CN110731920A

CN110731920A - Application of preparation in improving structure and function of dermis and true-epidermis junction

Info

Publication number: CN110731920A
Application number: CN201810804109.9A
Authority: CN
Inventors: 姜山山; 摩根多斯桑托斯; 蒋丹丹; 李慧; 章漳
Original assignee: Galan (group) Co Ltd
Current assignee: Galan (group) Co Ltd
Priority date: 2018-07-20
Filing date: 2018-07-20
Publication date: 2020-01-31

Abstract

The invention discloses application of preparations containing glacier water, which is applied to improving the structure and function of human skin dermis and/or true-epidermis junction, wherein the final volume percentage of the glacier water is 2.0-5.0%, and/or the final concentration of the myrobalan extract is 5-10 mug/mL.

Description

Application of preparation in improving structure and function of dermis and true-epidermis junction

Technical Field

The invention relates to the field of skin biology, in particular to application of a preparation in improving the structure and the function of dermis and true-epidermis junction.

Background

The skin is the largest organ of the human body and mainly plays a role in protecting the body, removing sweat, and feeling cold, heat and pressure. The skin covers the whole body, prevents external invasion, also keeps moisture, and has the functions of keeping warm, blocking and feeling; it protects various tissues and organs in the body from physical, mechanical, chemical and pathogenic microbial attack. The skin of human and higher animals is composed of three layers, epidermis, dermis, and subcutaneous tissue. With the aging, the skin is loose, dry and rough, the elasticity is reduced, wrinkles are increased, and other aging phenomena occur, and the skin aging is mainly divided into two forms of natural aging and photoaging.

The most prominent change in histology is the reduction in the structure and function of the dermal junction (the junction between the two layers), leading to a reduction in the transport of nutrients between the two layers, the thickness of the dermal layer is primarily affected by the reduction in collagen, elastin and hyaluronic acid content during life, the reduction in proliferation and the appearance of aging keratinocytes in the epidermal layer results in a gradual reduction in the rate of epidermal cell renewal, which takes 28 days for young people and 40-60 days for older people, affecting the barrier function, moisturization and repair of the skin.

Mineral spring water has been used for several decades in products for external use and simultaneously used as an adjuvant in dermatology in the treatment of skin moisturization, skin aging and skin regeneration after aggressive cosmetic procedures, in the past decades, numerous studies have demonstrated the biological efficacy of mineral spring water and have suggested that their chemical and thermal properties have a direct effect on skin cell physiology and homeostasis.national "natural national standards for drinking" define mineral water that mineral water naturally gushes out from deep underground or artificially uncovered, uncontaminated underground mineral water, contains amounts of mineral salts, trace elements or carbon dioxide gas.

Myrobalan is originally produced in India, Dian and the like, is distributed in Yunnan, east, West, Tibet and the like in China, is a common Tibetan medicine, is a very used medicine in traditional Chinese medicine, is named as Bijueya, arlara, Laoxing cloth and the like in the classic Tibetan medicine works of Jingzhuibena, is called frontier wood sweat, namely the king of the Tibetan medicine, is a dried mature fruit of Terminalia chebula (Terminalia chebula Retz.) or Terminalia tomentosa Retz (Terminalia chebula Retz. var. tomentocellula Kurt), contains tannins, mainly contains myrobalaminic acid (bullellac acid), Terminalia chebula acid (bullagic acid), 1,3, 6-triacylglycerol (1,3, 6-triocosyl-36), is not clinically used for treating chronic chebular dysentery, chronic chebular diarrhea, chronic chebulagic acid (bulleyin the skin, chronic chebulagic acid, chronic.

Disclosure of Invention

The application of the preparation is natural and safe, and has the effects of improving the health state of skin, enhancing moisture retention and elasticity, and resisting aging, moisture retention, whitening, elasticity and firmness.

In order to solve the technical problems, applications of preparations containing glacier water are provided, the applications are in the aspect of improving the structure and the function of human skin dermis and/or true-epidermis junction, the final volume percentage of the glacier water is 2.0-5.0%, and/or the final concentration of the myrobalan extract is 5-10 mug/mL.

Preferably, the final volume percentage of glacier water is 2.0%. More preferably, the medicine also comprises a myrobalan extract, and the final concentration of the myrobalan extract is 10 mug/mL.

Preferably, the myrobalan extract is prepared by a process comprising the steps of:

(1) crushing myrobalan to 20-60 meshes;

(2) ultrasonic extracting with 10 times volume of 70% ethanol at room temperature for more than 2 times, each time for more than 30min to obtain crude extractive solution;

(3) filtering the extracting solution obtained in the step (2) to obtain filtrate;

(4) mixing the above filtrates, and rotary evaporating to dryness to obtain fructus Chebulae extract.

More preferably, the frequency of the ultrasound is 400-550W, and the time of the ultrasound is 25-40 min; most preferably, the frequency of the ultrasound is 500W, and the time of the ultrasound is 30 min.

Preferably, in the step (3), the filtration is performed by performing preliminary filtration with gauze to obtain a primary filtrate, and then performing centrifugal filtration with 8000rpm/min for 10 min.

More preferably, the glacier water-containing preparation further comprises or more of a moisturizing active ingredient, an antioxidant active ingredient, an oil-controlling active ingredient, a whitening active ingredient and an anti-aging active ingredient.

Preferably, the anti-aging active ingredient is an anti-aging active ingredient having or more effects in increasing the thickness of dermis and increasing the secretion of dermal elastin of skin.

More preferably, the improvement in the structure and function of the dermis of human skin is an increase in the expression of type I collagen and/or hyaluronic acid or, more preferably, the improvement in the structure and function of the true-epidermal junction of human skin is an increase in the secretion of laminin 332, type VII collagen, and/or the expression of integrin β 1.

In the present invention, the term "formulation" refers to a liquid formulation, i.e., a liquid formulation composed of a drug dispersed in a liquid dispersion medium, particularly a solution type/suspension type/spray type liquid formulation as understood by those skilled in the art.

In the invention, pure glacier water with the concentration of 100% of glacier water mother liquor is prepared into the final volume percentages of 1%, 2% and 5% according to the experimental design. In the present invention, the final volume percentage refers to the actual volume percentage in the system at the time of application. Similarly, myrobalan extract was also prepared in the mother liquor at an initial concentration of 500. mu.g/mL, diluted to final concentrations of 5. mu.g/mL and 10. mu.g/mL according to the experimental design. The final concentration is the actual concentration in the system at the time of application, as understood by those skilled in the art.

In the present invention, the glacier water is taken from the multi-gitriptan-nima spring (elevation 5128 m) of himalayas, and each liter of the glacier water contains the following components: sr⁺0.2-0.5mg、K⁺0.5-10.0mg、Na⁺0.99-5.0mg、Ca²⁺14.0-30.0Mg and Mg² ⁺2.0-10.0 mg; the total soluble solid content is 60-150 mg/L; the pH of the glacier water was 7.4, and the deuterium content of the glacier water was 132 ppm. Himalayan glacier water was pretreated by filtration through a 0.22 μm filter and stored at 4 ℃ until use.

In the present invention, the anti-aging active ingredient may be an anti-aging active ingredient that conventionally acts in the art, and preferably, the anti-aging active ingredient is an anti-aging active ingredient having or more effects of increasing the thickness of the dermis and increasing the secretion of dermal elastin in the skin dermis.

As is known to those skilled in the art, the type I collagen forms the basic scaffold of the skin in the dermis, giving the skin its elasticity.

As is known to those skilled in the art, the hyaluronic acid has the ability to retain water in amounts up to 100 times its weight. Hyaluronic acid in the skin has physiological functions of retaining water, maintaining extracellular space, participating in the change process of keratin cells, receptor binding action, scavenging free radicals, and the like.

In the present invention, the repair active ingredient may be a repair active ingredient that has a conventional effect in the art, and is preferably a repair active ingredient having an effect of promoting the expression of laminin 332, collagen VII, and integrin β 1 at the true-epidermal junction.

As known to those skilled in the art, laminin 332 is , which is the major constituent protein of basement membrane of epithelial tissue, and has the functions of promoting adhesion, migration and cell differentiation of epithelial cells.

It is well known to those skilled in the art that the type VII collagen serves primarily as an anchor at the interface between the dermis and epidermis, which allows for a strong connection between the dermal and epidermal cells.

As is known to those skilled in the art, the integrin β 1 is type glycoprotein receptor family molecules located on the surface of cell membranes, which mainly mediate the adhesion of cells and extracellular matrix, so that the intracellular skeleton and the extracellular matrix are integrated into a whole, and the integrin is also involved in the adhesion of cells and cells to maintain the stable skin structure.

The 3D full-layer skin model is a skin tissue model which is constructed by adopting a matrix material with good biocompatibility and inoculating human dermal fibroblasts, epidermal keratinocytes and the like through an in-vitro culture technology and has an integral structure containing the epidermis, the dermis and the true epidermis at the joint, and highly reducing the three-dimensional structure and the function of human skin.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The invention has the positive effects that 1) the preparation containing glacier water is added to obviously promote the secretion of dermal layer type I collagen and hyaluronic acid, 2) the secretion of structural function related proteins of the dermis and the junction of the dermis and the true epidermis, such as laminin 332, integrin β 1 and VII collagen, is increased, and 3) the myrobalan extract is added to the preparation, so that the effects of resisting skin aging, moisturizing, locking water, repairing aged skin and achieving elasticity and firmness can be achieved.

Drawings

Fig. 1 is a graph of hyaluronic acid staining of a 3D full-thickness skin model in effect example 1, fig. 1A is a blank control group, fig. 1B is a 2% glacier water group, ocular lens x objective lens: 10X 10, and the arrows in FIG. 1A and FIG. 1B indicate areas stained positively with hyaluronic acid.

Fig. 2 is a bar graph of quantitative data analysis after normalization of the length of the junction of the true epidermis in the hyaluronic acid positive staining region in the blank control group and the 2% glacier water group after staining with hyaluronic acid in effect example 1, where P is less than 0.001 compared with the blank control group.

Fig. 3 is a staining pattern of laminin 332 of the 3D full-thickness skin model in effect example 1, fig. 3A is a blank control group, fig. 3B is a 2% glacier water group, ocular lens × objective lens: 10X 10, arrows in FIGS. 3A and 3B indicate areas stained positive for laminin 332.

Figure 4 is a bar graph of quantitative data analysis of laminin 332 positive stained areas normalized by the length of the true epidermal junction after staining laminin 332 in effect example 1, P <0.001 compared to the blank control.

Fig. 5 is a graph showing collagen VII staining of the 3D full-thickness skin model of effect example 1, fig. 5A is a blank control group, fig. 5B is a 2% glacier water group, ocular lens × objective lens: 10X 10, arrows in FIGS. 5A and 5B indicate areas positive for collagen VII staining.

Fig. 6 is a bar graph of quantitative data analysis of collagen VII positive stained areas normalized by the length of the true epidermal junction in the blank control group and 2% glacier water group after staining for collagen VII in effect example 1, where P is <0.001 compared to the blank control group.

Fig. 7 is a staining pattern of integrin β 1 in the 3D full-thickness skin model of effect example 1, fig. 7A is a blank control group, fig. 7B is a 2% glacier water group, eyepiece × objective lens: 10 × 10, and arrows in fig. 7A and 7B indicate positive regions for integrin β 1 staining.

Fig. 8 is a bar graph of quantitative data analysis after normalization of lengths of the junctions of the epidermis in the white space control group and the positive stained region of integrin β 1 in the 2% glacier water group after staining of integrin β 1 in effect example 1, where P is <0.001 compared to the white space control group.

Fig. 9 is a graph showing staining of type I collagen in the 3D full-thickness skin model of example 1, fig. 9A is a blank control group, fig. 9B is a 2% glacier water group, ocular lens × objective lens: 10X 10, and arrows in FIGS. 9A and 9B indicate the areas positive for collagen type I staining.

Fig. 10 is a bar graph of quantitative data analysis of collagen I positive stained areas normalized by the length of the true epidermal junction in the placebo group and the 2% glacier water group after staining for collagen I in effect example 1, where P is <0.01 compared to the placebo group.

FIG. 11 is a bar graph showing the effect of different concentrations of glacial water on the secretion of type I collagen in fibroblast cells cultured according to the DNA content of in example 4, comparing the quantitative data of type I collagen content in 1% glacial water, 2% glacial water and 5% glacial water with the blank control.

FIG. 12 is a bar graph showing the effect of 2% glacial water, 5. mu.g/mL myrobalan extract, 10. mu.g/mL myrobalan extract and 2% glacial water + 10. mu.g/mL myrobalan extract on the secretion of type I collagen in the course of fibroblast culture in Effect example 4, after classifying the DNA content into , comparing the quantitative data of type I collagen with those of blank control.

Detailed Description

The invention is further illustrated by the following examples , but is not intended to be limited thereby within the scope of the examples.

EXAMPLE 13D construction of full-thickness skin model

The three-dimensional recombinant human full-thickness skin model (3D full-thickness skin model) is also called tissue engineering skin, human skin substitute, etc. The three-dimensional recombinant human skin model is a skin tissue model which is constructed by adopting a matrix material with good biocompatibility and inoculating human dermal fibroblasts, epidermal keratinocytes and the like through an in-vitro culture technology and has an integral structure containing an epidermis, a dermis and a true epidermis junction, and highly reducing the three-dimensional structure and functions of human skin. The safety and efficacy of the method for evaluating glacier-containing water by using the 3D full-thickness skin model are improved.

1.1 skin cell culture

Original cultures of keratinocytes (NHEK) and fibroblasts (NHDF) were obtained from skin discarded by routine surgery from two healthy persons, following the declaration of helsinki (shanghai cell and tissue bank-SOBC, shanghai.) NHDF was isolated from the face of female donors aged 42 years, and NHEK was obtained from the foreskin of male donors aged 16 years.

1.2 in vitro human skin substitute culture

Preparation of dermal substitute (DE) NHDF at 2.5X 10 from adult donor (42 years old)⁵cells/cm²Was seeded on a chitosan-collagen-glycosaminoglycan matrix material (from BASF). The dermal substitute (DE) was grown at 37 deg.C in an environment of 5% CO2 for 21 days. Preparation of skin Substitute (SE) NHEK from donor (16 years old) at 2.5X 10⁵cells/cm²Was seeded on a 21 day DE. After 7 days of submerged culture, SE was lifted to the gas-liquid surface and samples were harvested at day 35 and used for histological and immunohistological studies.

In a 3D skin model, epidermal keratinocytes and dermal fibroblasts are co-cultured in a microenvironment simulating natural conditions of a human body to form a tissue engineering skin with components, structures and functions similar to those of a real human skin, a Masson (Masson) trichrome staining method is adopted to observe the microstructure of the in-vitro reconstructed skin model and the normal human skin, and the staining result shows that the in-vitro reconstructed skin model has dermis and epidermis with the same good tissue forms, an ultrasmall structure of a basal membrane zone of the reconstructed skin model is observed under a transmission electron microscope, and typical hemidesmosomes, tunica, a transparent layer, a compact layer and a reticular layer structure similar to the normal human skin are seen.

EXAMPLE 2 preparation of glacier Water formulation

Preparing glacier water: glacier water is obtained from the Dogqutnenyma spring (elevation 5128 m) of Himalayas mountain, and contains the following components per liter: sr⁺0.2-0.5mg、K⁺0.5-10.0mg、Na⁺0.99-5.0mg、Ca²⁺14.0-30.0Mg and Mg²⁺2.0-10.0 mg; the total soluble solid content is 60-150 mg/L; the pH of the glacier water was 7.4, and the deuterium content of the glacier water was 132 ppm. Himalayan glacier water was pretreated by filtration through a 0.22 μm filter and stored at 4 ℃ until use.

Preparing a myrobalan extract: taking 30g of myrobalan, crushing to 20-60 meshes, carrying out ultrasonic extraction for 2 times at room temperature by using 70% ethanol 500W with the volume being 10 times of that of the myrobalan, each time for 30min, combining filtrates, and carrying out rotary evaporation on the filtrates (rotary evaporator EYELA SB-1100, Shanghai Ailang apparatus Co., Ltd.) to dryness to obtain about 16.9g of myrobalan extract, and drying at 4 ℃ in a dark place.

Preparing glacier water preparation mother liquor: mixing glacial water and fructus Chebulae extract to obtain mother liquor containing fructus Chebulae extract and glacial water, and making into preparation method shown in Table 1.

TABLE 1 preparation of glacier-containing Water formulations

Example 3 Effect of glacial Water-containing formulations on 3D full-thickness skin model

Glacial water was added daily to the 3D full-thickness skin model medium at the final concentrations and final volume ratios of table 1, starting from media changes after NHDF inoculation (day ) until the end of the study (day 35). 35 days skin substitute cultures were incubated with no glacial water added as a blank control.

Example 4 histology, immunohistology and image acquisition and data analysis

The skin model was harvested on day 35 of all cell cultures, quickly transferred to neutral buffer 4% formalin for 24h and either embedded in paraffin or frozen in a freezing embedding medium at-20 ℃. The paraffin-embedded formalin-fixed samples were then cut into 5 μm sections.

The Masson trichrome staining method-after dewaxing and rehydrating the tissue slices, the tissue slices are stained with Wengelt's ferrohematoxylin staining solution, after rinsing with pure water, the solution of British scarlet-acid fuchsin is added dropwise, after rinsing with pure water, the solution of phosphotungstic acid is added dropwise, then the solution of brilliant green is added dropwise, and after rinsing in pure water, absolute ethyl alcohol and methylcyclohexane in sequence, the slices are sealed and subjected to conventional histological analysis.

Tissue sections at 5% H after immunohistochemical-thermoconductive antigen retrieval treatment₂O₂Non-specific binding was blocked in phosphate buffered PBS containing 5% BSA after which the sections were incubated with anti (see table 2) overnight at room temperature in PBS/BSA 5%. after incubation with peroxidase-bound secondary antibody (EnVision, Dakocytomation) hours, the antigen was detected by diaminobenzidine hydrochloride as substrate.

For immunofluorescence, labeling was performed on air-dried 5 μm frozen sections, incubated with anti-Alexa-546-conjugated secondary murine anti- (molecular Probe, Invitrogen) antibody (concentration: antibody/5% BSA-1/1000) for 1 hour at room temperature, nuclei were counterstained routinely with Dapi (Roche) (concentration: Dapi/5% BSA-1/500) as a negative control, anti was replaced by the corresponding IgG.

Hyaluronic Acid (HA) assay: tissue sections were incubated overnight with 3mg/mL of bio-acylated HA-binding protein (HABP) (Merck). After PBS washing, the combined HABP was visualized by applying the avidin-biotin complex technique according to the manufacturer's protocol.

TABLE 2 antibody used in immunohistochemical experiments

Immunofluorescent samples were visualized and obtained by TCS SP8 laser scanning confocal system (Leica). The eight-bit picture is saved in a Leica picture file format. 6 representative z-stack pictures were obtained under the same process but different conditions. Image processing and analysis was performed by MBF _ ImageJ software available for microscopy. The parameters collected were the total surface area of positive areas immunostained for hyaluronic acid, laminin 332, type VII collagen, integrin, and type I collagen.

All data are presented as mean ± standard deviation. All statistical data significance was assessed using a T-test run. Each set of data relating to 35 days himalaya glacier water treatment was compared to 35 sky white. Statistically significant differences are marked by the number:^nsP>0.05,*P<0.05,**P<0.01 and P<0.001。

Effect example 1 Effect on hyaluronic acid

In order to analyze the morphological and functional effects of the glacial water-containing formulation on the genuine-epidermal junction and dermal layers of the 3D skin model under aging conditions, immunohistological and image analyses were performed to investigate the expression of markers such as type VII collagen, laminin 332 and integrin β 1 at the genuine-epidermal junction and to explore the secretion of hyaluronic acid and the effects of glacial water on the dermal layers, the expression of markers such as type I collagen, etc.

The results are shown in fig. 1, which shows that the expression of hyaluronic acid in the 2% himalaya glacier water-treated group is significantly enhanced compared to the untreated control group, indicating that glacier water significantly increases the expression of markers related to anti-aging, moisturizing and elasticity of skin cells. Image processing and analysis (fig. 2) showed a significant increase in hyaluronic acid secretion of 144.7% in the 2% himalayan glacier water-treated group compared to the untreated group.

Effect example 2 true-epidermal morphology and adhesion

Basement membrane proteins closely related to skin layer adhesion, skin aging, such as laminin 332 and type VII collagen, were analyzed by immunostaining. As shown in fig. 3 and 5, the stained section images show that, 15 days after the epidermization (35 days in culture), the expression of laminin 332 and collagen VII in the untreated group is weak, and the staining observed is dotted, indicating the onset of morphogenesis of the epidermal basement membrane at the early stage of cell culture. In sharp contrast to the 2% himalayaglacier water-treated skin model, the expression of these proteins was greatly enhanced, allowing linear expression at the true-epidermal junction level and promoting maturation of the true-epidermal junction.

Laminin 332 is which is a major constituent protein of epithelial tissue basement membrane and has functions of promoting epithelial cell adhesion, migration, cell differentiation and the like.2% concentration glacial water can stimulate the expression of laminin 332 in a 3D reconstructed skin model (figure 3), and image processing and analysis (figure 4) show that the 2% himalayan glacial water-treated group has a 454.9% obviously increased expression of laminin 332 compared with the untreated group.

The VII type collagen is mainly used for anchoring at the junction of dermis and epidermis, so that firm connection is formed between the true epidermal cells. Glacier water at 2% concentration stimulated collagen VII expression in the 3D skin model (fig. 5), and image processing and analysis (fig. 6) showed a significant increase in collagen VII expression of 318.8% in the 2% himalayas glacier water-treated group compared to the untreated group.

The integrin is glycoprotein receptor family molecules positioned on the surface of a cell membrane, the molecules mainly mediate the adhesion of cells and an extracellular matrix, an intracellular framework and the extracellular matrix are integrated to form a whole, the integrin is simultaneously involved in the adhesion of the cells and the cells, and the skin structure is maintained to be stable, during the proliferation and differentiation processes, the expression of the integrin on the surface of basal layer cells is gradually reduced till the cell disappears, the cells are gradually migrated to the surface of the skin, finally keratinization and desquamation are realized, the expression of the integrin β 1 in a 3D skin model can be stimulated by glacial water with the concentration of 2% (figure 7), and image processing and analysis (figure 8) show that the expression of the integrin β 1 is obviously increased by 90.2% compared with that in a 2% himalaya glacier water-treated group.

The immunohistochemistry of these proteins was analyzed by step , which showed that the basement membrane protein expression and the morphogenesis of the genuine epidermis junction were statistically significantly increased.

Effect example 3 Effect on dermis layer

In the distribution of collagen in the whole body, more than 50% is distributed in the skin, wherein the collagen is mainly type I collagen, and the collagen mainly forms a basic bracket in the dermis layer, so that the skin has elasticity. Glacier water at 2% concentration can stimulate type I collagen expression in 3D skin models (figure 9). Image processing and analysis (figure 10) showed a significant increase in type I collagen expression of 53.3% in the 2% himalayan glacier water-treated group compared to the untreated group,

the above effects examples 1 to 3 show specific effect data in the following table 3.

Table 3 efficacy data

Item	Blank control group	Experimental group
			Concentration of	-	2% glacier water
Hyaluronic acid secretion amount (pixel)	102140.8	249977.6
			Laminin 332 expression (pixel)	1.42	7.88
Type VII collagen expression (pixel)	57.85	88.66
			Integrin β 1 expression (pixel)	27.88	34.01
Type I collagen expression (pixel)	2.08	8.71

Effect example 4 Effect on type I collagen expression during fibroblast culture

The influence of different concentrations of glacier water and myrobalan extract water on the secretion content of the type I collagen is evaluated after the DNA content is classified into (figures 11 and 12), no obvious difference is shown in 1% of himalaya glacier water on the secretion of the type I collagen, 2% of glacier water on the secretion of the type I collagen (14% is enhanced compared with a blank control), 5% of glacier water on the secretion of the type I collagen (31% is enhanced compared with the blank control), and 2% of himalaya glacier water and 10 mu g/mL of myrobalan extract compound on the secretion of the type I collagen (199% is significantly different compared with the blank control, 185% is significantly different compared with the 2% of glacier water, and 107% is significantly different compared with 10 mu g/mL of myrobalan extract).

The specific effect data described above can be seen in table 4 below.

Table 4 efficacy data of type I collagen expression at 2D cell level

In summary, immunohistological results show that 2% concentration of himalayan glacier water significantly enhances the expression of hyaluronic acid, which is deeply involved in skin moisturization and drastically decreases with aging of the skin, data show that 2% himalayan glacier water significantly enhances the expression of three major components, laminin 332, integrin β 1, type VII collagen, and morphogenesis of epidermal basement membrane, which are generally drastically reduced with aging, for the dermis layer, 2% concentration of glacier water significantly increases the thickness of the dermal papilla, secretion of type I collagen, promotes dermal maturation for the dermal basement membrane, and for cell level fibroblasts, the application of type I collagen secretion by 2% concentration of himalayan glacier water or 10 μ g/mL myrobalan extract has a significant promoting effect, but 2% concentration of himalayan glacier water +10 μ g/mL myrobalan extract exhibits a synergistic effect, shows a more excellent combined efficacy in promoting secretion of type I water and aging-inducing the primary moisturizing and anti-aging characteristics of the corium.

Claims

The application of preparations containing glacier water for improving the structure and function of human skin dermis and/or true-epidermis junction is characterized in that the final volume percentage of the glacier water is 2.0-5.0%, and/or the final concentration of the myrobalan extract is 5-10 mug/mL.
2. The use of claim 1, wherein the extract of myrobalan is prepared by a process comprising the steps of:

(1) crushing myrobalan to 20-60 meshes;

(2) ultrasonic extracting with 10 times volume of 70% ethanol at room temperature for more than 2 times, each time for more than 30min to obtain crude extractive solution;

(3) filtering the extracting solution obtained in the step (2) to obtain filtrate;

(4) mixing the above filtrates, and rotary evaporating to dryness to obtain fructus Chebulae extract.
3. The use of claim 2, wherein in the step (2), the frequency of the ultrasound is 400-550W, and the time of the ultrasound is 25-40 min; preferably, the frequency of the ultrasound is 500W, and the time of the ultrasound is 30 min.
4. The use of claim 2, wherein in step (3), the filtration is performed by performing a preliminary filtration with gauze to obtain a primary filtrate, and then performing a centrifugal filtration at 8000rpm/min for 10 min.
5. The use of , wherein the glacier water-containing formulation further comprises or more of a moisturizing active ingredient, an antioxidant active ingredient, an oil control active ingredient, a whitening active ingredient and an anti-aging active ingredient.
6. The use according to claim 5, wherein the anti-ageing active ingredient is an anti-ageing active ingredient having or more effects on increasing dermal thickness and increasing dermal elastin secretion in the skin.
7. The use according to any one of claims 1 to 4 at , wherein the improvement in the structure and function of the human skin dermis is a function of increasing type I collagen and/or hyaluronic acid expression.
8. The use according to claim 5, wherein the improvement in the structure and function of the dermis of human skin is a function of increasing the expression of type I collagen and/or hyaluronic acid.
9. The use of any one of of claims 1 to 4, wherein the improvement in the structure and function of the true-epidermal junction of human skin is an increase in the secretion of laminin 332, collagen VII, and/or integrin β 1 expression.
10. The use according to claim 8, wherein the improvement in the structure and function of the true-epidermal junction of human skin is a function of increasing laminin 332, collagen VII secretion, and/or integrin β 1 expression.