An ornamental peach petal nanovesicle, a preparation method and application thereof

Abstract

The application provides an ornamental peach petal nanocapsule and a preparation method and application thereof, and belongs to the technical field of biotechnology. The preparation method of the ornamental peach petal nanocapsule is as follows: collecting ornamental peach petals at a flower bud stage or an initial opening stage, and extracting the nanocapsule by adopting a direct centrifugation method or a sucrose density gradient centrifugation method. The ornamental peach petals at the flower bud stage or the initial opening stage are used as raw materials, the prepared nanocapsule has good biocompatibility, is rich in functional components such as polyphenols and lipids which are specific to ornamental peach petals, has good application potential in terms of antioxidation, anti-inflammation and skin quality improvement, and realizes high-value deep processing of the ornamental peach petals.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to an ornamental peach petal nanovesicle, its preparation method, and its application. Background Technology

[0002] Ornamental traits have been an important focus in peach breeding, leading to the successful selection of a number of ornamental peach varieties with different flower colors and characteristics. These varieties typically use cultivated peach (Prunus persica) as their main parent or are selected populations. However, during the breeding process, hybridization with native Chinese peach resources such as Chinese mountain peach (P. davidiana), smooth walnut (P. mira), Gansu peach (P. kansuensis), and Xinjiang peach (P. ferganensis) may have also been incorporated. Furthermore, because ornamental peaches are not primarily selected for their fresh consumption, greater genetic diversity has been preserved during the breeding process. Benefiting from their cold hardiness, ease of cultivation, and dual value for both ornamental and edible purposes, ornamental peaches are now widely distributed in both southern and northern China.

[0003] Although ornamental peaches are widely cultivated, their primary purpose is for aesthetic appeal, resulting in limited industrial development beyond seedling cultivation. This hinders the realization of higher economic benefits from their widespread planting across the country. Nanovesicles, on the other hand, are vesicle-like structures encased in a lipid bilayer at the nanoscale, formed during cellular life processes. In ornamental peach blossoms, they serve as important sites for the synthesis of substances for cellular life activities and as precursor organelles for the vacuoles responsible for blossom expansion. Thanks to their lipid bilayer structure, substances within nanovesicles are more easily transported within systems where cells or organelles share a lipid bilayer as the primary interface.

[0004] Therefore, nanovesicles, primarily derived from ornamental peach petals, are expected to provide important technical support for the high-value utilization of ornamental peach resources. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a method for preparing ornamental peach petal nanovesicles. The prepared ornamental peach petal nanovesicles have good biocompatibility and are rich in functional components such as polyphenols and lipids unique to ornamental peach petals. They have good application potential in anti-oxidation, anti-inflammation and skin improvement.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] A method for preparing ornamental peach petal nanovesicles involves collecting ornamental peach petals during the bud stage or early blooming stage, and extracting the nanovesicles using direct centrifugation or sucrose density gradient centrifugation.

[0008] Preferably, the ornamental peach petal is treated in darkness before extracting the nano vesicles.

[0009] Preferably, the darkness treatment is placing the ornamental peach petal at 30-40℃ with 85%-99% humidity for 12-16h.

[0010] Preferably, the direct centrifugation method comprises: crushing the frozen ornamental peach petal, mixing the petal powder with Tris-HCl buffer solution, homogenizing, centrifuging the homogenate at 650g, 3000g, 10000g and 15000g in sequence, taking the supernatant after each centrifugation for the next centrifugation, taking the supernatant after the last centrifugation, centrifuging at 100000g, collecting the precipitate, resuspending with PBS to obtain the nano vesicle suspension.

[0011] Preferably, the sucrose density gradient centrifugation method comprises: homogenizing the ornamental peach petal, centrifuging the homogenate at 1000g, collecting the supernatant, centrifuging the supernatant at 5000g, using sucrose density gradient centrifugation, the sucrose gradient is 8%, 30%, 45% and 60%, after centrifugation, taking the nano vesicle suspension from the junction of 30% and 45% sucrose.

[0012] Another object of the present application is to provide the ornamental peach petal nano vesicle obtained by the preparation method, which contains kaempferol derivatives and fatty acids.

[0013] Another object of the present application is to provide the use of the above preparation method or the above ornamental peach petal nano vesicle in the preparation of skin care products.

[0014] Preferably, the skin care product comprises whitening products, freckle-removing products, anti-inflammatory products, anti-wrinkle products and anti-aging products.

[0015] Another object of the present application is to provide a skin care product containing the above ornamental peach petal nano vesicle.

[0016] Preferably, the skin care product is any one of emulsion, cream and serum.

[0017] Compared with the prior art, the present application has the following beneficial effects:

[0018] The present application provides a preparation method of ornamental peach petal nano vesicle, which uses ornamental peach petal in the bud stage or early blooming stage as raw material, effectively utilizing ornamental peach petal resources. The ornamental peach petal nano vesicle provided by the present application has good biocompatibility, and is rich in functional ingredients such as polyphenols and lipids unique to ornamental peach petal, and has good application potential in antioxidant, anti-inflammatory and skin quality improvement, realizing high value-added deep processing of ornamental peach petal. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 : The six kaempferol (K1-K6) derivatives and six quercetin (Q1-Q6) derivatives detected in the petals of P. persica cv. Yingshun and their retention times (min);

[0020] Figure 2 : The relative contents of kaempferol derivatives in the nanovesicles prepared from the petals of different P. persica cultivars;

[0021] Figure 3 : The relative contents of quercetin derivatives in the nanovesicles prepared from the petals of different P. persica cultivars;

[0022] Figure 4 : The relative contents of several fatty acid derivatives in the nanovesicles prepared from the petals of different P. persica cultivars;

[0023] Figure 5 : The antioxidant activities of the nanovesicles prepared from the petals of different P. persica cultivars, where different capital letters represent significant differences in ABTS radical scavenging rates, and different small letters represent significant differences in DPPH radical scavenging rates;

[0024] Figure 6 : The inhibitory activities of the nanovesicles prepared from the petals of different P. persica cultivars on tyrosinase, where different letters represent significant differences between two groups;

[0025] Figure 7 : The inhibitory effects of the nanovesicles prepared from the petals of different P. persica cultivars on cell melanin synthesis, where ** indicates P<0.01;

[0026] Figure 8 : The inhibitory activities of the nanovesicles prepared from the petals of different P. persica cultivars on intracellular tyrosinase, where different letters represent significant differences between two groups;

[0027] Figure 9 : The effects of the nanovesicles prepared from the petals of different P. persica cultivars on the expression levels of MMP-1 in fibroblasts, where different letters represent significant differences between two groups;

[0028] Figure 10 : The effects of the nanovesicles prepared from the petals of different P. persica cultivars on the expression levels of Col 1 and HA in fibroblasts, where different capital letters represent significant differences in Col 1, and different small letters represent significant differences in HA;

[0029] Figure 11 : The effects of the nanovesicles prepared from the petals of different P. persica cultivars on NO content, where different letters represent significant differences between two groups;

[0030] Figure 12The influence of the nanovesicles prepared from different ornamental peach petals on the expression of TNF-α, wherein different letters represent significant differences between two groups. DETAILED DESCRIPTION

[0031] The application provides a preparation method of ornamental peach petal nanovesicles, wherein ornamental peach petals at the bud stage or the initial opening stage are collected, and the nanovesicles are extracted by using a direct centrifugation method or a sucrose density gradient centrifugation method. Researches show that the rapid combination of vesicles in petals and the rapid swelling of central vacuoles are the main reasons for the blooming of buds, the nanovesicles extracted from ornamental peach petals contain vacuole precursors, the nanovesicles extracted from ornamental peach petals at the bud stage or the initial opening stage are rich in flavonol compounds and various fatty acids unique to ornamental peach petals, the prepared nanovesicles have smaller particle sizes, more uniform sizes, higher yields and better stability.

[0032] As an implementable mode, after the peach flowers are collected, pistils, stamens, calyces and receptacles are removed, and the ornamental peach petals are reserved for extracting the nanovesicles.

[0033] In the application, the ornamental peach petals are treated in darkness before the nanovesicles are extracted, and the conditions for treating the ornamental peach petals in darkness are as follows: the temperature is 30-40 DEG C, the humidity is 85-99%, and the ornamental peach petals are placed in darkness for 12-16 hours. The temperature for treating the ornamental peach petals in darkness is preferably 32-38 DEG C, and more preferably 35 DEG C, and the humidity is preferably 88-95%, and more preferably 90%. The macromolecular carbohydrates contained in the buds can interfere with the preparation of the nanovesicles, after the ornamental peach petals are treated in darkness, the respiration of the peach flowers consumes the intracellular sugar powder, the content of the carbohydrates is reduced, especially the content of the starch is reduced, the formation of colloid by the starch and macromolecular substances in the extraction process is avoided, the abundance of the nanovesicles in the final prepared suspension is improved, and the content of impurities is reduced.

[0034] In the application, the direct centrifugation method comprises the following steps: the ornamental peach petals are crushed after being frozen, the petal powder is mixed with Tris-HCl buffer solution to form a homogenate, the homogenate is centrifuged at 650g, 3000g, 10000g and 15000g in sequence, the supernatant is taken out after each centrifugation and used for the next centrifugation, the supernatant is taken out after the last centrifugation, centrifuged at 100000g, the precipitate is collected, resuspended with PBS, and the nanovesicle suspension is obtained.

[0035] In the direct centrifugation method, the ornamental peach petals are preferably rapidly frozen using liquid nitrogen, and the ornamental peach petals are ground into powder in a frozen state; the petal powder is mixed with Tris-HCl buffer (100 mM, pH 7.4) at a ratio of 1:1 (w:v), and high-speed homogenization is performed at 4°C for 8-12 min, preferably 10 min, using a high-speed homogenizer; the homogenate is filtered using filter paper to remove solid particles to collect the homogenate liquid phase; the liquid homogenate liquid phase is sequentially centrifuged at 650 g, 3000 g, 10000 g and 15000 g for 15-25 min, preferably 20 min, at 4°C; the supernatant is centrifuged at 100000 g for 30-180 min, preferably 120 min, at 4°C; and the precipitate is used to prepare a nanovesicle suspension with PBS (100 mM, pH 6.8), and the nanovesicle suspension is rapidly frozen using liquid nitrogen and stored at -80°C.

[0036] In the present application, the nanovesicle suspension obtained by the direct centrifugation method is subjected to ultrafiltration centrifugation, which is performed by placing the nanovesicle suspension in an ultrafiltration tube with a molecular weight of 100 kDa and centrifuging at 4000-6000 g for 25-35 min; after centrifugation, 5 mL of Tris-HCl buffer is added to the ultrafiltration tube, and the mixture is centrifuged again at 4000-6000 g for 25-35 min. By ultrafiltration centrifugation, small molecular impurities such as pigments and monosaccharides in the system can be further removed, and the obtained ornamental peach petal nanovesicles are more pure.

[0037] In the present application, the sucrose density gradient centrifugation method comprises: homogenizing the ornamental peach petals, centrifuging the homogenate at 1000 g, centrifuging the supernatant at 5000 g, and using sucrose density gradient centrifugation to collect the supernatant; the sucrose gradient is 8%, 30%, 45% and 60%, and the nanovesicle suspension is obtained by suctioning at the junction of sucrose 30% and 45% after centrifugation.

[0038] In the sucrose density gradient centrifugation method, the ornamental peach petals are mixed with PBS (100 mM, pH 7.4) at a ratio of 1:1 (w:v), and high-speed homogenization is performed at 4°C for 8-12 min, preferably 10 min, using a high-speed homogenizer; the homogenate is centrifuged at 1000 g for 25-35 min, preferably 30 min, at 4°C, and the supernatant is centrifuged at 5000 g for 25-35 min, preferably 30 min, at 4°C; the sucrose density gradient centrifugal force parameter is 100000-12000 g, and the preferred centrifugal parameter is 120000 g; the centrifugation time is 110-130 min, preferably 120 min; and the nanovesicle suspension is rapidly frozen using liquid nitrogen and stored at -80°C.

[0039] The application also provides an ornamental peach petal nanovesicle, which is prepared by the above preparation method and contains a kaempferol derivative and a fatty acid. The ornamental peach petal nanovesicle contains flavonols and various saturated and unsaturated fatty acids, and the flavonols are mainly glycoside derivatives of kaempferol and quercetin. The ornamental peach petal is rich in kaempferol derivatives and quercetin derivatives, which have good anti-inflammatory and antioxidant effects. Meanwhile, the fatty acids contained in the ornamental peach petal can moisturize and hydrate the skin and improve the skin quality. The ornamental peach petal nanovesicle provided by the application retains these active substances and enhances the biocompatibility of the active substances in the form of nanovesicles.

[0040] The ornamental peach petal in the application is obtained from a Prunus plant used for ornamental purposes. Preferably, the ornamental peach petal is any one of Yingshun, Fietao, Zaofei Hong, Hanbai, Pinhong, Pinxia, Fenfeng Shanbi, Fenhuashanbi and Lengmeiren. The above peach petal varieties are preserved in the Germplasm Resource Garden of Changde Vocational and Technical College in Hunan.

[0041] The application also provides the use of the preparation method or the ornamental peach petal nanovesicle in the preparation of skin care products, such as whitening products, freckle-removing products, anti-inflammatory products, anti-wrinkle products and anti-aging products.

[0042] The application also provides a skin care product containing the ornamental peach petal nanovesicle, which has anti-inflammatory, antioxidant, anti-aging and skin quality improving effects. The skin care product can be in the form of a lotion, a cream or an essence.

[0043] The ornamental peach petal nanovesicle can inhibit the activity of tyrosinase, inhibit the synthesis of cell melanin, brighten the skin color, whiten and remove freckles. The ornamental peach petal nanovesicle can inhibit the expression of human matrix metalloproteinase 1 (MMP-1), promote the expression of collagen type I (Col I) and hyaluronic acid (HA), and has anti-wrinkle and anti-aging effects. The ornamental peach petal nanovesicle can scavenge free radicals and inhibit the expression of tumor necrosis factor (TNF-α), and has anti-inflammatory and anti-inflammatory effects.

[0044] Unless otherwise specified, the reagents, consumables and the like involved in the application can be obtained from commercial channels. If the specific experimental conditions are not specified, the experiments are usually carried out under conventional conditions or according to the conditions recommended by the reagent company.

[0045] The technical solutions provided by the application will be described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the application.

[0046] Example 1

[0047] The preparation method of the ornamental peach petal nanovesicle is as follows:

[0048] The flower petals of ornamental peach (P. persica) "Yingchun" at the flower bud stage (removing pistil, stamen, calyx and receptacle) were collected, frozen with liquid nitrogen, mixed with Tris-HCl buffer (100 mM, pH 7.4) at a ratio of 1:1 (w:v), then homogenized at high speed for 10 min at 4°C using a high-speed homogenizer, the homogenate was filtered with filter paper to remove solid particles, and the liquid phase was centrifuged at 650 g, 3000 g, 10000 g and 15000 g in turn, and the supernatant was taken after each centrifugation for the next centrifugation, each centrifugation lasted for 20 min, and the supernatant was collected after the last centrifugation, and centrifuged at 100000 g for 120 min, then the supernatant was sucked out, and the precipitate was gently blown with PBS (100 mM, pH 6.8) to prepare a nanovesicle suspension.

[0049] The nanovesicle suspension was placed in a 100 kDa ultrafiltration tube and centrifuged at 5000 g for 30 min, 5 mL of Tris-HCl buffer (100 mM, pH 7.4) was added to the ultrafiltration tube after centrifugation, and centrifuged again at 5000 g for 30 min to obtain the ultrafiltration nanovesicle suspension.

[0050] The ultrafiltration nanovesicle suspension was frozen with liquid nitrogen and stored at -80°C for standby. The centrifugation in this example was carried out at 4°C.

[0051] Example 2

[0052] The preparation method of the ornamental peach petal nanovesicle is as follows:

[0053] The flower petals of ornamental peach (P. persica) "Yingchun" at the flower bud stage (removing pistil, stamen, calyx and receptacle) were collected, mixed with PBS (100 mM, pH 7.4) at a ratio of 1:1 (w:v), then homogenized at high speed for 10 min at 4°C using a high-speed homogenizer, the homogenate was centrifuged at 1000 g for 30 min, the supernatant was collected and centrifuged at 5000 g for 30 min, and the nanovesicles prepared from the junction of 30% and 45% were collected by sucrose density gradient centrifugation (20 mM Tris-HCl, pH 7.2, sucrose gradient of 8%, 30%, 45% and 60%) for 2 h, then the nanovesicle suspension was frozen with liquid nitrogen and stored at -80°C for standby. The centrifugation in this example was carried out at 4°C.

[0054] Example 3

[0055] The petals of P. persica 'Yingchun' were collected at the stages of flower bud, early blooming and full bloom, respectively, and the nanovesicles were prepared using the method of Example 1. The particle size and Zeta potential of the nanovesicles were determined using a nanoparticle size analyzer and a Zeta potential analyzer. The results of three repeated samples of each stage of P. persica flowers showed that the average particle sizes of the nanovesicles extracted from the flower buds, early blooming and full bloom were 65.05 ± 4.13 nm, 81.65 ± 4.14 nm and 142.17 ± 6.31 nm, respectively. The Zeta potentials were -34.6 mV, -28.7 mV and -23.2 mV, respectively. It was shown that the nanovesicles extracted from the petals of P. persica collected at the stages of flower bud and early blooming had smaller particle sizes, more uniform sizes and better stability.

[0056] Example 4

[0057] The flower buds of P. persica 'Yingchun' were collected at the stage of flower bud, and the flower buds were placed in a dark condition with a humidity of 90% and a temperature of 35°C for 4 h, 8 h, 12 h, 16 h, 20 h and 24 h, respectively. Then the pistil, stamen, calyx and receptacle were removed, and the petals were collected for the preparation of nanovesicles. The preparation method was as follows: 10 g of petals were mixed with Tris-HCl buffer (100 mM, pH = 7.4) at a ratio of 1:1 (w:v), and then homogenized at 4°C for 10 min using a high-speed homogenizer. The homogenate was filtered using filter paper to remove solid particles. The liquid phase was sequentially centrifuged at 650 g, 3000 g, 10000 g and 15000 g for 20 min at 4°C. After each centrifugation, the precipitate was removed, and the supernatant was retained. The supernatant was centrifuged at 5000 g for 30 min using a 100 kDa ultrafiltration tube. Then 5 mL of Tris-HCl buffer was added, and the mixture was centrifuged again for 30 min. Nanovesicles of P. persica petals treated with different dark times were obtained.

[0058] The content of polyphenols in the nanovesicles was determined using the Folin-phenol method for semi-quantitative determination of the amount of nanovesicles. The results are shown in Table 1.

[0059] Table 1 shows that the content of polyphenols began to decrease significantly from the 16 h dark treatment, indicating that excessive dark treatment is not conducive to the preparation of nanovesicles. However, moderate dark treatment can reduce the content of carbohydrates in the petal material. In this example, the carbohydrate content of the petal material treated with dark for 12-16 h can be reduced by 30%-40% compared with the sample without dark treatment. Therefore, the preferred dark treatment time is 12-16 h.

[0060] Table 1 Effect of dark treatment on nanovesicles

[0061] Dark treatment time (h) Total polyphenol content in nanovesicles (mg / g) 4 8.20±0.28 8 8.10±0.19 12 8.20±0.31 16 7.99±0.25 20 6.74±0.25 24 5.34±0.16

[0062] Example 5

[0063] A method for preparing ornamental peach petal nanovesicles.

[0064] The flower buds of P. persica "Yingchun" were collected at the flower bud stage, and the flower buds were placed in darkness at 90% humidity and 35°C for 12 h, then the pistil, stamen, calyx and receptacle were removed, the petals were collected, frozen with liquid nitrogen, and then ground in a frozen state. The frozen petal powder was mixed with Tris-HCl buffer (100 mM, pH 7.4) at a ratio of 1:1 (w:v), and then homogenized at 4°C for 10 min using a high-speed homogenizer. The homogenate was filtered with filter paper to remove solid particles, and the liquid phase was centrifuged at 650g, 3000g, 10000g and 15000g, respectively. The supernatant was collected after each centrifugation and centrifuged again for 20 min. The supernatant was collected after the last centrifugation, and then centrifuged at 100000g for 120 min. The supernatant was aspirated, and the precipitate was gently blown with PBS (100 mM, pH 6.8) to prepare a nanovesicle suspension. The nanovesicle suspension was placed in a 100 kDa ultrafiltration tube and centrifuged at 5000g for 30 min at 4°C. After centrifugation, 5 mL of Tris-HCl buffer (100 mM, pH 7.4) was added to the ultrafiltration tube, and centrifuged again at 5000g for 30 min to obtain the ultrafiltrated nanovesicle suspension. The average particle size of the ultrafiltrated nanovesicles was 78.12 ± 3.33 nm, and the Zeta potential was -37.2 mV.

[0065] Example 6

[0066] Nanovesicles were extracted from the petals of the following ornamental peach cultivars: Yingshun, Fietao, Zaofei Hong, Hanbai, Pinhong, Pinxia, Fenhuangshanbi, Fenhuahuangshanbi, Lengmeiren. All ornamental peaches were picked at the bud stage, and the petals were separated after 12 h of darkness. Nanovesicles were extracted according to the method of Example 5. The extracted nanovesicles were added to 10 times 50% methanol (containing 1% formic acid), then broken up using ultrasonic oscillation, and the mixture was centrifuged at 4°C for 15 min (12000g). The supernatant was filtered through a 0.22 pm polytetrafluoroethylene membrane, and then high-resolution liquid chromatography-mass spectrometry was used for detection, with the following liquid chromatography-mass spectrometry conditions: The liquid phase detection conditions were as follows: mobile phase A was acetonitrile added with 0.1% formic acid, and mobile phase B was pure water added with 0.1% formic acid. The column temperature was 35°C, and the flow rate was 0.3 mL / min. The chromatographic column was Shimadzu Shim-Pack GIS (3.0 x 100 mm, 2 pm). The gradient elution program was as follows: 0 min, 95% B; 5 min, 95% B; 10 min, 90% B; 15 min, 40% B; 20 min, 40% B; 21 min, 95% B; and 3 min for equilibration. The mass spectrometry detection conditions were as follows: Q Exactive Orbitrap high-resolution mass spectrometer, equipped with a HESI ion source, ion transmission tube temperature 350°C, auxiliary gas temperature 300°C, flow rate 10 arb, sheath gas 45 arb; and spray voltage 3.5 kV. The results are shown in Table 2. Figures 1-4

[0067] A variety of flavonols and fatty acid compounds were identified in the nanovesicles extracted from the petals of the above-mentioned 9 ornamental peaches. Figure 1 Six kaempferol derivatives and six quercetin derivatives detected in the petals of ornamental peach Yingshun are shown, and the mass spectrometry results of each component are shown in Table 2. It can be found by semi-quantification that Figure 2 and Figure 3 The content of flavonols in the petals of ornamental peaches is very high, except for Fietao and Zaofei Hong. The petals of ornamental peaches also contain rich fatty acid components Figure 4 , and the highest content is in Hanbai, Fietao, and Zaofei Hong. These fatty acids are mainly dihydroxypalmitic acid, and Hanbai also contains a high content of undecenoic acid (10-DHA). These flavonols and fatty acid compounds have good biological activity, among which flavonols have antioxidant and anti-inflammatory effects, and fatty acids can have a moisturizing and water-retaining effect. This shows that the nanovesicles prepared from the petals of ornamental peaches have potential bioactive functions.

[0068] Table 2 Flavonols detected by high-resolution mass spectrometry

[0069]

[0070] ​

[0071] Example 7

[0072] The nanovesicle suspensions extracted from the ornamental peach petals of Yingshun, Feitao, Zaofeihong, Hanbai, Pinhong, Pinxia, Fenghongshanbi, Fenhuangshanbi, and Lengmeiren in Example 6 were diluted 10 times, respectively, and then 10 μL of each was mixed with 190 μL of DPPH free radicals (87 μM, ethanol solution) and ABTS (7 mM, aqueous solution) free radicals, respectively, and reacted for 30 min in the dark. The ability of the various nanovesicle extracts to scavenge the two free radicals was determined at 529 nm and 723 nm, respectively, using vitamin C as a positive control. Each group of experiments was repeated 3 times for averaging, and the data were analyzed using one-way ANOVA, and the data were expressed as mean ± standard deviation. The results are shown in Figure 5 .

[0073] The results show that all the nanovesicles have the ability to scavenge free radicals, and most of the nanovesicles are significantly stronger than vitamin C, among which Pinhong and Fenghongshanbi have the strongest ability to scavenge free radicals.

[0074] Example 8

[0075] 10 μL of the nanovesicle suspensions extracted from the ornamental peach petals of Yingshun, Feitao, Zaofeihong, Hanbai, Pinhong, Pinxia, Fenghongshanbi, Fenhuangshanbi, and Lengmeiren in Example 6 were mixed with an equal volume of tyrosinase PBS solution, and the enzyme concentration was 200 U / mL. After incubation for 10 min at 37°C with shaking, 8 times the volume of L-tyrosine PBS solution was added, and the L-tyrosine concentration was 20 mg / mL. After reaction for 10 min at 37°C, the absorbance of the entire reaction system was determined at 475 nm. The inhibition activity of tyrosinase was calculated according to the following formula:

[0076] Inhibition rate = 100% x (A0-A) / A0;

[0077] wherein A0 is the absorbance of the reaction after mixing tyrosinase with tyrosine without adding any arbutin or nanovesicles, A is the absorbance of the test group with the addition of nanovesicles or arbutin, and arbutin is used as a positive control compound. All absorbance values are subtracted from the background value of the reaction system (absorbance of the reaction system with the addition of only nanovesicles or arbutin without the addition of tyrosinase). Each group of experiments was repeated 3 times for averaging, and the data were analyzed using one-way ANOVA, and the data were expressed as mean ± standard deviation. The results are shown in Figure 6 .

[0078] The results show that all the nanovesicles from the petals of ornamental peach cultivars have tyrosinase inhibitory activity, and the nanovesicles from the petals of the cultivar Hanbai have the strongest inhibitory effect, followed by the cultivar Pinhong, and both of them are superior to the model compound arbutin. The cultivars Yingchun and Fenhongshanbi have inhibitory activities comparable to arbutin.

[0079] Example 9

[0080] The inhibitory effect of several nanovesicles prepared from the petals of ornamental peach cultivars according to Example 6 on the synthesis of melanin was tested using B16-F10 (CRL-6475) melanoma cells (ATCC) as a model.

[0081] The cells were inoculated in DMEM medium containing 10% fetal bovine serum, and the culture conditions were 37°C, 5% CO2, and 98% relative humidity. The cells in the logarithmic growth phase were trypsinized and inoculated in 6-well culture plates at a concentration of 1 x 10 6 After 8 h of culture, the supernatant was discarded, and the cells were washed once with PBS. Then, the nanovesicle suspensions prepared from the petals of the ornamental peach cultivars Yingchun, Bietao, Zaobihong, Hanbai, Pinhong, Pinxia, Fenhongshanbi, Fenhuashanbi, and Lengmeiren according to Example 6 were diluted 10-fold and added to the culture plates at 2 mL per well. Arbutin was used as a positive control, and DEME medium without any extract was used as a negative control. After 48 h of culture, the supernatant of the cultured cells was discarded, and the cells were collected after digestion, centrifuged at 1500 r / min for 10 min, and then 1 mL of 1 mol / L sodium hydroxide solution containing 10% DMSO was added. After incubation at 80°C for 1 h, the absorbance was measured at 405 nm using an enzyme marker. The inhibitory rate of the extract on melanin formation was calculated according to the following formula:

[0082] Melanin formation inhibition rate = 100% x (A1-A) / A1

[0083] wherein A1 is the absorbance of the melanin of the cells without the addition of any arbutin or nanovesicles, and A is the absorbance of the test group with the addition of nanovesicles or arbutin. Arbutin was used as a positive control compound, and all the absorbance values were subtracted by the system blank of the reaction system (the absorbance of the reaction system with the addition of only nanovesicles or arbutin). Each group of experiments was repeated 5 times to obtain the average value, and the data were analyzed using one-way ANOVA, and the data were expressed as the mean ± standard deviation.

[0084] The results are shown in Table 1. Figure 7 As shown in Table 1, the nanovesicles prepared from the petals of all the ornamental peach cultivars can significantly inhibit the synthesis of melanin in cells, and their inhibitory effect is significantly stronger than that of the model compound arbutin.

[0085] The cells treated by the above method were centrifuged and rinsed with PBS, then 40 μL of 1% Triton-X100 was added, then transferred to -80°C for 30 min, then to 37°C to completely melt, then 10 μL of L-levodopa solution (1 w / w%) was added. The reaction was carried out at 37°C for 30 min, then the absorbance of the system was measured at 450 nm wavelength. The inhibition rate of nanovesicles on intracellular melanin formation was calculated according to the following formula:

[0086] Intracellular melanin formation inhibition rate = 100% x (A3-A) / A3;

[0087] Wherein A3 is the absorbance of the tyrosinase reaction after extraction of the cells without adding any nanovesicles or compounds, A is the absorbance of the test group added with extract or arbutin, arbutin as a positive control compound, all absorbance values are subtracted from the system blank of the reaction system (absorbance of the reaction system only added with nanovesicles or arbutin). Each group of experiments was repeated 5 times to take the average, the data was analyzed by one-way ANOVA, and the data was expressed as mean ± standard deviation.

[0088] The results are shown in Figure 8 All the nanovesicles prepared from ornamental peach petals have an inhibitory effect on intracellular tyrosinase activity, among which the inhibition effect of Yingchun and Lengmei is consistent with that of the positive control compound arbutin, Pinxia and Fenhong Shanbi are weaker than arbutin, and the nanovesicles prepared from other several ornamental peach petals are stronger than arbutin, among which the inhibition activity of Hanbai is the best, more than 70%.

[0089] Example 10

[0090] The anti-aging effect of nanovesicles extracted from the petals of Yingchun, Hanbai, Pinxia and Fenhong Shanbi (extracted by the method in Example 5) was tested using human dermal fibroblasts (Wuhan Punsai Life Science).

[0091] Human dermal fibroblasts were cultured using fibroblast-specific medium (CM-H103, Wuhan Punsai Life Science), and were used for experiments when they were subcultured to the third generation. The cells were digested using trypsin, and were inoculated in a 6-well culture plate at a concentration of 0.5 x 10 6 After 24 hours of culture in the incubator (CO2 5%, 37°C, humidity 98%), the culture medium in the culture dish was discarded, then 1.0 mL of PBS was added, and the experimental group, positive control group (Ilomastat) and negative control group (control group) cells were irradiated using UVA (340 nm) at a dose of 10 J / cm 2, the blank group is not irradiated. Nanovesicles diluted 10-fold with the medium are added to the culture medium of the experimental group, Ilomastat (matrix metalloproteinase inhibitor) (1 nM) is added to the culture medium of the positive control group, and only the medium is added to the negative control group and the blank group. The total volume of the medium in each group is 2 mL, and then cultured for 24 h. After culturing, the cells are digested and centrifuged (2000 g, 20 min), and the supernatant is used to test the expression level of MMP-1 using a human matrix metalloproteinase 1 (MMP-1) kit. The cells obtained by centrifugation are blown into a suspension with PBS, and the concentration is counted as 1×10 6 cells / mL, then sonicated for 30 min, frozen quickly with liquid nitrogen, thawed at 37 °C and refrozen, and centrifuged (1000 g, 20 min) after repeated freezing and thawing three times. The supernatant is used to test the content of type I collagen (ColⅠ) using a ColⅠ test kit and the content of human hyaluronic acid (HA) using an ELISA kit. Each experiment in each group is repeated 4 times and averaged, and the data is analyzed using one-way analysis of variance. The data is expressed as the mean ± standard deviation. The results are as Figure 9 and Figure 10 shown.

[0092] The results show that compared with the control group, all nanovesicle extracts can significantly reduce the expression level of MMP-1, and the effect of Hanbai is close to that of the model compound Ilomastat. At the same time, in all nanovesicle treatment groups, the expression levels of Col 1 and HA are significantly higher than those of the control group, and the nanovesicles prepared from Yingchun and Hanbai are even higher than those of the blank group and the model compound Ilomastat group. These results indicate that the nanovesicles extracted from the petals of ornamental peach can effectively combat skin aging caused by ultraviolet irradiation and have a certain anti-wrinkle effect.

[0093] Example 11

[0094] The anti-inflammatory effect of nanovesicles extracted from the petals of Yingchun, Hanbai, Magenta and Pink Mountain Jasper (extracted by the method in Example 5) was tested using the cell line RAW246.7.

[0095] Cells were cultured using a medium of DMEM high glucose + 10% FBS and 1% double antibody, and the culture conditions were 37 °C, 5% carbon dioxide content and 98% relative humidity. Take 1×10 6The cells in the logarithmic growth phase of 1 x 106 / mL were inoculated into 96-well plates at 100 μL per well. After the cells adhered, lipopolysaccharide (LPS) was used to induce inflammation. The supernatant was removed, and the medium containing 1 μg / mL of lipopolysaccharide was added. The blank group was not added with lipopolysaccharide, the experimental group was added with 10 times diluted nano vesicle suspension, and the control group was only added with the medium containing LPS. After 24 h of culture, the supernatant was aspirated, and the levels of NO and TNF-α in the cell culture fluid were determined using an NO kit and a TNF-α ELISA kit, respectively. The results are shown in Figure 11 and Figure 12 .

[0096] The results show that, compared with the control group, the nano vesicles prepared from the petals of four ornamental peach varieties can significantly reduce the levels of NO and TNF-α, and the effect of Hanbai is the best. This indicates that the nano vesicles prepared from the petals of ornamental peach have the effects of anti-inflammation and inhibition of expression of inflammatory factors.

[0097] Example 12

[0098] Preparation method of emulsion containing nano vesicles of ornamental peach petals:

[0099] The following components were added to the water gel containing 0.5% gelatin to prepare a base emulsion: glycerol 1%, 1,3-propanediol 1%, hyaluronic acid 1%, L-ascorbic acid 5%, ethylene glycol phenyl ether 0.1%. Then, the nano vesicles prepared from the petals of Hanbai were diluted 100 times using the base emulsion. Thus, the emulsion containing nano vesicles of ornamental peach petals was obtained.

[0100] In this example, the preparation method of nano vesicles was the same as that in Example 5.

[0101] Example 13

[0102] Preparation method of cream containing nano vesicles of ornamental peach petals:

[0103] The following components were added to water to prepare a cream agent in terms of mass / volume percentage: nicotinamide 8%, glycerol 3%, palmitol 1.5%, glycerol 1%, 1,3-propanediol 1%, vitamin E 0.5%. Then, the nano vesicles prepared from the petals of Pinhong were compounded with the cream agent at a volume ratio of 1:100 to form a uniform cream agent. Thus, the cream containing nano vesicles of ornamental peach petals was obtained.

[0104] In this example, the preparation method of nano vesicles was the same as that in Example 5.

[0105] Example 14

[0106] Preparation method of cream containing nano vesicles of ornamental peach petals:

[0107] The following components are contained in the cream in mass / volume percentage: 1,3-propanediol 2%, polydimethylsiloxane 2%, bovine (hydroxypropyl tetrahydropyran triol) 2%, cyclohexylsiloxane 1%, glycerol 1%, and water. The nanovesicles prepared from the petals of pink mountain peony are then compounded with the cream at a volume ratio of 1:100 to form a uniform cream. A face cream containing nanovesicles of ornamental peach flower petals is obtained.

[0108] The method for preparing the nanovesicles in this example is the same as that in Example 5.

[0109] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for preparing ornamental peach petal nanovesicles, characterized by, The petal of ornamental peach at the stage of bud or initial opening is collected, and the nanovesicles are obtained by direct centrifugation or sucrose density gradient centrifugation; The petal of ornamental peach is collected, and the nanovesicles are obtained by direct centrifugation or sucrose density gradient centrifugation; The petal of ornamental peach is collected, and the nanovesicles are obtained by direct centrifugation or sucrose density gradient centrifugation; The petal of ornamental peach is collected, and the nanovesicles are obtained by direct centrifugation or sucrose density gradient centrifugation; The petal of ornamental peach is collected, and the nanovesicles are obtained by direct centrifugation or sucrose density gradient centrifugation; The petal of ornamental peach is collected, and the nanovesicles are obtained by direct centrifugation or sucrose density gradient centrifugation; 2. The ornamental peach petal nanovesicles obtained by the preparation method of claim 1, characterized in that, The petal of ornamental peach is collected, and the nanovesicles are obtained by direct centrifugation or sucrose density gradient centrifugation. The nanovesicles of the petal of ornamental peach contain kaempferol derivatives and fatty acids.

4. Use according to claim 3, characterized in that, 3. The use of the preparation method of claim 1 or the nanovesicles of the petal of ornamental peach of claim 2 in the preparation of skin care products.

5. A skin care product, characterized by, The skin care products include whitening products, freckle-removing products, anti-inflammatory products, anti-wrinkle products and anti-aging products.

6. The skin care product of claim 5, wherein, The skin care products include emulsions, creams and serums. The skin care products include emulsions, creams and serums.

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