Preparation method of Cistanche deserticola exosome-like nanovesicles and their application in breast enhancement products

Abstract

The present invention belongs to the technical field of daily chemical raw materials, and specifically relates to a preparation method of Cistanche deserticola exosome-like nanovesicles and their application in breast enhancement products. The product provided by the present invention contains at least Cistanche deserticola exosome-like nanovesicles, and Cistanche deserticola exosome-like nanovesicles are used as the main active ingredients. The Cistanche deserticola exosome-like nanovesicles are obtained after differential centrifugation and direct current electrophoresis. The protein content of the Cistanche deserticola exosome-like nanovesicles obtained by the present invention is as high as 42.6%, which can effectively increase the lipid content in cells, increase the volume of intracellular lipid droplets, and can also effectively improve the survival rate of 3T3-L1 cells, increase the number of fat cells, and have a good breast enhancement effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of daily chemical product raw materials, and particularly relates to a preparation method of Cistanche deserticola exosome-like nanovesicles and application thereof in breast enhancement products. Background Art

[0002] Breast augmentation, a crucial aspect of women's pursuit of beauty, has garnered significant attention since ancient times. With the advancement of technology, breast enhancement methods have evolved continuously, from surgical procedures to medications and now to physical and dietary enhancements. However, regardless of these evolving methods, safety, health, and effectiveness remain the unchanging themes of breast enhancement products.

[0003] In recent years, plant extracts have become the preferred raw materials for breast enhancement products due to their high safety. Patents such as CN112544986A, CN109662939A, and CN110585320A all use plant extracts as the main ingredients of their breast enhancement products. However, in the process of preparing plant extracts, existing technologies have overlooked an important factor that affects the effectiveness of breast enhancement products, namely the content of active ingredients in the prepared plant extracts. The required plant extracts are obtained only by traditional methods, and the content of bioactive ingredients such as proteins, sugars, terpenes, flavonoids, alkaloids, and steroids in the obtained plant extracts is not high. In addition, the use of traditional water extraction, organic solvent extraction, and other methods will destroy the lipid system in the plant extracts, making it difficult to obtain genetic materials such as microRNA that play a role in the plant source.

[0004] Cistanche deserticola, a parasitic plant that lives on the roots of the Haloxylon ammodendron tree in the desert, survives by absorbing nutrients and water from its host. It possesses significant medicinal value and is often known as the "desert ginseng." Furthermore, Cistanche deserticola is one of the most frequently used tonic herbs in kidney-tonifying and yang-strengthening prescriptions. Consequently, it is often used as an adjunct ingredient in various health products, including breast enhancement products.

[0005] However, to date, there has been no research on whether Cistanche deserticola truly has the effect of breast enhancement, and it is unknown whether it can be used as the main active ingredient in breast enhancement products. Summary of the Invention

[0006] In order to solve this technical problem, the present invention provides a preparation method of Cistanche deserticola exosome-like nanovesicles and the application of the same in breast enhancement products.

[0007] The first aspect to be protected by the present invention is to provide a method for preparing exosome-like nanovesicles from Cistanche deserticola, comprising the following steps:

[0008] (1) Washing, drying, and crushing the Cistanche deserticola to obtain Cistanche deserticola powder;

[0009] (2) adding water to the Cistanche deserticola powder obtained in (1) and beating the mixture into a pulp, collecting the supernatant after differential centrifugation, and obtaining a Cistanche deserticola centrifuge pretreatment solution;

[0010] (3) The Cistanche deserticola centrifugal pretreatment liquid obtained in (2) was treated with direct current, and the liquid at 1 / 8 to 1 / 3 of the length of the positive terminal was collected and decolorized to obtain Cistanche deserticola exosome-like nanovesicles.

[0011] In the preparation method of the exosome-like nanovesicles of Cistanche deserticola provided by the present invention, preferably, in (1), the drying temperature is 40-85°C, and the pulverization is performed by using an ultrafine grinder to pulverize the Cistanche deserticola to 800-1200 mesh.

[0012] Preferably, in (2), the mass of the added water is 5 to 40 times the mass of the Cistanche deserticola powder.

[0013] Preferably, the beating in (2) is performed by using a tissue homogenizer for 10 to 30 minutes.

[0014] Preferably, the differential centrifugation operation described in (2) is specifically as follows: first centrifugation at a centrifugal force of 2000-5000 g for 0.5-4 h, and then centrifugation at a centrifugal force of 6000-15000 g for 0.5-4 h.

[0015] More preferably, the centrifugation operation described in (2) is as follows: first centrifugation at a centrifugal force of 3000-5000 g for 0.5-1 h, and then centrifugation at a centrifugal force of 10000-12000 g for 0.5-1 h.

[0016] Preferably, the direct current treatment conditions described in (3) are: current 50-150 mA, and electrophoresis time 1-5 h.

[0017] More preferably, the direct current treatment conditions described in (3) are: current 80-130 mA, and electrophoresis time 1-3 h.

[0018] Preferably, in (3), a macroporous adsorption resin is used to decolorize the collected liquid, and the macroporous resin is D941.

[0019] As a further preferred embodiment, in (3), before using the macroporous adsorption resin for decolorization, the macroporous adsorption resin is pretreated, and the specific operation is as follows:

[0020] The macroporous adsorption resin was soaked in 95% ethanol by volume for 24 h, and then washed with deionized water until there was no alcohol smell. The washed resin was then soaked in 1M sodium hydroxide and 1M hydrochloric acid for 5 h each, and washed with deionized water until the pH value of the resin reached 7. Finally, the resin was soaked in 95% ethanol by volume for 12 h, and then washed with distilled water until there was no alcohol smell.

[0021] More preferably, the decolorization described in (3) is specifically performed as follows: the pretreated resin is placed in a conical flask, and the liquid to be decolorized is added thereto, and the flask is placed in a shaker and shaken at 20-25°C and 120-150 r / min for 20-26 h.

[0022] The second aspect of the present invention is to provide a product containing the above-mentioned Cistanche deserticola exosome-like nanovesicles, wherein the product contains at least the Cistanche deserticola exosome-like nanovesicles and uses the Cistanche deserticola exosome-like nanovesicles as the main active ingredient. In the product, the Cistanche deserticola exosome-like nanovesicles account for 1 to 10 parts and other excipients account for 90 to 99 parts, and the other excipients are selected from at least one of trehalose, mannitol, sorbitol, maltitol, and phosphate.

[0023] Furthermore, when the product is a solid powder, the present invention also provides a method for preparing the product, that is, based on the preparation of the above-mentioned Cistanche deserticola exosome-like nanovesicles, it also includes step (4): first using a phosphate buffer to adjust the pH of the Cistanche deserticola exosome-like nanovesicles obtained in (3) to 5.5~8.5, and then freeze-drying. The freeze-drying operation is specifically: first pre-freezing at -50~-40℃ for 5~10 h, heating the main partition to 40-60℃, and then freezing at -60~-50℃ for 48~72 h.

[0024] A third aspect of the present invention provides the use of the above-mentioned product containing Cistanche deserticola exosome-like nanovesicles in breast augmentation. When the product is solid, the use is specifically as follows: a small amount of powder is added to one volume of water to dissolve until it is in the form of an essence, and the powder is evenly applied to the breasts and gently patted, with the weight-to-volume ratio of the product to water being 1 g: 1-3 mL.

[0025] When the product is liquid, the specific application is: take 4-5 drops and apply evenly on the chest, and pat gently to promote absorption.

[0026] The beneficial effects of the present invention are:

[0027] (1) A new application of Cistanche deserticola exosome-like nanovesicles was developed, namely, the nanovesicles were used in breast enhancement products, showing good breast enhancement and breast firming effects; experimental results showed that the Cistanche deserticola exosome-like nanovesicles can effectively increase the lipid content in cells, increase the volume of intracellular lipid droplets, and effectively improve the survival rate of 3T3-L1 cells and increase the number of fat cells, thereby achieving a good breast enhancement effect;

[0028] (2) A method for preparing Cistanche deserticola exosome-like nanovesicles with a simple preparation process and high content of active ingredients is provided. The protein content of the Cistanche deserticola exosome-like nanovesicles obtained by the method of the present invention is as high as 42.6%, which is 5.5% higher than that of the traditional ultrafiltration centrifugation method. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the effect of the Cistanche deserticola exosome-like nanovesicles obtained in Example 1 of the present invention on cell survival rate at different concentrations;

[0030] Figure 2 The lipid content in 3T3-L1 cells was detected by Oil Red O staining of the Cistanche deserticola exosome-like nanovesicles obtained in Example 1 of the present invention at a concentration of 20 ug / mL;

[0031] Figure 3 This is a quantitative graph of the lipid content of the Cistanche deserticola exosome-like nanovesicles obtained in Example 1 of the present invention at a concentration of 20 ug / mL stained with Oil Red O. DETAILED DESCRIPTION

[0032] In order to enable those skilled in the art to better understand the present invention, the present invention will be further explained in conjunction with specific embodiments.

[0033] Example 1

[0034] A method for preparing Cistanche deserticola exosome-like nanovesicles comprises the following steps:

[0035] (1) After washing the Cistanche deserticola, dry it at 80°C and then grind it into 1000 mesh using an ultrafine grinder to obtain 10 g of Cistanche deserticola powder;

[0036] (2) Add 10 times the mass of distilled water to the Cistanche deserticola powder obtained in (1), beat with a tissue homogenizer for 10 minutes, centrifuge at 5000g for 0.5 hours, collect the supernatant, centrifuge the obtained supernatant at 10000g for 1 hour, and collect the supernatant again to obtain the Cistanche deserticola centrifuge pretreatment solution;

[0037] (3) The Cistanche deserticola centrifugal pretreatment liquid obtained in (2) was treated with a direct current of 100 mA for 3 h, the liquid at 1 / 4 of the length of the positive terminal was collected, and the liquid was decolorized with a D941 macroporous adsorption resin to obtain Cistanche deserticola exosome-like nanovesicles;

[0038] The specific operation of the decolorization treatment is as follows: take the pretreated macroporous adsorption resin into a 250 mL conical flask, add 50 mL of the liquid to be decolorized, place it in a shaker, and shake at 25°C and 130 r / min for 24 h;

[0039] The pretreatment operation of macroporous adsorption resin is as follows:

[0040] The macroporous adsorption resin was soaked in 95% ethanol by volume for 24 h, and then washed with deionized water until there was no alcohol smell. The washed resin was then soaked in 1M sodium hydroxide and 1M hydrochloric acid for 5 h each, and washed with deionized water until the pH value of the resin reached 7. Finally, the resin was soaked in 95% ethanol by volume for 12 h, and then washed with distilled water until there was no alcohol smell.

[0041] Example 2

[0042] The difference from Example 1 is that in (2), 40 times of distilled water was added to the Cistanche deserticola powder obtained in (1), and the mixture was slurried using a tissue homogenizer for 10 minutes.

[0043] Example 3

[0044] The difference from Example 1 is that: in (2), the supernatant is collected after centrifugation at 5000g for 0.5h, and the obtained supernatant is further centrifuged at 12000g for 1h.

[0045] Example 4

[0046] The difference from Example 1 is that: in (2), the supernatant is collected after centrifugation at 3000g for 1 hour, and the obtained supernatant is further centrifuged at 15000g for 1 hour.

[0047] Example 5

[0048] The difference from Example 1 is that: in (2), the supernatant is collected after centrifugation at 2000g for 0.5h, and the obtained supernatant is further centrifuged at 8000g for 1h.

[0049] Example 6

[0050] The difference from Example 1 is that in (3), the direct current treatment with a current intensity of 80 mA was carried out for 3 h.

[0051] Example 7

[0052] The difference from Example 1 is that in (3), the direct current treatment with a current intensity of 100 mA was performed for 2 h.

[0053] Comparative Example 1

[0054] The difference between this comparative example and Example 1 is that: in (2), 10 times the mass of distilled water is added to the Cistanche deserticola powder obtained in (1), and the mixture is slurried for 10 minutes using a tissue homogenizer. The mixture is first centrifuged at a centrifugal force of 3000g for 0.5 hours, and the supernatant is collected. The obtained supernatant is then centrifuged at a centrifugal force of 3000g for 1 hour, and the supernatant is collected again to obtain a Cistanche deserticola centrifugal pretreatment liquid.

[0055] Comparative Example 2

[0056] The biggest difference between this comparative example and Example 1 is that the current intensity in (3) is 20 mA, and the other steps and operations are the same as those in Example 1.

[0057] Comparative Example 3

[0058] The biggest difference between this comparative example and Example 1 is that the electrophoresis time in (3) is 0.1 h, and the other steps and operations are the same as those in Example 1.

[0059] Comparative Example 4

[0060] The biggest difference between this comparative example and Example 1 is that the current intensity in (3) is 180 mA, and the other steps and operations are the same as those in Example 1.

[0061] Comparative Example 5

[0062] The biggest difference between this comparative example and Example 1 is that the electrophoresis time in (3) is 10 h, and the other steps and operations are the same as in Example 1.

[0063] The protein content of the Cistanche deserticola exosome-like nanovesicles obtained in Examples 1-7 and Comparative Examples 1-5 is shown in Table 1 and the attached Figure 1 .

[0064] Among them, the protein content in Cistanche deserticola exosome-like nanovesicles was determined by BCA protein concentration assay method; the cell viability was determined by MTT cell viability assay method.

[0065] Table 1 Protein content of Cistanche deserticola exosome-like nanovesicles in each experimental group

[0066] Group Protein content of exosome-like nanovesicles (%) Cell survival rate (%) Example 1 42.6 201.65 Example 2 39.7 179.2 Example 3 39.4 182.84 Example 4 40.2 182.55 Example 5 37.6 173.67 Example 6 39.8 188.41 Example 7 38.4 176.49 Comparative Example 1 29.1 96.98 Comparative Example 2 31.9 79.23 Comparative Example 3 26.2 88.08 Comparative Example 4 28.5 71.54 Comparative Example 5 37.6 94.89

[0067] According to the data in the above table, the protein content extracted from the Cistanche deserticola exosome-like nanovesicles in Examples 1-3 is relatively high, and the highest protein content in Example 1 is as high as 42.6%. Furthermore, the present invention also tested the cell viability by the MTT method, and found that the cell viability of the examples was significantly higher than that of the control example, and the highest protein content can reach about 201.65%.

[0068] The breast enhancement effects of the Cistanche deserticola exosome-like nanovesicles obtained from the above experimental groups were investigated. The specific experimental procedures are as follows:

[0069] 3T3-L1 cells were cultured in vitro and divided into a blank group (normal culture group), a control group (acetyl hexapeptide-38, i.e., breast enhancement peptide group), and an example group (exosome-like nanovesicle sample treatment group). After adding different samples, the cells were treated for 72 h and the lipid content was measured. The results are shown in the attached figure. Figure 2-3 .

[0070] Attachment Figure 2-3 In the figure, from left to right are the lipid contents in the cells of the normal group, the control group (acetyl hexapeptide-38, i.e., breast enhancement peptide treatment group), and the example group (20ug / mL Cistanche deserticola exosome-like nanovesicle treatment group).

[0071] Obviously, the Cistanche deserticola exosome-like nanovesicles obtained in the present invention show excellent effects in increasing the cellular lipid content.

[0072] In addition, the present invention also provides a product containing the above-mentioned Cistanche deserticola exosome-like nanovesicles.

[0073] Experimental Example 1

[0074] The preparation method of the product containing Cistanche deserticola exosome-like nanovesicles is as follows:

[0075] Take 10 parts of the Cistanche deserticola exosome-like nanovesicles obtained in Example 1, add 2 parts of trehalose thereto, adjust the pH to 6.5 with phosphate buffer, place it in a vacuum freeze dryer and freeze-dry it, first pre-freeze it at -42°C for 8 h, and then freeze-dry it at -55°C for 48 h to obtain a freeze-dried powder containing Cistanche deserticola exosome-like nanovesicles.

[0076] Experimental Example 2

[0077] The difference from Experimental Example 1 is:

[0078] 5 parts of nanovesicles, 10 parts of trehalose, 70 parts of mannitol, and 20 parts of phosphate.

[0079] Experimental Example 3

[0080] The difference from Experimental Example 1 is:

[0081] 7 parts of nanovesicles, 50 parts of mannitol, 3 parts of sorbitol, 30 parts of maltitol and 10 parts of phosphate.

[0082] Experimental Example 4

[0083] The difference from Experimental Example 1 is:

[0084] 10 parts of nanovesicles, 60 parts of trehalose, and 30 parts of phosphate.

[0085] The final experiment showed that the different types of products mentioned above all showed good results in the process of lipid content verification.

Claims

1. An application of Cistanche deserticola exosome-like nanovesicles in the preparation of breast enhancement products, characterized in that: The nanovesicles are prepared by the following method: (1) Washing, drying, and crushing the Cistanche deserticola to obtain Cistanche deserticola powder; (2) adding water to the Cistanche deserticola powder obtained in (1) to make a pulp, collecting the supernatant after differential centrifugation to obtain a Cistanche deserticola centrifuge pretreatment solution; The differential centrifugation operation is specifically as follows: first centrifugation at a centrifugal force of 2000-5000 g for 0.5-4 h; then centrifugation at a centrifugal force of 6000-15000 g for 0.5-4 h; (3) The Cistanche deserticola centrifugal pretreatment liquid obtained in (2) was treated with direct current, and the liquid at 1 / 8 to 1 / 3 of the length of the positive terminal was collected and decolorized to obtain Cistanche deserticola exosome-like nanovesicles; the direct current treatment conditions were: current 50 to 150 mA, and electrophoresis time 1 to 5 h.

2. The use according to claim 1, characterized in that In (2), 5 to 40 times the mass of water is added to the Cistanche deserticola powder obtained in (1), and then the powder is homogenized in a tissue homogenizer for 10 to 30 minutes.

3. The use according to claim 1, characterized in that In (3), a macroporous adsorption resin is used to decolorize the collected liquid, and the macroporous resin is D941.

4. The use according to claim 1, wherein The product contains at least the Cistanche deserticola exosome-like nanovesicles, and uses the Cistanche deserticola exosome-like nanovesicles as the main active ingredient. In the product, the Cistanche deserticola exosome-like nanovesicles account for 1 to 10 parts, and other excipients account for 90 to 99 parts, and the other excipients are selected from at least one of trehalose, mannitol, sorbitol, maltitol, and phosphate.

5. The use according to claim 1, characterized in that When the product is a solid powder, it also includes (4): first using a phosphate buffer to adjust the pH of the Cistanche deserticola exosome-like nanovesicles obtained in (3) to 5.5~8.5, and then freeze-drying. The freeze-drying operation is specifically: first pre-freezing at -50~-40℃ for 5~10 hours, heating the main partition to 40-60℃, and then freezing at -60~-50℃ for 48~72 hours.

6. The use according to claim 1, wherein When the product is solid, the application is specifically as follows: dissolve the solid product in water until it is in the form of an essence, apply it evenly on the chest, and pat gently, with the weight-to-volume ratio of the product to water being 1g:1~3mL; When the product is liquid, the application is specifically: take 4 to 5 drops of liquid product and apply it evenly on the chest, and pat gently.

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