Liquorice-sourced external vesicle and skin care product applying external vesicle

Abstract

The invention provides a liquorice-sourced external vesicle. A preparation method of the liquorice-sourced external vesicle comprises the following steps: obtaining liquorice juice; filtering the liquorice juice for multiple times; and removing small-size fragments from the filtrate to obtain a solution containing the liquorice-sourced external vesicles. In the prior art, according to the size of vesicles, extraction methods such as ultracentrifugation, polymer precipitation, molecular size exclusion, ultra-sonar filtration or kit extraction are adopted, but all the methods are difficult to enlarge extraction and improve, and the extraction quality and the extraction quantity are limited. According to the method, based on the sizes of the vesicles, the plant external vesicles are rapidly obtained on a large scale by adopting a tangential flow ultrafiltration method, and the vesicles with a clearer size range are collected according to the commercially available sizes of membrane packages, so that biological effect analysis and subsequent ground production application are facilitated, and a basis is provided for clinical application.

Description

Technical Field

[0001] The present invention relates to a biological extracellular vesicle, in particular to an extracellular vesicle derived from licorice and a skin care product applying the extracellular vesicle. Background Art

[0002] Long-term and excessive ultraviolet radiation can cause skin oxidative stress. The accumulation of excessive reactive oxygen species (ROS) can not only damage the structures and functions of biological macromolecules such as proteins, DNA, and lipids, but also activate a series of signal transduction pathways by mitogen-activated protein kinase (MAPK), nuclear transcription factor (NF), transcription activator factor (ATF), etc., inducing skin cell apoptosis, degradation of extracellular matrix (ECM), and activation of tumor-related signals. Eventually, it leads to skin aging and deepening of wrinkles. The clinical manifestations are skin relaxation and pigmentation deepening. Currently, most of the treatment regimens adopted on the market are to add antioxidant bioactive substances to skin care products. Among them, exosomes have advantages such as small size, low immunity, and easy penetration through biological membranes, and have received more and more attention from researchers. However, the active ingredients and mechanisms of action are not yet clear, and there are difficulties in extraction and preparation.

[0003] Exosomes include EVs. EVs refer to natural nanoscale vesicles with membrane structures secreted by (animals, plants, microorganisms). They carry molecules such as nucleic acids, proteins, and lipids, and efficiently transmit intercellular signal regulators. According to the source and size, they can be divided into exosomes of animal cells, with a size of 30 - 150 nm, and microvesicles derived from plants or other microorganisms, with a size of less than 1000 nm. Therefore, according to the vesicle size, the existing extraction methods include ultracentrifugation, polymer precipitation, molecular size exclusion, ultrasonic nanofiltration, or extraction using kits. However, the above methods all have difficulties in scale-up extraction and improvement, and there are limitations in the extraction quality and quantity. Summary of the Invention

[0004] The present invention provides an extracellular vesicle derived from licorice and a skin care product applying the extracellular vesicle to achieve large-scale preparation of the extracellular vesicle derived from licorice.

[0005] The present invention provides an extracellular vesicle derived from licorice. The preparation method of the extracellular vesicle derived from licorice includes:

[0006] Obtain licorice juice;

[0007] Filter the licorice juice multiple times;

[0008] The filtrate is subjected to removal of small-sized fragments to obtain a solution containing exosomes derived from licorice.

[0009] Furthermore, the method for obtaining the licorice juice is as follows:

[0010] Take 1 part by weight of fresh licorice in 2 - 4 parts by weight of water;

[0011] Break the cell walls and extract the juice to obtain licorice juice.

[0012] Even further, the temperature of the water is 2 - 6°C.

[0013] Furthermore, the conditions for breaking the cell walls are: 2800 rpm, lasting for 2 minutes.

[0014] Furthermore, the multiple filtrations of the licorice juice include:

[0015] The licorice juice is filtered through a gauze to remove large particles;

[0016] Use a 300 - 400 mesh sieve to filter and obtain a preliminary filtrate;

[0017] Use a hollow fiber column to ultrafilter the preliminary filtrate to obtain a filtered liquid.

[0018] Even further, the use of a hollow fiber column to ultrafilter the preliminary filtrate to obtain a filtered liquid includes:

[0019] Set the pump flow rate to 500 mL / min, the transmembrane pressure to 0.23 bar, the transmembrane flux to 18 L / h, and use 3 L of water to wash the reflux liquid.

[0020] Even further, the membrane area of the hollow fiber column is 220 cm 2 .

[0021] Furthermore, the removal of small-sized fragments from the filtrate to obtain a solution containing exosomes derived from licorice includes:

[0022] Use membrane package ultrafiltration to remove fragments below 30 nm. The tested membrane package has a membrane area of 0.1 m 2 , the minimum operating volume is 30 mL, set the pump flow rate to 400 mL / min, the transmembrane pressure to 0.43 bar, the transmembrane flux to 12 L / h, and use 2 L of PBS to wash the filtered solution to obtain a solution containing exosomes derived from licorice.

[0023] The present invention also discloses a skin care product applying the above exosomes. The skin care product includes 50 - 100 parts by weight of a base and 5 - 50 parts by weight of a solution containing exosomes derived from licorice.

[0024] Furthermore, the base is one of an emulsion and a microemulsion.

[0025] Compared with the existing extraction processes such as ultracentrifugation in the prior art, based on the size of the vesicles, the present invention uses the tangential flow ultrafiltration method to obtain plant extracellular vesicles on a large scale and rapidly, and collects vesicles with a more definite size range through commercially available membrane package sizes, which helps to analyze biological effects and subsequent large-scale production applications, providing a basis for clinical applications. Description of the Drawings

[0026] Figure 1 It is the NTA result diagram of the extracellular vesicles derived from Glycyrrhiza uralensis Fisch. in Example 1 of the present invention ( Figure 1 A is the relationship diagram between the particle size and the planar size of the extracellular vesicles derived from Glycyrrhiza uralensis Fisch., Figure 1 B is the relationship diagram between the volume and the three-dimensional volume of the extracellular vesicles derived from Glycyrrhiza uralensis Fisch.);

[0027] Figure 2 It is the TEM result diagram of the extracellular vesicles derived from Glycyrrhiza uralensis Fisch. in Example 1 of the present invention;

[0028] Figure 3 It is the apoptosis result diagram of hacat cells in the normal group, model group, and treatment group after UV irradiation in Example 1 of the present invention;

[0029] Figure 4 It is the cutin change result diagram of the normal group, model group, and treatment group after UV irradiation in Example 1 of the present invention. Detailed Embodiments

[0030] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0031] In the embodiments of the present invention, the geographical indication product approved by the General Administration of Quality Supervision, Inspection and Quarantine, Baicheng Glycyrrhiza uralensis Fisch., is selected as the main source of Glycyrrhiza uralensis Fisch. extracellular vesicles. The roots of Baicheng Glycyrrhiza uralensis Fisch. are thick, uniform, with a flat appearance, a fragrant smell, and a strong sweet taste. It has the effects of invigorating the spleen and replenishing qi, clearing heat and detoxifying, resolving phlegm and relieving cough, alleviating pain and harmonizing medicinal properties when used as medicine for traditional rhizomes. It has been applied in the fields of food, cosmetics, etc. for a long time. Therefore, Baicheng Glycyrrhiza uralensis Fisch. is used as the source for extracting Glycyrrhiza uralensis Fisch. extracellular vesicles.

[0032] The currently reported method for preparing glycyrrhiza-derived extracellular vesicles mainly uses the method of ultracentrifugation. This method requires expensive instruments, has low processing efficiency, and there are also batch-to-batch differences in the quality of the obtained vesicles. For the remaining preparation methods, such as extraction using kits, other reagents need to be introduced and are difficult to remove. At the same time, there are also batch-to-batch differences and low extraction yields. The size exclusion preparation method also has the above problems. These problems have led to the difficulty in exerting the reported biological effects in clinical practice. Especially, its application in skin care products is greatly restricted. In this application, the tangential flow filtration method combining a hollow fiber column and a membrane package can well solve the above existing problems. A large number of glycyrrhiza-derived extracellular vesicles can be stably prepared. At the same time, through photo-damaged cells and animal skin models, it is verified that the extracted extracellular vesicles have biological effects such as antioxidant. Generally speaking, this preparation method can extract glycyrrhiza extracellular vesicles on a large scale and has good morphological characteristics and skin photo-damage repair activity.

[0033] Specifically, the present invention provides a glycyrrhiza-derived extracellular vesicle, and the preparation method of the glycyrrhiza-derived extracellular vesicle includes:

[0034] S1. Obtain glycyrrhiza juice;

[0035] S2. Filter the glycyrrhiza juice multiple times;

[0036] S3. Remove small-sized fragments from the filtrate to obtain a solution containing glycyrrhiza-derived extracellular vesicles.

[0037] Optionally, the method for obtaining glycyrrhiza juice in S1 is:

[0038] S101. Take 1 part by weight of fresh glycyrrhiza in 2 - 4 parts by weight of water;

[0039] S102. Break the cell wall and extract the juice as glycyrrhiza juice.

[0040] Particularly, the temperature of the water is 2 - 6 °C.

[0041] Particularly, the conditions for breaking the cell wall are: 2800 rpm, for 2 minutes.

[0042] Optionally, the multiple filtrations of the glycyrrhiza juice in S2 include:

[0043] S201. Filter the glycyrrhiza juice through a gauze to remove large particles;

[0044] S202. Use a 300 - 400 mesh sieve to filter and obtain a preliminary filtrate;

[0045] S203. Ultrafilter the preliminary filtrate using a hollow fiber column to obtain a filtered liquid.

[0046] Specifically, in S203, ultrafiltration of the initial liquid is performed using a hollow fiber column to obtain the filtered liquid, including:

[0047] Set the pump flow rate to 500 mL / min, the transmembrane pressure to 0.23 bar, the transmembrane flux to 18 L / h, and use 3 L of water to wash the reflux liquid.

[0048] Specifically, the membrane area of the hollow fiber column is 220 cm 2 .

[0049] Optionally, in S3, removal of small-sized fragments from the filtrate is performed to obtain a solution containing exosomes derived from licorice, including:

[0050] Use a membrane package for ultrafiltration to remove fragments below 30 nm. The tested membrane package area is 0.1 m 2 , the minimum operating volume is 30 mL, set the pump flow rate to 400 mL / min, the transmembrane pressure to 0.43 bar, the transmembrane flux to 12 L / h, and use 2 L of PBS to wash the filtered solution to obtain a solution containing exosomes derived from licorice.

[0051] The present invention also discloses a skin care product applying the above exosomes. The skin care product includes 50 - 100 parts by weight of a base and 5 - 50 parts by weight of a solution containing exosomes derived from licorice.

[0052] Among them, the base is one of an emulsion and a microemulsion.

[0053] To specifically illustrate the solution and effects of the present invention, the following examples are listed.

[0054] Example 1

[0055] Step 1. Wash the freshly picked Baicheng licorice to remove sediment. Add 3000 mL of 4°C water to every 1000 g of licorice, and use the juicing function with a blender at 2800 rpm for 2 minutes to obtain the juice.

[0056] Step 2. Filter the juice through a gauze to remove large particles, and then use a 400-mesh sieve to filter to obtain 3000 mL of the filtered initial liquid; ultrafilter the initial liquid using a hollow fiber column. The tested membrane area of the hollow fiber column is 220 cm 2 Set the pump flow rate to 500 mL / min, the transmembrane pressure to 0.23 bar, the transmembrane flux to 18 L / h, and it is possible to process 15 L of juice within one hour. Use 3 L of water to wash the reflux liquid.

[0057] Among them, a total of 18 L of the initial liquid can be processed in this step in one hour, which is equivalent to processing 5 Kg of Baicheng licorice in one hour.

[0058] Step 3. Take the liquid obtained in Step 2, and use membrane package ultrafiltration to remove fragments below 30 nm. The tested membrane package area is 0.1 m 2 , the minimum running volume is 30 mL, set the pump flow rate to 400 mL / min, the transmembrane pressure to 0.43 bar, the transmembrane flux to 12 L / h, and use 2 L of PBS to wash the filter.

[0059] Among them, through actual measurement, Step 3 can process 12 L of juice within 1 hour, which is equivalent to processing 12 L of the liquid obtained in Step 2 in total within 1 hour, and is equivalent to completing the processing of the outer vesicles of 5 Kg of licorice in 1.6 h. Moreover, this processing capacity can increase linearly with the increase of the membrane package area and the area of the hollow fiber column.

[0060] Example 1 of the present invention further includes a skin care device. The skin care product includes 100 parts by weight of emulsion and 20 parts by weight of a solution containing licorice-derived outer vesicles.

[0061] The specific preparation method is as follows: Take 20 parts by weight of the emulsion and place it in the licorice-derived outer vesicles, stir at 4 - 10 °C for 5 min, then add the remaining emulsion and stir for 10 min to obtain the skin care device.

[0062] Among them, the emulsion is specifically composed of 0.5 part of vitamin C, 1 part of inositol, 0.5 part of ginkgo biloba extract, 1 part of sialic acid, 2 parts of nicotinamide, and 80 - 120 parts of water.

[0063] Example 2

[0064] Step 1. Take freshly picked Baicheng licorice, wash away the sediment, add 2000 mL of 4 °C water to every 1000 g of licorice, use the juicing function, the blender at 2800 rpm for 2 minutes to obtain the juice;

[0065] Step 2. Filter the juice through a gauze to remove large particles, then use a 400 - mesh sieve to filter and obtain 2000 mL of the initial filtered liquid; Ultrafilter the initial liquid using a hollow fiber column. The tested membrane area of the hollow fiber column is 220 cm 2 Set the pump flow rate to 500 mL / min, the transmembrane pressure to 0.23 bar, the transmembrane flux to 18 L / h, and it can process 15 L of juice within one hour, and use 3 L of water to wash the reflux liquid.

[0066] Among them, this step can process 18 L of the initial liquid in total within 1 hour, which is equivalent to processing 5 Kg of Baicheng licorice in 1 hour.

[0067] Step 3. Take the liquid obtained in Step 2, and use membrane package ultrafiltration to remove fragments below 30 nm. The tested membrane package area is 0.1 m 2, the minimum operating volume is 30 mL, the pump flow rate is set at 400 mL / min, the transmembrane pressure is 0.43 bar, the transmembrane flux is 12 L / h, and 2 L of PBS is used to wash the filter.

[0068] Among them, through actual measurement, step 3 can process 12 L of juice within 1 hour, which is equivalent to a total of 12 L of the liquid obtained in step 2 processed in 1 hour, and it is equivalent that the outer vesicles of 5 Kg of licorice can be processed in 1.6 h, and this processing capacity can increase linearly with the increase of the membrane package area and the hollow fiber column area.

[0069] Example 3

[0070] Step 1. Take the freshly picked Baicheng licorice, wash the sediment, add 4000 mL of 4°C water to every 1000 g of licorice, perform the juicing function, the blender at 2800 rpm for 2 minutes to obtain the juice;

[0071] Step 2. The juice is filtered through a gauze to remove large particles, and then a 400-mesh sieve is used for filtration to obtain 4000 mL of the initial filtered liquid; the initial liquid is ultrafiltered using a hollow fiber column, and the membrane area of the hollow fiber column is measured to be 220 cm 2 Set the pump flow rate at 500 mL / min, the transmembrane pressure at 0.23 bar, the transmembrane flux at 18 L / h, and it can process 15 L of juice within one hour, and 3 L of water is used to wash the reflux liquid.

[0072] Among them, this step can process a total of 18 L of the initial liquid in 1 hour, which is equivalent to processing 5 Kg of Baicheng licorice in 1 hour.

[0073] Step 3. Take the liquid obtained in step 2, and use membrane package ultrafiltration to remove fragments below 30 nm. The membrane area of the membrane package is measured to be 0.1 m 2 , the minimum operating volume is 30 mL, the pump flow rate is set at 400 mL / min, the transmembrane pressure is 0.43 bar, the transmembrane flux is 12 L / h, and 2 L of PBS is used to wash the filter.

[0074] Among them, through actual measurement, step 3 can process 12 L of juice within 1 hour, which is equivalent to a total of 12 L of the liquid obtained in step 2 processed in 1 hour, and it is equivalent that the outer vesicles of 5 Kg of licorice can be processed in 1.6 h, and this processing capacity can increase linearly with the increase of the membrane package area and the hollow fiber column area.

[0075] Example 4

[0076] Step 1. Take the freshly picked Baicheng licorice, wash the sediment, add 3000 mL of 4°C water to every 1000 g of licorice, perform the juicing function, the blender at 2800 rpm for 2 minutes to obtain the juice;

[0077] Step 2. The juice extract is filtered through a gauze to remove large particles, and then a 400-mesh sieve is used for filtration to obtain 3000 mL of the initially filtered liquid. The initially filtered liquid is ultrafiltered using a hollow fiber column. The membrane area of the hollow fiber column is tested to be 220 cm 2 Set the pump flow rate at 500 mL / min, the transmembrane pressure at 0.23 bar, and the transmembrane flux at 18 L / h. It is possible to process 15 L of juice within one hour, and 3 L of water is used to wash the reflux liquid.

[0078] Among them, a total of 18 L of the initially filtered liquid can be processed in this step within one hour, which is equivalent to processing 5 Kg of Baicheng licorice within one hour.

[0079] Step 3. Take the liquid obtained in Step 2 and use a membrane package for ultrafiltration to remove fragments smaller than 30 nm. The membrane area of the membrane package is tested to be 0.1 m 2 , the minimum operating volume is 30 mL, set the pump flow rate at 400 mL / min, the transmembrane pressure at 0.43 bar, and the transmembrane flux at 12 L / h, and use 2 L of PBS to wash the filter.

[0080] Among them, through actual measurement, it can be achieved to process 12 L of juice within one hour in Step 3, which is equivalent to processing a total of 12 L of the liquid obtained in Step 2 within one hour, equivalent to completing the processing of the outer vesicles of 5 Kg of licorice within 1.6 h, and this processing capacity can increase linearly with the increase of the membrane package area and the hollow fiber column area.

[0081] Example 1 of the present invention further includes a skin care device. The skin care product includes 100 parts by weight of emulsion and 50 parts by weight of a solution containing licorice-derived outer vesicles.

[0082] The specific preparation method is as follows: Take 20 parts by weight of the emulsion and add it to the licorice-derived outer vesicles, stir at 4 - 10 °C for 5 min, then add the remaining emulsion and stir for 10 min to obtain the skin care device.

[0083] Taking Example 1 as an example, as Figure 1 、 2 shown, it can be seen that the method of Example 1 of the present invention can obtain a large number of vesicles between 50 - 250 nm directionally

[0084] To test the effect of Example 1, a light damage experiment is conducted using HaCaT cells. Specifically:

[0085] Culture HaCaT cells. When the growth density reaches about 80%, digest with trypsin and transfer to three culture plates;

[0086] Among them, the three culture plates are divided into a normal group, a model group, and a treatment group according to the experimental design. In the treatment group, add the licorice-derived outer vesicle solution obtained in Example 1 until the outer vesicle content in the solution in the culture plate is 10 8 / mL.

[0087] HaCaT cells were cultured in three culture plates for 4 hours;

[0088] In the normal group, no ultraviolet lamp intervention was performed. In the model group and the treatment group, ultraviolet lamp intervention was used (intervention time: 10 min, irradiation dose: 4 J / cm 2 ). Subsequently, within the next four hours, the model group and the extracellular vesicle group were irradiated with ultraviolet light;

[0089] After culturing for 24 hours, the cell survival rate was measured.

[0090] As Figure 3 shown, after using the extracellular vesicle solution derived from licorice obtained in Example 1, the ability of light irradiation recovery of cells can be effectively improved.

[0091] To test the effect of the skin care product in Example 1, the following mouse test was conducted:

[0092] Sixty 6-8-week-old male Balb / c mice were taken. After 7 days of environmental adaptation culture, the mice were divided into three groups, with 20 mice in each group, namely the normal group, the model group, and the treatment group. The hair on the backs of the mice was shaved and depilated with 2% sodium sulfide, and the area was 1.5 cm × 3 cm. After anesthetizing the mice in the model group and the treatment group with 1.5% isoflurane, the ultraviolet lamp was placed at a vertical height of 10 cm from the mice, and the power was turned up to 300 mJ / cm 2 for 5 min; this process was repeated for 28 consecutive days to establish a photo-damaged mouse model. Before irradiation, the skin care product of Example 1 was applied to the backs of the mice in the treatment group every day; before irradiation, the lotion of Example 1 (the lotion component without added vesicles) was applied to the backs of the mice in the control group every day.

[0093] The skin of the three groups of mice was tested. As a result, as Figure 4 shown, it can be seen that Figure 4 in the treatment group, the skin of mice can be effectively protected. Compared with the model group, the formation of skin keratinization can be effectively reduced, and the thickness of the stratum corneum of the skin of the mice in the treatment group is similar to that of the normal group, and even better than that of the normal group in some areas.

[0094] In summary, the method disclosed in the present invention can be applied to the licorice treatment process, can obtain a large amount of licorice extracellular vesicles in a short time, and after testing, the licorice extracellular vesicles still have good activity and can be applied to the field of sunscreen skin care.

[0095] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that after reading the specification of this application, they can still modify or equivalently replace the specific implementation manners of the present invention, but these modifications or changes do not depart from the protection scope of the pending claims of this invention application.

Claims

1. A licorice-derived extracellular vesicle, characterized in that: The preparation method of the licorice-derived exosomes comprises: Get licorice juice; The liquorice juice is filtered several times; The filtrate was subjected to removal of small-sized fragments to obtain a solution containing licorice-derived extracellular vesicles.

2. The licorice-derived extracellular vesicle according to claim 1, characterized in that: The method for obtaining the liquorice juice is as follows: Take 1 part by weight of fresh licorice and dissolve it in 2-4 parts by weight of water; The wall is broken and the juice is extracted to form licorice juice.

3. The licorice-derived extracellular vesicle according to claim 2, characterized in that: The temperature of the water is 2-6°C.

4. The licorice-derived extracellular vesicle according to claim 1, characterized in that: The wall breaking condition is: 2800 rpm, lasting for 2 minutes.

5. The licorice-derived extracellular vesicle according to claim 1, characterized in that: The method of filtering the liquorice juice multiple times comprises: Licorice juice was filtered through gauze to remove large particles; Use a 300-400 mesh sieve to filter and obtain the filtered initial liquid; The initial solution is ultrafiltered using a hollow fiber column to obtain a filtered liquid.

6. The licorice-derived extracellular vesicle according to claim 5, characterized in that: The method of ultrafiltration of the initial liquid using a hollow fiber column to obtain a filtered liquid comprises: Set the pump flow rate to 500 mL / min, the transmembrane pressure to 0.23 bar, the transmembrane flux to 18 L / h, and use 3 L of water to wash the reflux liquid.

7. The licorice-derived extracellular vesicle according to claim 6, characterized in that: The hollow fiber column membrane area is 220 cm 2 .

8. The licorice-derived extracellular vesicle according to claim 1, characterized in that: The step of removing small-sized fragments from the filtrate to obtain a solution containing licorice-derived exosomes comprises: The membrane package was used for ultrafiltration to remove fragments below 30 nm. The membrane area of ​​the test membrane was 0.1 m 2 , the minimum operating volume was 30 mL, the pump flow rate was set to 400 mL / min, the transmembrane pressure was 0.43 bar, the transmembrane flux was 12 L / h, and 2 L PBS was used to wash the filtrate solution to obtain a solution containing licorice-derived extracellular vesicles.

9. A skin care product using the exosome according to any one of claims 1 to 8, characterized in that: The skin care product comprises 50-100 parts by weight of a base and 5-50 parts by weight of a solution containing licorice-derived extracellular vesicles.

10. The skin care product according to claim 9, characterized in that: The base is one of an emulsion and a microemulsion.

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