Method for preparing catalpol 6-caffeic acid ester from flame tree flowers as well as product and application of catalpol 6-caffeic acid ester

Abstract

The invention relates to a method for preparing catalpol 6-caffeic acid ester from flammulina velutipes as well as a product and application of the catalpol 6-caffeic acid ester. The method comprises the following steps: S1, carrying out heating reflux extraction on the flammulina velutipes subjected to drying and crushing treatment by using a first solvent, filtering, and carrying out vacuum concentration to obtain a first extracting solution; s2, extracting the first extracting solution by using a second solvent as an extracting agent for 2-3 times, combining supernate, filtering and concentrating to obtain a second extracting solution; s3, resin is adopted as an adsorbent, water, a third solvent and a fourth solvent are adopted for elution, the elution part of the fourth solvent is collected and concentrated, and a refined product is obtained; and S4, adding a fifth solvent into the refined product, performing low-temperature precipitation, and after precipitation, performing filtration and reduced-pressure drying to obtain the catalpol 6-caffeic acid ester. The catalpol 6-caffeic acid ester prepared by the preparation method disclosed by the invention has the effects of inhibiting tyrosinase activity and repairing a skin barrier.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to a method for preparing catalpol 6-caffeate from flame tree flowers, and a product and application thereof. Background Art

[0002] The flame tree flower comes from the flame tree of the genus Spathodea in the Bignoniaceae family ( Spathodea Campanulata Beauv.) Dried flower buds. Literature reports that all parts of the flame tree are rich in bioactive substances and have multiple pharmacological effects. Flame tree leaves are often used to treat painful inflammation, constipation and dysentery; flowers and bark are often used to treat mental disorders, malaria, hemorrhoids, bacterial infections, HIV, poor blood circulation, gastrointestinal diseases, respiratory diseases, urogenital diseases, etc.; leaf ethanol extract has anticonvulsant effects; flame tree flower extract has the ability to absorb ultraviolet rays; in addition, flame tree bark decoction has anti-HIV activity. Phytochemical analysis shows that flame tree contains chemical components such as alkaloids, tannins, saponins, anthraquinone glycosides, caffeic acid, flavonoids, steroids, terpenes, alkaloids, tannins and glycosides. Verminoside is an cyclopentadiene ether terpenoid compound isolated from flame tree flowers, which has pharmacological activities such as antioxidant, anti-inflammatory, anti-ulcer and anti-tumor.

[0003] At present, the process of separating and extracting catalpol 6-caffeate mainly involves solvent extraction, macroporous resin elution and silica gel column chromatography. However, the above process has defects such as large loss of effective ingredients and poor industrial reproducibility, and is not suitable for industrial production. Chinese patent publication (CN108373488A) reported catalpol 6-caffeate derivatives and their preparation methods and applications, and provided a method for preparing catalpol-6 caffeate derivatives with n-butoxy groups. The derivatives prepared by this method have better tumor inhibition activity than the original compound catalpol 6-caffeate. At present, no reports have been found on the tyrosinase activity inhibition and skin barrier repair effects of catalpol 6-caffeate.

[0004] Therefore, it is urgent to propose a method for preparing catalpol 6-caffeate from flame tree flowers, as well as its products and applications, so as to solve the technical problem that the active ingredient catalpol 6-caffeate in flame tree flowers has not been efficiently extracted and applied to products with related functions such as tyrosinase activity inhibition and skin barrier repair. Summary of the invention

[0005] In view of the shortcomings of the prior art, the main purpose of the present invention is to propose a method for preparing catalpol 6-caffeate from the flower of the flame tree, and its product and application, aiming to solve at least one of the defects of the current extraction process of catalpol 6-caffeate, such as large loss of effective ingredients, poor industrial reproducibility, and unsuitability for industrial production, as well as the technical problem that catalpol 6-caffeate has not been applied to products with tyrosinase activity inhibition and skin barrier repair effects.

[0006] To achieve this object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a method for preparing catalpol 6-caffeate from flame tree flowers, the method comprising the following steps: Step S1: extracting the dried and crushed flame tree flower by heating and refluxing with a first solvent, filtering after the extraction, and concentrating the filtrate under reduced pressure to obtain a first extract; Step S2: extracting the first extract with a second solvent as an extractant, wherein the second solvent is a mixed solvent of ethyl acetate and dichloromethane, the extraction times are 2-3 times, combining the supernatants, filtering and concentrating, and obtaining a second extract; Step S3: using a resin as an adsorbent, eluting the second extract with water, a third solvent, and a fourth solvent respectively, collecting the eluted portion of the fourth solvent, and concentrating to obtain a refined product; Step S4: adding the fifth solvent to the refined product and standing at low temperature to precipitate, and then filtering and drying under reduced pressure to obtain catalpol 6-caffeate.

[0007] Preferably, in step S1, the first solvent is an ethanol aqueous solution with an ethanol content of 30-50wt%, and the heating reflux extraction is performed 2-3 times, and each extraction time is 2-3 hours.

[0008] It should be noted that the first solvent is preferably an ethanol aqueous solution with an ethanol content of 31-49wt%, and is also preferably an ethanol aqueous solution with an ethanol content of 32-48wt%, and is also preferably an ethanol aqueous solution with an ethanol content of 33-47wt%, and is also preferably an ethanol aqueous solution with an ethanol content of 34-46wt%, and is also preferably an ethanol aqueous solution with an ethanol content of 35-45wt%, and is also preferably an ethanol aqueous solution with an ethanol content of 36-44wt%, and is also preferably an ethanol aqueous solution with an ethanol content of 37-43wt%, and is also preferably an ethanol aqueous solution with an ethanol content of 38-42wt%, and is also preferably an ethanol aqueous solution with an ethanol content of 39-41wt%, and is also preferably an ethanol aqueous solution with an ethanol content of 40wt%. During the research process, it was found that an ethanol aqueous solution with an ethanol content of 40wt% can fully extract catalol 6-caffeic acid ester from flame tree flowers to the greatest extent with less impurities.

[0009] Preferably, in step S2, the mass ratio of ethyl acetate to dichloromethane in the mixed solvent is 2:1 to 4:1.

[0010] It should be noted that in step S2, the mass ratio of ethyl acetate to dichloromethane in the mixed solvent is preferably 3:1. During the research process, it was found that at this mass ratio, catalpol 6-caffeate in the flame tree flower can be extracted completely to the greatest extent.

[0011] Preferably, in step S3, the resin is selected from any one of HP20SS, SP20SS or SP207SS.

[0012] Preferably, in step S3, the third solvent is an ethanol aqueous solution with an ethanol content of 15-25wt%, and the fourth solvent is an ethanol aqueous solution with an ethanol content of 45-55wt%.

[0013] It should be noted that in step S3, the third solvent is preferably an ethanol aqueous solution with an ethanol content of 16-24wt%, an ethanol aqueous solution with an ethanol content of 17-23wt%, an ethanol aqueous solution with an ethanol content of 18-22wt%, an ethanol aqueous solution with an ethanol content of 19-21wt%, and is also preferably an ethanol aqueous solution with an ethanol content of 20wt%.

[0014] The fourth solvent is preferably an ethanol aqueous solution with an ethanol content of 46-54wt%, further preferably an ethanol aqueous solution with an ethanol content of 47-53wt%, further preferably an ethanol aqueous solution with an ethanol content of 48-52wt%, further preferably an ethanol aqueous solution with an ethanol content of 49-51wt%, further preferably an ethanol aqueous solution with an ethanol content of 50wt%.

[0015] Preferably, in step S4, in step S4, the fifth solvent is a mixed solvent of n-hexane and dichloromethane, the mass ratio of n-hexane to dichloromethane is 1:1 to 1:3, and the mass ratio of the refined product to the fifth solvent is 1:8 to 1:12.

[0016] It should be noted that the mass ratio of n-hexane to dichloromethane is preferably 1:2, and the mass ratio of the refined product to the fifth solvent is preferably 1:9 to 1:11, and further preferably 1:10.

[0017] Preferably, in step S4, the low-temperature standing time is 2-4 h, and the low-temperature standing temperature is 2-8°C.

[0018] It should be noted that the low-temperature standing temperature is preferably 3-7° C., further preferably 3-6° C., further preferably 3-5° C., further preferably 4° C. The standing time is preferably 3 h.

[0019] Preferably, in step S4, the content of the prepared catalpol 6-caffeate is not less than 90.00 wt%, wherein the structural formula of the catalpol 6-caffeate is: .

[0020] In a second aspect, the present invention also provides an application of catalpol 6-caffeate prepared by the above method in skin care products, characterized in that the types of the skin care products include: lotion, cream, water solution or gel, but the types of the skin care products are not limited to the above examples.

[0021] Preferably, the catalpol 6-caffeate has the effects of inhibiting tyrosinase activity and repairing the skin barrier.

[0022] Preferably, the catalpol 6-caffeate can inhibit tyrosinase activity and upregulate the expression of skin barrier repair genes, and the skin barrier repair genes include at least one of the FLG gene and the LOR gene.

[0023] Compared with the prior art, the present invention has at least the following beneficial effects: (1) The method for preparing catalpol 6-caffeate from the flower of the flame tree provided by the present invention can be used for mass production of high-content catalpol 6-caffeate to meet its use requirements in skin barrier repair and skin whitening scenarios.

[0024] (2) The present invention provides a method for preparing catalpol 6-caffeate from flame tree flowers, which is simple to operate and can greatly increase the content of catalpol 6-caffeate. The content of the prepared catalpol 6-caffeate is not less than 90.00 wt%, and has excellent industrial reproducibility, and is suitable for industrial production; specifically, in step S2, the mass ratio of ethyl acetate to dichloromethane in the mixed solvent is 2:1 to 4:1, and the setting of this ratio range enables the mixed solvent to efficiently extract the target component in the first extract; and, in step S3, resin is used as an adsorbent, which can enrich catalpol 6-caffeate to the greatest extent to obtain a refined product; in step S4, the fifth solvent is a mixed solvent of n-hexane and dichloromethane, the mass ratio of n-hexane to dichloromethane is 1:1 to 1:3, and the mass ratio of the refined product to the fifth solvent is 1:8 to 1:12, so that the target component can be precipitated in a crystalline form at low temperature, that is, the synergy of steps S2, S3 and step S4 can make the content of the prepared catalpol 6-caffeate not less than 90.00wt%.

[0025] (3) The catalpol 6-caffeate prepared by the above method provided by the present invention has the effects of inhibiting tyrosinase activity and repairing the skin barrier. The catalpol 6-caffeate has tyrosinase inhibitory activity, and the IC50 value is less than 0.85 mg / mL, and has good whitening potential; it upregulates the expression of skin barrier repair genes at a concentration of 15 μg / mL~1.2 mg / mL, and can significantly increase the expression of FLG gene and LOR gene. The upregulation rate of skin barrier repair genes is 48%-149%, and has a good effect of maintaining and repairing the skin barrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The liquid chromatogram of catalpol 6-caffeate prepared in Example 1 is shown; Figure 2 The results show that the catalpol 6-caffeate prepared in Test Example 1 13 C-NMR spectrum; Figure 3 The catalpol 6-caffeate prepared in Test Example 1 is shown. 1 H-NMR spectrum; Figure 4 The present invention shows a flow chart of a method for preparing catalpol 6-caffeate from flame tree flowers. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0028] The source information of relevant raw materials and materials is as follows: HaCaT cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences; culture conditions: DMEM culture medium, FBS fetal bovine serum with a volume percentage concentration of 10%).

[0029] The MTT assay kit was obtained from Xinbosheng Biotechnology Co., Ltd.; The CCK-8 test kit was sourced from Xinbosheng Biotechnology Co., Ltd.; Catalpol 6-caffeate standard was purchased from Sichuan Weikeqi Biotechnology Co., Ltd.; The raw materials, consumables or reagents involved in the present invention are all conventional products that can be purchased commercially. FLG gene: The Filaggrin (FLG) gene is located on human chromosome 1q21.3, and its translation expression product is closely related to maintaining the skin barrier and cell differentiation function.

[0030] LOR gene: Loricrin (LOR) gene is the main component of the keratinized membrane of terminally differentiated keratinocytes and plays an important role in the barrier function of the epidermis.

[0031] Example 1

[0032] A method for preparing catalpol 6-caffeate from flame tree flowers, the method comprising the following steps: Step S1: weigh 50 g of dried flame tree flowers, crush them, pass through a 40-mesh sieve, and place them in a reflux extraction device, use 40 wt% ethanol aqueous solution as the extraction solvent, control the solid-liquid ratio to 1:10 (g / mL), heat reflux extraction, the extraction temperature is 80°C, each extraction takes 3 hours, and the extraction is performed twice in total. After the extraction is completed, filter while hot, and concentrate under reduced pressure to recover ethanol to obtain a first extract; Step S2: extracting the first extract with a mixed solvent of ethyl acetate and dichloromethane as an extractant, wherein the mass ratio of ethyl acetate to dichloromethane is 3:1, and the number of extractions is 2 times, combining the supernatants, filtering, concentrating under reduced pressure, and recovering the second solvent to obtain a second extract; Step S3: using SP20SS resin as an adsorbent to elute the second extract, the elution steps are: 3BV of water elution, 5BV of 20wt% ethanol aqueous solution elution, 5BV of 50wt% ethanol aqueous solution elution, collecting the 50wt% ethanol aqueous solution elution part, concentrating to obtain a refined product; Step S4: Add 10 times the amount of a mixed solvent consisting of n-hexane and dichloromethane to the refined product, with a mass ratio of n-hexane to dichloromethane being 1:2, and place it in a refrigerator at 4°C for 3 h. After crystallization or precipitation, filter it, and dry it under reduced pressure to finally obtain 0.90 g of catalpol 6-caffeate.

[0033] After calculation, the yield was 1.80%. The purchased sample was used as the standard product. The content of catalpol 6-caffeate was detected by high performance liquid chromatography. A standard curve was drawn and the mass percentage content of catalpol 6-caffeate was measured to be 93.44%. The liquid chromatogram is shown in FIG. Figure 1 shown.

[0034] Example 2

[0035] A method for preparing catalpol 6-caffeate from flame tree flowers, the method involves steps similar to those in Example 1, the only difference being that the mass ratio of the extractant ethyl acetate to dichloromethane in step S2 is 2:1. Finally, 0.85 g of catalpol 6-caffeate is obtained, the calculated yield is 1.70%, and the content of catalpol 6-caffeate is detected by high performance liquid chromatography using a purchased sample as a standard product, and a standard curve is drawn, and the mass percentage content of catalpol 6-caffeate is measured to be 92.69%.

[0036] Example 3

[0037] A method for preparing catalpol 6-caffeate from flame tree flowers, the method involves steps similar to those in Example 1, the only difference being that the mass ratio of the extractant ethyl acetate to dichloromethane in step S2 is 4:1. Finally, 0.93 g of catalpol 6-caffeate is obtained, the calculated yield is 1.86%, and the content of catalpol 6-caffeate is detected by high performance liquid chromatography using a purchased sample as a standard product, and a standard curve is drawn, and the mass percentage content of catalpol 6-caffeate is measured to be 91.31%.

[0038] Example 4

[0039] A method for preparing catalpol 6-caffeate from flame tree flowers, the method involves steps similar to those in Example 1, the only difference being that in step S4, a mixed solvent consisting of n-hexane and dichloromethane is added to the refined product, the mass ratio of n-hexane to dichloromethane is 1:3, and 0.85 g of catalpol 6-caffeate is finally obtained, with a calculated yield of 1.70%. A purchased sample is used as a standard product, and the content of catalpol 6-caffeate is detected by high performance liquid chromatography, and a standard curve is drawn, and the mass percentage content of catalpol 6-caffeate is measured to be 90.41%.

[0040] Example 5

[0041] A method for preparing catalpol 6-caffeate from flame tree flowers, the method involving steps similar to those in Example 1, the only difference being that in step S4, a mixed solvent consisting of n-hexane and dichloromethane is added to the refined product, the mass ratio of n-hexane to dichloromethane is 1:1, and 0.86 g of catalpol 6-caffeate is finally obtained, with a calculated yield of 1.72%. A purchased sample is used as a standard, and the content of catalpol 6-caffeate is detected by high performance liquid chromatography, and a standard curve is drawn, and the mass percentage content of catalpol 6-caffeate is measured to be 91.19%.

[0042] Example 6

[0043] A method for preparing catalpol 6-caffeate from flame tree flowers, the steps involved in the method are similar to the steps in Example 1, the only difference is that in step S4, the precipitation temperature is 6°C and the mixture is refrigerated and left to stand for 4 hours, and finally 0.86 g of catalpol 6-caffeate is obtained, and the calculated yield is 1.71%. A purchased sample is used as a standard product, and the content of catalpol 6-caffeate is detected by high performance liquid chromatography, and a standard curve is drawn, and the mass percentage content of catalpol 6-caffeate is measured to be 93.03%.

[0044] Comparative Example 1 A method for preparing catalpol 6-caffeate from flame tree flowers, the method involves steps similar to those in Example 1, the only difference being that the mass ratio of the extractant ethyl acetate to dichloromethane in step S2 is 1:1. Finally, 0.72 g of catalpol 6-caffeate is obtained, the calculated yield is 1.44%, and the content of catalpol 6-caffeate is detected by high performance liquid chromatography using a purchased sample as a standard product, and a standard curve is drawn, and the mass percentage content of catalpol 6-caffeate is measured to be 81.79%.

[0045] Comparative Example 2 A method for preparing catalpol 6-caffeate from flame tree flowers, the method involves steps similar to those in Example 1, the only difference being that the mass ratio of the extractant ethyl acetate to dichloromethane in step S2 is 5:1. No crystallization or precipitation is produced, and 0.98 g of a refined product is obtained after concentration and drying, with a calculated yield of 1.95%. The content of catalpol 6-caffeate is detected by high performance liquid chromatography using a purchased sample as a standard, and a standard curve is drawn, and the mass percentage content of catalpol 6-caffeate is measured to be 74.41%.

[0046] Comparative Example 3 A method for preparing catalpol 6-caffeate from flame tree flowers, the method involves steps similar to those in Example 1, the only difference being that in step S4, a mixed solvent consisting of n-hexane and dichloromethane is added to a refined product, the mass ratio of n-hexane to dichloromethane is 2:1, 0.79 g of a refined product is obtained by concentration and drying, the calculated yield is 1.58%, a purchased sample is used as a standard, the content of catalpol 6-caffeate is detected by high performance liquid chromatography, a standard curve is drawn, and the mass percentage content of catalpol 6-caffeate is measured to be 82.01%.

[0047] Comparative Example 4 A method for preparing catalpol 6-caffeate from flame tree flowers, the method involves steps similar to those in Example 1, the only difference being that the extractant in step S2 is only ethyl acetate. Finally, no crystallization or precipitation was found, and 0.98 g of refined product was obtained after concentration and drying, with a calculated yield of 1.96%. The content of catalpol 6-caffeate was detected by high performance liquid chromatography using a purchased sample as a standard, and a standard curve was drawn, and the mass percentage content of catalpol 6-caffeate was measured to be 77.12%.

[0048] Comparative Example 5 A method for preparing catalpol 6-caffeate from flame tree flowers, the method involves steps similar to those in Example 1, the only difference being that the extractant in step S2 is only dichloromethane. Finally, no crystallization or precipitation is found, and 0.66 g of refined product is obtained after concentration and drying, with a calculated yield of 1.32%. The content of catalpol 6-caffeate is detected by high performance liquid chromatography using a purchased sample as a standard, and a standard curve is drawn, and the mass percentage content of catalpol 6-caffeate is measured to be 75.86%.

[0049] Comparative Example 6 A method for preparing catalpol 6-caffeate from flame tree flowers, the method involving steps similar to those in Example 1, the only difference being that in step S4, only n-hexane is added to the refined product, and finally no crystallization or precipitation is found. After concentration and drying, 1.07 g of refined product is obtained, and the calculated yield is 2.14%. A purchased sample is used as a standard product, and the content of catalpol 6-caffeate is detected by high performance liquid chromatography, and a standard curve is drawn, and the mass percentage content of catalpol 6-caffeate is measured to be 76.98%.

[0050] Comparative Example 7 A method for preparing catalpol 6-caffeate from flame tree flowers, the method involving steps similar to those in Example 1, the only difference being that in step S4, only dichloromethane is added to the refined product, and finally no crystal or precipitation is found. After concentration and drying, 1.10 g of the refined product is obtained, and the calculated yield is 2.20%. A purchased sample is used as a standard product, and the content of catalpol 6-caffeate is detected by high performance liquid chromatography, and a standard curve is drawn, and the mass percentage content of catalpol 6-caffeate is measured to be 76.71%.

[0051] Comparative Example 8 A method for preparing catalpol 6-caffeic acid ester from flame tree flowers. The steps involved in the method are similar to those in Example 1, except that the mixed solvent extraction step in step S2 is removed. Finally, no crystallization or precipitation is found. After concentration and drying, 1.29 g of refined product is obtained, and the calculated yield is 2.58%. A purchased sample is used as a standard product, and the content of catalpol 6-caffeic acid ester is detected by high performance liquid chromatography. A standard curve is drawn, and the mass percentage content of catalpol 6-caffeic acid ester is measured to be 65.28%. The yield is low and the purity of the obtained monomer is not high.

[0052] Comparative Example 9 A method for preparing catalpol 6-caffeate from flame tree flowers, the method involving steps similar to those in Example 1, the only difference being that the SP20SS resin used in step S3 is replaced by a conventional macroporous resin AB-8, and finally no crystallization or precipitation is found. After concentration and drying, 1.26 g of a refined product is obtained, and the calculated yield is 2.51%. A purchased sample is used as a standard product, and the content of catalpol 6-caffeate is detected by high performance liquid chromatography, and a standard curve is drawn, and the mass percentage content of catalpol 6-caffeate is measured to be 65.33%.

[0053] Comparative Example 10 A method for preparing catalpol 6-caffeate from flame tree flowers, the method involving steps similar to those in Example 1, the only difference being that in step S4, the crystallization temperature is refrigerated and left to stand for 1.5 hours at room temperature, and finally no crystallization or precipitation is found. After concentration and drying, 1.08 g of refined product is obtained, and the calculated yield is 2.15%. A purchased sample is used as a standard product, and the catalpol 6-caffeate content is detected by high performance liquid chromatography, and a standard curve is drawn, and the mass percentage content of the catalpol 6-caffeate is measured to be 81.88%.

[0054] Analysis of Examples 1-6 and Comparative Examples 1-10 shows that: (1) For Examples 1-3, other conditions were kept unchanged, and only the mass ratio of ethyl acetate to dichloromethane in step S2 was set to 3:1, 2:1, and 4:1, respectively. When the mass ratio of ethyl acetate to dichloromethane was 3:1, the mass percentage content of catalpol 6-caffeate was the highest, which was 93.44%; Comparing Example 1-3 with Comparative Example 1-2, the only difference between Comparative Example 1-2 and Example 1-3 is that the mass ratio of ethyl acetate to dichloromethane in step S2 is set outside the range of 2:1 to 4:1, and is 1:1 and 5:1, respectively. The results show that the mass percentage content of catalpol 6-caffeate in Comparative Example 1-2 is greatly reduced to only 81.79% and 74.41%; it can be seen that the mass ratio of ethyl acetate to dichloromethane in step S2 is 2:1 to 4:1, which is the optimal ratio range.

[0055] (2) For Examples 1, 4-5, other conditions were kept unchanged, and only the mass ratio of n-hexane to dichloromethane in step S4 was set to 1:2, 1:3, and 1:1, respectively. When the mass ratio of n-hexane to dichloromethane was 1:2, the mass percentage content of catalpol 6-caffeate was the highest, which was 93.44%; Comparing Examples 1 and 4-5 with Comparative Example 3, the only difference between Comparative Example 3 and Examples 1 and 4-5 is that the mass ratio of n-hexane to dichloromethane in step S4 is set outside the range of 1:1 to 1:3, which is 2:1. The results show that the mass percentage content of catalpol 6-caffeate in Comparative Example 3 is greatly reduced to only 82.01%. It can be seen that the mass ratio of n-hexane to dichloromethane in step S4 is 1:1 to 1:3, which is the optimal ratio range.

[0056] (3) Based on Examples 1-5 and Comparative Examples 1-3, it was unexpectedly found during the research process that when the mass ratio of ethyl acetate to dichloromethane in step S2 was set to 3:1 and the mass ratio of n-hexane to dichloromethane in step S4 was set to 1:2, the mass percentage content results of catalpol 6-caffeate were better than the corresponding results when the mass ratio of ethyl acetate to dichloromethane in step S2 was set to 2:1 or 4:1 and the mass ratio of n-hexane to dichloromethane in step S4 was set to 1:2, and at the same time, it was better than the corresponding results when the mass ratio of ethyl acetate to dichloromethane in step S2 was set to 3:1 and the mass ratio of n-hexane to dichloromethane in step S4 was set to 1:3 or 1:1; it can be seen that when the mass ratio of ethyl acetate to dichloromethane in step S2 is set to 3:1 and the mass ratio of n-hexane to dichloromethane in step S4 is set to 1:2, steps S2 and S4 can have a synergistic effect, so the scheme corresponding to Example 1 is a better scheme.

[0057] (4) For Examples 1-5 and Comparative Examples 4-7, it can be seen that when only ethyl acetate or dichloromethane is used in step S2, or when only n-hexane or dichloromethane is used in step S4, the mass percentage content of catalpol 6-caffeate (not exceeding 78%) is seriously inferior to that of Examples 1-5. The lower mass percentage content of catalpol 6-caffeate will lead to the whitening and barrier repair effects of the monomer in skin care products. Therefore, in the preparation method of the present application, it is necessary to use the compounded second solvent and the fifth solvent in steps S2 and S4.

[0058] (5) For Examples 1-5 and Comparative Examples 8-9, it can be seen that omitting step S2 or selecting the conventional macroporous resin AB-8 as the adsorbent in step S3 will result in failure to crystallize or a significant reduction in the mass percentage content of catalpol 6-caffeate, which will also affect the application of catalpol 6-caffeate in skin care products.

[0059] (6) For Example 6 and Comparative Example 10, it can be seen that the crystallization temperature set in step S4 is 2-8°C and the standing time is 2-4 h, which is more optimal. Outside this numerical range, the mass percentage content of catalpol 6-caffeic acid ester will be greatly reduced.

[0060] Test Example 1: NMR Identification of Catalpol 6-caffeate See attached Figure 2-3 , taking the catalpol 6-caffeate obtained in Example 1 as an example: it is a yellow solid powder, with the molecular formula: C 24 H 28 O 13 , ESI-MS (pos.): 547.18[M+Na] + ,ESI-MS(neg.):523[MH-] - , 1 H NMR (600 MHz, CD3OD): δ :5.13 (d, J = 9.3 Hz, 1H, H-1), 6.33 (dd, J = 6.0, 1.8 Hz, 1H, H-3), 4.98 (dd, J = 7.9, 1.3 Hz, 1H, H-6), 4.95 (dd, J = 6.0, 4.2 Hz, 1H, H-4), 2.62-2.51 (m, 2H, H-5, H-9), 3.66 (d, J = 13.2 Hz, 1H, H-7), 4.13 (d, J = 13.2 Hz, 1H, H-10a), 3.80 (d, J =13.2 Hz, 1H, H-10b), 4.75 (s, 1H, H-1'), 3.29 (dd, J = 6.7, 2.3 Hz, 1H, H-2'), 3.37 (d, J = 9.0 Hz, 1H, H-3'), 3.24 (td, J = 9.3, 8.7, 4.2 Hz, 2H, H-4', H-5'), 3.89 (dd, J =12.0, 2.2 Hz, 1H, H-6'a), 3.64-3.60 (m, 1H, H-6'b), 7.01 (d, J = 2.1 Hz, 1H, H-2''), 6.72 (d, J = 8.2 Hz, 1H, H-5''), 6.91 (dd, J = 8.2 2.1 Hz, 1H, H-6''), 7.56 (d, J =15.8 Hz, 1H, H-7''), 6.33 (dd, J = 6.0, 1.8 Hz, 1H, H-8''); 13C NMR (150 MHz, CD3OD): δ : 95.72 (C-1), 142.97 (C-3), 103.60 (C-4), 37.37 (C-5), 81.85 (C-6), 60.92 (C-7), 67.45 (C-8), 43.81 (C-9), 61.92 (C-10), 100.33 (C-1'), 75.47 (C-2'), 78.31 (C-3'), 72.38 (C-4'), 79.24 (C-5'), 63.53 (C-6'), 127.33 (C-1''), 115.32 (C-2''), 148.15(C-3''), 152.23 (C-4''), 117.27 (C-5''), 124.03 (C-6''), 148.61 (C-7''), 114.26 (C-8''), 169.77 (C-9'').

[0061] Test Example 2: Tyrosinase activity test 1. Experimental purpose: By inhibiting tyrosinase activity, measuring the change in absorbance level, calculating the IC50 value of the tyrosinase inhibition effect of the test sample, and evaluating the tyrosinase inhibition ability of the test sample by comparing it with the positive sample arbutin.

[0062] 2. Experimental Principle Tyrosinase is the key rate-limiting enzyme that converts tyrosine into melanin through a series of biochemical reactions. Under the catalysis of tyrosinase, it is converted into dopachrome, which has characteristic absorption at 475 nm, so the activity of tyrosinase can be measured by colorimetry.

[0063] 3. Test method: An α-arbutin control group and a sample group were set in a 96-well plate. Taking the catalpol 6-caffeate obtained in Examples 1-6 and Comparative Examples 1-2, 4-5, and 8-10 as examples, the mother liquor of catalpol 6-caffeate obtained in each group was prepared to be 4.0 mg / mL. The control group and the sample group were both prepared to a mother liquor concentration of 4 mg / mL, and 7 half-dilution concentrations were made.

[0064] The sample group includes a control group, a blank group, an experimental group, and a color-deducting group; the arbutin control group includes an experimental group and a blank group. The addition amount of each sample group is as follows: Control group B: 100 μL of tyrosine solution + 50 μL of tyrosinase + 50 μL of buffer solution; Blank group B0: 100 μL of tyrosine solution + 100 μL of buffer solution; Experimental group A: 100 μL of tyrosine solution + 50 μL of tyrosinase solution + 50 μL of samples of different concentrations; Color correction group A0: 100 μL of tyrosine solution + 50 μL of buffer + 50 μL of samples of different concentrations; The samples in groups B and B0 can be shared; The amount of arbutin added to the control group is as follows: Experimental group A: 100 μL tyrosine solution + 50 μL arbutin of different concentrations + 50 μL tyrosinase solution; A0 experimental group: 100 μL tyrosine solution + 50 μL arbutin of different concentrations + 50 μL buffer; Three replicate wells were set for each concentration. After adding the sample, the wells were allowed to react at room temperature for 30 min, and the absorbance was measured at 475 nm.

[0065] The calculation formula for tyrosinase activity inhibition rate (%IR) is: %IR=(1-(A-A0) / (B-B0))*100%.

[0066] 4. The experimental results show that catalpol 6-caffeate can inhibit tyrosinase activity and has good whitening potential. Since the catalpol 6-caffeate prepared in Examples 1-6 has a high mass percentage content, it exhibits better tyrosinase activity inhibition efficacy, see Table 1.

[0067] Table 1. Tyrosinase activity inhibition test results

[0068] Test Example 3: In vitro HaCaT cell skin barrier efficacy evaluation test 1. Test method: HaCaT cells in logarithmic growth phase with good morphology were inoculated into 24-well plates, and 1×10 5 Cells / well were incubated in an incubator for 24 h. A control group (untreated cells) and a sample group (taking catalpol 6-caffeate obtained in Example 1-6 and Comparative Examples 1-2, 4-5, and 8-10 as an example, the concentration of catalpol 6-caffeate was 120 μg / mL) were set up, and 3 replicates were set for each concentration. The cells were treated with drugs and continued to be incubated in an incubator at 37°C and 5% CO2 by volume for 24 h. 0.5 mL of lysis buffer was added to each well and lysed by pipetting. RNA was extracted and reverse transcribed to cDNA, and then fluorescent quantitative PCR was performed. 2 -ΔΔCT Methods were used to calculate the results. ComparativeCT (ΔΔCT), set the control group, the internal reference gene was β-actin, RQ=2 -ΔΔCTWhere ΔΔCT = ΔCttreated-ΔCtcontrol, ΔCttreated and ΔCtcontrol are 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 is the relative expression level of the target gene mRNA.

[0069] 2. The experimental results show that: due to the high mass percentage content of catalpol 6-caffeate prepared in Example 1-6, catalpol 6-caffeate has no cytotoxicity at a concentration of 120 μg / mL, and the expression of FLG and LOR genes is significantly upregulated, with significant skin barrier repair effect (see Table 2).

[0070] Table 2. FLG and LOR gene expression detection results

[0071] The applicant declares that the above is only a specific implementation mode 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 thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for preparing catalpol 6-caffeate from flame tree flowers, characterized in that: The method comprises the following steps: Step S1: extracting the dried and crushed flame tree flower by heating and refluxing with a first solvent, filtering after the extraction, and concentrating the filtrate under reduced pressure to obtain a first extract; Step S2: extracting the first extract with a second solvent as an extractant, wherein the second solvent is a mixed solvent of ethyl acetate and dichloromethane, the extraction times are 2-3 times, combining the supernatants, filtering and concentrating, and obtaining a second extract; Step S3: using a resin as an adsorbent, eluting the second extract with water, a third solvent, and a fourth solvent respectively, collecting the eluted portion of the fourth solvent, and concentrating to obtain a refined product; Step S4: adding the fifth solvent to the refined product and standing at low temperature to precipitate, and then filtering and drying under reduced pressure to obtain catalpol 6-caffeate.

2. A method for preparing catalpol 6-caffeate from flame tree flowers according to claim 1, characterized in that, In step S1, the first solvent is an ethanol aqueous solution with an ethanol content of 30-50wt%, and the heating reflux extraction is performed 2-3 times, and each extraction time is 2-3 hours.

3. A method for preparing catalpol 6-caffeate from flame tree flowers according to claim 1, characterized in that, In step S2, the mass ratio of ethyl acetate to dichloromethane in the mixed solvent is 2:1 to 4:

1.

4. A method for preparing catalpol 6-caffeate from flame tree flowers according to claim 1, characterized in that, In step S3, the resin model is selected from any one of HP20SS, SP20SS or SP207SS.

5. A method for preparing catalpol 6-caffeate from flame tree flowers according to claim 1, characterized in that, In step S3, the third solvent is an ethanol aqueous solution with an ethanol content of 15-25wt%, and the fourth solvent is an ethanol aqueous solution with an ethanol content of 45-55wt%.

6. A method for preparing catalpol 6-caffeate from flame tree flowers according to claim 1, characterized in that, In step S4, the fifth solvent is a mixed solvent of n-hexane and dichloromethane, the mass ratio of n-hexane to dichloromethane is 1:1 to 1:3, and the mass ratio of the refined product to the fifth solvent is 1:8 to 1:

12.

7. A method for preparing catalpol 6-caffeate from the flower of flame tree according to claim 1, characterized in that, In step S4, the low-temperature standing time is 2-4 hours, and the low-temperature standing temperature is 2-8°C.

8. A method for preparing catalpol 6-caffeate from flame tree flowers according to claim 1, characterized in that: In step S4, the content of the prepared catalpol 6-caffeic acid ester is not less than 90.00wt%, wherein the structural formula of the catalpol 6-caffeic acid ester is: 。 9. Use of catalpol 6-caffeate prepared by the method according to any one of claims 1 to 8 in skin care products, characterized in that: The types of the skin care products include: lotion, cream, water solution or gel.

10. A use according to claim 9, characterized in that The catalpol 6-caffeate has the effects of inhibiting tyrosinase activity and repairing the skin barrier.

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