CN117180164B

CN117180164B - Extraction method of saussurea involucrata purple callus vesicles and application of saussurea involucrata purple callus vesicles in preparation of antioxidant or skin whitening products

Info

Publication number: CN117180164B
Application number: CN202311474586.0A
Authority: CN
Inventors: 王夜宇; 张振兴; 张旭辉; 高琦
Original assignee: Beijing Yaojing Gene Technology Co ltd
Current assignee: Beijing Yaojing Gene Technology Co ltd
Priority date: 2023-11-08
Filing date: 2023-11-08
Publication date: 2024-01-30
Anticipated expiration: 2043-11-08
Also published as: CN117180164A

Abstract

The invention relates to an extraction method of saussurea involucrata purple callus vesicles and application thereof in preparing an antioxidant or whitening skin product, wherein the antioxidant or whitening skin product comprises at least one of I) to V): i) Skin products that promote the scavenging of DPPH radicals; II) skin products that inhibit ROS production; III) skin products that inhibit tyrosinase activity; IV) a skin product that inhibits the production of melanin in cells; v) a skin product that promotes cell proliferation and migration. The saussurea involucrata purple callus vesicles disclosed by the invention can promote the removal of DPPH free radicals, inhibit the generation of ROS, inhibit the activity of tyrosinase, inhibit the generation of melanin in cells, promote the proliferation and migration of cells, and have excellent effects on the aspects of antioxidation and whitening.

Description

Extraction method of saussurea involucrata purple callus vesicles and application of saussurea involucrata purple callus vesicles in preparation of antioxidant or skin whitening products

Technical Field

The invention belongs to the technical field of skin care products or medicines, and relates to an extraction method of saussurea involucrata purple callus vesicles and application thereof in preparing antioxidant or whitening skin products, wherein the skin products comprise the following components: i) Skin products that promote the scavenging of DPPH radicals; II) skin products that inhibit ROS production; III) skin products that inhibit tyrosinase activity; IV) a skin product that inhibits the production of melanin in cells; v) a skin product that promotes cell proliferation and migration.

Background

Saussurea involucrata is a common rare medicinal plant in the folk of mountain areas of China, and is a perennial herb plant of the genus saussurea of the family Compositae. As a rare traditional medicine, the remarkable curative effect of the medicine is receiving more and more attention. Several studies have shown that saussurea involucrata contains a variety of phenolic compounds including phenolic acids, flavonoids, lignans, etc. Some of which have been identified, for example: chlorogenic acid, umbelliferone, scopoletin, syringin, arctiin, rutin and quercetin. Pharmacological researches show that saussurea involucrata has good effects of resisting oxidation, resisting inflammation, easing pain, activating blood circulation to dissipate blood stasis, resisting aging, resisting radiation and the like.

Therefore, many medicinal, cosmetic, health-care and food products using saussurea involucrata as raw materials are made. The interest in saussurea involucrata has increased over the last 30 years, resulting in excessive harvesting of wild plants. Nowadays, saussurea involucrata is almost trace-free and has been listed as a national second-class protection wild plant. The saussurea involucrata cells containing a large amount of bioactive substances are obtained by a plant cell culture technology, are an effective way for solving the problem of wild saussurea involucrata resources, and are widely focused in academia and industry due to the advantages of no cultivated land occupation, no influence of natural environment, controllable production and the like.

The culture mode of saussurea involucrata cells mainly comprises callus culture, suspension cell culture and hairy root culture. These culture techniques have been mature, enabling cell lines to be obtained which contain higher levels of active substances, and enabling large-scale culture. Through systematic composition studies, more than 60 chemical compositions have been determined.

In recent years, research shows that plant cells can secrete a vesicle with the diameter of 30-500nm, and can selectively wrap a plurality of bioactive substances such as lipid protein, mRNA, miRNA and the like, thereby having the effects of clearly reducing the damage degree of tissues and promoting the morphological and functional repair of damaged tissues.

In the existing plant vesicle extraction scheme, the direct extraction from plant callus or whole plant has a plurality of defects. Firstly, a large amount of calli or plants are consumed for extracting plant vesicles, the culture period of the calli or the plants is very long, and the culture period of the calli or the plants is more than a few months and a plurality of years, and many rare plants cannot be picked under the national protection. Secondly, the callus or the whole plant is required to be minced, filter residues are sufficiently filtered, the operation is complicated and time-consuming, and the purity is low. In addition, due to the difference of plant osmotic pressure, phosphate Buffer (PBS) is adopted to possibly cause the rupture of exosome vesicle structures in the long-time extraction process due to the difference of osmotic pressure, so that the exosome extraction yield is reduced, and the effectiveness cannot be exerted.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides an extraction method of saussurea involucrata purple callus vesicles and application thereof in preparing antioxidant or whitening skin products.

The invention provides a method for separating and purifying the vesicles from saussurea involucrata purple callus culture fluid aiming at various defects existing in the extraction of the plant vesicles in the prior art, which greatly simplifies the extraction steps of the plant vesicles, improves the purity and yield of the vesicles, reduces the production cost and shortens the production time. The saussurea involucrata purple callus vesicles have more excellent effects than cell extracts, can promote the removal of DPPH free radicals, reduce the generation of ROS, inhibit the activity of tyrosinase, reduce the generation level of melanin, help the skin to effectively resist photooxidation, improve the complexion and enable the skin to be more whitened and shiny. The active substances in the saussurea involucrata purple callus vesicles are beneficial to exerting efficacy deeply from the inside of the skin, inhibiting subcutaneous pigment deposition and effectively improving the problem of dark and yellow skin. Can also enhance the cell activity to repair the damaged skin quickly, and ensure that the skin state is healthier and more stable.

Solution scheme

In order to achieve the above purpose, the present invention provides the following technical solutions:

in a first aspect, the invention provides an application of saussurea involucrata purple callus vesicles in preparing an antioxidant or whitening skin product.

Further, the antioxidant or whitening skin product comprises at least one of I) to V):

i) Skin products that promote the scavenging of DPPH radicals;

II) skin products that inhibit ROS production;

III) skin products that inhibit tyrosinase activity;

IV) a skin product that inhibits the production of melanin in cells;

v) a skin product that promotes cell proliferation and migration.

Further, the concentration of the saussurea involucrata purple callus vesicles in the product is 10 ⁷ ~10 ¹² Particles/mL, optionally 10 ⁹ ~10 ¹² Particles/mL, optionally 10 ¹⁰ ~10 ¹² particles/mL。

Further, the saussurea involucrata purple callus vesicles are derived from saussurea involucrata purple callus culture fluid.

Further, the saussurea involucrata purple callus is prepared by the following method:

1) Performing illumination culture on saussurea involucrata purple callus, and collecting culture solution;

2) Separating and obtaining saussurea involucrata purple callus vesicles from the culture solution in the step 1), wherein the separating method comprises the following steps: collecting supernatant, filtering to collect filtrate, concentrating the filtrate (optionally concentrating by tangential flow method), and purifying the concentrated solution to obtain saussurea involucrata purple callus vesicles.

In a second aspect, a method for extracting saussurea involucrata purple callus vesicles is provided, which comprises the following steps:

1) Carrying out illumination culture on saussurea involucrata purple callus vesicles, and collecting culture solution;

2) Separating and obtaining saussurea involucrata callus vesicles from the culture solution in the step 1), wherein the separating method comprises the following steps: collecting supernatant, filtering, collecting filtrate, concentrating (optionally concentrating by tangential flow method), and purifying to obtain saussurea involucrata callus vesicles.

In the first or second aspect, in step 2), centrifugation is used for collecting the supernatant, and the centrifugation condition is differential centrifugation: optionally, 300g-1000g, 2000g-4000g and 8000g-10000g are adopted for centrifugation in sequence, the supernatant is taken for subsequent centrifugation in the first two times, and the supernatant is taken for filtration after the third centrifugation.

In the first or second aspect described above, in step 2), the pore size of the filtered membrane is not less than 0.2. Mu.m, optionally not less than 0.45. Mu.m, optionally 0.45 μm to 1.5 μm or 0.8. Mu.m.

In the first or second aspect, in the step 2), the hollow fiber is used for concentration, the molecular weight cut-off of the hollow fiber is more than or equal to 100KD, alternatively 100 KD-800 KD, alternatively 100 KD-750 KD, alternatively 100 KD-300 KD.

In the first or second aspect, in the step 2), the concentration multiple is 50 to 200 times, or 80 to 150 times, or 100 to 150 times.

In the first or second aspect described above, in step 1), the saussurea involucrata purple callus is obtained by inducing young stem or leaf tissue of saussurea involucrata plants.

In the first or second aspect, the saussurea involucrata purple callus is obtained by induction, and the induction medium comprises: based on the MS culture medium, 18-22 g/L of sucrose, 3-5 g/L of agar, 3-4 mg/L of naphthylacetic acid and 2-3 mg/L of 6-benzylaminopurine are also added, and the pH value is 5.8-6.2 (preferably 20g/L of sucrose, 5g/L of agar, 3mg/L of naphthylacetic acid and 2mg/L of 6-benzylaminopurine are added, and the pH value is 6.0).

Further, in step 1), the medium for light culture is: based on the MS culture medium, 27-33 g/L of sucrose, 4-5 g/L of agar, 2-3 mg/L of naphthylacetic acid and 2-3 mg/L of 6-benzylaminopurine are also added, and the pH value is 4.8-5.2 (preferably 30g/L of sucrose, 4.5g/L of agar, 3mg/L of naphthylacetic acid and 2mg/L of 6-benzylaminopurine, and the pH value is 5.0).

Further, the callus induction culture method comprises the following steps: culturing for 14-18 days at 20-24 ℃ and illumination time: 7-9 hours/day, the illumination intensity: 58-62 mol/m.s;

and/or, the aseptic processing method of the saussurea involucrata tissue comprises the following steps: cleaning young stems and/or leaves of fresh saussurea involucrata plants, sterilizing with alcohol, sterilizing with mercuric chloride, rinsing with sterile water, and cutting into small pieces in a sterile operation.

Further, the aseptic processing method of the saussurea involucrata tissue comprises the following steps: cleaning young stems and/or leaves of fresh saussurea involucrata plants, soaking the young stems and/or the leaves in 75% alcohol for 15s for surface sterilization, soaking the young stems and/or the leaves subjected to alcohol sterilization treatment in 0.1% mercuric chloride for 4-6 min, rinsing the young stems and/or the leaves with sterile water for 3-8 times, and cutting the young stems and/or the leaves into small pieces with the length of 0.3-0.5 cm in a sterile operation.

Further, in the step 2), the illumination culture method of the saussurea involucrata purple callus comprises the following steps: culturing for 14-18 days at 20-24 ℃ and illumination time: 7-9 hours/day, the illumination intensity: 58-62 mol/m.s;

further, in step 2), the purification method includes: the concentrate was exchanged with PBS and purified by SEC purification column (purification effect is better, process scale-up is easier to achieve).

Further, the method also comprises a step 3) of freeze-drying the saussurea involucrata purple callus vesicles.

In a third aspect, at least one skin product of the following I) to V) is provided, wherein the skin product comprises saussurea involucrata purple callus vesicles extracted by the extraction method in the second aspect;

i) Skin products that promote the scavenging of DPPH radicals;

II) skin products that inhibit ROS production;

III) skin products that inhibit tyrosinase activity;

IV) a skin product that inhibits the production of melanin in cells;

v) a skin product that promotes cell proliferation and migration.

Optionally, the saussurea involucrataThe concentration of purple callus vesicles in the product is 10 ⁷ ~10 ¹² Particles/mL (optionally 10) ⁹ ~10 ¹² Particles/mL, optionally 10 ¹⁰ ~10 ¹² particles/mL)。

The preparation method of the saussurea involucrata purple callus vesicles comprises the following steps:

i) Purple callus culture of saussurea involucrata:

1) Selecting and asepsis the saussurea involucrata plant body:

a) Selecting young stems and leaves of a fresh plant of wild saussurea involucrata with the altitude of more than 3500m for 7 months, cleaning with water, and soaking in distilled water;

b) Soaking the cleaned young stems and leaves in 75% alcohol for 15s for surface sterilization;

c) Soaking young stems and leaves sterilized by alcohol in 0.1% mercuric chloride solution for 5min, and continuously lightly stirring in the soaking process to make the surfaces of plants fully contact with the mercuric chloride solution;

d) Rinsing the plant body soaked and sterilized by the mercuric chloride solution with distilled water subjected to high-temperature sterilization treatment for 5 times, rinsing the mercuric chloride solution adhered to the surface of the plant body, and then sucking the water of the plant body with sterile filter paper;

e) Shearing young stems and leaves into 0.3-0.5 cm by using sterile scissors, and inoculating 5 blocks of the surface sterilized material into an induction culture medium for culture under the sterile condition.

2) Inducing and culturing the purple calluses of saussurea involucrata:

performing callus induction culture on the saussurea involucrata tissue subjected to aseptic treatment in an induction culture medium for 16 days at a culture temperature of 22 ℃ for 8 hours/day under the illumination intensity of 60 mol/m.s, wherein the induction culture medium is as follows: basic culture medium MS+sucrose 20 g/L+agar 5 g/L+alpha-naphthylacetic acid 3 mg/L+6-benzylaminopurine 2mg/L, and pH value is 6.0.

3) Large-scale culture of saussurea involucrata purple callus:

purple callus generated by induction culture is selected and cultured in a large-scale culture medium. The large-scale culture medium is as follows: basic culture medium MS, sucrose 30g/L, agar 4.5g/L, alpha-naphthylacetic acid 3mg/L, 6-benzylaminopurine 2mg/L and pH value 5.0. Callus culture fluid was collected during the culture.

II) extraction of purple callus vesicles of saussurea involucrata

And collecting culture solution of the saussurea involucrata purple callus, and carrying out gradient centrifugation. Firstly, 300g-1000g are centrifuged, and the supernatant is called as supernatant I; then, centrifuging the supernatant I with 2000g-4000g, and taking the supernatant, namely the supernatant II; centrifuging the supernatant II at 8000g-10000g, and collecting supernatant, namely supernatant III (i.e. supernatant of third centrifugation); finally, the supernatant III is filtered by using a filter membrane with the diameter of 0.2 μm or 0.8 μm in sequence, and the filtrate is collected.

Concentrating the obtained filtrate by tangential flow method, specifically concentrating the filtrate to a certain volume by using hollow fiber with cut-off molecular weight of 100Kd/300Kd/500Kd/750Kd, and changing liquid with 5-10 times PBS to obtain concentrated solution with PBS solvent. And finally purifying the obtained concentrated solution through a SEC purification column to obtain the saussurea involucrata purple callus vesicles.

At present, no method for separating and purifying the vesicle from the saussurea involucrata purple callus culture solution is reported in the literature or patent, so the patent discloses a method for extracting the vesicle from the saussurea involucrata purple callus culture solution by combining tangential flow and column chromatography.

Advantageous effects

1) The invention realizes the large-scale industrialized production of the saussurea involucrata purple callus vesicles by combining tangential flow and column chromatography from the saussurea involucrata purple callus culture solution. A series of experiments prove that the saussurea involucrata purple callus vesicles have excellent effects on antioxidation and whitening.

2) The extraction method of the saussurea involucrata purple callus vesicles disclosed by the invention is simple and convenient, shortens the production period, reduces the production cost, improves the purity and the yield of the extracted vesicles, and is suitable for large-scale production. The saussurea involucrata purple callus vesicles are natural active plant components, have high biological safety and are environment-friendly.

3) The invention verifies the efficacy of saussurea involucrata purple callus vesicles and cell extracts through an in-vitro antioxidation experiment, and discovers that saussurea involucrata purple callus vesicles can promote the elimination of DPPH free radicals. The invention verifies the efficacy of saussurea involucrata purple callus vesicles and cell extracts through a cell experiment, and discovers that saussurea involucrata purple callus vesicles can inhibit the generation of ROS. The invention verifies the efficacy of saussurea involucrata purple callus vesicles and cell extracts through a cell experiment, and discovers that saussurea involucrata purple callus vesicles can inhibit the activity of tyrosinase. The invention verifies the efficacy of saussurea involucrata purple callus vesicles and cell extracts through a cell experiment, and discovers that saussurea involucrata purple callus vesicles can inhibit the generation of melanin in cells. The invention verifies the efficacy of saussurea involucrata purple callus vesicles and cell extracts through a cell experiment, and discovers that saussurea involucrata purple callus vesicles can promote cell proliferation and migration.

Drawings

One or more embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

Fig. 1: the invention tests transmission electron microscopy images of the purple saussurea involucrata vesicles extracted from hollow fibers with different cut-off molecular weights of example 1.

Fig. 2: SEC analysis chromatogram of the purple snow vesicle of test example 1 of the present invention.

Fig. 3: the ROS scavenging test of the Umbelliferae vesicle and cell extract of test example 1 of the present invention, wherein A is the Umbelliferae vesicle (10 ⁹ Particles/mL), B is the cell extract of saussurea involucrata (0.3 g/mL), C is the cell extract of saussurea involucrata (0.03 g/mL).

Fig. 4: DPPH radical scavenging test of the purple snow vesicle and cell extract of test example 2 of the present invention.

Fig. 5: determination of relative tyrosinase Activity of the purple snow vesicles and cell extracts of Experimental example 3 of the present invention.

Fig. 6: measurement of the relative melanin content of the purple snow vesicles and cell extracts of test example 4 of the present invention.

Fig. 7: experimental example 5 shows the results of the experiment for promoting human fibroblast migration of saussurea involucrata purple callus vesicles.

Detailed Description

For a better description of the invention, various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed description of certain aspects, features and embodiments of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. The present specification and embodiments are to be considered as exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to. Unless otherwise indicated, all reagents used hereinafter are commercial reagents in which the chemical reagents used are not less than analytically pure.

In the following examples, the culture method of saussurea involucrata purple callus is as follows:

purple callus culture of saussurea involucrata:

1) Selecting and asepsis the saussurea involucrata plant body:

2) Inducing and culturing the purple calluses of saussurea involucrata:

performing callus induction culture on the saussurea involucrata tissue subjected to aseptic treatment in an induction culture medium for 16 days at a culture temperature of 22 ℃ for 8 hours/day under illumination intensity: 60 mol/m.s; the induction culture medium is as follows: basic culture medium MS+sucrose 20 g/L+agar 5 g/L+alpha-naphthylacetic acid 3 mg/L+6-benzylaminopurine 2mg/L, and pH value is 6.0.

3) Large-scale culture of saussurea involucrata purple callus:

The callus culture fluid is separated according to different methods to obtain saussurea involucrata purple callus vesicles, some examples of which are as follows.

Example 1

5L of culture solution of the saussurea involucrata purple callus is collected, and then gradient centrifugation is carried out, wherein the gradient centrifugation method comprises the following steps: first 300g, centrifuge for 10min, collect supernatant and discard the pellet. Then 2000g of the collected supernatant was centrifuged for 20min, and the supernatant was collected and the precipitate was discarded. And finally 10000g of the collected supernatant is centrifuged for 30min, and the supernatant is collected.

The supernatant collected by the third centrifugation was filtered with a 0.8 μm filter.

Concentrating the filtered solution to 50mL by hollow fiber with cut-off molecular weight of 100Kd, changing the solution by using 250mL of 1xPBS, purifying the 50mL concentrated solution by SEC purification column to obtain saussurea involucrata purple callus vesicle 50mL, and measuring the particle concentration to 5.4X10 by Nanoparticle Tracking Analysis (NTA) ¹⁰ Particles/mL。

Example 2

Concentrating the filtered solution to 50mL by hollow fiber with a cutoff molecular weight of 300Kd, changing the solution by using 250mL of 1xPBS, and purifying the 50mL concentrated solution by SEC purification column to obtain 50mL of saussurea involucrata purple callus vesicle, and determining the particle concentration to be 4.9X10 by Nanoparticle Tracking Analysis (NTA) ¹⁰ Particles/mL。

Example 3

Concentrating the filtered solution to 50mL by hollow fiber with a cutoff molecular weight of 500Kd, changing the solution by using 250mL of 1xPBS, and purifying the 50mL concentrated solution by SEC purification column to obtain saussurea involucrata purple callus vesicle with particle concentration of 3.7X10 determined by Nanoparticle Tracking Analysis (NTA) ¹⁰ Particles/mL。

Example 4

Concentrating the filtered solution to 50mL by hollow fiber with cutoff molecular weight of 750Kd, changing the solution by using 250mL of 1xPBS, purifying the 50mL concentrated solution by SEC purification column to obtain saussurea involucrata purple callus vesicle 50mL, and measuring particle concentration by Nanoparticle Tracking Analysis (NTA) to 2.3X10 ¹⁰ Particles/mL。

Comparative example 1:

The supernatant collected by the third centrifugation was filtered with a 0.2 μm filter.

Concentrating the filtered solution to 50mL by hollow fiber with cut-off molecular weight of 100Kd, changing the solution by using 250mL of 1xPBS, purifying the 50mL concentrated solution by SEC purification column to obtain saussurea involucrata purple callus vesicle 50mL, and measuring the particle concentration to 8.1X10 by Nanoparticle Tracking Analysis (NTA) ⁹ Particles/mL。

Comparative example 2:

Concentrating the filtered solution to 50mL by hollow fiber with a cutoff molecular weight of 300Kd, changing the solution by using 250mL of 1xPBS, and purifying the 50mL concentrated solution by SEC purification column to obtain 50mL of saussurea involucrata purple callus vesicle, and measuring the particle concentration to 6.4X10 by Nanoparticle Tracking Analysis (NTA) ⁹ Particles/mL。

Comparative example 3:

Concentrating the filtered solution to 50mL by hollow fiber with a cutoff molecular weight of 500Kd, changing the solution by using 250mL of 1xPBS, and purifying the 50mL concentrated solution by SEC purification column to obtain saussurea involucrata purple callus vesicle with particle concentration of 5.7X10 determined by Nanoparticle Tracking Analysis (NTA) ⁹ Particles/mL。

Comparative example 4:

Concentrating the filtered solution to 50mL by hollow fiber with cutoff molecular weight of 750Kd, changing the solution by using 250mL of 1xPBS, and purifying the 50mL concentrated solution by SEC purification column to obtain saussurea involucrata purple callus vesicle 50mL, and measuring particle concentration to 4.9X10 by Nanoparticle Tracking Analysis (NTA) ⁹ Particles/mL。

Comparative example 5:

freeze-drying the cultured saussurea involucrata purple callus cells to obtain saussurea involucrata purple callus cell powder, wherein the freeze-drying method comprises the following steps: pre-freezing at-80 ℃ for 2 hours, and then putting the mixture into a freeze dryer for vacuum freeze drying for 16 hours. Weighing 20g of the freeze-dried saussurea involucrata purple callus cell powder, adding the powder into 400mL of 50% ethanol solution for treatment for 12h, then adding 1% pectase, 1% cellulase, adjusting pH to 4 by using citric acid and sodium hydroxide at 50 ℃, carrying out enzymolysis for 3h, carrying out heat reflux for 3h at 100 ℃, cooling, carrying out suction filtration by using a filter membrane with the thickness of 0.45 mu m, collecting filtrate, and carrying out freeze-drying again to obtain saussurea involucrata purple callus cell powder.

Test example 1

The results of lens electron microscopic observation of the vesicles extracted in examples 1 to 4 and comparative examples 1 to 4 are shown in fig. 1.

The results in fig. 1 show that the effect of selecting a filter membrane with a diameter of 0.8 μm is better in the experimental process, and the concentration of vesicle-like particles is reduced by selecting a filter membrane with a diameter of 0.2 μm for suction filtration, which means that a lot of vesicles are lost in the suction filtration process, and the filter membrane is easy to block in the suction filtration process due to small pore diameter, so that the operation complexity is increased by frequent replacement of the filter membrane.

In examples 1 to 4, the concentration effect of the four hollow fibers with different molecular weight cut-off of 100Kd/300Kd/500Kd/750Kd in the concentration process by using a tangential flow method (tangential flow method: means a filtration mode that the liquid flow direction is perpendicular to the filtration direction) is not greatly different, wherein the particle size distribution range of vesicles after concentration by using 100Kd hollow fibers is wider, and the distribution of 20nm to 200nm can be seen by a transmission electron microscope image, and the generated impurities are relatively more; the particle concentration is obviously reduced after the hollow fiber with 750kd is used for concentration, and the particle number is obviously reduced and the particle size is concentrated between 100nm and 160nm, so that the vesicle with the particle size lower than 100nm is almost completely lost; concentration with 500kd hollow fiber retains some small particle size vesicles but also loses more vesicles; therefore, the 300kd hollow fiber, namely the embodiment 2, has the best effect, the particle size is mainly concentrated at 50nm to 170nm, and the vesicle is hardly lost.

The vesicle particle concentrations obtained after purification of examples 1 to 4 and comparative examples 1 to 4 are shown in table 1:

。

the vesicle-like liquid of example 2 was subjected to SEC chromatography, and the result is shown in FIG. 2, and the SEC analysis chromatogram shows that the vesicle-like liquid extracted in example 2 has a uniform particle size.

Efficacy tests were performed using the vesicles obtained in example 2:

efficacy experiment

Test example 1: determination of ROS clearance of purple snow vesicles and cell extracts

Taking HaCaT cells (from ATCC) in logarithmic growth phase, inoculating into 12-well cell culture plate, and inoculating 2×10 per well ⁵ Cells were cultured for 24 hours to adhere the cells.

The purple snow vesicles obtained in example 2 were diluted to 10 with DMEM complete medium ⁹ Solutions of Particles/mL, i.e. 10 ⁹ The Particles/mL of the purple snow vesicle fluid.

The saussurea involucrata cell extract obtained in comparative example 5 was diluted with DMEM complete medium into 0.03g/mL and 0.3g/mL solutions, respectively, which were designated as 0.03g/mL and 0.3g/mL.

The culture medium in the 12-well cell culture plate was aspirated, the cells were washed once with PBS, and 1mL of complete medium (used as control and blank) and 1mL of 10 were added, respectively ⁹ The Particles/mL purple snow vesicle fluid or the purple snow cell extract solution with different concentrations (0.03 g/mL, 0.3 g/mL) (as experimental group).

After 24h incubation, the culture supernatant was aspirated, cells were digested with 400. Mu.L pancreatin per well, and stopped with 800. Mu.L LDMEM complete medium, as follows:

1) The cell suspension was transferred to a 1.5mL centrifuge tube.

2) Centrifuge at 500g for 5min and discard supernatant. 100 mu L of dye solution is added into the experimental group, the experimental group is blown and evenly mixed, the experimental group and the blank group are incubated for 60 minutes at 37 ℃ in a dark place, and the control group and the blank group are directly added with equal volume of PBS without adding the dye solution.

3) Each tube was washed with 900. Mu.LPBS, centrifuged at 500g for 5min and the supernatant discarded. 200. Mu.L of 200. Mu.M H was added to each of the experimental group and the control group ₂ O ₂ Incubating for 30min; blank group does not add H ₂ O ₂ An equal volume of medium was added for incubation.

4) Again, 900. Mu.LPBS was added for washing, 500g centrifuged for 5min and the supernatant discarded. Then 200 μlpbs was added for resuspension. The flow cytometer detects the average fluorescence intensity.

The formula for the clearance of ROS is as follows:

。

wherein: t is the fluorescence intensity of the experimental group sample; c is control group (+H) ₂ O ₂ Stimulus) fluorescence intensity; c0 is the fluorescence intensity of the blank.

The results are shown in FIG. 3 and Table 2.

TABLE 2 fluorescence intensity and ROS clearance for each of the experimental, blank and control groups

。

The results are shown in FIG. 3 and Table 2, and are obtained by H ₂ O ₂ Stimulating HaCaT cells to establish a ROS high expression cell model, and compared with a blank group, the control group is added with H ₂ O ₂ The stimulated fluorescence intensity is significantly enhanced. In experimental group and added with H ₂ O ₂ The difference in stimulated control group serves as an indicator of the ability of the sample to down-regulate ROS, the greater the difference, the better the ability of the sample to scavenge ROS. The fluorescence intensity is reduced to different degrees after the purple snow vesicle and the purple snow cell extract are added, which shows that the purple snow vesicle and the purple snow cell extract have ROS inhibition effects to different degrees, and compared with the purple snow cell extract with different concentrations, the red fluorescent dye has the advantages of 10 ⁹ The ROS clearance rate of the Particles/mL of the purple snow vesicles reaches 35.07%, and the inhibition effect is better。

Test example 2 measurement of DPPH radical scavenger of purple snow vesicle and cell extract

The DPPH reagent in the DPPH kit was first prepared as a 0.1mM ethanol solution. Then 10% Triton X-100 lysate was prepared.

The purple snow vesicles obtained in example 2 were diluted to 10 in sequence with PBS ⁸ 、10 ⁹ 、10 ¹⁰ The Particles/mL solution. The saussurea involucrata cell extract obtained in comparative example 5 was formulated into 0.03, 0.3, 3g/mL solutions with PBS in this order. 10% Triton X-100 lysate was added to the above 6 solutions to give a final Triton X-100 concentration of 1%. After mixing, incubation for 60min at room temperature, each solution was divided equally into experimental and blank groups: 200 mu L of the solution is respectively taken in each experimental group and 200 mu L of the ethanol solution of LDPPH is respectively added, and 200 mu L of the solution is respectively taken in each blank control group and 200 mu L of the ethanol is added. The control group was 200. Mu.LPBS with 200. Mu.l DPPH ethanol solution, each group was subjected to 3 parallel experiments, protected from light, incubated at room temperature for 30min, and absorbance at 517nm was measured using an enzyme-labeled instrument.

。

Wherein: a is the absorbance value of the experimental group; b is the absorbance value of the blank control group; c is the absorbance value of the control group;

as shown in fig. 4 and table 3, both the saussurea involucrata vesicles and saussurea involucrata cell extract had the effect of promoting DPPH radical scavenging, with the scavenging effect being more remarkable with increasing concentration. 10 ¹⁰ Compared with 3g/mL of the purple snow cell extract, the purple snow cell extract has equivalent free radical clearance rate, namely, the concentration of the purple snow cell extract needs to be 3g/mL when the purple snow cell extract is required to reach better free radical clearance rate, which can lead the additive amount of the purple snow cell extract in cosmetics to be too high, greatly influence the preparation of the cosmetics, and the concentration of the purple snow cell extract provided by the invention is 10 ¹⁰ The Particles/mL (about 30 mug/mL) can well promote the removal of DPPH free radicals and has good antioxidation effect.

TABLE 3 DPPH radical scavenging Rate of purple snow vesicles and purple snow cell extracts

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Test example 3 determination of relative tyrosinase Activity of purple snow vesicles and cell extracts

Taking B16 cells in logarithmic phase, inoculating into 6-well cell culture plate, inoculating 2×10 each well ⁵ Cells were cultured for 24 hours to adhere the cells.

Arbutin was formulated as a 3g/mL solution in 1640 complete medium.

The purple snow vesicles obtained in example 2 were diluted to 10 in this order with 1640 complete medium ⁸ 、10 ⁹ The Particles/mL solution. The saussurea involucrata cell extract obtained in comparative example 5 was diluted to 0.003, 0.03, 0.3g/mL of solutions in this order with 1640 complete medium. The culture solution in the 6-hole cell culture plate is sucked out, the cells are washed once by PBS, 2mL1640 complete culture medium (as a control group), arbutin solution (as a positive control group), different concentrations of the purple snow vesicle solution or the purple snow cell extract solution (as an experimental group) are respectively added, 3 parallel experiments are carried out on each group, the culture solution is replaced and then is put back into an incubator for continuous culture for 48 hours, then the culture solution is sucked out, the cells are washed once by PBS, 500 mu L pancreatin is added into each hole to digest the cells, 1000 mu L culture medium is added to terminate digestion, the cell suspension is transferred into a 1.5mL centrifuge tube for counting by a cell counter, and then the supernatant is discarded after centrifugation at 13000rpm for 5 minutes.

Absorbance values for each sample were measured according to the instructions of tyrosinase activity detection kit (purchased from beijing solebao technologies limited):

1) Adding an extracting solution in tyrosinase activity detection kit (purchased from Beijing Soy Bao technology Co., ltd.) into each group according to the proportion of 500 ten thousand cells/1 mL, breaking cells by ultrasonic waves (power 200w, ultrasonic waves for 3s, interval 10s, repeating 30 times), freeze thawing once at 80 ℃, centrifuging for 20min at 12000g and 4 ℃, taking supernatant, and placing on ice for testing.

2) Taking a 96-well plate, adding 20 mu L of a sample into each well, quickly blowing and uniformly mixing 180 mu L of a reagent I in a tyrosinase activity detection kit, measuring an absorbance value A1 at 475nm by using an enzyme-labeled instrument, quickly placing the mixture in a 37 ℃ incubator for 60min, quickly measuring an absorbance value A2 at 60min after taking out, and calculating delta A=A2-A1.

。

Wherein: Δa1 is the difference in absorbance of the experimental or positive control group; Δa2 is the absorbance difference for the control group;

as shown in FIG. 5 and Table 4, the relative tyrosinase activity was reduced by the addition of different concentrations of the purple snow vesicles as compared with the control group, and 10 ⁹ The relative tyrosinase activity of the vesicles of the saussurea involucrata type of the Particles/mL is as low as 50.36 percent, and the vesicles have better effect of inhibiting the tyrosinase activity than a positive control (79.36 percent). Whereas the saussurea involucrata cell extract did not show a significant function of inhibiting tyrosinase activity. The results show that the purple snow vesicles provided by the application have better effect of inhibiting tyrosinase activity than the purple snow cell extract, and have good whitening effect.

TABLE 4 relative tyrosinase activity of purple snow vesicles and purple snow extracts

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Test example 4 determination of relative melanin content of purple snow vesicle and cell extract

Taking B16 cells in logarithmic phase, inoculating into 12-well cell culture plate, inoculating 2×10 each well ⁴ Cells were cultured for 24 hours to adhere the cells. The purple snow vesicles obtained in example 2 were diluted to 10 with DMEM complete medium ⁹ The extracts of the cells of the saussurea involucrata obtained in comparative example 5 were diluted in the DMEM complete medium to 0.003, 0.03 and 0.3g/mL in this order, and the diluted saussurea involucrata vesicles and the cell extracts were used as experimental groups. Weigh oneQuantitative kojic acid was formulated as a 200. Mu.g/mL solution with DMEM complete medium as a positive control. The culture solution in the 12-well cell culture plate was aspirated, the cells were washed once with PBS, and 1mL of complete medium (as a control group), 200. Mu.g/mL of kojic acid solution (as a positive control group), and different concentrations of the purple snow vesicle solution or the purple snow cell extract solution (as an experimental group) were added, respectively. And after the liquid is changed, the culture medium is continuously put back into the incubator for culturing for 48 hours. Then 200. Mu.L of pancreatin was added to each well to digest the cells, and after centrifugation at 15000g for 10min, the supernatant was aspirated off with a pipette. After washing the cells once with 500. Mu.LPBS, they were centrifuged again. Then 200. Mu.L of 1mol/LNaOH was added and the mixture was placed in a water bath at 80℃for 60min to lyse the cells. After the end, 100. Mu.L of the liquid was pipetted into a 96-well plate and another 100. Mu.L of 1mol/LNaOH was used as a blank. Absorbance at 405nm was measured by an enzyme-labeled instrument.

。

Wherein: a is the absorbance value of the experimental group; b is the absorbance value of the control group; c is the absorbance value of the blank control group;

as shown in fig. 6 and table 5, both the purple snow vesicles and the purple snow cell extract have the effect of inhibiting melanin secretion and are better than the positive control. Compared with the extract of the saussurea involucrata cells, the saussurea involucrata vesicle is 10 ⁹ The relative melanin content of the purple snow vesicles of the Particles/mL is 79.85 percent, which is lower than that of the purple snow cell extract of 0.3g/mL, which indicates that the purple snow vesicles provided by the application have good effect of inhibiting melanin secretion, and can whiten and lighten skin.

TABLE 5 relative melanin content of purple snow vesicles and purple snow extracts

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Test example 5 measurement of cell migration promotion of purple snow vesicle and cell extract

Human skin fibroblasts are the most predominant cells in the dermis layer of the skin and have an important role in maintaining the elasticity and tone of the skin. The human skin fibroblast has strong protein synthesis capability, can synthesize and secrete a large amount of matrix components such as elastin, collagen, glycosaminoglycan, glycoprotein and the like, further generates elastic fibers, collagen fibers and reticular fibers, secretes various cell repair factors, and enables the skin to have strong renewal and self-repair capability.

Human skin fibroblasts were cultured at a rate of 2X 10 ⁵ Wells were seeded into 6-well plates. Cells were incubated at 37℃with 5% CO ₂ Culturing in an incubator for 24 hours. When the cell fusion rate reached 90%, human skin fibroblasts were scratched by streaking with a 200. Mu.L pipette tip perpendicular to the well plate, and cell fragments separated by the streaks were removed by washing with PBS to make no cells in the streak range, and used for observing whether the cells proliferated and migrated.

The experiments were grouped into control, purple snow vesicle and purple snow cell extract groups, each group having 3 duplicate wells. Diluting the extract of example 2 into 10 with DMEM complete medium ⁹ The Particles/mL of the purple snow vesicle solution. The saussurea involucrata cell extract obtained in comparative example 5 was diluted to 3g/mL and 0.3g/mL in this order with DMEM complete medium. 200 mu LDMEM complete culture medium is added into each hole of a control group, and the 10 parts of the purple snow vesicle group are added ⁹ The 3g/mL and 0.3g/mL of the purple snow cell extract solution are added to the purple snow cell extract group of the vesicle solution of the purple snow class of the Particles/mL. The scratch area was photographed 0h and 36h after scratch formation, and the migration of cells was observed.

As shown in FIG. 7, after 36 hours, 10 was added compared with the control group ⁹ The proliferation and migration of human skin fibroblasts of the Particles/mL purple snow vesicles are faster, while the proliferation and migration of cells of the purple snow cell extract solution added with 3g/mL purple snow cell extract solution are almost not, the cell activity is obviously reduced, and the cell migration effect of the purple snow cell extract solution added with 0.3g/mL is equivalent to that of a control group, so that the purple snow vesicles provided by the application can obviously promote the proliferation and migration of human skin fibroblasts, and can enhance the cell activity to enable damaged musclesThe skin is quickly repaired, so that the skin state is healthier and more stable.

While the foregoing description illustrates and describes preferred embodiments of the present invention, as aforesaid, it is to be understood that the invention is not limited to the forms disclosed herein but is not to be construed as limited to other embodiments, but is capable of use in various other combinations, modifications and environments and is capable of changes or modifications within the spirit of the invention described herein, either as a result of the foregoing teachings or as a result of the knowledge or skill of the relevant art. And that modifications and variations which do not depart from the spirit and scope of the invention are intended to be within the scope of the appended claims.

Claims

1. Application of saussurea involucrata purple callus vesicles in preparing antioxidant or whitening skin products;

the saussurea involucrata purple callus vesicles are derived from saussurea involucrata purple callus culture solution;

the saussurea involucrata purple callus vesicles are used as main active ingredients.

2. Use according to claim 1, characterized in that the antioxidant or whitening skin product comprises at least one of I) to V):

i) Skin products that promote the scavenging of DPPH radicals;

II) skin products that inhibit ROS production;

III) skin products that inhibit tyrosinase activity;

IV) a skin product that inhibits the production of melanin in cells;

v) a skin product that promotes cell proliferation and migration.

3. The use according to claim 1, wherein the concentration of saussurea involucrata purple callus vesicles in the product is 10 ⁷ ~10 ¹² particles/mL。

4. The use according to claim 1, wherein the Tianshan mountainThe concentration of the saussurea involucrata purple callus vesicles in the product is 10 percent ⁹ ~10 ¹² particles/mL。

5. The use according to claim 1, wherein the concentration of saussurea involucrata purple callus vesicles in the product is 10 ¹⁰ ~10 ¹² particles/mL。

6. The use according to claim 1, wherein said saussurea involucrata purple callus vesicles are prepared by the following method:

2) Separating and obtaining saussurea involucrata purple callus vesicles from the culture solution in the step 1), wherein the separating method comprises the following steps: collecting the supernatant, filtering and collecting filtrate, concentrating the filtrate, and purifying the concentrated solution to obtain the saussurea involucrata purple callus vesicles.

7. The method according to claim 6, wherein the pore size of the filter membrane is not less than 0.2 μm;

and/or concentrating by adopting hollow fibers, wherein the molecular weight cut-off of the hollow fibers is more than or equal to 100KD;

and/or in the step 2), the concentration multiple is 50-200 times.

8. The use according to claim 6, wherein the filter pore size is 0.45 μm to 1.5 μm;

and/or concentrating by adopting hollow fibers, wherein the cut-off molecular weight of the hollow fibers is 100 KD-750 KD;

and/or the concentration multiple is 80-150 times.

9. The method according to claim 6, wherein the pore size of the filter is 0.8. Mu.m.

10. The use according to claim 6, wherein the concentration is performed by using hollow fibers having a molecular weight cut-off of 100kd to 300kd.

11. The use according to claim 6, wherein in step 2) the concentration factor is 100-150.

12. The use according to claim 6, wherein in step 2) the supernatant is collected by centrifugation under differential centrifugation conditions: respectively centrifuging 300g-1000g, 2000g-4000g and 8000g-10000g, wherein the supernatant is taken for subsequent centrifugation in the first two times, and the supernatant is taken for filtration after the third centrifugation.

13. The use according to any one of claims 6 to 12, wherein in step 1) the saussurea involucrata purple callus is obtained by inducing young stem or leaf tissue of saussurea involucrata plants.

14. The use according to claim 13, wherein said saussurea involucrata purple callus is obtained by induction, the induction medium comprising: based on an MS culture medium, 18-22 g/L of sucrose, 3-5 g/L of agar, 3-4 mg/L of naphthylacetic acid and 2-3 mg/L of 6-benzylaminopurine are also added, and the pH value is 5.8-6.2.

15. The use according to claim 13, wherein in step 1) the medium for light culture is: based on an MS culture medium, 27-33 g/L of sucrose, 4-5 g/L of agar, 2-3 mg/L of naphthylacetic acid and 2-3 mg/L of 6-benzylaminopurine are also added, and the pH value is 4.8-5.2.