CN117205129B

CN117205129B - Extraction method of alpine leontopodium herb vesicles and application of alpine leontopodium herb vesicles in preparation of skin cell collagen regeneration and repair products

Info

Publication number: CN117205129B
Application number: CN202311474588.XA
Authority: CN
Inventors: 张振兴; 高琦; 张旭辉; 赵旺宇; 王夜宇
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: 2024-02-23
Anticipated expiration: 2043-11-08
Also published as: CN117205129A

Abstract

The invention relates to an extraction method of alpine leonurus vesicles and application thereof in preparing skin cell collagen regeneration and repair products, wherein the skin cell collagen regeneration products comprise products for promoting generation of type I collagen; and/or the skin repair product comprises a skin product that promotes migration of human fibroblasts and/or is anti-inflammatory, the Gao Shanhuo chorionic vesicles being present in the product at a concentration of 10 ⁸ ~10 ¹³ particles/mL; and/or, the alpine leontopodium vesicles are derived from alpine leontopodium callus culture fluid. The invention can realize large-scale industrialized production of the alpine leontopodium herb vesicles. The alpine leonurus vesicles of the invention can promote the generation of type I collagen, the migration of human fibroblasts, anti-inflammatory and the like.

Description

Extraction method of alpine leontopodium herb vesicles and application of alpine leontopodium herb vesicles in preparation of skin cell collagen regeneration and repair products

Technical Field

The invention belongs to the technical field of skin care products or medicines, and relates to an extraction method of alpine leonurus vesicles and application of the alpine leonurus vesicles in preparation of skin cell collagen regeneration and repair products.

Background

Mountain leontopodium faerie is produced in europe and is mostly grown in mountains at altitudes of 1400-3500 m, mainly in the Niu Si and alpine mountains. The herba Hyperici Japonici contains natural active substances such as orthoic acid, caffeic acid, p-hydroxy cinnamic acid ester and vanillic acid, chlorogenic acid and 3, 5-dicaffeoylquinic acid. These natural active substances have antioxidant, anti-inflammatory and cytoprotective activities. However, due to the severe conditions of alpine leontopodium growing and global climate change, and by mass exploitation, the alpine leontopodium population is continuously reduced. Alpine leontopodium has been listed as an endangered species in many european countries.

The vesicle-like particle size is between 40-150nm, secreted by most types of cells and transmits information between cells and organisms. Plant vesicles contain certain plant-specific DNA, RNA and other pharmacologically active molecules. Research shows that the plant vesicles contain 90% of active ingredients of plants and are essence in the essence of the effective ingredients of the plants.

The callus is naked cells after the cell walls are removed by a chemical method and a mechanical means, has cell totipotency, and can secrete vesicle like animal cells in the process of callus culture. The plant callus vesicles are different from traditional callus plant extracts, and the traditional callus plant extracts are extracted by using organic reagents and at high temperature. Organic reagents, high temperature and the like destroy some proteins and functional DNA and RNA in plant callus cells. Therefore, the plant callus vesicles have more components and have wider spectrum compared with the plant callus.

At present, the method for extracting plant vesicles mostly uses the whole plant to break up, and then extracts the plant vesicles by a differential centrifugation method. The differential centrifugation method has the advantages of complicated operation, long time consumption and low yield, the integrity of the vesicles is destroyed in the breaking process of the whole plant, the purity of the extracted vesicles is low, and a large number of plant vesicles are required to be obtained, so that the distribution of the plant is seriously destroyed.

The research shows that the culture supernatant of the calli of the alpine leontopodium is rich in a large number of plant vesicles secreted by the calli. The vesicle isolated from the callus culture solution of the alpine leontopodium can not only retain DNA, RNA and other pharmacological active molecules specific to the alpine leontopodium, but also reduce the damage of the alpine leontopodium plants. The vesicles of the alpine leontopodium are absorbed by skin tissues, and can induce and amplify secondary metabolites beneficial to the skin, promote the anti-aging and anti-inflammatory effects of the skin and achieve the effect of improving the skin.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides an extraction method of alpine leonurus vesicles and application of the method in preparation of skin cell collagen regeneration and repair products.

The invention provides a method for extracting alpine leontopodium herb vesicles from a alpine leontopodium herb callus culture solution for the first time and a method for extracting the alpine leontopodium herb vesicles from the alpine leontopodium herb callus culture solution by using a tangential flow column chromatography method. The alpine leonurus vesicles extracted from the alpine leonurus callus culture solution have high purity, high yield and short time consumption, and can be produced on a large scale. Meanwhile, the culture solution of the calli of the alpine leontopodium can be obtained in a large amount, and the culture solution has no damage to the alpine leontopodium plants. And the extracted alpine leonurus vesicles are subjected to a series of efficacy tests, and the effects of promoting the generation of type I collagen, the migration of human fibroblasts, anti-inflammatory and the like are found.

Solution scheme

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

in a first aspect, the present invention provides the use of a alpine leontopodium vesicles for the preparation of a skin cell collagen regeneration and/or skin repair product.

Further, the skin cell collagen regeneration product includes a product that promotes the production of type I collagen.

Further, the skin repair products include skin products that promote migration of human fibroblasts and/or are anti-inflammatory. Alternatively, anti-inflammatory includes inhibiting the production of cyclooxygenase-2.

Further, the concentration of Gao Shanhuo velvet grass vesicles in the product is 10 ⁸ ~10 ¹³ particles/mL; alternatively 10 ¹⁰ ~10 ¹² Particles/mL, optionally 10 ¹¹ ~10 ¹² particles/mL。

Further, the alpine leontopodium vesicles are derived from alpine leontopodium callus culture fluid.

Further, the Gao Shanhuo erigeron vesicles are extracted by the following method:

1) Inoculating the calli of the alpine leontopodium into a suspension culture medium, carrying out illumination culture, and collecting a culture solution;

2) Taking the culture solution in the step 1), separating to obtain supernatant containing the alpine leonurus vesicles, concentrating, and purifying to obtain the alpine leonurus callus vesicles.

In a second aspect, a method for extracting alpine leontopodium vesicles is provided, comprising the following steps:

In the first or second aspect, in the step 2), the means for obtaining the supernatant containing the alpine leonurus vesicles includes centrifugation, and filtering the centrifuged supernatant to obtain the supernatant containing the alpine leonurus vesicles.

In the first or second aspect, the filtering mode is suction filtration, and the pore size of the suction filtration membrane is more than or equal to 0.2 μm or more than or equal to 0.45 μm or 0.8 μm.

In the first or second aspect, the centrifugation conditions are: and (3) centrifuging the mixture for 25-45 min by 3000g-10000g or centrifuging the mixture for 30min by 10000 g.

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, or 100 KD-800 KD, or 100 KD-750 KD, or 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, in the step 2), the purification mode is column purification.

Optionally, in the step 1), the culture temperature is 20 ℃ to 25 ℃.

Further, in the step 1), the suspension medium is an MS medium, and the suspension medium may be replaced for 20 days.

Further, in the step 1), the alpine leontopodium callus is obtained by inducing and culturing tissue blocks of leaves or stems of the alpine leontopodium through an induction culture medium.

Wherein the induction medium is based on woody plant culture medium (such as WPM culture medium), and sucrose, enzymatic hydrolyzed casein, plant gel and inositol are added.

Further, in the step 1), the preparation method of the alpine leontopodium herb callus comprises the following steps: cutting leaves or stems of alpine leontopodium into small pieces, and carrying out induction culture by adopting an induction culture medium to obtain callus; the induction culture conditions are 20-25 ℃, and the light culture is carried out. Alternatively, the induction medium is changed for a period of 20 days.

Wherein the induction medium is based on a woody plant culture medium (such as WPM culture medium), and further comprises 30-60 g/L sucrose, 1-3 g/L enzymatic hydrolyzed casein, 2.0-4.2 g/L plant gel, 0.1-0.5 g/L inositol (preferably 40g/L sucrose, 1g/L enzymatic hydrolyzed casein, 3.3g/L plant gel, and 0.1g/L inositol).

Preferably, when the aperture of a filter membrane adopting suction filtration is 0.8 mu m and the cut-off molecular weight of the hollow fiber is 100 KD-800 KD (preferably 100 KD-300 KD, more preferably 300 KD), the number of the separated vesicle-like substances is more, and the efficiency is higher.

Further, agarose column Q-large Scale is used for column purification.

Further, the method also comprises a step 3) of freeze-drying the alpine leontopodium vesicles.

In a third aspect, a product for regeneration and repair of skin cell collagen is provided, comprising alpine leonurus vesicles extracted by the extraction method of the second aspect.

Further, the Gao Shanhuo erigeronsThe concentration of vesicles in the product was 10 ⁸ ~10 ¹³ Particles/mL (preferably 10) ⁹ ~10 ¹² particles/mL）。

The preparation method of the alpine leontopodium herb vesicle comprises the following steps:

i) callus culture solution for preparing alpine leontopodium

Culturing the alpine leontopodium herb: washing seeds of the alpine leontopodium herb for 30min by flowing water, immersing 2 drops of Tween-20, treating for 5-15 min by using a 4% sodium hypochlorite solution, then sterilizing for 30s by using 70% ethanol, washing for 3 times by using sterile distilled water, inoculating the treated seeds of the alpine leontopodium herb to an MS culture medium (microphone), and culturing under the condition of 20-25 ℃ and sufficient illumination.

Induction of alpine leontopodium callus: cutting leaves or stems of high mountain leontopodium into small pieces of 2-3mm under a sterile environment, inoculating into an induction culture medium for induction culture, wherein the induction culture medium takes woody plant culture medium (WPM) (Soy palao) as a basic culture medium, and an induction culture medium of 40g/L sucrose, 1g/L enzymatic hydrolysis casein, 3.3g/L plant gel and 0.1g/L inositol is added into the induction culture medium. Culturing at 20-25 deg.c under sufficient illumination for 20 days to replace inducing culture medium for one period and culturing the callus successively.

Suspension culture of alpine leontopodium callus: the induced calli of the alpine leontopodium herb are inoculated onto a suspension culture medium MS (microphone forest), placed on a shaking table, subjected to suspension culture at the temperature of 20-25 ℃ under the condition of sufficient illumination, the suspension culture medium is replaced for 20 days for a period, and the calli are subjected to secondary culture.

II) extraction of high mountain velveteen vesicles

Collecting culture solution of callus of herba Lespedezae Cuneatae, centrifuging for 30min by 3000g-10000g, and collecting supernatant, which is called supernatant I; the supernatant I was then filtered sequentially through 0.2 μm, 0.45 μm or 0.8 μm filters to give a supernatant II.

Concentrating supernatant II by tangential flow method, concentrating to a certain volume by using hollow fiber with cut-off molecular weight of 100Kd or hollow fiber with cut-off molecular weight of 300Kd or hollow fiber with cut-off molecular weight of 500Kd or hollow fiber with cut-off molecular weight of 750Kd, and washing with 1 XPBS 5-10 times of the volume to obtain concentrated solution. And finally purifying the obtained concentrated solution by using an agarose chromatographic column Q large Scale to obtain the required alpine leonurus vesicles. Molecular sieves (HipreO 16/60 Sephacryl S-500 HR) were used to identify purity of Gao Shanhuo chorine vesicles for separation and purification.

Advantageous effects

1) The invention combines tangential flow ultrafiltration, hollow fiber and chromatography in the culture solution of the alpine leontopodium callus to realize large-scale industrialized production of the alpine leontopodium vesicles. A series of experiments show that the alpine leontopodium vesicles have excellent effects in the aspects of promoting the generation of I-type collagen, the migration of human fibroblasts, anti-inflammatory and the like.

2) The invention provides a method for extracting alpine leontopodium herb vesicles from a alpine leontopodium herb callus culture solution for the first time and a method for extracting the alpine leontopodium herb vesicles from the alpine leontopodium herb callus culture solution by using a tangential flow column chromatography method. The alpine leonurus vesicles extracted from the alpine leonurus callus culture solution have high purity, high yield and short time consumption, and can be produced on a large scale. Meanwhile, the culture solution of the calli of the alpine leontopodium can be obtained in a large amount, and the culture solution has no damage to the alpine leontopodium plants. And the extracted alpine leonurus vesicles are subjected to a series of efficacy tests, and the effects of promoting the generation of type I collagen, the migration of human fibroblasts, anti-inflammatory and the like are found.

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 relates to a process flow chart for extracting and separating vesicle-like substances from a culture solution of a calli of alpine leontopodium herb.

Fig. 2: in different embodiments of test example 1, different cutoff hollow fibers extract transmission electron microscope pictures of alpine leontopodium vesicles.

Fig. 3: transmission electron microscope pictures of the alpine leontopodium vesicles extracted in comparative example 9.

Fig. 4: molecular sieve diagrams of vesicles-like extracts from the culture solution of the calli of alpine leontopodium and the whole strain of alpine leontopodium in test example 2 of the present invention. Wherein a is the extracted and purified alpine leontopodium herb vesicles from example 1 (alpine leontopodium herb callus culture medium), and b is the alpine leontopodium herb vesicles from comparative example 9 (alpine leontopodium herb whole plant extraction).

Fig. 5: the results of promotion of type I collagen production by alpine leontopodium vesicles of test example 1 of the present invention are shown.

Fig. 6: the results of the anti-inflammatory effect of the alpine leontopodium vesicles of test example 2 of the present invention are shown.

Fig. 7: experimental results of the promotion of human fibroblast migration by the alpine leontopodium vesicles of test example 3 of the present invention.

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, seeds of alpine leontopodium (purchased from the autumn seed manager of Zhen Lin under Zha Yu county)

The extraction flow of the alpine leonurus vesicles is shown in figure 1, and specifically:

in the following examples, the culture method of the alpine leontopodium callus is as follows:

culturing the alpine leontopodium herb: washing seeds of the alpine leontopodium herb for 30min by flowing water, immersing 2 drops of Tween-20, treating for 5-15 min by using a 4% sodium hypochlorite solution, then sterilizing for 30s by using 70% ethanol, washing for 3 times by using sterile distilled water, inoculating the treated seeds of the alpine leontopodium herb to an MS culture medium (purchased from a microphone), and culturing under the condition of 20-25 ℃ and sufficient illumination to obtain the alpine leontopodium herb plants.

Induction of alpine leontopodium callus: cutting the leaves or stems of the above-mentioned cultured alpine leontopodium into small pieces of 2-3mm under aseptic condition, inoculating into induction culture medium, and performing induction culture. The induction medium was based on woody plant culture medium (WPM) (available from Soy Corp.) and added with 40g/L sucrose (sigma), 1g/L casein enzymatically hydrolyzed (sigma), 3.3g/L plant gel (sigma), and 0.1g/L inositol (sigma). Culturing at 20-25 deg.c under sufficient illumination for 20 days to replace inducing culture medium for one period and culturing the callus successively.

Suspension culture of alpine leontopodium callus: the induced calli of the alpine leontopodium herb are inoculated onto a suspension culture medium MS (purchased from microphone), placed on a shaking table, subjected to suspension culture at the temperature of 20-25 ℃ under sufficient illumination, replaced by the suspension culture medium for 20 days, and subjected to subculture to obtain the culture solution of the calli of the alpine leontopodium herb.

Example 1

5L of culture solution of the alpine leontopodium callus is collected at one time, 10000g of collected supernatant is subjected to centrifugation for 30min, sediment is discarded, and the supernatant is collected. The supernatant was obtained by suction filtration through a 0.8 μm filter. The supernatant was concentrated to 50mL by a hollow fiber having a cut-off molecular weight of 100Kd, the supernatant was changed to 250mL of 1 XPBS, and finally the 50mL of the concentrate was purified by an agarose column Q-large Scale to obtain 50mL of alpine leonurus vesicles. NTA (nanoparticle tracking analyzer) measures the particle count of alpine leontopodium vesicles. Freeze drying to obtain high mountain herba Potentillae Anserinae vesicle powder.

Example 2

5L of culture solution of the alpine leontopodium callus is collected at one time, 10000g of collected supernatant is subjected to centrifugation for 30min, sediment is discarded, and the supernatant is collected. The supernatant was obtained by suction filtration through a 0.8 μm filter. The supernatant was concentrated to 50mL by a hollow fiber having a cut-off molecular weight of 300Kd, the supernatant was changed to 250mL of 1 XPBS, and finally the 50mL of the concentrate was purified by an agarose column Q-large Scale to obtain 50mL of alpine leonurus vesicles. NTA measures the particle count of the alpine leontopodium vesicles. Freeze drying to obtain high mountain herba Potentillae Anserinae vesicle powder.

Example 3

5L of culture solution of the alpine leontopodium callus is collected at one time, 10000g of collected supernatant is subjected to centrifugation for 30min, sediment is discarded, and the supernatant is collected. The supernatant was obtained by suction filtration through a 0.8 μm filter. The supernatant was concentrated to 50mL by a hollow fiber having a cutoff molecular weight of 500Kd, the supernatant was changed to 250mL of 1 XPBS, and finally the 50mL of the concentrate was purified by an agarose column Q-large Scale to obtain 50mL of alpine leonurus vesicles. NTA measures the particle count of the alpine leontopodium vesicles. Freeze drying to obtain high mountain herba Potentillae Anserinae vesicle powder.

Example 4

Collecting culture solution of callus of herba Lespedezae Cuneatae 5L at a time, centrifuging for 20min after 2000g of the collected supernatant, discarding the precipitate, collecting the supernatant, centrifuging for 30min after 10000g of the collected supernatant, discarding the precipitate, and collecting the supernatant. The supernatant was obtained by suction filtration through a 0.8 μm filter. The supernatant was concentrated to 50mL by a hollow fiber having a cutoff molecular weight of 750Kd, the supernatant was changed to 250mL of 1 XPBS, and finally the 50mL of the concentrate was purified by an agarose column Q-large Scale to obtain 50mL of alpine leonurus vesicles. NTA measures the particle count of the alpine leontopodium vesicles. Freeze drying to obtain high mountain herba Potentillae Anserinae vesicle powder.

Example 5

3mg of the lyophilized powder of the alpine leonurus vesicles of example 3 was added to 1mL of 1 XPBS and dissolved into 3mg/mL of the alpine leonurus vesicle solution.

Comparative example 1

The difference from example 1 is that the supernatant was obtained by suction filtration using a 0.2 μm filter, and the remaining operation conditions were the same as in example 1.

NTA measured the particle count of alpine leontopodium vesicles of comparative example 1. Freeze drying to obtain high mountain herba Potentillae Anserinae vesicle powder.

Comparative example 2

The difference from example 1 is that the supernatant was obtained by suction filtration using a 0.45 μm filter, and the remaining operation conditions were the same as in example 1.

NTA measured the particle count of alpine leontopodium vesicles of comparative example 2. Freeze drying to obtain high mountain herba Potentillae Anserinae vesicle powder.

Comparative example 3

The difference from example 2 is that the supernatant was obtained by suction filtration using a 0.2 μm filter, and the remaining operation conditions were the same as in example 2.

NTA measured the particle count of alpine leontopodium vesicles of comparative example 3. Freeze drying to obtain the high mountain leontopodium herb vesicle powder.

Comparative example 4

The difference from example 2 is that the supernatant was obtained by suction filtration using a 0.45 μm filter, and the remaining operation conditions were the same as in example 2.

NTA measured the particle count of alpine leontopodium vesicles of comparative example 4. Freeze drying to obtain high mountain herba Potentillae Anserinae vesicle powder.

Comparative example 5

The difference from example 3 is that the supernatant was obtained by suction filtration using a 0.2 μm filter, and the remaining operation conditions were the same as in example 3.

NTA measured the particle count of alpine leontopodium vesicles of comparative example 5. Freeze drying to obtain high mountain herba Potentillae Anserinae vesicle powder.

Comparative example 6

The difference from example 3 is that the supernatant was obtained by suction filtration using a 0.45 μm filter, and the remaining operation conditions were the same as in example 3.

NTA measured the particle count of alpine leontopodium vesicles of comparative example 6. Freeze drying to obtain high mountain herba Potentillae Anserinae vesicle powder.

Comparative example 7

The difference from example 4 is that the supernatant was obtained by suction filtration using a 0.2 μm filter, and the remaining operation conditions were the same as in example 4.

NTA measured the particle count of alpine leontopodium vesicles of comparative example 7. Freeze drying to obtain high mountain herba Potentillae Anserinae vesicle powder.

Comparative example 8

The difference from example 4 is that the supernatant was obtained by suction filtration using a 0.45 μm filter, and the remaining operation conditions were the same as in example 4.

NTA measured the particle count of alpine leontopodium vesicles of comparative example 8. Freeze drying to obtain high mountain herba Potentillae Anserinae vesicle powder.

Comparative example 9 (differential centrifugation)

200g of whole plant of the alpine leontopodium herb (whole plant with root) is taken, purified water is used for cleaning for 5-10 times until the alpine leontopodium herb is clean, and then 1 XPBS is added to 1L. Crushing and squeezing to obtain alpine leontopodium herb juice. The obtained juice was centrifuged at 300g for 10min, at 3000g for 20min, at 10000g for 30min and at 110000g for 90min, respectively. Collecting the precipitate, and re-suspending the precipitate in PBS to obtain whole plant of extracted alpine leonurus vesicles, and freeze-drying to obtain whole plant of extracted alpine leonurus vesicles powder.

Comparative example 10 (alpine leontopodium herb extract)

Freeze-drying the cultured alpine leontopodium callus cells to obtain alpine leontopodium callus cell powder, adding 10g of alpine leontopodium callus cell powder into 200mL of 50% ethanol solution for treatment for 12 hours, adding 1% pectase and 1% cellulase, regulating the PH to 4 by using citric acid and sodium hydroxide at the temperature of 50 ℃, carrying out enzymolysis for 3 hours, carrying out heat reflux for 3 hours at the temperature of 100 ℃, cooling, filtering with a 0.45 mu m filter membrane, collecting filtrate, and freeze-drying to obtain alpine leontopodium extract powder.

Comparative example 11

3mg of the alpine leontopodium herb extract powder of comparative example 10 was dissolved in 1mL of 1 XPBS to prepare a solution having a concentration of 3 mg/mL.

Test example 1

The vesicles extracted in examples 1 to 4 and comparative example 9 were subjected to lens observation.

The results of lens observation of the vesicles of examples 1-4 are shown in fig. 2, and fig. 2 shows that the transmission electron microscope pictures of the high mountain leontopodium herb vesicles extracted by the hollow fiber columns with different cut-off amounts are all cup-shaped vesicle structures, and conform to the vesicle structures.

The result of lens observation of the vesicle like material of comparative example 9 is shown in fig. 3, and fig. 3 shows that the vesicle like material of comparative example 9 is extracted from the whole plant of alpine leontopodium herb by differential centrifugation, and has high impurity content and unclear vesicle like structure.

It was demonstrated that the vesicles extracted in examples 1 to 4 were more complete in morphology and better in effect than comparative example 9, and the obtained alpine leonurus vesicles were high in purity.

Test example 2

The results of passing the purified alpine leontopodium vesicles of example 1 and the alpine leontopodium vesicles extracted from the whole plant of alpine leontopodium in comparative example 9 through a molecular sieve column (Hipreo 16/60 Sephacryl S-500 HR) are shown in FIG. 4.

FIG. 4 shows that the purified alpine leontopodium vesicles extracted from example 1 (alpine leontopodium callus culture broth) have no impurity peaks, while the alpine leontopodium vesicles extracted from comparative example 9 (alpine leontopodium whole plant) have impurity peaks.

In summary, as shown in fig. 2-4, the vesicle-like purity extracted from the alpine leontopodium callus culture solution by utilizing the filter membrane filtration with specific pore diameter and the hollow fiber is higher, and the method is suitable for mass production.

Test example 3

The particle numbers measured by NTA after the lyophilized powders obtained in examples 1 to 4 and comparative examples 1 to 8 were diluted with 1 XPBS by the same factor are shown in Table 1.

Table 1 shows the particle numbers measured by NTA of examples 1 to 4 and comparative examples 1 to 8

。

The results in Table 1 show that the four types of hollow fibers with different cut-off amounts of 100kd,300kd,500kd and 750kd are used for purifying the alpine leontopodium vesicles in examples 1-4 respectively, and the particle numbers are not very different, namely, the four types of hollow fibers with different cut-off amounts can be used for purifying the alpine leontopodium vesicles.

The results in Table 1 also show that the filtration with different pore sizes resulted in a larger difference in the numbers of alpine leonardite type vesicle particles when the filtration with different pore sizes was used in examples 1-4 and comparative examples 1-8. Even if the filter membranes with different pore diameters are used for clarifying the alpine velvet callus culture solution, the concentration of the alpine velvet grass vesicles is obviously influenced, and the effect of the embodiment 2 is relatively better.

Efficacy experiment

Test example 1: effect of alpine leontopodium vesicles on the Synthesis of type I collagen (Col 1a 1)

Type I collagen is a highly structured protein, and is composed of a three-stage helix structure consisting of three alpha-chain helices. Type i collagen is the main component of the skin structure, accounting for 75% of the skin weight, and it gives skin elasticity and tension, and keeps the skin tight and smooth. With age, the content of type I collagen gradually decreases, so that the skin becomes loose and wrinkles increase.

Cryopreserved human skin fibroblasts were seeded at a suitable density and passaged at least once prior to testing. Human skin fibroblasts were according to 2X 10 ⁵ Inoculating into multiple 6-well plates at 37deg.C and 5% CO ₂ Culturing in the culture medium for 24 hours, wherein the cell fusion degree reaches 45% -60%. After 24H cell attachment, the cells were divided into a blank group, a control group, a group of example 5, and a group of comparative example 11, each of which was repeated 3 times, wherein the blank group was not added with H ₂ O ₂ The remaining groups were added with 2mL of 200uMH per well ₂ O ₂ After 1h of stimulation, the cells were washed 3 times with PBS. Then, 200. Mu.L of the high mountain velvet grass vesicle solution of example 5 was added to each well of example 5, and 200. Mu.L of the high mountain velvet of comparative example 11 was added to each well of the group of comparative example 11Grass extract, control group per well added 200 μl of 1×pbs, after 24h incubation, cells were collected. After completion of the culture, the protein was eluted with a cell lysis buffer (0.1% Sodium Dodecyl Sulfate (SDS), 1% NP40, 150mM NaCl, 0.5% sodium deoxycholate, 50mM Tris-HCl (pH 7.5)), and then the amount of the protein was measured by a BCA (bicinchoninic acid) quantitative method. 20ug of protein (converted from the amount of protein detected) was separated by SDS-polyacrylamide gel electrophoresis, and then transferred to a PVDF membrane (i.e., polyvinylidene fluoride membrane). The PVDF membrane was then reacted with TBST (10 mM Tris HCl, pH8.0, 150mM NaCl,0.1% Tween 20) buffer containing 5% skim milk for 1 hour, and then reacted with primary antibody Col1a1 (available from CST) (dilution 1:1000) overnight at 4 ℃. Goat anti-rabbit IgG antibodies (from Santa Cruz, USA) were labeled with horseradish peroxidase as secondary antibodies (dilution 1:5000) and allowed to react for 1 hour at ambient temperature. Then, the sample was washed with TBST buffer for 30 minutes, developed by a chemiluminescent detection kit (ECL solution kit, amersham, UK), and then analyzed for Col1a1, the results of which are shown in FIG. 5.

FIG. 5 shows that the comparison between the control group and the blank group shows that the test sample is H ₂ O ₂ The content of type I collagen (Col 1a 1) in human skin fibroblasts under stimulation is obviously reduced. Example 5 group of human dermal fibroblasts in H ₂ O ₂ The content of type I collagen (Col 1a 1) in human skin fibroblasts was increased relative to the blank after the addition of the alpine leontopodium herb vesicles of example 5 under stimulation, and the content of type I collagen (Col 1a 1) was not increased by the addition of the alpine leontopodium herb callus extract of comparative example 11. Thus, the alpine leonurus vesicles can promote the generation of type I collagen (Col 1a 1) in human skin fibroblasts, and have an anti-aging function.

Test example 2: alpine leontopodium vesicles assay for anti-inflammatory effects by inhibiting cyclooxygenase-2 (Cox-2) production

Cyclooxygenase-2 (COX-2) is a less expressed inducible enzyme in normal tissues, and is expressed in large amounts when cells are stimulated by inflammation. COX-2 has long been thought to play an important role in the pathological process of inflammation as it responds rapidly to a range of pro-inflammatory mediators and cytokines.

Cryopreserved raw264.7 cells were seeded at a suitable density and passaged at least once prior to detection. Raw264.7 cells were according to 2X 10 ⁵ The wells were seeded in 6-well plates at 37℃with 5% CO ₂ After 24 hours of culture in the culture medium, the cell fusion degree reaches 45% -60%. 24h after cell attachment, the cells were divided into a blank group, a control group, a group of example 5, and a group of comparative example 11, each group was repeated 3 times, wherein the blank group was not added with lipopolysaccharide, and the rest groups were added with Lipopolysaccharide (LPS) at a final concentration of 1. Mu.g/mL per well, and PBS was washed 3 times after the completion of stimulation. Then, 200. Mu.L of the high mountain leontopodium polycephalum vesicle solution of example 5 was added to each well of example 5, 200. Mu.L of the high mountain leontopodium polycephalum extract of comparative example 11 was added to each well of comparative example 11, 200. Mu.L of 1 XPBS was added to each well of the control group, and after culturing for 24 hours, the cells were collected. After completion of the culture, the protein was eluted with a cell lysis buffer (0.1% Sodium Dodecyl Sulfate (SDS), 1% NP40, 150mM NaCl, 0.5% sodium deoxycholate, 50mM Tris-HCl (pH 7.5)), and the amount of the protein was measured by a BCA (bicinchoninic acid) quantitative method. The same amount of protein (converted from the amount of protein detected) was taken out for each group of 20ug, and after separation by SDS-polyacrylamide gel electrophoresis, the protein was transferred to a PVDF membrane, i.e., a polyvinylidene fluoride membrane. The PVDF membrane was then reacted with TBST (10mMTris HCl,pH8.0, 150mMNaCl,0.1%Tween (Tween) 20) buffer containing 5% skim milk for 1 hour and then reacted with primary antibody Cox-2 (available from CST) (dilution 1:1000) overnight at 4 ℃. Goat anti-rabbit IgG antibodies (from Santa Cruz, USA) were labeled with horseradish peroxidase as secondary antibodies (dilution 1:5000) and allowed to react for 1 hour at ambient temperature. Then, the sample was washed with TBST buffer for 30 minutes, and after development of color by a chemiluminescent detection kit (ECL solution kit, amersham, UK), cox-2 was analyzed, as shown in FIG. 6.

As can be seen from FIG. 6, the control group showed that the Raw264.7 cells produced Cox-2, i.e., the cells produced inflammation, under the stimulation of Lipopolysaccharide (LPS) compared with the control group, whereas the example 5 group showed that Cox-2 production was inhibited after the addition of the alpine leopard vesicles, i.e., the alpine leopard vesicles were able to inhibit the production of inflammation.

Test example 3: scratch test of high mountain leontopodium herb vesicles

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. The number and quality and activity of fibroblasts in the dermis layer directly determine the degree of aging of the skin.

Human skin fibroblasts were cultured at a rate of 2X 10 ⁵ The wells were seeded into 6 well plates. Cells were incubated at 37℃with 5% CO ₂ Culturing for 1 day. 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 migrated. Cell migration tests were performed in groups, which were divided into a control group, a group of example 5 and a group of comparative example 11, 3 replicates per well, 200. Mu.L of the alpine leonurus vesiculosus solution of example 5 was added per well of the group of example 5, 200. Mu.L of the alpine leonurus callus extract of comparative example 11 was added per well of the group of comparative example 11, and 200. Mu.L of 1 XPBS was added to the group of control. The scratch areas were photographed at 0h and 36h after scratch formation, and the migration of cells was observed as shown in fig. 7.

The results in fig. 7 show that the human skin fibroblasts of example 5 grew faster after 36 hours than the control and comparative example 11 groups. The alpine leontopodium vesicles can promote migration and growth of human skin fibroblasts, and have an anti-aging function.

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. A method for extracting alpine leontopodium vesicles for skin cell collagen regeneration and/or skin repair, which is characterized by comprising the following steps:

in the step 1), the alpine leontopodium callus is obtained by inducing and culturing tissue blocks of leaves or stems of the alpine leontopodium through an induction culture medium, and the induced alpine leontopodium callus is inoculated into a suspension culture medium;

the induction culture medium takes a WPM culture medium as a basic culture medium, and 30-60 g/L of sucrose, 1-3 g/L of enzymatic hydrolysis casein, 2.0-4.2 g/L of plant gel and 0.1-0.5 g/L of inositol are added;

the suspension culture medium is MS culture medium;

2) Taking the culture solution in the step 1), separating to obtain supernatant containing the alpine leonurus vesicles, concentrating, and purifying to obtain the alpine leonurus callus vesicles; wherein,

in the step 2), the mode of obtaining the supernatant containing the alpine leonurus vesicles comprises centrifuging, taking the centrifuged supernatant and filtering to obtain the supernatant containing the alpine leonurus vesicles;

in the step 2), the filtering mode is suction filtration, and the aperture of a suction filtration membrane is 0.8 mu m;

in the step 2), hollow fiber concentration is adopted, and the cut-off molecular weight of the hollow fiber is 100 KD-750 KD; the concentration multiple is 50-200 times;

in the step 2), the purification mode is chromatographic column purification.

2. The extraction method according to claim 1, wherein the centrifugation conditions are: centrifuging 3000g-10000g for 25-45 min.

3. The extraction method according to claim 1, wherein the centrifugation conditions are: centrifuge 10000g for 30min.

4. The extraction method according to claim 1, wherein in step 2), the hollow fiber has a molecular weight cut-off of 100kd to 300kd.

5. The extraction method according to claim 1, wherein in step 2), the concentration ratio is 80 to 150 times.

6. The extraction method according to claim 1, wherein in step 2), the concentration factor is 100 to 150 times.

7. Use of a alpine leontopodium type vesicle according to any one of claims 1 to 6 for the preparation of a skin cell collagen regeneration and/or skin repair product.

8. The use according to claim 7, wherein the skin cell collagen regeneration product comprises a product that promotes collagen type I production.

9. The use according to claim 7, wherein the skin repair product comprises a skin product that promotes migration of human fibroblasts and/or is anti-inflammatory.

10. The use according to claim 7, wherein anti-inflammatory comprises inhibiting the production of cyclooxygenase-2.

11. The use according to claim 7, wherein the Gao Shanhuo villus vesicles are present in the product at a concentration of 10 ⁸ ~10 ¹³ particles/mL。

12. The use according to claim 7, wherein said Gao Shanhuo erigeron vesicles are producedThe concentration in the product is 10 ¹⁰ ~10 ¹² particles/mL。

13. The use according to claim 7, wherein the Gao Shanhuo villus vesicles are present in the product at a concentration of 10 ¹¹ ~10 ¹² particles/mL。