A method for preparing catalpol 6-caffeate from Spathodea campanulata flowers, and its product and application

Abstract

The present invention relates to a method for preparing catalpol 6-caffeate from the flowers of Spathodea campanulata, and its product and application. The method comprises the following steps: Step S1: Heating and refluxing the dried and pulverized flowers of Spathodea campanulata with a first solvent, filtering and then concentrating under reduced pressure to obtain a first extract; Step S2: Using a second solvent as an extractant to extract the first extract, the extraction times being 2-3 times, combining the supernatant and filtering and concentrating to obtain a second extract; Step S3: Using a resin as an adsorbent, eluting with water, a third solvent, and a fourth solvent respectively, collecting the fraction eluted with the fourth solvent, and concentrating to obtain a refined product; Step S4: Adding a fifth solvent to the refined product and precipitating at a low temperature, filtering and drying under reduced pressure after precipitation to obtain catalpol 6-caffeate. The catalpol 6-caffeate prepared by the present invention has the effects of inhibiting tyrosinase activity and repairing the skin barrier.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and more specifically, relates to a method for preparing catalpol 6-caffeate from Spathodea campanulata flowers, its products and applications. Background Art

[0002] The flowers of Spathodea campanulata come from the dried flower buds of Spathodea of the family Bignoniaceae ( Spathodea Campanulata Beauv.). Literature reports that various parts of Spathodea campanulata contain rich bioactive substances and have various pharmacological effects. The leaves of Spathodea campanulata are often used to treat painful inflammation, constipation, dysentery, etc.; the flowers and bark are often used to treat mental disorders, malaria, hemorrhoids, bacterial infections, HIV, poor blood circulation, gastrointestinal diseases, respiratory diseases, urogenital system diseases, etc.; the ethanol extract of the leaves has an anticonvulsant effect; the extract of Spathodea campanulata flowers has the ability to absorb ultraviolet rays; in addition, the decoction of Spathodea campanulata bark has anti-HIV activity. Phytochemical analysis shows that Spathodea campanulata contains chemical components such as alkaloids, tannins, saponins, anthraquinone glycosides, caffeic acid, flavonoids, steroids, terpenoids, alkaloids, tannins and glycosides. Catalpol 6-caffeate (Verminoside) is a iridoid compound isolated from the flowers of Spathodea campanulata and has pharmacological activities such as antioxidant, anti-inflammatory, ulcer inhibition, and antitumor.

[0003] At present, the processes for separating and extracting catalpol 6-caffeate mainly involve solvent extraction, macroporous resin elution and silica gel column chromatography. However, the above processes have defects such as large loss of active ingredients and poor industrial reproducibility, and are not suitable for industrial production. Chinese Patent Publication (CN108373488A) reported catalpol 6-caffeate derivatives and their preparation methods and applications, and provided a preparation method of a catalpol-6 caffeate derivative with a n-butoxy group, and the derivative prepared by this method has better tumor inhibitory activity than the original compound catalpol 6-caffeate. At present, no reports on the inhibition of tyrosinase activity and the efficacy of skin barrier repair of catalpol 6-caffeate have been found.

[0004] Therefore, there is an urgent need to propose a method for preparing catalpol 6-caffeate from Spathodea campanulata flowers, its products and applications to solve the technical problems that the active ingredient catalpol 6-caffeate in Spathodea campanulata flowers has not been efficiently extracted and applied to products with related effects such as inhibition of tyrosinase activity and skin barrier repair at the present stage. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the main object of the present invention is to provide a method for preparing catalpol 6-caffeate from Spathodea campanulata flowers, as well as its products and applications, aiming to solve at least one of the technical problems that the existing extraction process of catalpol 6-caffeate has large losses of active ingredients, poor industrial reproducibility, is not suitable for industrial production, and catalpol 6-caffeate has not been applied to products with tyrosinase activity inhibition and skin barrier repair effects.

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

[0007] In the first aspect, the present invention provides a method for preparing catalpol 6-caffeate from Spathodea campanulata flowers, and the method comprises the following steps:

[0008] Step S1: Heat and reflux the dried and pulverized Spathodea campanulata flowers with a first solvent, filter after extraction, and concentrate the filtrate under reduced pressure to obtain a first extract;

[0009] Step S2: Use a second solvent as an extraction agent to extract the first extract, the second solvent is a mixed solvent of ethyl acetate and dichloromethane, the number of extractions is 2-3 times, combine the supernatant and filter and concentrate to obtain a second extract;

[0010] Step S3: Use resin as an adsorbent, elute the second extract with water, a third solvent, and a fourth solvent respectively, collect the elution part of the fourth solvent, and concentrate to obtain a refined product;

[0011] Step S4: Add a fifth solvent to the refined product, let it stand at low temperature to precipitate, then filter and dry under reduced pressure to obtain catalpol 6-caffeate.

[0012] Preferably, in step S1, the first solvent is an ethanol aqueous solution with an ethanol content of 30-50 wt%, and the heat reflux extraction is carried out 2-3 times, and the extraction time for each time is 2-3 hours.

[0013] It should be noted that the first solvent is preferably an aqueous ethanol solution with an ethanol content of 31-49 wt%, more preferably an aqueous ethanol solution with an ethanol content of 32-48 wt%, more preferably an aqueous ethanol solution with an ethanol content of 33-47 wt%, more preferably an aqueous ethanol solution with an ethanol content of 34-46 wt%, more preferably an aqueous ethanol solution with an ethanol content of 35-45 wt%, more preferably an aqueous ethanol solution with an ethanol content of 36-44 wt%, more preferably an aqueous ethanol solution with an ethanol content of 37-43 wt%, more preferably an aqueous ethanol solution with an ethanol content of 38-42 wt%, more preferably an aqueous ethanol solution with an ethanol content of 39-41 wt%, and most preferably an aqueous ethanol solution with an ethanol content of 40 wt%. During the research process, it was found that an aqueous ethanol solution with an ethanol content of 40 wt% can extract catalpol 6-caffeate from the flowers of Spathodea campanulata completely to the greatest extent with less impurity content.

[0014] Preferably, in step S2, the mass ratio of ethyl acetate to dichloromethane in the mixed solvent is 2:1 to 4:1.

[0015] It should be noted that in step S2, the mass ratio of ethyl acetate to dichloromethane in the mixed solvent is more preferably 3:1. During the research process, it was found that catalpol 6-caffeate in the flowers of Spathodea campanulata can be extracted completely to the greatest extent at this mass ratio.

[0016] Preferably, in step S3, the resin type is selected from any one of HP20SS, SP20SS or SP207SS.

[0017] Preferably, in step S3, the third solvent is an aqueous ethanol solution with an ethanol content of 15-25 wt%, and the fourth solvent is an aqueous ethanol solution with an ethanol content of 45-55 wt%.

[0018] It should be noted that in step S3, the third solvent is preferably an aqueous ethanol solution with an ethanol content of 16-24 wt%, 17-23 wt%, 18-22 wt%, 19-21 wt%, and most preferably an aqueous ethanol solution with an ethanol content of 20 wt%.

[0019] The fourth solvent is preferably an aqueous ethanol solution with an ethanol content of 46-54 wt%, more preferably an aqueous ethanol solution with an ethanol content of 47-53 wt%, more preferably an aqueous ethanol solution with an ethanol content of 48-52 wt%, more preferably an aqueous ethanol solution with an ethanol content of 49-51 wt%, and most preferably an aqueous ethanol solution with an ethanol content of 50 wt%.

[0020] Preferably, in step S4, the fifth solvent is a mixed solvent of n-hexane and dichloromethane, and the mass ratio of n-hexane to dichloromethane is 1:1 to 1:3. The mass ratio of the refined product to the fifth solvent is 1:8 to 1:12.

[0021] 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 more preferably 1:10.

[0022] Preferably, in step S4, the low-temperature standing time is 2 - 4 h, and the low-temperature standing temperature is 2 - 8 °C.

[0023] It should be noted that the low-temperature standing temperature is preferably 3 - 7 °C, more preferably 3 - 6 °C, more preferably 3 - 5 °C, and most preferably 4 °C. The standing time is preferably 3 h.

[0024] Preferably, in step S4, the content of the prepared catalpol 6-caffeate is not less than 90.00 wt%, and the structural formula of the catalpol 6-caffeate is:

[0025] 。

[0026] In a second aspect, the present invention also provides an application of the catalpol 6-caffeate prepared by the above method in skin care products, characterized in that the types of the skin care products include: emulsion, cream, aqueous solution or gel, but the types of the skin care products are not limited to the above examples.

[0027] Preferably, the catalpol 6-caffeate has the effects of inhibiting tyrosinase activity and repairing the skin barrier.

[0028] Preferably, the catalpol 6-caffeate can inhibit tyrosinase activity and up-regulate the expression of skin barrier repair genes, and the skin barrier repair genes include at least one of FLG gene and LOR gene.

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

[0030] (1) The method for preparing catalpol 6-caffeate from Spathodea campanulata flowers provided by the present invention can be used for batch production of high-content catalpol 6-caffeate to meet the usage requirements in the scenarios of skin barrier repair and skin whitening.

[0031] (2)The method for preparing catalpol 6-caffeate from Spathodea campanulata flowers provided by the present invention is simple in operation, 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, being 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 components in the first extract. Moreover, in step S3, resin is used as the adsorbent to enrich catalpol 6-caffeate to the greatest extent and obtain a refined product. In step S4, the fifth solvent is a mixed solvent of n-hexane and dichloromethane, and the mass ratio of n-hexane to dichloromethane is 1:1 to 1:3. The setting of the mass ratio of the refined product to the fifth solvent being 1:8 to 1:12 enables the target components to precipitate in the form of crystals at low temperature, that is, the synergy of steps S2, S3, and S4 can make the content of the prepared catalpol 6-caffeate not less than 90.00 wt%.

[0032] (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 values are all less than 0.85 mg / mL, having good whitening potential. At a concentration of 15 μg / mL to 1.2 mg / mL, it upregulates the expression of skin barrier repair genes, can significantly increase the expression of FLG gene and LOR gene, and the upregulation rate of skin barrier repair genes is 48% - 149%, having a good effect of maintaining and repairing the skin barrier. Description of the Drawings

[0033] Figure 1 Shows the liquid chromatography diagram of the catalpol 6-caffeate prepared in Example 1;

[0034] Figure 2 Shows the 13 C-NMR spectrum of the catalpol 6-caffeate prepared in Test Example 1;

[0035] Figure 3 Shows the 1 H-NMR spectrum of the sample containing catalpol 6-caffeate prepared in Test Example 1;

[0036] Figure 4 Shows the process flow diagram of the method for preparing catalpol 6-caffeate from Spathodea campanulata flowers of the present application. Detailed Embodiments

[0037] The technical solution of the present invention will be further described below in conjunction with the accompanying 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 rights of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0038] The source information of relevant raw materials and materials is as follows:

[0039] HaCaT cells (Shanghai Institute of Cells, Chinese Academy of Sciences; culture conditions: DMEM culture medium, fetal bovine serum (FBS) with a volume percentage concentration of 10%).

[0040] The MTT detection kit is from Xinbosheng Biotechnology Co., Ltd.;

[0041] The CCK-8 detection kit is from Xinbosheng Biotechnology Co., Ltd.;

[0042] The catalpol 6-caffeate standard was purchased from Sichuan Victy Biotech Co., Ltd.;

[0043] The raw materials, consumables or reagents involved in the present invention are all conventional products that can be obtained through commercial purchase. Term explanations:

[0044] 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.

[0045] 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 epidermal barrier function.

[0046] Example 1

[0047] A method for preparing catalpol 6-caffeate from the flowers of Spathodea campanulata, the method comprising the following steps:

[0048] Step S1: Weigh 50 g of dried Spathodea campanulata flowers, pulverize them, pass them through a 40-mesh sieve, place them in a reflux extraction device, use a 40 wt% ethanol aqueous solution as the extraction solvent, control the solid-liquid ratio to 1:10 (g / mL), heat and reflux for extraction, the extraction temperature is 80 °C, each extraction is for 3 hours, and a total of 2 extractions are carried out. After the extraction is completed, filter while it is hot and concentrate under reduced pressure to recover ethanol to obtain a first extraction solution;

[0049] Step S2: Use a mixed solvent of ethyl acetate and dichloromethane as the extractant to extract the first extraction solution. The mass ratio of ethyl acetate to dichloromethane is 3:1, the number of extractions is 2 times, combine the supernatant, filter, concentrate under reduced pressure, and recover the second solvent to obtain a second extraction solution;

[0050] Step S3: Use SP20SS resin as an adsorbent to elute the second extract. The elution steps are as follows: wash with water for 3BV, elute with 20wt% ethanol aqueous solution for 5BV, elute with 50wt% ethanol aqueous solution for 5BV, collect the eluate eluted with 50wt% ethanol aqueous solution, and concentrate to obtain the refined product;

[0051] Step S4: Add a mixed solvent composed of 10 times the amount of n-hexane and dichloromethane to the refined product. The mass ratio of n-hexane to dichloromethane is 1:2. Place it in a refrigerator at 4°C and let it stand still for 3 h. After crystallization or precipitation, filter and dry under reduced pressure to finally obtain 0.90 g of catalpol 6-caffeate.

[0052] After calculation, the yield is 1.80%. Using the purchased sample as the standard product, the content of catalpol 6-caffeate was detected by high performance liquid chromatography, and a standard curve was made. The mass percentage content of catalpol 6-caffeate was measured to be 93.44%. The liquid chromatography diagram is as Figure 1 shown.

[0053] Example 2

[0054] A method for preparing catalpol 6-caffeate from the flowers of Spathodea campanulata. The steps involved in the method are similar to those in Example 1, except that the mass ratio of ethyl acetate to dichloromethane as the extractant in Step S2 is 2:1. Finally, 0.85 g of catalpol 6-caffeate was obtained. The yield was calculated to be 1.70%. Using the purchased sample as the standard product, the content of catalpol 6-caffeate was detected by high performance liquid chromatography, and a standard curve was made. The mass percentage content of catalpol 6-caffeate was measured to be 92.69%.

[0055] Example 3

[0056] A method for preparing catalpol 6-caffeate from the flowers of Spathodea campanulata. The steps involved in the method are similar to those in Example 1, except that the mass ratio of ethyl acetate to dichloromethane as the extractant in Step S2 is 4:1. Finally, 0.93 g of catalpol 6-caffeate was obtained. The yield was calculated to be 1.86%. Using the purchased sample as the standard product, the content of catalpol 6-caffeate was detected by high performance liquid chromatography, and a standard curve was made. The mass percentage content of catalpol 6-caffeate was measured to be 91.31%.

[0057] Example 4

[0058] A method for preparing catalpol 6-caffeate from the flowers of Spathodea campanulata, the steps involved in this method are similar to those in Example 1, with the only difference being that in step S4, a mixed solvent composed of n-hexane and dichloromethane is added to the refined product, and the mass ratio of n-hexane to dichloromethane is 1:3. Finally, 0.85 g of catalpol 6-caffeate is obtained, and the calculated yield is 1.70%. Using an externally purchased sample as the standard, the content of catalpol 6-caffeate is detected by high performance liquid chromatography, a standard curve is made, and the mass percentage content of catalpol 6-caffeate is measured to be 90.41%.

[0059] Example 5

[0060] A method for preparing catalpol 6-caffeate from the flowers of Spathodea campanulata, the steps involved in this method are similar to those in Example 1, with the only difference being that in step S4, a mixed solvent composed of n-hexane and dichloromethane is added to the refined product, and the mass ratio of n-hexane to dichloromethane is 1:1. Finally, 0.86 g of catalpol-6 caffeate is obtained, and the calculated yield is 1.72%. Using an externally purchased sample as the standard, the content of catalpol 6-caffeate is detected by high performance liquid chromatography, a standard curve is made, and the mass percentage content of catalpol 6-caffeate is measured to be 91.19%.

[0061] Example 6

[0062] A method for preparing catalpol 6-caffeate from the flowers of Spathodea campanulata, the steps involved in this method are similar to those in Example 1, with the only difference being that in step S4, it is refrigerated and left standing for 4 h under the condition that the precipitation temperature is 6°C. Finally, 0.86 g of catalpol 6-caffeate is obtained, and the calculated yield is 1.71%. Using an externally purchased sample as the standard, the content of catalpol 6-caffeate is detected by high performance liquid chromatography, a standard curve is made, and the mass percentage content of catalpol 6-caffeate is measured to be 93.03%.

[0063] Comparative Example 1

[0064] A method for preparing catalpol 6-caffeate from the flowers of Spathodea campanulata, the steps involved in this method are similar to those in Example 1, with the only difference being that in step S2, the mass ratio of the extraction agent ethyl acetate to dichloromethane is 1:1. Finally, 0.72 g of catalpol 6-caffeate is obtained, and the calculated yield is 1.44%. Using an externally purchased sample as the standard, the content of catalpol 6-caffeate is detected by high performance liquid chromatography, a standard curve is made, and the mass percentage content of catalpol 6-caffeate is measured to be 81.79%.

[0065] Comparative Example 2

[0066] A method for preparing catalpol 6-caffeate from Spathodea campanulata flowers. The steps involved in this method are similar to those in Example 1, with the only difference being that in step S2, the mass ratio of ethyl acetate to dichloromethane as the extractant is 5:1. No crystallization or precipitation occurred. After concentration and drying, 0.98 g of the refined product was obtained. The yield was calculated to be 1.95%. Using an externally purchased sample as the standard, the content of catalpol 6-caffeate was detected by high-performance liquid chromatography, a standard curve was made, and the mass percentage content of catalpol 6-caffeate was measured to be 74.41%.

[0067] Comparative Example 3

[0068] A method for preparing catalpol 6-caffeate from Spathodea campanulata flowers. The steps involved in this method are similar to those in Example 1, with the only difference being that in step S4, a mixed solvent composed of n-hexane and dichloromethane was added to the refined product, and the mass ratio of n-hexane to dichloromethane was 2:1. After concentration and drying, 0.79 g of the refined product was obtained. The yield was calculated to be 1.58%. Using an externally purchased sample as the standard, the content of catalpol 6-caffeate was detected by high-performance liquid chromatography, a standard curve was made, and the mass percentage content of catalpol 6-caffeate was measured to be 82.01%.

[0069] Comparative Example 4

[0070] A method for preparing catalpol 6-caffeate from Spathodea campanulata flowers. The steps involved in this method are similar to those in Example 1, with the only difference being that in step S2, the extractant was only ethyl acetate. Finally, no crystallization or precipitation was found. After concentration and drying, 0.98 g of the refined product was obtained. The yield was calculated to be 1.96%. Using an externally purchased sample as the standard, the content of catalpol 6-caffeate was detected by high-performance liquid chromatography, a standard curve was made, and the mass percentage content of catalpol 6-caffeate was measured to be 77.12%.

[0071] Comparative Example 5

[0072] A method for preparing catalpol 6-caffeate from Spathodea campanulata flowers. The steps involved in this method are similar to those in Example 1, with the only difference being that in step S2, the extractant was only dichloromethane. Finally, no crystallization or precipitation was found. After concentration and drying, 0.66 g of the refined product was obtained. The yield was calculated to be 1.32%. Using an externally purchased sample as the standard, the content of catalpol 6-caffeate was detected by high-performance liquid chromatography, a standard curve was made, and the mass percentage content of catalpol 6-caffeate was measured to be 75.86%.

[0073] Comparative Example 6

[0074] A method for preparing catalpol 6-caffeate from the flowers of Spathodea campanulata, the steps involved in this method are 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%. Using the purchased sample as the standard product, the content of catalpol 6-caffeate is detected by high performance liquid chromatography, a standard curve is made, and the mass percentage content of catalpol 6-caffeate is measured to be 76.98%.

[0075] Comparative Example 7

[0076] A method for preparing catalpol 6-caffeate from the flowers of Spathodea campanulata, the steps involved in this method are 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 crystallization or precipitation is found. After concentration and drying, 1.10 g of refined product is obtained, and the calculated yield is 2.20%. Using the purchased sample as the standard product, the content of catalpol 6-caffeate is detected by high performance liquid chromatography, a standard curve is made, and the mass percentage content of catalpol 6-caffeate is measured to be 76.71%.

[0077] Comparative Example 8

[0078] A method for preparing catalpol 6-caffeate from the flowers of Spathodea campanulata, the steps involved in this method are similar to those in Example 1, the only difference being that the mixed solvent extraction step in step S2 is removed, and 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%. Using the purchased sample as the standard product, the content of catalpol 6-caffeate is detected by high performance liquid chromatography, a standard curve is made, and the mass percentage content of catalpol 6-caffeate is measured to be 65.28%. The yield is low and the purity of the obtained monomer is not high.

[0079] Comparative Example 9

[0080] A method for preparing catalpol 6-caffeate from the flowers of Spathodea campanulata, the steps involved in this method are similar to those in Example 1, the only difference being that the SP20SS resin used in step S3 is replaced with the conventional macroporous resin AB-8, and finally no crystallization or precipitation is found. After concentration and drying, 1.26 g of refined product is obtained, and the calculated yield is 2.51%. Using the purchased sample as the standard product, the content of catalpol 6-caffeate is detected by high performance liquid chromatography, a standard curve is made, and the mass percentage content of catalpol 6-caffeate is measured to be 65.33%.

[0081] Comparative Example 10

[0082] A method for preparing catalpol 6-caffeate from the flowers of Spathodea campanulata. The steps involved in this method are similar to those in Example 1, with the only difference being that in step S4, the crystallization temperature is refrigerated and left standing for 1.5 h at room temperature. Finally, no crystallization or precipitation is found. After concentration and drying, 1.08 g of refined product is obtained. The calculated yield is 2.15%. Using the purchased sample as the standard product, the content of catalpol 6-caffeate is detected by high performance liquid chromatography, and a standard curve is made. The mass percentage content of catalpol 6-caffeate is measured to be 81.88%.

[0083] Analysis of Examples 1-6 and Comparative Examples 1-10 shows that:

[0084] (1) For Examples 1-3, with other conditions unchanged, only the mass ratio of ethyl acetate to dichloromethane in step S2 is set to 3:1, 2:1, and 4:1 respectively. Among them, when the mass ratio of ethyl acetate to dichloromethane is 3:1, the measured mass percentage content of catalpol 6-caffeate is the highest, which is 93.44%.

[0085] Comparing Examples 1-3 with Comparative Examples 1-2, the difference between Comparative Examples 1-2 and Examples 1-3 is only that the mass ratio of ethyl acetate to dichloromethane in step S2 is set outside the range of 2:1 to 4:1, which are 1:1 and 5:1 respectively. The results show that the mass percentage content of catalpol 6-caffeate in Comparative Examples 1-2 decreases significantly, only being 81.79% and 74.41%. It can be seen that the mass ratio of ethyl acetate to dichloromethane in step S2 of 2:1 to 4:1 is the optimal ratio range.

[0086] (2) For Examples 1, 4-5, with other conditions unchanged, only the mass ratio of n-hexane to dichloromethane in step S4 is set to 1:2, 1:3, and 1:1 respectively. Among them, when the mass ratio of n-hexane to dichloromethane is 1:2, the measured mass percentage content of catalpol 6-caffeate is the highest, which is 93.44%.

[0087] Comparing Examples 1, 4-5 with Comparative Example 3, the difference between Comparative Example 3 and Examples 1, 4-5 is only 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 decreases significantly, only being 82.01%. It can be seen that the mass ratio of n-hexane to dichloromethane in step S4 of 1:1 to 1:3 is the optimal ratio range.

[0088] (3)Combining 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 simultaneously, 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 were also 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, a synergistic effect can occur between step S2 and step S4, so the corresponding scheme of Example 1 is a better scheme.

[0089] (4)For Examples 1-5 and Comparative Examples 4-7, it can be seen that only ethyl acetate or dichloromethane was used in step S2; or only n-hexane or dichloromethane was used in step S4, and the mass percentage content results of catalpol 6-caffeate (not exceeding 78%) were significantly worse than those of Examples 1-5. And a lower mass percentage content of catalpol 6-caffeate will result in 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 a compounded second solvent and fifth solvent in step S2 and step S4.

[0090] (5)For Examples 1-5 and Comparative Examples 8-9, it can be seen that omitting step S2 or selecting the adsorbent in step S3 as the conventional macroporous resin AB-8 will lead to inability to crystallize or a significant reduction in the mass percentage content of catalpol 6-caffeate, and will also affect the application of catalpol 6-caffeate in skin care products.

[0091] (6)For Example 6 and Comparative Example 10, it can be seen that it is better to set the crystallization temperature in step S4 to 2-8 °C and the static time to 2-4 h. Outside this numerical range, the mass percentage content of catalpol 6-caffeate will be significantly reduced.

[0092] Test Example 1: NMR Identification of Catalpol 6-Caffeate

[0093] See Appendix Figures 2 - 3 , taking the catalpol 6-caffeate obtained in Example 1 as an example: it is a yellow solid powder, molecular formula: C 24 H 28 O 13 , ESI-MS (pos.): 547.18 [M+Na] + , ESI-MS(neg.): 523 [M-H-] - ,1 1H NMR (600 MHz, CD3OD): δ : 5.13 (d, J J = 9.3 Hz, 1H, H-1), 6.33 (dd, J J = 6.0, 1.8 Hz, 1H, H-3), 4.98 (dd, J J = 7.9, 1.3 Hz, 1H, H-6), 4.95 (dd, J J = 6.0, 4.2 Hz, 1H, H-4), 2.62 - 2.51 (m, 2H, H-5, H-9), 3.66 (d, J J = 13.2 Hz, 1H, H-7), 4.13 (d, J J = 13.2 Hz, 1H, H-10a), 3.80 (d, J J = 13.2 Hz, 1H, H-10b), 4.75 (s, 1H, H-1'), 3.29 (dd, J J = 6.7, 2.3 Hz, 1H, H-2'), 3.37 (d, J J = 9.0 Hz, 1H, H-3'), 3.24 (td, J J = 9.3, 8.7, 4.2 Hz, 2H, H-4', H-5'), 3.89 (dd, J J = 12.0, 2.2 Hz, 1H, H-6'a), 3.64 - 3.60 (m, 1H, H-6'b), 7.01 (d, J J = 2.1 Hz, 1H, H-2''), 6.72 (d, J J = 8.2 Hz, 1H, H-5''), 6.91 (dd, J J = 8.2 2.1 Hz, 1H, H-6''), 7.56 (d, J J = 15.8 Hz, 1H, H-7''), 6.33 (dd, J J = 6.0, 1.8 Hz, 1H, H-8''); 13 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'').

[0094] Test Example 2: Tyrosinase Activity Test

[0095] 1. Experimental Purpose: By inhibiting the activity of tyrosinase, measuring the change in absorbance level, calculating the IC50 value of the inhibitory effect of the sample to be tested on tyrosinase, and evaluating the strength of the tyrosinase inhibitory ability of the test sample by comparing with the positive sample arbutin.

[0096] 2. Experimental Principle

[0097] Tyrosinase is the key rate-limiting enzyme for the generation of melanin from tyrosine through a series of biochemical reactions. Tyrosine

[0098] is converted into dopachrome under the catalytic action of tyrosinase, and this substance has a characteristic absorption at 475 nm. Therefore, the activity of tyrosinase can be measured by colorimetry.

[0099] 3. Test Method: Set up an α-arbutin control group and a sample group in a 96-well plate. Taking the catalpol 6-caffeate obtained in Examples 1-6 and Comparative Examples 1-2, 4-5, 8-10 as an example, prepare a stock solution of catalpol 6-caffeate obtained in each group at a concentration of 4.0 mg / mL. Both the control group and the sample group are prepared at a concentration of 4 mg / mL, which is the stock solution concentration, and serially diluted seven-fold.

[0100] The sample group is set with a control group, a blank group, an experimental group, and a color correction group; the arbutin control group is set with an experimental group and a blank group. The addition amounts of each group in the sample group are as follows:

[0101] Control Group B: 100 μL of tyrosine solution + 50 μL of tyrosinase + 50 μL of buffer solution;

[0102] Blank Group B0: 100 μL of tyrosine solution + 100 μL of buffer solution;

[0103] Experimental group A: 100 μL tyrosine solution + 50 μL tyrosinase solution + 50 μL samples with different concentrations;

[0104] Color removal group A0: 100 μL tyrosine solution + 50 μL buffer solution + 50 μL samples with different concentrations;

[0105] Samples in groups B and B0 can be shared;

[0106] The addition amounts in the arbutin control group are as follows:

[0107] Experimental group A: 100 μL tyrosine solution + 50 μL arbutin with different concentrations + 50 μL tyrosinase solution;

[0108] Experimental group A0: 100 μL tyrosine solution + 50 μL arbutin with different concentrations + 50 μL buffer solution;

[0109] Set 3 replicate wells for each concentration. After adding the samples, react at room temperature for 30 min, and measure the absorbance at 475 nm.

[0110] Calculation formula for the inhibition rate of tyrosinase activity (%IR): %IR = (1 - (A - A0) / (B - B0)) * 100%.

[0111] 4. Experimental results show that catalpol 6 - caffeate can inhibit the activity of tyrosinase, has good whitening potential, and due to the high mass percentage content of catalpol 6 - caffeate prepared in Examples 1 - 6, its inhibitory effect on tyrosinase activity is relatively excellent. See Table 1.

[0112] Table 1. Detection results of tyrosinase activity inhibition

[0113]

[0114] Test Example 3: In vitro evaluation test of the skin barrier efficacy of HaCaT cells

[0115] 1. Test method: Select HaCaT cells in the logarithmic growth phase with good morphology and inoculate them into a 24 - well plate, 1×10 5Cells / well were incubated in an incubator for 24 h. A control group (untreated cells) and a sample group (using catalpol 6-caffeate obtained in Examples 1-6 and Comparative Examples 1-2, 4-5, 8-10 as an example, with a catalpol 6-caffeate concentration of 120 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 CO2 incubator at 37 °C with a volume percentage of 5% 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. Comparative CT (ΔΔCT), with a control group set up, and the internal reference gene was β-actin, RQ = 2 -ΔΔCT where ΔΔCT = ΔCt treated - ΔCt control, and ΔCt treated and ΔCt control are the Ct differences between the target gene and the reference gene in the experimental group and the control group respectively, that is, ΔCt = CT target - CT β-actin. The RQ value is the relative expression level of the target gene mRNA.

[0116] 2. The experimental results showed that: and because the mass percentage content of catalpol 6-caffeate prepared in Examples 1-6 was high, catalpol 6-caffeate had no cytotoxicity at a concentration of 120 μg / mL and the expression of FLG and LOR genes was significantly up-regulated, and the skin barrier repair effect was significant (see Table 2).

[0117] Table 2. Detection results of FLG and LOR gene expression

[0118]

[0119] The applicant declares that the above description 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 any person 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 method for preparing catalpol 6-caffeate from the flowers of Spathodea campanulata, characterized in that, The method includes the following steps: Step S1: Heat and reflux the dried and pulverized flame tree flowers with a first solvent, filter after extraction, and concentrate the filtrate under reduced pressure to obtain a first extract; Step S2: Use a second solvent as an extractant to extract the first extract. The second solvent is a mixed solvent of ethyl acetate and dichloromethane, and the extraction is carried out 2 - 3 times. Combine the supernatant and filter and concentrate to obtain a second extract. The mass ratio of ethyl acetate to dichloromethane in the mixed solvent is 2:1 to 4:1; Step S3: Use resin as an adsorbent, elute the second extract with water, a third solvent, and a fourth solvent respectively, collect the elution part of the fourth solvent, and concentrate to obtain a refined product; The resin model is selected from any one of HP20SS, SP20SS, or SP207SS; Step S4: Add a fifth solvent to the refined product and let it stand still at a low temperature to precipitate, then filter and dry under reduced pressure to obtain catalpol 6 - caffeate with a content of not less than 90.00 wt%, where the fifth solvent is a mixed solvent of n - hexane and dichloromethane, and the mass ratio of n - hexane to dichloromethane is 1:1 to 1:3; the mass ratio of the refined product to the fifth solvent is 1:8 to 1:12; the low - temperature standing time is 2 - 4 h, and the low - temperature standing temperature is 2 - 8°C.

2. The method for preparing catalpol 6-caffeate from the flowers of Spathodea campanulata according to claim 1, wherein, In step S1, the first solvent is an aqueous ethanol solution with an ethanol content of 30 - 50 wt%, and the heat and reflux extraction is carried out 2 - 3 times, with each extraction time being 2 - 3 hours.

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

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