Preparation method of cistanche deserticola exosome-like nano vesicles and application of cistanche deserticola exosome-like nano vesicles in breast enlarging products

Abstract

The invention belongs to the technical field of raw materials of daily chemical products, and particularly relates to a preparation method of cistanche exosome-like nano-vesicles and application of the cistanche exosome-like nano-vesicles in breast enlarging products. The product provided by the invention at least contains the cistanche deserticola exosome-like nano-vesicles, the cistanche deserticola exosome-like nano-vesicles are used as main active ingredients, and the cistanche deserticola exosome-like nano-vesicles are obtained after differential centrifugation and direct current electrophoresis treatment. The protein content of the Cistanche deserticola exosome-like nano-vesicles obtained by the invention is up to 42.6%, the intracellular lipid content can be effectively increased, the intracellular lipid droplet volume can be increased, the survival rate of 3T3-L1 cells can be effectively increased, the number of fat cells can be increased, and the Cistanche deserticola exosome-like nano-vesicles have a good breast enlarging effect.

Description

Technical Field

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

[0002] Breast augmentation, as an important aspect of women's pursuit of beauty, has attracted much attention since ancient times. With the development of science and technology, breast augmentation methods are constantly being updated, from the initial surgical breast augmentation, to the later drug breast augmentation, to the current physical breast augmentation, dietary breast augmentation, etc., breast augmentation methods are becoming increasingly diverse. However, no matter how the methods change, safety, health and effectiveness are always the unchanging themes of breast augmentation products.

[0003] In recent years, plant extracts have become the preferred raw materials for breast enhancement products due to their high safety. For example, patents CN112544986A, CN109662939A, CN110585320A, etc. all use plant extracts as the main ingredients of their breast enhancement products. However, in the process of preparing plant extracts, the existing technologies have ignored an important factor that affects the effect of breast enhancement products, that is, the content of active ingredients in the prepared plant extracts. The required plant extracts are obtained only by traditional methods. The content of biologically active ingredients such as proteins, sugars, terpenes, flavonoids, alkaloids, steroids, etc. 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 extract, resulting in difficulty in obtaining genetic materials such as microRNA that play a role in plant sources.

[0004] Cistanche deserticola is a parasitic plant that lives on the roots of the Haloxylon ammodendron tree in the desert. It absorbs nutrients and water from the host Haloxylon ammodendron tree to survive. It has extremely high medicinal value and is known as the "desert ginseng". In addition, Cistanche deserticola is also one of the most frequently used tonic drugs in prescriptions for tonifying the kidney and strengthening yang. Therefore, Cistanche deserticola is often used as an auxiliary ingredient in various health products and even breast enhancement products.

[0005] However, to date, there has been no research on whether Cistanche deserticola really 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 method for preparing Cistanche deserticola exosome-like nanovesicles and 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 Cistanche deserticola exosome-like nanovesicles, comprising the following steps: (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, and obtaining a Cistanche deserticola centrifugal pretreatment liquid; (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.

[0008] In the method for preparing the above-mentioned Cistanche exosome-like nanovesicles provided by the present invention, preferably, in (1), the drying temperature is 40-85° C., and the pulverization is performed by using an ultrafine pulverizer to pulverize the Cistanche to 800-1200 meshes.

[0009] Preferably, in (2), the mass of the added water is 5 to 40 times the mass of the Cistanche deserticola powder.

[0010] Preferably, the beating in (2) is performed by using a tissue homogenizer for 10 to 30 minutes.

[0011] Preferably, the differential centrifugation operation described in (2) is specifically: first centrifuging at a centrifugal force of 2000-5000 g for 0.5-4 h, and then centrifuging at a centrifugal force of 6000-15000 g for 0.5-4 h.

[0012] 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.

[0013] Preferably, the direct current treatment conditions described in (3) are: current 50-150 mA, and electrophoresis time 1-5 h.

[0014] More preferably, the direct current treatment conditions described in (3) are: current 80-130 mA, and electrophoresis time 1-3 h.

[0015] Preferably, in (3), a macroporous adsorption resin is used to decolorize the collected liquid, and the macroporous resin is D941.

[0016] As a further preferred embodiment, in (3), before using the macroporous adsorption resin for decolorization, the macroporous adsorption resin is first pretreated, and the specific operation is as follows: 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 was 7. Finally, the resin was soaked in 95% ethanol by volume for 12 h, and washed with distilled water until there was no alcohol smell for use.

[0017] 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.

[0018] The second aspect of the present invention is to provide a product containing the above-mentioned Cistanche exosome-like nanovesicles, wherein the product at least contains the Cistanche exosome-like nanovesicles, and the Cistanche exosome-like nanovesicles are used as the main active ingredient. In the product, the Cistanche exosome-like nanovesicles account for 1 to 10 parts, and other auxiliary materials account for 90 to 99 parts, and the other auxiliary materials are selected from at least one of trehalose, mannitol, sorbitol, maltitol, and phosphate.

[0019] Furthermore, when the product is a solid powder, the present invention also provides a method for preparing the product, that is, on the basis of preparing the above-mentioned Cistanche exosome-like nanovesicles, it also includes step (4): first using a phosphate buffer to adjust the pH of the Cistanche exosome-like nanovesicles obtained in (3) to 5.5-8.5, and then freeze-drying, wherein 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.

[0020] The third aspect of the present invention is to provide the use of the above-mentioned product containing Cistanche exosome-like nanovesicles in breast augmentation. When the product is solid, the application is specifically: take a small amount of powder and add one volume of water to dissolve it into an essence state, evenly apply it on the chest, and gently pat it, and the weight volume ratio of the product to water is 1g:1~3mL; When the product is liquid, the application is specifically: take 4-5 drops and apply evenly on the chest, and pat gently to promote absorption.

[0021] The beneficial effects of the present invention are: (1) A new application of Cistanche 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 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; (2) A method for preparing Cistanche exosome-like nanovesicles with a simple preparation process and high content of active ingredients is provided. The protein content of the Cistanche 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

[0022] Figure 1 The effect of the Cistanche exosome-like nanovesicles obtained in Example 1 of the present invention on cell survival rate at different concentrations; Figure 2 The lipid content in 3T3-L1 cells was detected by Oil Red O staining of the Cistanche exosome-like nanovesicles obtained in Example 1 of the present invention at a concentration of 20 ug / mL; Figure 3 This is a quantitative graph of the lipid content of the Cistanche 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

[0023] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific implementation methods.

[0024] Example 1 A method for preparing Cistanche deserticola exosome-like nanovesicles comprises the following steps: (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; (2) adding 10 times the mass of distilled water to the Cistanche deserticola powder obtained in (1), beating the powder with a tissue homogenizer for 10 min, centrifuging the powder at 5000 g for 0.5 h, collecting the supernatant, centrifuging the supernatant at 10000 g for 1 h, and collecting the supernatant again to obtain a Cistanche deserticola centrifugal pretreatment solution; (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 the 1 / 4 length of the positive terminal was collected, and decolorized with a D941 macroporous adsorption resin to obtain Cistanche deserticola exosome-like nanovesicles; 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 it at 25°C and 130 r / min for 24 h; The pretreatment of macroporous adsorption resin is as follows: 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 was 7. Finally, the resin was soaked in 95% ethanol by volume for 12 h, and washed with distilled water until there was no alcohol smell for use.

[0025] Example 2 The difference from Example 1 is that in (2), 40 times 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.

[0026] Example 3 The difference from Example 1 is that in (2), the supernatant is collected after centrifugation at 5000 g for 0.5 h, and the obtained supernatant is further centrifuged at 12000 g for 1 h.

[0027] Example 4 The difference from Example 1 is that in (2), the supernatant is collected after centrifugation at 3000 g for 1 h, and the obtained supernatant is further centrifuged at 15000 g for 1 h.

[0028] Example 5 The difference from Example 1 is that in (2), the supernatant is collected after centrifugation at 2000 g for 0.5 h, and the obtained supernatant is further centrifuged at 8000 g for 1 h.

[0029] Example 6 The difference from Example 1 is that in (3), the direct current treatment with a current intensity of 80 mA is carried out for 3 h.

[0030] Example 7 The difference from Example 1 is that in (3), the direct current treatment with a current intensity of 100 mA is carried out for 2 h.

[0031] Comparative Example 1 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 powder obtained in (1), and the mixture is slurried for 10 min using a tissue homogenizer. The mixture is first centrifuged at 3000 g for 0.5 h and the supernatant is collected. The obtained supernatant is then centrifuged at 3000 g for 1 h and the supernatant is collected again to obtain a Cistanche centrifugal pretreatment liquid.

[0032] Comparative Example 2 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.

[0033] Comparative Example 3 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.

[0034] Comparative Example 4 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.

[0035] Comparative Example 5 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 those in Example 1.

[0036] The protein content of the Cistanche exosome-like nanovesicles obtained in Examples 1-7 and Comparative Examples 1-5 is specifically shown in Table 1 and Attached Figure 1 .

[0037] Among them, the protein content in the Cistanche exosome-like nanovesicles was determined by the BCA protein concentration determination method; the cell viability was determined by the MTT cell viability detection method.

[0038] Table 1 Protein content of Cistanche exosome-like nanovesicles in each experimental group 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 According to the data in the above table, the protein content extracted from the Cistanche exosome-like nanovesicles in Examples 1-3 is relatively high, and the highest content in Example 1 is as high as 42.6%; further, the present invention detected the cell survival rate by the MTT method, and found that the cell survival rate of the example was significantly higher than that of the control example, and the highest could reach about 201.65%.

[0039] The breast enhancement effects of the Cistanche exosome-like nanovesicles obtained from the above experimental groups were explored. The specific experimental operations are as follows: 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 determined. The results are shown in the attached Figure 2-3 .

[0040] Attached 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).

[0041] Obviously, the Cistanche exosome-like nanovesicles obtained in the present invention show excellent effects in increasing the cellular lipid content.

[0042] In addition, the present invention also provides a product containing the above-mentioned Cistanche exosome-like nanovesicles.

[0043] Experimental Example 1 The preparation method of the product containing Cistanche exosome-like nanovesicles is as follows: Take 10 parts of the Cistanche 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 exosome-like nanovesicles.

[0044] Experimental Example 2 The difference from Experimental Example 1 is: 5 parts of nanovesicles, 10 parts of trehalose, 70 parts of mannitol, and 20 parts of phosphate.

[0045] Experimental Example 3 The difference from Experimental Example 1 is: 7 parts of nanovesicles, 50 parts of mannitol, 3 parts of sorbitol, 30 parts of maltitol and 10 parts of phosphate.

[0046] Experimental Example 4 The difference from Experimental Example 1 is: 10 parts of nanovesicles, 60 parts of trehalose, and 30 parts of phosphate.

[0047] The final experiment showed that the above-mentioned different types of products all showed good results in the process of lipid content verification.

Claims

1. A method for preparing Cistanche deserticola exosome-like nanovesicles, characterized in that: The steps include: (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, and obtaining a Cistanche deserticola centrifuge pretreatment liquid; (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 end was collected and decolorized to obtain Cistanche deserticola exosome-like nanovesicles.

2. The preparation method 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 blended with a tissue homogenizer for 10 to 30 minutes.

3. The preparation method according to claim 1, characterized in that: In (2), the differential centrifugation operation 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.

4. The preparation method according to claim 1, characterized in that: (3) The treatment conditions of medium direct current are: current 50~150mA, electrophoresis time 1~5h.

5. The preparation method 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.

6. A product containing the Cistanche deserticola exosome-like nanovesicles according to claim 1, characterized in that: The product at least contains the Cistanche exosome-like nanovesicles, and uses the Cistanche exosome-like nanovesicles as the main active ingredient. In the product, the Cistanche exosome-like nanovesicles account for 1 to 10 parts, and other auxiliary materials account for 90 to 99 parts. The other auxiliary materials are selected from at least one of trehalose, mannitol, sorbitol, maltitol, and phosphate.

7. A method for preparing a product as claimed in claim 6, 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 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.

8. Use of a product containing the Cistanche deserticola exosome-like nanovesicles according to claim 1 in breast augmentation, characterized in that: 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 it gently, and the weight-to-volume ratio of the product to water is 1g:1~3mL; When the product is liquid, the application is specifically: absorb 4 to 5 drops of liquid product and apply evenly on the chest, and pat gently.

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