A method for preparing Jacaranda flower extract, its product and applications

Abstract

This invention discloses a Jacaranda flower extract, its preparation method, and its applications, belonging to the field of pharmaceutical technology. The preparation method includes: drying and pulverizing Jacaranda flowers, extracting them by heating and reflux with an ethanol-water solution, concentrating the extract under reduced pressure to obtain a first crude extract; mixing the first crude extract with an aqueous two-phase mixture for extraction, concentrating the upper phase under reduced pressure to obtain a second crude extract; purifying and eluting the second crude extract using a macroporous resin, decolorizing the eluent, and drying to obtain the Jacaranda flower extract. The Jacaranda flower extract obtained by this method has clearly defined active ingredients, exhibits significant photoprotective effects against UVB, and demonstrates significant repair effects after UVB photodamage. It is highly safe and can be used as an active ingredient in skincare products for sun protection.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and more specifically, relates to a method for preparing Jacaranda flower extract, its products, and applications. Background Technology

[0002] Jacaranda Jacaranda mimosifolia Jacaranda (D. Don) is a deciduous tree belonging to the genus Jacaranda in the family Bignoniaceae. Its leaves are opposite, with leaflets that are elliptic-lanceolate to elliptic-rhomboid, with an acute apex and a cuneate base. The flowers are blue, with a tubular calyx and a slender, blue corolla tube with rounded lobes. Jacaranda flowers contain flavonoids, phenolic compounds, polysaccharides, triterpenoids, and other active ingredients. The extracted bioactive substances possess various pharmacological effects, including antioxidant, anti-inflammatory, soothing, repairing, moisturizing, and skin damage healing properties.

[0003] Currently, jacaranda flower extract is listed in international cosmetic catalogs and is used in cosmetics abroad. CN117100657A invention patent describes a method combining supercritical carbon dioxide extraction and microwave extraction to prepare a jacaranda flower extract with moisturizing, soothing, and anti-inflammatory effects. Japanese patent JP5276857B2 discloses a whitening agent that incorporates jacaranda extract, which inhibits melanin production. The aforementioned jacaranda flower extracts have complex compositions, making accurate quality control difficult and thus challenging to ensure their safety and efficacy.

[0004] However, as a monomeric compound isolated from jacaranone-β-D-glucopyranoside, there are no reports in the existing technology regarding the process of preparing an extract containing jacaranone-β-D-glucopyranoside from jacaranone flowers, nor are there any reports on the sun protection efficacy of jacaranone-β-D-glucopyranoside extract from jacaranone flowers.

[0005] Therefore, it is of great significance to develop a Jacaranda flower extract with clearly defined active ingredients, photoprotective and sun damage repair effects, which can be used in cosmetics and pharmaceutical auxiliary products. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a method for preparing Jacaranda flower extract, its products and applications, and in particular, to provide a method for preparing Jacaranda flower extract with clearly defined active ingredients, sun protection effects and strong safety, its products and applications.

[0007] To achieve the above objectives, in a first aspect, the present invention provides a method for preparing Jacaranda flower extract, the method comprising the following steps: Step S1: Extract the dried and pulverized Jacaranda flowers by heating and reflux with an ethanol aqueous solution. After extraction, filter the solution and concentrate and dry the filtrate to obtain the first crude extract. Step S2: Mix the first crude extract with an aqueous two-phase extractant for extraction. After extraction, take the upper phase, concentrate and dry it to obtain the second crude extract. Step S3: Dissolve the second crude extract in pure water and elute it using a macroporous resin column to obtain the eluent; Step S4: Decolorize the eluent, filter under reduced pressure, concentrate and dry to obtain Jacaranda flower extract.

[0008] The method for preparing Jacaranda flower extract disclosed in this invention creatively combines aqueous two-phase extraction, macroporous resin purification and enrichment, and decolorization techniques. This effectively removes highly polar impurities such as polysaccharides and amino acids, resulting in an extract with clearly defined and high concentrations of active ingredients, exhibiting excellent anti-UV damage and post-UV damage repair effects. Furthermore, the Jacaranda flower extract demonstrates good safety and can be used as an active ingredient in skincare products and pharmaceutical adjuvants, providing sun protection.

[0009] Furthermore, in step S1, the volume percentage of ethanol in the ethanol-water solution is 40%-60%, for example: 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0010] It should be noted that the inventors have creatively discovered that using an ethanol aqueous solution with a volume percentage of 40%-60% for reflux extraction can effectively increase the content of active ingredients in Jacaranda flowers and its sun protection effect.

[0011] Furthermore, in step S1, the mass ratio of the ethanol aqueous solution to Jacaranda flowers is (10-15):1, wherein the specific point values ​​in (10-15) can be independently selected as 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, etc. Other specific point values ​​within this range can be selected, and will not be elaborated here.

[0012] Furthermore, in step S1, the heating reflux extraction is performed 1-3 times, with each extraction lasting 1-3 hours (e.g., 1.5 hours, 2 hours, 2.5 hours). After extraction, the mixture is filtered and the filtrates are combined.

[0013] Furthermore, in step S2, the upper phase of the aqueous two-phase extractant is an aqueous ethanol solution with a volume percentage of 30%-50%, and the lower phase of the aqueous two-phase extractant is an aqueous solution of a water-soluble salt with a mass percentage of 20%-30%.

[0014] Preferably, the volume percentage of ethanol in the upper phase is, for example, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0015] Preferably, the mass percentage content of water-soluble salts in the lower phase is, for example, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0016] Furthermore, in step S2, the water-soluble salt in the lower phase of the aqueous two-phase extractant is a combination of ammonium sulfate and dipotassium hydrogen phosphate, wherein the mass ratio of ammonium sulfate to dipotassium hydrogen phosphate is 1:1 to 3:1, for example, 1:1, 2:1, 3:1, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0017] It should be noted that using a combined salt solution in the lower phase during the aqueous two-phase extraction process can effectively increase the content of active ingredients and the sun protection efficacy of Jacaranda flower extract.

[0018] Furthermore, in step S3, the macroporous resin includes AB-8 macroporous resin or D101 type macroporous resin.

[0019] Furthermore, in step S3, the purification and elution steps include: after adsorption is complete, first elute with water for 4-6 BV, then elute with an ethanol aqueous solution with a volume percentage of 10%-20% for 5-10 BV, then elute with an ethanol aqueous solution with a volume percentage of 40%-60% for 5-10 BV, and collect the eluent with the ethanol aqueous solution with a volume percentage of 40%-60%.

[0020] It should be noted that using the specific elution procedure described above, namely eluting with water first and then with an ethanol aqueous solution, combined with a specific concentration of ethanol aqueous solution, can effectively increase the content of active ingredients in Jacaranda flower extract and its sun protection efficacy.

[0021] Preferably, the 4-6 BV can be 4 BV, 5 BV, 6 BV, etc.

[0022] Preferably, the 5-10 BV can be 5 BV, 6 BV, 7 BV, 8 BV, 9 BV, 10 BV, etc.

[0023] Preferably, the volume percentage of ethanol in the 10%-20% ethanol aqueous solution can be selected as 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, etc. Other specific values ​​within this range can also be selected, and will not be elaborated here.

[0024] Preferably, the volume percentage of the ethanol aqueous solution with a volume percentage of 40%-60% can be selected from 40%, 45%, 50%, 55%, 60%, etc. Other specific values ​​within this range can also be selected, and will not be elaborated here.

[0025] Furthermore, in step S4, the decolorizing agent is a compound of activated carbon and diatomaceous earth, and the mass ratio of activated carbon to diatomaceous earth is 1:1 to 3:1; for example, 1:1, 2:1, 3:1, etc. Other specific values ​​within this range can be selected, which will not be elaborated here.

[0026] Furthermore, in step S4, the decolorization treatment temperature is 40℃-60℃ and the duration is 30min-60min. Specific values ​​within the above range can be selected, and will not be elaborated here.

[0027] Secondly, the present invention provides a Jacaranda flower extract, which is prepared by the aforementioned method for preparing Jacaranda flower extract. The active ingredient in the Jacaranda flower extract includes jacaranda ketone-β-D-glucopyranoside, and the mass percentage content of jacaranda ketone-β-D-glucopyranoside in the Jacaranda flower extract is greater than 20%.

[0028] Thirdly, this invention proposes the application of Jacaranda flower extract in the preparation of skin care products with UVB photoprotection and / or UV damage repair effects, wherein the sun protection products include: skin care water, lotion, essence, isolation cream, spray or gel.

[0029] Compared with the prior art, the present invention has at least the following beneficial effects: (1) This invention discloses a method for preparing Jacaranda flower extract, its products, and applications, clarifying that the Jacaranda flower extract contains the active ingredient jacaranda ketone-β-D-glucopyranoside, enriching the research system of Jacaranda active ingredients. Furthermore, the preparation method of this invention employs a synergistic process of "heating and reflux, aqueous two-phase extraction, macroporous resin separation, and composite decolorization," wherein a recyclable ethanol aqueous solution is used as the main solvent, significantly reducing environmental pollution; the aqueous two-phase extraction uses a water-soluble salt system, leaving no toxic solvent residue, and the salts can be recovered, reducing waste; the compound decolorizing agent is a natural mineral material, causing no secondary pollution, suitable for continuous industrial production, and solving industrialization problems.

[0030] (2) This invention discloses a method for preparing Jacaranda flower extract, its products, and applications. The obtained extract has clearly defined active ingredients, including jacaranda ketone-β-D-glucopyranoside, with a mass percentage content of jacaranda ketone-β-D-glucopyranoside greater than 20%. The extract exhibits excellent UV damage protection and post-UV damage repair effects, providing significant sun protection. It is gentler and safer than traditional chemical sunscreens. Furthermore, the extract can be widely used in various sunscreen products such as lotions and creams, demonstrating strong compatibility and promoting the high-value utilization of Jacaranda, thus benefiting both economic and social aspects. Attached Figure Description

[0031] Figure 1 The liquid chromatogram of the Jacaranda flower extract prepared in Example 1; Figure 2 The mass spectrum of monomer compound 1 in the Jacaranda flower extract prepared in Example 1; Figure 3 This is the 1H NMR spectrum of monomer compound 1 in the Jacaranda flower extract prepared in Example 1. Detailed Implementation

[0032] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified illustrations 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 is determined by the claims.

[0033] Unless otherwise specified, the reagents and consumables used in the following embodiments were purchased from conventional reagent manufacturers in the art; unless otherwise specified, the experimental methods and techniques used were conventional methods and techniques in the art.

[0034] The ammonium sulfate and dipotassium hydrogen phosphate used in the following examples were purchased from Shanghai Yuanye Biotechnology Co., Ltd., and the activated carbon and diatomaceous earth were purchased from Shandong Xiya Chemical Co., Ltd.

[0035] Example 1

[0036] This embodiment provides a method for preparing Jacaranda flower extract, the preparation method comprising the following steps: Step S1: Take 50 g of dried and pulverized Jacaranda flowers and extract them by heating and refluxing with 500 g of 50% ethanol aqueous solution for 2 h. Repeat the extraction twice. After extraction, combine and filter the extracts. Concentrate the filtrate under reduced pressure to obtain the first crude extract. Step S2: The first crude extract obtained in step S1 is mixed with an aqueous two-phase extractant and stirred for 30 minutes. After extraction, the upper phase is taken and concentrated under reduced pressure to obtain the second crude extract. The upper phase of the aqueous two-phase extractant is 500 mL of ethanol aqueous solution with a volume percentage of 40%, and the lower phase is 250 mL of salt solution of ammonium sulfate and dipotassium hydrogen phosphate with a mass ratio of 2:1 and a mass percentage of 25%. Step S3: Dissolve the second crude extract obtained in step S2 in 5 times the amount of pure water, and separate and elute it using an AB-8 macroporous resin column. The elution steps are as follows: elute with water for 5 BV, elute with 15% ethanol aqueous solution for 8 BV, and elute with 50% ethanol aqueous solution for 6 BV. Collect the 50% ethanol aqueous solution eluent to obtain the eluent. Step S4: The eluent is decolorized using a decolorizing agent, the amount of which is 0.5% of the mass of the eluent. The decolorizing agent is a mixture of activated carbon and diatomaceous earth in a 2:1 ratio. The decolorization is carried out at a decolorization temperature of 50 °C for 40 min with stirring. After decolorization, the mixture is filtered under reduced pressure, concentrated and dried to obtain Jacaranda flower extract.

[0037] Example 2

[0038] This embodiment provides a method for preparing Jacaranda flower extract, the preparation method comprising the following steps: Step S1: Take 50g of dried and pulverized Jacaranda flowers and extract them by heating and refluxing with 500g of 40% ethanol aqueous solution for 3 hours. Repeat the extraction twice. After extraction, combine and filter the extracts. Concentrate the filtrate under reduced pressure to obtain the first crude extract. Step S2: Mix the first crude extract obtained in step S1 with an aqueous two-phase extractant and stir for 30 min. After extraction, take the upper phase and concentrate under reduced pressure to obtain the second crude extract. The upper phase of the aqueous two-phase extractant is 500 mL of ethanol aqueous solution with a volume percentage of 30%, and the lower phase is 250 mL of salt solution of ammonium sulfate and dipotassium hydrogen phosphate with a mass ratio of 3:1 and a mass percentage of 20%. Step S3: Dissolve the second crude extract obtained in step S2 in 5 times the amount of pure water, and separate and elute it using a D101 macroporous resin column. The elution steps are: elution with water for 4 BV, elution with 20% ethanol aqueous solution for 7 BV, and elution with 60% ethanol aqueous solution for 8 BV. Collect the 60% ethanol aqueous solution eluent to obtain the eluent. Step S4: The eluent is decolorized using a decolorizing agent, the amount of which is 0.3% of the mass of the eluent. The decolorizing agent is a mixture of activated carbon and diatomaceous earth in a 1:1 ratio. The decolorization is carried out at a decolorization temperature of 60 °C for 30 min with stirring. After decolorization, the mixture is filtered under reduced pressure, concentrated and dried to obtain Jacaranda flower extract.

[0039] Example 3

[0040] This embodiment provides a method for preparing Jacaranda flower extract, the preparation method comprising the following steps: Step S1: Take 50g of dried and pulverized Jacaranda flowers and extract them by heating and refluxing with 500g of 60% ethanol aqueous solution for 1 hour. Repeat the extraction 3 times. After extraction, combine and filter the extracts. Concentrate the filtrate under reduced pressure to obtain the first crude extract. Step S2: The first crude extract obtained in step S1 is mixed with an aqueous two-phase extractant and stirred for 30 minutes. After extraction, the upper phase is taken and concentrated under reduced pressure to obtain the second crude extract. The upper phase of the aqueous two-phase extractant is 500 mL of 50% ethanol aqueous solution, and the lower phase is 250 mL of 30% ammonium sulfate and dipotassium hydrogen phosphate salt solution with a mass ratio of 1:1. Step S3: Dissolve the second crude extract obtained in step S2 in 5 times the amount of pure water, and separate and elute it using an AB-8 macroporous resin column. The elution steps are: elution with water for 6 BV, elution with 10% ethanol aqueous solution for 6 BV, and elution with 40% ethanol aqueous solution for 9 BV. Collect the 40% ethanol aqueous solution eluted to obtain the eluent. Step S4: The eluent is decolorized using a decolorizing agent at a concentration of 1% of the eluent mass. The decolorizing agent is a mixture of activated carbon and diatomaceous earth in a 3:1 ratio. The decolorization process is carried out at a decolorization temperature of 40 °C for 30 min with stirring. After decolorization, the mixture is filtered under reduced pressure, concentrated, and dried to obtain Jacaranda flower extract.

[0041] Comparative Example 1 This embodiment provides a method for preparing Jacaranda flower extract, which differs from Example 1 only in that the volume percentage of ethanol and water in step S1 is 30%.

[0042] Comparative Example 2 This embodiment provides a method for preparing Jacaranda flower extract, which differs from Example 1 only in that the volume percentage of ethanol and water in step S1 is 70%.

[0043] Comparative Example 3 This embodiment provides a method for preparing Jacaranda flower extract, which differs from Example 1 only in that the lower phase in step S2 is a 250 mL ammonium sulfate salt solution with a mass percentage content of 25%.

[0044] Comparative Example 4 This embodiment provides a method for preparing Jacaranda flower extract, which differs from Example 1 only in that the lower phase in step S2 is a 250 mL solution of 25% by mass dipotassium hydrogen phosphate.

[0045] Comparative Example 5 This embodiment provides a method for preparing Jacaranda flower extract, which differs from Example 1 only in that the step of rinsing with water to remove 5 BV is omitted in step S3.

[0046] Comparative Example 6 This embodiment provides a method for preparing Jacaranda flower extract, which differs from Example 1 only in that the step of eluting 8 BV with a 15% ethanol aqueous solution in step S3 is omitted.

[0047] Comparative Example 7 This embodiment provides a method for preparing Jacaranda flower extract, which differs from Example 1 only in that the decolorizing agent in step S4 is activated carbon, a single decolorizing agent.

[0048] Comparative Example 8 This embodiment provides a method for preparing Jacaranda flower extract, which differs from Example 1 only in that the decolorizing agent in step S4 is a single decolorizing agent, diatomaceous earth.

[0049] Comparative Example 9 This embodiment provides a method for preparing Jacaranda flower extract, which differs from Example 1 only in that the step S2 aqueous two-phase extraction is omitted.

[0050] Comparative Example 10 This embodiment provides a method for preparing Jacaranda flower extract, which differs from Example 1 only in that the macroporous resin enrichment step in step S3 is omitted.

[0051] Comparative Example 11 This embodiment provides a method for preparing Jacaranda flower extract, which differs from Example 1 only in that the decolorization step S4 is omitted.

[0052] Test Example 1 The active ingredients of the Jacaranda flower extract obtained in Example 1 were analyzed by HPLC, and the results are shown in the attached figure. Figure 1 As shown, compound 1 is the main component.

[0053] The preparative liquid was used to separate and purify compound 1 from Example 1. The chromatographic column used was a Shim-pack GIS with an inner diameter of 20 mm and a length of 250 mm. The column packing material was octadecylsilane-bonded silica with a particle size of 5 μm. The preparative liquid chromatography conditions were as follows: mobile phase A was water, and mobile phase B was acetonitrile, with gradient elution. The gradient elution program was as follows: 0-10 min, mobile phase A: 90%~75%, mobile phase B: 10%~25%; 10-25 min, mobile phase A: 75%~68%, mobile phase B: 25%~32%; 25-30 min, mobile phase A: 68%~50%, mobile phase B: 32%~50%; 30-40 min, mobile phase A: 50%~20%, mobile phase B: 50%~80%. The elution flow rate was 8 mL / min, and the detection wavelength was 230 nm, yielding monomer compound 1 with a purity greater than 95%. Its NMR structure was identified, and the mass spectrum is shown below. Figure 2 As shown, the data is as follows, including the proton NMR spectrum. Figure 3 As shown in the figure, the data is shown below, and the structure of monomer compound 1 is determined as shown in formula (I).

[0054] Compound 1: Jacaranda ketone-β-D-glucopyranoside, yellow oily liquid, molecular formula: C 14 H 18 O9, ESI-MS (pos.) m / z: δ (ppm): 353.0839[M+Na] + , 1 H NMR (600 MHz, CD3OD): δ: 6.98 (m, 5H, 5-OH), 6.07 (m, 4H, H-2', H-3', H-5', H-6'), 4.47 (s, 4H, H-Glu), 3.35 (1H, H-1), 2.68 (dd, 2H, H-7'), 2.65 (s, 2H, H-6); (I) The content of jacaranda ketone-β-D-glucopyranoside in the Jacaranda flower extracts obtained in Examples 1-3 and Comparative Examples 9-11 was determined by HPLC, as shown in Table 1.

[0055] Table 1.

[0056] As shown in Table 1, the jacaranda flower extracts prepared in Examples 1-3 using the specific method of this invention are rich in compound 1 (jacaranda ketone-β-D-glucopyranoside), and its mass percentage content is greater than 20%. In Comparative Example 9, the aqueous two-phase extraction step S2 was omitted, and the mass percentage content of compound 1 (jacaranda ketone-β-D-glucopyranoside) in the prepared jacaranda flower extract was 14.32%. In Comparative Example 10, the macroporous resin enrichment step S3 was omitted, and the mass percentage content of compound 1 (jacaranda ketone-β-D-glucopyranoside) in the prepared jacaranda flower extract was 9.28%. In Comparative Example 11, the decolorization step S4 was omitted, and the mass percentage content of compound 1 (jacaranda ketone-β-D-glucopyranoside) in the prepared jacaranda flower extract was 18.37%. The mass percentage content of compound 1 (jacaranda ketone-β-D-glucopyranoside) in the Jacaranda flower extracts prepared in the other comparative examples was inferior to that in comparative examples 1-3, and not all of them are shown in Table 1.

[0057] It should also be noted that, according to testing, no coumarin or anthraquinone compounds that would affect the application of the jacaranda flower extract prepared by the specific method of this invention were detected in the jacaranda flower extract.

[0058] Test Example 2: Security Test The safety of Jacaranda flower extracts prepared in Examples 1-3 was tested according to the 2015 edition of the "Cosmetic Safety Technical Specifications". The results are shown in Table 2.

[0059] Table 2.

[0060] The results showed that the Jacaranda flower extract prepared by this invention was not irritating to the eyes, skin, or caused any skin allergies or phototoxicity. Its safety met the requirements of the Cosmetic Safety Technical Specifications and was highly safe.

[0061] Test Example 3: Sunscreen Efficacy Test (1) Protective effect of UVB irradiation on keratinocytes Prolonged exposure to ultraviolet (UV) radiation can easily cause age spots, wrinkles, and skin laxity, primarily due to UVB rays. Pre-UV exposure followed by sample intervention, then UV irradiation, and finally culturing keratinocytes for 24 hours to assess their morphology and survival, is an important method for evaluating the photoprotective performance (i.e., pre-UVB damage protection efficacy) of the added drug.

[0062] Blank control group: 100 μL of well-shaped cells were added to a 96-well cell culture plate, with a cell suspension density of 1 × 10⁻⁶ cells / well. 5 The cells were cultured at a concentration of 5% per mL until they reached about 80% of the culture plate, and then cultured at 37°C for 24 hours in CO2 at a volume percentage of 5%.

[0063] Model control group: 100 μL of well-morphologically healthy cells were added to a 96-well cell culture plate, with a cell suspension density of 1 × 10⁻⁶ cells / well. 5 cell / mL. When the cells reached approximately 80% confluence with the culture plate, they were simultaneously irradiated with UVB (irradiation dose 30 mJ / cm²). 2 The cells were then cultured for 24 hours in a fresh complete culture medium containing 5% CO2 at 37°C.

[0064] Experimental group: 100 μL of well-morphologically healthy cells were added to a 96-well cell culture plate, with a cell suspension density of 1 × 10⁻⁶ cells / well. 5 Cells / mL. When the cells reached approximately 80% confluence in the culture plate, 120 μg / mL of Jacaranda mimosifolia flower extract was added before light irradiation for 2 hours; followed by UVB irradiation (irradiation dose: 30 mJ / cm²). 2 The cells were then cultured for 24 hours in a fresh complete culture medium containing 5% CO2 at 37°C.

[0065] Detection method: A) Add 100 μL of cell suspension to the center of each 96-well cell culture plate, with a cell density of 1 × 10⁻⁶ cells. 5 The cell / mL cell suspension was prepared with complete culture medium; the 96-well cell culture plate was placed in an incubator and the cells were cultured for 24 hours at 37°C with 5% CO2 (v / v). B) Set up a blank control group, a model control group, and an experimental group. In the experimental group, the sample types according to the serial numbers shown in Table 2 were incubated separately, and a corresponding sample concentration was set for each sample type. Each sample type was pretreated for 2 h, and then irradiated with UVB at a dose of 30 mJ / cm². 2 ; C) After culturing cells for 24 h, the 96-well cell culture plate was replaced with complete medium containing CCK8 and incubated for 2 h. The absorbance at a wavelength of 450 nm was measured using an ELISA reader to obtain the optical density values ​​OD1 of the blank control group and OD2 of the experimental group.

[0066] D) Experimental data were processed using GraphPad and statistical analysis was performed using One-way ANOVA (and nonparametric) Turkey: Compare all pairs of columns. Each experiment was repeated at least 3 times to obtain the keratinocyte survival rate.

[0067] In this test case, the keratinocyte survival rates of the Jacaranda flower extract samples prepared in the model control group, Examples 1-3, and Comparative Examples 1-9 are shown in Table 3.

[0068] Table 3. Group Keratinocyte survival rate (%) Model control group 45.60% Example 1 86.64% Example 2 85.89% Example 3 85.23% Comparative Example 1 65.28% Comparative Example 2 56.75% Comparative Example 3 76.22% Comparative Example 4 73.87% Comparative Example 5 78.56% Comparative Example 6 63.52% Comparative Example 7 76.52% Comparative Example 8 72.20% Comparative Example 9 61.15% Comparative Example 10 43.23% Comparative Example 11 69.21% As shown in Table 3, the jacaranda flower extract prepared by the specific method of this invention has a significant protective effect on keratinocytes under UVB irradiation.

[0069] Comparing Examples 1-3 and Comparative Examples 1-2, it can be seen that the concentration of ethanol in the aqueous ethanol solution during heating and reflux extraction significantly affects the protective effect of the extract on keratinocytes under UVB irradiation. Comparing Examples 1-3 and Comparative Examples 3-4, it can be seen that the use of a combination of ammonium sulfate and dipotassium hydrogen phosphate in aqueous two-phase extraction can make the Jacaranda flower extract have a more significant protective effect on keratinocytes under UVB irradiation. Comparing Examples 1-3 and Comparative Examples 5-6, it can be seen that specific elution procedures and their specific operations significantly affect the protective effect of the extract on keratinocytes under UVB irradiation. Comparing Examples 1-3 and Comparative Examples 9-11, it can be seen that the aqueous two-phase extraction step, the macroporous resin enrichment step, and the decolorization step all significantly affect the protective effect of the extract on keratinocytes under UVB irradiation.

[0070] (2) In vitro repair effect of keratinocytes against ultraviolet damage Model building and sample processing Blank control group: 100 μL of well-shaped cells were added to a 96-well cell culture plate, with a cell suspension density of 1 × 10⁻⁶ cells / well. 5 The cells were cultured at a concentration of 5% CO2 per mL until they reached about 80% of the culture plate. Then, the cells were cultured at 37 °C for 24 hours.

[0071] Model control group: 100 μL of well-morphologically healthy cells were added to a 96-well cell culture plate, with a cell suspension density of 1 × 10⁻⁶ cells / well. 5 cell / mL. When the cells reach approximately 80% confluence with the culture plate, simultaneously irradiate with UVB (irradiation dose 30 mJ / cm²). 2After replacing the culture medium with a new complete medium, the cells were cultured for 24 hours at 37°C with a CO2 concentration of 5% (v / v).

[0072] Experimental group: 100 μL of well-morphologically healthy cells were added to a 96-well cell culture plate, with a cell suspension density of 1 × 10⁻⁶ cells / well. 5 Cells / mL, when the cells have grown to about 80% of the culture plate, are then irradiated with UVB (irradiation dose: 30 mJ / cm²). 2 After that, 120 μg / mL of Jacaranda flower extract was added and the cells were treated for 2 h. After replacing the medium with fresh complete medium, the cells were cultured for 24 h at 37°C with 5% CO2 (v / v).

[0073] Detection method: A) Add 100 μL of cell suspension to the center of each 96-well cell culture plate, with a cell density of 1×10⁻⁶ cells. 5 The cell count was 10 ... B) Set up a blank control group, a model control group, and an experimental group. After replacing PBS, irradiate with UVB light (irradiation dose 30 mJ / cm²). 2 In the experimental group, the test samples prepared with different culture media were incubated with different sample types in different numbered groups as shown in Table 3, and different sample concentrations were set for each sample type. The cells were cultured for 24 hours at 37 °C with CO2 at a volume percentage concentration of 5%. C) Replace the 96-well cell culture plate with complete medium containing CCK8, incubate for 2 hours, and measure the absorbance at a wavelength of 450 nm using an ELISA reader to obtain the optical density value OD1 of the blank control group and the optical density value OD2 of the experimental group. D) Experimental data were processed using GraphPad and statistical analysis was performed using One-way ANOVA (and nonparametric) Turkey: Compare all pairs of cells. A blank control group, a model control group, and an experimental group were set up. Each experiment was repeated at least 3 times to obtain the keratinocyte survival rate.

[0074] In this test case, the keratinocyte survival rates of the Jacaranda flower extract samples prepared in the model control group, Examples 1-3, and Comparative Examples 1-9 are shown in Table 4.

[0075] Table 4. Group Keratinocyte survival rate (%) Model group 44.16% Example 1 78.54% Example 2 76.63% Example 3 72.28% Comparative Example 1 55.63% Comparative Example 2 51.02% Comparative Example 3 67.26% Comparative Example 4 65.34% Comparative Example 5 68.59% Comparative Example 6 63.22% Comparative Example 7 68.27% Comparative Example 8 65.43% Comparative Example 9 58.28% Comparative Example 10 49.36% Comparative Example 11 63.78% As shown in Table 4, the jacaranda flower extract prepared by the specific method of the present invention has a significant in vitro repair effect on UV damage to keratinocytes.

[0076] Comparative Examples 1-3 and 1-2 show that the concentration of ethanol in the aqueous ethanol solution during reflux extraction significantly affects the in vitro keratinocyte UV damage repair effect of the extract. Comparative Examples 1-3 and 3-4 show that the combined use of ammonium sulfate and dipotassium hydrogen phosphate in aqueous two-phase extraction can make the Jacaranda flower extract have a more significant in vitro keratinocyte UV damage repair effect. Comparative Examples 1-3 and 5-6 show that specific elution procedures and their specific operations significantly affect the in vitro keratinocyte UV damage repair effect of the extract. Comparative Examples 1-3 and 9-11 show that the aqueous two-phase extraction step, the macroporous resin enrichment step, and the decolorization step all significantly affect the in vitro keratinocyte UV damage repair effect of the extract.

[0077] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing an extract of Paulownia elongata flowers, characterized by, The preparation method includes the following steps: Step S1: Extract the dried and pulverized Jacaranda flowers by heating and reflux with an ethanol aqueous solution. After extraction, filter the solution and concentrate and dry the filtrate to obtain the first crude extract. Step S2: Mix the first crude extract with an aqueous two-phase extractant for extraction. After extraction, take the upper phase, concentrate and dry it to obtain the second crude extract. Step S3: Dissolve the second crude extract in pure water and elute it using a macroporous resin column to obtain the eluent; Step S4: Decolorize the eluent, filter under reduced pressure, concentrate and dry to obtain Jacaranda flower extract.

2. The method of claim 1, wherein the preparation of the extract of the flower of Paulownia elongata is characterized by, In step S1, the volume percentage of ethanol in the aqueous ethanol solution is 40% to 60%.

3. The method for preparing Jacaranda flower extract according to claim 1, characterized in that, In step S1, the mass ratio of the ethanol aqueous solution to the Jacaranda flower raw material is (10-15):1, the number of heating and reflux extractions is 1-3 times, the extraction time for each extraction is 1-3 hours, and the extracted materials are combined and filtered after extraction.

4. The method for preparing Jacaranda flower extract according to claim 1, characterized in that, In step S2, the upper phase of the aqueous two-phase extractant is an aqueous ethanol solution with a volume percentage of 30% to 50%, and the lower phase is an aqueous solution containing a mass percentage of 20% to 30% water-soluble salts.

5. The method for preparing Jacaranda flower extract according to claim 4, characterized in that, In step S2, the water-soluble salt is a combination of ammonium sulfate and dipotassium hydrogen phosphate, wherein the mass ratio of ammonium sulfate to dipotassium hydrogen phosphate is (1-3):

1.

6. The method for preparing Jacaranda flower extract according to claim 1, characterized in that, In step S3, the macroporous resin is AB-8 or D101; The purification and elution steps include: after adsorption is complete, first elute with water for 4-6 BV, then elute with 10%-20% ethanol aqueous solution for 5-10 BV, then elute with 40%-60% ethanol aqueous solution for 5-10 BV, and collect the 40%-60% ethanol aqueous solution eluent.

7. The method for preparing Jacaranda flower extract according to claim 1, characterized in that, In step S4, the decolorization process involves using a decolorizing agent at a concentration of 0.1%-1% of the eluent.

8. The method for preparing Jacaranda flower extract according to claim 7, characterized in that, The decolorizing agent is a compound of activated carbon and diatomaceous earth, with a mass ratio of activated carbon to diatomaceous earth of (1-3):1; the decolorization treatment temperature is 40℃-60℃, and the treatment time is 30min-60min.

9. A Jacaranda flower extract, characterized in that, Prepared by the preparation method according to any one of claims 1-8, the active ingredient in the Jacaranda flower extract includes jacaranda ketone-β-D-glucopyranoside, wherein the mass percentage content of jacaranda ketone-β-D-glucopyranoside in the Jacaranda flower extract is greater than 20%.

10. The use of the Jacaranda flower extract as described in claim 9 in the preparation of skin care products with UVB photoprotection and / or UV damage repair effects.

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