CN108992369B

CN108992369B - Hyaluronic acid, preparation method and application thereof

Info

Publication number: CN108992369B
Application number: CN201810707842.9A
Authority: CN
Inventors: 李榕榕; 江俊杰; 于淼
Original assignee: Shandong Ts Biotech Co ltd
Current assignee: Shandong Ts Biotech Co ltd
Priority date: 2018-07-02
Filing date: 2018-07-02
Publication date: 2021-07-30
Anticipated expiration: 2038-07-02
Also published as: CN108992369A

Abstract

The invention relates to hyaluronic acid, a preparation method and application thereof, wherein the hyaluronic acid has a weight average molecular weight of 5-3000 kDa, a number average molecular weight of 5-3000 kDa, a viscosity average molecular weight of 5-3000 kDa and a polydispersity of 7-15. The invention also relates to a method for producing said product and to the use thereof.

Description

Hyaluronic acid, preparation method and application thereof

Technical Field

The invention relates to the field of cosmetics, and particularly relates to hyaluronic acid, a preparation method thereof and application thereof in preparing cosmetics.

Background

Hyaluronic Acid (HA), also known as uronic acid, hyaluronic acid, and azululosic acid, is a linear macromolecular polysaccharide composed of alternating D-glucuronic acid and N-acetyl-D-glucosamine. Hyaluronic acid is present in connective tissues of vertebrates, such as hyaline cartilage, synovial joint fluid and skin tissues (dermis and epidermis), and is involved in many physiological processes, such as adhesion, development, Cell movement, cancer, angiogenesis and wound healing (Laurent t.c. and framer j.r.e., 1992, fasseb j.6: 2397-.

Traditional HA development HAs focused on novel applications for studying the physicochemical properties of HA. For example, the use of hyaluronic acid in the range of 1000Da to 3000kDa has been reported so far. Other studies include pretreatment of HA, new efficacy brought by new formulations, chemical modification and crosslinking of HA to give it unique properties. The physiological mechanisms of HA are studied in a physiologically and medically biased direction. For example, how HA produces physiological activity through cellular receptors, transdermal permeability of various HA and corresponding clinical effects.

In addition, it has been reported in the art that hyaluronic acid having a high molecular weight (200kDa to 3500kDa) has an excellent moisturizing effect and a lubricating effect in a physical friction state, and can be advantageously used for pharmaceuticals or cosmetics. However, the high molecular weight also makes hyaluronic acid difficult to be absorbed by the human body, and the moisturizing effect is not durable after the application surface is in contact with water. In response, hyaluronic acid comprising low molecular weight fractions has been developed in the art. It is currently known that HA of the high molecular weight fraction (average molecular weight between 1MDa and 1.5MDa) provides excellent moisturizing properties to cosmetic compositions, while HA of the low molecular weight fraction (average molecular weight between 20kDa and 400kDa) shows anti-wrinkle properties due to its ability to penetrate the skin barrier. In this regard, hyaluronic acid compositions having these two molecular weight fractions have been provided in the art (CN 101123942A). However, further studies on the ratio of hyaluronic acid of these two different molecular weight fractions are still needed to achieve a balance of short-term and long-term effects.

Disclosure of Invention

In a first aspect, the present invention provides a hyaluronic acid having a weight average molecular weight of 5-3000 kDa, a number average molecular weight of 5-3000 kDa, a viscosity average molecular weight of 5-3000 kDa, and having a polydispersity of 7-15.

In a second aspect, the present invention provides a cosmetic composition comprising hyaluronic acid according to the first aspect.

In a third aspect, the invention relates to the use of said hyaluronic acid for the manufacture of a cosmetic product.

Advantageous effects

The present inventors have intensively studied and found that, although the prior art teaches that the low molecular weight fraction has an anti-wrinkle effect and the high molecular weight fraction has a moisturizing effect, not only apparent effects complement each other but also synergistic effects of the two fractions can achieve both rapid onset and long-term maintenance of the effects when the different molecular weight fractions are combined. In particular, the inventors have found that when PD is in the range of 1.5-2.9 and the molecular weight is in the range of 10-80kDa, the main effects of hyaluronic acid are in cell repair and wound healing, promotion of cell and nutrient growth (especially elastin synthesis), improvement of skin barrier integrity and increase of skin elasticity. When the PD is in the range of 3.0-15.0 and the molecular weight is in the range of 5kDa-3000kDa, hyaluronic acid has the effects of instantly lifting, improving skin firmness and elasticity. And when the PD is in the range of 7.0-30.0 and the molecular weight range is 5-3000 kDa, the hyaluronic acid can realize long-term water locking and moisture retention, long-term wrinkle resistance and improvement of skin smoothness and elasticity.

Thus, for the sake of a balance of transient and long-lasting effects, polydisperse hyaluronic acids with PD in the range of 7.0-15.0 are chosen by the present invention. According to the embodiments of the present invention, hyaluronic acid having such characteristics exhibits excellent physiological activity and transdermal permeability; in a test of a subject, the cosmetic prepared from the hyaluronic acid with the characteristics has excellent effects of resisting wrinkles, moisturizing, improving skin elasticity and the like, and simultaneously has excellent instantaneous and long-acting effects.

Drawings

FIG. 1 is a graph of the results of different product prototype skin moisture test screens based on the preliminary screen.

Figure 2 is a graph of TEWL skin water loss for different product prototypes according to the preliminary screening.

FIG. 3 is a chromatogram of hyaluronic acid product C (Cube3 transiently).

FIG. 4 is a chromatogram of hyaluronic acid product D (Difference showa).

Fig. 5 is a graph showing the change in moisture content of the stratum corneum after applying the product Cube3 instantaneously for 30min and 120min according to effect example 1.

Fig. 6 is a graph showing changes in skin water loss after applying the product Cube3 instantaneously for 30min and 120min according to effect example 1.

Fig. 7 is a graph showing the rate of increase in skin elasticity after instantaneous bloom using the product Cube3 according to effect example 1.

Fig. 8 is a graph showing the rate of increase in skin smoothness after the instant waviness of the product Cube3 was applied, according to effect example 1.

Fig. 9 is a graph showing the rate of change in the number of skin wrinkles after the instant bloom in which the product Cube3 was applied, according to effect example 1.

Fig. 10 is a score of anti-wrinkle efficacy of lifting after conua administration of product Cube3 by a practitioner according to effect example 1.

FIG. 11 is a graph showing the skin moisture increase rate after Difference showa in the application of the product according to effect example 2.

FIG. 12 is a graph showing the rate of increase in skin elasticity after Difference showa in the application of the product according to effect example 2.

FIG. 13 is a graph showing the improvement in skin smoothness after Difference showa in the application of the product according to effect example 2.

FIG. 14 is a graph showing the rate of change in skin roughness after Difference showa of the product applied, in accordance with Effect example 2.

FIG. 15 is a graph showing the rate of change in the number of skin wrinkles after Difference showa of application of the product according to Effect example 2.

FIG. 16 is a graph showing the rate of change in skin scales after Difference showa in the application of the product according to effect example 2.

FIG. 17 is a graph showing the rate of change in volume of skin wrinkles after Difference showa of the product applied, according to Effect example 2.

Figures 18A-18C are graphs of efficacy scores by practitioners following administration of product Difference according to effect example 2.

Detailed Description

The invention provides Hyaluronic Acid (HA) with a novel proportion. Through in vitro (cellular level) and systemic physiological studies on subjects, we developed, prepared and screened HA products with excellent physiological activity and transdermal permeability; clinical tests of human bodies prove that the product has excellent effects of resisting wrinkles, preserving moisture, improving skin elasticity and the like, and has excellent instant and long-acting effects.

The term "hyaluronic acid" as used herein, also known as hyaluronan, has a meaning well known in the art. The basic unit units of hyaluronic acid are repeating disaccharide units of N-acetyl-D-glucosamine (GlcNAc) and D-glucuronic acid (GlcUA) linked together by alternating β -1,4 and β -1,3 glycosidic linkages forming a linear structure. Since hyaluronic acid is often present in the form of inorganic salts, in the art the term "hyaluronic acid" also includes inorganic salts thereof, such as sodium hyaluronate, potassium hyaluronate, ammonium hyaluronate, magnesium hyaluronate, calcium hyaluronate, zinc hyaluronate, cobalt hyaluronate, and the like.

The term "Polydispersity (PD)" as used herein characterizes in the art the width of the molecular weight distribution curve in terms of the ratio of the weight average molecular weight Mw, which is the total weight of all polymer molecules in the sample divided by the total number of molecules, to the number average molecular weight Mn, which is the weight average molecular weight of each molecule multiplied by its corresponding molecular weight, i.e., the weight statistically averaged molecular weight. In general, a smaller PD value indicates a narrower molecular weight distribution range. Further, the term "viscosity average molecular weight" (Mv) is an average molecular weight measured by a solution viscosity method, which is measured, for example, see CN 103124558B.

The respective average molecular weights (e.g., weight average molecular weight, number average molecular weight, and viscosity average molecular weight) of hyaluronic acid can be determined using standard methods in the art, such as Ueno et al, 1988, chem. pharm. Bull.36, 4971-4975; wyatt, 1993, anal.chim.acta 272: 1 to 40; and those described by Wyatt Technologies, 1999, Light carving University DAWN Course Manual and DAWN EOS Manual, Wyatt Technology Corporation, Santa Barbara, California.

In some embodiments, the hyaluronic acid of the invention has a molecular weight in the range of 5kDa to 3000kDa, in particular a weight average molecular weight of 5kDa to 3000kDa, a number average molecular weight of 5kDa to 3000kDa and a viscosity average molecular weight of 5kDa to 3000kDa, preferably a weight average molecular weight of 30kDa to 1000kDa, a number average molecular weight of 20kDa to 150kDa and a viscosity average molecular weight of 30kDa to 600kDa, with Mw/Mn between 7 and 15.

In some embodiments, the hyaluronic acid of the invention has a polydispersity of 7-15. In some embodiments, the hyaluronic acid of the invention has a polydispersity of 8-12.

The hyaluronic acid-containing cosmetic composition of the present invention can be used as an active ingredient of cosmetics to achieve skin care. The cosmetic containing hyaluronic acid of the present invention is not particularly limited, and examples thereof include a toner, a cream, a face lotion, a pack, a gel, and an external soft capsule formulation, such as a cleanser for skin, a toner, a vanishing cream, a cold cream, an emulsion, a pack, a foundation, a nail cosmetic such as a blusher, a lipstick, and a nail dresser, an eye cosmetic such as mascara, a shampoo for hair, a hair conditioner, a hair dresser, a shaving lotion, and a tooth brushing agent.

Examples

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

Analytical method

Molecular weight range, weight average molecular weight (Mw), number average molecular weight (Mn), viscosity average molecular weight (Mv), and dispersion coefficient (PD) of hyaluronic acid product: the measurement temperature was 30 ℃ and the mobile phase was 0.1M sodium nitrate ultrapure water solution, and the flow rate was 0.6mL/min, using a type 1260 Gel Permeation Chromatograph (GPC) from Agilent and a G1362A differential detector, and the measurement was performed according to the size exclusion method of "Chinese pharmacopoeia" 2015 edition. The weight average molecular weight (Mw), the number average molecular weight (Mn), the viscosity average molecular weight (Mv) and the dispersion coefficient (PD) of the obtained product are calculated by GPC software by using the dextrans with different molecular weights as standard substances and drawing a standard curve.

Viscosity of 1% aqueous solution: the viscosity of the aqueous solution was measured by a rotor-type rotational viscometer according to the viscometry in the Chinese pharmacopoeia 2015 edition to have a mass fraction of 1 w/v%.

Example 1 preliminary screening

In order to develop a better performing hyaluronic acid product, the inventors first prepared HA in different PD and molecular weight ranges.

Preparation of HA

The hyaluronic acid is prepared by fermenting and purifying streptococcus zooepidemicus (Stertococcus zoepidemicus, purchased from ATCC, with the catalog number of HA-116ATCC39920), and the specific method is as follows:

culture medium

Slant medium (g/L): heart brain extract powder (BHI)36, glucose 12, yeast powder 12, agar powder 24, pH 7.2.

Seed medium (g/L): 40 parts of glucose, 20 parts of yeast powder, 2.0 parts of magnesium sulfate heptahydrate, 0.1 part of manganese sulfate tetrahydrate, 2.0 parts of monopotassium phosphate and 20 parts of calcium carbonate, and the pH value is 7.0.

Fermentation medium (g/L): 20 parts of yeast powder, 6.2 parts of disodium hydrogen phosphate, 1.3 parts of potassium sulfate, 140 parts of glucose, 2.0 parts of magnesium sulfate heptahydrate and 7.0 parts of pH.

Culture method

Slant culture: the inoculated slant is placed in a constant temperature incubator with the temperature of 37 plus or minus 0.2 ℃ for 16 plus or minus 0.5h for shake flask inoculation.

Seed culture: inoculating the cultured slant seeds into a 500mL triangular flask filled with 50mL seed culture medium for culture at the rotating speed of a shaking table of 200r/min at the temperature of 37 +/-0.2 ℃ for 14-16 h.

Fermentation culture: inoculating the seed culture medium into a full-automatic fermentation tank KFT-7L (Biostat B2 nd generation 1000009844, Sartorius, Germany) according to the inoculation amount of 10%, filling 3.5L of the fermentation culture medium into the tank, stirring at the rotating speed of 200 +/-10 r/min, ventilating at the air flow of 1.0vvm and the temperature of 37 +/-0.2 ℃, detecting the pH on line by utilizing a pH electrode, and adding 5mol/L NaOH solution by an automatic feeding pump to adjust so as to maintain the pH change within 7.0 +/-0.1. And (3) detecting the dissolved oxygen concentration on line by using a dissolved oxygen electrode, and sampling once every 2h for detection and analysis.

Extracting and purifying

Taking a proper amount of fermentation liquor, placing the fermentation liquor in a stainless steel tank (a discharge port is connected with a plate-and-frame filter), adding water until the concentration is about 0.5%, stirring the mixed liquor at the stirring speed of 200 +/-10 r/min and the temperature of 60 ℃, mixing for about 30 minutes, after uniform mixing, adding 6mol/L HCl to adjust the pH value to 1.5-2.6, then continuing stirring at the temperature, and adjusting the stirring and discharging time (about 2-13 h) according to the table 1 so as to keep the stirring state in the tank during discharging. Discharging, filtering until the light transmittance of the filtrate reaches 99.9% (550nm) to obtain mixed filtrate O1, placing in a stainless tank, slowly adding 5mol/L NaOH to adjust the pH to 7, keeping stirring at the stirring speed of 200 +/-10 r/min to obtain mixed filtrate O2. The mixed filtrate O2 was taken, precipitated with 70% ethanol, and stirred at a rate of 50. + -. 5r/min to obtain a mixed solution O3. Taking the mixed liquid O3, centrifuging for 1 hour at the speed of 4000r/min, discarding the supernatant, taking the precipitate of the lower layer, drying, turning the plate every 2 hours, taking the dried sample to measure the drying weight loss, drying to constant weight, crushing, and sieving by a sieve of 80 meshes to obtain 4 hyaluronic acid products: a (Mw 1460kDa, Mn 97.8kDa, PD 8.86), E (Mw 1255kDa, Mn 1014kDa, PD 1.24), K (Mw 60.4kDa, Mn 31.4kDa, PD 1.92).

TABLE 1

Product numbering	pH	Mixing time (h)	Discharge time (h)
				A	2.5	3	12
C	2.3	4	6
				E	2.6	4.5	0.8
K	1.8	10	1
				B	2.4	3	4
D	2.5	3	8
				H	2.0	8	0.8

Each product was formulated as a cream according to table 2, and a blank control cream was formulated.

TABLE 2

The preparation method of the blank cream comprises the following steps: weighing xanthan gum and water, fully dissolving and uniformly mixing, adding EDTA disodium, heating to about 85 ℃ to serve as phase A, weighing cyclopentadimethylsiloxane, isononyl isononanoate, PEG-6 stearate, PEG-32 stearate and glycerol stearate, heating to about 90 ℃ to completely dissolve, serving as phase B, slowly adding phase B into phase A at a similar temperature, adding while stirring, homogenizing for 3-5 minutes, stirring and cooling, and adding a methylchloroisothiazolinone, methylisothiazolinone, magnesium nitrate, magnesium chloride mixture and triethanolamine when the temperature is lower than 50 ℃.

Hyaluronic acid prepared according to table 1 was added to the blank control cream in different proportions for short-term stratum corneum moisture content and transdermal moisture loss testing.

15 healthy women of 25 to 60 years old were selected as subjects and the effect of the administration of the product of the present invention was evaluated. The study was carried out at 21. + -. 1 ℃ and 50. + -. 5% humidity. Test areas (3cm x 3cm coated area per product) and control areas were selected on the face, respectively. Subjects were asked to not wash their face for at least 8 hours prior to taking the measurement and not to apply any product to the area involved in the test. The test and control areas were evaluated for short term stratum corneum moisture content at the beginning of the study and 30min and 120min after product application. The amount of product and blank control cream used remained the same for each test.

Skin surface hydration was evaluated by stratum corneum moisture measurement using a corneometer (corneometer, Courage & Khazaka). Skin stratum corneum moisture measurements measure the capacitance of the stratum corneum and thus reflect the relative stratum corneum moisture content. The results of the measurements are shown in fig. 1, and the product No. C, K has excellent skin moisturization capacity in the short term.

Referring to this preliminary screening range, 3 more products were prepared: b (Mw 1374kDa, Mn 319kDa, PD 4.3), D (Mw 1012kDa, Mn 98.2kDa, PD 10.3) and H (Mw 255kDa, Mn 189kDa, PD 1.35). For product B, D, H and E prepared above, the transdermal water loss was measured before, 30 minutes, and 120 minutes of use in 20 women. Evaluation of the skin moisture protection layer was performed using the percutaneous water loss (TEWL) test. The transcutaneous water loss (TEWL) test measures the water loss value and thus reflects the water lost to the outside of the body. The measurement was carried out using a skin moisture loss tester, Tewameter TM300(Courage & Khazaka). The greater the TEWL value, the more water lost to the body and the weaker the skin barrier function. The results of the measurements are shown in fig. 2, and the product No. B, D has excellent capability of reducing the loss of moisture through skin in the short term.

Through preliminary screening, C, K, B, D, four products with excellent short-term effects were selected. Next, the permeability of these four products was compared with that of ordinary commercially available low molecular weight HA and high molecular weight HA.

The permeability of the four products was measured by human reconstituted epidermis model (RHE Skinethic Laboratories, France) and compared with commercially available low molecular HA (commercially available product 1, Mw 255kDa, PD 1.35) and high molecular HA (commercially available product 2, Mw 1805kDa, PD 1.5) and the components in the skin model were measured by ELISA, and the results showed that the permeability of the four products was better than that of the commercially

available products

1 and 2 at equivalent concentrations at 0.5% and 1.0% in 24 hours (table 3).

TABLE 3 results of various HA Permeability tests

The efficacy of the four products is further confirmed by cell tests.

1) Test for promoting cell metabolism

The effects of these four products on cell metabolism were measured in vitro. Cellular metabolism was evaluated by the amount of protein synthesized by the cells after HA treatment, and the higher the amount of protein synthesized, the stronger the metabolism.

1mL of a solution having a density of 5X 10⁵one/mL suspension of keratinocytes (PC2011, Cat. No. PC3011, Guangdong Boxi Biotech Co., Ltd.) was seeded into wells of 12-well plates at 37 ℃ in 5% CO₂Culturing in a constant temperature incubator (Shanghai-Hengchun scientific instruments Co., Ltd., BPH-9162) for 24h to allow the cells to be attached to the wall completely. These 4 products were diluted to 0.6% in culture medium (IMDM with 20mM glutamine, 10% fetal bovine serum) and a dilution without the HA to be tested was used as a negative control. 1mL of each of the different HA and control dilutions was added to the corresponding sample and blank well plates to obtain a final concentration of 0.3% of sample solution before cell culture. After further culturing for 48h, the cells were taken upThe serum was tested for protein synthesis. The amount of protein synthesized was determined by colorimetry (Folin's phenol reaction). Under alkaline condition, copper ions and protein form chelate to catalyze tyrosine and tryptophan residue to be oxidized to generate blue compound, and the intensity of the color represents the protein concentration. The results are expressed in mg protein/ml and are shown in Table 4.

TABLE 4 results of different HA accelerated cell metabolism tests

Grouping	Total protein (mg/mL)	100X Total protein (mg/mL)
			Blank group	0.94	94.39
C	1.08	107.68
			K	1.04	103.67
B	0.98	98.16
			D	1.06	106.08

The result shows that the product C, D, K has excellent effect of promoting cell metabolism.

2) Damaged cell repair assay

The anti-damaging effects of these four products on cells were measured in vitro. 10 μ L of the mixture was mixed at a density of 5X 10⁵Fibroblasts (PC2031, Cat. No. PC3031, Guangdong Boxi Biotech Co., Ltd.) were seeded at 37 ℃ in wells of a 96-well plate at 5% CO₂Culturing in a constant temperature incubator (SANYO, MCO-15AC) with 20mM glutamine added in IMDM and 10% fetal calf serum for 24 h. Adding 1.6mM hydrogen peroxide and 2% HA to be detected into a fresh culture medium simultaneously to serve as an experimental group, independently adding 1.6mM hydrogen peroxide to serve as a positive control and untreated cells to serve as a negative control, adding 10 mu L of sample liquid and 10 mu L of positive and negative control liquid into the culture liquid of the pore plates of the corresponding sample group and the corresponding control group respectively (the final concentration of the sample is 1 percent at the moment), continuing to culture for 24h, adding 20 mu L of MTT (Mecanum Biochemical technology Co., Ltd., Shanghai, C10069452) into each pore, removing the culture medium after culturing for 4h, adding 150 mu L of DMSO (Tianjin Mao Daimao chemical reagent factory, 20170301), and detecting the absorbance value under the wavelength of 490nm by using a microplate reader (Thermo, MULTISKAN GO). Cell viability is expressed as a percentage. Experiment the test was repeated 3 times to take the average. The results are shown in Table 5.

Table 5 different HA repair test results on damaged cells

The results show that product C, D, K has excellent effect of repairing damaged cells.

3) Elastin synthesis assay

The effect of these four products on elastin synthesis was measured in vitro. The amount of elastin extracted from the biological source is determined by quantitative dye binding to the basic and non-polar amino acid sequences of mammalian elastin. The dye used is 5,10,15, 20-tetraphenyl-23, 23-porphine Tetrasulfonate (TPPS), which is useful for measuring three forms of elastin: 1) soluble recombinant elastin; 2) a lathyrus type elastin; 3) insoluble elastin, a component after solubilization of elastin polypeptide, [ alpha-elastin; kappa-elastin ].

1mL of a solution having a density of 1.5X 10⁶one/mL fibroblast (PC2031, Cat. No. PC3031, Guangdong Boxi Biotech Co., Ltd.) suspension was seeded into wells of 12-well plates at 37 ℃ with 5% CO₂Culturing in a constant temperature incubator (Shanghai-Hengchun scientific instruments Co., Ltd., BPH-9162) for 24 h. These 4 products were diluted to 2% in medium (IMDM plus 20mM glutamine, 10% fetal bovine serum) and an equal volume of complete medium was added to the blank. 1mL of different HA and control dilutions were added to the corresponding sample and blank well plates to obtain a final concentration of 1% sample solution followed by cell culture. After further culturing for 24 hours, cell supernatants were collected and assayed for elastin using elastin kit (CMS50691.1, Shanghai Jiwei Biotech Co., Ltd.). The results are shown in Table 6.

TABLE 6 results of different HA vs elastin synthesis assays

The results show that the product C, D has excellent effect of promoting elastin synthesis.

Through the above screening and verification, we initially selected product C (Mw 867kDa, Mn 97.8kDa, Mv 482kDa, PD 8.86, viscosity of 1% aqueous solution 730mpa.s), D (Mw 1012kDa, Mn 98.2kDa, Mv 540kDa, PD 10.3, viscosity of 1% aqueous solution 910mpa.s), and respectively named it as cube3 instant wawa and difference showa, and the chromatograms are shown in fig. 3-4. C, D hyaluronic acid was added to the blank control cream at a concentration of 0.5 w/w% to give products 1-2.

Effect example 1: product 1 in vivo efficacy evaluation

Short term stratum corneum moisture content and transdermal water loss

The moisture content of the stratum corneum and the percutaneous water loss after application of product 1 were evaluated in the same way as in the preliminary screening. The results are shown in fig. 5-6, and it can be seen that product 1 significantly improved the stratum corneum moisture content in 30 and 120 minutes. Product 1 significantly reduced transdermal water loss after 30 minutes of administration, with a more significant effect after 120 minutes.

Skin elasticity, smoothness, number of wrinkles and depth of wrinkles test

10 healthy women of 30-50 years old were selected as subjects and the effect of the administration of the product of the present invention was evaluated. The study was carried out at 21. + -. 1 ℃ and 50. + -. 5% humidity. Test and control areas (3cm x 3cm each) were selected on the face. Subjects were asked to not wash their face for at least 8 hours prior to taking the measurement and not to apply any product to the area involved in the test. Skin elasticity, smoothness, number of wrinkles were evaluated on the test and control areas at the beginning of the study and 0.5 hours (T0.5), 2 hours (T2) and 4 hours (T4) after use of the product. The amount of product and blank control cream used remained the same for each test.

The apparatus used for measuring the skin elasticity is Cutomer dual MPA580, and the method is an instrumental method.

The skin smoothness was determined using Visioscan as the instrument and SeSm (surface evaluation of smoothness) values as the instrument.

Skin wrinkle number test Visioscan was used, the method being instrumental.

The results are shown in FIGS. 7-9. The use of product 1 significantly improves skin elasticity and smoothness and reduces the number of wrinkles.

Professional doctor scoring

10 healthy women of 30-50 years old were selected as subjects and the effect of the administration of the product of the present invention was evaluated. The study was carried out at 21. + -. 1 ℃ and 50. + -. 5% humidity. Test and control areas (3cm x 3cm each) were selected on the face, respectively. Subjects were asked not to wash their face and not to apply product to the area involved in the test for at least 8 hours prior to taking the measurement. The skin condition of the subjects was scored by the same dermatologist at the beginning of the study and 30min (T0.5), 2h (T2) and 4h (T4) after use of the product, with scores of 1-4 corresponding to the following:

fraction 1: no lifting action: the skin was relaxed and fine lines were visible;

and 2, fraction: there is a slight lifting action: the skin was relaxed and the fine lines visible were reduced;

score 3: moderate lifting effect: the skin is relaxed, and visible fine wrinkles are obviously reduced;

and 4, fraction: has excellent pulling effect: the skin is lifted and fine wrinkles are not seen.

The results are shown in figure 10, where product 1 had a lifting effect on the skin compared to the control.

Elastin assay

The effect of cube3 on elastin synthesis was measured in vitro using the same method as the primary screen. As a result, as shown in table 7, cube3 exhibited an excellent elastin synthesis-promoting effect.

TABLE 7

Effect example 2: product 2 efficacy evaluation

Long term stratum corneum moisture content

20 healthy women of 30-50 years old were selected as subjects and the long-term stratum corneum moisture content of the skin after application of the product of the invention was evaluated. The study was carried out at 21. + -. 1 ℃ and 50. + -. 5% humidity. Test and control areas (3cm x 3cm coated area) were selected on the face, respectively. The subjects were asked to not apply the product to the area involved in the test for at least 1 day prior to taking the measurement. The test and control areas were coated once daily and the short term stratum corneum moisture content was assessed at the beginning of the

study

15, 30 and 60 days after each test sample was used. Evaluation was performed in the same manner as in the primary screening. The amount of product and blank control cream used remained the same for each test.

The results are shown in FIG. 11. It can be seen that the water content of the stratum corneum was significantly improved 15 days after application of product 2.

Skin elasticity, smoothness, number and depth of wrinkles, skin scale test

20 healthy women of 30-50 years old were selected as subjects and the effect of the administration of the product of the present invention was evaluated. The study was carried out at 21. + -. 1 ℃ and 50. + -. 5% humidity. Test and control areas were selected separately on the face (3cm x 3cm coated area per product). The subjects were asked to not apply the product to the area involved in the test for at least 1 day prior to taking the measurement. The test and control areas were coated once a day and evaluated for skin elasticity, smoothness, number of wrinkles and wrinkle depth and skin scale at the start of the study after 15, 30 and 60 days using each test sample. The evaluation of skin elasticity, smoothness, number of wrinkles and depth of wrinkles was performed in the same manner as in effect example 1. The amount of product and blank control cream used remained the same for each test.

The assessment of skin scales was carried out by instrumental methods, using Visioscan to analyze the surface scales SESc (surface evaluation of scaliness). The principle of the method is as follows: the skin surface is shot by a skin mirror consisting of a uniform annular ultraviolet light (UVA) illuminating light source and a black and white high-resolution CCD camera, the image is transmitted to a host computer for digital processing, and the host computer is connected with a PC (personal computer) through a USB (universal serial bus) line to input the skin image into software for analysis. The image is displayed with gray scale values ranging from 0 to 255, wrinkles appear dark black in the image phase, the exfoliated stratum corneum appears bright white, and the software can analyze various parameters of the skin surface. The unique high-resolution ultraviolet light shooting system is used for directly shooting the surface of the active skin and analyzing the surface morphology, the roughness and the dryness degree of the skin, and the method is a method which is simple, economical and accurate to operate in the efficacy test of the cosmetics. Also can be used for testing the efficacy of skin drugs and objective clinical diagnosis of dermatologists.

The results are shown in FIGS. 12-17. Application of product 2 significantly improved skin elasticity and smoothness and reduced the number and depth of wrinkles. Moreover, product 2 is able to significantly reduce the phenomenon of desquamation of the skin.

Professional doctor scoring

20 healthy women of 30 to 50 years old were selected as subjects, and the effects 15 days, 30 days, and 60 days after the application of the product 2 were evaluated by the same professional dermatologist according to a method similar to effect example 1. The scores are shown in fig. 18A-18C. Compared to the blank control, the dermatologist confirmed that the cream containing 0.5% of the HA of the present invention had visibly distinct smoothing, wrinkle-reducing, and firming effects.

Damaged cell repair assay

The anti-damage effect of difference showa on cells was measured in vitro in the same manner as in the primary screening. As a result, as shown in Table 8, difference Showa has an excellent effect of repairing damaged cells.

TABLE 8

In conclusion, the HA prepared according to the present invention can improve the moisture content of the stratum corneum for a short and long period of time, can remarkably improve the elasticity and smoothness of the skin, and reduce the number and depth of wrinkles, and can also remarkably reduce the desquamation phenomenon of the skin. In-vitro cell experiments prove that the HA prepared by the invention also HAs excellent effects of promoting elastin synthesis and repairing damaged cells.

Claims

1. A hyaluronic acid having a weight average molecular weight of 30kDa to 3000kDa, a number average molecular weight of 20kDa to 150kDa, and a viscosity average molecular weight of 30kDa to 600kDa, and having a polydispersity index of 8 to 12.

2. The hyaluronic acid of claim 1, which has a weight average molecular weight of between 30kDa and 1000 kDa.

3. The hyaluronic acid of claim 1, which has a polydispersity of 8.86.

4. The hyaluronic acid of claim 1, which has a polydispersity of 10.3.

5. The hyaluronic acid of any of claims 1-4, having a viscosity of 700-1000 mPa.s in a 1% aqueous solution.

6. Cosmetic composition comprising hyaluronic acid according to any of claims 1-5.

7. Use of the hyaluronic acid of any of claims 1-5 for the manufacture of a cosmetic product.