Apple nano-vesicle as well as preparation method and application thereof

Abstract

The invention provides an apple nano-vesicle as well as a preparation method and application thereof, and belongs to the technical field of biology. According to the preparation method of the apple nano-vesicles, young apple fruits 14-60 days after flowering are collected as raw materials, and the nano-vesicles are extracted by adopting a direct centrifugation method or a sucrose density gradient centrifugation method. The nano-vesicles are extracted by taking young apple fruits as raw materials, so that wastes generated by fruit thinning in a field management process of an orchard are effectively utilized, and resource waste caused by a large amount of waste fruits generated by fruit thinning is avoided. The prepared apple nano-vesicles are uniform in particle size, small in size and good in stability, have good whitening, skin-care, anti-wrinkle and anti-aging effects, and can realize high-added-value deep processing of young apple fruits generated by fruit thinning.

Description

Technical Field

[0001] The present invention belongs to the technical field of biotechnology, and in particular relates to an apple nanocapsule and a preparation method and application thereof. Background Art

[0002] Apple is one of the most productive fruits in the world. In the management of apple orchards, fruit thinning is one of the essential field management to ensure the yield and quality of apples. However, the fruit thinning process will produce a large number of discarded young apples, which are often discarded without any valuable transformation. In order to improve the economic benefits of orchards, it is imperative to carry out high value-added deep processing of young apples produced by fruit thinning.

[0003] At present, the utilization of byproducts from apple orchard management mainly focuses on the extraction of pectin, polyphenols and other substances, but the market for such products is saturated and the application scenarios are limited. As a research hotspot in recent years, "nanovesicles" can be used as an important breakthrough in the deep processing of young apple fruits. Nanovesicles refer to a type of vesicular structure wrapped in a double lipid layer at the nanoscale formed during the life activities of cells. They are of great significance to the entire cell life activities. On the one hand, the vesicles secreted by cells can be used as "exosomes" for intercellular material transfer and signal communication. At the same time, the vesicles in the cells can also be responsible for the material transport between various organelles or directly develop into mature organelles. During the fruit development process of apple plants, there will also be many vesicle-like structures in the pulp cells. These vesicles often eventually converge to form a large central vacuole, and this process is also accompanied by the ripening of the fruit. As the main organelle for the storage of secondary metabolites in apple plants, the vacuole and its precursor vesicle-like structure are also the key medium for the synthesis and transport of these compounds. At the same time, thanks to the double-layer lipid membrane structure of the nanovesicles, the substances in the vesicles are easier to transfer in systems such as cells or organelles with double-layer lipid membranes as the main interface.

[0004] Therefore, nanovesicles mainly derived from Malus plants are expected to become a new way to utilize the secondary metabolites of Malus plants. They also point out a new comprehensive utilization channel for the waste generated by fruit thinning during orchard management. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a method for preparing apple nanovesicles, wherein the prepared apple nanovesicles have antioxidant, whitening, anti-aging and improvement effects. The present invention can improve the utilization rate of waste generated during apple thinning and increase the economic value per unit area of ​​the orchard.

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

[0007] The invention provides a method for preparing apple nanovesicles. Young apple fruits 14 to 60 days after flowering are collected as raw materials, and the nanovesicles are extracted by direct centrifugation or sucrose density gradient centrifugation.

[0008] Preferably, the nanovesicles are extracted from the young apple fruits after being treated in darkness, and the conditions for the dark treatment are: at 30-40° C., 75%-95% humidity, and placed in darkness for 10-24 hours.

[0009] Preferably, the nanovesicles are destarched using an α-amylase solution.

[0010] Preferably, the direct centrifugation method comprises: freezing young apple fruits and then crushing them, mixing the fruit 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 buffer to obtain a nanovesicle suspension.

[0011] Preferably, the density gradient centrifugation method includes: homogenizing young apple fruits, 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 apple nanovesicles obtained by the preparation method, wherein the apple nanovesicles contain dihydrochalcone compounds.

[0013] Another object of the present invention is to provide the preparation method or the use of the apple nanovesicles in the preparation of skin care products.

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

[0015] Another object of the present invention is to provide a skin care product, comprising the above-mentioned apple 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 apple nanovesicles, wherein the nanovesicles are obtained by extracting young apple fruits 14 to 60 days after flowering as raw materials, effectively utilizing the young apple fruits produced during the thinning of fruits in the orchard, avoiding waste of resources during the field management of apples, and improving the unit economic value of the orchard. The apple nanovesicles provided by the present invention have uniform particle size, small size, good stability, and good biocompatibility. After dark pretreatment and amylase pretreatment during processing, the carbohydrates in the young apple fruits that interfere with the extraction of nanovesicles are greatly reduced, the extraction rate of nanovesicles is improved, and the young apple fruits can be compatible with ultrafiltration centrifugation and density gradient centrifugation at the same time, so as to achieve simple and rapid preparation. And the apple nanovesicles finally prepared are rich in apple characteristic polyphenolic compounds, especially whitening active ingredients such as phloretin. The prepared apple young fruit nanovesicles have excellent whitening and anti-wrinkle effects. They can not only inhibit the activity of tyrosinase, but also reduce the synthesis of melanin in cells, and the effect is better than that of the model compound arbutin; at the same time, the apple young fruit nanovesicles can also reduce the expression of MMP-1 in human cells, and promote the synthesis of type I collagen (ColⅠ) and hyaluronic acid (HA), which has the effect of anti-aging and wrinkle removal, and improving skin quality. The present invention realizes the high added value comprehensive utilization of apple field management waste, and the final product has good application prospects in whitening and freckle removal, skin care and wrinkle removal, and improving skin quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : Distribution of vesicle size extracted from young apple fruits at different developmental stages, DDA indicates days after anthesis;

[0020] Figure 2 : Flow chart of the preparation of destarch nanovesicle suspension in Example 5;

[0021] Figure 3 :The free radical scavenging ability of nanovesicles prepared from different young apple fruits, where different capital letters represent significant differences in ABTS free radical scavenging rate, and different lowercase letters represent significant differences in DPPH free radical scavenging rate;

[0022] Figure 4 : The inhibitory activity of nanovesicles prepared from different young apple fruits on tyrosinase, where different letters represent significant differences between the two groups;

[0023] Figure 5 : The inhibitory effect of nanovesicles prepared from young fruits of various apple plants on cellular melanin synthesis, where ** indicates P < 0.01;

[0024] Figure 6 :The inhibitory activity of nanovesicles prepared from young fruits of various apple plants on intracellular tyrosinase, where different letters represent significant differences between the two groups;

[0025] Figure 7 :The effect of nanovesicles prepared from different young apple fruits on the expression of MMP-1 in human fibroblasts, where different letters represent significant differences between the two groups;

[0026] Figure 8 :The effects of nanovesicles prepared from different young apple fruits on the expression levels of Col 1 and HA in human fibroblasts, where different capital letters represent significant differences in Col 1, and different lowercase letters represent significant differences in HA. DETAILED DESCRIPTION

[0027] The present invention provides a method for preparing apple nanovesicles, wherein young apple fruits 14 to 60 days after flowering are collected as raw materials, and the nanovesicles are extracted by direct centrifugation or sucrose density gradient centrifugation. The present invention has found that as the time after flowering increases, the particle size of the nanovesicles extracted from the young apple fruits increases, and the absolute value of the Zeta potential decreases. If the collection time after flowering is too early, the volume of the young apple fruits is too small, and the extracted nanovesicles are few. The present invention selects young apple fruits 14 to 60 days after flowering, and the extracted nanovesicles have uniform particle size, small size, good stability, and high extraction rate of nanovesicles.

[0028] In the present invention, the apple young fruit is treated in the dark before extracting the nanovesicles. The conditions for the dark treatment of the apple young fruit are: the temperature is 30-40°C, the humidity is 75%-95%, and the apple young fruit is placed in the dark for 10-24 hours. The temperature of the dark treatment of the present invention is preferably 32-38°C, more preferably 35°C, the humidity is preferably 78%-85%, more preferably 80%, and the apple young fruit is preferably placed in the dark for 15-22 hours, more preferably 20 hours. A large amount of starch will accumulate in the apple young fruit, and the high molecular weight starch will interfere with the preparation of nanovesicles. After the apple young fruit is treated in the dark, the starch in the fruit is consumed by the respiration of the fruit cells themselves, reducing the macromolecular substances, avoiding the formation of colloids between starch and macromolecular substances during the extraction process, and the extracted nanovesicles have smaller particle size, and can improve the purity of nanovesicles in the nanovesicle suspension.

[0029] In the present invention, the nanovesicles are destarched using an α-amylase solution, wherein the concentration of the α-amylase (EC 3.2.1.1) solution is 0.1 to 5 U / mL, preferably 0.5 to 2 U / mL, and more preferably 1 U / mL. The nanovesicles obtained in the present invention are nanovesicle suspensions, and the added mass of the amylase solution is 0.5% to 1.5% of the mass of the nanovesicle suspension to be treated, preferably 0.8% to 1.2%, and more preferably 1%. As an practicable manner, the present invention adds an α-amylase solution to a nanovesicle suspension at 15° C., stirs at a speed of 8 to 12 rmp for 15 to 25 minutes to obtain a mixed solution, and then ultrafiltration centrifuges the mixed solution. The ultrafiltration centrifugation comprises: placing the mixed solution in a 100 kDa ultrafiltration tube for centrifugation at 4000 to 6000 g for 25 to 35 minutes; after the centrifugation, 5 mL of Tris-HCl buffer is added to the ultrafiltration tube, and centrifuged again at 4000 to 6000 g for 25 to 35 minutes to finally obtain a destarched nanovesicle suspension.

[0030] In the present invention, the direct centrifugation method comprises: crushing young apple fruits after freezing, mixing fruit 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.

[0031] In the direct centrifugation method of the present invention, after the core of the young apple fruit is removed, it is preferably quick-frozen using liquid nitrogen, and the young apple fruit is ground into powder in a frozen state; the fruit powder is mixed with Tris-HCl buffer (100mM, pH 7.4) at a ratio of 5:4 (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 100000-12000g, preferably at 100000g, the centrifugation time is 30-180min, preferably for 120min, and the centrifugation temperature is 4°C; the precipitate is centrifuged using PBS (100mM, pH 6.8) preparing a nanovesicle suspension, which was quickly frozen with liquid nitrogen and stored at -80°C.

[0032] 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 pigments and other small molecules, so that the purity of the obtained apple nanovesicles is better.

[0033] In the present invention, the sucrose density gradient centrifugation method comprises: homogenizing young apple fruits, 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.

[0034] In the sucrose density gradient centrifugation method of the present invention, the core of the young apple fruit is removed and the fruit is mixed with PBS (100mM, pH7.4) at a ratio of 1:0.5 (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, and 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 centrifugation is set to 100000-12000g, preferably 120000g, and the centrifugation time is 30-180min, preferably for 120min; the nanovesicle suspension is quick-frozen using liquid nitrogen and stored at -80°C.

[0035] The present invention also provides apple nanovesicles prepared by the above method, wherein the apple nanovesicles contain dihydrochalcone compounds.

[0036] Polyphenols are the main phytochemical components in apples, mainly including five polyphenol groups, flavanols, flavonols, phenolic acids, dihydrochalcone and flavonoids, among which dihydrochalcone compounds are the highest content and richest type of secondary metabolites in apple plants. Apple polyphenols can reduce cell damage caused by aging as a whole and play a role in preventing various diseases. In the apple nanocapsule provided by the present invention, a variety of polyphenol compounds were identified, including five major categories: flavanols, flavonols, anthocyanins, phenolic acids and dihydrochalcone. The apple nanocapsule of the present invention retains the original polyphenol components in apples. These polyphenols generally have the effects of anti-oxidation and improving inflammation, among which dihydrochalcone compounds also have clear whitening and freckle removal effects.

[0037] The apple fruitlets described in the present invention are fruitlets of plants of the genus Malus, including cultivated species, native species and ornamental species, preferably any one of M. domestica 'Fuji', M. domestica 'Golden Delicious', M. 'Royalty', M. 'Donghongguo', M. 'Sparkler', M. 'Otterson' and M. micromalus. All apples were collected from the germplasm resource garden of the College of Horticulture of Northwest Agriculture and Forestry University.

[0038] The present invention also provides a preparation method of the apple nanovesicles or an application of the apple nanovesicles in the preparation of skin care products, wherein the skin care products include whitening products, freckle removal products, anti-wrinkle products and anti-aging products.

[0039] The present invention also provides a skin care product, which contains the above-mentioned apple nanocapsule, and has the effects of whitening, anti-oxidation, anti-aging, wrinkle removal and improving skin quality. The skin care product of the present invention is any one of lotion, cream and essence.

[0040] 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.

[0041] 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.

[0042] Example 1

[0043] A method for preparing apple nanovesicles:

[0044] Young Fuji apple fruits 30 days after flowering were collected, the cores were removed, and they were quick-frozen using liquid nitrogen. Then, they were ground into powder in the frozen state. The frozen fruit powder was mixed with Tris-HCl buffer (100 mM, pH 7.4) in a ratio of 5:4 (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, 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 min. After the last centrifugation, the supernatant was collected and centrifuged at 100000g for 120 min. The supernatant was aspirated, and the precipitate was gently blown using PBS (100 mM, pH 6.8) to prepare a nanovesicle suspension.

[0045] 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.

[0046] 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.

[0047] Example 2

[0048] A method for preparing apple nanovesicles:

[0049] Fuji apple young fruits 30 days after flowering were collected, the cores were removed, and mixed with PBS (100mM, pH=7.4) at a ratio of 1:0.5 (w:v), and homogenized at high speed for 10min at 4°C 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, and then the prepared nanocapsules were drawn from the junction of 30% and 45%, and the nanocapsules suspension was quickly frozen in liquid nitrogen, stored at -80°C, and used for standby. The centrifugation in this embodiment was all carried out at 4°C.

[0050] Example 3

[0051] Fuji apple young fruits were collected at 14, 30, 60, 80 and 180 days after flowering, and the cores were removed. Nanovesicles were prepared using the method of Example 1, and the particle size and Zeta potential of the nanovesicles were measured using a nanoparticle size analyzer and a Zeta potential analyzer. The apples at each stage were independently repeated for three groups, and the results were as follows. Figure 1 shown.

[0052] The results showed that the average particle size of nanovesicles extracted from apples at 14, 30, 60, 80 and 180 days after flowering was 76.06±3.13nm, 76.23±3.35nm, 82.71±4.01nm, 101.90±4.01±4.29nm and 131.95±5.62nm, respectively. The Zeta potential was -39.6mV, -38.2mV, -35.7mV, -29.6mV, -26.6mV( Figure 1 ). It shows that the nanovesicles extracted from apple fruitlets 14 to 60 days after flowering have uniform particle size, small size and good stability. Among them, the nanovesicles obtained by high-speed centrifugation from apple fruitlets 30 days after flowering are more numerous and of better quality, and the volume of 30-day-old fruitlets is relatively large, which is more economical to use for extracting nanovesicles.

[0053] Example 4

[0054] The starch content of Fuji apples at 14, 30, 60, 80 and 180 days after flowering was determined by enzyme hydrolysis. The results showed that the starch content of young apples at 14, 30, 60, 80 and 180 days after flowering was 8.5±1.2mg / g, 12.4±1.5mg / g, 47.8±5.2mg / g, 58.4±3.6mg / g and 11.2±2.3mg / g, respectively. This indicates that the starch content of apples increased and then decreased during their development.

[0055] The young apple fruits were picked 30 days after flowering and placed in dark conditions with a humidity of 80% and a temperature of 35°C for 8h, 10h, 12h, 16h, 20h and 24h, respectively. The starch content of the young apple fruits after dark treatment for different periods of time was 11.2±1.6mg, 9.8±1.5mg, 8.2±1.2mg, 7.8±1.3mg, 6.9±1.0mg and 6.8±1.1mg, respectively, indicating that the starch content in young apple fruits can be significantly reduced by dark treatment.

[0056] Apples 30 days after flowering were treated with darkness (80% humidity, 35°C, 20h) or without treatment, and then used to prepare nanovesicles. The preparation method is as follows: the young fruits were cored and 10 g was taken respectively. The frozen fruit powder was mixed with Tris-HCl buffer (100 mM, pH 7.4) in a ratio of 5:4 (w:v), and 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. The liquid phase was centrifuged at 650g, 3000g, 10000g and 15000g for 20 min respectively at 4°C. The precipitate was removed after each centrifugation and the supernatant was retained. The supernatant obtained from the last centrifugation was centrifuged at 5000g for 30 min using a 100 kDa ultrafiltration tube, and then 5 mL of Tris-HCl buffer was added and centrifuged again at 5000g for 30 min to obtain a nanovesicle suspension. The starch content was determined using the iodine-potassium iodide method (GB / T15683-2008).

[0057] The results showed that after dark treatment, 0.7 mL of nanovesicle suspension was obtained, the starch content was 0.18 mg / mL, and the average particle size of the nanovesicles was 75.67±2.21 nm. Without dark treatment, 1.0 mL of nanovesicle suspension was obtained, the starch content was 0.61 mg / mL, and the average particle size of the nanovesicles was 75.63±3.57 nm. This shows that after dark treatment, the starch content in the extracted material can be greatly reduced, and the particle size distribution of the extracted nanovesicles is narrower. Without dark treatment, the retained liquid during ultrafiltration contains colloids formed by starch and macromolecular substances, so the retained liquid collected without dark treatment has a large volume, but the abundance of nanovesicles is low.

[0058] Example 5

[0059] Fuji apple young fruits 30 days after flowering were collected and placed in a dark condition with a humidity of 80% and a temperature of 35°C for 20 hours. The ultrafiltered nanovesicle suspension was prepared by the method of Example 1. 1U / mL of α-amylase solution (EC 3.2.1.1) was added at 15°C. The added mass of the α-amylase solution was 1% of the mass of the ultrafiltered nanovesicle suspension. The mixture was stirred at a low speed for 20 minutes (10 rpm) to obtain a mixed solution. The mixed solution was then placed in a 100 kDa ultrafiltration tube and centrifuged at 5000g for 30 minutes at 4°C. After the centrifugation, 5 mL of Tris-HCl buffer (100 mM, pH 7.4) was added to the ultrafiltration tube, and the mixture was centrifuged again at 5000g for 30 minutes to obtain a destarched nanovesicle suspension (the preparation flow chart is shown in FIG. 1 ). Figure 2 The diameter and Zeta potential of the nanovesicles were measured by a particle size analyzer, which were 78.23±4.03nm and -39.1mV, respectively.

[0060] Example 6

[0061] The method in Example 5 was adopted to prepare the destarch nanovesicle suspension using young fruits of Fuji apple, Huang Yuanshuai apple, Wangzu apple, Donghong fruit, Diamond crabapple, Otterson red flesh apple and Xifu crabapple as raw materials.

[0062] The same volume of methanol (containing 1% formic acid by mass) was added to the obtained de-starch nanovesicle suspension, and then ultrasonic vibration was used for 30 minutes to obtain a mixed solution, which was centrifuged at 4°C for 15 minutes (12000g), and the supernatant was filtered through a 0.22μm filter membrane, and then high-resolution liquid mass spectrometry was used for determination. The liquid mass spectrometry conditions were as follows: mobile phase A was acetonitrile with 0.1% formic acid added, and mobile phase B was pure water with 0.1% formic acid added. The column temperature was 35°C, and the flow rate was 0.3mL / min. The chromatographic column was Inertsil ODS-3 (4.6mm×250mm, 5.0μm). The gradient elution program was 95% A (0min), 85% A (25min), 78% A (42min), 64% A (60min), 95% A (65min), and equilibrated for 5min. The mass spectrometry detection conditions are as follows: Applied Biosystems API 2000 triple quadrupole mass spectrometer, equipped with ESI ion source, using positive ion mode detection, scanning range 180-800 m / z, nebulizer gas 50 psi, curtain gas 20 psi, heating gas 50 psi, heating temperature 450°C, spray voltage 3.8 kV, all gases are high-purity nitrogen, and the secondary spectrum fragmentation energy is 30 V.

[0063] A variety of polyphenolic compounds were identified from apple nanovesicles, including five categories: flavanols, flavonols, anthocyanidins, phenolic acids and dihydrochalcone, with a total of 20 species, as shown in Table 1.

[0064] Table 1 Types of polyphenolic compounds in nanovesicles

[0065]

[0066] As shown in Table 1, a total of 20 polyphenols were absolutely identified in the nanovesicles extracted from the young fruits of Fuji, Huang Yuanshuai, Wangzu, Donghongguo, Diamond, Red Flesh Apple, and Xifu Begonia 30 days after flowering. Among them, flavanols, flavonols and phenolic acid compounds were not much different between cultivated varieties (Fuji, Huang Yuanshuai), ornamental begonias (Wangzu, Diamond), red flesh apples and native species (Otterson; Xifu Begonia); anthocyanidins were only detected in two ornamental begonias (Wangzu, Diamond) and red flesh apples, and phlorizin and its main glycoside derivative phlorizin in the dihydrochalcone compounds were detected in all apple compounds; 3-hydroxyphlorizin was only detected in Donghongguo, Diamond, Otterson and Xifu Begonia, and its glycoside derivative (3-hydroxyphlorizin) was mainly detected in two ornamental begonias, Donghongguo and Diamond.

[0067] Example 7

[0068] Antioxidant effect of nanovesicles from young apple fruits.

[0069] The method in Example 5 was adopted to prepare the destarch nanovesicle suspension using young fruits of Fuji apple, Huang Yuanshuai apple, Wangzu apple, Donghong fruit, Diamond crabapple, Otterson red flesh apple and Xifu crabapple as raw materials.

[0070] The nanovesicle suspensions prepared from Fuji apple, Huang Yuanshuai apple, Wangzu apple, Donghong fruit, Diamond crabapple, Red meat apple Otterson and Xifu crabapple were diluted 10 times, and then 10 μL of each was mixed with 190 μL of DPPH free radical (87 μM, ethanol solution) and 190 μL of ABTS free radical (7 mM, aqueous solution), 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 the figure. Figure 3 shown.

[0071] Figure 3 The results showed that the nanovesicles prepared by Otterson from Royal Apple and Red Flesh Apple had the strongest free radical scavenging ability, followed by Malus sinensis. The other two ornamental crabapples (Winter Red Fruit and Diamond Crabapple) also had good scavenging ability. The scavenging ability of the two cultivated apples (Fuji and Huang Yuanshuai) was weaker, but all the nanovesicles were stronger than vitamin C.

[0072] Example 8

[0073] The inhibitory effect of nanovesicles in young apple fruit on tyrosinase.

[0074] The method in Example 5 was adopted to prepare the destarch nanovesicle suspension using young fruits of Fuji apple, Huang Yuanshuai apple, Wangzu apple, Donghong fruit, Diamond crabapple, Otterson red flesh apple and Xifu crabapple as raw materials.

[0075] Take 10 μL of the nanovesicle suspension prepared from Fuji apple, Huang Yuanshuai apple, Wangzu apple, Donghong fruit, Diamond crabapple, Red meat apple Otterson and Xifu crabapple, respectively, and mix with an equal volume of tyrosinase PBS solution, the enzyme concentration is 200U / mL. Mix and shake and incubate at 37°C for 10 minutes, then add 8 times the volume of the enzyme solution PBS solution, the PBS solution contains L-tyrosine, wherein the L-tyrosine concentration 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 as follows: Inhibition rate = 100% × (A0-A) / A0.

[0076] 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 corresponding reaction system (absorbance of the reaction system with only nanovesicles or arbutin added but no tyrosinase added). Each group of experiments was repeated 6 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 4 shown.

[0077] Figure 4 The results showed that all the nanovesicles in young apple fruits had tyrosinase inhibitory activity, and the inhibitory effect of the nanovesicles extracted from winter red fruit was better than that of the positive compound arbutin, while the inhibitory effects of the nanovesicles extracted from young fruits of three species of crabapple, namely royal crabapple, diamond crabapple and western crabapple, were close to that of arbutin, and the inhibitory effects of the nanovesicles extracted from the other three cultivated apple varieties were weaker than that of arbutin.

[0078] Example 9

[0079] Whitening effect of nanovesicles in young apple fruit.

[0080] The method in Example 5 was adopted to prepare the destarch nanovesicle suspension using young fruits of Fuji apple, Huang Yuanshuai apple, Wangzu apple, Donghong fruit, Diamond crabapple, Otterson red flesh apple and Xifu crabapple as raw materials.

[0081] B16-F10 (CRL-6475) melanoma cells (ATCC CRL-6475) were used as a model to test the inhibitory effect of apple fruit nanovesicles on melanin synthesis. The cells were inoculated in DMEM medium containing 10% fetal bovine serum at 37°C, 5% CO2 content, and 98% relative humidity. Cells in the logarithmic growth phase were digested with trypsin and 1×10 6 / mL concentration was inoculated in a 6-well culture plate, 2mL per well. After culturing for 8 hours, the supernatant was discarded and rinsed once with PBS. The nanovesicle suspension prepared from Fuji, Huang Yuanshuai, Wangzu, Donghongguo, Diamond Begonia, Red Meat Apple Otterson and Xifu Begonia was diluted 10 times and then added to the above culture plate, 2mL per well. Arbutin was used as a positive control, and the final concentration of arbutin was 100μg / mL. 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 1mL of 1mol / L sodium hydroxide solution containing 10% DMSO was added. After incubation in a water bath at 80℃ for 1 hour, the absorbance was measured at 405nm using an enzyme reader. The inhibition rate of melanin formation was calculated according to the following formula:

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

[0083] Where A1 is the absorbance of cellular melanin without any arbutin or nanovesicles, A is the absorbance of the test group with nanovesicles or arbutin, arbutin is used as a positive control compound, and all absorbance values ​​are subtracted from the system blank of the corresponding reaction system (absorbance of the 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. The results are shown in Figure 5 shown.

[0084] 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 thereto. 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 intracellular tyrosinase was calculated according to the following formula:

[0085] Intracellular tyrosinase inhibition rate = 100% × (A3-A) / A3;

[0086] A3 is the absorbance of tyrosinase reaction after cell extraction without any nanovesicles or arbutin, A is the absorbance of the test group with nanovesicles or arbutin, arbutin is used as a positive control compound, and all absorbance values ​​are subtracted from the system blank of the corresponding reaction system (absorbance of the 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 ANOVA, and the data were expressed as mean ± standard deviation. The results are shown in Figure 6 shown.

[0087] Figure 5 The results showed that all nanovesicles extracted from apple plants had significant melanin synthesis inhibitory activity, which was much stronger than arbutin.

[0088] Figure 6 The results showed that all nanovesicles extracted from young fruits of apple plants had inhibitory activity against intracellular tyrosinase, and the inhibitory activities of selenium-rich, yellow marshmallow, winter red fruit, and crabapple were stronger than those of the model compound arbutin. The inhibitory activities of diamond crabapple and red flesh apple Otterson were close to arbutin, and only royal crabapple was weaker than the model compound.

[0089] It can be seen that the apple nanovesicles prepared by the present invention have good whitening potential.

[0090] Example 10

[0091] Anti-aging effects of nanovesicles from young apple fruits.

[0092] The method in Example 5 was adopted to prepare the destarch nanovesicle suspension using young fruits of Fuji apple, Huang Yuanshuai apple, Wangzu apple, Donghong fruit, Diamond crabapple, Otterson red flesh apple and Xifu crabapple as raw materials.

[0093] Human dermal fibroblasts (Wuhan Pronocell Life Science) were used to test the anti-aging effect of apple fruit nanovesicles. Fibroblast-specific culture medium (CM-H103, Wuhan Pronocell Life Science) was used for culture. When the cells were subcultured to the third generation, they were used for experiments. The cells were digested with trypsin and the concentration was 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, and negative control group were irradiated with UVA (340 nm) at a radiation dose of 10 J / cm 2 , the blank group was not irradiated. The nanovesicle suspensions prepared from Fuji, Huang Yuanshuai, Wangzu, Donghongguo, Diamond Begonia, Red Meat Apple Otterson and Xifu Begonia were diluted 10 times with culture medium, and the 10-fold diluted nanovesicle suspensions were added to the culture medium of the experimental group, Ilomastat (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, and then cultured for 24 hours. After culture, the cells were digested and centrifuged (2000g, 20min), and the supernatant was tested for the expression of MMP-1 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 Figure 7 and Figure 8 shown.

[0094] Figure 7 and Figure 8The results showed that compared with the irradiated group, all nanovesicle extracts can significantly reduce the expression of MMP-1, among which Fuji and Huang Yuanshuai can reduce the expression of MMP-1 to normal levels (the same as the blank group), while the nanovesicles extracted from several other crabapples and red meat apples have a stronger inhibitory effect on MMP-1. At the same time, in all nanovesicle treatment groups, the expression of Col 1 and HA is significantly higher than that of the control group, even higher than that of the blank group and the positive control compound (Ilomastat), especially the nanovesicles of two cultivated apples (Fuji and Huang Yuanshuai) and a native crabapple (Xifu crabapple), which have the best effect on promoting the expression of Col1 and HA. These results show that the apple young fruit nanovesicles prepared by the present invention can effectively resist skin aging due to ultraviolet irradiation and have a certain anti-wrinkle effect.

[0095] Embodiment 11

[0096] An emulsion containing nanovesicles from young fruits of the genus Malus.

[0097] The following components, calculated by mass / volume percentage, are added to a hydrocolloid containing 0.5% gelatin to prepare a base emulsion. The nanovesicles prepared from young winter red fruit are then diluted 100 times with the base emulsion. The emulsion containing apple nanovesicles is obtained.

[0098] The preparation method of nanovesicles in this example is the same as that in Example 5.

[0099] Example 12

[0100] A facial cream containing nanovesicles from young fruits of apple plants.

[0101] 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 young Fuji fruit and the cream are compounded in a volume ratio of 1:100 to form a uniform cream. That is, a facial cream containing apple nanovesicles is obtained.

[0102] The preparation method of nanovesicles in this example is the same as that in Example 5.

[0103] Embodiment 13

[0104] A facial cream containing nanovesicles from young fruits of apple plants.

[0105] The composition contains the following components in terms of mass / volume percentage: 2% 1,3-propylene glycol, 2% polydimethylsiloxane, 2% hydroxypropyl tetrahydropyrantriol, 1% cyclohexasiloxane, and 1% glycerol, which are compounded with water to form a cream. Then, the nanovesicles prepared from young Fuji fruits are compounded with the cream in a volume ratio of 1:100 to form a uniform cream. That is, a facial cream containing apple nanovesicles is obtained.

[0106] The preparation method of nanovesicles in this example is the same as that in Example 5.

[0107] 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 apple nanovesicles, characterized in that: Young apple fruits 14 to 60 days after flowering are collected as raw materials, 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 nanovesicles are extracted from the young apple fruits after they are treated in darkness, and the conditions for the dark treatment are: placed in darkness at 30-40° C. and 75%-95% humidity for 10-24 hours.

3. The preparation method according to claim 1 or 2, characterized in that: The nanovesicles are destarched using an α-amylase solution.

4. The preparation method according to claim 1, characterized in that: The direct centrifugation method comprises: freezing young apple fruits and then crushing them, mixing the fruit 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 a buffer to obtain a nanovesicle suspension.

5. The preparation method according to claim 1, characterized in that: The sucrose density gradient centrifugation method comprises: homogenizing young apple fruits, 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 apple nanovesicles obtained by the preparation method according to any one of claims 1 to 5, characterized in that: The apple nanocapsule contains dihydrochalcone compounds.

7. Use of the preparation method according to any one of claims 1 to 5 or the apple 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-wrinkle products and anti-aging products.

9. A skin care product, characterized in that: The skin care product comprises the apple nanovesicles described in 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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