阅读说明：本技术 一种含冰川水的制剂、制备方法及其应用 (preparation containing glacier water, and its preparation method and application ) 是由 姜山山 摩根多斯桑托斯 蒋丹丹 李慧 章漳 于 2018-07-20 设计创作，主要内容包括：本发明公开了一种含冰川水的制剂,其中所述制剂含有冰川水和诃子提取物,所述冰川水最终体积百分比2.0-5.0％,所述诃子提取物终浓度为5-10μg/mL。本发明还公开了所述的含冰川水的制剂的制备方法；以及所述的含冰川水的制剂的应用,所述应用为在改善人体皮肤表皮内稳态及屏障功能上的应用。本发明的含冰川水的制剂天然、安全,具有激发表皮细胞的增殖与分化、表皮更快的再生与修复能力,以及促进屏障功能修复的效果,在改善人体皮肤表皮内稳态及皮肤屏障功能上有很高的应用价值。(The invention discloses preparations containing glacier water, wherein the preparations contain glacier water and myrobalan extract, the final volume percentage of the glacier water is 2.0-5.0%, and the final concentration of the myrobalan extract is 5-10 mug/mL.)

1, preparations containing glacier water, which is characterized by comprising glacier water and a myrobalan extract, wherein the final volume percentage of the glacier water is 2.0-5.0%, and the final concentration of the myrobalan extract is 5-10 mu g/mL.

2. A glacial water-containing formulation according to claim 1, wherein the final volume percentage of glacial water is 2.0% and the final concentration of myrobalan extract is 10 μ g/mL.

3. A glacial water-containing formulation according to claim 1, wherein said 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.

4. The glacier water-containing preparation according to claim 3, wherein the frequency of the ultrasound in the step (2) is 400-550W, and the time of the ultrasound is 25-40 min.

5. A glacial water-containing formulation according to claim 4, wherein in step (2), the frequency of said ultrasound is 500W and the duration of said ultrasound is 30 min.

6. The glacial water-containing preparation according to claim 3, wherein in the step (3), the filtration is performed by performing a preliminary filtration with gauze to obtain an initial filtrate, and then performing a centrifugal filtration at 8000rpm/min for 10 min.

7. A method of preparing a glacial water-containing preparation according to any of claims 1-6, wherein the preparation is prepared by mixing fructus Chebulae extract with glacial water.

8, use of a preparation containing glacier water for improving homeostasis and barrier function of human skin epidermis, wherein the preparation contains glacier water, and the final volume percentage of the glacier water is 1.0-5.0%.

9. Use of a glacier water containing formulation according to claim 8 wherein the final volume percentage of glacier water is 2.0%.

10. Use of a glacier water-containing preparation according to claim 9, characterized in that the preparation further contains a myrobalan extract with a final concentration of 5-10 μ g/mL.

Technical Field

The invention relates to the field of skin biology, in particular to preparations containing glacier water, a preparation method and application thereof, and especially application thereof in improving homeostasis and barrier function of human skin epidermis.

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.

Histologically, , the most prominent change, is the structural and functional challenge of the dermoepidermal junction (DEJ), leading to reduced transport of nutrients between the two layers, the thickness of the dermis layer is mainly affected by the reduced levels of collagen, elastin, and hyaluronic acid during life, the reduced proliferation and appearance of aging keratinocytes in the epidermis results in a gradual reduction in the rate of epidermal cell renewal, epidermal 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.

Myrobalan is originally produced in India, Burma and the like, is distributed in Yunnan, east, west, Tibet and the like in China, is a common Tibetan medicine, is a very used traditional Chinese medicine, is named as Bijueya, arlara, Laoxing cloth and the like in the Mongolian name of the Tibetan medicine classic works, namely Jingzhuibena, namely the King of the Tibetan medicine, is a dry mature fruit of Terminalia chebula (Terminalia chebula Retz.) or Terminalia tomentosa Retz (Terminalia chebula Retz. var. tomentosa Kurt) in the family of Combretaceae, contains tannins, mainly contains myrobalac acid (butinic acid), Terminalia chebulagic acid (balagic acid), 1,3, 6-trigalloylglucose (1,3, 6-triogastic-83), is mainly used for treating chronic chebulagic diarrhea, chronic chebulagic disease, chronic chebulagic diarrhea, chronic chebulagic acid (Chebulagic acid), chronic chebulagic acid, chronic che.

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 over the years, 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 drinking natural national standards define mineral water as mineral water that naturally gushes out from deep underground or artificially uncovered uncontaminated, containing amounts of mineral salts, trace elements or carbon dioxide gas.

Disclosure of Invention

The invention aims to provide glacier water-containing preparations, a preparation method and application thereof, in particular to application thereof in improving homeostasis and barrier function of human skin epidermis.

In order to solve the technical problems, the invention provides preparations containing glacier water, which contain glacier water and a myrobalan extract, wherein the final volume percentage of the glacier water is 1.0-5.0%, and the final concentration of the myrobalan extract is 5-10 mug/mL.

Preferably, the final volume percentage of the glacier water is 2.0%, and the final concentration of the myrobalan extract is 10 mug/mL.

More preferably, the myrobalan extract is prepared by the following method, and the method comprises the following steps:

(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.

Preferably, in the step (2), the frequency of the ultrasonic treatment is 400-550W, and the time of the ultrasonic treatment is 25-40 min. More preferably, in the step (2), the frequency of the ultrasound is 500W, and the time of the ultrasound is 30 min.

More preferably, in the step (3), the filtration is performed by performing a preliminary filtration with gauze to obtain a primary filtrate, and then performing a centrifugal filtration with 8000rpm/min for 10 min. The scrim used in embodiments of the present invention is preferably a double layer scrim.

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 as applied, 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 order to solve the technical problems, the invention provides a preparation method of preparations containing glacier water, which is prepared by uniformly mixing the myrobalan extract with the concentration and the glacier water according to the volume percentage.

In order to solve the technical problems, the invention also provides application of the preparation containing glacier water, wherein the application is the application on improving the homeostasis and the barrier function of the epidermis of the human body skin, the preparation contains the glacier water, and the final volume percentage of the glacier water is 1.0-5.0%.

Preferably, the final volume percentage of the glacier water is 2.0%.

More preferably, the preparation also contains myrobalan extract, and the final concentration of the myrobalan extract is 5 mug/mL or 10 mug/mL.

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 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 glacier water-containing preparation are evaluated by using a 3D full-thickness skin model.

The positive progress effects of the invention are as follows: 1) the preparation containing 2.0% glacier water can obviously promote the proliferation of epidermal cells and the increase of epidermal thickness; 2) the preparation containing 2.0% glacier water has obvious promotion effect on the expression level of silk polymerized protein of important factors (natural moisturizing factors) of epidermal barrier function; 3) the glacier water and the myrobalan extract have the effects of resisting skin aging, preserving moisture, locking water and tightening.

Drawings

FIG. 1 is a comparison of the morphology of normal human skin and a 3D skin model after glacier water treatment in example 1; FIG. 1A is a 3D skin model of a 2% glacier water treatment group, and FIG. 1B is a normal human skin tissue structure; hematoxylin and eosin staining was performed using paraffin sections, fig. 1A is ocular x objective: 10 × 10, fig. 1B eyepiece × objective: 10X 25.

Fig. 2 shows the epidermis and dermis morphology of the 3D full-thickness skin model stained by masson trichrome staining in example 1, fig. 2A is a blank control, fig. 2B is a 2% glacial water group, fig. 2C is a 5% glacial water group, eyepiece × objective: 10 × 10.

Fig. 3 is a bar graph of quantitative data analysis of epidermal thickness images of the blank control group, the 2% glacial water group, and the 5% glacial water group after mason trichrome staining in effect example 1, where the epidermal thickness of the 2% glacial water group was changed (μm) compared to the blank control group and P was < 0.001.

Fig. 4 is a 3D full-thickness skin model Ki-67 immunostaining plot of effect example 1, fig. 4A is a blank control, fig. 4B is a 2% glacier water plot, fig. 4C is a 5% glacier water plot, eyepiece x objective: 10X 10, arrows in FIG. 4A, FIG. 4B and FIG. 4C indicate Ki-67 immunostaining-positive areas.

Fig. 5 is a bar graph of quantitative data analysis of proliferation index of epidermal cells characterized by Ki-67 staining in blank control, 2% glacier water, and 5% glacier water after Ki-67 immunostaining in effect example 1, where P is <0.01 compared to blank control.

Fig. 6 is a graph of 3D full-thickness skin model silk polyprotein immunostaining in effect example 1, fig. 6A is a blank control, fig. 6B is a 2% glacier water group, fig. 6C is a 5% glacier water group, eyepiece x objective: 10X 10, arrows in FIG. 6A, FIG. 6B and FIG. 6C indicate areas positive for silk fibroin immunostaining.

Fig. 7 is a histogram of quantitative data analysis of silk fibroin positive staining regions normalized by the length of the true epidermal junction in the blank control group, the 2% glacial water group, and the 5% glacial water group after immunostaining with silk fibroin in example 1, wherein P is less than 0.01 for the 2% glacial water group compared to the blank control group.

FIG. 8 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. 9 is a bar graph showing the quantitative data of type I collagen content in comparison with blank control, which is obtained by evaluating 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 type I collagen secretion content according to DNA content classified into in fibroblast culture of example 4.

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.