar solvent ethanol aqueous solution. In addition, at low temperatures, while removing pigments with ethanol water, the active ingredient catalpol 6-caffeate can be retained to the greatest extent. Combining the filtration operation in step S1 can basically remove the anthocyanins contained in the flame tree flowers and remove a part of the fat-soluble pigments and impurities. In step S4, the decolorizing agent obtained by compounding activated carbon and silica gel can further remove the remaining fat-soluble carotenoids and other impurities. Step S1 and step S4 can act synergistically to make the decolorizing effect more significant and retain the active ingredient catalpol 6-caffeate in the flame tree flower extract to the greatest extent.

[0017] Preferably, the operating conditions of the low-temperature ultrasonic cold extraction in step S1 are: ultrasonic power 250 - 400w, extraction times 2 - 3 times, and extraction temperature 2 - 8°C.

[0018] It should be noted that the ultrasonic power is also preferably 260 - 390W, also preferably 270 - 380W, also preferably 280 - 370W, also preferably 290 - 360W, also preferably 300 - 350W, also preferably 310 - 340W, also preferably 320 - 330W, and also preferably 320W.

[0019] The extraction temperature is also preferably 3 - 7°C, also preferably 4°C, also preferably 5°C, and also preferably 6°C.

[0020] Preferably, the macroporous resin in step S3 is selected from any one of D101, AB-8, HP-20, HPD-100, and MCI-gel CHP 20P.

[0021] Preferably, the mass ratio of the compounding of activated carbon and silica gel in step S4 is 1:1 to 3:1.

[0022] Preferably, the mass ratio of the compounding of activated carbon and silica gel in step S4 is 2:1.

[0023] Preferably, the silica gel in step S4 is replaced by zeolite molecular sieve.

[0024] Preferably, the pore size numerical range of the zeolite molecular sieve in step S4 is 0.5 - 2nm.

[0025] In a third aspect, the present invention provides an application of a flame tree flower extract in skin care products. The flame tree flower extract contains the active ingredient catalpol 6-caffeate, and the extract is used to up-regulate the expression of skin barrier repair factors.

[0026] Preferably, the skin barrier repair factors include at least one of the FLG gene, the LOR gene, the Claudin4 gene, and the Claudin5 gene.

[0027] Preferably, in an in vitro skin barrier model, when the concentration of the flame tree flower extract is 60 μg / ml, the up-regulation rate of the FLG gene expression is 33%-51%, and the up-regulation rate of the LOR gene expression is 44%-61%.

[0028] Preferably, in a 3D skin barrier model, when the concentration of the flame tree flower extract is 600 μg / ml, the up-regulation rate of the FLG gene expression is 15%-26%, the up-regulation rate of the LOR gene expression is 47%-62%, the up-regulation rate of the Claudin4 gene expression is 225%-260%, and the up-regulation rate of the Claudin5 gene expression is 10%-28%.

[0029] Compared with the prior art, the present invention has at least the following beneficial effects:

[0030] (1) The preparation method of a flame tree flower extract provided by the present invention is simple in operation, low in cost, green and environmentally friendly, can be industrially produced, and can ensure a high yield and an active ingredient content (catalpol 6-caffeate) to meet the usage requirements for its skin barrier repair efficacy.

[0031] (2) For the preparation method of a flame tree flower extract provided by the present invention, a decolorization process combining a pre-decolorization process for the flame tree flower raw material and a post-decolorization process for the extract is adopted. In addition, in the operation steps of the post-decolorization process for the extract, a decolorizing agent obtained by compounding activated carbon and silica gel is further used, which can comprehensively achieve a significant decolorization effect without affecting the content of the active ingredient; the silica gel can be further replaced by zeolite molecular sieve to further enhance the physical adsorption effect on pigment molecules. The content of catalpol 6-caffeate in the flame tree flower extract prepared by the preparation method is not less than 41%.

[0032] (3) Application of an extract of Spathodea campanulata flowers in skin care products. It is found for the first time that the extract of Spathodea campanulata flowers has the effect of repairing the skin barrier, specifically manifested as follows: in an in vitro skin barrier model, the extract of Spathodea campanulata flowers can significantly improve the expression of skin barrier repair genes FLG gene and LOR gene at a concentration of 60 μg / ml. The promotion rate of the FLG gene is 33%-51%, and the promotion rate of the LOR gene is 44%-61%. In a 3D skin barrier model, the extract of Spathodea campanulata flowers can significantly improve the expression of skin barrier repair factors: FLG gene, LOR gene, Claudin4 gene, and Claudin5 gene at a concentration of 600 μg / ml. The promotion rate of the FLG gene expression is 15%-26%, the promotion rate of the LOR gene expression is 47%-62%, the promotion rate of the Claudin4 gene expression is 225%-260%, and the promotion rate of the Claudin5 gene expression is 10%-28%. The highest up-regulation rate of skin barrier repair factors is 260%, showing a good effect of maintaining and repairing the skin barrier. Description of the Drawings

[0033] Figure 1 Shows the chromatogram of the extract of Spathodea campanulata flowers prepared in Example 1;

[0034] Figure 2 Shows the schematic diagram of the preparation method steps of the extract of Spathodea campanulata flowers of the present application. Detailed Embodiments

[0035] The technical solution of the present invention will be further described below with reference to the drawings and through specific embodiments. However, the following examples are only simple examples of the present invention and do not represent or limit the scope of the protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0036] The source information of relevant raw materials and materials is as follows:

[0037] HaCaT cells (Shanghai Institute of Cells, Chinese Academy of Sciences; culture conditions: DMEM culture medium, fetal bovine serum with a volume percentage concentration of 10%);

[0038] The MTT detection kit is from Xinbosheng Biotechnology Co., Ltd.;

[0039] The CCK-8 detection kit is from Xinbosheng Biotechnology Co., Ltd.;

[0040] The catalpol 6-caffeate standard product is purchased from Sichuan Weikeqi Biotechnology Co., Ltd.;

[0041] The raw materials, consumables or reagents involved in the present invention are all conventional products that can be obtained through commercial purchase.

[0042] Term Explanation:

[0043] FLG gene: The filaggrin (FLG) gene is located on human chromosome 1q21.3, and its translation expression product is closely related to maintaining skin barrier and cell differentiation functions.

[0044] LOR gene: The loricrin (LOR) gene is a major component of the cornified envelope of terminally differentiated keratinocytes and plays an important role in the barrier function of the epidermis.

[0045] Claudin4 gene: The Claudin4 gene is located on human chromosome 7q11.23, and the protein it expresses is a tight junction protein that forms tight junctions between cells and regulates intercellular permeability; tight junctions are a type of intercellular junction in epithelial and endothelial cell layers and play a key role in maintaining tissue integrity and barrier function.

[0046] Claudin5 gene: The Claudin5 gene is located on human chromosome 22q11.21, and the protein it expresses is a tight junction protein that is particularly important in the tight junctions of brain endothelial cells and is crucial for maintaining the integrity of the blood-brain barrier.

[0047] See Figure 2 , the present invention provides a preparation method of a flame tree flower extract, and the preparation method includes the following steps:

[0048] Step S1: Subject the dried and pulverized flame tree flowers to low-temperature ultrasonic cold extraction with a first solvent, filter, and dry the filter residue for later use;

[0049] Step S2: Subject the dried filter residue to heating and reflux extraction with a second solvent, filter, repeat the operation 2-3 times, combine the filtrates, and concentrate the filtrates to obtain a first extract;

[0050] Step S3: Use macroporous resin as an adsorbent, elute the first extract with water, a third solvent, and a fourth solvent, collect the portion eluted with the fourth solvent, and concentrate to obtain a second extract;

[0051] Step S4: Decolorize the second extract with a decolorizing agent, which is obtained by compounding activated carbon and silica gel, and the addition amount of the decolorizing agent accounts for 0.1%-1% of the mass of the second extract, heat and stir, and filter to obtain a third extract;

[0052] Step S5: Concentrate and dry the third extract to obtain a flame tree flower extract.

[0053] Example 1

[0054] Prepare a flame tree flower extract:

[0055] Step S1: Weigh 50 g of dried Spathodea campanulata flowers, crush them to obtain Spathodea campanulata flower powder, and subject the Spathodea campanulata flower powder to low-temperature ultrasonic cold extraction with 98 wt% ethanol aqueous solution. The ultrasonic power is 320 w, the extraction time is 2 h / time, the extraction frequency is 2 times, the extraction temperature is 4 °C, filter, recover ethanol, and dry the filter residue for later use;

[0056] Step S2: Place the filter residue in a reflux extraction device, use 40 wt% ethanol aqueous solution as the extraction solvent, control the solid-liquid ratio to be 1:10 (g / ml), heat for reflux extraction, the extraction temperature is 80 °C, extract for 2 hours each time, extract 2 times in total, combine the two extraction solutions, filter while it is hot and concentrate under reduced pressure to obtain a concentrated solution;

[0057] Step S3: Separate and elute the concentrated solution with an AB-8 type macroporous resin column. The elution steps are: water elution for 5 BV, 20 wt% ethanol aqueous solution elution for 9 BV, 50 wt% ethanol aqueous solution elution for 6 BV, collect the part eluted with 50 wt% ethanol water to obtain an eluate;

[0058] Step S4: Add a decolorizing agent to the eluate for decolorization treatment. The dosage of the decolorizing agent is 1% of the solution mass. The decolorizing agent includes activated carbon and silica gel with a mass ratio of 2:1. Heat to 50 °C and treat for 30 min, then filter under reduced pressure to obtain a filtrate;

[0059] Step S5: Concentrate the filtrate under reduced pressure at 55 °C and freeze-dry to finally obtain 2.420 g of Spathodea campanulata flower extract. The calculated yield is 4.84%. The mass percentage content of catalpol 6-caffeate in the Spathodea campanulata flower extract is measured to be 45.82%. Prepare the extract into a 4 mg / ml, 30 wt% 1,3-butanediol solution, and the solution state is a light yellow clear solution.

[0060] Appendix Figure 1 It is the chromatogram of the Spathodea campanulata flower extract prepared in Example 1.

[0061] Example 2

[0062] Example 2 is similar to Example 1 in the steps adopted, with the only difference being that the cold extraction temperature in Step S1 is 2 °C. Finally, 2.475 g of Spathodea campanulata flower extract is obtained. The calculated yield is 4.95%. The mass percentage content of catalpol 6-caffeate in the Spathodea campanulata flower extract is measured to be 43.84%. Prepare the extract into a 4 mg / ml, 30 wt% 1,3-butanediol solution, and the solution state is a light yellow clear solution.

[0063] Example 3

[0064] Example 3 was similar to Example 1 in steps, except that the cold extraction temperature in step S1 was 8°C, and finally 2.315 g of the extract of Spathodea campanulata was obtained. The calculated yield was 4.63%. The mass percentage content of catalpol 6-caffeate in the extract of Spathodea campanulata was measured to be 42.21%. The extract was formulated into a 4 mg / ml, 30 wt% 1,3-butanediol solution, and the solution state was a light yellow clear solution.

[0065] Example 4

[0066] Example 4 was similar to Example 1 in steps, except that the mass ratio of activated carbon and silica gel included in the decolorizing agent in step S4 was set to 1:1. Finally, 2.460 g of the extract of Spathodea campanulata was obtained. The calculated yield was 4.92%. The mass percentage content of catalpol 6-caffeate in the extract of Spathodea campanulata was measured to be 41.47%. The extract was formulated into a 4 mg / ml, 30 wt% 1,3-butanediol solution, and the solution state was a light yellow clear solution.

[0067] Example 5

[0068] Example 5 was similar to Example 1 in steps, except that the mass ratio of activated carbon and silica gel included in the decolorizing agent in step S4 was set to 3:1. Finally, 2.155 g of the extract of Spathodea campanulata was obtained. The calculated yield was 4.31%. The mass percentage content of catalpol 6-caffeate in the extract of Spathodea campanulata was measured to be 41.34%. The extract was formulated into a 4 mg / ml, 30 wt% 1,3-butanediol solution, and the solution state was a light yellow clear solution.

[0069] Comparative Example 1

[0070] Comparative Example 1 was similar to Example 1 in steps, except that the extraction temperature in step S1 was 24°C (room temperature). Finally, 1.865 g of the extract of Spathodea campanulata was obtained. The calculated yield was 3.73%. The mass percentage content of catalpol 6-caffeate in the extract of Spathodea campanulata was measured to be 36.54%. The extract was formulated into a 4 mg / ml, 30 wt% 1,3-butanediol solution, and the solution state was a light yellow clear solution.

[0071] Comparative Example 2

[0072] Comparative Example 2 was similar to Example 1 in steps, except that the mass ratio of activated carbon and silica gel included in the decolorizing agent in step S4 was set to 4:1. Finally, 2.135 g of the extract of Spathodea campanulata was obtained. The calculated yield was 4.27%. The mass percentage content of catalpol 6-caffeate in the extract of Spathodea campanulata was measured to be 35.11%. The extract was formulated into a 4 mg / ml, 30 wt% 1,3-butanediol solution, and the solution state was a dark yellow clear solution.

[0073] Comparative Example 3

[0074] Comparative Example 3 was similar to Example 1 in steps, except that the mass ratio of activated carbon and silica gel included in the decolorizing agent in step S4 was set to 1:2. Finally, 2.820 g of the flame tree flower extract was obtained, and the calculated yield was 5.64%. The mass percentage content of catalpol 6-caffeate in the flame tree flower extract was measured to be 37.63%. The extract was formulated into a 4 mg / ml, 30 wt% 1,3-butanediol solution, and the solution state was a dark yellow clear solution.

[0075] Comparative Example 4

[0076] Comparative Example 4 was similar to Example 1 in steps, except that the percentage of the amount of the decolorizing agent added in step S4 in the mass of the second extract was 2%. Finally, 1.785 g of the flame tree flower extract was obtained, and the calculated yield was 3.57%. The mass percentage content of catalpol 6-caffeate in the flame tree flower extract was measured to be 30.27%. The extract was formulated into a 4 mg / ml, 30 wt% 1,3-butanediol solution, and the solution state was a light yellow clear solution.

[0077] Comparative Example 5

[0078] Comparative Example 5 was similar to Example 1 in steps, except that the ultrasonic low-temperature cold extraction step in step S1 was removed. Finally, 3.250 g of the flame tree flower extract was obtained, and the calculated yield was 6.50%. The mass percentage content of catalpol 6-caffeate in the flame tree flower extract was measured to be 34.32%. The extract was formulated into a 4 mg / ml, 30 wt% 1,3-butanediol solution, and the solution state was a dark red-brown clear solution.

[0079] Comparative Example 6

[0080] Comparative Example 6 was similar to Example 1 in steps, except that the decolorizing agent in step S4 was set to single activated carbon. Finally, 2.545 g of the flame tree flower extract was obtained, and the calculated yield was 5.09%. The mass percentage content of catalpol 6-caffeate in the flame tree flower extract was measured to be 33.21%. The extract was formulated into a 4 mg / ml, 30 wt% 1,3-butanediol solution, and the solution state was a red-brown clear solution.

[0081] Comparative Example 7

[0082] Comparative Example 7 was similar to Example 1 in the steps adopted, with the only difference being the removal of the decolorizing agent described in step S4. Finally, 3.120 g of the flame tree flower extract was obtained, and the calculated yield was 6.24%. The mass percentage content of catalpol 6-caffeate in the flame tree flower extract was measured to be 37.53%. The extract was formulated into a 4 mg / ml, 30 wt% 1,3-butanediol solution, and the solution state was a dark red-brown clear solution.

[0083] Analysis of Examples 1-5 and Comparative Examples 1-7 shows that:

[0084] (1) For Examples 1-3, with other conditions unchanged, only the cold extraction temperatures in step S1 were set at 4°C, 2°C, and 8°C respectively. Among them, when the extraction temperature in step S1 was 4°C, the mass percentage content of catalpol 6-caffeate measured in the flame tree flower extract was the highest, at 45.82% (the corresponding values in Examples 2-3 were 43.84% and 42.21% respectively);

[0085] Comparing Examples 1-3 with Comparative Example 1, the only difference between Comparative Example 1 and Examples 1-3 was that the extraction temperature in step S1 was set outside the range of 2-8°C, at 24°C (room temperature). The results showed that the mass percentage content of catalpol 6-caffeate measured in the flame tree flower extract in Comparative Example 1 was 36.54%, significantly lower than the corresponding values in Examples 1-3; it can be seen that the cold extraction temperature of 2-8°C in step S1 was a preferable range;

[0086] The only difference between Comparative Example 5 and Examples 1-3 was the removal of the ultrasonic low-temperature cold extraction step in step S1. The results showed that the mass percentage content of catalpol 6-caffeate measured in the flame tree flower extract in Comparative Example 5 was 34.32%, much lower than the corresponding values in Examples 1-3; and the color of the extract solution in Comparative Example 5 was darker than that in Examples 1-3; it can be seen that the cold extraction step in step S1 would directly affect the obtained amount of the target product and the color of the extract solution.

[0087] (2) For Examples 1, 4-5, with other conditions unchanged, only the mass ratios of activated carbon and silica gel included in the decolorizing agent in step S4 were set at 2:1, 1:1, and 3:1 respectively, and the dosage of the decolorizing agent was 1% of the solution mass. Among them, when the mass ratio of activated carbon and silica gel included in the decolorizing agent in step S4 was 2:1, the mass percentage content of catalpol 6-caffeate measured in the flame tree flower extract was the highest, at 45.82% (the corresponding values in Examples 4-5 were 41.47% and 41.34% respectively);

[0088] Examples 1, 4 - 5 were compared with Comparative Examples 2 - 3. The only difference between Comparative Examples 2 - 3 and Examples 1, 4 - 5 was that the mass ratio of activated carbon and silica gel included in the decolorizing agent in Step S4 was set outside the range of 1:1 to 3:1, being 4:1 and 1:2 respectively. The results showed that the mass percentage contents of catalpol 6 - caffeate measured in the extracts of flame tree flower in Comparative Examples 2 - 3 were 35.11% and 37.63% respectively, which were significantly lower than the corresponding values in Examples 1, 4 - 5. It can be seen that the optimal range of the mass ratio of activated carbon and silica gel included in the decolorizing agent in Step S4 is 1:1 to 3:1.

[0089] The only difference between Comparative Example 4 and Examples 1, 4 - 5 was that the dosage of the decolorizing agent in Step S4 was set to 2% of the solution mass, exceeding the numerical range of 0.1% - 1%. The results showed that the mass percentage content of catalpol 6 - caffeate measured in the extract of flame tree flower in Comparative Example 4 was 30.27%, which was significantly lower than the corresponding values in Examples 1, 4 - 5. It can be seen that the percentage of the decolorizing agent dosage in the solution mass in Step S4 should not be too high, and 0.1% - 1% is preferable.

[0090] The only difference between Comparative Examples 6 - 7 and Examples 1, 4 - 5 was that the decolorizing agent in Step S4 was single activated carbon or the decolorizing step of the decolorizing agent was cancelled. The results showed that the mass percentage contents of catalpol 6 - caffeate measured in the extracts of flame tree flower in Comparative Examples 6 - 7 were 33.21% and 37.53% respectively, which were significantly lower than the corresponding values in Examples 1, 4 - 5. And the color of the extract solution was darker than that of the extract solutions in Examples 1, 4 - 5. It can be seen that the decolorizing step of the decolorizing agent in Step S4 cannot be cancelled, and the decolorizing effect of the compound decolorizing agent is better.

[0091] In addition, combining the above - mentioned analysis content to comprehensively evaluate Examples 1 - 5 and Comparative Examples 1 - 7, there is a synergistic effect between Step S1 and Step S4, which is mainly reflected in the selection of the cold extraction temperature in Step S1 and the setting of the compounding ratio of the decolorizing agent and the dosage of the decolorizing agent in Step S4. The synergy of these two operation steps enables the extracts in Examples 1 - 5 to be formulated into a 1,3 - butanediol solution with a concentration of 4mg / ml and a mass fraction of 30wt%, and the solution state is a light yellow clear solution. That is, the extracts in Examples 1 - 5 are suitable for further application in the field of skin care products. The main defects of Comparative Examples 1 - 7 are mainly low content of catalpol 6 - caffeate in the extracts, incomplete decolorization, etc. When the extracts are formulated into a 1,3 - butanediol solution with a concentration of 4mg / ml and a mass fraction of 30wt%, the solution state is a deep yellow, dark red - brown or red - brown clear solution, which constitutes a limitation for applying the extracts to the field of skin care products.

[0092] Test Example 1: Evaluation of in vitro skin barrier efficacy

[0093] 1. Test method: HaCaT cells in the logarithmic growth phase with good morphology were seeded in 24-well plates at a density of 1×10 5 cells / well and incubated in an incubator for 24 h. A control group (untreated cells) and a sample group (taking the extracts containing catalpol-6-caffeate obtained in Examples 1-5 and Comparative Examples 1-7 as an example, with an extract concentration of 60 μg / ml) were set up. Three replicates were set for each concentration. The cells were treated with the drug and continued to be incubated in a 37°C, 5% CO2 incubator for 24 h. 0.5 mL of lysis buffer was added to each well and pipetted to lyse the cells. RNA was extracted, reverse transcribed into cDNA, and then fluorescence quantitative PCR was performed for detection. The 2^(-ΔΔCT) method was used for result calculation.

[0094] Comparative CT (ΔΔCT), a control group was set up, the internal reference gene was β-actin, and RQ = 2^(-ΔΔCT), where ΔΔCT = ΔCttreated - ΔCtcontrol, and ΔCttreated and ΔCtcontrol were the Ct differences between the target gene and the reference gene in the experimental group and the control group, respectively, that is, ΔCt = CTtarget - CTβ-actin. The RQ value was the relative expression level of the target gene mRNA.

[0095] 2. The experimental results showed that: Since the extracts prepared in Examples 1-5 had a high mass percentage content of catalpol 6-caffeate, the extracts had no cytotoxicity at a concentration of 60 μg / ml and significantly up-regulated the expression of FLG and LOR genes, and had a significant skin barrier repair effect (see Table 1).

[0096] Table 1. Detection results of FLG and LOR gene expression in the cell experiment model

[0097]

[0098]

[0099] Test Example 2: Evaluation of 3D skin barrier efficacy

[0100] 1. Experimental materials: The 3D skin model used in this test was the Episkin large-pore skin model, purchased from Shanghai Anfunuo Biotechnology Co., Ltd.

[0101] 2. Test method: Using an in vitro reconstituted normal 3D epidermal model, a damaged 3D skin model constructed by chemical stimulation, the extracts containing catalpol-6-caffeate obtained in Examples 1-5 and Comparative Examples 1-7 were incubated, and the skin was collected for immunofluorescence detection; QRT-PCR gene detection, and the experimental results were comprehensively evaluated.

[0102] 3. Evaluation criteria: The expression of FLG / PCR genes was detected by QRT-PCR. The results showed that the expression levels of the corresponding FLG gene, LOR gene, Claudin4 gene, and Claudin5 gene in Examples 1-5 were high, and the skin barrier repair function was good (see Table 2).

[0103] Table 2. Detection results of the expression of FLG gene, LOR gene, Claudin4 gene, and Claudin5 gene under 3D skin model

[0104]

[0105]

[0106] The applicant declares that the above is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A flame tree flower extract, characterized in that, The flame tree flower extract contains the active ingredient catalpol 6-caffeate, and the mass percentage content of catalpol 6-caffeate in the flame tree flower extract is not less than 41%.

2. A preparation method of the flame tree flower extract as described in claim 1, characterized in that, The preparation method includes the following steps: Step S1: Subject the dried and pulverized flame tree flowers to low-temperature ultrasonic cold extraction with a first solvent, filter, and dry the filter residue for later use; Step S2: Heat and reflux extract the dried filter residue with a second solvent, filter, repeat the operation 2-3 times, combine the filtrates, and concentrate the filtrates to obtain a first extract; Step S3: Use macroporous resin as an adsorbent, elute the first extract with water, a third solvent, and a fourth solvent, collect the part eluted with the fourth solvent, and concentrate to obtain a second extract; Step S4: Decolorize the second extract with a decolorizing agent, which is obtained by compounding activated carbon and silica gel. The addition amount of the decolorizing agent accounts for 0.1%-1% of the mass of the second extract. Heat, stir, and filter to obtain a third extract; Step S5: Concentrate and dry the third extract to obtain the flame tree flower extract.

3. The preparation method of a flame tree flower extract according to claim 2, characterized in that, The first solvent in Step S1 is an ethanol aqueous solution with the mass percentage content of ethanol greater than 95%; the second solvent in Step S2 is an ethanol aqueous solution with the mass percentage content of ethanol being 35%-45%; the third solvent in Step S3 is an ethanol aqueous solution with the mass percentage content of ethanol being 15%-25%; the fourth solvent in Step S3 is an ethanol aqueous solution with the mass percentage content of ethanol being 40%-60%.

4. The preparation method of a flame tree flower extract according to claim 2, wherein The operating conditions of the low-temperature ultrasonic cold extraction in Step S1 are: ultrasonic power 250-400w, extraction times 2-3 times, and extraction temperature 2-8°C.

5. The preparation method of a flame tree flower extract according to claim 2, characterized in that, The macroporous resin in Step S3 is selected from any one of D101, AB-8, HP-20, HPD-100, and MCI-gel CHP 20P.

6. The preparation method of a flame tree flower extract according to claim 2, characterized in that, The mass ratio of the compounding of activated carbon and silica gel in Step S4 is 1:1 to 3:

1.

7. Application of a flame tree flower extract prepared by the preparation method according to any one of claims 2-6 in skin care products, wherein the flame tree flower extract is used to up-regulate the expression of skin barrier repair factors.

8. Use of the flame tree flower extract according to claim 7 in skin care products, characterized in that, The skin barrier repair factors include at least one of FLG gene, LOR gene, Claudin4 gene, and Claudin5 gene.

9. Use of the flame tree flower extract according to claim 7 in skin care products, characterized in that, In an in vitro skin barrier model, under the condition of a flame tree flower extract concentration of 60 μg / ml, the expression promotion rate of the FLG gene is 33%-51%, and the expression promotion rate of the LOR gene is 44%-61%.

10. Use of the flame tree flower extract according to claim 7 in skin care products, characterized in that, In a 3D skin barrier model, under the condition of a flame tree flower extract concentration of 600 μg / ml, the expression promotion rate of the FLG gene is 15%-26%, the expression promotion rate of the LOR gene is 47%-62%, the expression promotion rate of the Claudin4 gene is 225%-260%, and the expression promotion rate of the Claudin5 gene is 10%-28%.

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