CN108703931B

CN108703931B - Composition with moisturizing effect and preparation method thereof

Info

Publication number: CN108703931B
Application number: CN201810988013.2A
Authority: CN
Inventors: 敢小双; 邓黎丽; 董银卯; 孟宏; 李丽
Original assignee: Infinitus China Co Ltd
Current assignee: Infinitus China Co Ltd
Priority date: 2018-08-28
Filing date: 2018-08-28
Publication date: 2021-03-30
Anticipated expiration: 2038-08-28
Also published as: CN108703931A; FR3085275A1; FR3085275B1

Abstract

The invention belongs to the field of daily chemical products, and discloses a composition with a moisturizing effect, a preparation method thereof and a skin care product. The composition with the moisturizing effect is prepared from dendrobium officinale, tremella, bamboo fungus and radix ophiopogonis, wherein the mass ratio of the dendrobium officinale to the tremella to the bamboo fungus to the radix ophiopogonis is (1-4) to (1-4). The dendrobium officinale, the tremella, the bamboo fungus and the radix ophiopogonis are matched and used together in proportion, and the moisturizing effect is superior to that of each single component. The composition can be applied to skin external skin care products in various dosage forms such as gel, facial cleanser, cream, emulsion and the like.

Description

Composition with moisturizing effect and preparation method thereof

Technical Field

The invention belongs to the field of daily chemical products, and particularly relates to a composition with a moisturizing effect, a preparation method thereof and a skin care product.

Background

The skin is divided into epidermis, dermis and subcutaneous fat. The outermost layer of the epidermis is the stratum corneum, which maintains a certain water content and moisture of the skin due to the water absorption and barrier functions of the stratum corneum itself and the action of natural moisturizing factors, i.e., amino acids, lactate, saccharides, etc., contained in the stratum corneum, and the normal stratum corneum generally contains 10% to 30% of water to maintain the softness and elasticity of the skin. However, with age, the moisture content of the stratum corneum gradually decreases, and when the moisture content of the stratum corneum is less than 10%, the skin becomes dry, tight, rough, desquamated, and the like. Generally, the moisture content of human skin varies with age, season, environment, health condition, etc., and when the moisture content of the skin is low, the skin is not only rough and dull to affect the beauty but also the skin is accelerated to age, and symptoms such as itching, redness, stabbing pain, sensitivity, etc. may appear. Therefore, maintaining moisture in the skin is not only for beauty but also for maintaining the health of the skin. The skin is in direct contact with the external environment at any time, and if the skin is not protected, the skin is more or less lack of water, so that the appearance of the skin is directly influenced. The primary conditions for a healthy, moist, elastic, glossy skin are sufficient moisture in the skin and good moisture retention of the skin.

There are many substances related to moisture retention in skin, wherein aquaporin AQP3 is a transport protein for transporting water, glycerol, urea and other small molecular substances across membranes on cell membranes, and participates in skin hydration, skin barrier and other functions; the zonulin ZO-1 has the function of closing the intercellular space, is a barrier for preventing the diffusion of substances, and separates the cells from external body fluid; the filaggrin FLG and several related proteins involved in the epidermal differentiation process have important barrier functions, can prevent water loss, and furthest reduce the invasion of allergens and microorganisms; loricrin is the main component of the horny cell seal, can strengthen the horny cell seal, enhance the permeability barrier, and interact with intermediate fiber filaments to increase the flexibility of the horny cell seal structure. However, some external factors cause the skin to produce an inflammatory response, and excessive inflammatory factors such as TNF-alpha are produced, which increases skin permeability, decreases integrity, and affects skin barrier function.

The existing moisturizer on the market mainly solves the problem of dry skin through a 'water locking and moisturizing' way. With the growing concern of consumers on their own health and safety in recent years, materials derived from natural sources, having excellent effects and being edible in daily life are preferred to many biochemically synthesized cosmetic efficacy materials. Therefore, it is necessary to develop a safer and more effective product with moisturizing effect.

Disclosure of Invention

In view of the above, the present invention provides a composition with a moisturizing effect, wherein the active ingredients are all natural active ingredients, and the combination of the active ingredients achieves the effects of locking water and replenishing water, so as to achieve a safe and effective moisturizing effect.

In order to realize the purpose of the invention, the invention adopts the following technical scheme:

a composition with moisture keeping effect is prepared from herba Dendrobii, Tremella, Dictyophora Indusiata (Vent. Ex pers) Fisch and radix Ophiopogonis.

Wherein, the Dendrobium officinale (Dendrobium officinale) of the Orchidaceae, the Dendrobium genus perennial epiphytic herbaceous plant is mainly distributed in southwest and south China. The "Shen nong Ben Cao Jing" as early as the Qin Han period is called dendrobium officinale "which is mainly used for treating diseases of middle-jiao, removing arthralgia, descending qi, tonifying internal organs, strengthening yin and making intestines thick and fat after long-term use"; in Bencao gang mu, Li Shizhen considers that Shi dendron is the top grade of the root channel, sweet, light and slightly developed, mainly treats injuries, removes arthralgia, lowers qi, tonifies the interior, calms the stomach, grows muscles, benefits intelligence, removes fright and prolongs life. Researches show that the dendrobium officinale is rich in polysaccharide, and the dendrobium officinale is superior to dendrobium nobile in the quantity and content of alkaloid components. The amino acids contained in the dendrobium officinale mainly comprise aspartic acid, glutamic acid, glycine, valine and leucine, and the latter two are 'essential amino acids' of a human body. Pharmacological studies show that the dendrobium officinale polysaccharide has the effects of resisting oxidation, reducing blood sugar, enhancing immunity, resisting tumors and the like.

Tremella fuciformis (Tremella fuciformis) is an edible fungus and a medicinal fungus which have extremely high economic value and are extremely precious in China, and is a traditional food and medicine dual-purpose product. It contains carbohydrate, protein, vitamins and amino acids, and is effective in tonifying body, strengthening body resistance, and consolidating constitution. The polysaccharide is the most important component of the tremella and accounts for 60-70% of the dry weight of the tremella.

Bamboo fungus (Dictyophora indusiata), also known as "Zhusheng", is a generic name for a large group of fungi belonging to the genus Dictyophora. It is recorded in dietetic Ben Cao, Yun, by the name of bamboo straw mat: ' Ci Zhu forest grows straw mat in summer and rains, looks like a deer horn, is white and is edible. Dictyophora Indusiata has effects of nourishing, strengthening body, invigorating qi, nourishing brain, tranquilizing, invigorating qi, nourishing yin, moistening lung, relieving cough, clearing heat, and promoting diuresis. Dictyophora Indusiata (Vent. Ex pers) Fisch has effects of protecting liver and reducing fat accumulation in abdominal wall, so it is commonly called as "oil scraping". Modern researches also prove that the dictyophora indusiata has the effects of inhibiting tumors, resisting oxidation and inhibiting bacteria.

Radix Ophiopogonis (Ophiogon japonicus (Linn.f.) Ker-Gawl) is sweet, slightly bitter, and slightly cold in nature. Has the effects of nourishing yin, moistening lung, benefiting stomach, promoting fluid production, clearing away heart-fire, relieving restlessness and the like. Modern pharmacological studies show that radix Ophiopogonis also has effects of resisting oxidation, reducing blood sugar, relieving fatigue, resisting arrhythmia, and resisting bacteria. The ophiopogonpolysaccharide is one of the commonly used cosmetic raw materials, and has the main functions of moisturizing and delaying aging.

Preferably, the mass ratio of the dendrobium officinale, the tremella, the bamboo fungus and the radix ophiopogonis is (1-4) to (1-4).

In some embodiments, the mass ratio of the dendrobium officinale, the tremella fuciformis, the bamboo fungus and the radix ophiopogonis is 4:3:4: 3.

In some embodiments, the mass ratio of the dendrobium officinale, the tremella fuciformis, the bamboo fungus and the radix ophiopogonis is 3:4:3: 4.

In some embodiments, the mass ratio of the dendrobium officinale, the tremella fuciformis, the bamboo fungus and the radix ophiopogonis is 1:1:1: 1.

The invention also provides a preparation method of the composition, which comprises the steps of crushing and sieving the raw materials, adding water for extraction, and then sequentially carrying out hot fine filtration and cold fine filtration to collect the extract.

Preferably, the sieving is 100 mesh sieving.

Preferably, the water is added for extraction according to the feed-liquid ratio of 1 (75-85), and the extraction is carried out at the temperature of 55-65 ℃ for 110-130 min. In some embodiments, the water extraction is performed by adding water according to a feed-to-liquid ratio of 1:80, and extracting at 60 ℃ for 120 min.

The invention also comprises a soaking process before extraction. The soaking is beneficial to fully dissolving out the active ingredients, and can also shorten the decoction time of the traditional Chinese medicine, and avoid the loss and the excessive damage of the active ingredients of the medicine caused by overlong decoction time.

Preferably, the soaking time is 0.5 h.

Preferably, the hot fine filtration is to put a paper board H60 in a Buchner funnel, inject warm extracting solution for soaking, open the suction filtration and negative pressure suction to ensure that the paper board is tightly attached to the funnel but the whole paper board is wet, and obtain clear and transparent extracting solution. Wherein the pore size of the filter plate for heat fine filtration is preferably 1 μm.

The preparation method of the composition further comprises a step of cold fine filtration after the hot fine filtration so as to remove part of precipitate possibly caused by temperature reduction.

Preferably, the cold fine filtration is to place a paper board H70 in a Buchner funnel, inject warm extracting solution, open the suction filtration and negative pressure suction to ensure that the paper board is tightly attached to the funnel but the whole paper board is wet, so as to obtain clear and transparent extracting solution. The pore size of the filter plate for cold fine filtration is preferably 0.45 μm.

The preparation method of the composition controls the turbidity of the extracting solution to be less than or equal to 2.0FNU after cold fine filtration.

The composition can be applied to skin external skin care products in various dosage forms such as gel, facial cleanser, cream, emulsion and the like.

The invention also provides a skin care product with moisturizing effect, which comprises the composition with moisturizing effect and a substrate.

In some embodiments, the amount of the composition in the skin care product is preferably 0.25 wt%.

Further, in some embodiments the substrate is comprised of glycerol, 1, 2-pentanediol, hydroxyethyl acrylate/sodium acryloyldimethyl taurate copolymer, phenoxyethanol/ethylhexylglycerol, and water.

The matrix can be prepared as follows:

(1) heating glycerol, 1, 2-pentanediol and water as phase A raw materials to 80 deg.C, mixing well for use

(2) Stirring and cooling, adding hydroxyethyl acrylate/sodium acryloyldimethyl taurate copolymer (SEPINOV EMT 10) as phase B raw material at 60 deg.C, and stirring;

(3) cooling under stirring, adding phenoxyethanol/ethylhexyl glycerol (EUXYL PE 9010) as phase C material below 40 deg.C, and stirring;

(4) stirring and cooling, discharging at 38 ℃ to obtain the matrix.

The extract of the composition is dried to be in a powder state, dissolved by adding water and mixed and stirred with a matrix to obtain the skin care product with the moisturizing effect. Preferably, the composition powder is added in an amount of 0.25 wt%.

The invention also provides a moisturizing method, and the composition or the skin care product containing the composition is applied to the surface of skin. Such as applying gels, creams, lotions containing the compositions of the present invention; or cleaning the face or other skin parts by using the facial cleanser containing the composition; or a skin care product consisting of the above-mentioned substrate and the composition of the present invention.

Therefore, the invention provides a composition with a moisturizing effect, a preparation method thereof and a skin care product. The composition with the moisturizing effect is prepared from dendrobium officinale, tremella, bamboo fungus and radix ophiopogonis, wherein the mass ratio of the dendrobium officinale to the tremella to the bamboo fungus to the radix ophiopogonis is (1-4) to (1-4). The dendrobium officinale, the tremella, the bamboo fungus and the radix ophiopogonis are matched and used together in proportion, and the moisturizing effect is superior to that of each single component. The composition can be applied to skin external skin care products in various dosage forms such as gel, facial cleanser, cream, emulsion and the like.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows the comparison of moisture absorption rates of the respective components;

FIG. 2 shows the comparison results of the moisture retention rates of the respective components;

FIG. 3 shows the results of comparing the amount of change in moisture content in examples;

FIG. 4 shows the results of comparing the amount of change in moisture loss in the examples;

FIG. 5 shows cell viability curves of the effective substance (g) at different concentrations in test example 3;

FIG. 6 shows fluorescence patterns of the assay of the effective substance (g) Loricrin protein in test example 3;

FIG. 7 is a fluorescence chart showing detection of an effective substance (g) Filaggrin protein in test example 3;

FIG. 8 is a fluorescence chart showing detection of an effective substance (g) Zonula Occluden-1 protein in test example 3;

FIG. 9 is a fluorescence chart showing detection of the effective substance (g) Aquaporin 3 protein in test example 3;

FIG. 10 is a graph showing the tendency of the secretion amount of TNF α, an inflammation-related factor, as an effective substance (g) in test example 3.

Detailed Description

The invention discloses a composition with a moisturizing effect and a preparation method and application thereof. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and products of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.

In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available.

Example 1

Weighing Dendrobium officinale in a clean and dry beaker, adding deionized water according to a material-liquid ratio of 1:80, soaking in the beaker for 0.5h, heating and stirring, and extracting at 60 ℃ for 120 min. Then, carrying out heat fine filtration, placing a paper board H60(1 mu m) in a Buchner funnel, injecting warm water (extracting solution) for soaking thoroughly, opening a suction filtration negative pressure pump to ensure that the paper board is tightly attached to the funnel but the whole paper board is wet, and obtaining a clear and bright extracting solution. Then, cold fine filtration is carried out, a paper board H70(0.45 mu m) is placed in a Buchner funnel, warm water (extracting solution) is injected for soaking thoroughly, suction filtration and negative pressure suction are opened to ensure that the paper board is tightly attached to the funnel but the whole paper board is wet, and clear and transparent extracting solution (the turbidity is controlled to be less than or equal to 2.0FNU) is obtained.

Example 2

Weighing Tremella, pretreating the raw materials, extracting, heat fine filtering and cold fine filtering according to the method of example 1 to obtain clear and transparent extractive solution (turbidity is controlled to be less than or equal to 2.0 FNU).

Example 3

Weighing Dictyophora Indusiata, pretreating raw materials according to the method of example 1, extracting, heat fine filtering and cold fine filtering to obtain clear and transparent extractive solution (turbidity is controlled to be less than or equal to 2.0 FNU).

Example 4

Weighing radix Ophiopogonis, pretreating, extracting, heat filtering and cold filtering the raw materials according to the method of example 1 to obtain clear and transparent extractive solution (turbidity is controlled to be less than or equal to 2.0 FNU).

Example 5

Weighing herba Dendrobii, Tremella, Dictyophora Indusiata and radix Ophiopogonis at a ratio of 4:3:4:3 in a clean and dry beaker, adding deionized water at a ratio of 1:80, soaking for 0.5h, heating and stirring, and extracting at 60 deg.C for 120 min. Then, carrying out heat fine filtration, placing a paper board H60(1 mu m) in a Buchner funnel, injecting warm water (extracting solution) for soaking thoroughly, opening a suction filtration negative pressure pump to ensure that the paper board is tightly attached to the funnel but the whole paper board is wet, and obtaining a clear and bright extracting solution. Then, cold fine filtration is carried out, a paper board H70(0.45 mu m) is placed in a Buchner funnel, warm water (extracting solution) is injected for soaking thoroughly, suction filtration and negative pressure suction are opened to ensure that the paper board is tightly attached to the funnel but the whole paper board is wet, and clear and transparent extracting solution (the turbidity is controlled to be less than or equal to 2.0FNU) is obtained.

Example 6

Weighing herba Dendrobii, Tremella, Dictyophora Indusiata and radix Ophiopogonis at a ratio of 3:4:3:4 in a clean and dry beaker, soaking in deionized water for 0.5h at a ratio of 1 (80 + -5) in the beaker, heating and stirring, and extracting at 60 + -5 deg.C for 120 + -10 min. Then, carrying out heat fine filtration, placing a paper board H60(1 mu m) in a Buchner funnel, injecting warm water for soaking thoroughly, opening the suction filtration and negative pressure suction to ensure that the paper board is tightly attached to the funnel but the whole paper board is wet, and obtaining a clear and transparent extracting solution. Then, cold fine filtration is carried out, a paper board H70(0.45 mu m) is placed in a Buchner funnel, warm water is injected for thorough soaking, suction filtration and negative pressure pumping are opened to ensure that the paper board is tightly attached to the funnel but the whole paper board is wet, and clear and transparent extracting solution (the turbidity is controlled to be less than or equal to 2.0FNU) is obtained.

Example 7

Weighing herba Dendrobii, Tremella, Dictyophora Indusiata and radix Ophiopogonis at a ratio of 1:1:1:1 in a clean and dry beaker, soaking in deionized water at a ratio of 1 (80 + -5) in the beaker for 0.5h, heating and stirring, and extracting at 60 + -5 deg.C for 120 + -10 min. Then, carrying out heat fine filtration, placing a paper board H60(1 mu m) in a Buchner funnel, injecting warm water for soaking thoroughly, opening the suction filtration and negative pressure suction to ensure that the paper board is tightly attached to the funnel but the whole paper board is wet, and obtaining a clear and transparent extracting solution. Then, cold fine filtration is carried out, a paper board H70(0.45 mu m) is placed in a Buchner funnel, warm water is injected for thorough soaking, suction filtration and negative pressure pumping are opened to ensure that the paper board is tightly attached to the funnel but the whole paper board is wet, and clear and transparent extracting solution (the turbidity is controlled to be less than or equal to 2.0FNU) is obtained.

Example 8

The dendrobium officinale, the dictyophora phalloidea and the radix ophiopogonis are weighed according to the ratio of 2:1:1, and the raw materials are pretreated, extracted, subjected to heat fine filtration and cold fine filtration according to the method in the example 1 to obtain clear and transparent extract (the turbidity is controlled to be less than or equal to 2.0 FNU).

Example 9

The dendrobium officinale and the bamboo fungus are weighed according to the proportion of 2:1, and the raw materials are pretreated, extracted, subjected to hot fine filtration and cold fine filtration according to the method in the embodiment 1 to obtain clear and transparent extract (the turbidity is controlled to be less than or equal to 2.0 FNU).

Test example 1 measurement of moisture absorption and Retention Performance

1) Test materials

Ammonium sulfate, plate and glass drier

2) And a detection method

1. And respectively freeze-drying the dendrobium officinale, the tremella, the dictyophora phalloidea and the radix ophiopogonis extracting solutions prepared in the embodiments 1-4 to obtain freeze-dried powder. The freeze-drying conditions were: the temperature is-76 ℃, the pressure is 60-70mTor, and the time is 24 h.

2. Solid samples were weighed directly from the lyophilized powder, three replicates per sample (dry sample weight recorded as M1).

3. First, each dish containing the sample was placed in a desiccator having a relative humidity of 81% and made by a saturated solution of ammonium sulfate, and the sample was weighed after the sample reached equilibrium for 96 hours (the weight of the sample after water absorption was recorded as M2), and the moisture absorption rate of each sample was calculated from the difference in weight between the sample before and after the placement.

The moisture absorption rate calculation formula is as follows:

moisture absorption rate (%) (M2-M1)/M1%

4. The sample, which had reached moisture absorption equilibrium as described above, was placed in a desiccator with a relative humidity of 46% which was achieved by a saturated solution of potassium acetate. After the samples were allowed to stand for 96 hours until the samples were dehydrated and equilibrated, the weight of each sample was weighed (the weight of the sample after absorbing water was recorded as M3), and the moisture retention rate of each sample was calculated.

The moisturizing rate calculation formula is as follows:

moisture retention rate (%) {1- [ (M2-M1) - (M3-M1)/(M2-M1) ] } × 100%

3) And the results obtained

The moisture absorption and moisture retention results for each component are shown in FIGS. 1-2 and tables 1-2.

Table 1 moisture absorption results for each component

Name (R)	Rate of moisture absorption
		Tremella fuciformis berk	58.89％±0.0141
Bamboo fungus	41.84％±0.0169
		Ophiopogon japonicus	23.69％±0.0142
DendrobiumDendrobium officinale Kimura et Migo	23.64％±0.00541

TABLE 2 moisture retention results for each component

Name (R)	Moisture retention rate
		Ophiopogon japonicus	38.76％±0.0637
DendrobiumDendrobium officinale Kimura et Migo	30.73％±0.0117
		Bamboo fungus	24.19％±0.0107
Tremella fuciformis berk	13.24％±0.00684

From the above results it can be seen that:

(1) in a hygroscopicity experiment, tremella, dictyophora phalloidea, ophiopogon root and dendrobium officinale are selected;

(2) in the moisture retention experiment, the dwarf lilyturf tuber, the dendrobium officinale, the bamboo fungus and the tremella are used.

Test example 2 measurement of human body moisturizing efficacy

1) Effective substance

(a) Example 1 freeze-drying an extract of dendrobium officinale to obtain a freeze-dried powder;

(b) example 2 freeze drying of tremella extract to obtain lyophilized powder;

(c) example 3 freeze-drying bamboo fungus extract to obtain freeze-dried powder;

(d) example 4 freeze-drying an extract of Ophiopogon japonicus to obtain a lyophilized powder;

(e) example 5 the composition is prepared and the extract is freeze dried to obtain freeze dried powder;

(f) example 6 the composition is prepared and the extract is freeze dried to obtain a lyophilized powder;

(g) example 7 the composition is prepared and the extract is freeze dried to obtain a lyophilized powder;

(h) example 8 the composition is prepared and the extract is freeze dried to obtain a lyophilized powder;

(i) example 9 the composition is prepared and the extract is freeze dried to obtain a lyophilized powder.

The above freeze-drying conditions were: the temperature is-76 ℃, the pressure is 60-70mTor, and the time is 24 h.

2) Substrate

The formulation of the matrix is shown in Table 3 in parts by mass.

Table 3 base formula table

The preparation method of the matrix comprises the following steps:

(1) heating the phase A raw material to 80 ℃, and uniformly mixing for later use;

(2) stirring and cooling, adding the B-phase raw material at 60 ℃, and stirring uniformly;

(3) stirring and cooling, adding the C-phase raw material below 40 ℃, and stirring uniformly;

(4) stirring and cooling, discharging at 38 ℃ to obtain the matrix.

3) Test sample

According to the mass part, 0.25 part of each effective substance (freeze-dried powder obtained by freeze drying) is added into 9.75 parts of water, and is mixed with 90 parts of the matrix and stirred uniformly.

4) Moisture content test

The moisture content was measured by the CORNEOMETER-capacitance method. The result is expressed by a change rate of a set humidity Measurement Value (MMV). The skin moisture content was measured by the test subjects after using the product for 30min, 1h, 2h and 4 h. The experimental apparatus is as follows: corneometer CM825, CK, germany.

The calculation formula of the moisture content variation is as follows: the moisture content variation is Tn-T0, wherein: t0 is the data acquisition value of the skin substrate when the sample is not smeared; tn is the data collected for each time point for the sample set. The higher the moisture content variation, the more obvious the water replenishing effect is. The results are shown in FIG. 3 and Table 4.

TABLE 4 moisture content Change results

Note: analyzing by a Duncan's multiple range test method, wherein the same column is marked with different capital letters to indicate that the difference between groups is obvious (P < 0.01); marked with different lower case letters to indicate significant differences between groups (P < 0.05); the same letter designations do not significantly differ between groups (P > 0.05). The positive control was 0.1% hyaluronic acid, and the matrix was as shown in table 3.

The above moisture content test results show that:

(1) data measured after sample application for 5min show that the difference among 11 samples is very obvious (P <0.01), and f > g > e > h > d > c > positive > i > b > a > matrix, and the formula is superior to a single formula;

(2) data measured after sample application for 30min show that the difference among 11 samples is very obvious (P <0.01), and f > g > e > h > i > d > b > c > a > positive matrix, and the formula is superior to the single formula;

(3) data measured after sample application for 60min show that the difference among 11 samples is very obvious (P <0.01), and h > g > f > e > i > c > d > b > a > positive matrix, and the formula is superior to the single formula;

(4) data measured after 120min of plating showed significant differences (P <0.05) among the 11 samples, and e > g > f > d > i > c > positive > b > h > a > matrix;

(5) the data measured after 240min of proofing showed no significant difference between c and i and b and e (P > 0.05);

the results show that the moisturizing effect of the composition is better than that of the single component, and the sample and the matrix have very obvious difference (P < 0.01).

5) Water Dispersion loss test

The subjects and test environment were the same as the skin moisture assay evaluation.

Test the change of the Water vapor pressure of the surface of the adjacent skin is measured according to the diffusion principle found by A.Fick, the Water Loss of the surface of the skin is measured, and the Water Loss is expressed by TEWL (Trans-Epidermal Water Loss) and is given in g/hm²The lower the value, the more obvious the effect of preventing the water loss of the sample is, and the more perfect the skin barrier function is. The subjects measured the rate of change of skin water loss after using the samples for 30min, 1h, 2h and 4 h. The experimental apparatus is as follows: tewameter TM300, CK, Germany.

The calculation formula of the change rate of the skin water dispersion loss is as follows: (Tn-T0)/T0 × 100, wherein: t0 is the data acquisition value of the skin substrate when the sample is not smeared; tn is the data collected for each time point for the sample set. The lower the negative value of the change rate of the water dispersion loss, the more obvious the water locking effect is. The results are shown in FIG. 4 and Table 5.

TABLE 5 Water Dispersion Rate results

Note: analyzing by a Duncan's multiple range test method, wherein the same column is marked with different capital letters to indicate that the difference between groups is obvious (P < 0.01); marked with different lower case letters to indicate significant differences between groups (P < 0.05); the same letter designations do not significantly differ between groups (P > 0.05).

The above-mentioned moisture loss experimental results show:

(1) after sample application, water dispersion loss data are measured at 1h, 2h and 4h, and the difference among 11 samples is found to be extremely obvious (P is less than 0.01);

(2) after 4h stabilization, the compositions of the present invention were found to have better water loss prevention than the single formulations.

Test example 3 epidermal cell-based detection of the content of barrier and moisturizing-related proteins

1) Effective substance

(g) Example 7 the composition is prepared and the extract is freeze dried to obtain a lyophilized powder; the freeze-drying conditions were: the temperature is-76 ℃, the pressure is 60-70mTor, and the time is 24 h.

2) Test sample

Diluting the effective substance (lyophilized powder obtained by freeze drying) with solvent or water according to dilution requirement of each cell experimental sample.

3) MTT assay

The MTT method is also called MTT colorimetric method, and is a method for detecting cell survival and growth. The detection principle is that succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT into water-insoluble blue-purple crystalline Formazan (Formazan) and deposit the blue-purple crystalline Formazan in the cells, and dead cells do not have the function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and its light absorption value is measured at 540 or 720nm wavelength by ELISA detector, which can indirectly reflect living cell number. Therefore, in order to determine the maximum safe dose for moisturizing efficacy in the cell experiment, it was ensured that the cells remained at a high survival rate without affecting the outcome judgment. The samples were set from high to low at 7 dosing concentrations, and cytotoxicity test experiments were performed on keratinocytes, and the results are shown in fig. 5 and table 6.

The experimental cells were: keratinocyte culture fluid (institute of tissue engineering).

TABLE 6 cytotoxicity assay results for active principles

Sample concentration (ppm)	Survival rate (%)
		25	102.54±6.34
50	101.64±0.58
		100	109.64±8.31
200	101.31±8.68
		500	99.27±0.76
1000	89.99±1.43
		2000	83.25±8.23
Negative control (4% DMSO)	20.67±2.51
		Blank group	100.00±2.40

The results of the cytotoxicity test experiment show that:

the cell survival rate is more than 90% under the administration dosage of 1000 ppm. Therefore, the administration dose of 1000ppm or less can be selected for the subsequent cell experiment.

4) Epidermal cell-based barrier and moisturization-related protein content detection

Based on the results of the MTT assay, the maximum safe dose of the sample on keratinocytes (SC) was determined to be 1000 ppm. On the basis of the test results, on the basis of keratinocytes, an experiment for detecting the contents of loricrin (Loricin), mitogen (Filaggrin), Zonula occluden-1 (Zonula occluden-1) and Aquaporin (Aquaporin 3) was performed, and an experiment for inhibiting the secretion of inflammation-related factor (TNF α) was performed under UVB stimulation. The above experiment employed immunofluorescence to detect the expression levels of loricrin (Loricin), mitogen (Filaggrin), Zonula occluden-1 (Zonula occluden-1), and Aquaporin (Aquaporin 3) in keratinocytes.

The principle of the immunofluorescence method is as follows: the binding between the antibody and the antigen is highly specific. The antibody is used to detect the same kind of antigen substance in the tissue or cell. Since the complex of antigen and antibody is colorless, it is necessary to perform quantitative staining by cellular immunofluorescence. The green fluorescence intensity in the photograph of the cells that excited green fluorescence in blue light was analyzed using Image-pro Plus (IPP) software, and the fluorescence intensity per unit area was calculated. The mean optical density value (IOD/Area) was used as an index for protein expression determination. The higher the IOD/Area, the higher the protein expression level.

The experimental samples are all water-soluble, and the positive control tretinoin is dissolved in DMSO, so two groups of negative controls, namely SC (epidermal cell culture fluid) and SC (epidermal cell culture fluid containing 1 ‰ DMSO), are set in the experiment.

The fluorescence profiles for each protein are shown in FIGS. 6 to 9, and the data are shown in Table 7.

TABLE 7 epidermal cell-based detection of barrier and moisturizing-related protein content

Note: statistical analysis was performed using the T-Test method pair, indicating significant differences between groups (P <0.05) compared to the negative control (SC) group; indicates that the difference between groups was significant (P < 0.01).

The detection results of the barrier and moisture retention related protein content of the epidermal cells show that:

compared with an effective substance negative control (SC) group, the effective substance has a remarkable (P <0.05) improvement effect on the expression quantity of the keratinocyte skin barrier related Loricrin protein and Filaggrin protein under the administration dosage of 500 ppm. Has very obvious (P <0.01) improving effect on the expression quantity of Zonula Occludens-1 protein and Aquaporin 3 protein related to keratinocyte skin moisturizing.

5) Experiment for inhibiting TNF-alpha secretion of inflammation-related factor

Several external factors can cause the skin to produce an inflammatory response, producing an excess of inflammatory factors such as TNF- α, which can increase skin permeability, decrease integrity, and affect skin barrier function. The effects of the effective substances on relieving inflammation and enhancing skin barrier are evaluated by detecting the influence of the effective substances on tumor necrosis factor (TNF-alpha). And carrying out UVB irradiation on the negative control group, the sample group and the positive control group according to the experimental requirements, establishing a cell model generated by inflammatory factors, and testing by adopting a Human TNF-alpha ELISA detection kit.

The experimental samples are all water-soluble, the positive control dexamethasone is dissolved in DMSO, so two blank control groups are set in the experiment, namely SC (epidermal cell culture solution) without ultraviolet irradiation and SC (epidermal cell culture solution containing 1 thousandth DMSO). The method comprises the steps of utilizing ultraviolet radiation to cause cell damage, establishing an inflammatory factor hypersecretion model, using the model as a negative control of a sample group, and setting two groups of negative control groups, namely SC (epidermal cell culture fluid) applying ultraviolet radiation and SC (epidermal cell culture fluid containing 1 thousandth DMSO) applying ultraviolet radiation.

The results are shown in FIG. 10 and Table 8.

TABLE 8 TNF-alpha assay results for inflammation-related factors

Sample name	TNF-alpha concentration (pg/mL)
		Blank control group 1(SC, 1% DMSO, UVB-)	40.42±7.32^Aa
Negative control group 1(SC, 1% DMSO, UVB +)	84.41±10.17^Bb
		Positive control (0.1% dexamethasone)	59.22±17.34^Bc
Blank control group 2(SC, UVB-)	43.69±4.19^Aa
		Negative control group 2(SC, UVB +)	60.73±2.3^Ab
500ppm active principle	55.96±8.85^Aa

Note: statistical analysis is carried out by adopting a T-Test method, and the same column is marked with different capital letters to show that the difference between groups is obvious (P < 0.01); marked with different lower case letters to indicate significant differences between groups (P < 0.05); the same letter designations do not significantly differ between groups (P > 0.05).

The detection result of the inflammation-related factor TNF-alpha shows that:

compared with a blank control group 1(SC, 1% DMSO, UVB-), the secretion amount of inflammatory factors TNF-alpha of the negative control group 1(SC, 1% DMSO, UVB +) is remarkably increased (P is less than 0.01), and the UVB irradiation condition is effective; compared with the negative control group 1(SC, 1% DMSO, UVB +), the positive control group (dexamethasone, 0.1%, UVB +) has obvious inhibition effect (P <0.05) on the secretion of the inflammatory factor TNF-alpha.

Compared with a blank control group 2(SC, UVB-), the secretion amount of inflammatory factors TNF-alpha of the negative control group 2(SC, UVB +) is obviously increased (P <0.05), which indicates that the UVB irradiation condition is effective; compared with the negative control group 2(SC, UVB +), the effective substance has an inhibiting effect on the secretion amount of the inflammation-related factor TNF-alpha under the administration dosage of 500 ppm.

Test example 4 evaluation of safety

1) Effective substance

Example 7 the composition produced an extract (polysaccharide, 3 mg/mL. gtoreq.3).

2) Test sample

And diluting the effective substances with a solvent or water according to the concentration requirements of each safety experiment.

3) Haemolysis test of erythrocytes

The effective substance was diluted with PBS (phosphate buffered saline) to 40% effective substance. The test results are evaluated by measuring the absorbance of the leakage of hemoglobin from freshly isolated red blood cells incubated with the test substance under standard conditions, designed on the basis of the principle that chemicals can damage the cell membrane. The higher the hemolysis rate, the greater the irritation. The stimulation intensity of the sample with slight stimulation can be predicted through the result of the erythrocyte hemolysis test, the method is quick and efficient, and the stimulation of the plant raw materials can be quickly and accurately predicted. The results are shown in Table 9.

TABLE 940% effective substance erythrocyte hemolysis test result

The results of the above-mentioned erythrocyte hemolysis experiment show that:

the hemolysis rate of the 40% active was 0%, indicating no irritation of the composition described in example 7.

4) Multiple skin irritation test in animals

Diluting the effective components with distilled water to obtain 60% effective components. 24h before the experiment, the back hairs on two sides of the spine of the animal are cut off, the hair removal ranges are respectively 3cm multiplied by 3cm, about 0.5g of the tested object is taken and smeared on the skin on one side, the smearing range is 2.5cm multiplied by 2.5cm, 1 time/d smearing is carried out, 14d smearing is carried out continuously, and the skin on the other side is used as a control group. Shearing hair before each smearing from day 2, removing residual test substances with warm water or non-irritant solvent, observing the result after 1h, grading according to a skin irritation/corrosion test grading table of technical Specification for cosmetic safety (2015 edition), calculating average integral of each animal every day after the experiment is finished, and determining skin irritation strength. The higher the integral, the greater the irritation. The results are shown in Table 10.

TABLE 1060% actives animal multiple skin irritation test results

Note: erythema/edema a column 0/4 shows that each animal had a daily integral of 0 and the number of experimental animals per day was 4. The calculation formula is as follows:

the results of the multiple skin irritation tests of the animals show that:

and (3) applying 60% of effective substances for 14 days, wherein the integral mean value of each animal per day is 0, and judging the result to be nonirritating.

5) Eye irritation test in animals

Diluting the effective components with distilled water to obtain 80% effective components. Dripping 80% of the effective components into conjunctival sac of rabbit eye, and performing clinical examination and scoring without washing eye within 24 hr. The scores should be recorded for observation of the cornea, iris, conjunctiva, and other damaging effects. And (4) judging the result according to the grading standard of eye damage/eye irritation reaction grading of technical Specification for cosmetic safety (2015 edition). The higher the integral, the greater the irritation. The results are shown in Table 11.

Table 1180% actives animal eye irritation test results

The results of the animal eye irritation test show that:

80% of effective substances are dripped into conjunctival sac of rabbit eye, no irritation reaction is seen on conjunctiva, iris and cornea for 1h, 24h, 48h and 72h, and no irritation is judged as a result.

6) Phototoxicity test for neutral Red uptake of 3T3

The phototoxicity experiment of 3T3 neutral red uptake was performed using the stock solution of the active substance. Neutral red is a weak cationic dye that readily penetrates cell membranes by nonionic diffusion and accumulates in the cytosol. Under the action of certain chemical substances and external conditions, irreversible changes such as change of sensitivity of cell surfaces or lysosome membranes can be caused to cause increase of fragility of lysosomes, and the capacity of cells to absorb neutral red is reduced. Whether the chemical substance has phototoxicity or not is judged by measuring the change of the neutral red absorption capacity of the 3T3 fibroblasts after the combined action of the chemical substance and ultraviolet irradiation. Experimental parameters PIF (light stimulus factor) and MPE (mean light effect) were used to evaluate phototoxicity, the criteria are shown in table 12, and the results are shown in table 13.

TABLE 12 in vitro determination criteria for phototoxicity test of 3T3 neutral Red uptake

Note:

1. the positive control OD540 values are all larger than 0.4, and the positive control chlorpromazine hydrochloride meets the following conditions, which indicates that the experiment is true.

IC50(+ UV) was in the range of 0.1-3.0. mu.g/mL.

IC50(-UV) was in the range of 7.0-90.0. mu.g/ml.

PIF≥6

2. PIF (light stimulation factor) ═ IC50(-UV)/IC50(+ UV)

3. MPE (mean light effect) cytotoxicity concentration response curves obtained by comparing the concentration response curves obtained with and without illumination (+ UV) on a concentration grid, the contamination concentration range being chosen within the concentration range common to the illumination (+ UV) and non-illumination (-UV) experiments. The average light effect (MPE) is found using a special software "PHOTO 32".

TABLE 13 phototoxicity test results for the 3T3 neutral Red uptake of the active ingredient

Note: *1: it also does not exhibit cytotoxicity when the highest concentration value allowed (1000.0. mu.g/mL) is reached;

+: phototoxicity is expected;

and (2) preparing: no phototoxicity is expected.

The above results of the 3T3 neutral red uptake phototoxicity experiment show that:

the IC50 of the raw liquid of the effective substance is more than 1000.0 mug/mL under the conditions of no light and light, the PIF value is all 1, and the MPE value is less than 0.1. According to the phototoxicity result evaluation standard table 9, the active ingredient stock solution has no phototoxicity.

In summary, the composition of example 7 has a hemolysis rate of 0%, no hemolysis irritation, no skin and eye irritation, no phototoxicity, and high safety.

The safety tests of the compositions described in examples 5 and 6 were carried out according to the above-mentioned method, and the results were similar to those of the composition described in example 7, and the compositions described in examples 5 and 6 were non-hemolytic irritant, non-skin and eye irritants, non-phototoxic, and highly safe.

Claims

1. A composition with a moisturizing effect is characterized by being prepared from dendrobium officinale, tremella fuciformis, bamboo fungus and radix ophiopogonis, wherein the mass ratio of the dendrobium officinale to the tremella fuciformis to the bamboo fungus to the radix ophiopogonis is (1-4) to (1-4);

pulverizing herba Dendrobii, Tremella, Dictyophora Indusiata (Vent. Ex pers) Fisch and radix Ophiopogonis, sieving, extracting with water, sequentially performing hot fine filtration and cold fine filtration, and collecting extractive solution;

the sieving is to sieve through a 100-mesh sieve; the water extraction is to add water according to the material-liquid ratio of 1 (75-85), soak for 0.5h, and extract for 110-130 min at 55-65 ℃;

placing a paper board H60 in a Buchner funnel, injecting warm extracting solution for soaking, opening a suction filtration negative pressure pump to ensure that the paper board is tightly attached to the funnel but the whole paper board is wet, and obtaining clear and transparent extracting solution; and (3) placing a paper board H70 in a Buchner funnel, injecting warm extracting solution, opening a suction filtration negative pressure pump to ensure that the paper board is tightly attached to the funnel but the whole paper board is wet, and obtaining clear and transparent extracting solution.

2. The composition of claim 1, wherein the mass ratio of the dendrobium officinale to the tremella fuciformis to the dictyophora indusiata to the ophiopogon japonicus is 4:3:4: 3.

3. The composition of claim 1, wherein the mass ratio of the dendrobium officinale to the tremella fuciformis to the dictyophora indusiata to the ophiopogon japonicus is 3:4:3: 4.

4. The composition of claim 1, wherein the mass ratio of the dendrobium officinale to the tremella fuciformis to the dictyophora indusiata to the ophiopogon japonicus is 1:1:1: 1.

5. A process for preparing the composition as claimed in any one of claims 1 to 4, wherein the raw materials are crushed, sieved, extracted with water and then subjected to heat fine filtration and cold fine filtration in sequence to collect the extract;

6. A skin care product having a moisturizing effect comprising the composition of any one of claims 1-4 and a base.

7. A skin care product according to claim 6 wherein said composition is present in an amount of 0.25% by weight.

8. A skin care product according to claim 6 or 7 wherein the matrix consists of glycerol, 1, 2-pentanediol, hydroxyethyl acrylate/sodium acryloyldimethyl taurate copolymer, phenoxyethanol/ethylhexylglycerol and water.

9. Use of a composition according to any one of claims 1 to 4 in the preparation of a skin care product having skin moisturising properties.