An apple nanovesicle, its preparation method and application

Abstract

The present invention provides an apple nanovesicle, a preparation method thereof and an application thereof, belonging to the field of biotechnology. In the preparation method of the apple nanovesicle of the present invention, apple young fruits 14 to 60 days after flowering are collected as raw materials, and the nanovesicles are obtained by direct centrifugation or sucrose density gradient centrifugation. The present invention extracts nanovesicles from apple young fruits, effectively utilizes the waste generated by fruit thinning in the orchard field management process, and avoids a large amount of waste fruits caused by fruit thinning, resulting in waste of resources. The prepared apple nanovesicles have uniform particle size, small size, good stability, and have good whitening and skin care, anti-wrinkle and anti-aging effects, and can realize high-value deep processing of apple young fruits generated by fruit thinning.

Description

Technical Field

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

[0002] Apples are fruits with a leading yield worldwide. In the process of managing apple orchards, in order to ensure the yield and quality of apples, fruit thinning is one of the essential field management measures. However, a large number of discarded young apple fruits are generated during the fruit thinning process, and these resources are often discarded without valuable conversion. To improve the economic benefits of orchards, it is imperative to carry out high-value deep processing of the young apple fruits generated by fruit thinning.

[0003] Currently, the utilization of by-products in apple orchard management mainly focuses on the extraction of substances such as pectin and polyphenols. However, the market for such products is saturated and their application scenarios are limited. "Nanovesicles", as a research hotspot in recent years, can be an important breakthrough for the deep processing of young apple fruits. Nanovesicles refer to a class of vesicular structures wrapped by a bilayer lipid layer at the nanoscale formed during the life activities of cells, which are of great significance for the entire cell life activities. On the one hand, the vesicles secreted by cells can act as "exosomes" for intercellular material transfer and signal communication, and at the same time, the vesicles in the cell 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 are also numerous vesicular structures in the pulp cells, and these vesicles often eventually converge to form a central large vacuole, which is also accompanied by the ripening of the fruit. The vacuole, as the main organelle for storing secondary metabolites in apple plants, the precursor vesicular structures are also the key mediators for the synthesis and transport of these compounds. At the same time, due to the bilayer lipid membrane structure of nanovesicles, the substances inside the vesicles are more likely to be transferred in systems with bilayer lipid membranes such as cells or organelles as the main interfaces.

[0004] Therefore, nanovesicles mainly derived from apple plants are expected to become a new way for the utilization of secondary metabolites of apple plants, and at the same time, it also points out a new comprehensive utilization channel for the waste generated by fruit thinning during the orchard management process. Summary of the Invention

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

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

[0007] The present invention provides a method for preparing apple nanovesicles. Apple young fruits collected 14 - 60 days after flowering are used as raw materials, and the nanovesicles are obtained by direct centrifugation or sucrose density gradient centrifugation.

[0008] Preferably, the apple young fruits are subjected to dark treatment before extracting the nanovesicles. The conditions for the dark treatment are: placing at 30 - 40 °C, with a humidity of 75% - 95%, and under dark conditions for 10 - 24 h.

[0009] Preferably, the nanovesicles are de-starchified using α-amylase solution.

[0010] Preferably, the direct centrifugation method includes: freezing and pulverizing the apple young fruits, mixing the fruit powder with Tris-HCl buffer solution and homogenizing, centrifuging the liquid part of the homogenate successively at 650 g, 3000 g, 10000 g, and 15000 g, taking the supernatant after each centrifugation for the next centrifugation, taking the supernatant after the last centrifugation, centrifuging at 100000 g, collecting the precipitate, and resuspending with buffer solution to obtain a nanovesicle suspension.

[0011] Preferably, the density gradient centrifugation method includes: homogenizing the apple young fruits, centrifuging the homogenate at 1000 g, collecting the supernatant and centrifuging at 5000 g, collecting the supernatant and performing sucrose density gradient centrifugation, where the sucrose gradients are 8%, 30%, 45%, and 60%, and sucking at the junction of 30% and 45% sucrose after centrifugation to obtain a nanovesicle suspension.

[0012] Another object of the present invention is to provide the apple nanovesicles obtained by the above preparation method, and the apple nanovesicles contain dihydrochalcone compounds.

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

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

[0015] Another object of the present invention is to provide a skin care product, and the skin care product contains the above 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. The nanovesicles are extracted from young apple fruits 14 - 60 days after flowering, effectively utilizing the young apple fruits generated during the fruit thinning process in orchards, avoiding waste of resources during apple field management, and increasing the unit economic value of orchards. The apple nanovesicles provided by the present invention have uniform particle size, small size, good stability, and good biocompatibility. Through dark pretreatment and amylase pretreatment during processing, the carbohydrates in young apple fruits that interfere with the extraction of nanovesicles are greatly reduced, the extraction rate of nanovesicles is increased, enabling young apple fruits to be compatible with both ultrafiltration centrifugation and density gradient centrifugation methods, and achieving simple and rapid preparation. Moreover, the finally prepared apple nanovesicles are rich in apple characteristic polyphenolic compounds, especially whitening active ingredients such as phloretin. The prepared nanovesicles from young apple fruits 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, with an effect superior to that of the model compound arbutin. At the same time, the nanovesicles from young apple fruits can also reduce the expression level of MMP-1 in human cells and promote the synthesis of type I collagen (ColⅠ) and hyaluronic acid (HA), achieving the effects of anti-aging and wrinkle removal and improving skin quality. The present invention realizes the high-value comprehensive utilization of apple field management waste, and the final product has good application prospects in aspects such as whitening and freckle removal, skin care and wrinkle removal, and skin quality improvement. Description of the Drawings

[0019] Figure 1 : Particle size distribution diagram of vesicles extracted from young apple fruits at different developmental stages, where DDA represents the number of days after flowering;

[0020] Figure 2 : Flow chart for the preparation of the starch-removed nanovesicle suspension in Example 5;

[0021] Figure 3 : Free radical scavenging ability of nanovesicles prepared from different young apple fruits. 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 : Inhibitory activity of nanovesicles prepared from different young apple fruits against tyrosinase. Different letters represent significant differences between the two groups;

[0023] Figure 5 : Inhibitory effect of nanovesicles prepared from young fruits of various apple species on cellular melanin synthesis, where ** represents P < 0.01;

[0024] Figure 6 : Inhibitory activity of nanovesicles prepared from young fruits of various apple species against intracellular tyrosinase. Different letters represent significant differences between the two groups;

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

[0026] Figure 8 : Effects of nanovesicles prepared from different apple young 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 implementation manners

[0027] The present invention provides a method for preparing apple nanovesicles. Apple young fruits collected 14 - 60 days after flowering are used as raw materials, and the nanovesicles are obtained by direct centrifugation or sucrose density gradient centrifugation. The present invention studies and finds that with the extension of the time after flowering, the particle size of the nanovesicles extracted from apple young fruits increases, and the absolute value of the Zeta potential decreases. If the collection time after flowering is too early, the volume of the apple young fruits is too small, and the amount of nanovesicles extracted is small. The present invention selects apple young fruits 14 - 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 fruits are subjected to dark treatment before extracting the nanovesicles. The conditions for the dark treatment of the apple young fruits are: the temperature is 30 - 40 °C, the humidity is 75% - 95%, and the apple young fruits are placed in the dark for 10 - 24 h. The temperature for the dark treatment in 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 fruits are preferably placed in the dark for 15 - 22 h, more preferably 20 h. A large amount of starch will accumulate in the apple young fruits, and the large molecular weight starch will interfere with the preparation of nanovesicles. After the apple young fruits are subjected to dark treatment in the present invention, the starch in the fruits is consumed through the respiration of the fruit cells themselves, reducing 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 de-starched using an α-amylase solution. The concentration of the α-amylase (EC 3.2.1.1) solution is 0.1 - 5 U / mL, preferably 0.5 - 2 U / mL, and more preferably 1 U / mL. The nanovesicles obtained in the present invention are a nanovesicle suspension. The added mass of the amylase solution is 0.5% - 1.5% of the mass of the nanovesicle suspension to be treated, preferably the added amount is 0.8% - 1.2%, and more preferably 1%. As an implementable method, in the present invention, at 15 °C, an α-amylase solution is added to the nanovesicle suspension, and stirred at a rotation speed of 8 - 12 rmp for 15 - 25 min to obtain a mixed solution, and then the mixed solution is ultrafiltered and centrifuged. The ultrafiltration and centrifugation are as follows: the mixed solution is placed in an ultrafiltration tube with a molecular weight cut-off of 100 kDa and centrifuged, the centrifugation conditions are 4000 - 6000 g, and the centrifugation time is 25 - 35 min; after centrifugation, 5 mL of Tris-HCl buffer solution is added to the ultrafiltration tube and centrifuged again, the centrifugation conditions are 4000 - 6000 g, and the centrifugation time is 25 - 35 min, and finally a de-starched nanovesicle suspension is obtained.

[0030] In the present invention, the direct centrifugation method includes: freezing young apple fruits and then crushing them, mixing the fruit powder with Tris-HCl buffer solution and homogenizing, successively centrifuging the liquid phase of the homogenate at 650 g, 3000 g, 10000 g, and 15000 g, taking the supernatant for the next centrifugation each time after centrifugation, taking the supernatant after the last centrifugation, centrifuging at 100000 g, collecting the precipitate, and resuspending it with PBS to obtain a nanovesicle suspension.

[0031] In the direct centrifugation method of the present invention, after removing the fruit cores of the young apple fruits, it is preferably quick-frozen using liquid nitrogen, and the young apple fruits are ground into powder in a frozen state; the fruit powder is mixed with Tris-HCl buffer solution (100 mM, pH 7.4) in a ratio of 5:4 (w:v), and homogenized at high speed for 8 - 12 min at 4 °C using a high-speed homogenizer, preferably homogenized for 10 min, and the homogenate is filtered through filter paper to remove solid particles and the liquid phase of the homogenate is collected; the liquid phase of the homogenate is successively centrifuged at 650 g, 3000 g, 10000 g, and 15000 g for 15 - 25 min, preferably centrifuged for 20 min, and the centrifugation temperature is 4 °C; the supernatant is centrifuged at 100000 - 12000 g, preferably centrifuged at 100000 g, and the centrifugation time is 30 - 180 min, preferably centrifuged for 120 min, and the centrifugation temperature is 4 °C; the precipitate is made into a nanovesicle suspension using PBS (100 mM, pH 6.8), and the nanovesicle suspension is quick-frozen using liquid nitrogen and stored at -80 °C.

[0032] In the present invention, the nano-vesicle suspension obtained by the direct centrifugation method is subjected to ultrafiltration centrifugation, and the ultrafiltration centrifugation is as follows: the nano-vesicle suspension is placed in an ultrafiltration tube with a molecular weight cut-off of 100 kDa and centrifuged, the centrifugation conditions are 4000 - 6000 g, and the centrifugation time is 25 - 35 min; after centrifugation, 5 mL of Tris-HCl buffer is added to the ultrafiltration tube and centrifuged again, the centrifugation conditions are 4000 - 6000 g, and the centrifugation time is 25 - 35 min. Through ultrafiltration centrifugation, pigments and other small molecule substances can be further removed, making the obtained apple nano-vesicles have better purity.

[0033] In the present invention, the sucrose density gradient centrifugation method includes: homogenizing young apple fruits, centrifuging the homogenate at 1000 g, collecting the supernatant and centrifuging it at 5000 g, collecting the supernatant and using sucrose density gradient centrifugation, the sucrose gradients are 8%, 30%, 45% and 60%, and the nano-vesicle suspension is obtained by aspirating at the interface between 30% and 45% of sucrose after centrifugation.

[0034] In the sucrose density gradient centrifugation method of the present invention, after removing the fruit cores, the young apple fruits are mixed with PBS (100 mM, pH 7.4) at a ratio of 1:0.5 (w:v), and homogenized at a high speed for 8 - 12 min at 4°C using a high-speed homogenizer, preferably homogenized for 10 min; the homogenate is centrifuged at 1000 g for 25 - 35 min, preferably centrifuged for 30 min, the centrifugation temperature is 4°C, the supernatant is collected and centrifuged at 5000 g for 25 - 35 min, preferably centrifuged for 30 min, the centrifugation temperature is 4°C; the centrifugal force parameter for the sucrose density gradient centrifugation is set to 100000 - 12000 g, preferably the centrifugation parameter is 120000 g, the centrifugation time is 30 - 180 min, preferably centrifuged for 120 min; the nano-vesicle suspension is quickly frozen with liquid nitrogen and stored at -80°C.

[0035] The present invention also provides apple nano-vesicles prepared by the above method, and the apple nano-vesicles contain dihydrochalcone compounds.

[0036] Polyphenols are the main phytochemical components in apples, mainly including five polyphenol groups, flavanols, flavonols, phenolic acids, dihydrochalcones and flavonoids. Among them, dihydrochalcone compounds are a large class of secondary metabolites with the highest content and very rich variety in apple plants. Apple polyphenols can generally reduce cell damage caused by aging and play a role in preventing various diseases. Multiple polyphenol compounds identified in the apple nano-vesicles provided by the present invention include: five major categories of flavanols, flavonols, anthocyanins, phenolic acids and dihydrochalcones. The apple nano-vesicles of the present invention retain the original polyphenol components in apples, and these polyphenols generally have antioxidant and anti-inflammatory effects. Among them, dihydrochalcone compounds also have a clear effect of whitening and freckle removal.

[0037] In the present invention, the apple young fruits are young fruits of plants of the genus Malus, including cultivated species, wild species, and ornamental varieties, preferably any one of Fuji apple (M. domestica 'Fuji'), Golden Delicious apple (M. domestica 'Golden Delicious'), Royalty apple (M. 'Royalty'), Donghongguo apple (M. 'Donghongguo'), Sparkler crabapple (M. 'Sparkler'), Otterson apple (M. 'Otterson'), and Micromalus crabapple (M. micromalus). All apples were collected from the germplasm resource nursery of the College of Horticulture, Northwest A&F University.

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

[0039] The present invention also provides a skin care product, which contains the above-mentioned apple nanovesicles, and the skin care product has the effects of whitening, antioxidant, 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 indicated, they are usually carried out according to conventional conditions or the conditions recommended by the reagent company.

[0041] The technical solutions provided by the present invention will be 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] Collect the young fruits of Fuji apples 30 days after flowering, remove the fruit cores, quickly freeze them with liquid nitrogen, and then grind them into powder in the frozen state. Mix the frozen fruit powder with Tris-HCl buffer (100 mM, pH 7.4) at a ratio of 5:4 (w:v), and then use a high-speed homogenizer to homogenize at high speed for 10 min at 4°C. The homogenate is filtered through filter paper to remove solid particles. The liquid phase is centrifuged successively at 650 g, 3000 g, 10000 g, and 15000 g, and the supernatant is taken for the next centrifugation after each centrifugation. Each centrifugation lasts for 20 min. After the last centrifugation, collect the supernatant and centrifuge it at 100000 g at high speed for 120 min. Aspirate the supernatant, and gently pipette the precipitate with PBS (100 mM, pH 6.8) to make a nanovesicle suspension.

[0045] The nanovesicle suspension was placed in an ultrafiltration tube with a molecular weight cut-off of 100 kDa and centrifuged at 5000 g for 30 min. After centrifugation, 5 mL of Tris-HCl buffer (100 mM, pH 7.4) was added to the ultrafiltration tube, and then centrifuged again at 5000 g for 30 min to obtain the ultrafiltered nanovesicle suspension.

[0046] The ultrafiltered nanovesicle suspension was snap-frozen in liquid nitrogen and stored at -80 °C for later use. All centrifugations in this example were carried out at 4 °C.

[0047] Example 2

[0048] A method for preparing apple nanovesicles:

[0049] Young fruits of Fuji apples at 30 days after flowering were collected, the cores were removed, and they were mixed with PBS (100 mM, pH = 7.4) at a ratio of 1:0.5 (w:v). They were homogenized at high speed using a high-speed homogenizer at 4 °C for 10 min. The homogenate was centrifuged at 1000 g for 30 min, and the supernatant was collected and centrifuged at 5000 g for 30 min. The collected supernatant was subjected to sucrose density gradient centrifugation (20 mM Tris-HCl, pH 7.2, sucrose gradients of 8%, 30%, 45% and 60%) for 2 h. Then, the prepared nanovesicles were aspirated from the interface between 30% and 45%. The nanovesicle suspension was snap-frozen in liquid nitrogen and stored at -80 °C for later use. All centrifugations in this example were carried out at 4 °C.

[0050] Example 3

[0051] Young fruits of Fuji apples at 14, 30, 60, 80 and 180 days after flowering were collected respectively, the cores were removed, and the method of Example 1 was used to prepare nanovesicles. The particle size and Zeta potential of the nanovesicles were measured using a nanoparticle size analyzer and a Zeta potential analyzer. Each stage of apples was independently repeated three times, and the results are as Figure 1 shown.

[0052] The results showed that the average particle sizes of the nanovesicles extracted from apples at 14, 30, 60, 80 and 180 days after flowering were 76.06 ± 3.13 nm, 76.23 ± 3.35 nm, 82.71 ± 4.01 nm, 101.90 ± 4.01 ± 4.29 nm and 131.95 ± 5.62 nm respectively. The Zeta potentials were -39.6 mV, -38.2 mV, -35.7 mV, -29.6 mV, -26.6 mV ( Figure 1 ). It was shown that the nanovesicles extracted from young fruits of apples from 14 days to 60 days after flowering had uniform particle sizes, small sizes and good stability. Among them, young fruits of apples at 30 days after flowering yielded more nanovesicles of good quality through high-speed centrifugation, and the young fruits at 30 days had a relatively larger volume, making them more economical for extracting nanovesicles.

[0053] Example 4

[0054] The starch content of Fuji apples was measured at 14, 30, 60, 80, and 180 days after flowering using the enzymatic hydrolysis method. The results showed that the starch contents in the young apple fruits at 14, 30, 60, 80, and 180 days after flowering were 8.5 ± 1.2 mg / g, 12.4 ± 1.5 mg / g, 47.8 ± 5.2 mg / g, 58.4 ± 3.6 mg / g, and 11.2 ± 2.3 mg / g, respectively, indicating that the starch content increased and then decreased during the apple development process.

[0055] After picking the young apple fruits at 30 days after flowering, they were placed in the dark at 80% humidity and 35°C for 8 h, 10 h, 12 h, 16 h, 20 h, and 24 h, respectively, and then their starch contents were measured again. After the young apple fruits were treated in the dark for different times, the starch contents were 11.2 ± 1.6 mg, 9.8 ± 1.5 mg, 8.2 ± 1.2 mg, 7.8 ± 1.3 mg, 6.9 ± 1.0 mg, and 6.8 ± 1.1 mg, respectively, indicating that the starch content in the young apple fruits could be significantly reduced by dark treatment.

[0056] The apples at 30 days after flowering were treated with darkness (80% humidity, 35°C, 20 h) and without treatment, respectively, and then used to prepare nanovesicles. The preparation method was as follows: the young fruits were pitted, and 10 g was taken from each. The frozen fruit powder was mixed with Tris-HCl buffer (100 mM, pH 7.4) at 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 through filter paper to remove solid particles. The liquid phase was centrifuged at 650 g, 3000 g, 10000 g, and 15000 g for 20 min in sequence at 4°C. After each centrifugation, the precipitate was removed and the supernatant was retained. The supernatant obtained from the last centrifugation was centrifuged at 5000 g for 30 min using a 100 kDa ultrafiltration tube, and then 5 mL of Tris-HCl buffer was added and centrifuged again at 5000 g for 30 min to obtain a nanovesicle suspension. The starch content was measured using the iodine-potassium iodide method (GB / T 15683-2008).

[0057] The results showed that after dark treatment, 0.7 mL of nanovesicle suspension was obtained. The measured 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 measured starch content was 0.61 mg / mL, and the average particle size of the nanovesicles was 75.63 ± 3.57 nm. It indicated that after dark treatment, the starch content in the extraction material could be significantly reduced, and the particle size distribution of the extracted nanovesicles was narrower. Without dark treatment, the liquid retained during ultrafiltration contained a colloid formed by starch and macromolecular substances. Therefore, the volume of the retained liquid collected without dark treatment was large, but the abundance of nanovesicles was low.

[0058] Example 5

[0059] The young fruits of Fuji apples 30 days after flowering were collected and placed in the dark at a humidity of 80% and a temperature of 35 °C for 20 h. Then, the method of Example 1 was adopted to prepare the ultrafiltered nanovesicle suspension. At 15 °C, 1 U / mL of α-amylase solution (EC 3.2.1.1) was added. The added mass of the α-amylase solution was 1% of the mass of the ultrafiltered nanovesicle suspension. The mixture was stirred slowly for 20 min (10 rmp) to obtain a mixed solution. Then, the mixed solution was placed in a 100 kDa ultrafiltration tube and centrifuged at 5000 g 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 it was centrifuged again at 5000 g for 30 min to obtain a starch-free nanovesicle suspension (the preparation flow chart is as Figure 2 shown). The iodine-potassium iodide method was used for determination, and no starch was detected. The particle size and Zeta potential of the nanovesicles were measured using a particle size analyzer, which were 78.23 ± 4.03 nm and -39.1 mV, respectively.

[0060] Example 6

[0061] The method in Example 5 was adopted to prepare starch-free nanovesicle suspensions using the young fruits of Fuji apples, Golden Delicious apples, Royalty apples, Winter Red fruits, Diamond Begonia, Red-fleshed apple Otterson, and Malus micromalus Makino as raw materials.

[0062] To the obtained de-starch nanovesicle suspension, add an equal volume of methanol (containing 1% formic acid by mass fraction), then use ultrasonic oscillation for 30 min to obtain a mixture. The mixture is centrifuged at 4 °C for 15 min (12,000 g), and the supernatant is filtered through a 0.22 μm filter membrane, and then analyzed by high-resolution liquid chromatography-mass spectrometry. The liquid chromatography-mass spectrometry conditions are as follows: Mobile phase A is acetonitrile containing 0.1% formic acid, and mobile phase B is pure water containing 0.1% formic acid. The column temperature is 35 °C, and the flow rate is 0.3 mL / min. The chromatographic column is Inertsil ODS-3 (4.6 mm × 250 mm, 5.0 μm). The gradient elution program is as follows: 95% A (0 min), 85% A (25 min), 78% A (42 min), 64% A (60 min), 95% A (65 min), and equilibrate for 5 min. The mass spectrometry detection conditions are as follows: Applied Biosystems API 2000 triple quadrupole mass spectrometer, equipped with an ESI ion source, detected in positive ion mode, the scanning range is 180 - 800 m / z, the nebulizing gas is 50 psi, the curtain gas is 20 psi, the heating gas is 50 psi, the heating temperature is 450 °C, the spray voltage is 3.8 kV, all gases are high-purity nitrogen, and the fragmentation energy of the secondary spectrum is 30 V.

[0063] A variety of polyphenolic compounds were identified from apple nanovesicles, including flavanols, flavonols, anthocyanins, phenolic acids, and dihydrochalcones, a total of 20 species, and the results are shown in Table 1.

[0064] Table 1 Types of polyphenolic compounds in nanovesicles

[0065]

[0066] As can be seen from Table 1, a total of 20 polyphenols were absolutely characterized in the nanovesicles extracted from the young fruits of Fuji, Golden Delicious, Royal Gala, Winter Red Fruit, Diamond Crabapple, Red Flesh Apple, and Malus micromalus Makino after 30 days of flowering. Among them, the contents of flavanols, flavonols, and phenolic acids showed little difference among the cultivated varieties (Fuji, Golden Delicious), ornamental crabapples (Royal Gala, Diamond), red flesh apple, and the original species (Otterson; Malus micromalus Makino). Anthocyanin compounds were only detected in two ornamental crabapples (Royal Gala, Diamond) and red flesh apple. Phloridzin and its main glycoside derivative phloridzin were detected in all apple compounds among the dihydrochalcone compounds; 3-hydroxyphloretin was only detected in Winter Red Fruit, Diamond, Otterson, and Malus micromalus Makino, and its glycoside derivative (3-hydroxyphloridzin) was mainly in the two ornamental crabapples, Winter Red Fruit and Diamond.

[0067] Example 7

[0068] Antioxidant effect of apple young fruit nanovesicles.

[0069] The method in Example 5 was adopted, and nanovesicle suspensions without starch were prepared using the young fruits of Fuji apples, Golden Delicious apples, Royalty apples, Winter Red Fruits, Diamond Crabapples, Red-fleshed apple Otterson, and Chinese flowering crabapples as raw materials.

[0070] The nanovesicle suspensions prepared from Fuji apples, Golden Delicious apples, Royalty apples, Winter Red Fruits, Diamond Crabapples, Red-fleshed apple Otterson, and Chinese flowering crabapples were diluted 10 times respectively. Then, 10 μL of each was taken and mixed with 190 μL of DPPH free radicals (87 μM, ethanol solution) and 190 μL of ABTS free radicals (7 mM, aqueous solution) respectively, and reacted in the dark for 30 min. 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 averaged. The data were analyzed using one-way analysis of variance and presented as mean ± standard deviation. The results are as Figure 3 shown.

[0071] Figure 3 The results showed that the nanovesicles prepared from Royalty apples and Red-fleshed apple Otterson had the strongest ability to scavenge free radicals, followed by Chinese flowering crabapples. The other two ornamental crabapples (Winter Red Fruits and Diamond Crabapples) also had good scavenging ability. The scavenging ability of the two cultivated apples (Fuji and Golden Delicious) was weak, but all the nanovesicles were stronger than vitamin C.

[0072] Example 8

[0073] Effect of apple young fruit nanovesicles on inhibiting tyrosinase.

[0074] The method in Example 5 was adopted, and nanovesicle suspensions without starch were prepared using the young fruits of Fuji apples, Golden Delicious apples, Royalty apples, Winter Red Fruits, Diamond Crabapples, Red-fleshed apple Otterson, and Chinese flowering crabapples as raw materials.

[0075] 10 μL of the nanovesicle suspensions prepared from Fuji apples, Golden Delicious apples, Royalty apples, Winter Red Fruits, Diamond Crabapples, Red-fleshed apple Otterson, and Chinese flowering crabapples were taken respectively and mixed with an equal volume of tyrosinase PBS solution, and the enzyme concentration was 200 U / mL. They were incubated with shaking at 37 °C for 10 min, and then PBS solution with 8 times the volume of the enzyme solution was added respectively. The PBS solution contained L-tyrosine, and the concentration of L-tyrosine was 20 mg / mL. The reaction was carried out at 37 °C for 10 min, and then the absorbance of the whole reaction system was measured at 475 nm. The inhibitory activity against tyrosinase was calculated according to the following formula: Inhibition rate = 100% × (A0 - A) / A0.

[0076] Among them, A0 is the absorbance of the reaction after mixing tyrosinase and tyrosine without adding any arbutin or nanovesicles. A is the absorbance of the test group adding nanovesicles or arbutin. Arbutin is used as a positive control compound. All absorbance values are subtracted by the background value of the corresponding reaction system (the absorbance of the reaction system adding only nanovesicles or arbutin without adding tyrosinase). Each group of experiments was repeated 6 times and averaged. The data was analyzed using one-way ANOVA and presented as mean ± standard deviation. The results are as Figure 4 shown.

[0077] Figure 4 The results showed that all the nanovesicles from young apple fruits had tyrosinase inhibitory activity. The nanovesicles extracted from Donghongguo had a better inhibitory effect than the positive compound arbutin. The inhibitory effects of the nanovesicles extracted from the young fruits of three kinds of Chinese flowering crabapples, namely Wangzu, Diamond Begonia, and Malus micromalus, were close to that of arbutin. The inhibitory effects of the nanovesicles extracted from the other three cultivated apples were weaker than that of arbutin.

[0078] Example 9

[0079] Whitening effect of nanovesicles from young apple fruits.

[0080] Adopt the method in Example 5 to prepare a de-starched nanovesicle suspension using the young fruits of Fuji apple, Golden Delicious apple, Wangzu apple, Donghongguo, Diamond Begonia, Red-fleshed apple Otterson, and Malus micromalus as raw materials.

[0081] Use B16-F10 (CRL-6475) melanoma cells (ATCC CRL-6475) as a model to test the inhibitory effect of nanovesicles from young apple fruits on melanin synthesis. The cells were inoculated in DMEM medium containing 10% fetal bovine serum, and the culture conditions were 37 °C, 5% CO2 content, and 98% relative humidity. The cells in the logarithmic growth phase were digested with trypsin and inoculated in a 6-well culture plate at a concentration of 1×10 6 cells / mL, 2 mL per well. After culturing for 8 h, the supernatant was discarded and the cells were rinsed once with PBS. The nanovesicle suspensions prepared from Fuji, Golden Delicious, Wangzu, Donghongguo, Diamond Begonia, Red-fleshed apple Otterson, and Malus micromalus were diluted 10 times and then added to the above culture plate, 2 mL per well. Arbutin was used as a positive control with a final concentration of 100 μg / mL, and the DEME medium without adding any extract was used as a negative control. After culturing for 48 h, the supernatant of the cultured cells was discarded, the cells were digested and collected, centrifuged at 1500 r / min for 10 min, the supernatant was aspirated, 1 mL of 1 mol / L sodium hydroxide solution containing 10% DMSO was added, incubated in a water bath at 80 °C for 1 h, and the absorbance was measured at 405 nm using an enzyme-linked immunosorbent assay. 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 melanin in cells without adding any arbutin or nanovesicles, A is the absorbance of the test group adding nanovesicles or arbutin, and arbutin is used as a positive control compound. All absorbance values are subtracted by the system blank of the corresponding reaction system (the absorbance of the system only adding nanovesicles or arbutin). Each group of experiments is repeated 5 times and averaged. The data is analyzed using one-way ANOVA and presented in the form of mean ± standard deviation. The results are as Figure 5 shown.

[0084] The cells treated by the above method are centrifuged, rinsed with PBS, then 40 μL of 1% Triton-X100 is added, and then transferred to -80 °C for freezing for 30 min, and then placed at 37 °C until completely melted. Subsequently, 10 μL of L-DOPA solution (1 w / w%) is added thereto. React at 37 °C for 30 min, and then measure the absorbance of the system at a wavelength of 450 nm. The inhibition rate of tyrosinase in cells is calculated according to the following formula:

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

[0086] Where A3 is the absorbance after the reaction of tyrosinase extracted from cells without adding any nanovesicles or arbutin, A is the absorbance of the test group adding nanovesicles or arbutin, and arbutin is used as a positive control compound. All absorbance values are subtracted by the system blank of the corresponding reaction system (the absorbance of the system only adding nanovesicles or arbutin). Each group of experiments is repeated 5 times and averaged. The data is analyzed using one-way ANOVA and presented in the form of mean ± standard deviation. The results are as Figure 6 shown.

[0087] Figure 5 The results show that all the nanovesicles extracted from Malus plants have obvious melanin synthesis inhibition activity, and are far stronger than arbutin.

[0088] Figure 6 The results show that all the nanovesicles extracted from young fruits of Malus plants have the inhibition activity of intracellular tyrosinase. The inhibition activities of Se-rich, Golden Delicious, Donghongguo, and Malus micromalus are stronger than that of the model compound arbutin. The inhibition activities of Malus baccata 'Diamond' and Malus pumila 'Red Flesh Otterson' are close to that of arbutin, and only Malus spectabilis is weaker than the model compound.

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

[0090] Example 10

[0091] Anti-aging effect of nano-vesicles from young apple fruits.

[0092] Adopt the method in Example 5, and use the young fruits of Fuji apples, Golden Delicious apples, Royalty apples, Winter Red fruits, Diamond Begonia, Red-fleshed apple Otterson and Micromalus spectabilis as raw materials to prepare a starch-free nano-vesicle suspension.

[0093] Use human dermal fibroblasts (Wuhan Punosai Life Science & Technology) to test the anti-aging effect of nano-vesicles from young apple fruits. Culture them using a special fibroblast medium (CM-H103, Wuhan Punosai Life Science & Technology), and use them for experiments when subcultured to the third generation. Digest the cells with trypsin, inoculate them in a 6-well culture plate at a concentration of 0.5×10 6 cells / mL, then place them in an incubator (5% CO2, 37 °C, humidity 98%) and culture for 24 hours. Then discard the medium in the culture dish, and add 1.0 mL of PBS. Irradiate the cells in the experimental group, positive control group and negative control group with UVA (340 nm), and the radiation dose is 10 J / cm 2 , and the blank group is not irradiated. Dilute the nano-vesicle suspensions prepared from Fuji, Golden Delicious, Royalty, Winter Red, Diamond Begonia, Red-fleshed apple Otterson and Micromalus spectabilis 10 times with the medium. Add the diluted nano-vesicle suspensions to the experimental group culture medium respectively, add Ilomastat (1 nM) to the positive control group medium, and only add the medium to the negative control group and the blank group. Then culture for 24 h. After culturing, digest and centrifuge the cells (2000 g, 20 min), and use a human matrix metalloproteinase 1 (MMP-1) kit to test the MMP-1 expression level in the supernatant. Resuspend the centrifuged cells with PBS, count the concentration as 1×10 6 cells / mL, then ultrasonically treat for 30 min, then quickly freeze with liquid nitrogen, thaw at 37 °C and then refreeze. After repeated freeze-thawing three times, centrifuge (1000 g, 20 min), and use a type I collagen (ColⅠ) test kit to detect the ColⅠ content in the supernatant, and use a human hyaluronic acid (HA) ELISA Kit to detect the HA content. Each experiment is repeated 4 times and the average is taken, and the data is analyzed using one-way ANOVA. The data is expressed as the mean ± standard deviation, and the results are as Figure 7 and Figure 8 shown.

[0094] Figure 7 and Figure 8The results showed that, compared with the irradiation group, all the nano-vesicle extracts could significantly reduce the expression level of MMP-1. Among them, the extracts from Fuji and Golden Delicious apples could reduce the expression level of MMP-1 to the normal level (the same as the blank group), while the nano-vesicles extracted from several other Chinese flowering crabapples and red-fleshed apples had a stronger inhibitory effect on MMP-1. At the same time, in all the nano-vesicle treatment groups, the expression levels of Col 1 and HA were significantly higher than those in the control group, even higher than those in the blank group and the positive control compound (Ilomastat). In particular, the nano-vesicles from two cultivated apples (Fuji and Golden Delicious) and one native Chinese flowering crabapple (Malus micromalus) had the best effect on promoting the expression of Col1 and HA. These results indicate that the nano-vesicles of young apple fruits prepared in the present invention can effectively combat skin aging caused by ultraviolet irradiation and have a certain anti-wrinkle effect.

[0095] Example 11

[0096] An emulsion containing nano-vesicles of young fruits of Malus plants.

[0097] It contains the following components in terms of mass / volume percentage: 1% glycerol, 1% 1,3-propanediol, 1% hyaluronic acid, 5% L-ascorbic acid, 0.1% ethylene glycol phenyl ether, which are added to a hydrocolloid containing 0.5% gelatin to prepare a base emulsion. Then, the nano-vesicles prepared from the young fruits of Winter Red Fruit are diluted 100 times with the base emulsion. Thus, the emulsion containing apple nano-vesicles is obtained.

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

[0099] Example 12

[0100] A cream containing nano-vesicles of young fruits of Malus plants.

[0101] It contains the following components in terms of mass / volume percentage: 8% niacinamide, 3% glycerol, 1.5% cetyl alcohol, 1% glycerol, 1% 1,3-propanediol, 0.5% vitamin E, which are compounded with water to form a cream. Then, the nano-vesicles prepared from the young fruits of Fuji are compounded with the cream in a volume ratio of 1:100 to form a uniform cream. Thus, the cream containing apple nano-vesicles is obtained.

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

[0103] Example 13

[0104] A cream containing nano-vesicles of young fruits of Malus plants.

[0105] It contains the following components in terms of mass / volume percentage: 2% of 1,3-propanediol, 2% of polydimethylsiloxane, 2% of boswellia serrata extract (hydroxypropyltetrahydropyrantriol), 1% of cyclohexasiloxane, 1% of glycerol, and is formulated into a cream by compounding with water. Then, the nanovesicles prepared from Fuji young 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 the nanovesicles in this example is the same as that in Example 5.

[0107] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing apple nanovesicles, characterized in that, The young apples collected 14 to 60 days after flowering are used as raw materials, and the direct centrifugation method is adopted to extract and obtain the nano vesicles; The nano vesicles are extracted after the young apples are subjected to dark treatment, and the conditions for the dark treatment are: placed at 35 °C and 80% humidity in the dark for 20 h; The starch of the nano vesicles is removed by using an α-amylase solution, and the concentration of the α-amylase solution is 1 U / mL; The direct centrifugation method includes: freezing and crushing the young apples, homogenizing after mixing the apple powder with Tris-HCl buffer solution, centrifuging the liquid part of the homogenate at 650 g, 3000 g, 10000 g and 15000 g for 20 min in sequence, taking the supernatant for the next centrifugation after each centrifugation, taking the supernatant after the last centrifugation, centrifuging at 100000 g for 120 min, collecting the precipitate, and resuspending with buffer solution to obtain a nano vesicle suspension.

2. The apple nanovesicles obtained by the preparation method according to claim 1, characterized in that, The apple nano vesicles contain dihydrochalcone compounds.

3. Use of the apple nanovesicles according to claim 2 in the preparation of skin care products, characterized in that, The skin care products include whitening products, freckle-removing products, anti-wrinkle products and anti-aging products.

4. A skin care product, characterized in that, The skin care product contains the apple nano vesicles described in claim 2.

5. The skin care product according to claim 4, wherein The skin care product is any one of lotion, cream and essence.

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