CN113069376A

CN113069376A - Skin micro-ecological regeneration and restoration essence and preparation method thereof

Info

Publication number: CN113069376A
Application number: CN202110320389.8A
Authority: CN
Inventors: 陈阳; 吕敏; 殷淑菁
Original assignee: Hangzhou Sanjiang Shangyu Biotechnology Co ltd
Current assignee: Hangzhou Sanjiang Shangyu Biotechnology Co ltd
Priority date: 2021-03-25
Filing date: 2021-03-25
Publication date: 2021-07-06

Abstract

The invention relates to a skin care product, and particularly relates to skin micro-ecological regeneration and restoration essence and a preparation method thereof. The essence comprises a concentrated solution of a menstrual blood stem cell factor, human albumin, a moisturizing component, an antiallergic component and a Hamamelis virginiana extract, wherein the antiallergic component is a yeast fermentation product filtrate, a schizosaccharomyces cerevisiae fermentation product lysate and a giant kelp extract. The essence comprises the following components in percentage by weight: 20 to 35 percent of concentrated solution of blood stem cell factor, 2 to 5 percent of human albumin, 0.1 to 5 percent of filtrate of yeast fermentation product, 0.05 to 10 percent of lysate of second split yeast fermentation product, 1 to 10 percent of giant kelp extract, 0.05 to 10 percent of hamamelis extract and 1 to 5 percent of moisture-keeping component. The invention provides a skin micro-ecological regeneration repair essence for repairing and resisting aging of skin by utilizing menstrual stem cells and a preparation method thereof.

Description

Skin micro-ecological regeneration and restoration essence and preparation method thereof

Technical Field

The invention relates to a skin care product, and particularly relates to skin micro-ecological regeneration and restoration essence and a preparation method thereof.

Background

Menstrual blood-derived mesenchymal stem cells are stem cells isolated from female menstrual blood, and were obtained from menstrual blood of healthy women by the american research group for the first time, and among known human cells, the menstrual blood-derived mesenchymal stem cells are stem cells having the strongest self-replicating and directional differentiation ability, are derived from endometrium, and are excluded by menstrual blood.

The menstrual blood stem cells have multidirectional differentiation potential, and can be differentiated into cells such as fat cells, muscle cells, osteoblasts, chondrocytes, vascular endothelium, nerve cells and the like after induction culture. It can be successfully differentiated into osteoblasts and smooth muscle cells under the induction of an osteogenic induction culture medium and a smooth muscle induction culture medium. Santamaria et al induce endometrial stem cells into cells with insulin secretion activity, and still have the function of secreting insulin after being transplanted into a mouse model, can maintain the blood sugar level of mice to be stable, and reduce the occurrence of diabetic complications. Wolff et al induced endometrial stem cells in vitro into dopaminergic neurons, which secreted dopamine in vitro and had electrophysiological properties of central neurons, transplanted into immunocompetent Parkinsonian model brains, and showed significant increases in striatal concentrations of dopamine and its metabolites.

The discovery of the hematopoietic stem cells undoubtedly brings a new revolution to reproductive medicine. The menstrual blood stem cells can be repeatedly obtained from menstrual blood, and have wide clinical application prospects in the aspects of tissue regeneration treatment and the like due to the convenient material acquisition and the biological characteristics of the menstrual blood stem cells. It not only becomes a new model for better understanding the pathophysiology of uterus, but also can become a new means for improving endometrium and treating uterine infertility in the near future. However, the research on the endometrial stem cells is more and more biased to the disease mechanism, such as endometriosis, endometrial cancer, infertility, endometrial hyperplasia and the like.

However, in fact, the stem cells are the stem cells with the strongest capacity of self-replication and directional differentiation, the differentiation potential of the stem cells is better than that of the embryonic stem cells, and the cytokines of the stem cells also have the physiological repair or substitute for body injury, pathological changes or aging.

As is known, a large amount of bacteria are bred on the skin of a human face when the skin of the face is exposed outside for a long time, under the irradiation of ultraviolet rays, or stays overnight for a long time, or under the influence of some adverse environments, so that inflammation is caused, the skin is mostly damaged to a certain extent, and a series of skin problems such as skin redness, large pores, acne growing and the like occur.

Disclosure of Invention

In order to overcome the defects of the background technology, the invention provides a skin micro-ecological regeneration repair essence for repairing and resisting aging of skin by utilizing menstrual stem cells and a preparation method thereof.

The technical scheme adopted by the invention is as follows: the skin microecological regeneration and repair essence comprises a concentrated solution of a blood stem cell factor, human albumin, a moisturizing component, an antiallergic component, a Hamamelis virginiana extract and distilled water, wherein the antiallergic component comprises a yeast fermentation product filtrate, a schizosaccharomyces cerevisiae fermentation product lysate and a giant kelp extract.

Preferably, the essences comprise, by weight: 20 to 35 percent of concentrated solution of blood stem cell factor, 2 to 5 percent of human albumin, 0.1 to 5 percent of filtrate of yeast fermentation product, 0.05 to 10 percent of lysate of second split yeast fermentation product, 1 to 10 percent of giant kelp extract, 0.05 to 10 percent of hamamelis extract and 1 to 5 percent of moisture-keeping component.

Preferably, the moisturizing component comprises propylene glycol, butylene glycol, glycerin, hyaluronic acid and trehalose.

A preparation method of skin micro-ecological regeneration and repair essence comprises the following steps:

(1) preparing a concentrated solution of the blood stem cell factor:

A. collecting menstrual blood from the second day to the third day of the menstrual period of the female by using a menstrual cup; pouring the collected menstrual blood into a sterile container filled with a preservation solution, fully and uniformly mixing the menstrual blood and the preservation solution, preserving and standing at the temperature of 0-4 ℃ for 5-12 hours, and fully sterilizing the menstrual blood;

B. transporting the menstrual blood soaked in the menstrual blood preservation solution to a laboratory, transferring the menstrual blood soaked in the menstrual blood preservation solution to a centrifuge tube in an operation table, centrifuging at 2000-2500 rpm for the first time and centrifuging for 10-15min, and removing supernatant to obtain a primary liquid I; adding a cleaning solution into the primary liquid I, fully and uniformly mixing, centrifuging at 1500-;

C. precipitating the main liquid III, adding 0.25% pancreatin into the precipitate, standing at 37 ℃ for 1-3min, adding stop solution to stop digestion, quickly shaking, blowing cells, sucking out cell suspension, then adding physiological saline to wash, centrifuging, collecting the precipitate, using a complete culture medium to resuspend the precipitate, inoculating the precipitate into the complete culture medium to perform P1 generation culture, and continuously culturing until P4 generation-P6 generation when the cells grow to 70% -90% fusion;

D. adding 1% of calcium gluconate and 0.02% of prothrombin complex into the P2-P6 generation cell culture solution, standing at room temperature for 1 hour, filtering and concentrating through a microfiltration membrane and an ultrafiltration membrane to obtain a concentrated solution of the stem cells;

(2) preparation of a diluent: preparing human albumin, a moisture-preserving component, an anti-allergic component and a hamamelis virginiana extract by using distilled water according to a proportion, mixing and stirring at the rotating speed of 200-;

(3) and (3) mixing the concentrated solution of the menstrual blood stem cells obtained in the step (1) with the diluted solution obtained in the step (2) in proportion, and shaking for 5-10min uniformly to obtain the skin micro-ecological regeneration and restoration essence containing the menstrual blood stem cell factors.

Preferably, the stop solution comprises 92-94% (v/v) of alpha-MEM culture medium, 3.5-5% (v/v) of fetal bovine serum, 1.2-1.5% (w/v) of thiamine hydrochloride and 1.3-1.5% (w/v) of 4-hydroxyethyl piperazine ethanesulfonic acid.

Preferably, the preservation solution is a preservation solution containing antibiotics, sodium citrate and heparin sodium.

The invention has the beneficial effects that: the concentrated solution of the blood stem cell factor can obviously play roles of repairing and resisting aging after being added into skin care products, and improve the skin problem caused by skin injury, and the yeast fermentation product filtrate, the schizosaccharomyces cerevisiae fermentation product lysate, the light black kelp extract and the hamamelis virginiana extract are mutually matched and synergistically give effect to effectively inhibit inflammation quoted by bacteria and inhibit the growth of pathogenic flora so as to play a role in preventing allergy, so that the essence solution is used for a long time, the damaged skin is repaired, the aging is delayed, the repaired skin can be effectively inhibited, the growth of the pathogenic flora is prevented, the skin barrier is effectively protected, and the skin is on the basis of repairing, the skin is always in a healthy and good state under the dual guarantee of repair and defense, and the microecological balance is kept.

Detailed Description

The menstrual blood stem cells have the effects of physiologically repairing or replacing body injury, pathological changes or aging, have very strong repairing and regenerating functions on injuries caused by the surface of the skin, and can obviously play the roles of repairing and aging resistance and improve skin problems caused by the injuries of the skin after the concentrated solution of the menstrual blood stem cell factors is added into skin care products.

In the invention, the yeast fermentation product filtrate is prepared by selecting good cereals (preferably rice) and naturally fermenting by natural yeast, and is rich in various active ingredients required by skin, such as oligosaccharide, organic acid, polypeptide, amino acid and the like. The skin care product can be effectively and quickly absorbed and utilized by the metabolic activity of skin probiotics, but not by common pathogenic bacteria (such as propionibacterium acnes and staphylococcus aureus) in the skin, so that the healthy growth of probiotic groups in the skin is facilitated, the growth and reproduction of pathogenic bacteria are competitively inhibited, and a good probiotic ecological environment beneficial to the skin is established.

The lysate of the yeast fermentation product of the invention can generate small molecules such as vitamin B group, mineral substances, amino acids and the like which are beneficial to skin care, and the yeast fermentation product filtrate is combined to strengthen the metabolism of the horny layer and effectively inhibit the secretion of grease.

The effective components of the giant kelp extract disclosed by the invention are polysaccharide and various minerals, and the giant kelp extract can promote metabolism, balance metabolism, improve immune function, supplement minerals and the like.

The hamamelis virginiana extract disclosed by the invention has the main effects of promoting the skin to absorb yeast fermentation product filtrate, schizosaccharomyces cerevisiae fermentation product lysate, light black kelp extract, stem cell concentrated solution and moisturizing molecules, further improving the repair and defense functions of the facial skin, and meanwhile, the hamamelis virginiana contains a allergy-softening factor, so that the balance of skin metabolism can be kept.

According to the invention, the blood stem cell factor is combined with the yeast fermentation product filtrate, the schizosaccharomyces cerevisiae fermentation product lysate and the giant kelp extract, so that the damaged skin is repaired, the aging is delayed, the repaired skin can be effectively inhibited, the growth of pathogenic flora is prevented, the skin barrier can be effectively protected, the skin has a better defense function on the basis of repair, the repair and the defense are realized, the skin is always in a healthy and good state under the double guarantee of repair and defense, and the balance of microecology is kept.

The essence comprises the following components in percentage by weight: 20 to 35 percent of concentrated solution of blood stem cell factor, 2 to 5 percent of human albumin, 0.1 to 5 percent of filtrate of yeast fermentation product, 0.05 to 10 percent of lysate of second split yeast fermentation product, 1 to 10 percent of giant kelp extract, 0.05 to 10 percent of hamamelis extract and 1 to 5 percent of moisture-keeping component.

In the present invention, the content of human albumin is 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%.

In the present invention, the content of the concentrated menstrual blood stem cell factor solution is 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%.

In the invention, the content of the yeast fermentation product filtrate is 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%.

In the present invention, the content of the secondary split yeast fermentation product lysate is 0.05%, 0.1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%.

In the present invention, the content of the giant kelp extract is 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%.

In the present invention, the content of the hamamelis virginiana extract is 0.05%, 0.1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%.

In the present invention, the content of the moisture-retaining component is 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%.

The moisture keeping component comprises propylene glycol, butanediol, glycerol, hyaluronic acid and trehalose.

(1) preparing a concentrated solution of the blood stem cell factor:

A. collecting menstrual blood from the second day to the third day of the menstrual period of the female by using a menstrual cup; pouring the collected menstrual blood into a sterile container filled with a preservation solution, fully and uniformly mixing the menstrual blood and the preservation solution, preserving and standing for 12 hours at the temperature of 4 ℃, and fully sterilizing the menstrual blood; before collection, the collected object needs to be confirmed to have no infectious diseases or allergic diseases;

B. transporting the menstrual blood soaked in the menstrual blood preservation solution to a laboratory, transferring the menstrual blood soaked in the menstrual blood preservation solution to a centrifuge tube in an operation table, centrifuging at 2000-2500 rpm for the first time and centrifuging for 10min, and removing supernatant to obtain a primary liquid I; adding a cleaning solution into the primary liquid I, fully mixing uniformly, centrifuging at 1500-; centrifuging collected menstrual blood preserving fluid for multiple times, and removing supernatant fluid to obtain a main fluid with higher concentration;

C. precipitating the main liquid III, adding 0.25% pancreatin into the precipitate, standing at 37 ℃ for 2min, adding stop solution to stop digestion, quickly shaking, blowing cells, sucking out cell suspension, adding physiological saline to wash, centrifuging, collecting the precipitate, re-suspending the precipitate by using a complete culture medium, inoculating the precipitate to the complete culture medium to perform P1 generation culture, and continuously culturing until the cells grow to 70% -90% fusion, wherein the cell suspension is cultured to P4 generation-P6 generation; the step is mainly to passage primary cells and add pancreatin for dispersing tissues;

D. adding 1% of calcium gluconate and 0.02% of prothrombin complex into the P2-P6 generation cell culture solution, standing at room temperature for 1 hour, filtering and concentrating through a microfiltration membrane and an ultrafiltration membrane to obtain a concentrated solution of the stem cells; in principle, the cell culture solution in step C can be directly formed into a concentrated solution after being filtered and mixed with a diluent, but in the experimental process, it is found that the addition of the calcium gluconate and prothrombin complex in the cell culture solution can play a role in activating cells, so that the PDGF-BB concentration is higher.

(2) Preparation of a diluent: preparing human albumin, a moisture-preserving component, an anti-allergic component and distilled water in proportion, mixing and stirring at the rotation speed of 200-;

The stop solution comprises 92-94% (v/v) of alpha-MEM culture medium, 3.5-5% (v/v) of fetal bovine serum, 1.2-1.5% (w/v) of thiamine hydrochloride and 1.3-1.5% (w/v) of 4-hydroxyethyl piperazine ethanesulfonic acid.

The preservation solution is a preservation solution containing antibiotics, sodium citrate and heparin sodium.

The present invention will be further described with reference to the following examples.

The first embodiment is as follows:

(1) preparing a concentrated solution of the blood stem cell factor:

A. collecting menstrual blood from the second day to the third day of the menstrual period of the female by using a menstrual cup; pouring the collected menstrual blood into a sterile container filled with a preservation solution, fully and uniformly mixing the menstrual blood and the preservation solution, preserving and standing for 6 hours at the temperature of 4 ℃, and fully sterilizing the menstrual blood;

D. adding 1% calcium gluconate and 0.02% prothrombin complex into the heavy suspension precipitate, standing at room temperature for 0.5-1 hr, filtering with microfiltration membrane and ultrafiltration membrane, and concentrating to obtain concentrated solution of stem cells;

(2) preparation of a diluent: preparing 3% of human albumin, 3% of yeast fermentation product filtrate, 3% of schizosaccharomyces fermentation product lysate, 3% of light black kelp extract, 5% of hamamelis virginiana extract and 3% of moisture retention component by using distilled water in proportion, mixing and stirring at the rotating speed of 200-400r/min at the temperature of 30-60 ℃ for 10-20min, and filtering through micropores with 500 meshes of 300-60 ℃ to obtain diluent;

(3) and (3) mixing the 20% of the concentrated solution of the menstrual blood stem cells obtained in the step (1) with the 80% of the diluent obtained in the step (2), and shaking for 5-10min uniformly to obtain the skin micro-ecological regeneration and restoration essence containing the menstrual blood stem cell factors.

Example two:

(1) preparing a concentrated solution of the blood stem cell factor:

A. collecting menstrual blood from the second day to the third day of the menstrual period of the female by using a menstrual cup; pouring the collected menstrual blood into a sterile container filled with a preservation solution, fully and uniformly mixing the menstrual blood and the preservation solution, preserving and standing for 10 hours at the temperature of 4 ℃, and fully sterilizing the menstrual blood;

D. adding 1% of calcium gluconate and 0.02% of prothrombin complex into the heavy suspension precipitate, standing at room temperature for 1 hour, and filtering and concentrating through a microfiltration membrane and an ultrafiltration membrane to obtain a concentrated solution of the stem cells;

(2) preparation of a diluent: preparing 2% of human albumin, 2% of yeast fermentation product filtrate, 2% of schizosaccharomyces fermentation product lysate, 4% of light black kelp extract, 1% of hamamelis virginiana extract and 1% of moisture-preserving component by using distilled water in proportion, mixing and stirring at 45 ℃ at the rotating speed of 200 plus 400r/min for 10-20min, and filtering through micropores of 300 plus 500 meshes to obtain diluent;

(3) and (3) mixing 25% of the concentrated solution of the menstrual blood stem cells obtained in the step (1) with 75% of the diluent obtained in the step (2), and shaking for 5-10min uniformly to obtain the skin micro-ecological regeneration and restoration essence containing the menstrual blood stem cell factors.

Example three:

(1) preparing a concentrated solution of the blood stem cell factor:

(2) preparation of a diluent: preparing 3% of human albumin, 3% of yeast fermentation product filtrate, 5% of schizosaccharomyces cerevisiae fermentation product lysate, 5% of light black kelp extract, 5% of hamamelis virginiana extract and 3% of moisture-retaining component by using distilled water in proportion, mixing and stirring at 50 ℃ at the rotating speed of 200-400r/min for 10-20min, and filtering through micropores with the size of 300-500 meshes to obtain diluent;

(3) and (3) mixing the 30% of the concentrated solution of the menstrual blood stem cells obtained in the step (1) with the 70% of the diluent obtained in the step (2), and shaking for 5-10min uniformly to obtain the skin micro-ecological regeneration and restoration essence containing the menstrual blood stem cell factors.

Example four:

(1) preparing a concentrated solution of the blood stem cell factor:

A. collecting menstrual blood from the second day to the third day of the menstrual period of the female by using a menstrual cup; pouring the collected menstrual blood into a sterile container filled with a preservation solution, fully and uniformly mixing the menstrual blood and the preservation solution, preserving and standing for 12 hours at the temperature of 4 ℃, and fully sterilizing the menstrual blood;

(2) preparation of a diluent: preparing 4% of human albumin, 4% of yeast fermentation product filtrate, 3% of schizosaccharomyces fermentation product lysate, 2% of light black kelp extract, 4% of hamamelis virginiana extract and 3% of moisture-preserving component by using distilled water in proportion, mixing and stirring at 45 ℃ at the rotating speed of 200 plus 400r/min for 10-20min, and filtering through micropores of 300 plus 500 meshes to obtain diluent;

(3) and (3) mixing the 35% of the concentrated solution of the menstrual blood stem cells obtained in the step (1) with the 65% of the diluent obtained in the step (2), and shaking for 5-10min uniformly to obtain the skin micro-ecological regeneration and restoration essence containing the menstrual blood stem cell factors.

Testing of the essence:

searching 40 testers with dark faces, reddened skins or large pores with strong grease secretion or loose skins, randomly dividing the testers into four groups, continuously using the essence liquid in the first embodiment to the essence liquid in the fifth embodiment for 30 days, observing the skin conditions of the testers, and grading according to a table 1 to obtain a table 2:

table 1 shows the scoring criteria after 30 days of use

Facial condition	Fractional interval (points)
		Dark and rough face, vigorous secretion of oil and fat, no obvious improvement	1-2
The skin becomes smooth obviously, has slight oil secretion and slight improvement	3-5
		Smooth skin, no obvious oil secretion, and obvious improvement	5-8
Smooth and white skin, no oil secretion and obvious improvement	9-10

Table 2 serum efficacy test scores

	Number of people	Number of days	Scoring
				Example one	15	30	7.9
Example two	15	30	8.2
				EXAMPLE III	15	30	9.2
Example four	15	30	8.8

Tests show that the essence in the protection range of the application has the effects of repairing and defending, and obviously improving the skin quality, and in the third embodiment, the proportions of the concentrated solution and the diluted solution are respectively 30% and 70%, and the proportions of the human albumin, the yeast fermentation product filtrate, the schizosaccharomyces cerevisiae fermentation product lysate, the light black kelp extract, the hamamelis virginiana extract and the moisturizing component are all in the middle, so that the best effect is achieved.

The essence of the invention is preferably packaged in a container with a volume of less than 3ml and refrigerated at about 0 ℃.

The skilled person should understand that: although the invention has been described in terms of the above specific embodiments, the inventive concept is not limited thereto and any modification applying the inventive concept is intended to be included within the scope of the patent claims.

Claims

1. The skin microecological regeneration repair essence is characterized by comprising the following components in parts by weight: the essence comprises a concentrated solution of a menstrual blood stem cell factor, human albumin, a moisturizing component, an antiallergic component and a Hamamelis virginiana extract, wherein the antiallergic component is a yeast fermentation product filtrate, a schizosaccharomyces cerevisiae fermentation product lysate and a giant kelp extract.

2. The skin microecological rejuvenation repair essence according to claim 1, which is characterized in that: the essence comprises the following components in percentage by weight: 20 to 35 percent of concentrated solution of blood stem cell factor, 2 to 5 percent of human albumin, 0.1 to 5 percent of filtrate of yeast fermentation product, 0.05 to 10 percent of lysate of second split yeast fermentation product, 1 to 10 percent of giant kelp extract, 0.05 to 10 percent of hamamelis extract and 1 to 5 percent of moisture-keeping component.

3. The skin microecological rejuvenation repair essence according to claim 2, which is characterized in that: the moisture keeping component comprises propylene glycol, butanediol, glycerol, hyaluronic acid and trehalose.

4. A preparation method of skin micro-ecological regeneration and restoration essence is characterized by comprising the following steps: which comprises the following steps:

(1) preparing a concentrated solution of the blood stem cell factor:

A. collecting menstrual blood from the second day to the third day of the menstrual period of the female by using a menstrual cup; pouring the collected menstrual blood into a sterile container filled with a preservation solution, fully and uniformly mixing the menstrual blood and the preservation solution, preserving and standing for 5-12 hours at the temperature of 4 ℃, and fully sterilizing the menstrual blood;

5. The preparation method of the skin micro-ecological rejuvenation and restoration essence as claimed in claim 4, wherein: the stop solution comprises 92-94% (v/v) of alpha-MEM culture medium, 3.5-5% (v/v) of fetal bovine serum, 1.2-1.5% (w/v) of thiamine hydrochloride and 1.3-1.5% (w/v) of 4-hydroxyethyl piperazine ethanesulfonic acid.

6. The method for preparing the skin micro-ecological rejuvenation and restoration essence as claimed in claim 4 or 5, wherein: the preservation solution is a preservation solution containing antibiotics, sodium citrate and heparin sodium.