Preparation method and application of butterfly bean flower extract

Abstract

The invention provides a preparation method and application of a butterfly bean flower extract, and relates to the technical field of biology. The preparation method comprises the following steps: extracting dried petals of the butterfly bean flowers by using an extraction solvent under an ultrasonic condition, the material-liquid ratio of the dried petals of the butterfly bean flowers to the extraction solvent is 1: (15-25) g / mL, and the ultrasonic temperature is 50-70 DEG 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. According to the preparation method, the types of active ingredients in the butterfly bean flower extract can be increased, the content of flavonoid ingredients is increased, and the whitening and antioxidant effects are improved.

Description

Technical Field

[0001] The 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 Fabaceae Leguminosae Clitoria Clitoria Linn. Butterfly pea Clitoria ternatea L. The main active ingredients of butterfly pea flower extract include anthocyanins and flavonoids. Anthocyanins are mainly based on delphinidin, which is combined with glucose at its 3, 3', and 5' sites to form delphinidin glucoside, and then acylated with coumaric acid molecules, which gives butterfly pea flower pigments high stability. In addition, it also contains flavonoids such as myricetin, quercetin, kaempferol, myricetin-3-glucoside, quercetin-3-glucoside, and kaempferol glucoside. Butterfly pea flower extract can be directly added to sunscreens, lotions, facial masks, and essences. Butterfly pea flower extract has good antioxidant, anti-inflammatory, and anti-allergic skin protection effects. Its delphinidin anthocyanin components can reduce and repair the damage of ultraviolet radiation to skin cells. Myricetin and quercetin can significantly reduce the level of reactive oxygen 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 Name: A Method for Extracting Butterfly Pea Flowers) discloses the extraction of butterfly pea flowers using an ethanol / water solvent, but it only examines the best extraction method for anthocyanins and ignores the extraction of flavonoids, which may result in the inability to effectively extract the functional ingredients in butterfly pea flowers for whitening or anti-oxidation. In addition, the melanin inhibition mechanism of whitening ingredients in cosmetics is currently single, and most products only target tyrosinase, ignoring the effects of pathways such as oxidative stress (ROS). Flavonoids have antioxidant, anti-inflammatory, whitening and anti-aging effects. More fully extracting the active ingredients in butterfly pea flowers, especially flavonoids, can not only improve the whitening effect of butterfly pea flower extracts, but also inhibit ROS and enhance the whitening effect through anti-oxidation.

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

[0006] The object of the present invention is 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 flavonoid components.

[0007] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions: In the first aspect, a preparation method of butterfly pea flower extract is provided. The preparation method includes: extracting dried petals of butterfly pea flower with an extraction solvent under ultrasonic conditions, where the material-liquid ratio of the dried petals of butterfly pea flower 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.

[0008] In the second aspect, a butterfly pea flower extract prepared by the preparation method described in the first aspect is provided.

[0009] In the third aspect, an application of the butterfly pea flower extract described in the second aspect in the preparation of products for whitening and / or antioxidant is provided.

[0010] In the fourth aspect, a product for whitening and / or antioxidant is provided. The product for whitening and / or antioxidant contains the butterfly pea flower extract described in the second aspect.

[0011] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a preparation method of butterfly pea flower extract. By using an aqueous solution containing polyethylene glycol as the extraction solvent and extracting under ultrasonic conditions, the types of active ingredients in the butterfly pea flower extract are increased, and the content of flavonoid components is improved. The newly added compounds are as follows: herba hedyotis diffusae I, lonicerin, kaempferitrin, typhonoside, isorhamnetin-3-O-neohesperidoside, narcissin, eupatorin, swertiamarin, nicotinamide. Nicotinamide has a definite whitening effect; glycoside compounds have skin protection effects such as antioxidant, anti-inflammatory, and anti-allergic effects, which can increase the whitening efficacy of the butterfly pea flower extract. In addition, compared with extraction with water alone, polyethylene glycol / water extraction can increase the content of effective ingredients such as vitamin C, astragalin, and isoquercitrin. In the preferred embodiment, the flavonoid content in the butterfly pea flower extract can reach 36.56%, and the content of astragalin can reach 2.48 mg / g.

[0012] By investigating the effects of inhibiting tyrosinase activity, antioxidant, and inhibiting melanin production, and comparing 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 inhibitory effects on tyrosinase activity and melanin production than the butterfly pea flower extract extracted with water alone, and also has higher inhibitory effects on ROS production and antioxidant ability than the butterfly pea flower extract extracted with water alone.

[0013] The butterfly pea flower extract obtained by the preparation method of the present invention is a safe and non-irritating whitening active ingredient, which realizes the effect of reducing the generation and distribution of melanin granules by down-regulating the expression of tyrosinase, inhibiting tyrosinase activity, and inhibiting the excessive production of reactive oxygen species. Description of the Drawings

[0014] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0015] Figure 1 It is the total ion chromatogram of the butterfly pea flower extract obtained by PEG200 / water extraction in Example 1. Figure 2 It is the content of vitamin C in the butterfly pea flower extract obtained by PEG200 / water extraction and water extraction only in Example 1. Figure 3 It is the content of astragalin in the butterfly pea flower extract obtained by PEG200 / water extraction and water extraction only in Example 1. Figure 4 It is the content of isoquercitrin in the butterfly pea flower extract obtained by PEG200 / water extraction and water extraction only in Example 1. Figure 5 It is the effect of the butterfly pea flower extract obtained by PEG200 / water extraction at different concentrations in Example 2 on the activity of HaCaT / B16 cells, n = 6. Figure 6 It is the effect of the butterfly pea flower extract obtained by different extraction methods in Example 3 on the tyrosinase activity of HaCaT / B16 cells, n = 3, *p < 0.05, ****p < 0.0001, compared with the control group; ##p < 0.01, compared with the butterfly pea flower extract obtained by water extraction for the butterfly pea flower extract obtained by PEG200 / water extraction. Figure 7 It is the microscopic image of melanin after treating HaCaT / B16 cells with the butterfly pea flower extract obtained by different extraction methods in Example 4. Figure 8 It is the photo of the melanin precipitate after cell lysis and centrifugation after treating HaCaT / B16 cells with the butterfly pea flower extract obtained by different extraction methods in Example 4. Figure 9 It is the expression level of cell TYR (tyrosinase) after treating HaCaT / B16 cells with the butterfly pea flower extract obtained by different extraction methods in Example 5, n = 3, **p < 0.01, ***p < 0.001, compared with the control group; #p < 0.05, compared with the butterfly pea flower extract obtained by water extraction for the butterfly pea flower extract obtained by PEG200 / water extraction. Figure 10 It is the photo of each experimental group after ROS staining after treating cells with the butterfly pea flower extract obtained by different extraction methods in Example 6. Specific Embodiments

[0016] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0017] In this article, when an equivalent, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper limit preferred values and lower limit preferred values, it should be understood that all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value are specifically disclosed, regardless of whether the range is separately disclosed. For example, when the range "1 to 5" is disclosed, the described range should be interpreted as including ranges "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 in this article, unless otherwise stated, the range is intended to include its end values and all integers and fractions within the range.

[0018] In this article, unless otherwise specified, each reaction or operation step can be carried out in sequence or not in sequence.

[0019] In this article, unless otherwise specified, any numbering is used to distinguish one entity or act from another entity or act, rather than necessarily requiring or implying any actual such relationship, sequence, or importance between these entities or acts, such as numbering first, second, etc.

[0020] In this article, "and / or" is used to indicate that either or both of the described situations may occur. For example, A and / or B includes (A and B) and (A or B).

[0021] In this article, unless otherwise specified, "optionally", "optional", "selectable", or "selected" means that the subsequent described event or circumstance may but does not have to occur, and this description includes the occasions where the event or circumstance occurs or does not occur.

[0022] In this article, the term "comprising" or "including" means including the described elements, integers, or steps, but does not exclude any other elements, integers, or steps.

[0023] In this article, the terms "respectively independent", "each... independently selected from", "each... independently selected from", and "independently selected from" can be interchanged and should be understood in a broad sense, which means that among a class of objects, the specific characteristics of each specific object do not affect each other, and among this class of objects, the specific characteristics of any two objects can be the same or different.

[0024] In a first aspect, a method for preparing a butterfly pea flower extract is provided. The preparation method includes: extracting dried petals of butterfly pea flowers under ultrasonic conditions using an extraction solvent, where 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 material-liquid ratio of the dried petals of butterfly pea flowers 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; the ultrasonic temperature is 50 - 70 °C, for example, but not limited to, 50, 55, 60, 65, or 70 °C, preferably 60 °C.

[0025] In an optional embodiment, the concentration of polyethylene glycol in water is 0.1 g / mL, the material-liquid ratio of the dried petals of butterfly pea flowers to the extraction solvent is 1:25 g / mL, and the ultrasonic temperature is 60 °C.

[0026] In an optional embodiment, the ultrasonic conditions further include: the ultrasonic time is 30 - 90 min, for example, but not limited to, 30, 40, 50, 60, 70, 80, or 90 min; the ultrasonic power is 50 - 150 W, for example, but not limited to, 50, 80, 100, 120, or 150 W.

[0027] In an optional embodiment, the ultrasonic conditions further include: the ultrasonic time is 60 min, and the ultrasonic power is 100 W.

[0028] In an optional embodiment, the dried petals of butterfly pea flowers are dried petal powder of butterfly pea flowers.

[0029] In an optional embodiment, the preparation method further includes centrifuging after extraction to obtain a supernatant, and freeze-drying the supernatant to obtain the butterfly pea flower extract.

[0030] In an optional embodiment, the average molecular weight of the polyethylene glycol is 200 - 1000, for example, but not limited to, polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, or polyethylene glycol 1000, preferably polyethylene glycol 200 (PEG200).

[0031] In some specific embodiments, the preparation method includes removing the stamens, calyces, and pedicels of Clitoria ternatea flowers to obtain Clitoria ternatea petals; drying and pulverizing the Clitoria ternatea petals to obtain Clitoria ternatea flower powder; using the extraction solvent for ultrasonic-assisted hot water extraction, and the operating conditions are as follows: the concentration of polyethylene glycol 200 in the extraction solvent is 0.1 g / mL, the material-liquid ratio of Clitoria ternatea flower powder to the extraction solvent is 1:25, the ultrasonic temperature is 60°C, the ultrasonic time is 60 min, the ultrasonic power is 100 W, and the supernatant is obtained by centrifugation after extraction.

[0032] In a second aspect, there is also provided a Clitoria ternatea flower extract prepared by the preparation method described in the first aspect.

[0033] In an optional embodiment, the Clitoria ternatea flower extract is a lyophilized product.

[0034] In a third aspect, there is provided the use of the Clitoria ternatea flower extract described in the second aspect in the preparation of products for whitening and / or antioxidant.

[0035] In an optional embodiment, in the products for whitening and / or antioxidant, the Clitoria ternatea flower extract is a lyophilized product.

[0036] In an optional embodiment, in the products for whitening and / or antioxidant, the working concentration of the lyophilized Clitoria ternatea flower extract does not exceed 50 μg / mL, and for example, it can be, but is not limited to, not exceeding 30 μg / mL, not exceeding 35 μg / mL, not exceeding 40 μg / mL, not exceeding 45 μg / mL, or not exceeding 50 μg / mL.

[0037] In an optional embodiment, in the products for whitening and / or antioxidant, the working concentration of the lyophilized Clitoria ternatea flower extract is 25 μg / mL.

[0038] In an optional embodiment, the Clitoria ternatea flower extract is the sole therapeutic agent in the products for whitening and / or antioxidant.

[0039] In an optional embodiment, the Clitoria ternatea flower extract is combined with at least one other whitening and / or antioxidant ingredient to prepare products for whitening and / or antioxidant. When the Clitoria ternatea flower extract is used in combination with one or more other ingredients, it can be administered simultaneously with the one or more additional ingredients. In some such embodiments, the Clitoria ternatea flower extract and the additional ingredients can be administered simultaneously as part of the same composition. In other embodiments, the Clitoria ternatea flower extract used in combination with other ingredients does not need to be administered simultaneously or in the same composition as the ingredient. That is, one or more ingredients administered after and / or before the Clitoria ternatea flower extract in the present invention are also considered to be "used in combination" with the Clitoria ternatea flower extract, that is, the Clitoria ternatea flower extract and other ingredients are administered by different administration methods.

[0040] In alternative embodiments, the subjects of the whitening and / or antioxidant product 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.

[0041] In a fourth aspect, a whitening and / or antioxidant product is further provided, and the whitening and / or antioxidant product includes the Clitoria ternatea extract described in the second aspect.

[0042] In alternative embodiments, the whitening and / or antioxidant product includes the freeze-dried Clitoria ternatea extract.

[0043] In alternative embodiments, the working concentration of the freeze-dried Clitoria ternatea extract in the whitening and / or antioxidant product does not exceed 50 μg / mL, and for example, it can be, but is not limited to, not exceeding 30 μg / mL, not exceeding 35 μg / mL, not exceeding 40 μg / mL, not exceeding 45 μg / mL, or not exceeding 50 μg / mL.

[0044] In alternative embodiments, the working concentration of the freeze-dried Clitoria ternatea extract in the whitening and / or antioxidant product is 25 μg / mL.

[0045] In alternative embodiments, the whitening and / or antioxidant product further contains optional excipients acceptable in the art, and the excipients include, but are not limited to, solvents, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, anti-caking agents, flavoring agents, bacteriostatic agents, suspending agents, coating agents, film-forming agents, fragrances, thickening agents, anti-adhesives, antioxidants, antioxidant synergists, chelating agents, pH regulators, adsorbents, plasticizers, surfactants, thickening agents, clathrates, protectants, moisturizers, softeners, absorbents, diluents, release regulators, pressure-sensitive adhesives, hardeners, hollow capsules, matrices, and drug carrier materials, or one or more of them.

[0046] In alternative embodiments, the whitening and / or antioxidant product includes, but is not limited to, topical agents, oral agents, or injectable agents.

[0047] In alternative embodiments, the whitening and / or antioxidant product includes a topical agent, and the topical agent includes, but is not limited to, pastes, ointments, sprays, gels, liniments, paints, film-forming agents, patches, cataplasms, or membranes.

[0048] In alternative embodiments, the whitening and / or antioxidant product includes cosmetics.

[0049] In alternative embodiments, the cosmetics include, but are not limited to, facial cleanser, cleansing cream, cleansing foam, lotion, essence, emulsion, cream or facial mask.

[0050] The present invention will be further illustrated by specific examples below. However, it should be understood that these examples are only for more detailed illustration and should not be construed as limiting the present invention in any way.

[0051] Example 1 Investigation on the extraction process of Clitoria ternatea flowers: I. Experimental method: (1) Design of the extraction process of Clitoria ternatea flowers: Remove the stamens, sepals and pedicels from the Clitoria ternatea flowers to obtain Clitoria ternatea petals; dry and crush the Clitoria ternatea petals to obtain Clitoria ternatea flower powder; weigh the pretreated Clitoria ternatea flower powder, and use polyethylene glycol 200 (PEG 200) and water as extraction solvents, and use ultrasonic-assisted hot water extraction. The operating conditions are as follows: set the PEG 200 concentration, the material-liquid ratio of Clitoria ternatea flower powder and extraction solvent, and the ultrasonic temperature according to Table 2; the ultrasonic time for each group is 60 min, and the ultrasonic power is 100 W. The extract is centrifuged at 4200 rpm for 15 min, and the supernatant is taken and freeze-dried.

[0052] Investigate the effects of polyethylene glycol concentration, material-liquid ratio and ultrasonic temperature on the contents of total flavonoids and cyanidin-3-O-glucoside in the extract of Clitoria ternatea flowers. Use single factor to set experimental groups. Refer to Table 1, and the specific experimental conditions are shown in Table 2. In addition, water is used as a single extraction solvent for comparison.

[0053] Table 1 Single factor investigation table

[0054] Table 2 Experimental conditions for each experimental group

[0055] Extract a total of 9 batches of Clitoria ternatea flower extracts according to the parameters recorded in experimental groups A-1~3, B-1~3 and C-1~3.

[0056] (2) Enrichment of chemical components: The components of the butterfly pea flower extracts obtained by PEG200 / water extraction (experimental groups A / B / C-1~3) and water extraction were identified using a UPLC chromatograph (Vanquish) and an Orbitrap Fusion mass spectrometer with a UPLC Hypersilgold column. Mobile phase A: 0.01% formic acid, mobile phase B: 0.1% formic acid-acetonitrile, gradient elution is shown in Table 3; injection volume: 5 μL; flow rate: 200 μL / min. The mass spectrometry parameters were set as follows: positive ion: 3.5 kV, negative ion: 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 was adopted, resolution: 60000; scan range: 100-1000 m / z; mass error: 10 ppm. The measured compounds were confirmed through the PubChem database and their structures were downloaded, and the files were saved in SDF format. The total ion chromatogram of the liquid chromatography-mass spectrometry of the butterfly pea flower extract obtained by PEG200 / water extraction is as Figure 1 shown.

[0057] Table 3 Conditions of HPLC mobile phase

[0058] A total of 65 compounds were identified from the components extracted by PEG200 / water through comparison with the database in positive and negative ion modes. According to the scoring results of mzVault and mzCloud, compounds with scores greater than 80 were screened, and the information of secondary fragment ions in the literature was referred to, including astragalin, isoquercitrin, morin, rutin, hyperoside, kaempferol-3-O-rutinoside, kaempferol, haliricin I, quercetin, mogroside V, L-proline, atractylodin, trigonelline hydrochloride, kaempferol-7-O-β-D-glucoside, L-phenylalanine, lonicerin, p-coumaric acid, kaempferitrin, typhaneoside, vitamin C, cyanidin-3-O-glucoside, luteoloside, myricetin, isorhamnetin-3-O-neohesperidoside, narcissin, luteolin, coumarin, quercitrin, herbacetin, camelloside B, eupatilin, nicotinamide, silybin. Among them, flavonoid components include astragalin, isoquercitrin, morin, rutin, hyperoside, kaempferol-3-O-rutinoside, kaempferol, haliricin I, quercetin, kaempferol-7-O-β-D-glucoside, lonicerin, kaempferitrin, typhaneoside, cyanidin-3-O-glucoside, luteoloside, myricetin, isorhamnetin-3-O-neohesperidoside, narcissin, luteolin, quercitrin, herbacetin, camelloside B, eupatilin, silybin.

[0059] From the results, it can be seen that the types of flavonoid components in the butterfly pea flower extract obtained by PEG200 / water extraction increased, especially flavonoid glycosides.

[0060] Compared with water alone as the extraction solvent, the new compounds extracted by PEG200 / water are as follows: cyperine I, honeysuckle glycoside, kaempferol glycoside, typhala glycoside, isorhamnetin-3-O-neohesperidin, narcissin, zedoaria flavonoids, tannin, and niacinamide. Niacinamide has a clear whitening effect; glycoside compounds have antioxidant, anti-inflammatory, anti-allergic and other skin protection effects, which can increase the whitening effect of butterfly pea flower extract.

[0061] (3) Comparison of the total flavonoids content and astragaloside 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: Table 4 Total flavonoids content and astragaloside content in butterfly pea flower extracts obtained by PEG200 / water extraction and water extraction

[0062] As can be seen from Table 4, after PEG200 / water extraction, with the increase of PEG 200 concentration, the total flavonoids content first increased and then decreased, and the highest was 35.97% at 0.1 g / mL; when the solid-liquid ratio was 1:25, the total flavonoids content was the highest; when the ultrasonic temperature was 60℃, the total flavonoids content was the highest. In terms of the content of astragaloside, the PEG 200 concentration was the highest at 0.2 g / mL, but there was no significant improvement compared with 0.1 g / mL; similarly, when the solid-liquid ratio was 1:25 and the ultrasonic temperature was 60℃, the astragaloside content was the highest. In summary, the optimal process was selected when the PEG 200 concentration was 0.1 g / mL, the solid-liquid ratio was 1:25g / mL, and the ultrasonic temperature was 60℃. After re-extraction, the total flavonoids and astragaloside contents in the extract were detected, and it was found that the total flavonoids content was 36.56% and the astragaloside content was 2.48 mg / g.

[0063] (4) Compare the contents of vitamin C, astragaloside and isoquercitrin in the butterfly pea flower extract obtained by the optimal process in step (3) above and the butterfly pea flower extract extracted only by water. The only difference between the extraction process of butterfly pea flower extracted only by 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.

[0064] In the following Examples 2 to 6, the extraction method of butterfly pea flower extract using PEG200 / water as the solvent is as follows: Remove the stamens, sepals and pedicels from the butterfly pea flowers to obtain butterfly pea petals; dry and crush the butterfly pea petals to obtain butterfly pea flower powder; weigh the pretreated butterfly pea flower powder, use PEG 200 and water as the extraction solvent, and perform ultrasonic-assisted hot water extraction. The operating conditions are as follows: the concentration of PEG 200 is 0.1 g / mL, the solid-liquid ratio of the butterfly pea flower powder to the extraction solvent is 1:25 g / mL, the ultrasonic temperature is 60 °C, the ultrasonic time for each group is 60 min, and the ultrasonic 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 butterfly pea flower extract using only water and the extraction method of butterfly pea flower extract using PEG200 / water as the solvent is only that the extraction solvent does not contain PEG200, and the other parameters are the same.

[0065] Example 2 CCK8 experiment: Establish a co-culture system of HaCaT (Human immortalized keratinocyte cell line) and B16 cells (Mouse Melanoma Cell Line B16), with HaCaT:B16 cells = 2:1. After the HaCaT and B16 cells adhere to the wall, the control group (culture system) does not add drugs and is subjected to liquid change treatment. The experimental groups are respectively intervened with butterfly pea flower extract extracted with 3.125, 6.25, 12.5, 25, 50, 100, 200, 400 and 800 μg / mL PEG200 / water prepared with complete medium for 24 h, and 6 replicate wells are set in each group. After 24 h, the old liquid is removed, 10 μL of CCK8 solution is added to each well, and it is cultured in the dark at 37 °C for 2 h. The supernatant is discarded, and the absorbance is measured at 450 nm with an enzyme-labeled instrument and the cell viability is calculated. The results are as Figure 5 shown. When the concentration of the butterfly pea flower extract extracted with PEG200 / water ≤ 50 μg / mL, the viability of the HaCaT / B16 mixed cells is higher than 85%, indicating its high biocompatibility and being a safe cosmetic raw material. Therefore, the butterfly pea flower extract with a concentration of 25 μg / mL is selected for the subsequent cell experiments in Examples 3 to 6.

[0066] Example 3 Determination of intracellular tyrosinase activity: A co - culture model of HaCaT and B16 cells was constructed using six - well plates. After the cells were completely adherent, the following experimental groups were set: the experimental groups were treated with 25 μg / mL PEG200 / water and water - extracted Clitoria ternatea extract respectively; the blank control group only changed the culture medium; the positive control group used 50 μg / mL vitamin C as the intervention drug. All experimental groups were set with 3 biological replicates. After 48 h of drug intervention, the reaction was terminated under ice - bath conditions. After removing the culture medium, the cells were washed twice with pre - cooled PBS buffer. 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 min, it was re - dissolved at 4 °C and centrifuged at 12,000 rpm and 4 °C for 20 min. 10 μL of the supernatant was taken for protein quantification using the BCA method. At the same time, 90 μL of the supernatant was transferred to a 96 - well plate, 10 μL of 10 mmol / L L - dopa substrate solution was added, and it was incubated at 37 °C in the dark until a brownish - black color reaction appeared. Immediately, the absorbance value was measured at a wavelength of 490 nm using an enzyme - linked immunosorbent assay (ELISA) reader. The following formulas were used for data analysis: Relative tyrosinase activity=(OD490 of the experimental group) / (protein concentration of the corresponding sample) Tyrosinase activation rate (%)=(relative tyrosinase activity of the experimental group / relative tyrosinase activity of the blank group)×100%.

[0067] The results were as Figure 6 shown: After the use of vitamin C, the tyrosinase activity of B16 cells decreased significantly compared with the control group. After the use of water and PEG200 / water - extracted Clitoria ternatea extract, the tyrosinase activity also decreased significantly compared with the control group. In addition, the ability of PEG200 / water - extracted Clitoria ternatea extract to inhibit tyrosinase activity was significantly higher than that of water - extracted Clitoria ternatea. The results showed that the inhibitory effect of PEG200 / water - extracted Clitoria ternatea extract on the tyrosinase activity of HaCaT / B16 cells was higher than that of water - extracted Clitoria ternatea extract.

[0068] Example 4 Determination of melanin content: The preparation process of cell samples was the same as the tyrosinase determination method in Example 3. After 48 h of drug intervention, samples were collected on ice: the culture supernatant was discarded, and the cells were washed twice with pre - cooled PBS buffer to remove residual culture medium. The distribution and content of melanin in each group of cells were observed under a microscope. 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 min, it was re - dissolved at 4 °C and centrifuged at 12,000 rpm and 4 °C for 20 min. The imageJ image analysis system was used to quantitatively detect the melanin precipitation area at the bottom of the tube, and the ImageJ software was used to analyze the color - developing area and gray - scale value of the precipitate.

[0069] Under the microscope, compared with the control group, the B16 cells in the treatment group with butterfly pea flower extract extracted by vitamin C and PEG200 / water secreted less melanin ( Figure 7 ). After the cell administration was completed, the cells were broken and centrifuged, and obvious black precipitates could be observed in the control group cells. After the use of vitamin C, the synthesis of cell melanin decreased significantly. After the use of the butterfly pea flower extract extracted by PEG200 / water, the melanin synthesis also decreased significantly compared with the control group; however, the ability of the butterfly pea flower extract extracted by water to inhibit melanin production was not obvious ( Figure 8 ). The results showed that the ability of the butterfly pea flower extract extracted by PEG200 / water to inhibit melanin production in B16 cells was higher than that of the butterfly pea flower extract extracted by water, and it was a potential whitening raw material.

[0070] Example 5 Fluorescent quantitative PCR was used to detect the expression of TYR gene in the HaCaT / B16 cell co-culture system: The preparation process of cell samples was the same as that of the tyrosinase determination method in Example 3. After the administration was completed, the total cell RNA was extracted by the Trizol method, and the cDNA was reverse transcribed and synthesized using the Prime Script TM RT reagent Kit with gDNA Eraser (perfect Real Time) kit, and the steps were strictly operated according to the instructions. The TB Green ® Premix ExTaqTM II (Tli RNaseH Plus) kit was used for Real Time PCR analysis to detect the expression of related mRNAs. 2- ΔΔ The Ct method was used for calculation and data analysis. GAPDH was used as an internal reference control. All primers were synthesized by Sangon Biotech Co., Ltd., and the primer sequences are listed in Table 5.

[0071] Table 5 Fluorescent quantitative PCR primer sequences

[0072] Compared with the control group, the butterfly pea flower extract extracted by PEG200 / water could significantly inhibit the expression of TYR (tyrosinase) ( Figure 9 ), and the effect was close to that of vitamin C. The butterfly pea flower extract extracted by water could also down-regulate the expression of TYR, but the effect was significantly lower than that of the butterfly pea flower extract extracted by PEG200 / water. It was shown that the butterfly pea flower extract extracted by PEG200 / water could down-regulate the expression of tyrosinase and inhibit the activity of tyrosinase, thereby inhibiting the production of melanin, and the effect was stronger than that of the butterfly pea flower extract obtained by water extraction alone.

[0073] Example 6 ROS staining: After the B16 cells were completely adherent, the following experimental groups were set up: the experimental groups were respectively given 25 μg / mL PEG200 / water-extracted Clitoria ternatea extract and water-extracted Clitoria ternatea extract; the blank control group only changed the culture medium; the positive control group used 50 μg / mL vitamin C as the intervention drug. After the administration, the cells were stimulated with UVB for 30 min. For ROS staining, 5 μM CellROX-green dye (Invitrogen) was added to the complete medium at 37°C for 30 min, and then live imaging was performed.

[0074] After UVB treatment, the generation of ROS in the cells increased significantly. After vitamin C administration, the ROS expression level returned to the normal level. After the use of water and PEG200 / water-extracted Clitoria ternatea extract, the generation of ROS also decreased significantly compared with the model group. However, the ability of PEG200 / water-extracted Clitoria ternatea extract to inhibit ROS generation was stronger ( Figure 10 ). The results showed that PEG200 / water-extracted Clitoria ternatea extract had a significant ability to inhibit ROS generation and antioxidant activity, which was another mechanism of its whitening effect.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions 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: The method comprises extracting dried butterfly pea flower petals by 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 the polyethylene glycol in water is 0.05-0.2 g / mL.

2. The preparation method according to claim 1, characterized in that 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 ultrasonic conditions also include: ultrasonic time of 30 to 90 min, and ultrasonic power of 50 to 150 W.

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

5. 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.

6. The preparation method according to any one of claims 1 to 5, characterized in that The average molecular weight of the polyethylene glycol is 200-1000.

7. The preparation method according to claim 5, characterized in that The method comprises 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 ultrasonic-assisted hot water 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 butterfly pea flower powder to the 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.

8. The butterfly pea flower extract prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the butterfly pea flower extract according to claim 8 in the preparation of whitening and / or anti-oxidation products.

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

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