Ornamental peach petal nano-vesicle as well as preparation method and application thereof

Abstract

The invention provides an ornamental peach petal nano-vesicle as well as a preparation method and application thereof, and belongs to the technical field of biology. The preparation method of the ornamental peach petal nano-vesicles comprises the following steps: collecting ornamental peach petals in a bud period or an initial blooming period, and extracting by adopting a direct centrifugation method or a sucrose density gradient centrifugation method to obtain the nano-vesicles. The ornamental peach petals in the bud period or the initial blooming period are used as raw materials, and the prepared nano-vesicles have good biocompatibility, are rich in specific polyphenol, lipid and other functional ingredients of the ornamental peach petals, have good application potential in the aspects of oxidation resistance, inflammation resistance, skin improvement and the like, and have good application prospects. And high-added-value deep processing of the ornamental peach petals is realized.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an ornamental peach petal nanovesicle and a preparation method and application thereof. Background Art

[0002] As an important breeding direction for peaches, ornamental traits have been successfully selected and bred. A number of ornamental peach varieties with different flower colors and characteristics have been successfully bred. These ornamental peach varieties usually use cultivated peach (Prunus persica) as the main parent or breeding group species, but in the breeding process, they may also be hybridized with Chinese native peach resources such as Chinese mountain peach (P. davidiana), light walnut (P. mira), Gansu peach (P. kansuensis), Xinjiang peach (P. ferganensis), etc. At the same time, since ornamental peaches are not selected for fresh eating as the main breeding trait, more genetic diversity has been preserved in the breeding process of ornamental peaches. Thanks to the advantages of ornamental peaches being cold-resistant, easy to plant, and edible, they have been widely distributed in southern and northern China.

[0003] Although ornamental peaches are widely planted, they are mainly for viewing purposes. Therefore, there are few related industries to develop and utilize them except for seedling cultivation, which makes it difficult for ornamental peaches widely planted across the country to gain higher economic benefits. Nanovesicles refer to a type of bubble-like structure wrapped in a double lipid layer at the nanoscale formed during the life activities of cells. In ornamental peach flowers, on the one hand, they can serve as an important place for the synthesis of substances for life activities at the cellular level, and on the other hand, they are also the precursor organelles of vacuoles responsible for the swelling of peach flowers. Thanks to the double lipid membrane structure of nanovesicles, the substances in the vesicles are more easily transferred in systems with double lipid membranes as the main interface, such as cells or organelles.

[0004] Therefore, nanovesicles with ornamental peach petals as the main source are expected to provide important technical support for the high value-added utilization of ornamental peach resources. Summary of the invention

[0005] In view of this, the purpose of the present 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 ingredients such as polyphenols and lipids unique to ornamental peach petals, and have good application potential in anti-oxidation, anti-inflammatory and improving skin quality.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] A method for preparing ornamental peach petal nanovesicles comprises collecting ornamental peach petals at the bud stage or the initial opening stage, and extracting and obtaining the nanovesicles by direct centrifugation or sucrose density gradient centrifugation.

[0008] Preferably, the nanovesicles are extracted from the ornamental peach petals after being treated in darkness.

[0009] Preferably, the dark treatment is placed in dark conditions at 30-40° C. and 85%-99% humidity for 12-16 hours.

[0010] Preferably, the direct centrifugation method includes: freezing the ornamental peach petals and then crushing them, mixing the petal powder with Tris-HCl buffer and then homogenizing them, centrifuging the homogenate liquid phase 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, and resuspending it with PBS to obtain a nanovesicle suspension.

[0011] Preferably, the sucrose density gradient centrifugation method includes: homogenizing the ornamental peach petals, centrifuging the homogenate at 1000g, collecting the supernatant and centrifuging at 5000g, collecting the supernatant and subjecting it to sucrose density gradient centrifugation, wherein the sucrose gradient is 8%, 30%, 45% and 60%, and after centrifugation, aspirating at the junction of 30% and 45% sucrose to obtain a nanovesicle suspension.

[0012] Another object of the present invention is to provide an ornamental peach petal nanovesicle obtained by the preparation method, wherein the ornamental peach petal nanovesicle contains kaempferol derivatives and fatty acids.

[0013] Another object of the present invention is to provide the above-mentioned preparation method or the use of the above-mentioned ornamental peach petal nanovesicles in the preparation of skin care products.

[0014] Preferably, the skin care products include whitening products, freckle removal products, anti-inflammatory products, anti-wrinkle products and anti-aging products.

[0015] Another object of the present invention is to provide a skin care product, which comprises the above-mentioned ornamental peach petal nanovesicles.

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

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

[0018] The present invention provides a method for preparing an ornamental peach petal nanovesicle, which uses ornamental peach petals in the bud stage or the initial opening stage as raw materials, and effectively utilizes ornamental peach petal resources. The ornamental peach petal nanovesicle provided by the present invention has good biocompatibility, is rich in functional components such as polyphenols and lipids that are unique to ornamental peach petals, has good application potential in anti-oxidation, anti-inflammatory and improving skin quality, and realizes high added value deep processing of ornamental peach petals. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 :Six kaempferol (K1-K6) derivatives and six quercetin (Q1-Q6) derivatives detected in the petals of the ornamental peach jasmine and their retention times (minutes);

[0020] Figure 2 : Relative contents of kaempferol compounds in nanovesicles prepared from different ornamental peach petals;

[0021] Figure 3 : Relative contents of quercetin-like compounds in nanovesicles prepared from different ornamental peach petals;

[0022] Figure 4 : Relative contents of several fatty acid compounds in nanovesicles prepared from different ornamental peach petals;

[0023] Figure 5 :Antioxidant activity of nanovesicles prepared from various ornamental peach petals, where different capital letters represent significant differences in ABTS free radical scavenging rates, and different lowercase letters represent significant differences in DPPH free radical scavenging rates;

[0024] Figure 6 : The inhibitory activity of nanovesicles prepared from different ornamental peach petals on tyrosinase, where different letters represent significant differences between the two groups;

[0025] Figure 7 : The inhibitory effect of nanovesicles prepared from different ornamental peach petals on cellular melanin synthesis, where ** indicates P < 0.01;

[0026] Figure 8 : The inhibitory activity of nanovesicles prepared from different ornamental peach petals on intracellular tyrosinase, where different letters represent significant differences between the two groups;

[0027] Fig. 9 :The effect of nanovesicles prepared from different ornamental peach petals on the expression of MMP-1 in fibroblasts, where different letters represent significant differences between the two groups;

[0028] Fig.10 :The effects of nanovesicles prepared from different ornamental peach petals on the expression of Col 1 and HA in fibroblasts, where different capital letters represent significant differences in Col 1, and different lowercase letters represent significant differences in HA;

[0029] Fig.11 :The effect of nanovesicles prepared from different ornamental peach petals on NO content, where different letters represent significant differences between the two groups;

[0030] Fig.12:Effects of nanovesicles prepared from different ornamental peach petals on the expression of TNF-α, where different letters represent significant differences between the two groups. DETAILED DESCRIPTION

[0031] The present invention provides a method for preparing nanovesicles of ornamental peach petals, wherein ornamental peach petals at the bud stage or the initial opening stage are collected, and the nanovesicles are extracted by direct centrifugation or sucrose density gradient centrifugation. Studies have found that the blooming of flower buds is mainly caused by the rapid merging of vesicles in the petals and the rapid expansion of the central vacuole. The nanovesicles extracted from the ornamental peach petals as raw materials contain vacuole precursors. The nanovesicles extracted from the 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 flowers, and the prepared nanovesicles have smaller particle sizes, more uniform sizes, higher yields, and better stability.

[0032] As a feasible method, after collecting peach flowers, the pistils, stamens, calyx and receptacle are removed, and the ornamental peach petals are retained for extracting nanovesicles.

[0033] In the present invention, the ornamental peach petals are treated in darkness before extracting the nanovesicles. The conditions for the dark treatment of the ornamental peach petals are: a temperature of 30 to 40°C, a humidity of 85 to 99%, and the ornamental peach petals are placed in darkness for 12 to 16 hours. The temperature of the dark treatment of the present invention is preferably 32 to 38°C, more preferably 35°C, and the humidity is preferably 88 to 95%, more preferably 90%. The high molecular weight carbohydrates contained in the flower buds will interfere with the preparation of nanovesicles. After the ornamental peach petals are treated in darkness, the respiration of the peach flowers consumes the sugar powder in the cells, reducing the content of carbohydrates, especially reducing the content of starch, avoiding the formation of colloids between starch and macromolecular substances during the extraction process, improving the abundance of nanovesicles in the finally prepared suspension, and reducing the content of impurities.

[0034] In the present invention, the direct centrifugation method comprises: freezing and then crushing the ornamental peach petals, mixing the petal powder with Tris-HCl buffer and then homogenizing, centrifuging the homogenate liquid phase 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, and resuspending it with PBS to obtain a nanovesicle suspension.

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

[0036] In the present invention, the nanovesicle suspension obtained by direct centrifugation is subjected to ultrafiltration centrifugation, and the ultrafiltration centrifugation is as follows: the nanovesicle suspension is placed in a 100kDa ultrafiltration tube for centrifugation, the centrifugation condition is 4000-6000g, and the centrifugation time is 25-35min; after the centrifugation, 5mL of Tris-HCl buffer is added to the ultrafiltration tube, and centrifugation is performed again, the centrifugation condition is 4000-6000g, and the centrifugation time is 25-35min. The ultrafiltration centrifugation can further remove small molecular impurities such as pigments and monosaccharides in the system, so that the obtained ornamental peach petal nanovesicles are purer.

[0037] In the present invention, the sucrose density gradient centrifugation method comprises: homogenizing the ornamental peach petals, centrifuging the homogenate at 1000g, collecting the supernatant and centrifuging at 5000g, collecting the supernatant and subjecting it to sucrose density gradient centrifugation, wherein the sucrose gradient is 8%, 30%, 45% and 60%, and after centrifugation, a nanovesicle suspension is obtained by aspirating at the junction of 30% and 45% sucrose.

[0038] In the sucrose density gradient centrifugation method of the present invention, the ornamental peach petals are mixed with PBS (100mM, pH7.4) in a ratio of 1:1 (w:v), and a high-speed homogenizer is used for high-speed homogenization at 4°C for 8-12min, preferably for 10min; the homogenate is centrifuged at 1000g for 25-35min, preferably for 30min, and the centrifugation temperature is 4°C; the supernatant is collected and centrifuged at 5000g for 25-35min, preferably for 30min, and the centrifugation temperature is 4°C; the centrifugal force parameter of the sucrose density gradient is 100000-12000g, the preferred centrifugation parameter is 120000g, and the centrifugation time is 110-130min, preferably 120min; the nanovesicle suspension is quick-frozen using liquid nitrogen and stored at -80°C.

[0039] The present invention also provides an ornamental peach petal nanovesicle, which is prepared by the above-mentioned preparation method, and contains kaempferol derivatives and fatty acids. The ornamental peach petal nanovesicles described in the present invention are rich in flavonol compounds and a variety of saturated and unsaturated fatty acids, wherein the flavonol compounds are mainly glycoside derivatives of kaempferol and quercetin. Ornamental peach petals are rich in kaempferol derivatives and quercetin derivatives, and these compounds have good anti-inflammatory and antioxidant effects; at the same time, the fatty acids rich in ornamental peach petals can moisturize the skin and improve skin quality. The ornamental peach petal nanovesicles provided by the present invention retain these active substances and enhance their biocompatibility in the form of vesicles.

[0040] The ornamental peach petals of the present invention are from ornamental varieties of Prunus plants of the Rosaceae family, and the preferred ornamental peach varieties are any one of Yingchun, Feitao, Zaofeihong, Hanbai, Fuminghong, Pinxia, ​​Fenhongshanbi, Fenhuashanbi and Lengmeiren. The above-mentioned peach varieties of the present invention are all stored in the germplasm resource garden of Hunan Changde Vocational and Technical College.

[0041] The present invention also provides the preparation method or the use of the ornamental peach petal nanovesicles in the preparation of skin care products, and the skin care products include whitening products, freckle removal products, anti-inflammatory products, anti-wrinkle products and anti-aging products.

[0042] The present invention also provides a skin care product, comprising the above-mentioned ornamental peach petal nanovesicles, and having the effects of anti-inflammatory, anti-oxidation, anti-aging and improving skin quality. The skin care product of the present invention can be in the form of lotion, cream and essence.

[0043] The ornamental peach petal nanovesicles of the present invention can inhibit tyrosinase activity and the synthesis of cellular melanin, and have the effects of brightening skin color and whitening and removing spots; the ornamental peach petal nanovesicles can inhibit the expression of human matrix metalloproteinase 1 (MMP-1), and promote the expression of type I collagen (ColⅠ) and hyaluronic acid (HA), and have anti-wrinkle and anti-aging effects; the ornamental peach petal nanovesicles can scavenge free radicals and inhibit the expression of tumor necrosis factor (TNF-α), and have the effects of anti-inflammatory and improving skin inflammation.

[0044] Unless otherwise specified, the reagents, consumables, etc. involved in the present invention can be obtained from commercial channels. If the specific experimental conditions are not specified, they are usually carried out under conventional conditions or under the conditions recommended by the reagent company.

[0045] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0046] Example 1

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

[0048] The petals of the ornamental peach (P. persica) "Yingchun" in the bud stage (excluding the pistil, stamens, calyx and receptacle) were collected and quickly frozen with liquid nitrogen. The frozen petal powder was mixed with Tris-HCl buffer (100mM, pH 7.4) at a ratio of 1:1 (w:v), and then homogenized at high speed for 10 minutes 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 650g, 3000g, 10000g and 15000g in sequence. After each centrifugation, the supernatant was taken for the next centrifugation, each centrifugation for 20 minutes. After the last centrifugation, the supernatant was collected and centrifuged at 100000g for 120 minutes. The supernatant was aspirated and the precipitate was gently blown with PBS (100mM, pH 6.8) to make a nanovesicle suspension.

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

[0050] The nanovesicle suspension after ultrafiltration was quickly frozen in liquid nitrogen and stored at -80°C for later use. The centrifugation in this example was performed at 4°C.

[0051] Example 2

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

[0053] The petals of the ornamental peach (P.persica) "Yingchun" in the bud stage were collected (without pistil, stamen, calyx and receptacle), mixed with PBS (100mM, pH 7.4) in a ratio of 1:1 (w:v), homogenized at 4°C for 10min using a high-speed homogenizer, the homogenate was centrifuged at 1000g for 30min, the supernatant was collected and centrifuged at 5000g for 30min, the supernatant was collected and centrifuged using sucrose density gradient centrifugation (20mM Tris-HCl, pH 7.2, sucrose gradient of 8%, 30%, 45% and 60%) for 2h, then the prepared nanovesicles were drawn from the 30% and 45% junction, the nanovesicle suspension was quickly frozen in liquid nitrogen, stored at -80°C for standby use. The centrifugation in the present embodiment was all carried out at 4°C.

[0054] Example 3

[0055] The petals of ornamental peach (P.persica) "Welcome to Spring" were collected at the bud stage, the initial opening stage, and the full bloom stage, and nanovesicles were prepared using the method of Example 1. The particle size and Zeta potential of the nanovesicles were measured using a nanoparticle sizer and a Zeta potential analyzer. The ornamental peach samples of each stage were repeated in three groups. The results showed that the average particle size of the nanovesicles extracted from the bud stage, the initial opening stage, and the full bloom stage were 65.05±4.13nm, 81.65±4.14nm, and 142.17±6.31nm, respectively. The Zeta potential was -34.6mV, -28.7mV, and -23.2mV, respectively. It shows that the nanovesicles extracted from the ornamental peach petals collected at the bud stage and the initial opening stage have a smaller particle size, a more uniform size, and better stability.

[0056] Example 4

[0057] The buds of ornamental peach (P. persica) "Yingchun" were collected during the peach bud stage, and the 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 preparing nanovesicles. The preparation method is as follows: 10 g of each was taken, and the petals were mixed with Tris-HCl buffer (100 mM, pH = 7.4) at a ratio of 1:1 (w:v) Then, a high-speed homogenizer was used for high-speed homogenization at 4°C for 10 minutes. The homogenate was filtered with filter paper to remove solid particles. The liquid phase was centrifuged at 650g, 3000g, 10000g and 15000g for 20 minutes each, and the centrifugation temperature was 4°C. After each centrifugation, the precipitate was removed and the supernatant was retained. The supernatant was centrifuged at 5000g for 30 minutes using a 100kDa ultrafiltration tube, and then 5mL of Tris-HCl buffer was added and centrifuged again for 30 minutes. Ornamental peach petal nanovesicles treated with different dark times were obtained.

[0058] The polyphenol content 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] The results in Table 1 show that the polyphenol content begins to decrease significantly from the 16th hour of dark treatment, which indicates that too long dark treatment is not conducive to the preparation of nanovesicles. However, moderate dark treatment can reduce the carbohydrate content in the petal material. In this embodiment, when the dark treatment is 12 to 16 hours, the carbohydrate content in the petal material can be reduced by 30% to 40% compared with the sample without dark treatment. Therefore, the optimal dark treatment time is 12 to 16 hours.

[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 buds of ornamental peach (P. persica) "Yingchun" were collected during the peach bud stage, and the buds were placed in a dark condition with a humidity of 90% and a temperature of 35°C for 12 h. The pistils, stamens, calyx and receptacle were then removed, and the petals were collected and quick-frozen with liquid nitrogen. Then, they were ground in a frozen state, and the frozen petal powder was mixed with Tris-HCl buffer (100 mM, pH 7.4) in a ratio of 1:1 (w:v). Then, a high-speed homogenizer was used for high-speed homogenization at 4°C for 10 min. The homogenate was filtered with filter paper to remove solid particles. The liquid phase was centrifuged at 650g, 3000g, 10000g and 15000g in sequence. After each centrifugation, the supernatant was taken for the next centrifugation, and each centrifugation was 20 min. After the last centrifugation, the supernatant was collected and centrifuged at 100000g for 120 min. The supernatant was aspirated and the precipitate was precipitated with PBS (100 mM, pH 6.8) Gently blow to prepare a nanovesicle suspension, place the nanovesicle suspension in a 100 kDa ultrafiltration tube and centrifuge at 5000 g for 30 min at 4° C. After the centrifugation, add 5 mL of Tris-HCl buffer (100 mM, pH 7.4) to the ultrafiltration tube, centrifuge again at 5000 g for 30 min to obtain an ultrafiltered nanovesicle suspension. The average particle size of the ultrafiltered nanovesicles is 78.12 ± 3.33 nm, and the Zeta potential is -37.2 mV.

[0065] Example 6

[0066] Nanovesicles were extracted from the petals of the following varieties of ornamental peaches, Yingchun, Feitao, Zaofeihong, Hanbai, Fuchshong, Pinxia, ​​Fenhongshanbi, Fenhuashanbi, and Lengmeiren. All ornamental peaches were picked at the bud stage, and the petals were separated after 12 hours of dark treatment, and nanovesicles were extracted according to the method of Example 5. The extracted nanovesicles were added with 10 times 50% methanol (containing 1% formic acid), and then crushed by ultrasonic vibration. The mixture was centrifuged at 4°C for 15min (12000g), and the supernatant was filtered through a 0.22μm polytetrafluoroethylene filter membrane, and then measured by high-resolution liquid chromatography-mass spectrometry. The liquid chromatography-mass spectrometry conditions were as follows: wherein the liquid phase detection conditions were acetonitrile with 0.1% formic acid added to mobile phase A, and pure water with 0.1% formic acid added to mobile phase B. The column temperature was 35°C and the flow rate was 0.3mL / min. The chromatographic column was Shimadzu Shim-Pack GIS (3.0×100mm, 2μm). The gradient elution program was: 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 of equilibrium. The mass spectrometry detection conditions were as follows: Thermo Fisher Q Exactive Orbitrap high-resolution mass spectrometer, equipped with HESI ion source, ion transfer tube temperature 350°C, auxiliary gas temperature 300°C, flow rate 10 arb, sheath gas: 45 arb; spray voltage 3.5 kV. The results are as follows Figure 1 to Figure 4 shown.

[0067] A variety of flavonols and fatty acid compounds were identified in the nanovesicles extracted from the petals of the above nine ornamental peach species. Figure 1 Six kaempferol derivatives and six quercetin derivatives detected in ornamental peach petals are shown in Table 2. The mass spectrometry results of each component are shown in Table 2. Semi-quantitative results show that ( Figure 2 and Figure 3 ), except for the red peach and early red peach, the other peach flowers have high content of flavonol compounds. The ornamental peach petals also contain rich fatty acid components ( Figure 4 ), with the highest content in Hanbai, Feitao and Zaofeihong. The main fatty acid in these fatty acids is dihydroxypalmitic acid. In addition, Hanbai also contains a high level of undecylenic acid (10-DHA). These flavonols and fatty acid compounds have good biological activities, among which flavonols have antioxidant and anti-inflammatory effects, and fatty acids can play a role in moisturizing and retaining water. This shows that the nanovesicles prepared from ornamental peach petals have potential biological activity functions.

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

[0069]

[0070]

[0071] Example 7

[0072] The nanovesicle suspension extracted from the ornamental peach petals of Yingchun, Feitao, Zaofeihong, Hanbai, Fuchsia, Pinxia, ​​Fenhongshanbi, Fenhuashanbi, and Lengmeiren in Example 6 was diluted 10 times, and then 10 μL of each was taken and mixed with 190 μL of DPPH free radical (87 μM, ethanol solution) and ABTS (7 mM, aqueous solution) free radical, respectively, and reacted in the dark for 30 minutes. The ability of various nanovesicle extracts to scavenge the two free radicals was measured at 529 nm and 723 nm, respectively, and vitamin C was used as a positive control. Each group of experiments was repeated 3 times and the average was taken. The data were analyzed using one-way analysis of variance, and the data were expressed as mean ± standard deviation. The results are shown in Figure 5 shown.

[0073] The results showed that all nanovesicles had the ability to scavenge free radicals, and most of them were significantly stronger than vitamin C, among which fuchsin and pink mountain blue had the strongest ability to scavenge free radicals.

[0074] Example 8

[0075] Take 10 μL of the nanovesicle suspension extracted from the ornamental peach petals of Yingchun, Feitao, Zaofeihong, Hanbai, Fuchsia, Pinxia, ​​Fenhongshanbi, Fenhuashanbi, and Lengmeiren in Example 6, and mix with an equal volume of tyrosinase PBS solution, the enzyme concentration is 200U / mL. Incubate with mixing and shaking at 37°C for 10 minutes, and then add 8 times the volume of the enzyme solution in PBS solution of L-tyrosine, wherein the concentration of L-tyrosine is 20mg / mL. React at 37°C for 10 minutes, and then measure the absorbance of the entire reaction system at 475nm. The inhibitory activity of tyrosinase is calculated according to the following formula:

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

[0077] Where A0 is the absorbance of the reaction after tyrosinase and tyrosine are mixed without any arbutin or nanovesicles, A is the absorbance of the test group with nanovesicles or arbutin added, arbutin is used as a positive control compound, and all absorbance values ​​are subtracted from the background value of the reaction system (absorbance of the reaction system with only nanovesicles or arbutin added but no tyrosinase added). Each group of experiments was repeated 3 times and the average was taken. The data were analyzed using one-way analysis of variance, and the data are expressed as mean ± standard deviation. The results are shown in Figure 6 shown.

[0078] The results showed that all the nano-vesicles extracted from ornamental peach petals had tyrosinase inhibitory activity, among which the nano-vesicles extracted from Hanbai had the strongest inhibitory effect, followed by Fuchsia, and both of them were better than the model compound arbutin. The inhibitory activity of Yingchun and Fenhongshanbi was comparable to arbutin.

[0079] Example 9

[0080] B16-F10 (CRL-6475) melanoma cells (ATCC) were used as a model to test the inhibitory effects of several ornamental peach petal nanovesicles prepared in Example 6 on melanin synthesis.

[0081] The cells were inoculated in DMEM medium containing 10% fetal bovine serum and cultured at 37°C, 5% CO2 and 98% relative humidity. The cells in the logarithmic growth phase were digested with trypsin and cultured at 1×10 6 The concentration of 1000 μg / mL was inoculated in a 6-well culture plate, 2 mL per well. After culturing for 8 hours, the supernatant was discarded, rinsed once with PBS, and then the nanovesicle suspension extracted from the ornamental peach petals of Yingchun, Feitao, Zaofeihong, Hanbai, Fuchsia, Pinxia, ​​Fenhongshanbi, Fenhuashanbi, and Lengmeiren in Example 6 was diluted 10 times and then added to the culture plate, 2 mL per well. Arbutin was used as a positive control, and DEME medium without any extract was used as a negative control. After culturing for 48 hours, the supernatant of the cultured cells was discarded, the cells were collected after digestion, centrifuged at 1500r / min for 10 minutes, the supernatant was aspirated, and 1 mL of 1 mol / L sodium hydroxide solution containing 10% DMSO was added. After incubation in a water bath at 80°C for 1 hour, the absorbance was measured at 405nm using an enzyme marker, and the inhibition rate of the extract on melanin formation was calculated according to the following formula:

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

[0083] Among them, A1 is the absorbance of cell melanin without adding any arbutin or nanovesicles, A is the absorbance of the test group with nanovesicles or arbutin added, arbutin is used as a positive control compound, and all absorbance values ​​are subtracted from the system blank of the reaction system (the absorbance of the reaction system with only nanovesicles or arbutin added). Each group of experiments was repeated 5 times and the average was taken. The data were analyzed using one-way analysis of variance, and the data were expressed as mean ± standard deviation.

[0084] The results are as follows Figure 7 As shown, the nanovesicles prepared from the petals of all ornamental peach varieties can significantly inhibit the synthesis of intracellular melanin, and their inhibitory effects are significantly stronger than those of the model compound arbutin.

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

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

[0087] Among them, A3 is the absorbance of the tyrosinase reaction after the cell extraction without adding any nanovesicles or compounds, A is the absorbance of the test group with the addition of extracts or arbutin, arbutin is used as a positive control compound, and all absorbance values ​​are subtracted from the system blank of the reaction system (absorbance of the reaction system with only nanovesicles or arbutin added). Each group of experiments was repeated 5 times and the average was taken. The data were analyzed using one-way analysis of variance, and the data were expressed as mean ± standard deviation.

[0088] The results are as follows Figure 8 As shown, all the nanovesicles prepared from ornamental peach petals have an inhibitory effect on intracellular tyrosinase activity, among which the inhibitory effects of Yingchun and Lengmeiren are consistent with the positive control compound arbutin, Pinxia and Fenhongshanbi are weaker than arbutin, and the inhibitory effects of nanovesicles prepared from several other ornamental peach petals are stronger than arbutin, among which Hanbai has the best inhibitory activity, exceeding 70%.

[0089] Example 10

[0090] Human dermal fibroblasts (Wuhan Pronocell Life Science) were used to test the anti-aging effects of nanovesicles extracted from the petals of Jasmine, White Jasmine, Pink Jasmine and Pink Jasmine (extracted using the method in Example 5).

[0091] Human dermal fibroblasts were cultured in a fibroblast-specific medium (CM-H103, Wuhan Pronocell Life Science Co., Ltd.) and used in experiments when they were subcultured to the third generation. The cells were digested with trypsin and incubated at 0.5 × 10 6 The cells were inoculated into a 6-well culture plate at a concentration of 100 / mL and then placed in an incubator (CO25%, 37°C, humidity 98%) for 24 hours. The culture medium in the culture dish was discarded and 1.0 mL of PBS was added. The cells in the experimental group, positive control group (Ilomastat) and negative control group (control group) were irradiated with UVA (340 nm) at a radiation dose of 10 J / cm 2, the blank group was not irradiated. Nanovesicles diluted 10 times with culture medium were added to the culture medium of the experimental group, Ilomastat (matrix metalloproteinase inhibitor) (1nM) was added to the culture medium of the positive control group, and only culture medium was added to the negative control group and the blank group. The total volume of the culture medium in each group was 2mL, and then cultured for 24h. After culture, the cells were digested and centrifuged (2000g, 20min), and the supernatant was tested for MMP-1 expression using a human matrix metalloproteinase 1 (MMP-1) kit. The cells obtained by centrifugation were blown into a suspension with PBS, and the concentration count was 1×10 6 / mL, then ultrasonically treated for 30 minutes, then quickly frozen with liquid nitrogen, thawed at 37°C and refrozen again, repeated freezing and thawing three times and centrifuged (1000g, 20min), the supernatant was tested for ColⅠ content using type Ⅰ collagen (ColⅠ) test kit, and human hyaluronic acid (HA) ELISA Kit was used to test HA content. Each group of experiments was repeated 4 times and the average was taken. The data were analyzed using one-way analysis of variance, and the data were expressed as mean ± standard deviation. The results are shown in Fig. 9 and Fig.10 shown.

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

[0093] Embodiment 11

[0094] The anti-inflammatory effect of nanovesicles extracted from the petals of Jasmine, White Jasmine, Pink Jasmine and Pink Jasmine (extracted using the method described in Example 5) was tested using the cell line RAW246.7.

[0095] The cells were cultured in DMEM high glucose + 10% FBS and 1% double antibody culture medium at 37°C, 5% carbon dioxide content and 98% relative humidity. 6Cells in the logarithmic growth phase of 100 μg / mL were inoculated into a 96-well plate, with 100 μL per well. After the cells adhered to the wall, lipopolysaccharide (LPS) was used to induce inflammation. The supernatant was removed and a culture medium containing 1 μg / mL of lipopolysaccharide was added. No lipopolysaccharide was added to the blank group, and a 10-fold diluted nanovesicle suspension was added to the experimental group. The control group only added a culture medium containing LPS. After 24 hours of culture, the supernatant was aspirated and the levels of NO and TNF-α in the cell culture medium were determined using a NO kit and a TNF-α ELISA kit, respectively. The results are shown in Fig.11 and Fig.12 shown.

[0096] The results showed that compared with the control group, the nanovesicles prepared from the four types of ornamental peach petals could significantly reduce the levels of NO and TNF-α, among which Hanbai had the best effect. This indicates that the nanovesicles prepared from ornamental peach petals have anti-inflammatory effects and inhibit the expression of inflammatory factors.

[0097] Example 12

[0098] Preparation method of emulsion containing ornamental peach petal nanovesicles:

[0099] The following components, measured by mass / volume percentage, are added to a hydrocolloid containing 0.5% gelatin to prepare a base emulsion. The nanovesicles prepared from the flower petals of the cold white peach are then diluted 100 times with the base emulsion. The emulsion containing the nanovesicles of the ornamental peach petals is obtained.

[0100] The preparation method of nanovesicles in this example is the same as that in Example 5.

[0101] Embodiment 13

[0102] Preparation method of facial cream containing ornamental peach petal nanovesicles:

[0103] The composition contains the following components in terms of mass / volume percentage: niacinamide 8%, glycerin 3%, palmityl alcohol 1.5%, glycerin 1%, 1,3-propylene glycol 1%, vitamin E 0.5%, and is compounded with water to form a cream. Then, the nanovesicles prepared from magenta petals are compounded with the cream in a volume ratio of 1:100 to form a uniform cream. Thus, a facial cream containing nanovesicles from ornamental peach petals is obtained.

[0104] The preparation method of nanovesicles in this example is the same as that in Example 5.

[0105] Embodiment 14

[0106] Preparation method of facial cream with ornamental peach petal nanovesicles:

[0107] The composition contains the following components in terms of mass / volume percentage: 1,3-propylene glycol 2%, polydimethylsiloxane 2%, hydroxypropyl tetrahydropyrantriol 2%, cyclohexasiloxane 1%, glycerol 1%, and water to form a cream. Then, the nanovesicles prepared from pink mountain green petals are compounded with the cream in a volume ratio of 1:100 to form a uniform cream. That is, a facial cream containing nanovesicles of ornamental peach petals is obtained.

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

[0109] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing ornamental peach petal nanovesicles, characterized in that: Ornamental peach petals at the bud stage or the initial opening stage are collected, and the nanovesicles are extracted by direct centrifugation or sucrose density gradient centrifugation.

2. The preparation method according to claim 1, characterized in that: The ornamental peach petals are treated in darkness before the nanovesicles are extracted.

3. The preparation method according to claim 2, characterized in that: The dark treatment is to place the mixture in the dark at 30-40° C. and 85%-99% humidity for 12-16 hours.

4. The preparation method according to claim 1, characterized in that: The direct centrifugation method comprises: freezing and then crushing the ornamental peach petals, mixing the petal powder with Tris-HCl buffer and then homogenizing, centrifuging the homogenate liquid phase 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, and resuspending it with PBS to obtain a nanovesicle suspension.

5. The preparation method according to claim 1, characterized in that: The sucrose density gradient centrifugation method comprises: homogenizing the ornamental peach petals, centrifuging the homogenate at 1000g, collecting the supernatant and centrifuging at 5000g, collecting the supernatant and subjecting it to sucrose density gradient centrifugation, wherein the sucrose gradient is 8%, 30%, 45% and 60%, and after centrifugation, a nanovesicle suspension is obtained by aspirating at the junction of 30% and 45% sucrose.

6. The ornamental peach petal nanovesicle obtained by the preparation method according to any one of claims 1 to 5, characterized in that: The ornamental peach petal nanovesicles contain kaempferol derivatives and fatty acids.

7. Use of the preparation method according to any one of claims 1 to 5 or the ornamental peach petal nanovesicles according to claim 6 in the preparation of skin care products.

8. The use according to claim 7, characterized in that: The skin care products include whitening products, freckle removal products, anti-inflammatory products, anti-wrinkle products and anti-aging products.

9. A skin care product, characterized in that: The skin care product comprises the ornamental peach petal nanovesicles according to claim 6.

10. The skin care product according to claim 9, characterized in that: The skin care product is any one of lotion, cream and essence.

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