Preparation method and application of butterfly pea flower extract

Abstract

The present invention provides a preparation method and application of a butterfly pea flower extract, relating to the field of biotechnology. The preparation method comprises: extracting dried butterfly pea flower petals using an extraction solvent under ultrasonic conditions, with the material-to-liquid ratio of the dried butterfly pea flower petals to the extraction solvent being 1:(15-25) g / mL, and the ultrasonic temperature being 50-70°C. The extraction solvent is an aqueous solution containing polyethylene glycol, with the concentration of polyethylene glycol in the water being 0.05-0.2 g / mL. This preparation method can increase the number of active ingredients in the butterfly pea flower extract, improve the content of flavonoids, and enhance the whitening and antioxidant effects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a preparation method of a butterfly pea flower extract and application thereof. Background Art

[0002] The following statements merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] Butterfly pea flower, also known as blue butterfly, butterfly flower bean, belongs to the leguminosae family Leguminosae butterfly pea Clitoria Linn. butterfly pea Clitoriaternatea L. The main active ingredients of butterfly pea flower extract include anthocyanins and flavonoids. Anthocyanins are primarily based on delphinidin, which is bound to glucose at the 3, 3', and 5' positions to form delphinidin glucoside, which is then acylated intramolecularly with coumaric acid, giving butterfly pea flower pigment its high stability. Other flavonoids include myricetin, quercetin, kaempferol, myricetin-3-glucoside, quercetin-3-glucoside, and kaempferol glucoside. Butterfly pea flower extract can be added directly to sunscreens, lotions, facial masks, and serums. Butterfly pea flower extract exhibits excellent antioxidant, anti-inflammatory, and anti-allergic skin-protecting properties. Its delphinidin anthocyanin can reduce and repair UV radiation damage to skin cells. Myricetin and quercetin can significantly reduce reactive oxygen species levels in endothelial cells, thereby reducing oxidative stress damage to vascular endothelial cells. However, there are few reports on the use of flavonoids in butterfly pea flower extract in cosmetics.

[0004] CN115414404A (Invention Title: A Method for Extracting Butterfly Pea Flowers) discloses the extraction of butterfly pea flowers using an ethanol / water solvent. However, this method only examines the optimal extraction method for anthocyanins, ignoring the extraction of flavonoids. This may result in ineffective extraction of the butterfly pea flower's active ingredients for whitening or antioxidant properties. Furthermore, current whitening ingredients in cosmetics have a limited melanin-inhibiting mechanism, with most products targeting only tyrosinase and ignoring the effects of pathways such as oxidative stress (ROS). Flavonoids have antioxidant, anti-inflammatory, whitening, and anti-aging properties. More fully extracting the active ingredients from butterfly pea flowers, especially flavonoids, would not only enhance the whitening effect of butterfly pea flower extract but also inhibit ROS, enhancing the whitening effect through antioxidant activity.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The present invention aims to provide a method for preparing a butterfly pea flower extract, so as to increase the types of active ingredients in the butterfly pea flower extract and improve the content of flavonoids.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] In a first aspect, a method for preparing a butterfly pea flower extract is provided, the preparation method comprising: extracting dried butterfly pea flower petals using an extraction solvent under ultrasonic conditions, wherein the solid-liquid ratio of the dried butterfly pea flower petals to the extraction solvent is 1:(15~25) g / mL, and the ultrasonic temperature is 50~70°C; the extraction solvent is an aqueous solution containing polyethylene glycol, and the concentration of polyethylene glycol in water is 0.05~0.2 g / mL.

[0009] In a second aspect, a butterfly pea flower extract prepared by the preparation method described in the first aspect is provided.

[0010] In a third aspect, the invention provides the use of the butterfly pea flower extract described in the second aspect in the preparation of whitening and / or antioxidant products.

[0011] In a fourth aspect, a product for whitening and / or anti-oxidation is provided, wherein the product for whitening and / or anti-oxidation comprises the butterfly pea flower extract described in the second aspect.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The present invention provides a method for preparing a butterfly pea flower extract. By using an aqueous solution containing polyethylene glycol as the extraction solvent and extracting under ultrasonic conditions, the active ingredients in the butterfly pea flower extract are increased and the content of flavonoids is enhanced. The newly added compounds are as follows: scutellarin I, honeysuckle glycoside, kaempferol, typhalaenoside, isorhamnetin-3-O-neohesperidin, narcissin, zedoaria flavonoids, tansyrin, and niacinamide. Niacinamide has a clear whitening effect; glycosides have antioxidant, anti-inflammatory, and anti-allergic skin protective properties, which can enhance the whitening efficacy of the butterfly pea flower extract. Furthermore, compared to extraction with water alone, the polyethylene glycol / water extraction method can increase the content of active ingredients such as vitamin C, astragalin, and isoquercitrin. In a preferred embodiment, the flavonoid content of the butterfly pea flower extract can reach 36.56%, and the astragalin content can reach 2.48 mg / g.

[0014] By examining the efficacy of inhibiting tyrosinase activity, anti-oxidation and inhibiting melanin production, and comparing it with the butterfly pea flower extract extracted with water alone, the butterfly pea flower extract obtained by the method of the present invention has higher inhibition of tyrosinase activity and melanin production than the butterfly pea flower extract obtained by water extraction alone, and also has higher ROS production inhibition and anti-oxidation ability than the butterfly pea flower extract obtained by water extraction alone.

[0015] The butterfly pea flower extract obtained by the preparation method of the present invention is a safe and non-irritating whitening active raw material. It reduces the generation and distribution of melanin particles by downregulating the expression of tyrosinase, inhibiting tyrosinase activity, and inhibiting the excessive production of reactive oxygen species. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 The total ion current of the butterfly pea flower extract obtained by PEG200 / water extraction in Example 1;

[0018] Figure 2 The content of vitamin C in the butterfly pea flower extract obtained by PEG200 / water extraction and water extraction alone in Example 1;

[0019] Figure 3 The content of astragalin in the butterfly pea flower extract obtained by PEG200 / water extraction and water extraction alone in Example 1;

[0020] Figure 4 is the content of isoquercetin in the butterfly pea flower extract obtained by PEG200 / water extraction and water extraction alone in Example 1;

[0021] Figure 5 Effects of different concentrations of PEG200 / water-extracted butterfly pea flower extract on HaCaT / B16 cell activity in Example 2, n=6;

[0022] Figure 6 Effects of butterfly pea flower extracts extracted by different methods on tyrosinase activity in HaCaT / B16 cells in Example 3, n=3, *p<0.05, ****p<0.0001, compared with the control group; ##p<0.01, compared with the PEG200 / water-extracted butterfly pea flower extract and the water-extracted butterfly pea flower extract;

[0023] Figure 7 The bottom image is the melanin micrograph of HaCaT / B16 cells after being treated with butterfly pea flower extracts extracted in different ways in Example 4;

[0024] Figure 8 This is a photo of melanin precipitation after cell lysis and centrifugation after HaCaT / B16 cells were treated with butterfly pea flower extracts using different extraction methods in Example 4;

[0025] Figure 9The expression of TYR (tyrosinase) in HaCaT / B16 cells after treatment with butterfly pea flower extracts extracted using different methods in Example 5, n=3, **p<0.01, ***p<0.001, compared with the control group; #p<0.05, compared with the PEG200 / water butterfly pea flower extract and the water butterfly pea flower extract;

[0026] Figure 10 These are photos of ROS staining in each experimental group after cells were treated with butterfly pea flower extracts using different extraction methods in Example 6. DETAILED DESCRIPTION

[0027] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0028] Herein, when an amount, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood to specifically disclose all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed alone. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted as including the range "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its end values ​​and all integers and fractions within the range.

[0029] Herein, unless otherwise stated, various reactions or operation steps may or may not be performed in sequence.

[0030] In this document, unless otherwise stated, arbitrary numbering is used to distinguish one entity or action from another entity or action, and does not necessarily require or imply any actual relationship, order, or importance between these entities or actions, such as numbering first, second, etc.

[0031] As used herein, "and / or" is used to indicate that one or both of the stated situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0032] As used herein, unless otherwise stated, "optionally," "optional," "optional," or "optional" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0033] Herein, the terms "comprise" or "comprising" are intended to imply the inclusion of stated elements, integers or steps, but not the exclusion of any other elements, integers or steps.

[0034] As used herein, the terms "respectively independently", "each independently selected from", "each independently selected from", and "independently selected from" are interchangeable and should be understood in a broad sense, meaning that within a class of objects, the specific characteristics of each specific object do not affect each other, and within this class of objects, the specific characteristics of any two objects may be the same or different.

[0035] In a first aspect, a method for preparing a butterfly pea flower extract is provided, the method comprising: extracting dried butterfly pea flower petals using an extraction solvent under ultrasonic conditions, wherein the extraction solvent is an aqueous solution containing polyethylene glycol, and the concentration of polyethylene glycol in water is 0.05-0.2 g / mL, for example, but not limited to 0.05, 0.1, 0.15, or 0.2 g / mL, preferably 0.1 g / mL. The solid-liquid ratio of the butterfly pea flower dried petals to the extraction solvent is 1:(15-25) g / mL, for example, but not limited to 1:15, 1:18, 1:20, 1:22, or 1:25 g / mL, preferably 1:25 g / mL; and the ultrasonic temperature is 50-70°C, for example, but not limited to 50, 55, 60, 65, or 70°C, preferably 60°C.

[0036] In an optional embodiment, the concentration of polyethylene glycol in water is 0.1 g / mL, the solid-liquid ratio of dried butterfly pea flower petals and extraction solvent is 1:25 g / mL, and the ultrasonic temperature is 60°C.

[0037] In an optional embodiment, the ultrasonic conditions further include: ultrasonic time of 30 to 90 min, for example, but not limited to 30, 40, 50, 60, 70, 80 or 90 min; ultrasonic power of 50 to 150 W, for example, but not limited to 50, 80, 100, 120 or 150 W.

[0038] In an optional embodiment, the ultrasonic conditions further include: ultrasonic time of 60 min, ultrasonic power of 100W.

[0039] In an optional embodiment, the butterfly pea flower dried petals are butterfly pea flower dried petals powder.

[0040] In an optional embodiment, the preparation method further includes centrifuging after extraction to obtain a supernatant, and freeze-drying the supernatant as the butterfly pea flower extract.

[0041] In an optional embodiment, the average molecular weight of the polyethylene glycol is 200-1000, such as but not limited to polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600 or polyethylene glycol 1000, preferably polyethylene glycol 200 (PEG200).

[0042] In some specific embodiments, the preparation method includes removing the stamens, calyx and pedicel of butterfly pea flower to obtain butterfly pea petals; drying and crushing the butterfly pea petals to obtain butterfly pea flower powder; using the extraction solvent to perform ultrasound-assisted extraction, and the operating conditions are as follows: the concentration of polyethylene glycol 200 in the extraction solvent is 0.1 g / mL, the solid-liquid ratio of butterfly pea flower powder and extraction solvent is 1:25, the ultrasonic temperature is 60°C, the ultrasonic time is 60 min, the ultrasonic power is 100W, and the supernatant is obtained by centrifugation after extraction.

[0043] In the second aspect, a butterfly pea flower extract prepared by the preparation method described in the first aspect is also provided.

[0044] In an optional embodiment, the butterfly pea flower extract is a lyophilized agent.

[0045] In a third aspect, the invention provides the use of the butterfly pea flower extract described in the second aspect in the preparation of whitening and / or antioxidant products.

[0046] In an optional embodiment, the butterfly pea flower extract in the whitening and / or anti-oxidation product is a lyophilized agent.

[0047] In an optional embodiment, in the whitening and / or anti-oxidation product, the working concentration of the butterfly pea flower extract in the lyophilized agent does not exceed 50 μg / mL, for example, it can be but is not limited to not more than 30 μg / mL, not more than 35 μg / mL, not more than 40 μg / mL, not more than 45 μg / mL or not more than 50 μg / mL.

[0048] In an optional embodiment, in the whitening and / or anti-oxidation product, the working concentration of the butterfly pea flower extract in the lyophilized agent is 25 μg / mL.

[0049] In an optional embodiment, the butterfly pea flower extract is the only therapeutic agent in the whitening and / or anti-oxidation product.

[0050] In an optional embodiment, the butterfly pea flower extract is used in conjunction with at least one other for whitening and / or antioxidant ingredients to prepare a whitening and / or antioxidant product. When the butterfly pea flower extract is used in conjunction with one or more other ingredients, it can be administered simultaneously with the one or more other ingredients. In some such embodiments, the butterfly pea flower extract and other ingredients can be administered simultaneously as a part of the same composition. In other embodiments, the butterfly pea flower extract used in conjunction with other ingredients does not need to be administered simultaneously or is administered in the same composition with the ingredient. That is, one or more ingredients administered after and / or before the butterfly pea flower extract in the present invention are also considered to be "used in conjunction" with the butterfly pea flower extract, that is, the butterfly pea flower extract and other ingredients are administered by different modes of administration.

[0051] In an optional embodiment, the subjects of the whitening and / or antioxidant products include but are not limited to mammals, and the mammals include but are not limited to humans, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, goats, sheep, cows, pigs, horses or monkeys.

[0052] In a fourth aspect, a whitening and / or antioxidant product is also provided, wherein the whitening and / or antioxidant product comprises the butterfly pea flower extract described in the second aspect.

[0053] In an optional embodiment, the whitening and / or antioxidant product includes the butterfly pea flower extract in a freeze-dried form.

[0054] In an optional embodiment, the working concentration of the butterfly pea flower extract in the lyophilized agent in the whitening and / or antioxidant product does not exceed 50 μg / mL, for example, it can be but is not limited to not more than 30 μg / mL, not more than 35 μg / mL, not more than 40 μg / mL, not more than 45 μg / mL or not more than 50 μg / mL.

[0055] In an optional embodiment, the working concentration of the butterfly pea flower extract in the lyophilized agent in the whitening and / or antioxidant product is 25 μg / mL.

[0056] In an optional embodiment, the whitening and / or antioxidant product further contains optional excipients acceptable in the art, including but not limited to solvents, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, anti-caking agents, flavoring agents, antibacterial agents, suspending agents, coating agents, film-forming agents, fragrances, viscosity enhancers, anti-adhesion agents, antioxidants, antioxidant synergists, chelating agents, pH regulators, adsorbents, plasticizers, surfactants, thickeners, inclusion agents, protective agents, moisturizers, softeners, absorbents, diluents, release regulators, pressure-sensitive adhesives, hardeners, hollow capsules, matrices and drug carrier materials. One or more.

[0057] In an optional embodiment, the whitening and / or antioxidant products include but are not limited to external use agents, oral agents or injections.

[0058] In an optional embodiment, the whitening and / or antioxidant product includes an external agent, which includes but is not limited to a paste, an ointment, a spray, a gel, a liniment, a paint, a film, a patch, an ointment or a film.

[0059] In an optional embodiment, the whitening and / or antioxidant product includes cosmetics.

[0060] In an optional embodiment, the cosmetics include but are not limited to cleansing milk, cleansing cream, cleansing foam, lotion, essence, emulsion, facial cream or facial mask.

[0061] The present invention is further described below by way of specific examples. However, it should be understood that these examples are merely provided for more detailed description and are not to be construed as limiting the present invention in any form.

[0062] Example 1

[0063] Investigation on the extraction process of butterfly pea flower:

[0064] 1. Experimental methods:

[0065] (1) Butterfly pea flower extraction process design:

[0066] The stamens, calyx, and pedicel of butterfly pea flowers were removed to obtain butterfly pea petals. The petals were dried and crushed to obtain butterfly pea flower powder. The pretreated butterfly pea flower powder was weighed and extracted with polyethylene glycol 200 (PEG 200) and water as the extraction solvent. Ultrasound-assisted extraction was performed under the following operating conditions: PEG 200 concentration, material-liquid ratio of butterfly pea flower powder to extraction solvent, and ultrasonic temperature were set according to Table 2. The ultrasonication time for each group was 60 minutes, and the ultrasonic power was 100 W. The extract was centrifuged at 4200 rpm for 15 minutes, and the supernatant was collected and freeze-dried.

[0067] The effects of polyethylene glycol concentration, material-to-liquid ratio, and ultrasonic temperature on the total flavonoids and astragalin content in butterfly pea flower extract were investigated. A single-factor experimental group was used (see Table 1). Detailed experimental conditions are detailed in Table 2. Water was used as the sole extraction solvent for comparison.

[0068] Table 1 Single factor investigation table

[0069]

[0070] Table 2 Experimental conditions of each experimental group

[0071]

[0072] A total of 9 batches of butterfly pea flower extracts were extracted according to the parameters recorded in experimental groups A-1~3, experimental groups B-1~3, and experimental groups C-1~3.

[0073] (2) Enrichment of chemical components:

[0074] Components of the butterfly pea flower extracts obtained from PEG200 / water extraction (experimental groups A / B / C-1–3) and water extraction were identified using a UPLC Hypersilgold column and an Orbitrap Fusion mass spectrometer. Mobile phase A: 0.01% formic acid, mobile phase B: 0.1% formic acid-acetonitrile, gradient elution (see Table 3); injection volume: 5 μL; flow rate: 200 μL / min. Mass spectrometry parameters were as follows: positive ionization: 3.5 kV, negative ionization: 3.0 kV; sheath gas (Arb): 40 Aux; auxiliary gas (Arb): 10 Aux; purge gas (Arb): 0 Aux; ion transfer tube temperature: 320°C, vaporizer temperature: 350°C; full scan mode: resolution: 60,000; scan range: 100–1000 m / z; mass tolerance: 10 ppm. The measured compounds were confirmed by PubChem database and their structures were downloaded. The files were saved in SDF format. Figure 1 shown.

[0075] Table 3 HPLC mobile phase conditions

[0076]

[0077] By comparing the database in positive and negative ion modes, a total of 65 compounds were identified from the components extracted by PEG200 / water. According to the scoring results of mzVault and mzCloud, compounds with scores greater than 80 were screened and compared with the information of secondary fragment ions in the literature, including astragalin, isoquercetin, morin, rutin, hyperoside, kaempferol-3-O-rutinoside, kaempferol, clethrin I, quercetin, and Luo Han Han. Flavouring, L-proline, atractylodesin, trigonelline hydrochloride, kaempferol-7-O-β-D-glucoside, L-phenylalanine, honeysuckle glycoside, p-hydroxycinnamic acid, kaempferol, typhalaenoside, vitamin C, cyanidin-3-O-glucoside, luteolin, myricetin, isorhamnetin-3-O-neohesperidin, narcissin, luteolin, coumarin, quercetin, herbaceous glycoside, camelliaside B, zearalenone, niacinamide, tannin. The flavonoid components include astragalin, isoquercetin, morin, rutin, hyperoside, kaempferol-3-O-rutinoside, kaempferol, cyperidin I, quercetin, kaempferol-7-O-β-D-glucoside, honeysuckle, kaempferol, typhaneuroside, cyanidin-3-O-glucoside, luteolin, myricetin, isorhamnetin-3-O-neohesperidin, narcissin, luteolin, quercetin, herbaceous glycoside, camelliaside B, zedoaria flavonoids, and tansylide.

[0078] The results showed that the types of flavonoid components in the butterfly pea flower extract obtained by PEG200 / water extraction increased, especially flavonoid glycosides.

[0079] Compared to water alone, the PEG200 / water extraction yielded the following compounds: scutellarin I, honeysuckle glycoside, kaempferol, typhalaenoside, isorhamnetin-3-O-neohesperidin, narcissin, zedoaria flavonoids, tansylated glycoside, and niacinamide. Niacinamide has a clear whitening effect; glycosides possess antioxidant, anti-inflammatory, and anti-allergic properties, enhancing the whitening efficacy of butterfly pea flower extract.

[0080] (3) Comparison of the total flavonoids content and astragalin content in butterfly pea flower extracts obtained by PEG200 / water extraction (experimental groups A / B / C-1~3) and water extraction. The results are shown in Table 4:

[0081] Table 4 Total flavonoids and astragalin content in butterfly pea flower extracts obtained by PEG200 / water extraction and water extraction

[0082]

[0083] As shown in Table 4, after PEG 200 / water extraction, the total flavonoid content first increased and then decreased with increasing PEG 200 concentration, reaching a maximum of 35.97% at 0.1 g / mL. The highest total flavonoid content was achieved at a solid-liquid ratio of 1:25 and at an ultrasonic temperature of 60°C. Regarding astragalin content, the highest concentration was achieved at a PEG 200 concentration of 0.2 g / mL, but there was no significant improvement compared to 0.1 g / mL. Similarly, the highest astragalin content was achieved at a solid-liquid ratio of 1:25 and an ultrasonic temperature of 60°C. In conclusion, a PEG 200 concentration of 0.1 g / mL, a solid-liquid ratio of 1:25 g / mL, and an ultrasonic temperature of 60°C were selected as the optimal extraction process. After re-extraction, the total flavonoid and astragalin contents of the extract were determined to be 36.56% and 2.48 mg / g, respectively.

[0084] (4) Compare the content of vitamin C, astragalin and isoquercetin in the butterfly pea flower extract obtained by the optimal process in step (3) above and the butterfly pea flower extract extracted only with water. The only difference between the butterfly pea flower extraction process using only water and the optimal process is that the extraction solvent does not contain PEG200. The results are as follows Figure 2 、 Figure 3 and Figure 4 shown.

[0085] In the following Examples 2 to 6, the extraction method for extracting butterfly pea flower extract using PEG200 / water as a solvent is as follows: the butterfly pea flower is removed from the stamens, calyx, and pedicel to obtain butterfly pea petals; the butterfly pea petals are dried and crushed to obtain butterfly pea flower powder; the pretreated butterfly pea flower powder is weighed, PEG 200 and water are used as extraction solvents, and ultrasound-assisted extraction is performed under the following operating conditions: PEG 200 concentration is 0.1 g / mL, the solid-liquid ratio of butterfly pea flower powder to extraction solvent is 1:25 g / mL, the ultrasound temperature is 60°C, the ultrasound time for each group is 60 min, and the ultrasound power is 100 W. The extract is centrifuged at 4200 rpm for 15 min, the supernatant is taken, and the supernatant is freeze-dried. The difference between the extraction method of extracting butterfly pea flower extract using only water and the extraction method of extracting butterfly pea flower extract using PEG200 / water as a solvent is that the extraction solvent does not contain PEG200, and the other parameters are the same.

[0086] Example 2

[0087] CCK8 experiment:

[0088] A co-culture system of HaCaT (Human immortalized keratinocyte cell line) and B16 cells (Mouse Melanoma Cell Line B16) was established with a HaCaT:B16 cell ratio of 2:1. After HaCaT and B16 cells adhered to the wall, the control group (culture system) was not treated with drugs and the medium was changed. The experimental groups were treated with 3.125, 6.25, 12.5, 25, 50, 100, 200, 400, and 800 µg / mL PEG200 / water-extracted butterfly pea flower extracts prepared in complete culture medium for 24 hours, with 6 replicate wells set up for each group. After 24 hours, the old medium was removed and 10 µL of CCK8 solution was added to each well. The cells were incubated at 37°C in the dark for 2 hours. The supernatant was discarded, and the absorbance at 450 nm was measured using a microplate reader to calculate the cell viability. The results are shown in Figure 2. Figure 5 As shown, when the concentration of PEG200 / water-extracted butterfly pea flower extract was ≤50 μg / mL, the viability of the HaCaT / B16 mixed cells exceeded 85%, indicating its high biocompatibility and its potential as a safe cosmetic raw material. Therefore, 25 μg / mL of butterfly pea flower extract was selected for the subsequent cell experiments in Examples 3-6.

[0089] Example 3

[0090] Determination of intracellular tyrosinase activity:

[0091] A co-culture model of HaCaT and B16 cells was established in six-well plates. After cells were fully adhered, the following experimental groups were assigned: experimental groups were treated with 25 μg / mL PEG200 / water and water-extracted butterfly pea flower extract, respectively; a blank control group received only medium replacement; and a positive control group received 50 μg / mL vitamin C as an intervention drug. Three biological replicates were performed for each experimental group. After 48 hours of drug treatment, the reaction was terminated in an ice bath, the medium was removed, and the cells were washed twice with pre-chilled PBS. Whole-cell lysis was performed by adding 100 μL of RIPA lysis buffer to each well. After rapid freezing at -80°C for 30 minutes, the cells were reconstituted at 4°C and centrifuged at 12,000 rpm for 20 minutes at 4°C. Take 10 μL of supernatant and quantify protein using the BCA assay. Simultaneously, transfer 90 μL of supernatant to a 96-well plate and add 10 μL of 10 mmol / L L-DOPA substrate solution. Incubate at 37°C in the dark until a brown color reaction develops. Immediately measure the absorbance at 490 nm on a microplate reader. Data analysis is performed using the following formula:

[0092] Relative tyrosinase activity = (OD490 of experimental group) / (corresponding sample protein concentration)

[0093] Tyrosinase activation rate (%) = (relative tyrosinase activity of the experimental group / relative tyrosinase activity of the blank group) × 100%.

[0094] The results are as follows Figure 6 As shown, after vitamin C administration, tyrosinase activity in B16 cells decreased significantly compared to the control group. Tyrosinase activity also decreased significantly after administration of water and PEG200 / water-extracted butterfly pea flower extracts compared to the control group. Furthermore, the PEG200 / water-extracted butterfly pea flower extract had a significantly higher inhibitory effect on tyrosinase activity than the water-extracted butterfly pea flower extract. These results indicate that the PEG200 / water-extracted butterfly pea flower extract was more potent than the water-extracted butterfly pea flower extract in inhibiting tyrosinase activity in HaCaT / B16 cells.

[0095] Example 4

[0096] Melanin content determination:

[0097] The preparation process for cell samples was the same as the tyrosinase assay method in Example 3. After 48 hours of drug intervention, samples were collected on ice: the culture supernatant was discarded, and the cells were washed twice with pre-chilled PBS buffer to remove residual culture medium. The distribution and content of melanin in each group of cells were observed microscopically. 100 μL of RIPA lysis buffer was added to each well for whole-cell lysis. After rapid freezing and lysis at -80°C for 30 minutes, the cells were re-dissolved at 4°C and centrifuged at 12,000 rpm at 4°C for 20 minutes. The melanin precipitate area at the bottom of the tube was quantitatively detected using the ImageJ image analysis system, and the color development area and grayscale value of the precipitate were analyzed using ImageJ software.

[0098] Under the microscope, compared with the control group, the B16 cells in the group treated with vitamin C and PEG200 / water-extracted butterfly pea flower extract secreted less melanin ( Figure 7 After the cell administration, the cells were broken and centrifuged, and it was observed that the cells in the control group had obvious black precipitates. After the use of vitamin C, the cell melanin synthesis decreased significantly. After the use of PEG200 / water-extracted butterfly pea flower extract, the melanin synthesis also decreased significantly compared with the control group; however, the ability of the water-extracted butterfly pea flower extract to inhibit melanin production was not obvious ( Figure 8 The results showed that the PEG200 / water-extracted butterfly pea flower extract had a higher ability to inhibit the production of melanin in B16 cells than the water-extracted butterfly pea flower extract, and is a potential whitening raw material.

[0099] Example 5

[0100] Fluorescence quantitative PCR detection of TYR gene expression in HaCaT / B16 cell co-culture system:

[0101] The cell sample preparation process was the same as the tyrosinase assay described in Example 3. After administration, total cellular RNA was extracted using the Trizol method, and cDNA was reverse transcribed using the Prime Script™ RT reagent Kit with gDNA Eraser (perfect Real Time) kit, all procedures strictly following the manufacturer's instructions. Real-time PCR analysis was performed using the TB Green® Premix ExTaq™ II (Tli RNaseH Plus) kit to detect the expression of relevant mRNAs. ΔΔ The Ct method was used for calculation and data analysis. GAPDH was used as an internal control. All primers were synthesized by Sangon Biotech Co., Ltd., and the primer sequences are listed in Table 5.

[0102] Table 5 Fluorescence quantitative PCR primer sequences

[0103]

[0104] Compared with the control group, the PEG200 / water-extracted butterfly pea flower extract significantly inhibited the expression of TYR (tyrosinase) ( Figure 9 ), with an effect similar to that of vitamin C. Water-extracted butterfly pea flower extract also downregulated TYR expression, but the effect was significantly lower than that of the PEG200 / water-extracted butterfly pea flower extract. This suggests that the PEG200 / water-extracted butterfly pea flower extract can downregulate tyrosinase expression and inhibit tyrosinase activity, thereby inhibiting melanin production, with a stronger effect than the butterfly pea flower extract extracted with water alone.

[0105] Example 6

[0106] ROS staining:

[0107] After B16 cells were fully adhered, the following experimental groups were assigned: experimental groups were treated with either 25 μg / mL PEG200 / water-extracted butterfly pea flower extract or water-extracted butterfly pea flower extract; a blank control group received only medium replacement; and a positive control group received 50 μg / mL vitamin C as an intervention drug. Following treatment, cells were stimulated with UVB for 30 minutes. For ROS staining, 5 μM CellROX-green dye (Invitrogen) was added to complete culture medium at 37°C for 30 minutes, followed by live imaging.

[0108] After UVB treatment, ROS production in cells increased significantly. After vitamin C administration, ROS expression levels returned to normal levels. After the use of water and PEG200 / water butterfly pea flower extracts, ROS production also decreased significantly compared to the model group. However, the PEG200 / water butterfly pea flower extract had a stronger ability to inhibit ROS production ( Figure 10 The results showed that the PEG200 / water-extracted butterfly pea flower extract had significant ROS inhibition and antioxidant capabilities, which is another mechanism of its whitening effect.

[0109] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a butterfly pea flower extract, characterized in that: include: Extracting dried butterfly pea flower petals using an extraction solvent under ultrasonic conditions, wherein the solid-liquid ratio of the butterfly pea flower petals to the extraction solvent is 1:(15-25) g / mL, and the ultrasonic temperature is 50-70°C; the extraction solvent is an aqueous solution containing polyethylene glycol, and the concentration of the polyethylene glycol in water is 0.05-0.2 g / mL; the average molecular weight of the polyethylene glycol is 200; and the ultrasonic conditions further include: ultrasonic time of 30-90 min and ultrasonic power of 50-150 W; The butterfly pea flower extract contains celery glycoside I, honeysuckle glycoside, kaempferol glycoside, cattail glycoside, isorhamnetin-3-O-neohesperidin, narcissin, zedoaria flavonoids, tansylide, nicotinamide, vitamin C, astragaloside and isoquercetin.

2. The preparation method according to claim 1, wherein The solid-liquid ratio of the dried butterfly pea flower petals to the extraction solvent is 1:25; and / or the concentration of polyethylene glycol in water is 0.1 g / mL; and / or the ultrasonic temperature is 60°C.

3. The preparation method according to claim 1, characterized in that The butterfly pea flower dried petals are butterfly pea flower dried petals powder.

4. The preparation method according to claim 1, characterized in that The preparation method further comprises centrifuging after extraction to obtain a supernatant, and freeze-drying the supernatant to serve as the butterfly pea flower extract.

5. The preparation method according to claim 4, wherein The method comprises removing butterfly pea flower stamens, calyxes and pedicels to obtain butterfly pea flower petals; drying and crushing the butterfly pea flower petals to obtain butterfly pea flower powder; and using the extraction solvent for ultrasound-assisted extraction, wherein the operating conditions are as follows: the concentration of polyethylene glycol 200 in the extraction solvent is 0.1 g / mL, the solid-liquid ratio of the butterfly pea flower powder to the extraction solvent is 1:25, the ultrasound temperature is 60°C, the ultrasound time is 60 min, the ultrasound power is 100 W, and after extraction, centrifugation is performed to obtain a supernatant.

6. The butterfly pea flower extract prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the butterfly pea flower extract according to claim 6 in the preparation of whitening and / or antioxidant products.

8. A product for whitening and / or anti-oxidation, characterized in that: The invention relates to a butterfly pea flower extract according to claim 6.

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